Synthesis of N-peptide-6-amino-D-luciferin Conjugates

PubMed Central

Kovács, Anita K.; Hegyes, Péter; Szebeni, Gábor J.; Nagy, Lajos I.; Puskás, László G.; Tóth, Gábor K.

2018-01-01

A general strategy for the synthesis of N-peptide-6-amino-D-luciferin conjugates has been developed. The applicability of the strategy was demonstrated with the preparation of a known substrate, N-Z-Asp-Glu-Val-Asp-6-amino-D-luciferin (N-Z-DEVD-aLuc). N-Z-DEVD-aLuc was obtained via a hybrid liquid/solid phase synthesis method, in which the appropriately protected C-terminal amino acid was coupled to 6-amino-2-cyanobenzothiazole and the resulting conjugate was reacted with D-cysteine in order to get the protected amino acid-6-amino-D-luciferin conjugate, which was then attached to resin. The resulting loaded resin was used for the solid-phase synthesis of the desired N-peptide-6-amino-D-luciferin conjugate without difficulties, which was then attested with NMR spectroscopy and LC-MS, and successfully tested in a bioluminescent system. PMID:29725588

Synthesis of N-peptide-6-amino-D-luciferin Conjugates.

PubMed

Kovács, Anita K; Hegyes, Péter; Szebeni, Gábor J; Nagy, Lajos I; Puskás, László G; Tóth, Gábor K

2018-01-01

A general strategy for the synthesis of N -peptide-6-amino-D-luciferin conjugates has been developed. The applicability of the strategy was demonstrated with the preparation of a known substrate, N -Z-Asp-Glu-Val-Asp-6-amino-D-luciferin ( N -Z-DEVD-aLuc). N -Z-DEVD-aLuc was obtained via a hybrid liquid/solid phase synthesis method, in which the appropriately protected C-terminal amino acid was coupled to 6-amino-2-cyanobenzothiazole and the resulting conjugate was reacted with D-cysteine in order to get the protected amino acid-6-amino-D-luciferin conjugate, which was then attached to resin. The resulting loaded resin was used for the solid-phase synthesis of the desired N -peptide-6-amino-D-luciferin conjugate without difficulties, which was then attested with NMR spectroscopy and LC-MS, and successfully tested in a bioluminescent system.

Derivatization of peptides as quaternary ammonium salts for sensitive detection by ESI-MS.

PubMed

Cydzik, Marzena; Rudowska, Magdalena; Stefanowicz, Piotr; Szewczuk, Zbigniew

2011-06-01

A series of model peptides in the form of quaternary ammonium salts at the N-terminus was efficiently prepared by the solid-phase synthesis. Tandem mass spectrometric analysis of the peptide quaternary ammonium derivatives was shown to provide sequence confirmation and enhanced detection. We designed the 2-(1,4-diazabicyclo[2.2.2] octylammonium)acetyl quaternary ammonium group which does not suffer from neutral losses during MS/MS experiments. The presented quaternization of 1,4-diazabicyclo[2.2.2]octane (DABCO) by iodoacetylated peptides is relatively easy and compatible with standard solid-phase peptide synthesis. This methodology offers a novel sensitive approach to analyze peptides and other compounds. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.

Comparison of the solid-phase fragment condensation and phase-change approaches in the synthesis of salmon I calcitonin.

PubMed

Gatos, D; Tzavara, C

2001-02-01

Salmon I calcitonin was synthesized using both phase-change and conventional solid-phase fragment condensation (SPFC) approaches, utilizing the Rink amide linker (Fmoc-amido-2,4-dimethoxybenzyl-4-phenoxyacetic acid) combined with 2-chlorotrityl resin and the Fmoc/tBu(Trt)-based protection scheme. Phase-change synthesis, performed by the selective detachment of the fully protected C-terminal 22-mer peptide-linker from the resin and subsequent condensation in solution with the N-terminal 1-10 fragment, gave a product of slightly less purity (85 vs. 92%) than the corresponding synthesis on the solid-phase. In both cases salmon I calcitonin was easily obtained in high purity.

Application of 2-chlorotrityl resin in solid phase synthesis of (Leu15)-gastrin I and unsulfated cholecystokinin octapeptide. Selective O-deprotection of tyrosine.

PubMed

Barlos, K; Gatos, D; Kapolos, S; Poulos, C; Schäfer, W; Yao, W Q

1991-12-01

The carboxyl terminal dipeptide amide, Fmoc-Asp-Phe-NH2, of gastrin and cholecystokinin (CCK) has been attached in high yield through its free side chain carboxyl group to the acid labile 2-chlorotrityl resin. The obtained peptide resin ester has been applied in the solid phase synthesis of partially protected (Leu15)-gastrin I utilising Fmoc-amino acids. Quantitative cleavage of this peptide from resin, with the t-butyl type side chain protection intact is achieved using mixtures of acetic acid/trifluoroethanol/dichloromethane. Under the same conditions complete detritylation of the tyrosine phenoxy function occurs simultaneously. Thus, the solid-phase synthesis of peptides selectively deprotected at the side chain of tyrosine is rendered possible by the use of 2-chlorotrityl resin and Fmoc-Tyr(Trt)-OH. The efficiency of this approach has been proved by the subsequent high-yield synthesis of three model peptides and the CCK-octapeptide.

Microwave-assisted solid-phase peptide synthesis of the 60-110 domain of human pleiotrophin on 2-chlorotrityl resin.

PubMed

Friligou, Irene; Papadimitriou, Evangelia; Gatos, Dimitrios; Matsoukas, John; Tselios, Theodore

2011-05-01

A fast and efficient microwave-assisted solid phase peptide synthesis (MW-SPPS) of a 51mer peptide, the main heparin-binding site (60-110) of human pleiotrophin (hPTN), using 2-chlorotrityl chloride resin (CLTR-Cl) following the 9-fluorenylmethyloxycarbonyl/tert-butyl (Fmoc/tBu) methodology and with the standard N,N'-diisopropylcarbodiimide/1-hydroxybenzotriazole (DIC/HOBt) coupling reagents, is described. An MW-SPPS protocol was for the first time successfully applied to the acid labile CLTR-Cl for the faster synthesis of long peptides (51mer peptide) and with an enhanced purity in comparison to conventional SPPS protocols. The synthesis of such long peptides is not trivial and it is generally achieved by recombinant techniques. The desired linear peptide was obtained in only 30 h of total processing time and in 51% crude yield, in which 60% was the purified product obtained with 99.4% purity. The synthesized peptide was purified by reversed phase high performance liquid chromatography (RP-HPLC) and identified by electrospray ionization mass spectrometry (ESI-MS). Then, the regioselective formation of the two disulfide bridges of hPTN 60-110 was successfully achieved by a two-step procedure, involving an oxidative folding step in dimethylsulfoxide (DMSO) to form the Cys(77)-Cys(109) bond, followed by iodine oxidation to form the Cys(67)-Cys(99) bond.

The Use of Aryl Hydrazide Linkers for the Solid Phase Synthesis of Chemically Modified Peptides

DOE Office of Scientific and Technical Information (OSTI.GOV)

Woo, Y; Mitchell, A R; Camarero, J A

2006-11-03

Since Merrifield introduced the concept of solid phase synthesis in 1963 for the rapid preparation of peptides, a large variety of different supports and resin-linkers have been developed that improve the efficiency of peptide assembly and expand the myriad of synthetically feasible peptides. The aryl hydrazide is one of the most useful resin-linkers for the synthesis of chemically modified peptides. This linker is completely stable during Boc- and Fmoc-based solid phase synthesis and yet it can be cleaved under very mild oxidative conditions. The present article reviews the use of this valuable linker for the rapid and efficient synthesis ofmore » C-terminal modified peptides, head-to-tail cyclic peptides and lipidated peptides.« less

Synthesis of peptide .alpha.-thioesters

DOEpatents

Camarero, Julio A [Livermore, CA; Mitchell, Alexander R [Livermore, CA; De Yoreo, James J [Clayton, CA

2008-08-19

Disclosed herein is a new method for the solid phase peptide synthesis (SPPS) of C-terminal peptide .alpha. thioesters using Fmoc/t-Bu chemistry. This method is based on the use of an aryl hydrazine linker, which is totally stable to conditions required for Fmoc-SPPS. When the peptide synthesis has been completed, activation of the linker is achieved by mild oxidation. The oxidation step converts the acyl-hydrazine group into a highly reactive acyl-diazene intermediate which reacts with an .alpha.-amino acid alkylthioester (H-AA-SR) to yield the corresponding peptide .alpha.-thioester in good yield. A variety of peptide thioesters, cyclic peptides and a fully functional Src homology 3 (SH3) protein domain have been successfully prepared.

Strategies for the solid-phase diversification of poly-L-proline-type II peptide mimic scaffolds and peptide scaffolds through guanidinylation.

PubMed

Flemer, Stevenson; Wurthmann, Alexander; Mamai, Ahmed; Madalengoitia, José S

2008-10-03

A strategy for the solid-phase diversification of PPII mimic scaffolds through guanidinylation is presented. The approach involves the synthesis N-Pmc-N'-alkyl thioureas as diversification reagents. Analogues of Fmoc-Orn(Mtt)-OH can be incorporated into a growing peptide chain on Wang resin. Side chain deprotection with 1% TFA/CH2Cl2 followed by EDCI-mediated reaction of N-Pmc-N'-alkyl thioureas with the side chain amine affords arginine analogues with modified guanidine head groups. The scope, limitations, and incidental chemistry are discussed.

Solid-Phase Synthesis of Difficult Purine-Rich PNAs through Selective Hmb Incorporation: Application to the Total Synthesis of Cell Penetrating Peptide-PNAs.

PubMed

Tailhades, Julien; Takizawa, Hotake; Gait, Michael J; Wellings, Don A; Wade, John D; Aoki, Yoshitsugu; Shabanpoor, Fazel

2017-01-01

Antisense oligonucleotide (ASO)-based drug development is gaining significant momentum following the recent FDA approval of Eteplirsen (an ASO based on phosphorodiamidate morpholino) and Spinraza (2'- O -methoxyethyl-phosphorothioate) in late 2016. Their attractiveness is mainly due to the backbone modifications which have improved the in vivo characteristics of oligonucleotide drugs. Another class of ASO, based on peptide nucleic acid (PNA) chemistry, is also gaining popularity as a platform for development of gene-specific therapy for various disorders. However, the chemical synthesis of long PNAs, which are more target-specific, remains an ongoing challenge. Most of the reported methodology for the solid-phase synthesis of PNA suffer from poor coupling efficiency which limits production to short PNA sequences of less than 15 residues. Here, we have studied the effect of backbone modifications with Hmb (2-hydroxy-4-methoxybenzyl) and Dmb (2,4-dimethoxybenzyl) to ameliorate difficult couplings and reduce "on-resin" aggregation. We firstly synthesized a library of PNA dimers incorporating either Hmb or Dmb and identified that Hmb is superior to Dmb in terms of its ease of removal. Subsequently, we used Hmb backbone modification to synthesize a 22-mer purine-rich PNA, targeting dystrophin RNA splicing, which could not be synthesized by standard coupling methodology. Hmb backbone modification allowed this difficult PNA to be synthesized as well as to be continued to include a cell-penetrating peptide on the same solid support. This approach provides a novel and straightforward strategy for facile solid-phase synthesis of difficult purine-rich PNA sequences.

Solid-phase synthesis of self-assembling multivalent π-conjugated peptides

DOE PAGES

Sanders, Allix M.; Kale, Tejaswini S.; Katz, Howard E.; ...

2017-02-07

Here, we present a completely solid-phase synthetic strategy to create three- and four-fold peptide-appended π-electron molecules, where the multivalent oligopeptide presentation is dictated by the symmetries of reactive handles placed on discotic π-conjugated cores. Carboxylic acid and anhydride groups were viable amidation and imidation partners, respectively, and oligomeric π-electron discotic cores were prepared through Pd-catalyzed cross-couplings. Due to intermolecular hydrogen bonding between the three or four peptide axes, these π-peptide hybrids self-assemble into robust one-dimensional nanostructures with high aspect ratios in aqueous solution. The preparation of these systems via solid-phase methods will be detailed along with their self-assembly properties, asmore » revealed by steady-state spectroscopy and transmission electron microscopy and electrical characterization using field-effect transistor measurements.« less

Solid-phase synthesis of difficult purine-rich PNAs through selective Hmb incorporation: Application to the total synthesis of cell penetrating peptide-PNAs

NASA Astrophysics Data System (ADS)

Tailhades, Julien; Takizawa, Hotake; Gait, Michael J.; Wellings, Don A.; Wade, John D.; Aoki, Yoshitsugu; Shabanpoor, Fazel

2017-10-01

Antisense oligonucleotide (ASO)-based drug development is gaining significant momentum following the recent FDA approval of Eteplirsen (an ASO based on phosphorodiamidate morpholino) and Spinraza (2’-O-methoxyethyl-phosphorothioate) in late 2016. Their attractiveness is mainly due to the backbone modifications which have improved the in vivo characteristics of oligonucleotide drugs. Another class of ASO, based on peptide nucleic acid (PNA) chemistry, is also gaining popularity as a platform for development of gene-specific therapy for various disorders. However, the chemical synthesis of long PNAs, which are more target-specific, remains an ongoing challenge. Most of the reported methodology for the solid-phase synthesis of PNA suffer from poor coupling efficiency which limits production to short PNA sequences of less than 15 residues. Here we have studied the effect of backbone modifications with Hmb (2-hydroxy-4-methoxybenzyl) and Dmb (2,4-dimethoxybenzyl) to ameliorate difficult couplings and reduce “on-resin” aggregation. We firstly synthesized a library of PNA dimers incorporating either Hmb or Dmb and identified that Hmb is superior to Dmb in terms of its ease of removal. Subsequently, we used Hmb backbone modification to synthesize a 22-mer purine-rich PNA, targeting dystrophin RNA splicing, which could not be synthesized by standard coupling methodology. Hmb backbone modification allowed this difficult PNA to be synthesized as well as to be continued to include a cell-penetrating peptide on the same solid support. This approach provides a novel and straightforward strategy for facile solid-phase synthesis of difficult purine-rich PNA sequences.

Solid-Phase Synthesis of Difficult Purine-Rich PNAs through Selective Hmb Incorporation: Application to the Total Synthesis of Cell Penetrating Peptide-PNAs

PubMed Central

Tailhades, Julien; Takizawa, Hotake; Gait, Michael J.; Wellings, Don A.; Wade, John D.; Aoki, Yoshitsugu; Shabanpoor, Fazel

2017-01-01

Antisense oligonucleotide (ASO)-based drug development is gaining significant momentum following the recent FDA approval of Eteplirsen (an ASO based on phosphorodiamidate morpholino) and Spinraza (2′-O-methoxyethyl-phosphorothioate) in late 2016. Their attractiveness is mainly due to the backbone modifications which have improved the in vivo characteristics of oligonucleotide drugs. Another class of ASO, based on peptide nucleic acid (PNA) chemistry, is also gaining popularity as a platform for development of gene-specific therapy for various disorders. However, the chemical synthesis of long PNAs, which are more target-specific, remains an ongoing challenge. Most of the reported methodology for the solid-phase synthesis of PNA suffer from poor coupling efficiency which limits production to short PNA sequences of less than 15 residues. Here, we have studied the effect of backbone modifications with Hmb (2-hydroxy-4-methoxybenzyl) and Dmb (2,4-dimethoxybenzyl) to ameliorate difficult couplings and reduce “on-resin” aggregation. We firstly synthesized a library of PNA dimers incorporating either Hmb or Dmb and identified that Hmb is superior to Dmb in terms of its ease of removal. Subsequently, we used Hmb backbone modification to synthesize a 22-mer purine-rich PNA, targeting dystrophin RNA splicing, which could not be synthesized by standard coupling methodology. Hmb backbone modification allowed this difficult PNA to be synthesized as well as to be continued to include a cell-penetrating peptide on the same solid support. This approach provides a novel and straightforward strategy for facile solid-phase synthesis of difficult purine-rich PNA sequences. PMID:29094037

A new strategy for the preparation of peptide-targeted technetium and rhenium radiopharmaceuticals. The automated solid-phase synthesis, characterization, labeling, and screening of a peptide-ligand library targeted at the formyl peptide receptor.

PubMed

Stephenson, Karin A; Banerjee, Sangeeta Ray; Sogbein, Oyebola O; Levadala, Murali K; McFarlane, Nicole; Boreham, Douglas R; Maresca, Kevin P; Babich, John W; Zubieta, Jon; Valliant, John F

2005-01-01

A new solid-phase synthetic methodology was developed that enables libraries of peptide-based Tc(I)/Re(I) radiopharmaceuticals to be prepared using a conventional automated peptide synthesizer. Through the use of a tridentate ligand derived from N-alpha-Fmoc-l-lysine, which we refer to as a single amino acid chelate (SAAC), a series of 12 novel bioconjugates [R-NH(CO)ZLF(SAAC)G, R = ethyl, isopropyl, n-propyl, tert-butyl, n-butyl, benzyl; Z = Met, Nle] that are designed to target the formyl peptide receptor (FPR) were prepared. Construction of the library was carried out in a multiwell format on an Advanced ChemTech 348 peptide synthesizer where multi-milligram quantities of each peptide were isolated in high purity without HPLC purification. After characterization, the library components were screened for their affinity for the FPR receptor using flow cytometry where the K(d) values were found to be in the low micromolar range (0.5-3.0 microM). Compound 5j was subsequently labeled with (99m)Tc(I) and the product isolated in high radiochemical yield using a simple Sep-Pak purification procedure. The retention time of the labeled compound matched that of the fully characterized Re-analogue which was prepared through the use of the same solid-phase synthesis methodology that was used to construct the library. The work reported here is a rare example of a method by which libraries of peptide-ligand conjugates and their rhenium complexes can be prepared.

A Laboratory Preparation of Aspartame Analogs Using Simultaneous Multiple Parallel Synthesis Methodology

ERIC Educational Resources Information Center

Qvit, Nir; Barda, Yaniv; Gilon, Chaim; Shalev, Deborah E.

2007-01-01

This laboratory experiment provides a unique opportunity for students to synthesize three analogues of aspartame, a commonly used artificial sweetener. The students are introduced to the powerful and useful method of parallel synthesis while synthesizing three dipeptides in parallel using solid-phase peptide synthesis (SPPS) and simultaneous…

Synthesis and binding studies of Alzheimer ligands on solid support.

PubMed

Rzepecki, Petra; Geib, Nina; Peifer, Manuel; Biesemeier, Frank; Schrader, Thomas

2007-05-11

Aminopyrazole derivatives constitute the first class of nonpeptidic rationally designed beta-sheet ligands. Here we describe a double solid-phase protocol for both synthesis and affinity testing. The presented solid-phase synthesis of four types of hybrid compounds relies on the Fmoc strategy and circumvents subsequent HPLC purification by precipitating the final product from organic solution in pure form. Hexa- and octapeptide pendants with internal di- and tetrapeptide bridges are now amenable in high yields to combinatorial synthesis of compound libraries for high-throughput screening purposes. Solid-phase peptide synthesis (SPPS) on an acid-resistant PAM allows us, after PMB deprotection, to subject the free aminopyrazole binding sites in an immobilized state to on-bead assays with fluorescence-labeled peptides. From the fluorescence emission intensity decrease, individual binding constants can be calculated via reference curves by simple application of the law of mass action. Gratifyingly, host/guest complexation can be monitored quantitatively even for those ligands, which are almost insoluble in water.

Synthesis of Sulfotyrosine-Containing Peptides by Incorporating Fluorosulfated Tyrosine Using an Fmoc-Based Solid-Phase Strategy.

PubMed

Chen, Wentao; Dong, Jiajia; Li, Suhua; Liu, Yu; Wang, Yujia; Yoon, Leonard; Wu, Peng; Sharpless, K Barry; Kelly, Jeffery W

2016-01-26

Tyrosine O-sulfation is a common protein post-translational modification that regulates many biological processes, including leukocyte adhesion and chemotaxis. Many peptides with therapeutic potential contain one or more sulfotyrosine residues. We report a one-step synthesis for Fmoc-fluorosulfated tyrosine. An efficient Fmoc-based solid-phase peptide synthetic strategy is then introduced for incorporating the fluorosulfated tyrosine residue into peptides of interest. Standard simultaneous peptide-resin cleavage and removal of the acid-labile side-chain protecting groups affords the crude peptides containing fluorosulfated tyrosine. Basic ethylene glycol, serving both as solvent and reactant, transforms the fluorosulfated tyrosine peptides into sulfotyrosine peptides in high yield. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Heat-enhanced peptide synthesis on Teflon-patterned paper.

PubMed

Deiss, Frédérique; Yang, Yang; Matochko, Wadim L; Derda, Ratmir

2016-06-14

In this report, we describe the methodology for 96 parallel organic syntheses of peptides on Teflon-patterned paper assisted by heating with an infra-red lamp. SPOT synthesis is an important technology for production of peptide arrays on a paper-based support for rapid identification of peptide ligands, epitope mapping, and identification of bio-conjugation reactions. The major drawback of the SPOT synthesis methodology published to-date is suboptimal reaction conversion due to mass transport limitations in the unmixed reaction spot. The technology developed in this report overcomes these problems by changing the environment of the reaction from static to dynamic (flow-through), and further accelerating the reaction by selective heating of the reaction support in contact with activated amino acids. Patterning paper with Teflon allows for droplets of organic solvents to be confined in a zone on the paper array and flow through the paper at a well-defined rate and provide a convenient, power-free setup for flow-through solid-phase synthesis and efficient assembly of peptide arrays. We employed an infra-red (IR) lamp to locally heat the cellulosic support during the flow-through delivery of the reagents to each zone of the paper-based array. We demonstrate that IR-heating in solid phase peptide synthesis shortened the reaction time necessary for amide bond formation down to 3 minutes; in some couplings of alpha amino acids, conversion rates increased up to fifteen folds. The IR-heating improved the assembly of difficult sequences, such as homo-oligomers of all 20 natural amino acids.

Synthesis and Primary Characterization of Self-Assembled Peptide-Based Hydrogels

PubMed Central

Nagarkar, Radhika P.; Schneider, Joel P.

2009-01-01

Summary Hydrogels based on peptide self-assembly form an important class of biomaterials that find application in tissue engineering and drug delivery. It is essential to prepare peptides with high purity to achieve batch-to-batch consistency affording hydrogels with reproducible properties. Automated solid-phase peptide synthesis coupled with optimized Fmoc (9-fluorenylmethoxycarbonyl) chemistry to obtain peptides in high yield and purity is discussed. Details of isolating a desired peptide from crude synthetic mixtures and assessment of the peptide’s final purity by high-performance liquid chromatography and mass spectrometry are provided. Beyond the practical importance of synthesis and primary characterization, techniques used to investigate the properties of hydrogels are briefly discussed. PMID:19031061

Automated Fmoc-based solid-phase synthesis of peptide thioesters with self-purification effect and application in the construction of immobilized SH3 domains.

PubMed

Mende, Franziska; Beisswenger, Michael; Seitz, Oliver

2010-08-18

Peptide thioesters are important building blocks in the total synthesis of proteins and protein domains via fragment ligation. However, synthetic access of peptide thioesters still is a bottleneck of this powerful ligation chemistry. The commonly used methods for the Fmoc-based synthesis of peptide thioesters involve nonautomated solution steps that have to be performed after the solid-phase assembly of the peptide. Usually, HPLC purification is required. Herein, a method that enables crude peptides to be used in divergent native chemical ligations reactions is described. We present an Fmoc-based solid-phase synthesis of peptide thioesters with self-purification which facilitates access to these important building blocks, since the often cumbersome HPLC purification can be avoided. Fmoc-protected amino acids are coupled on a safety catch sulfonamide resin. The self-purifying effect is achieved through the combination of (a) N-terminal coupling of a cleavable cyclization linker and subsequent backbone-to-side chain cyclization, (b) activation of the sulfonamide linkage by alkylation, (c) thiolysis for the selective detachment of truncation products, and (d) TFA cleavage for the liberation of the desired peptide thioester in unprotected form. We have previously shown a method wherein cyclization was performed after carboxymethylation of the sulfonamide. However, the automation of this method was difficult and side reactions at methionine residues hampered the general applicability. The new design involves peptide synthesis on a modified carboxy-functionalized sulfonamide linker, a substantially milder activation of the sulfonamide bond and the use of monomethoxytrityl as well as 2-phenyl-isopropyl protecting groups. This approach solved the problems with methionine containing peptides and enabled the complete automation of the self-purifying synthesis of peptide thioesters. The study also addressed problems in the synthesis of difficult peptides. Aggregated truncation products can resist extraction and contaminate full-length thioesters obtained after TFA cleavage. It is shown that significant enhancements of the purity were achieved when mild acidic extractions were included in the wash protocols after thiolysis. The potential of the method was demonstrated in the parallel synthesis of 20-40 amino acid long peptide thioesters, which were obtained in excellent purities. The thioesters and cysteinyl peptides were used without purification in the assembly of immobilized SH3 protein domains of SHO1 in yeast. A cysteine scan by native chemical ligation suggested single amino acid to cysteine substitutions that (a) confer useful ligation yields, (b) support correct folding, and (c) sustain the function of the folded protein domain. The chemical synthesis of the SH3-domain of SHO1 succeeded in highest yields when cysteine placements at positions S23, F24, and E36 were avoided. The synthetic SH3 mutants were examined in a binding assay, which indicated that N27C, L30C, and D34C mutations provide functional SH3-domain.

Synthesis of Mikto-Arm Star Peptide Conjugates.

PubMed

Koo, Jin Mo; Su, Hao; Lin, Yi-An; Cui, Honggang

2018-01-01

Mikto-arm star peptide conjugates are an emerging class of self-assembling peptide-based structural units that contain three or more auxiliary segments of different chemical compositions and/or functionalities. This group of molecules exhibit interesting self-assembly behavior in solution due to their chemically asymmetric topology. Here we describe the detailed procedure for synthesis of an ABC Mikto-arm star peptide conjugate in which two immiscible entities (a saturated hydrocarbon and a hydrophobic and lipophobic fluorocarbon) are conjugated onto a short β-sheet forming peptide sequence, GNNQQNY, derived from the Sup35 prion, through a lysine junction. Automated and manual Fmoc-solid phase synthesis techniques are used to synthesize the Mikto-arm star peptide conjugates, followed by HPLC purification. We envision that this set of protocols can afford a versatile platform to synthesize a new class of peptidic building units for diverse applications.

Total chemical synthesis of proteins without HPLC purification† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6sc01883a Click here for additional data file.

PubMed Central

Loibl, S. F.; Harpaz, Z.; Zitterbart, R.

2016-01-01

The total chemical synthesis of proteins is a tedious and time-consuming endeavour. The typical steps involve solid phase synthesis of peptide thioesters and cysteinyl peptides, native chemical ligation (NCL) in solution, desulfurization or removal of ligation auxiliaries in the case of extended NCL as well as many intermediary and final HPLC purification steps. With an aim to facilitate and improve the throughput of protein synthesis we developed the first method for the rapid chemical total on-resin synthesis of proteins that proceeds without a single HPLC-purification step. The method relies on the combination of three orthogonal protein tags that allow sequential immobilization (via the N-terminal and C-terminal ends), extended native chemical ligation and release reactions. The peptide fragments to be ligated are prepared by conventional solid phase synthesis and used as crude materials in the subsequent steps. An N-terminal His6 unit permits selective immobilization of the full length peptide thioester onto Ni-NTA agarose beads. The C-terminal peptide fragment carries a C-terminal peptide hydrazide and an N-terminal 2-mercapto-2-phenyl-ethyl ligation auxiliary, which serves as a reactivity tag for the full length peptide. As a result, only full length peptides, not truncation products, react in the subsequent on-bead extended NCL. After auxiliary removal the ligation product is liberated into solution upon treatment with mild acid, and is concomitantly captured by an aldehyde-modified resin. This step allows the removal of the most frequently observed by-product in NCL chemistry, i.e. the hydrolysed peptide thioester (which does not contain a C-terminal peptide hydrazide). Finally, the target protein is released with diluted hydrazine or acid. We applied the method in the synthesis of 46 to 126 amino acid long MUC1 proteins comprising 2–6 copies of a 20mer tandem repeat sequence. Only three days were required for the parallel synthesis of 9 MUC1 proteins which were obtained in 8–33% overall yield with 90–98% purity despite the omission of HPLC purification. PMID:28451120

Solid-phase synthesis of a nucleopeptide from the linking site of adenovirus-2 nucleoprotein, -Ser(p5'CATCAT)-Gly-Asp-. Convergent versus stepwise strategy.

PubMed Central

Robles, J; Pedroso, E; Grandas, A

1995-01-01

The synthesis of a nucleopeptide with the sequence -Ser(p5'CATCAT)-Gly-Asp- has been undertaken by either convergent or stepwise solid-phase strategies, both of which use base-labile permanent protecting groups. The coupling of phosphitylated protected peptides onto oligonucleotide-resins did not afford the desired nucleopeptide, which was nevertheless obtained after oligonucleotide elongation at the hydroxyl group of the resin-bound peptide and deprotection under mild basic conditions. A preliminary study on the stability of different nucleopeptides to bases is also reported. PMID:7479079

2-Methyltetrahydrofuran and cyclopentyl methyl ether for green solid-phase peptide synthesis.

PubMed

Jad, Yahya E; Acosta, Gerardo A; Khattab, Sherine N; de la Torre, Beatriz G; Govender, Thavendran; Kruger, Hendrik G; El-Faham, Ayman; Albericio, Fernando

2016-02-01

2-MeTHF and CPME were evaluated as greener alternatives for the most employed solvents in peptide synthesis. The ability of these solvents to dissolve amino acid derivatives and a range of coupling reagents were evaluated as well as the swelling of polystyrene and polyethylene glycol resins. In addition, racemization and coupling efficiencies were also determined. We concluded that the use of 2-MeTHF with combination of DIC/OxymaPure gave the lowest racemization level during stepwise synthesis of Z-Phg-Pro-NH2 and the highest purity during SPPS of Aib-enkephalin pentapeptide (H-Tyr-Aib-Aib-Phe-Leu-NH2).

Preparation of the very acid-sensitive Fmoc-Lys(Mtt)-OH. Application in the synthesis of side-chain to side-chain cyclic peptides and oligolysine cores suitable for the solid-phase assembly of MAPs and TASPs.

PubMed

Aletras, A; Barlos, K; Gatos, D; Koutsogianni, S; Mamos, P

1995-05-01

N alpha-9-Fluorenylmethoxycarbonyl-N epsilon-4=methyltrityl-lysine, [Fmoc-Lys(Mtt)-OH], was prepared in two steps from lysine, in 42% overall yield. The N epsilon-Mtt function can be quantitatively removed upon treatment with 1% TFA in dichloromethane or with a 1:2:7 mixture of acetic acid/trifluoroethanol/dichloromethane for 30 min and 1 h at room temperature, respectively. Under these conditions, groups of the tert-butyl type and peptide ester bonds to TFA-labile resins, such as the 2-chlorodiphenylmethyl- and the Wang-resin, remained intact. The utility of the new derivative in peptide synthesis has been exemplified with the synthesis of a cyclic cholecystokinin analog. As an example of further application, five types of lysine cores suitable for the solid-phase synthesis of one, two or three epitopes containing antigenic peptides or template-assembled synthetic proteins have been synthesized on Merrifield, Wang and 2-chlorodiphenylmethyl resin.

Easy parallel screening of reagent stability, quality control, and metrology in solid phase peptide synthesis (SPPS) and peptide couplings for microarrays

DOE PAGES

Achyuthan, Komandoor E.; Wheeler, David R.

2015-08-27

Evaluating the stability of coupling reagents, quality control (QC), and surface functionalization metrology are all critical to the production of high quality peptide microarrays. We describe a broadly applicable screening technique for evaluating the fidelity of solid phase peptide synthesis (SPPS), the stability of activation/coupling reagents, and a microarray surface metrology tool. This technique was used to assess the stability of the activation reagent 1-{[1-(Cyano-2-ethoxy-2-oxo-ethylidenaminooxy)dimethylamino-morpholinomethylene]}methaneaminiumHexafluorophosphate (COMU) (Sigma-Aldrich, St. Louis, MO, USA) by SPPS of Leu-Enkephalin (YGGFL) or the coupling of commercially synthesized YGGFL peptides to (3-aminopropyl)triethyoxysilane-modified glass surfaces. Coupling efficiency was quantitated by fluorescence signaling based on immunoreactivity of themore » YGGFL motif. It was concluded that COMU solutions should be prepared fresh and used within 5 h when stored at ~23 °C and not beyond 24 h if stored refrigerated, both in closed containers. Caveats to gauging COMU stability by absorption spectroscopy are discussed. Commercial YGGFL peptides needed independent QC, due to immunoreactivity variations for the same sequence synthesized by different vendors. This technique is useful in evaluating the stability of other activation/coupling reagents besides COMU and as a metrology tool for SPPS and peptide microarrays.« less

Easy parallel screening of reagent stability, quality control, and metrology in solid phase peptide synthesis (SPPS) and peptide couplings for microarrays

DOE Office of Scientific and Technical Information (OSTI.GOV)

Achyuthan, Komandoor E.; Wheeler, David R.

Evaluating the stability of coupling reagents, quality control (QC), and surface functionalization metrology are all critical to the production of high quality peptide microarrays. We describe a broadly applicable screening technique for evaluating the fidelity of solid phase peptide synthesis (SPPS), the stability of activation/coupling reagents, and a microarray surface metrology tool. This technique was used to assess the stability of the activation reagent 1-{[1-(Cyano-2-ethoxy-2-oxo-ethylidenaminooxy)dimethylamino-morpholinomethylene]}methaneaminiumHexafluorophosphate (COMU) (Sigma-Aldrich, St. Louis, MO, USA) by SPPS of Leu-Enkephalin (YGGFL) or the coupling of commercially synthesized YGGFL peptides to (3-aminopropyl)triethyoxysilane-modified glass surfaces. Coupling efficiency was quantitated by fluorescence signaling based on immunoreactivity of themore » YGGFL motif. It was concluded that COMU solutions should be prepared fresh and used within 5 h when stored at ~23 °C and not beyond 24 h if stored refrigerated, both in closed containers. Caveats to gauging COMU stability by absorption spectroscopy are discussed. Commercial YGGFL peptides needed independent QC, due to immunoreactivity variations for the same sequence synthesized by different vendors. This technique is useful in evaluating the stability of other activation/coupling reagents besides COMU and as a metrology tool for SPPS and peptide microarrays.« less

Hydrogen Bonded Squaramide-Based Foldable Module Induces Both β- and α-Turns in Hairpin Structures of α-Peptides in Water.

PubMed

Martínez, Luís; Martorell, Gabriel; Sampedro, Ángel; Ballester, Pablo; Costa, Antoni; Rotger, Carmen

2015-06-19

A novel tertiary squaramido-based reverse-turn module SQ is reported, and its conformational properties are evaluated. This module is easily incorporated into a α-peptide sequence by conventional solid-phase peptide synthesis. The structure characterization of the hybrid squaramido-peptide 4 is described, showing that the turn segment induces the formation of hairpin structures in water through the formation of both αSQ- and βSQ-turns.

Incorporation of a Bio-Active Reverse-Turn Heterocycle into a Peptide Template Using Solid-Phase Synthesis to Probe Melanocortin Receptor Selectivity and Ligand Conformations by 2D 1H NMR

PubMed Central

Singh, Anamika; Wilczynski, Andrzej; Holder, Jerry R.; Witek, Rachel M.; Dirain, Marvin L.; Xiang, Zhimin; Edison, Arthur S.; Haskell-Luevano, Carrie

2011-01-01

Using a solid-phase synthetic approach, a bioactive reverse turn heterocyclic was incorporated into a cyclic peptide template to probe melanocortin receptor potency and ligand structural conformations. The five melanocortin receptor isoforms (MC1R-MC5R) are G-protein coupled receptors (GPCRs) that are regulated by endogenous agonists and antagonists. This pathway is involved in pigmentation, weight, and energy homeostasis. Herein, we report novel analogues of the chimeric AGRP-melanocortin peptide template integrated with a small molecule moiety to probe the structural and functional consequences of the core His-Phe-Arg-Trp peptide domain using a reverse-turn heterocycle. A series of six compounds are reported that result in inactive to full agonists with nM potency. Biophysical structural analysis [2D 1H NMR and computer-assisted molecular modeling (CAMM)] were performed on selected analogues, resulting in the identification that these peptide-small molecule hybrids possessed increased flexibility and fewer discrete conformational families as compared to the reference peptide and result in a novel template for further structure-function studies. PMID:21306168

Monosaccharides as Scaffolds for the Synthesis of Novel Compounds

NASA Astrophysics Data System (ADS)

Murphy, Paul V.; Velasco-Torrijos, Trinidad

This chapter focuses on monosaccharides and scaffolds their derivatives as scaffolds for the synthesis of primarily bioactive compounds. Such carbohydrate derivatives have been designed to modulate mainly protein-protein and peptide-protein interactions although modulators of carbohydrate-protein and carbohydrate-nucleic acid interactions have also been of interest. The multiple hydroxyl groups that are present on saccharides have made pyranose, furanose and iminosugars ideal templates or scaffolds to which recognition or pharmacophoric groups can be grafted to generate novel compounds for medicinal chemistry. The synthesis of compounds for evaluations require strategies for regioselective reactions of saccharide hydroxyl groups and use of orthogonally stable protecting groups. Syntheses have been carried out on the solid phase and in solution. Also the use of uronic acids, amino sugars and sugar amino acids has facilitated the synthesis of peptidomimetics and prospecting libraries as they enable, through presence of amino or carboxylic acid groups, chemoselective approaches to be employed in solution and on solid phase. Sugar amino acids are readily incorporated, as peptide isosteres, to generate sugar-peptide hybrids or for the synthesis of novel carbopeptoids . The synthesis of new cyclic compounds, derived in part from saccharides, and their application as scaffolds is an emerging area and recent examples include spirocyclic compounds, benzodiazepine-saccharide hybrids and macrolide-saccharide hybrids. Potent bioactive saccharide derivatives have been identified that include enzyme inhibitors , somatostatin receptor ligands, integrin ligands, anti-viral compounds, shiga toxin inhibitors and cell growth inhibitors. Some saccharide derivatives have demonstrated improved cellular permeability when compared with peptides and are in clinical trials.

Scope and Limitations of Fmoc Chemistry SPPS-Based Approaches to the Total Synthesis of Insulin Lispro via Ester Insulin.

PubMed

Dhayalan, Balamurugan; Mandal, Kalyaneswar; Rege, Nischay; Weiss, Michael A; Eitel, Simon H; Meier, Thomas; Schoenleber, Ralph O; Kent, Stephen B H

2017-01-31

We have systematically explored three approaches based on 9-fluorenylmethoxycarbonyl (Fmoc) chemistry solid phase peptide synthesis (SPPS) for the total chemical synthesis of the key depsipeptide intermediate for the efficient total chemical synthesis of insulin. The approaches used were: stepwise Fmoc chemistry SPPS; the "hybrid method", in which maximally protected peptide segments made by Fmoc chemistry SPPS are condensed in solution; and, native chemical ligation using peptide-thioester segments generated by Fmoc chemistry SPPS. A key building block in all three approaches was a Glu[O-β-(Thr)] ester-linked dipeptide equipped with a set of orthogonal protecting groups compatible with Fmoc chemistry SPPS. The most effective method for the preparation of the 51 residue ester-linked polypeptide chain of ester insulin was the use of unprotected peptide-thioester segments, prepared from peptide-hydrazides synthesized by Fmoc chemistry SPPS, and condensed by native chemical ligation. High-resolution X-ray crystallography confirmed the disulfide pairings and three-dimensional structure of synthetic insulin lispro prepared from ester insulin lispro by this route. Further optimization of these pilot studies could yield an efficient total chemical synthesis of insulin lispro (Humalog) based on peptide synthesis by Fmoc chemistry SPPS. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

A Convenient Approach to Synthesizing Peptide C-Terminal N-Alkyl Amides

PubMed Central

Fang, Wei-Jie; Yakovleva, Tatyana; Aldrich, Jane V.

2014-01-01

Peptide C-terminal N-alkyl amides have gained more attention over the past decade due to their biological properties, including improved pharmacokinetic and pharmacodynamic profiles. However, the synthesis of this type of peptide on solid phase by current available methods can be challenging. Here we report a convenient method to synthesize peptide C-terminal N-alkyl amides using the well-known Fukuyama N-alkylation reaction on a standard resin commonly used for the synthesis of peptide C-terminal primary amides, the PAL-PEG-PS (Peptide Amide Linker-polyethylene glycol-polystyrene) resin. The alkylation and oNBS deprotection were conducted under basic conditions and were therefore compatible with this acid labile resin. The alkylation reaction was very efficient on this resin with a number of different alkyl iodides or bromides, and the synthesis of model enkephalin N-alkyl amide analogs using this method gave consistently high yields and purities, demonstrating the applicability of this methodology. The synthesis of N-alkyl amides was more difficult on a Rink amide resin, especially the coupling of the first amino acid to the N-alkyl amine, resulting in lower yields for loading the first amino acid onto the resin. This method can be widely applied in the synthesis of peptide N-alkyl amides. PMID:22252422

Native Chemical Ligation Strategy to Overcome Side Reactions during Fmoc-Based Synthesis of C-Terminal Cysteine-Containing Peptides.

PubMed

Lelièvre, Dominique; Terrier, Victor P; Delmas, Agnès F; Aucagne, Vincent

2016-03-04

The Fmoc-based solid phase synthesis of C-terminal cysteine-containing peptides is problematic, due to side reactions provoked by the pronounced acidity of the Cα proton of cysteine esters. We herein describe a general strategy consisting of the postsynthetic introduction of the C-terminal Cys through a key chemoselective native chemical ligation reaction with N-Hnb-Cys peptide crypto-thioesters. This method was successfully applied to the demanding peptide sequences of two natural products of biological interest, giving remarkably high overall yields compared to that of a state of the art strategy.

Facile rhenium-peptide conjugate synthesis using a one-pot derived Re(CO)3 reagent.

PubMed

Chanawanno, Kullapa; Kondeti, Vinay; Caporoso, Joel; Paruchuri, Sailaja; Leeper, Thomas C; Herrick, Richard S; Ziegler, Christopher J

2016-03-21

We have synthesized two Re(CO)3-modified lysine complexes (1 and 2), where the metal is attached to the amino acid at the Nε position, via a one-pot Schiff base formation reaction. These compounds can be used in the solid phase synthesis of peptides, and to date we have produced four conjugate systems incorporating neurotensin, bombesin, leutenizing hormone releasing hormone, and a nuclear localization sequence. We observed uptake into human umbilical vascular endothelial cells as well as differential uptake depending on peptide sequence identity, as characterized by fluorescence and rhenium elemental analysis.

The 4-pyridylmethyl ester as a protecting group for glutamic and aspartic acids: 'flipping' peptide charge states for characterization by positive ion mode ESI-MS.

PubMed

Garapati, Sriramya; Burns, Colin S

2014-03-01

Use of the 4-pyridylmethyl ester group for side-chain protection of glutamic acid residues in solid-phase peptide synthesis enables switching of the charge state of a peptide from negative to positive, thus making detection by positive ion mode ESI-MS possible. The pyridylmethyl ester moiety is readily removed from peptides in high yield by hydrogenation. Combining the 4-pyridylmethyl ester protecting group with benzyl ester protection reduces the number of the former needed to produce a net positive charge and allows for purification by RP HPLC. This protecting group is useful in the synthesis of highly acidic peptide sequences, which are often beset by problems with purification by standard RP HPLC and characterization by ESI-MS. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

Automatic procedures for the synthesis of difficult peptides using oxyma as activating reagent: A comparative study on the use of bases and on different deprotection and agitation conditions.

PubMed

Caporale, A; Doti, N; Monti, A; Sandomenico, A; Ruvo, M

2018-04-01

Solid-Phase Peptide Synthesis (SPPS) is a rapid and efficient methodology for the chemical synthesis of peptides and small proteins. However, the assembly of peptide sequences classified as "difficult" poses severe synthetic problems in SPPS for the occurrence of extensive aggregation of growing peptide chains which often leads to synthesis failure. In this framework, we have investigated the impact of different synthetic procedures on the yield and final purity of three well-known "difficult peptides" prepared using oxyma as additive for the coupling steps. In particular, we have comparatively investigated the use of piperidine and morpholine/DBU as deprotection reagents, the addition of DIPEA, collidine and N-methylmorpholine as bases to the coupling reagent. Moreover, the effect of different agitation modalities during the acylation reactions has been investigated. Data obtained represent a step forward in optimizing strategies for the synthesis of "difficult peptides". Copyright © 2018 Elsevier Inc. All rights reserved.

Comparative use of benzhydrylamine and chloromethylated resins in solid-phase synthesis of carboxamide terminal peptides. Synthesis of oxytocin derivatives

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hruby, V.J.; Upson, D.A.; Agarwal, N.S.

1977-10-28

Specifically deuterated derivatives of the peptide hormone oxytocin were synthesized by the solid-phase method of peptide synthesis using either the standard chloromethylated resin or the benzhydrylamine resin as the support for the syntheses, and a comparison of the overall efficiency of the syntheses on the two resins was made. (1-Hemi-DL-(..beta..,..beta..-/sup 2/H/sub 2/) cystine) oxytocin was synthesized using the standard chloromethylated resin, and the two diastereomers were separated and purified by partition chromatography and gel filtration in an overall yield of about 30%. (1-Hemi-DL-(..cap alpha..-/sup 2/H/sub 1/) cystine) oxytocin was prepared using the benzhydrylamine resin to prepare the nonapeptide resin precursor,more » but otherwise using essentially identical conditions as used for the synthesis on the chloromethylated resin. Again the two diastereomers were separated and purified by partition chromatography and gel filtration. The overall yield of purified diastereomers under the best conditions was about 49%. For the synthesis of the latter compounds, S-3,4-dimethylbenzyl protecting groups were used to introduce the cysteine residues. The overall yields of the peptide hormone derivatives prepared on the benzhydrylamine resin were substantially improved if HF reactions were run at lower temperatures (0/sup 0/C rather than 25/sup 0/C), and if the S-3,4-dimethylbenzyl rather than the S-benzyl group was used for cysteine protection. Reproducible procedures for preparing benzhydrylamine resins with amino substitution levels of 0.15-0.45 mmol of amino group/g of resin were developed.« less

Synthesis of Natural Cyclopentapeptides Isolated from Dianthus chinensis.

PubMed

Zhang, Shengping; Amso, Zaid; De Leon Rodriguez, Luis M; Kaur, Harveen; Brimble, Margaret A

2016-07-22

The first syntheses of the naturally occurring cyclic peptides dianthin I (1), pseudostellarin A (2), and heterophyllin J (3) are described. The linear protected peptide precursors were prepared efficiently via Fmoc-solid-phase synthesis and subsequently cyclized in solution under dilute conditions. The structures of the synthetic cyclopentapeptides were confirmed by NMR spectroscopy and mass spectrometry and were in agreement with the literature data reported for the natural products.

The self-assembly of redox active peptides: Synthesis and electrochemical capacitive behavior.

PubMed

Piccoli, Julia P; Santos, Adriano; Santos-Filho, Norival A; Lorenzón, Esteban N; Cilli, Eduardo M; Bueno, Paulo R

2016-05-01

The present work reports on the synthesis of a redox-tagged peptide with self-assembling capability aiming applications in electrochemically active capacitive surfaces (associated with the presence of the redox centers) generally useful in electroanalytical applications. Peptide containing ferrocene (fc) molecular (redox) group (Ac-Cys-Ile-Ile-Lys(fc)-Ile-Ile-COOH) was thus synthesized by solid phase peptide synthesis (SPPS). To obtain the electrochemically active capacitive interface, the side chain of the cysteine was covalently bound to the gold electrode (sulfur group) and the side chain of Lys was used to attach the ferrocene in the peptide chain. After obtaining the purified redox-tagged peptide, the self-assembly and redox capability was characterized by cyclic voltammetry (CV) and electrochemical impedance-based capacitance spectroscopy techniques. The obtained results confirmed that the redox-tagged peptide was successfully attached by forming an electroactive self-assembled monolayer onto gold electrode. The design of redox active self-assembly ferrocene-tagged peptide is predictably useful in the development of biosensor devices precisely to detect, in a label-free platform, those biomarkers of clinical relevance. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 357-367, 2016. © 2016 Wiley Periodicals, Inc.

Structure and Membrane Interactions of the Antibiotic Peptide Dermadistinctin K by Multidimensional Solution and Oriented 15N and 31P Solid-State NMR Spectroscopy

PubMed Central

Verly, Rodrigo M.; Moraes, Cléria Mendonça de; Resende, Jarbas M.; Aisenbrey, Christopher; Bemquerer, Marcelo Porto; Piló-Veloso, Dorila; Valente, Ana Paula; Almeida, Fábio C.L.; Bechinger, Burkhard

2009-01-01

DD K, a peptide first isolated from the skin secretion of the Phyllomedusa distincta frog, has been prepared by solid-phase chemical peptide synthesis and its conformation was studied in trifluoroethanol/water as well as in the presence of sodium dodecyl sulfate and dodecylphosphocholine micelles or small unilamellar vesicles. Multidimensional solution NMR spectroscopy indicates an α-helical conformation in membrane environments starting at residue 7 and extending to the C-terminal carboxyamide. Furthermore, DD K has been labeled with 15N at a single alanine position that is located within the helical core region of the sequence. When reconstituted into oriented phosphatidylcholine membranes the resulting 15N solid-state NMR spectrum shows a well-defined helix alignment parallel to the membrane surface in excellent agreement with the amphipathic character of DD K. Proton-decoupled 31P solid-state NMR spectroscopy indicates that the peptide creates a high level of disorder at the level of the phospholipid headgroup suggesting that DD K partitions into the bilayer where it severely disrupts membrane packing. PMID:19289046

Facile solid-phase synthesis of sulfated tyrosine-containing peptides: total synthesis of human big gastrin-II and cholecystokinin (CCK)-39.

PubMed

Kitagawa, K; Aida, C; Fujiwara, H; Yagami, T; Futaki, S; Kogire, M; Ida, J; Inoue, K

2001-01-12

Chemical synthesis of tyrosine O-sulfated peptides is still a laborious task for peptide chemists because of the intrinsic acid-lability of the sulfate moiety. An efficient cleavage/deprotection procedure without loss of the sulfate is the critical difficulty remaining to be solved for fluoren-9-ylmethoxycarbonyl (Fmoc)-based solid-phase synthesis of sulfated peptides. To overcome the difficulty, TFA-mediated solvolysis rates of a tyrosine O-sulfate [Tyr(SO3H)] residue and two protecting groups, tBu for the hydroxyl group of Ser and 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) for the guanidino group of Arg, were examined in detail. The desulfation obeyed first-order kinetics with a large entropy (59.6 J.K-1.mol-1) and enthalpy (110.5 kJ.mol-1) of activation. These values substantiated that the desulfation rate of the rigidly solvated Tyr(SO3H) residue was strongly temperature-dependent. By contrast, the SN1-type deprotections were less temperature-dependent and proceeded smoothly in TFA of a high ionizing power. Based on the large rate difference between the desulfation and the SN1-type deprotections in cold TFA, an efficient deprotection protocol for the sulfated peptides was developed. Our synthetic strategy for Tyr(SO3H)-containing peptides with this effective deprotection protocol is as follows: (i) a sulfated peptide chain is directly constructed on 2-chlorotrityl resin with Fmoc-based solid-phase chemistry using Fmoc-Tyr(SO3Na)-OH as a building block; (ii) the protected peptide-resin is treated with 90% aqueous TFA at 0 degree C for an appropriate period of time for the cleavage and deprotection. Human cholecystokinin (CCK)-12, mini gastrin-II (14 residues), and little gastrin-II (17 residues) were synthesized with this method in 26-38% yields without any difficulties. This method was further applied to the stepwise synthesis of human big gastrin-II (34 residues), CCK-33 and -39. Despite the prolonged acid treatment (15-18 h at 0 degree C), the ratios of the desulfated peptides were less than 15%, and the pure sulfated peptides were obtained in around 10% yields.

Quantitatively identifying the roles of interfacial water and solid surface in governing peptide adsorption.

PubMed

Xu, Zhijun; Yang, Xiao; Wei, Qichao; Zhao, Weilong; Cui, Beiliang; Yang, Xiaoning; Sahai, Nita

2018-06-11

Understanding the molecular mechanism of protein adsorption on solids is critical to their applications in materials synthesis and tissue engineering. Though the water phase at the surface/water interface has been recognized as three types: free water in the bulk region, intermediate water phase and surface-bound water layers adjacent to the surface, the roles of the water and surface in determining the protein adsorption are not clearly identified, particularly at the quantitative level. Herein, we provide a methodology involving the combination of microsecond strengthen sampling simulation and force integration to quantitatively characterize the water-induced contribution and the peptide-surface interactions into the adsorption free energy. Using hydroxyapatite and graphene surfaces as examples, we demonstrate how the distinct interfacial features dominate the delicate force balance between these two thermodynamics parameters, leading to surface preference/resistance to peptide adsorption. Specifically, the water layer provides sustained repelling force against peptide adsorption, as indicated by a monotonic increase in the water-induced free energy profile, whereas the contribution to the free energy from the surface effect is thermodynamically favorable, thus acting as the dominant driving force for peptide adsorptions. More importantly, the revealed adsorption mechanism is critically dictated by the distribution of water phase at the solid/water interface, which plays a crucial role in establishing the force balance between the interactions of the peptide with the water layer and the surface. For the HAP surface, the charged peptide exhibits strong binding affinity to the surface, which is ascribed to the controlling contribution of peptide-surface interaction in the intermediate water phase and the surface-bound water layers are observed as the origin of bioresistance of solid surfaces towards the adsorption of charge-neutral peptides. The preferred peptide adsorption on the graphene, however, is dominated by the surface-induced component at the water layers adjacent to the surface. Our results further elucidate that the intermediate water phase significantly shortens the effective range of the surface dispersion force to guide the diffusion of the peptide to the interface, in sharp contrast to the observation in interfacial systems involving the strong water-surface interaction.

Peptide chemistry toolbox - Transforming natural peptides into peptide therapeutics.

PubMed

Erak, Miloš; Bellmann-Sickert, Kathrin; Els-Heindl, Sylvia; Beck-Sickinger, Annette G

2018-06-01

The development of solid phase peptide synthesis has released tremendous opportunities for using synthetic peptides in medicinal applications. In the last decades, peptide therapeutics became an emerging market in pharmaceutical industry. The need for synthetic strategies in order to improve peptidic properties, such as longer half-life, higher bioavailability, increased potency and efficiency is accordingly rising. In this mini-review, we present a toolbox of modifications in peptide chemistry for overcoming the main drawbacks during the transition from natural peptides to peptide therapeutics. Modifications at the level of the peptide backbone, amino acid side chains and higher orders of structures are described. Furthermore, we are discussing the future of peptide therapeutics development and their impact on the pharmaceutical market. Copyright © 2018 Elsevier Ltd. All rights reserved.

Synthesis and characterization of natural and modified antifreeze glycopeptides: glycosylated foldamers.

PubMed

Nagel, Lilly; Plattner, Carolin; Budke, Carsten; Majer, Zsuzsanna; DeVries, Arthur L; Berkemeier, Thomas; Koop, Thomas; Sewald, Norbert

2011-08-01

In Arctic and Antarctic marine regions, where the temperature declines below the colligative freezing point of physiological fluids, efficient biological antifreeze agents are crucial for the survival of polar fish. One group of such agents is classified as antifreeze glycoproteins (AFGP) that usually consist of a varying number (n = 4-55) of [AAT]( n )-repeating units. The threonine side chain of each unit is glycosidically linked to β-D: -galactosyl-(1 → 3)-α-N-acetyl-D: -galactosamine. These biopolymers can be considered as biological antifreeze foldamers. A preparative route for stepwise synthesis of AFGP allows for efficient synthesis. The diglycosylated threonine building block was introduced into the peptide using microwave-enhanced solid phase synthesis. By this versatile solid phase approach, glycosylated peptides of varying sequences and lengths could be obtained. Conformational studies of the synthetic AFGP analogs were performed by circular dichroism experiments (CD). Furthermore, the foldamers were analysed microphysically according to their inhibiting effect on ice recrystallization and influence on the crystal habit.

Synthesis of a ratiometric fluorescent peptide sensor for the highly selective detection of Cd2+.

PubMed

Li, Yan; Li, Lianzhi; Pu, Xuewei; Ma, Guolin; Wang, Erqiong; Kong, Jinming; Liu, Zhipeng; Liu, Yangzhong

2012-06-15

A novel ratiometric fluorescent peptidyl chemosensor (Dansyl-Cys-Pro-Gly-Cys-Trp-NH(2), D-P5) for metal ions detection has been synthesized via Fmoc solid-phase peptide synthesis. The chemosensor exhibited a high selectivity for Cd(2+) over other metal ions including competitive transition and Group I and II metal ions in neutral pH. The fluorescence emission intensity of D-P5 was significantly enhanced in the presence of Cd(2+) by fluorescent resonance energy transfer (FRET) and chelation enhanced fluorescence (CHEF) effects. The binding stoichiometry, detection limit, binding affinity, reversibility and pH sensitivity of the sensor for Cd(2+) were investigated. Copyright © 2012 Elsevier Ltd. All rights reserved.

Biodegradable copolymers carrying cell-adhesion peptide sequences.

PubMed

Proks, Vladimír; Machová, Lud'ka; Popelka, Stepán; Rypácek, Frantisek

2003-01-01

Amphiphilic block copolymers are used to create bioactive surfaces on biodegradable polymer scaffolds for tissue engineering. Cell-selective biomaterials can be prepared using copolymers containing peptide sequences derived from extracellular-matrix proteins (ECM). Here we discuss alternative ways for preparation of amphiphilic block copolymers composed of hydrophobic polylactide (PLA) and hydrophilic poly(ethylene oxide) (PEO) blocks with cell-adhesion peptide sequences. Copolymers PLA-b-PEO were prepared by a living polymerisation of lactide in dioxane with tin(II)2-ethylhexanoate as a catalyst. The following approaches for incorporation of peptides into copolymers were elaborated. (a) First, a side-chain protected Gly-Arg-Gly-Asp-Ser-Gly (GRGDSG) peptide was prepared by solid-phase peptide synthesis (SPPS) and then coupled with delta-hydroxy-Z-amino-PEO in solution. In the second step, the PLA block was grafted to it via a controlled polymerisation of lactide initiated by the hydroxy end-groups of PEO in the side-chain-protected GRGDSG-PEO. Deprotection of the peptide yielded a GRGDSG-b-PEO-b-PLA copolymer, with the peptide attached through its C-end. (b) A protected GRGDSG peptide was built up on a polymer resin and coupled with Z-carboxy-PEO using a solid-phase approach. After cleavage of the delta-hydroxy-PEO-GRGDSG copolymer from the resin, polymerisation of lactide followed by deprotection of the peptide yielded a PLA-b-PEO-b-GRGDSG block copolymer, in which the peptide is linked through its N-terminus.

Chemical synthesis and orexigenic activity of rat/mouse relaxin-3.

PubMed

Hossain, Mohammed Akhter; Smith, Craig M; Ryan, Philip J; Büchler, Elena; Bathgate, Ross A D; Gundlach, Andrew L; Wade, John D

2013-06-01

The insulin-like peptide, relaxin-3 was first identified just a decade ago via a genomic database search and is now recognized to be a key neuropeptide with several roles including the regulation of arousal, stress responses and neuroendocrine homeostasis. It also has significant potential as a drug to treat stress and obesity. Its actions are mediated via its cognate G protein-coupled receptor, RXFP3, which is found in abundant numbers in the brain. However, much remains to be determined with respect to the mechanism of neurological action of this peptide. Consequently, the chemical synthesis of the rat and mouse (which share identical primary structures) two-chain, three disulfide peptide was undertaken and the resulting peptide subjected to detailed in vitro and in vivo assay. Use of efficient solid-phase synthesis methods provided the two regioselectively S-protected A- and B-chains which were readily combined via sequential disulfide bond formation. The synthetic rat/mouse relaxin-3 was obtained in high purity and good overall yield. It demonstrated potent orexigenic activity in rats in that central intracerebroventricular infusion led to significantly increased food intake and water drinking.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Camarero, J A; Hackel, B J; de Yoreo, J J

C-terminal peptide thioesters are key intermediates for the synthesis/semisynthesis of proteins and for the production of cyclic peptides by native chemical ligation. They can be synthetically prepared by solid-phase peptide synthesis (SPPS) methods or biosynthetically by protein splicing techniques. Until recently, the chemical synthesis of C-terminal a-thioester peptides by SPPS was largely restricted to the Boc/Benzyl methodology because of the poor stability of the thioester bond to the basic conditions employed for the deprotection of the N{sup {alpha}}-Fmoc group. In the present work, we describe a new method for the SPPS of C-terminal thioesters by Fmoc/t-Bu chemistry. This method ismore » based on the use of an aryl hydrazide linker, which is totally stable to the Fmoc-SPPS conditions. Once the peptide synthesis has been completed, activation of the linker can be achieved by mild oxidation. This step transforms the hydrazide group into a highly reactive diazene intermediate which can react with different H-AA-SEt to yield the corresponding {alpha}-thioester peptide in good yields. This method has been successfully used for the generation of different thioester peptides, circular peptides and a fully functional SH3 protein domain.« less

Chemical methods for peptide and protein production.

PubMed

Chandrudu, Saranya; Simerska, Pavla; Toth, Istvan

2013-04-12

Since the invention of solid phase synthetic methods by Merrifield in 1963, the number of research groups focusing on peptide synthesis has grown exponentially. However, the original step-by-step synthesis had limitations: the purity of the final product decreased with the number of coupling steps. After the development of Boc and Fmoc protecting groups, novel amino acid protecting groups and new techniques were introduced to provide high quality and quantity peptide products. Fragment condensation was a popular method for peptide production in the 1980s, but unfortunately the rate of racemization and reaction difficulties proved less than ideal. Kent and co-workers revolutionized peptide coupling by introducing the chemoselective reaction of unprotected peptides, called native chemical ligation. Subsequently, research has focused on the development of novel ligating techniques including the famous click reaction, ligation of peptide hydrazides, and the recently reported α-ketoacid-hydroxylamine ligations with 5-oxaproline. Several companies have been formed all over the world to prepare high quality Good Manufacturing Practice peptide products on a multi-kilogram scale. This review describes the advances in peptide chemistry including the variety of synthetic peptide methods currently available and the broad application of peptides in medicinal chemistry.

Rapid Flow-Based Peptide Synthesis

PubMed Central

Simon, Mark D.; Heider, Patrick L.; Adamo, Andrea; Vinogradov, Alexander A.; Mong, Surin K.; Li, Xiyuan; Berger, Tatiana; Policarpo, Rocco L.; Zhang, Chi; Zou, Yekui; Liao, Xiaoli; Spokoyny, Alexander M.; Jensen, Klavs F.

2014-01-01

A flow-based solid phase peptide synthesis methodology that enables the incorporation of an amino acid residue every 1.8 minutes under automatic control, or every three minutes under manual control, is described. This is accomplished by passing a stream of reagent through a heat exchanger, into a low volume, low backpressure reaction vessel, and through a UV detector. These features enable the continuous delivery of heated solvents and reagents to the solid support at high flow rate, maintaining a maximal concentration of reagents in the reaction vessel, quickly exchanging reagents, and eliminating the need to rapidly heat reagents after they have been added to the vessel. The UV detector enables continuous monitoring of the process. To demonstrate the broad applicability and reliability of this method, it was employed in the total synthesis of a small protein, as well as dozens of peptides. The quality of the material obtained with this method is comparable to traditional batch methods, and, in all cases, the desired material was readily purifiable via RP-HPLC. The application of this method to the synthesis of the 113 residue B. amyloliquefaciens RNase and the 130 residue pE59 DARPin is described in the accompanying manuscript. PMID:24616230

Printing Peptide arrays with a complementary metal oxide semiconductor chip.

PubMed

Loeffler, Felix F; Cheng, Yun-Chien; Muenster, Bastian; Striffler, Jakob; Liu, Fanny C; Ralf Bischoff, F; Doersam, Edgar; Breitling, Frank; Nesterov-Mueller, Alexander

2013-01-01

: In this chapter, we discuss the state-of-the-art peptide array technologies, comparing the spot technique, lithographical methods, and microelectronic chip-based approaches. Based on this analysis, we describe a novel peptide array synthesis method with a microelectronic chip printer. By means of a complementary metal oxide semiconductor chip, charged bioparticles can be patterned on its surface. The bioparticles serve as vehicles to transfer molecule monomers to specific synthesis spots. Our chip offers 16,384 pixel electrodes on its surface with a spot-to-spot pitch of 100 μm. By switching the voltage of each pixel between 0 and 100 V separately, it is possible to generate arbitrary particle patterns for combinatorial molecule synthesis. Afterwards, the patterned chip surface serves as a printing head to transfer the particle pattern from its surface to a synthesis substrate. We conducted a series of proof-of-principle experiments to synthesize high-density peptide arrays. Our solid phase synthesis approach is based on the 9-fluorenylmethoxycarbonyl protection group strategy. After melting the particles, embedded monomers diffuse to the surface and participate in the coupling reaction to the surface. The method demonstrated herein can be easily extended to the synthesis of more complicated artificial molecules by using bioparticles with artificial molecular building blocks. The possibility of synthesizing artificial peptides was also shown in an experiment in which we patterned biotin particles in a high-density array format. These results open the road to the development of peptide-based functional modules for diverse applications in biotechnology.

Photoinduced Thiol-ene Chemistry Applied to the Synthesis of Self-Assembling Elastin-Inspired Glycopeptides.

PubMed

Piccirillo, Germano; Pepe, Antonietta; Bedini, Emiliano; Bochicchio, Brigida

2017-02-21

Synthetic (glyco)peptides inspired by proteins able to self-assemble are appealing biomaterials in the field of tissue engineering and regenerative medicine. Herein, for the first time, taking advantage of thiol-ene chemistry coupled to solid-phase peptide synthesis, a self-assembling peptide inspired by elastin protein was bioconjugated to three carbohydrates in order to obtain the corresponding glycopeptides. They were studied at the molecular and supramolecular level. The results show that the carbohydrate influences the molecular conformation of the glycopeptide and its self-aggregation properties as well. As future perspective, the results could enable us to tune the final self-aggregation properties of the glycopeptide by changing the sugar moiety. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Facile solid-phase synthesis of C-terminal peptide aldehydes and hydroxamates from a common Backbone Amide-Linked (BAL) intermediate.

PubMed

Gazal, S; Masterson, L R; Barany, G

2005-12-01

C-Terminal peptide aldehydes and hydroxamates comprise two separate classes of effective inhibitors of a number of serine, aspartate, cysteine, and metalloproteases. Presented here is a method for preparation of both classes of peptide derivatives from the same resin-bound Weinreb amide precursor. Thus, 5-[(2 or 4)-formyl-3,5-dimethoxyphenoxy]butyramido-polyethylene glycol-polystyrene (BAL-PEG-PS) was treated with methoxylamine hydrochloride in the presence of sodium cyanoborohydride to provide a resin-bound methoxylamine, which was efficiently acylated by different Fmoc-amino acids upon bromo-tris-pyrrolidone-phosphonium hexafluorophosphate (PyBrOP) activation. Solid-phase chain elongation gave backbone amide-linked (BAL) peptide Weinreb amides, which were cleaved either by trifluoroacetic acid (TFA) in the presence of scavengers to provide the corresponding peptide hydroxamates, or by lithium aluminum hydride in tetrahydrofuran (THF) to provide the corresponding C-terminal peptide aldehydes. With several model sequences, peptide hydroxamates were obtained in crude yields of 68-83% and initial purities of at least 85%, whereas peptide aldehydes were obtained in crude yields of 16-53% and initial purities in the range of 30-40%. Under the LiAlH4 cleavage conditions used, those model peptides containing t-Bu-protected aspartate residues underwent partial side chain reduction to the corresponding homoserine-containing peptides. Similar results were obtained when working with high-load aminomethyl-polystyrene, suggesting that this chemistry will be generally applicable to a range of supporting materials.

Site-specific synthesis of Amadori-modified peptides on solid phase.

PubMed

Frolov, Andrej; Singer, David; Hoffmann, Ralf

2006-06-01

Glycation of peptides and proteins is a slow chemical reaction of reducing sugars modifying the amino groups. The first intermediates of this nonenzymatic glycosylation are the Amadori products that can undergo further chemical reactions, finally leading to advanced glycation end products (AGEs). The formation of AGEs was not only linked to aging of tissues and organs in general but also to several diseases such as diabetes mellitus and Alzheimer's disease. Because of the importance of these modifications and their potential use as diagnostic markers, a global postsynthetic approach on solid phase was developed. The peptides were synthesized by Fmoc/(t)Bu-chemistry, with the lysine residue to be modified being protected with the very acid-labile methyltrityl group. Incubation of the peptides with D-glucose in DMF at elevated temperatures resulted in product yields of 35%. Neighboring residues with bulky protecting groups reduced the yields only slightly. The major by-products were the unmodified peptide and an oxidation product. Whereas the unmodified peptide eluted before the glycated peptide, all other by-products eluted later in RP-HPLC, allowing simple purification.

Chemical synthesis of membrane proteins by the removable backbone modification method.

PubMed

Tang, Shan; Zuo, Chao; Huang, Dong-Liang; Cai, Xiao-Ying; Zhang, Long-Hua; Tian, Chang-Lin; Zheng, Ji-Shen; Liu, Lei

2017-12-01

Chemical synthesis can produce membrane proteins bearing specifically designed modifications (e.g., phosphorylation, isotope labeling) that are difficult to obtain through recombinant protein expression approaches. The resulting homogeneously modified synthetic membrane proteins are valuable tools for many advanced biochemical and biophysical studies. This protocol describes the chemical synthesis of membrane proteins by condensation of transmembrane peptide segments through native chemical ligation. To avoid common problems encountered due to the poor solubility of transmembrane peptides in almost any solvent, we describe an effective procedure for the chemical synthesis of membrane proteins through the removable-backbone modification (RBM) strategy. Two key steps of this protocol are: (i) installation of solubilizing Arg4-tagged RBM groups into the transmembrane peptides at any primary amino acid through Fmoc (9-fluorenylmethyloxycarbonyl) solid-phase peptide synthesis and (ii) native ligation of the full-length sequence, followed by removal of the RBM tags by TFA (trifluoroacetic acid) cocktails to afford the native protein. The installation of RBM groups is achieved by using 4-methoxy-5-nitrosalicyladehyde by reduction amination to incorporate an activated O-to-N acyl transfer auxiliary. The Arg4-tag-modified membrane-spanning peptide segments behave like water-soluble peptides to facilitate their purification, ligation and mass characterization.

Solid-Phase Synthesis of Molecularly Imprinted Polymer Nanoparticles with a Reusable Template - "Plastic Antibodies".

PubMed

Poma, Alessandro; Guerreiro, Antonio; Whitcombe, Michael J; Piletska, Elena V; Turner, Anthony P F; Piletsky, Sergey A

2013-06-13

Molecularly Imprinted Polymers (MIPs) are generic alternatives to antibodies in sensors, diagnostics and separations. To displace biomolecules without radical changes in infrastructure in device manufacture, MIPs should share their characteristics (solubility, size, specificity and affinity, localized binding domain) whilst maintaining the advantages of MIPs (low-cost, short development time and high stability) hence the interest in MIP nanoparticles. Herein we report a reusable solid-phase template approach (fully compatible with automation) for the synthesis of MIP nanoparticles and their precise manufacture using a prototype automated UV photochemical reactor. Batches of nanoparticles (30-400 nm) with narrow size distributions imprinted with: melamine (d = 60 nm, K d = 6.3 × 10 -8 m), vancomycin (d = 250 nm, K d = 3.4 × 10 -9 m), a peptide (d = 350 nm, K d = 4.8 × 10 -8 m) and proteins have been produced. Our instrument uses a column packed with glass beads, bearing the template. Process parameters are under computer control, requiring minimal manual intervention. For the first time we demonstrate the reliable re-use of molecular templates in the synthesis of MIPs (≥ 30 batches of nanoMIPs without loss of performance). NanoMIPs are produced template-free and the solid-phase acts both as template and affinity separation medium.

Solid-Phase Synthesis of Molecularly Imprinted Polymer Nanoparticles with a Reusable Template – “Plastic Antibodies”

PubMed Central

Poma, Alessandro; Guerreiro, Antonio; Whitcombe, Michael J.; Piletska, Elena V.; Turner, Anthony P.F.; Piletsky, Sergey A.

2016-01-01

Molecularly Imprinted Polymers (MIPs) are generic alternatives to antibodies in sensors, diagnostics and separations. To displace biomolecules without radical changes in infrastructure in device manufacture, MIPs should share their characteristics (solubility, size, specificity and affinity, localized binding domain) whilst maintaining the advantages of MIPs (low-cost, short development time and high stability) hence the interest in MIP nanoparticles. Herein we report a reusable solid-phase template approach (fully compatible with automation) for the synthesis of MIP nanoparticles and their precise manufacture using a prototype automated UV photochemical reactor. Batches of nanoparticles (30-400 nm) with narrow size distributions imprinted with: melamine (d = 60 nm, Kd = 6.3 × 10−8 m), vancomycin (d = 250 nm, Kd = 3.4 × 10−9 m), a peptide (d = 350 nm, Kd = 4.8 × 10−8 m) and proteins have been produced. Our instrument uses a column packed with glass beads, bearing the template. Process parameters are under computer control, requiring minimal manual intervention. For the first time we demonstrate the reliable re-use of molecular templates in the synthesis of MIPs (≥ 30 batches of nanoMIPs without loss of performance). NanoMIPs are produced template-free and the solid-phase acts both as template and affinity separation medium. PMID:26869870

Proline Editing: A General and Practical Approach to the Synthesis of Functionally and Structurally Diverse Peptides. Analysis of Steric versus Stereoelectronic Effects of 4-Substituted Prolines on Conformation within Peptides

PubMed Central

Pandey, Anil K.; Naduthambi, Devan; Thomas, Krista M.; Zondlo, Neal J.

2013-01-01

Functionalized proline residues have diverse applications. Herein we describe a practical approach, proline editing, for the synthesis of peptides with stereospecifically modified proline residues. Peptides are synthesized by standard solid-phase-peptide-synthesis to incorporate Fmoc-Hydroxyproline (4R-Hyp). In an automated manner, the Hyp hydroxyl is protected and the remainder of the peptide synthesized. After peptide synthesis, the Hyp protecting group is orthogonally removed and Hyp selectively modified to generate substituted proline amino acids, with the peptide main chain functioning to “protect” the proline amino and carboxyl groups. In a model tetrapeptide (Ac-TYPN-NH2), 4R-Hyp was stereospecifically converted to 122 different 4-substituted prolyl amino acids, with 4R or 4S stereochemistry, via Mitsunobu, oxidation, reduction, acylation, and substitution reactions. 4-Substituted prolines synthesized via proline editing include incorporated structured amino acid mimetics (Cys, Asp/Glu, Phe, Lys, Arg, pSer/pThr), recognition motifs (biotin, RGD), electron-withdrawing groups to induce stereoelectronic effects (fluoro, nitrobenzoate), handles for heteronuclear NMR (19F:fluoro; pentafluorophenyl or perfluoro-tert-butyl ether; 4,4-difluoro; 77SePh) and other spectroscopies (fluorescence, IR: cyanophenyl ether), leaving groups (sulfonate, halide, NHS, bromoacetate), and other reactive handles (amine, thiol, thioester, ketone, hydroxylamine, maleimide, acrylate, azide, alkene, alkyne, aryl halide, tetrazine, 1,2-aminothiol). Proline editing provides access to these proline derivatives with no solution phase synthesis. All peptides were analyzed by NMR to identify stereoelectronic and steric effects on conformation. Proline derivatives were synthesized to permit bioorthogonal conjugation reactions, including azide-alkyne, tetrazinetrans-cyclooctene, oxime, reductive amination, native chemical ligation, Suzuki, Sonogashira, cross-metathesis, and Diels-Alder reactions. These proline derivatives allowed three parallel bioorthogonal reactions to be conducted in one solution. PMID:23402492

PNA-encoded chemical libraries.

PubMed

Zambaldo, Claudio; Barluenga, Sofia; Winssinger, Nicolas

2015-06-01

Peptide nucleic acid (PNA)-encoded chemical libraries along with DNA-encoded libraries have provided a powerful new paradigm for library synthesis and ligand discovery. PNA-encoding stands out for its compatibility with standard solid phase synthesis and the technology has been used to prepare libraries of peptides, heterocycles and glycoconjugates. Different screening formats have now been reported including selection-based and microarray-based methods that have yielded specific ligands against diverse target classes including membrane receptors, lectins and challenging targets such as Hsp70. Copyright © 2015 Elsevier Ltd. All rights reserved.

Development of a Hypersensitive Periodate-Cleavable Amino Acid that is Methionine- and Disulfide-Compatible and its Application in MHC Exchange Reagents for T Cell Characterisation

PubMed Central

Amore, Alessia; Wals, Kim; Koekoek, Evelyn; Hoppes, Rieuwert; Toebes, Mireille; Schumacher, Ton N M; Rodenko, Boris; Ovaa, Huib

2013-01-01

Incorporation of cleavable linkers into peptides and proteins is of particular value in the study of biological processes. Here we describe the synthesis of a cleavable linker that is hypersensitive to oxidative cleavage as the result of the periodate reactivity of a vicinal amino alcohol moiety. Two strategies directed towards the synthesis of a building block suitable for solid-phase peptide synthesis were developed: a chemoenzymatic route, involving l-threonine aldolase, and an enantioselective chemical route; these led to α,γ-diamino-β-hydroxybutanoic acids in diastereoisomerically mixed and enantiopure forms, respectively. Incorporation of the 1,2-amino alcohol linker into the backbone of a peptide generated a conditional peptide that was rapidly cleaved at very low concentrations of sodium periodate. This cleavable peptide ligand was applied in the generation of MHC exchange reagents for the detection of antigen-specific T cells in peripheral blood cells. The extremely low concentration of periodate required to trigger MHC peptide exchange allowed the co-oxidation of methionine and disulfide residues to be avoided. Conditional MHC reagents hypersensitive to periodate can now be applied without limitations when UV irradiation is undesired or less practical. PMID:23280887

Biomedical Applications of Organometal-Peptide Conjugates

NASA Astrophysics Data System (ADS)

Metzler-Nolte, Nils

Peptides are well suited as targeting vectors for the directed delivery of metal-based drugs or probes for biomedical investigations. This chapter describes synthetic strategies for the preparation of conjugates of medically interesting peptides with covalently bound metal complexes. Peptides that were used include neuropeptides (enkephalin, neuropeptide Y, neurotensin), uptake peptides (TAT and poly-Arg), and intracellular localization sequences. To these peptides, a whole variety of transition metal complexes has been attached in recent years by solid-phase peptide synthesis (SPPS) techniques. The metal complex can be attached to the peptide on the resin as part of the SPPS scheme. Alternatively, the metal complex may be attached to the peptide as a postsynthetic modification. Advantages as well as disadvantages for either strategy are discussed. Biomedical applications include radiopharmaceutical applications, anticancer and antibacterial activity, metal-peptide conjugates as targeted CO-releasing molecules, and metal-peptide conjugates in biosensor applications.

NIR-Cyanine Dye Linker: a Promising Candidate for Isochronic Fluorescence Imaging in Molecular Cancer Diagnostics and Therapy Monitoring.

PubMed

Komljenovic, Dorde; Wiessler, Manfred; Waldeck, Waldemar; Ehemann, Volker; Pipkorn, Ruediger; Schrenk, Hans-Hermann; Debus, Jürgen; Braun, Klaus

2016-01-01

Personalized anti-cancer medicine is boosted by the recent development of molecular diagnostics and molecularly targeted drugs requiring rapid and efficient ligation routes. Here, we present a novel approach to synthetize a conjugate able to act simultaneously as an imaging and as a chemotherapeutic agent by coupling functional peptides employing solid phase peptide synthesis technologies. Development and the first synthesis of a fluorescent dye with similarity in the polymethine part of the Cy7 molecule whose indolenine-N residues were substituted with a propylene linker are described. Methylating agent temozolomide is functionalized with a tetrazine as a diene component whereas Cy7-cell penetrating peptide conjugate acts as a dienophilic reaction partner for the inverse Diels-Alder click chemistry-mediated ligation route yielding a theranostic conjugate, 3-mercapto-propionic-cyclohexenyl-Cy7-bis-temozolomide-bromide-cell penetrating peptide. Synthesis route described here may facilitate targeted delivery of the therapeutic compound to achieve sufficient local concentrations at the target site or tissue. Its versatility allows a choice of adequate imaging tags applicable in e.g. PET, SPECT, CT, near-infrared imaging, and therapeutic substances including cytotoxic agents. Imaging tags and therapeutics may be simultaneously bound to the conjugate applying click chemistry. Theranostic compound presented here offers a solid basis for a further improvement of cancer management in a precise, patient-specific manner.

NIR-Cyanine Dye Linker: a Promising Candidate for Isochronic Fluorescence Imaging in Molecular Cancer Diagnostics and Therapy Monitoring

PubMed Central

Komljenovic, Dorde; Wiessler, Manfred; Waldeck, Waldemar; Ehemann, Volker; Pipkorn, Ruediger; Schrenk, Hans-Hermann; Debus, Jürgen; Braun, Klaus

2016-01-01

Personalized anti-cancer medicine is boosted by the recent development of molecular diagnostics and molecularly targeted drugs requiring rapid and efficient ligation routes. Here, we present a novel approach to synthetize a conjugate able to act simultaneously as an imaging and as a chemotherapeutic agent by coupling functional peptides employing solid phase peptide synthesis technologies. Development and the first synthesis of a fluorescent dye with similarity in the polymethine part of the Cy7 molecule whose indolenine-N residues were substituted with a propylene linker are described. Methylating agent temozolomide is functionalized with a tetrazine as a diene component whereas Cy7-cell penetrating peptide conjugate acts as a dienophilic reaction partner for the inverse Diels-Alder click chemistry-mediated ligation route yielding a theranostic conjugate, 3-mercapto-propionic-cyclohexenyl-Cy7-bis-temozolomide-bromide-cell penetrating peptide. Synthesis route described here may facilitate targeted delivery of the therapeutic compound to achieve sufficient local concentrations at the target site or tissue. Its versatility allows a choice of adequate imaging tags applicable in e.g. PET, SPECT, CT, near-infrared imaging, and therapeutic substances including cytotoxic agents. Imaging tags and therapeutics may be simultaneously bound to the conjugate applying click chemistry. Theranostic compound presented here offers a solid basis for a further improvement of cancer management in a precise, patient-specific manner. PMID:26722379

Solid-phase total synthesis of cherimolacyclopeptide E and discovery of more potent analogues by alanine screening.

PubMed

Shaheen, Farzana; Rizvi, Tania S; Musharraf, Syed G; Ganesan, A; Xiao, Kai; Townsend, Jared B; Lam, Kit S; Choudhary, M Iqbal

2012-11-26

Cherimolacyclopeptide E (1) is a cyclic hexapeptide obtained from Annona cherimola, reported to be cytotoxic against the KB (human nasopharyngeal carcinoma) cell line. The solid-phase total syntheses of this cyclic peptide and its analogues were accomplished by employing FMOC/tert-butyl-protected amino acids and the Kenner sulfonamide safety-catch linker. The synthetic peptide 1 was found to be weakly cytotoxic against four cell lines (MOLT-4, Jurkat T lymphoma, MDA-MB-231, and KB). Analogues 3 and 7, where glycine at positions 2 and 6 of the parent compound was replaced by Ala, exhibited enhanced cytotoxicity against KB (3, IC50 6.3 μM; 7, IC50 7.8 μM) and MDA-MB-231 breast cancer cells (3, IC50 10.2 μM; 7, IC50 7.7 μM), thereby suggesting possible selective targeting of these cancer cells by these peptides. The spectral data of synthetic peptide 1 was found to be similar to that reported for the natural product. However, a striking difference in biological activity was noted, which warrants the re-evaluation of the original natural product for purity and the existence of conformational differences.

Unexpected opioid activity profiles of analogues of the novel peptide kappa opioid receptor ligand CJ-15,208.

PubMed

Aldrich, Jane V; Kulkarni, Santosh S; Senadheera, Sanjeewa N; Ross, Nicolette C; Reilley, Kate J; Eans, Shainnel O; Ganno, Michelle L; Murray, Thomas F; McLaughlin, Jay P

2011-09-05

An alanine scan was performed on the novel κ opioid receptor (KOR) peptide ligand CJ-15,208 to determine which residues contribute to the potent in vivo agonist activity observed for the parent peptide. These cyclic tetrapeptides were synthesized by a combination of solid-phase peptide synthesis of the linear precursors, followed by cyclization in solution. Like the parent peptide, each of the analogues exhibited agonist activity and KOR antagonist activity in an antinociceptive assay in vivo. Unlike the parent peptide, the agonist activity of the potent analogues was mediated predominantly, if not exclusively, by μ opioid receptors (MOR). Thus analogues 2 and 4, in which one of the phenylalanine residues was replaced by alanine, exhibited both potent MOR agonist activity and KOR antagonist activity in vivo. These peptides represent novel lead compounds for the development of peptide-based opioid analgesics. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Rapid and scalable synthesis of innovative unnatural α,β or γ-amino acids functionalized with tertiary amines on their side-chains.

PubMed

Schneider, Séverine; Ftouni, Hussein; Niu, Songlin; Schmitt, Martine; Simonin, Frédéric; Bihel, Frédéric

2015-07-07

We report a selective ruthenium catalyzed reduction of tertiary amides on the side chain of Fmoc-Gln-OtBu derivatives, leading to innovative unnatural α,β or γ-amino acids functionalized with tertiary amines. Rapid and scalable, this process allowed us to build a library of basic unnatural amino acids at the gram-scale and directly usable for liquid- or solid-phase peptide synthesis. The diversity of available tertiary amines allows us to modulate the physicochemical properties of the resulting amino acids, such as basicity or hydrophobicity.

Practical multipeptide synthesis: dedicated software for the definition of multiple, overlapping peptides covering polypeptide sequences.

PubMed

Heegaard, P M; Holm, A; Hagerup, M

1993-01-01

A personal computer program for the conversion of linear amino acid sequences to multiple, small, overlapping peptide sequences has been developed. Peptide lengths and "jumps" (the distance between two consecutive overlapping peptides) are defined by the user. To facilitate the use of the program for parallel solid-phase chemical peptide syntheses for the synchronous production of multiple peptides, amino acids at each acylation step are laid out by the program in a convenient standard multi-well setup. Also, the total number of equivalents, as well as the derived amount in milligrams (depend-ending on user-defined equivalent weights and molar surplus), of each amino acid are given. The program facilitates the implementation of multipeptide synthesis, e.g., for the elucidation of polypeptide structure-function relationships, and greatly reduces the risk of introducing mistakes at the planning step. It is written in Pascal and runs on any DOS-based personal computer. No special graphic display is needed.

On-resin conversion of Cys(Acm)-containing peptides to their corresponding Cys(Scm) congeners.

PubMed

Mullen, Daniel G; Weigel, Benjamin; Barany, George; Distefano, Mark D

2010-05-01

The Acm protecting group for the thiol functionality of cysteine is removed under conditions (Hg(2+)) that are orthogonal to the acidic milieu used for global deprotection in Fmoc-based solid-phase peptide synthesis. This use of a toxic heavy metal for deprotection has limited the usefulness of Acm in peptide synthesis. The Acm group may be converted to the Scm derivative that can then be used as a reactive intermediate for unsymmetrical disulfide formation. It may also be removed by mild reductive conditions to generate unprotected cysteine. Conversion of Cys(Acm)-containing peptides to their corresponding Cys(Scm) derivatives in solution is often problematic because the sulfenyl chloride reagent used for this conversion may react with the sensitive amino acids tyrosine and tryptophan. In this protocol, we report a method for on-resin Acm to Scm conversion that allows the preparation of Cys(Scm)-containing peptides under conditions that do not modify other amino acids. (c) 2010 European Peptide Society and John Wiley & Sons, Ltd.

Antimicrobial activities of amphiphilic peptides covalently bonded to a water-insoluble resin.

PubMed Central

Haynie, S L; Crum, G A; Doele, B A

1995-01-01

A series of polymer-bound antimicrobial peptides was prepared, and the peptides were tested for their antimicrobial activities. The immobilized peptides were prepared by a strategy that used solid-phase peptide synthesis that linked the carboxy-terminal amino acid with an ethylenediamine-modified polyamide resin (PepsynK). The acid-stable, permanent amide bond between the support and the nascent peptide renders the peptide resistant to cleavage from the support during the final acid-catalyzed deprotection step in the synthesis. Select immobilized peptides containing amino acid sequences that ranged from the naturally occurring magainin to simpler synthetic sequences with idealized secondary structures were excellent antimicrobial agents against several organisms. The immobilized peptides typically reduced the number of viable cells by > or = 5 log units. We show that the reduction in cell numbers cannot be explained by the action of a soluble component. We observed no leached or hydrolyzed peptide from the resin, nor did we observe any antimicrobial activity in soluble extracts from the immobilized peptide. The immobilized peptides were washed and reused for repeated microbial contact and killing. These results suggest that the surface actions by magainins and structurally related antimicrobial peptides are sufficient for their lethal activities. PMID:7726486

Enzymatic and chemical synthesis of new anticoagulant peptides.

PubMed

Origone, Anabella; Bersi, Grisel; Illanes, Andrés; Sturniolo, Héctor; Liggieri, Constanza; Guzmán, Fanny; Barberis, Sonia

2018-06-08

In this study we report the enzymatic synthesis of N-α-[Carbobenzyloxy]-Tyr-Gln-Gln (Z-YQQ), a new anticoagulant tripeptide. It was obtained using phytoproteases from the stems and petioles of Asclepias curassavica L. as catalyst in an aqueous-organic biphasic system formed by 50% (v/v) ethyl acetate and 0.1 M Tris - HCl buffer pH 8. The resulting peptide was compared with the analogous peptide Tyr-Gln-Gln (YQQ) produced by solid-phase chemical synthesis. The in vitro anticoagulant activity of the above mentioned peptides was determined using Wiener Lab Test (Wiener, Argentina). The toxicological activity of the peptides was also determined. The enzymatically synthesized Z-YQQ peptide acted on the extrinsic pathway of the coagulation cascade, delaying the conversion time of prothrombin to thrombin and fibrinogen to fibrin by 136% and 50%, respectively, with respect to the controls. The chemically synthesized YQQ peptide acted specifically on the intrinsic pathway of the coagulation cascade, affecting factors VIII, IX, XI and XII from such cascade, and increasing the coagulation time by 105% with respect to the control. The results suggest that two new anticoagulant peptides (Z-YQQ and YQQ) can be useful for safe pharmaceutical applications. Nevertheless, some aspects related to peptide production should be optimized. This article is protected by copyright. All rights reserved. © 2018 American Institute of Chemical Engineers.

Hmb(off/on) as a switchable thiol protecting group for native chemical ligation.

PubMed

Qi, Yun-Kun; Tang, Shan; Huang, Yi-Chao; Pan, Man; Zheng, Ji-Shen; Liu, Lei

2016-05-04

A new thiol protecting group Hmb(off/on) is described, which has a switchable activity that may be useful in the chemical synthesis of proteins. When placed on the side chain of Cys, Cys(Hmb(off)) is stable to trifluoroacetic acid (TFA) in the process of solid-phase peptide synthesis. When Cys(Hmb(off)) is treated with neutral aqueous buffers, it is cleanly converted to acid-labile Cys(Hmb(on)), which can later be fully deprotected by TFA to generate free Cys. The utility of Cys(Hmb(off/on)) is demonstrated by the chemical synthesis of an erythropoietin segment, EPO[Cys(98)-Arg(166)]-OH through native chemical ligation.

Synthesis and characterization of an 111In-labeled peptide for the in vivo localization of human cancers expressing the urokinase-type plasminogen activator receptor (uPAR)

PubMed Central

Liu, Dijie; Overbey, Douglas; Watkinson, Lisa; Giblin, Michael F.

2009-01-01

This study describes the synthesis and preliminary biologic evaluation of an 111Inlabeled peptide antagonist of the urokinase-type plasminogen activator receptor (uPAR) as a potential probe for assessing metastatic potential of human breast cancer in vivo. The peptide (NAc-dD-CHA-F-dS-dR-Y-L-W-S-βAla)2-K-K(DOTA)-NH2 was synthesized and conjugated with the DOTA chelating moiety via conventional Solid-Phase Peptide Synthesis (SPPS), purified by reversed-phase HPLC, and characterized by MALDI-TOF MS and receptor binding assay. In vitro receptor binding studies demonstrated an IC50 of 240 ± 125 nM for the peptide, compared with IC50’s of 0.44 ± 0.02 and 0.75 ± 0.01 nM for the amino terminal fragment (ATF) of the urokinase-type plasminogen activator (uPA) and full-length uPA, respectively. In vivo biodistribution studies were carried out using SCID mice bearing MDA-MB-231 human breast cancer xenografts. Biodistribution data was collected at 1, 4, and 24 hr post-injection of 111In-DOTA-peptide, and compared with data obtained using a scrambled control peptide, as well as with data obtained using wild-type ATF radiolabeled with I-125. Biodistribution studies showed rapid elimination of the 111In-labeled peptide from the blood pool, with 0.12 ± 0.06% ID/g remaining in blood at 4 hr pi. Elimination was seen primarily via the renal/urinary route, with 83.9 ± 2.2%ID in the urine at the same timepoint. Tumor uptake at this time was 0.53 ± 0.11%ID/g, resulting in tumor: blood and tumor: muscle ratios of 4.2 and 9.4, respectively. Uptake in tumor was significantly higher than that obtained using a scrambled control peptide that showed no specific binding to uPAR (p < 0.05). In vitro and ex vivo results both suggested that the magnitude of tumor-specific binding was reduced in this model by endogenous expression of uPA. The results indicate that radiolabeled peptide uPAR antagonists may find application in the imaging and therapy of uPAR-expressing breast cancers in vivo. PMID:19354275

A nickel tripeptide as a metallodithiolate ligand anchor for resin-bound organometallics.

PubMed

Green, Kayla N; Jeffery, Stephen P; Reibenspies, Joseph H; Darensbourg, Marcetta Y

2006-05-17

The molecular structure of the acetyl CoA synthase enzyme has clarified the role of individual nickel atoms in the dinickel active site which mediates C-C and C-S coupling reactions. The NiN2S2 portion of the biocatalyst (N2S2 = a cysteine-glycine-cysteine or CGC4- tripeptide ligand) serves as an S-donor ligand comparable to classical bidentate ligands operative in organometallic chemistry, ligating the second nickel which is redox and catalytically active. Inspired by this biological catalyst, the synthesis of NiN2S2 metalloligands, including the solid-phase synthesis of resin-bound Ni(CGC)2-, and sulfur-based derivatization with W(CO)5 and Rh(CO)2+ have been carried out. Through comparison to analogous well-characterized, solution-phase complexes, Attenuated Total Reflectance FTIR spectroscopy establishes the presence of unique heterobimetallic complexes, of the form [Ni(CGC)]M(CO)x, both in solution and immobilized on resin beads. This work provides the initial step toward exploitation of such an evolutionarily optimized nickel peptide as a solid support anchor for hybrid bioinorganic-organometallic catalysts.

Dynamic covalent chemistry enables formation of antimicrobial peptide quaternary assemblies in a completely abiotic manner

NASA Astrophysics Data System (ADS)

Reuther, James F.; Dees, Justine L.; Kolesnichenko, Igor V.; Hernandez, Erik T.; Ukraintsev, Dmitri V.; Guduru, Rusheel; Whiteley, Marvin; Anslyn, Eric V.

2018-01-01

Naturally occurring peptides and proteins often use dynamic disulfide bonds to impart defined tertiary/quaternary structures for the formation of binding pockets with uniform size and function. Although peptide synthesis and modification are well established, controlling quaternary structure formation remains a significant challenge. Here, we report the facile incorporation of aryl aldehyde and acyl hydrazide functionalities into peptide oligomers via solid-phase copper-catalysed azide-alkyne cycloaddition (SP-CuAAC) click reactions. When mixed, these complementary functional groups rapidly react in aqueous media at neutral pH to form peptide-peptide intermolecular macrocycles with highly tunable ring sizes. Moreover, sequence-specific figure-of-eight, dumbbell-shaped, zipper-like and multi-loop quaternary structures were formed selectively. Controlling the proportions of reacting peptides with mismatched numbers of complementary reactive groups results in the formation of higher-molecular-weight sequence-defined ladder polymers. This also amplified antimicrobial effectiveness in select cases. This strategy represents a general approach to the creation of complex abiotic peptide quaternary structures.

Synthesis of linear and cyclic peptide-PEG-lipids for stabilization and targeting of cationic liposome-DNA complexes.

PubMed

Ewert, Kai K; Kotamraju, Venkata Ramana; Majzoub, Ramsey N; Steffes, Victoria M; Wonder, Emily A; Teesalu, Tambet; Ruoslahti, Erkki; Safinya, Cyrus R

2016-03-15

Because nucleic acids (NAs) have immense potential value as therapeutics, the development of safe and effective synthetic NA vectors continues to attract much attention. In vivo applications of NA vectors require stabilized, nanometer-scale particles, but the commonly used approaches of steric stabilization with a polymer coat (e.g., PEGylation; PEG=poly(ethylene glycol)) interfere with attachment to cells, uptake, and endosomal escape. Conjugation of peptides to PEG-lipids can improve cell attachment and uptake for cationic liposome-DNA (CL-DNA) complexes. We present several synthetic approaches to peptide-PEG-lipids and discuss their merits and drawbacks. A lipid-PEG-amine building block served as the common key intermediate in all synthetic routes. Assembling the entire peptide-PEG-lipid by manual solid phase peptide synthesis (employing a lipid-PEG-carboxylic acid) allowed gram-scale synthesis but is mostly applicable to linear peptides connected via their N-terminus. Conjugation via thiol-maleimide or strain-promoted (copper-free) azide-alkyne cycloaddition chemistry is highly amenable to on-demand preparation of peptide-PEG-lipids, and the appropriate PEG-lipid precursors are available in a single chemical step from the lipid-PEG-amine building block. Azide-alkyne cycloaddition is especially suitable for disulfide-bridged peptides such as iRGD (cyclic CRGDKGPDC). Added at 10 mol% of a cationic/neutral lipid mixture, the peptide-PEG-lipids stabilize the size of CL-DNA complexes. They also affect cell attachment and uptake of nanoparticles in a peptide-dependent manner, thereby providing a platform for preparing stabilized, affinity-targeted CL-DNA nanoparticles. Copyright © 2016 Elsevier Ltd. All rights reserved.

Mixture-based combinatorial libraries from small individual peptide libraries: a case study on α1-antitrypsin deficiency.

PubMed

Chang, Yi-Pin; Chu, Yen-Ho

2014-05-16

The design, synthesis and screening of diversity-oriented peptide libraries using a "libraries from libraries" strategy for the development of inhibitors of α1-antitrypsin deficiency are described. The major buttress of the biochemical approach presented here is the use of well-established solid-phase split-and-mix method for the generation of mixture-based libraries. The combinatorial technique iterative deconvolution was employed for library screening. While molecular diversity is the general consideration of combinatorial libraries, exquisite design through systematic screening of small individual libraries is a prerequisite for effective library screening and can avoid potential problems in some cases. This review will also illustrate how large peptide libraries were designed, as well as how a conformation-sensitive assay was developed based on the mechanism of the conformational disease. Finally, the combinatorially selected peptide inhibitor capable of blocking abnormal protein aggregation will be characterized by biophysical, cellular and computational methods.

Synthesis, characterization and inhibitory activities of (4-N3[3,5-3H]Phe10)PKI(6-22)amide and its precursors: photoaffinity labeling peptides for the active site of cyclic AMP-dependent protein kinase.

PubMed

Katz, B M; Lundquist, L J; Walsh, D A; Glass, D B

1989-06-01

PKI(6-22)amide is a 17 residue peptide corresponding to the active portion of the heat-stable inhibitor of cAMP-dependent protein kinase. The peptide is a potent (Ki = 1.6 nM), competitive inhibitor of the enzyme. The photoreactive peptide analog (4-azidophenylalanine10)PKI(6-22)amide was synthesized in both its non-radiolabeled and tritiated forms by chemical modification of precursor peptides that were prepared by stepwise solid-phase synthesis. (4-Amino[3,5-3H]phenylalanine10)PKI(6-22)amide, the precursor for the radiolabeled arylazide peptide, was obtained by catalytic reduction of the corresponding peptide containing the 3,5-diiodo-4-aminophenylalanine residue at position 10. The purified PKI peptides were analyzed by HPLC, amino acid analysis, and u.v. spectra. In the dark, (4-azidophenylalanine10)PKI(6-22)amide inhibited the catalytic subunit of cAMP-dependent protein kinase with a Ki value of 2.8 nM. The photoreactivity of the arylazide peptide was demonstrated by time-dependent u.v. spectral changes on exposure to light. Photolysis of the catalytic subunit (4-azido[3,5-3H]phenylalanine10)PKI(6-22)amide complex resulted in specific covalent labeling of the enzyme. The data indicate that this peptide is a useful photoaffinity labeling reagent for the active site of the protein kinase.

C-terminal peptide extension via gas-phase ion/ion reactions

PubMed Central

Peng, Zhou; McLuckey, Scott A.

2015-01-01

The formation of peptide bonds is of great importance from both a biological standpoint and in routine organic synthesis. Recent work from our group demonstrated the synthesis of peptides in the gas-phase via ion/ion reactions with sulfo-NHS reagents, which resulted in conjugation of individual amino acids or small peptides to the N-terminus of an existing ‘anchor’ peptide. Here, we demonstrate a complementary approach resulting in the C-terminal extension of peptides. Individual amino acids or short peptides can be prepared as reagents by incorporating gas phase-labile protecting groups to the reactive C-terminus and then converting the N-terminal amino groups to the active ketenimine reagent. Gas-phase ion/ion reactions between the anionic reagents and doubly protonated “anchor” peptide cations results in extension of the “anchor” peptide with new amide bond formation at the C-terminus. We have demonstrated that ion/ion reactions can be used as a fast, controlled, and efficient means for C-terminal peptide extension in the gas phase. PMID:26640400

Hydrophobic benzyl amines as supports for liquid-phase C-terminal amidated peptide synthesis: application to the preparation of ABT-510.

PubMed

Matsumoto, Emiko; Fujita, Yuko; Okada, Yohei; Kauppinen, Esko I; Kamiya, Hidehiro; Chiba, Kazuhiro

2015-09-01

C-terminal amidation is one of the most common modification of peptides and frequently found in bioactive peptides. However, the C-terminal modification must be creative, because current chemical synthetic techniques of peptides are dominated by the use of C-terminal protecting supports. Therefore, it must be carried out after the removal of such supports, complicating reaction work-up and product isolation. In this context, hydrophobic benzyl amines were successfully added to the growing toolbox of soluble tag-assisted liquid-phase peptide synthesis as supports, leading to the total synthesis of ABT-510 (2). Although an ethyl amide-forming type was used in the present work, different types of hydrophobic benzyl amines could also be simply designed and prepared through versatile reductive aminations in one step. The standard acidic treatment used in the final deprotection step for peptide synthesis gave the desired C-terminal secondary amidated peptide with no epimerization. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.

Solid-phase-assisted synthesis of targeting peptide-PEG-oligo(ethane amino)amides for receptor-mediated gene delivery.

PubMed

Martin, Irene; Dohmen, Christian; Mas-Moruno, Carlos; Troiber, Christina; Kos, Petra; Schaffert, David; Lächelt, Ulrich; Teixidó, Meritxell; Günther, Michael; Kessler, Horst; Giralt, Ernest; Wagner, Ernst

2012-04-28

In the forthcoming era of cancer gene therapy, efforts will be devoted to the development of new efficient and non-toxic gene delivery vectors. In this regard, the use of Fmoc/Boc-protected oligo(ethane amino)acids as building blocks for solid-phase-supported assembly represents a novel promising approach towards fully controlled syntheses of effective gene vectors. Here we report on the synthesis of defined polymers containing the following: (i) a plasmid DNA (pDNA) binding domain of eight succinoyl-tetraethylenpentamine (Stp) units and two terminal cysteine residues; (ii) a central polyethylene glycol (PEG) chain (with twenty-four oxyethylene units) for shielding; and (iii) specific peptides for targeting towards cancer cells. Peptides B6 and c(RGDfK), which bind transferrin receptor and α(v)β(3) integrin, respectively, were chosen because of the high expression of these receptors in many tumoral cells. This study shows the feasibility of designing these kinds of fully controlled vectors and their success for targeted pDNA-based gene transfer. This journal is © The Royal Society of Chemistry 2012

The effect of selective D- or N(alpha)-methyl arginine substitution on the activity of the proline-rich antimicrobial peptide, Chex1-Arg20.

NASA Astrophysics Data System (ADS)

Li, Wenyi; Sun, Zhe; O'Brien-Simpson, Neil M.; Otvos, Laszlo; Reynolds, Eric C.; Hossain, Mohammed A.; Separovic, Frances; Wade, John D.

2017-01-01

In vivo pharmacokinetics studies have shown that the proline-rich antimicrobial peptide, A3-APO, which is a discontinuous dimer of the peptide, Chex1-Arg20, undergoes degradation to small fragments at positions Pro6-Arg7 and Val19-Arg20. With the aim of minimizing or abolishing this degradation, a series of Chex1-Arg20 analogues were prepared via Fmoc/tBu solid phase peptide synthesis with D-arginine or, in some cases, peptide backbone N-methylated arginine, substitution at these sites. All the peptides were tested for antibacterial activity against the Gram-negative bacterium Klebsiella pneumoniae. The resulting activity of position-7 substitution of Chex1-Arg20 analogues showed that arginine-7 is a crucial residue for maintaining activity against K. pneumoniae. However, arginine-20 substitution had a much less deleterious effect on the antibacterial activity of the peptide. Moreover, none of these peptides displayed any cytotoxicity to HEK and H-4-II-E mammalian cells. These results will aid the development of more effective and stable PrAMPs via judicious amino acid substitutions.

Synthesis and biological properties of enzyme-resistant analogues of substance P.

PubMed

Sandberg, B E; Lee, C M; Hanley, M R; Iversen, L L

1981-02-01

Six analogues of substance P were synthesized with the aim of developing a metabolically stable peptide that would retain the biological activity of substance P. A recently isolated and characterized substance-P-degrading enzyme from human brain with a high specificity for substance P described in the preceding paper in this journal was used as a model for the enzymatic inactivation of substance P. The synthetic analogues were designed to protect the peptide bonds on the carboxyl side of residues 6, 7 and 8 of substance P, which represent the sites of cleavage by substance-P-degrading enzyme. To test for increased enzymatic resistance, the analogues were incubated with the enzyme, the digests were separated on a high-performance liquid chromatography reverse-phase column and the peptide fragments were collected and identified by amino acid analysis. Of the analogues described, an heptapeptide analogue of residues 5-11, less than Glu-Gln-Phe-MePhe-MeGly-Leu-MetNH2, showed almost complete resistance both towards substance-P-degrading enzyme and to degradation on exposure to rat hypothalamic slices. This analogue was about a third as potent as substance P in competing for binding to receptor sites for this peptide in rat brain membranes and a tenth as potent in eliciting contractions of the guinea pig ileum. The peptides were synthesized using the solid-phase technique with polydimethylacrylamide as a solid support and the coupling was achieved with pre-formed symmetrical anhydrides in dimethylacetamide. Fluorenylmethyloxycarbonyl was used as an alpha-amino protecting group in conjunction with t-butyloxycarbonyl as an epsilon-amino protecting group. Ammoniolytic cleavage from the resin was followed by stepwise elution from an SP-Sephadex column, deprotection with trifluoroacetic acid and chromatography on a Bio-Rex 70 ion-exchanger. The peptides were finally purified on a semi-preparative reverse-phase column.

An efficient synthesis of the constrained peptidomimetic 2-oxo-3-(N-9-fluorenyloxycarbonylamino)-1-azabicyclo[4.3.0]nonane-9-carboxylic acid from pyroglutamic acid.

PubMed

Mandal, Pijus Kumar; Kaluarachchi, Kumar K; Ogrin, Douglas; Bott, Simon G; McMurray, John S

2005-11-25

[reaction: see text] Azabicyclo[X.Y.0]alkane amino acids are rigid dipeptide mimetics that are useful tools for structure-activity studies in peptide-based drug discovery. Herein, we report an efficient synthesis of three diastereomers of 9-tert-butoxycarbonyl-2-oxo-3-(N-tert-butoxycarbonylamino)-1-azabicyclo[4.3.0]nonane (3S,6S,9S, 3S,6R,9R, and 3S,6R,9S). Methyl N-Boc-pyroglutamate is cleaved with vinylmagnesium bromide to produce an acyclic gamma-vinyl ketone. Michael addition of N-diphenylmethyleneglycine tert-butyl ester produces the N-Boc-delta-oxo-alpha,omega-diaminoazelate intermediate, which, on hydrogenloysis, gives the fused ring system. Acidolytic deprotection followed by Fmoc-protection provided building blocks suitable for solid-phase synthesis.

Group A Streptococcal vaccine candidate: contribution of epitope to size, antigen presenting cell interaction and immunogenicity.

PubMed

Zaman, Mehfuz; Chandrudu, Saranya; Giddam, Ashwini K; Reiman, Jennifer; Skwarczynski, Mariusz; McPhun, Virginia; Moyle, Peter M; Batzloff, Michael R; Good, Michael F; Toth, Istvan

2014-12-01

Utilize lipopeptide vaccine delivery system to develop a vaccine candidate against Group A Streptococcus. Lipopeptides synthesized by solid-phase peptide synthesis-bearing carboxyl (C)-terminal and amino (N)-terminal Group A Streptococcus peptide epitopes. Nanoparticles formed were evaluated in vivo. Immune responses were induced in mice without additional adjuvant. We demonstrated for the first time that incorporation of the C-terminal epitope significantly enhanced the N-terminal epitope-specific antibody response and correlated with forming smaller nanoparticles. Antigen-presenting cells had increased uptake and maturation by smaller, more immunogenic nanoparticles. Antibodies raised by vaccination recognized isolates. Demonstrated the lipopeptidic nanoparticles to induce an immune response which can be influenced by the combined effect of epitope choice and size.

Conformation-Based Design and Synthesis of Apratoxin A Mimetics Modified at the α,β-Unsaturated Thiazoline Moiety.

PubMed

Onda, Yuichi; Masuda, Yuichi; Yoshida, Masahito; Doi, Takayuki

2017-08-10

We have demonstrated design, synthesis, and biological evaluation of apratoxin A mimetics. In the first generation, the moCys moiety was replaced with seven simple amino acids as their 3D structures can be similar to that of apratoxin A. Apratoxins M1-M7 were synthesized using solid-phase peptide synthesis and solution-phase macrolactamization. Apratoxin M7, which contains a piperidinecarboxylic acid moiety, exhibited potent cytotoxicity against HCT-116 cells. In the second generation, substitution of each amino acid residue in the tripeptide Tyr(Me)-MeAla-MeIle moiety in apratoxin M7 led to the development of the highly potent apratoxin M16 possessing biphenylalanine (Bph) instead of Tyr(Me), which exhibited an IC 50 value of 1.1 nM against HCT-116 cells. Moreover, compared to apratoxin A, apratoxin M16 exhibited a similarly high level of growth inhibitory activity against various cancer cell lines. The results indicate that apratoxin M16 could be a potential candidate as an anticancer agent.

GAP Peptide Synthesis via Design of New GAP Protecting Group: An Fmoc/tBu Synthesis of Thymopentin Free from Polymers, Chromatography and Recrystallization

PubMed Central

Seifert, Cole W.; Paniagua, Armando; White, Gabrielle A.; Cai, Lucy

2016-01-01

A novel method for Fmoc/tBu solution-phase peptide synthesis and the development of a new benzyl-type GAP protecting group is reported. This new GAP protecting group is utilized in place of a polymer support, facilitating C→N Fmoc peptide synthesis without chromatography, recrystallization, or polymer supports. The GAP group can be added and removed in high yield, and was used to synthesize over 1 gram of the immunostimulant, thymopentin, in high overall yield (83%) and purity (99%). PMID:28663711

Chemical synthesis, 3D structure, and ASIC binding site of the toxin mambalgin-2.

PubMed

Schroeder, Christina I; Rash, Lachlan D; Vila-Farrés, Xavier; Rosengren, K Johan; Mobli, Mehdi; King, Glenn F; Alewood, Paul F; Craik, David J; Durek, Thomas

2014-01-20

Mambalgins are a novel class of snake venom components that exert potent analgesic effects mediated through the inhibition of acid-sensing ion channels (ASICs). The 57-residue polypeptide mambalgin-2 (Ma-2) was synthesized by using a combination of solid-phase peptide synthesis and native chemical ligation. The structure of the synthetic toxin, determined using homonuclear NMR, revealed an unusual three-finger toxin fold reminiscent of functionally unrelated snake toxins. Electrophysiological analysis of Ma-2 on wild-type and mutant ASIC1a receptors allowed us to identify α-helix 5, which borders on the functionally critical acidic pocket of the channel, as a major part of the Ma-2 binding site. This region is also crucial for the interaction of ASIC1a with the spider toxin PcTx1, thus suggesting that the binding sites for these toxins substantially overlap. This work lays the foundation for structure-activity relationship (SAR) studies and further development of this promising analgesic peptide. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A solid-phase combinatorial approach for indoloquinolizidine-peptides with high affinity at D(1) and D(2) dopamine receptors.

PubMed

Molero, Anabel; Vendrell, Marc; Bonaventura, Jordi; Zachmann, Julian; López, Laura; Pardo, Leonardo; Lluis, Carme; Cortés, Antoni; Albericio, Fernando; Casadó, Vicent; Royo, Miriam

2015-06-05

Ligands acting at multiple dopamine receptors hold potential as therapeutic agents for a number of neurodegenerative disorders. Specifically, compounds able to bind at D1R and D2R with high affinity could restore the effects of dopamine depletion and enhance motor activation on degenerated nigrostriatal dopaminergic systems. We have directed our research towards the synthesis and characterisation of heterocycle-peptide hybrids based on the indolo[2,3-a]quinolizidine core. This privileged structure is a water-soluble and synthetically accessible scaffold with affinity for diverse GPCRs. Herein we have prepared a solid-phase combinatorial library of 80 indoloquinolizidine-peptides to identify compounds with enhanced binding affinity at D2R, a receptor that is crucial to re-establish activity on dopamine-depleted degenerated GABAergic neurons. We applied computational tools and high-throughput screening assays to identify 9a{1,3,3} as a ligand for dopamine receptors with nanomolar affinity and agonist activity at D2R. Our results validate the application of indoloquinolizidine-peptide combinatorial libraries to fine-tune the pharmacological profiles of multiple ligands at D1 and D2 dopamine receptors. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Mimicking of Arginine by Functionalized N(ω)-Carbamoylated Arginine As a New Broadly Applicable Approach to Labeled Bioactive Peptides: High Affinity Angiotensin, Neuropeptide Y, Neuropeptide FF, and Neurotensin Receptor Ligands As Examples.

PubMed

Keller, Max; Kuhn, Kilian K; Einsiedel, Jürgen; Hübner, Harald; Biselli, Sabrina; Mollereau, Catherine; Wifling, David; Svobodová, Jaroslava; Bernhardt, Günther; Cabrele, Chiara; Vanderheyden, Patrick M L; Gmeiner, Peter; Buschauer, Armin

2016-03-10

Derivatization of biologically active peptides by conjugation with fluorophores or radionuclide-bearing moieties is an effective and commonly used approach to prepare molecular tools and diagnostic agents. Whereas lysine, cysteine, and N-terminal amino acids have been mostly used for peptide conjugation, we describe a new, widely applicable approach to peptide conjugation based on the nonclassical bioisosteric replacement of the guanidine group in arginine by a functionalized carbamoylguanidine moiety. Four arginine-containing peptide receptor ligands (angiotensin II, neurotensin(8-13), an analogue of the C-terminal pentapeptide of neuropeptide Y, and a neuropeptide FF analogue) were subject of this proof-of-concept study. The N(ω)-carbamoylated arginines, bearing spacers with a terminal amino group, were incorporated into the peptides by standard Fmoc solid phase peptide synthesis. The synthesized chemically stable peptide derivatives showed high receptor affinities with Ki values in the low nanomolar range, even when bulky fluorophores had been attached. Two new tritiated tracers for angiotensin and neurotensin receptors are described.

SOLID-LIQUID PHASE TRANSFER CATALYZED SYNTHESIS OF CINNAMYL ACETATE-KINETICS AND ANALYSIS OF FACTORS AFFECTING THE REACTION IN A BATCH REACTOR

EPA Science Inventory

The use of solid-liquid phase transfer catalysis has an advantage of carrying out reaction between two immiscible substrates, one in solid phase and the other in liquid phase, with high selectivity and at relatively low temperatures. In this study we investigated the synthesis ci...

Selection of staphylococcal enterotoxin B (SEB)-binding peptide using phage display technology

DOE Office of Scientific and Technical Information (OSTI.GOV)

Soykut, Esra Acar; Dudak, Fahriye Ceyda; Boyaci, Ismail Hakki

In this study, peptides were selected to recognize staphylococcal enterotoxin B (SEB) which cause food intoxication and can be used as a biological war agent. By using commercial M13 phage library, single plaque isolation of 38 phages was done and binding affinities were investigated with phage-ELISA. The specificities of the selected phage clones showing high affinity to SEB were checked by using different protein molecules which can be found in food samples. Furthermore, the affinities of three selected phage clones were determined by using surface plasmon resonance (SPR) sensors. Sequence analysis was realized for three peptides showing high binding affinitymore » to SEB and WWRPLTPESPPA, MNLHDYHRLFWY, and QHPQINQTLYRM amino acid sequences were obtained. The peptide sequence with highest affinity to SEB was synthesized with solid phase peptide synthesis technique and thermodynamic constants of the peptide-SEB interaction were determined by using isothermal titration calorimetry (ITC) and compared with those of antibody-SEB interaction. The binding constant of the peptide was determined as 4.2 {+-} 0.7 x 10{sup 5} M{sup -1} which indicates a strong binding close to that of antibody.« less

Mechanism of Formation of Li 7 P 3 S 11 Solid Electrolytes through Liquid Phase Synthesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Yuxing; Lu, Dongping; Bowden, Mark

Crystalline Li7P3S11 is a promising solid electrolyte for all solid state lithium/lithium ion batteries. A controllable liquid phase synthesis of Li7P3S11 is more desirable compared to conventional mechanochemical synthesis, but recent attempts suffer from reduced ionic conductivities. Here we elucidate the formation mechanism of crystalline Li7P3S11 synthesized in the liquid phase (acetonitrile, or ACN). We conclude that the crystalline Li7P3S11 forms through a two-step reaction: 1) formation of solid Li3PS4∙ACN and amorphous Li2S∙P2S5 phases in the liquid phase; 2) solid-state conversion of the two phases. The implication of this two-step reaction mechanism to the morphology control and the transport propertiesmore » of liquid phase synthesized Li7P3S11 is identified and discussed.« less

Design, Synthesis and Biological Evaluation of Novel Peptide-Like Analogues as Selective COX-2 Inhibitors

PubMed Central

Ahmaditaba, Mohammad Ali; Houshdar Tehrani, Mohammad Hassan; Zarghi, Afshin; Shahosseini, Sorayya; Daraei, Bahram

2018-01-01

A new series of peptide-like derivatives containing different aromatic amino acids and possessing pharmacophores of COX-2 inhibitors as SO2Me or N3 attached to the para position of an end phenyl ring was synthesized for evaluation as selective cyclooxygenase-2 (COX-2) inhibitors. The synthetic reactions were based on the solid phase peptide synthesis method using Wang resin. One of the analogues, i.e., compound 2d, as the representative of these series was recognized as the most effective and the highest selective COX-2 inhibitor with IC50 value of 0.08 μM and COX-2 selectivity index of 351.2, among the other synthesized compounds. Molecular docking study was operated to determine possible binding models of compound 2d to COX-2 enzyme. The study showed that the p-azido-phenyl fragment of 2d occupied inside the secondary COX-2 binding site (Arg513, and His90). The structure-activity relationships acquired disclosed that compound 2d with 4-(azido phenyl) group as pharmacophore and histidine as amino acid gives the essential geometry to provide inhibition of the COX-2 enzyme with high selectivity. Compound 2d can be a good candidate for the development of new hits of COX-2 inhibitors.

Recent advances in racemic protein crystallography.

PubMed

Yan, Bingjia; Ye, Linzhi; Xu, Weiliang; Liu, Lei

2017-09-15

Solution of the three-dimensional structures of proteins is a critical step in deciphering the molecular mechanisms of their bioactivities. Among the many approaches for obtaining protein crystals, racemic protein crystallography has been developed as a unique method to solve the structures of an increasing number of proteins. Exploiting unnatural protein enantiomers in crystallization and resolution, racemic protein crystallography manifests two major advantages that are 1) to increase the success rate of protein crystallization, and 2) to obviate the phase problem in X-ray diffraction. The requirement of unnatural protein enantiomers in racemic protein crystallography necessitates chemical protein synthesis, which is hitherto accomplished through solid phase peptide synthesis and chemical ligation reactions. This review highlights the fundamental ideas of racemic protein crystallography and surveys the harvests in the field of racemic protein crystallography over the last five years from early 2012 to late 2016. Copyright © 2017. Published by Elsevier Ltd.

Unexpected Hydrolytic Instability of N-Acylated Amino Acid Amides and Peptides

PubMed Central

2015-01-01

Remote amide bonds in simple N-acyl amino acid amide or peptide derivatives 1 can be surprisingly unstable hydrolytically, affording, in solution, variable amounts of 3 under mild acidic conditions, such as trifluoroacetic acid/water mixtures at room temperature. This observation has important implications for the synthesis of this class of compounds, which includes N-terminal-acylated peptides. We describe the factors contributing to this instability and how to predict and control it. The instability is a function of the remote acyl group, R2CO, four bonds away from the site of hydrolysis. Electron-rich acyl R2 groups accelerate this reaction. In the case of acyl groups derived from substituted aromatic carboxylic acids, the acceleration is predictable from the substituent’s Hammett σ value. N-Acyl dipeptides are also hydrolyzed under typical cleavage conditions. This suggests that unwanted peptide truncation may occur during synthesis or prolonged standing in solution when dipeptides or longer peptides are acylated on the N-terminus with electron-rich aromatic groups. When amide hydrolysis is an undesired secondary reaction, as can be the case in the trifluoroacetic acid-catalyzed cleavage of amino acid amide or peptide derivatives 1 from solid-phase resins, conditions are provided to minimize that hydrolysis. PMID:24617596

The remarkable stability of chimeric, sialic acid-derived alpha/delta-peptides in human blood plasma.

PubMed

Saludes, Jonel P; Natarajan, Arutselvan; DeNardo, Sally J; Gervay-Hague, Jacquelyn

2010-05-01

Peptides are labile toward proteolytic enzymes, and structural modifications are often required to prolong their metabolic half-life and increase resistance. One modification is the incorporation of non-alpha-amino acids into the peptide to deter recognition by hydrolytic enzymes. We previously reported the synthesis of chimeric alpha/delta-peptides from glutamic acids (Glu) and the sialic acid derivative Neu2en. Conformational analyses revealed these constructs adopt secondary structures in water and may serve as conformational surrogates of polysialic acid. Polysialic acid is a tumor-associated polysaccharide and is correlated with cancer metastasis. Soluble polysialic acid is rapidly cleared from the blood limiting its potential for vaccine development. One motivation in developing structural surrogates of polysialic acid was to create constructs with increased bioavailability. Here, we report plasma stability profiles of Glu/Neu2en alpha/delta-peptides. DOTA was conjugated at the peptide N-termini by solid phase peptide synthesis, radiolabeled with (111)In, incubated in human blood plasma at 37 degrees C, and their degradation patterns monitored by cellulose acetate electrophoresis and radioactivity counting. Results indicate that these peptides exhibit a long half-life that is two- to three-orders of magnitude higher than natural alpha-peptides. These findings provide a viable platform for the synthesis of plasma stable, sialic acid-derived peptides that may find pharmaceutical application.

Synthesis of tritium labelled endomorphin II and its stability in the radioreceptor assay

NASA Astrophysics Data System (ADS)

Tömböly, Cs.; Spetea, M.; Borsodi, A.; Tóth, G.

1999-01-01

Endomorphine I (Tyr-Pro-Trp-Phe-NH2) and endomorphin II (Tyr-Pro-Phe-Phe-NH2) are recently isolated neuropeptides. They have the highest specificity and affinity for the μ-opiate receptor among all the endogenous substances so far described, and they may be natural ligands for this receptor [1]. We prepared the [3H] endomorphin II with high specific radioactivity (53.4 Ci/mmol) by catalytic dehalotritiation. The precursor [(3,5-I2)Tyr1]-endomorphin II was synthesized by solid phase peptide synthesis using Boc chemistry. Labelled endomorphin II was used to investigate its binding properties in rat brain membrane. The stability of [3H]endomorphin II toward enzymatic degradation in membrane preparation was examined by RP-HPLC and by using a radioactivity detector.

Molecular-level understanding of protein adsorption at the interface between water and a strongly interacting uncharged solid surface.

PubMed

Penna, Matthew J; Mijajlovic, Milan; Biggs, Mark J

2014-04-09

Although protein adsorption on solids is of immense relevance, experimental limitations mean there is still a remarkable lack of understanding of the adsorption mechanism, particularly at a molecular level. By subjecting 240+ molecular dynamics simulations of two peptide/water/solid surface systems to statistical analysis, a generalized molecular level mechanism for peptide adsorption has been identified for uncharged surfaces that interact strongly with the solution phase. This mechanism is composed of three phases: (1) biased diffusion of the peptide from the bulk phase toward the surface; (2) anchoring of the peptide to the water/solid interface via interaction of a hydrophilic group with the water adjacent to the surface or a strongly interacting hydrophobic group with the surface; and (3) lockdown of the peptide on the surface via a slow, stepwise and largely sequential adsorption of its residues, which we term 'statistical zippering'. The adsorption mechanism is dictated by the existence of water layers adjacent to the solid and orientational ordering therein. By extending the solid into the solution by ~8 Å and endowing it with a charged character, the water layers ensure the peptide feels the effect of the solid at a range well beyond the dispersion force that arises from it, thus inducing biased diffusion from afar. The charging of the interface also facilitates anchoring of the peptide near the surface via one of its hydrophilic groups, allowing it time it would otherwise not have to rearrange and lockdown. Finally, the slowness of the lockdown process is dictated by the need for the peptide groups to replace adjacent tightly bound interfacial water.

Fluorous tagging strategy for solution-phase synthesis of small molecules, peptides and oligosaccharides

PubMed Central

Zhang, Wei

2005-01-01

The purification of reaction mixtures is a slow process in organic synthesis, especially during the production of large numbers of analogs and compound libraries. Phase-tag methods such as solid-phase synthesis and fluorous synthesis, provide efficient ways of addressing the separation issue. Fluorous synthesis employs functionalized perfluoroalkyl groups attached to substrates or reagents. The separation of the resulting fluorous molecules can be achieved using strong and selective fluorous liquid-liquid extraction, fluorous silica gel-based solid-phase extraction or high-performance liquid chromatography. Fluorous technology is a novel solution-phase method, which has the advantages of fast reaction times in homogeneous environments, being readily adaptable to literature conditions, having easy intermediate analysis, and having flexibility in reaction scale and scope. In principle, any synthetic methods that use a solid-support could be conducted in solution-phase by replacing the polymer linker with a corresponding fluorous tag. This review summarizes the progress of fluorous tags in solution-phase synthesis of small molecules, peptides and oligosaccharides. PMID:15595439

Conformational and Functional Effects Induced by D- and L-Amino Acid Epimerization on a Single Gene Encoded Peptide from the Skin Secretion of Hypsiboas punctatus

PubMed Central

de Magalhães, Mariana T. Q.; Barbosa, Eder A.; Prates, Maura V.; Verly, Rodrigo M.; Munhoz, Victor Hugo O.; de Araújo, Ivan E.; Bloch, Carlos

2013-01-01

Skin secretion of Hypsiboas punctatus is the source of a complex mixture of bioactive compounds where peptides and small proteins prevail, similarly to many other amphibians. Among dozens of molecules isolated from H. punctatus in a proteomic based approach, we report here the structural and functional studies of a novel peptide named Phenylseptin (FFFDTLKNLAGKVIGALT-NH2) that was purified as two naturally occurring D- and L-Phes configurations. The amino acid epimerization and C-terminal amidation for both molecules were confirmed by a combination of techniques including reverse-phase UFLC, ion mobility mass spectrometry, high resolution MS/MS experiments, Edman degradation, cDNA sequencing and solid-phase peptide synthesis. RMSD analysis of the twenty lowest-energy 1H NMR structures of each peptide revealed a major 90° difference between the two backbones at the first four N-terminal residues and substantial orientation changes of their respective side chains. These structural divergences were considered to be the primary cause of the in vitro quantitative differences in antimicrobial activities between the two molecules. Finally, both molecules elicited equally aversive reactions in mice when delivered orally, an effect that depended entirely on peripheral gustatory pathways. PMID:23565145

Natural Proline-Rich Cyclopolypeptides from Marine Organisms: Chemistry, Synthetic Methodologies and Biological Status.

PubMed

Fang, Wan-Yin; Dahiya, Rajiv; Qin, Hua-Li; Mourya, Rita; Maharaj, Sandeep

2016-10-26

Peptides have gained increased interest as therapeutics during recent years. More than 60 peptide drugs have reached the market for the benefit of patients and several hundreds of novel therapeutic peptides are in preclinical and clinical development. The key contributor to this success is the potent and specific, yet safe, mode of action of peptides. Among the wide range of biologically-active peptides, naturally-occurring marine-derived cyclopolypeptides exhibit a broad range of unusual and potent pharmacological activities. Because of their size and complexity, proline-rich cyclic peptides (PRCPs) occupy a crucial chemical space in drug discovery that may provide useful scaffolds for modulating more challenging biological targets, such as protein-protein interactions and allosteric binding sites. Diverse pharmacological activities of natural cyclic peptides from marine sponges, tunicates and cyanobacteria have encouraged efforts to develop cyclic peptides with well-known synthetic methods, including solid-phase and solution-phase techniques of peptide synthesis. The present review highlights the natural resources, unique structural features and the most relevant biological properties of proline-rich peptides of marine-origin, focusing on the potential therapeutic role that the PRCPs may play as a promising source of new peptide-based novel drugs.

Natural Proline-Rich Cyclopolypeptides from Marine Organisms: Chemistry, Synthetic Methodologies and Biological Status

PubMed Central

Fang, Wan-Yin; Dahiya, Rajiv; Qin, Hua-Li; Mourya, Rita; Maharaj, Sandeep

2016-01-01

Peptides have gained increased interest as therapeutics during recent years. More than 60 peptide drugs have reached the market for the benefit of patients and several hundreds of novel therapeutic peptides are in preclinical and clinical development. The key contributor to this success is the potent and specific, yet safe, mode of action of peptides. Among the wide range of biologically-active peptides, naturally-occurring marine-derived cyclopolypeptides exhibit a broad range of unusual and potent pharmacological activities. Because of their size and complexity, proline-rich cyclic peptides (PRCPs) occupy a crucial chemical space in drug discovery that may provide useful scaffolds for modulating more challenging biological targets, such as protein-protein interactions and allosteric binding sites. Diverse pharmacological activities of natural cyclic peptides from marine sponges, tunicates and cyanobacteria have encouraged efforts to develop cyclic peptides with well-known synthetic methods, including solid-phase and solution-phase techniques of peptide synthesis. The present review highlights the natural resources, unique structural features and the most relevant biological properties of proline-rich peptides of marine-origin, focusing on the potential therapeutic role that the PRCPs may play as a promising source of new peptide-based novel drugs. PMID:27792168

Synthesis of the very acid-sensitive Fmoc-Cys(Mmt)-OH and its application in solid-phase peptide synthesis.

PubMed

Barlos, K; Gatos, D; Hatzi, O; Koch, N; Koutsogianni, S

1996-03-01

S-4-methoxytrityl cysteine was synthesized and converted into the corresponding Fmoc-Cys(Mmt)-OH by its reaction with Fmoc-OSu. As compared to the corresponding Fmoc-Cys(Trt)-OH, the S-Mmt-function was found to be considerably more acid labile. Quantitative S-Mmt-removal occurs selectively in the presence of groups of the tert butyl type and S-Trt by treatment with 0.5-1.0% TFA. The new derivative was successfully utilized in the SPPS of Tyr1-somatostatin on 2-chlorotrityl resin. In this synthesis groups of the Trt-type were exclusively used for amino acid side-chain protection. Quantitative cleavage from the resin and complete deprotection was performed by treatment with 3% TFA in DCM-TES (95:5) for 30 min at RT. We observed no reduction of tryptophan under these conditions.

Synthesis of Bipartite Tetracysteine PNA Probes for DNA In Situ Fluorescent Labeling.

PubMed

Fang, Ge-Min; Seitz, Oliver

2017-12-24

"Label-free" fluorescent probes that avoid additional steps or building blocks for conjugation of fluorescent dyes with oligonucleotides can significantly reduce the time and cost of parallel bioanalysis of a large number of nucleic acid samples. A method for the synthesis of "label-free" bicysteine-modified PNA probes using solid-phase synthesis and procedures for sequence-specific DNA in situ fluorescent labeling is described here. The concept is based on the adjacent alignment of two bicysteine-modified peptide nucleic acids on a DNA target to form a structurally optimized bipartite tetracysteine motif, which induces a sequence-specific fluorogenic reaction with commercially available biarsenic dyes, even in complex media such as cell lysate. This unit will help researchers to quickly synthesize bipartite tetracysteine PNA probes and carry out low-cost DNA in situ fluorescent labeling experiments. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Structure based design and synthesis of peptide inhibitor of human LOX-12: in vitro and in vivo analysis of a novel therapeutic agent for breast cancer.

PubMed

Singh, Abhay Kumar; Singh, Ratnakar; Naz, Farhat; Chauhan, Shyam Singh; Dinda, Amit; Shukla, Abhay Anand; Gill, Kamaldeep; Kapoor, Vaishali; Dey, Sharmistha

2012-01-01

Human breast cancer cell proliferation involves a complex interaction between growth factors, steroid hormones and peptide hormones. The interaction of growth factors, such as epidermal growth factor (EGF), with their receptors on breast cancer cells can lead to the hydrolysis of phospholipids and release of fatty acid such as arachidonic acid, which can be further metabolized by cyclooxygenase (COX) and lipoxygenase (LOX) pathways to produce prostaglandins. The high concentration of prostaglandins has been associated with chronic inflammatory diseases and several types of human cancers. This is due to the over expression COX, LOX and other inflammatory enzymes. Ten peptides were designed and synthesized by solid phase peptide synthesis and analyzed in vitro for enzyme inhibition. Out of these peptides, YWCS had shown significant inhibitory effects. The dissociation constant (K(D)) was determined by surface plasmon resonance (SPR) analysis and was found to be 3.39 × 10(-8) M and 8.6 × 10(-8) M for YWCS and baicalein (positive control), respectively. The kinetic constant Ki was 72.45 × 10(-7) M as determined by kinetic assay. The peptide significantly reduced the cell viability of estrogen positive MCF-7 and estrogen negative MDA-MB-231 cell line with the half maximal concentration (IC(50)) of 75 µM and 400 µM, respectively. The peptide also induced 49.8% and 20.8% apoptosis in breast cancer cells MCF-7 and MDA-MB-231, respectively. The YWCS was also found to be least hemolytic at a concentration of 358 µM. In vivo studies had shown that the peptide significantly inhibits tumor growth in mice (p<0.017). This peptide can be used as a lead compound and complement for ongoing efforts to develop differentiation therapies for breast cancer.

Peptide-based, two-fluorophore, ratiometric probe for quantifying mobile zinc in biological solutions.

PubMed

Zhang, Daniel Y; Azrad, Maria; Demark-Wahnefried, Wendy; Frederickson, Christopher J; Lippard, Stephen J; Radford, Robert J

2015-02-20

Small-molecule fluorescent sensors are versatile agents for detecting mobile zinc in biology. Capitalizing on the abundance of validated mobile zinc probes, we devised a strategy for repurposing existing intensity-based sensors for quantitative applications. Using solid-phase peptide synthesis, we conjugated a zinc-sensitive Zinpyr-1 derivative and a zinc-insensitive 7-hydroxycoumarin derivative onto opposite ends of a rigid P9K peptide scaffold to create HcZ9, a ratiometric fluorescent probe for mobile zinc. A plate reader-based assay using HcZ9 was developed, the accuracy of which is comparable to that of atomic absorption spectroscopy. We investigated zinc accumulation in prostatic cells and zinc levels in human seminal fluid. When normal and tumorigenic cells are bathed in zinc-enriched media, cellular mobile zinc is buffered and changes slightly, but total zinc levels increase significantly. Quantification of mobile and total zinc levels in human seminal plasma revealed that the two are positively correlated with a Pearson's coefficient of 0.73.

A New Synthetic Peptide with In vitro Antibacterial Potential Against Escherichia coli O157:H7 and Methicillin-Resistant Staphylococcus aureus (MRSA).

PubMed

Prada, Y A; Guzmán, F; Rondón, P; Escobar, P; Ortíz, C; Sierra, D A; Torres, R; Mejía-Ospino, E

2016-09-01

In this work, we performed the rational design of a cationic antimicrobial peptide, GIBIMPY4, using the software DEPRAMPs developed at the GIBIM research group. GIBIMPY4 has a length of 17 amino acids, it is amphipathic, its structure is α-helix and it has a net charge of (+5). Solid-phase peptide synthesis was performed using the Fmoc strategy in acid medium. The primary structure was confirmed by MALDI-TOF mass spectrometry. The antimicrobial activity of the peptide was evaluated by broth microdilution method by measuring optical density in 96-well microplates. The minimal inhibitory concentration of GIBIMPY4 to kill 50 % of the bacterial cells (MIC50) was 6.20 ± 0.02 µM for MRSA and 4.55 ± 0.02 µM for E. coli O157:H7, while also reporting a bacteriostatic effect for the later. GIBIMPY4 activity was sensitive to salt concentration in E. coli but insignificant effect in its activity against MRSA. The peptide seems to be a broad-spectrum antimicrobial agent based on the results against Gram-positive and Gram-negative bacteria and was specific for bacterial cells E. coli O157:H7 with index of specificity equal to 9.01 in vitro assays.

In situ click chemistry: from small molecule discovery to synthetic antibodies

PubMed Central

Agnew, Heather D.; Lai, Bert; Lee, Su Seong; Lim, Jaehong; Nag, Arundhati; Pitram, Suresh; Rohde, Rosemary; Heath, James R.

2013-01-01

Advances in the fields of proteomics, molecular imaging, and therapeutics are closely linked to the availability of affinity reagents that selectively recognize their biological targets. Here we present a review of Iterative Peptide In Situ Click Chemistry (IPISC), a novel screening technology for designing peptide multiligands with high affinity and specificity. This technology builds upon in situ click chemistry, a kinetic target-guided synthesis approach where the protein target catalyzes the conjugation of two small molecules, typically through the azide–alkyne Huisgen cycloaddition. Integrating this methodology with solid phase peptide libraries enables the assembly of linear and branched peptide multiligands we refer to as Protein Catalyzed Capture Agents (PCC Agents). The resulting structures can be thought of as analogous to the antigen recognition site of antibodies and serve as antibody replacements in biochemical and cell-based applications. In this review, we discuss the recent progress in ligand design through IPISC and related approaches, focusing on the improvements in affinity and specificity as multiligands are assembled by target-catalyzed peptide conjugation. We compare the IPISC process to small molecule in situ click chemistry with particular emphasis on the advantages and technical challenges of constructing antibody-like PCC Agents. PMID:22836343

Development of a seaweed derived platelet activating factor acetylhydrolase (PAF-AH) inhibitory hydrolysate, synthesis of inhibitory peptides and assessment of their toxicity using the Zebrafish larvae assay.

PubMed

Fitzgerald, Ciarán; Gallagher, Eimear; O'Connor, Paula; Prieto, José; Mora-Soler, Leticia; Grealy, Maura; Hayes, Maria

2013-12-01

The vascular inflammatory role of platelet activating factor acetylhydrolase (PAF-AH) is thought to be due to the formation of lysophosphatidyl choline and oxidized non-esterified fatty acids. This enzyme is considered a promising therapeutic target for the prevention of atherosclerosis and there is a need to expand the available chemical templates of PAF-AH inhibitors. This study demonstrated how natural PAF-AH inhibitory peptides were isolated and characterized from the red macroalga Palmaria palmata. The dried powdered alga was hydrolyzed using the food grade enzyme papain, and the resultant peptide containing fraction generated using RP-HPLC. Several oligopeptides were identified as potential PAF-AH inhibitors following bio-guided fractionation, and the amino acid sequences of these oligopeptides were confirmed by Q-TOF-MS and microwave-assisted solid phase de novo synthesis. The most promising PAF-AH inhibitory peptide had the amino acid sequence NIGK and a PAF-AH IC50 value of 2.32 mM. This peptide may constitute a valid drug template for PAF-AH inhibitors. Furthermore the P. palmata hydrolysate was nontoxic when assayed using the Zebrafish toxicity model at a concentration of 1mg/ml. Copyright © 2013 Elsevier Inc. All rights reserved.

Synthesis of Tc-99m labeled 1,2,3-triazole-4-yl c-met binding peptide as a potential c-met receptor kinase positive tumor imaging agent.

PubMed

Kim, Eun-Mi; Joung, Min-Hee; Lee, Chang-Moon; Jeong, Hwan-Jeong; Lim, Seok Tae; Sohn, Myung-Hee; Kim, Dong Wook

2010-07-15

The mesenchymal-epithelial transition factor (c-Met), which is related to tumor cell growth, angiogenesis and metastases, is known to be overexpressed in several tumor types. In this study, we synthesized technetium-99m labeled 1,2,3-triazole-4-yl c-Met binding peptide (cMBP) derivatives, prepared by solid phase peptide synthesis and the 'click-to-chelate' protocol for the introduction of tricarbonyl technetium-99m, as a potential c-Met receptor kinase positive tumor imaging agent, and evaluated their in vitro c-Met binding affinity, cellular uptake, and stability. The (99m)Tc labeled cMBP derivatives ([(99m)Tc(CO)(3)]12, [(99m)Tc(CO)(3)]13, and [(99m)Tc(CO)(3)]14) were prepared in 85-90% radiochemical yields. The cold surrogate cMBP derivatives, [Re(CO)(3)]12, [Re(CO)(3)]13, and [Re(CO)(3)]14, were shown to have high binding affinities (0.13 microM, 0.06 microM, and 0.16 microM, respectively) to a purified cMet/Fc chimeric recombinant protein. In addition, the in vitro cellular uptake and inhibition studies demonstrated the high specific binding of these (99m)Tc labeled cMBP derivatives ([(99m)Tc(CO)(3)]12-14) to c-Met receptor positive U87MG cells. 2010 Elsevier Ltd. All rights reserved.

Synthesis of solid solutions of perovskites

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dambekalne, M.Y.; Antonova, M.K.; Perro, I.T.

The authors carry out thermographic studies, using a derivatograph, in order to understand the nature of the processes taking place during the synthesis of solid solutions of perovskites. Based on the detailed studies on the phase transformations occurring in the charges of the PSN-PMN solid solutions and on the selection of the optimum conditions for carrying out their synthesis, the authors obtained a powder containing a minimum quantity of the undesirable pyrochlore phase and by sintering it using the hot pressing method, they produced single phase ceramic specimens containing the perovskite phase alone with a density close to the theoreticalmore » value and showing zero apparent porosity and water absorption.« less

Solution-Phase Synthesis of Dipeptides: A Capstone Project That Employs Key Techniques in an Organic Laboratory Course

ERIC Educational Resources Information Center

Marchetti, Louis; DeBoef, Brenton

2015-01-01

A contemporary approach to the synthesis and purification of several UV-active dipeptides has been developed for the second-year organic laboratory. This experiment exposes students to the important technique of solution-phase peptide synthesis and allows an instructor to highlight the parallel between what they are accomplishing in the laboratory…

Solid-Phase and Microwave-Assisted Syntheses of 2,5-Diketopiperazines: Small Molecules with Great Potential

PubMed Central

O'Neill, J.C.; Blackwell, H. E.

2008-01-01

Diketopiperazines (DKPs) are a well-known class of heterocycles that have recently emerged as a promising biologically active scaffold. Solid-phase organic synthesis has become an important tool in the combinatorial exploration of these privileged structures, expediting the synthesis and, therefore, the discovery of active compounds. To date, certain DKPs have shown potent activities against a range of diseases and biological phenomena, including bacterial infections, various cancers, asthma, infertility, premature labor, and HIV. Recent applications of solid-phase DKP synthesis, with a particular focus on cyclative cleavage and microwave-assisted reactions, are highlighted herein. PMID:18288948

Synthesis, Biophysical, and Pharmacological Evaluation of the Melanocortin Agonist AST3-88: Modifications of Peptide Backbone at Trp 7 Position Lead to a Potent, Selective, and Stable Ligand of the Melanocortin 4 Receptor (MC4R)

PubMed Central

2015-01-01

The melanocortin-3 (MC3R) and melanocortin-4 (MC4R) receptors are expressed in the brain and are implicated in the regulation of food intake and energy homeostasis. The endogenous agonist ligands for these receptors (α-, β-, γ-MSH and ACTH) are linear peptides with limited receptor subtype selectivity and metabolic stability, thus minimizing their use as probes to characterize the overlapping pharmacological and physiological functions of the melanocortin receptor subtypes. In the present study, an engineered template, in which the peptide backbone was modified by a heterocyclic reverse turn mimetic at the Trp7 residue, was synthesized using solid phase peptide synthesis and characterized by a β-galactosidase cAMP based reporter gene assay. The functional assay identified a ∼5 nM mouse MC4R agonist (AST3-88) with more than 50-fold selectivity over the mMC3R. Biophysical studies (2D 1H NMR spectroscopy and molecular dynamics) of AST3-88 identified a type VIII β-turn secondary structure spanning the pharmacophore domain stabilized by the intramolecular interactions between the side chains of the His and Trp residues. Enzymatic studies of AST3-88 revealed enhanced stability of AST3-88 over the α-MSH endogenous peptide in rat serum. Upon central administration of AST3-88 into rats, a decreased food intake response was observed. This is the first study to probe the in vivo physiological activity of this engineered peptide-heterocycle template. These findings advance the present knowledge of pharmacophore design for potent, selective, and metabolically stable melanocortin ligands. PMID:25141170

Augmenting Influenza-Specific T Cell Memory Generation with a Natural Killer T Cell-Dependent Glycolipid-Peptide Vaccine.

PubMed

Anderson, Regan J; Li, Jasmine; Kedzierski, Lukasz; Compton, Benjamin J; Hayman, Colin M; Osmond, Taryn L; Tang, Ching-Wen; Farrand, Kathryn J; Koay, Hui-Fern; Almeida, Catarina Filipa Dos Santos Sa E; Holz, Lauren R; Williams, Geoffrey M; Brimble, Margaret A; Wang, Zhongfang; Koutsakos, Marios; Kedzierska, Katherine; Godfrey, Dale I; Hermans, Ian F; Turner, Stephen J; Painter, Gavin F

2017-11-17

The development of a universal vaccine for influenza A virus (IAV) that does not require seasonal modification is a long-standing health goal, particularly in the context of the increasing threat of new global pandemics. Vaccines that specifically induce T cell responses are of considerable interest because they can target viral proteins that are more likely to be shared between different virus strains and subtypes and hence provide effective cross-reactive IAV immunity. From a practical perspective, such vaccines should induce T cell responses with long-lasting memory, while also being simple to manufacture and cost-effective. Here we describe the synthesis and evaluation of a vaccine platform based on solid phase peptide synthesis and bio-orthogonal conjugation methodologies. The chemical approach involves covalently attaching synthetic long peptides from a virus-associated protein to a powerful adjuvant molecule, α-galactosylceramide (α-GalCer). Strain-promoted azide-alkyne cycloaddition is used as a simple and efficient method for conjugation, and pseudoproline methodology is used to increase the efficiency of the peptide synthesis. α-GalCer is a glycolipid that stimulates NKT cells, a population of lymphoid-resident immune cells that can provide potent stimulatory signals to antigen-presenting cells engaged in driving proliferation and differentiation of peptide-specific T cells. When used in mice, the vaccine induced T cell responses that provided effective prophylactic protection against IAV infection, with the speed of viral clearance greater than that seen from previous viral exposure. These findings are significant because the vaccines are highly defined, quick to synthesize, and easily characterized and are therefore appropriate for large scale affordable manufacture.

Synthetic procedure for N-Fmoc amino acyl-N-sulfanylethylaniline linker as crypto-peptide thioester precursor with application to native chemical ligation.

PubMed

Sakamoto, Ken; Sato, Kohei; Shigenaga, Akira; Tsuji, Kohei; Tsuda, Shugo; Hibino, Hajime; Nishiuchi, Yuji; Otaka, Akira

2012-08-17

N-sulfanylethylanilide (SEAlide) peptides 1, obtainable using Fmoc-based solid-phase peptide synthesis (Fmoc SPPS), function as crypto-thioesters in native chemical ligation (NCL), yielding a wide variety of peptides/proteins. Their acylating potential with N-terminal cysteinyl peptides 2 can be tuned by the presence or absence of phosphate salts, leading to one-pot/multifragment ligation, operating under kinetically controlled conditions. SEAlide peptides have already been shown to be promising for use in protein synthesis; however, a widely applicable method for the synthesis of N-Fmoc amino acyl-N-sulfanylethylaniline linkers 4, required for the preparation of SEAlide peptides, is unavailable. The present study addresses the development of efficient condensation protocols of 20 naturally occurring amino acid derivatives to the N-sulfanylethylaniline linker 5. N-Fmoc amino acyl aniline linkers 4 of practical use in NCL chemistry, except in the case of the proline- or aspartic acid-containing linker, were successfully synthesized by coupling of POCl(3)- or SOCl(2)-activated Fmoc amino acid derivatives with sodium anilide species 6, without accompanying racemization and loss of side-chain protection. Furthermore, SEAlide peptides 7 possessing various C-terminal amino acids (Gly, His, Phe, Ala, Asn, Ser, Glu, and Val) were shown to be of practical use in NCL chemistry.

A dimethyl ketal-protected benzoin-based linker suitable for photolytic release of unprotected peptides.

PubMed

Chumachenko, Nataliya; Novikov, Yehor; Shoemaker, Richard K; Copley, Shelley D

2011-11-18

Photolabile 3',5'-dimethoxybenzoin-based linkers are advantageous for a variety of solid-phase synthetic procedures and manipulations of biomolecules because UV irradiation in aqueous media provides fast and essentially quantitative release of tethered molecules, while generating unreactive side products. Practical applications of previously reported linkers are compromised to some extent by the 1,3-dithiane protection of the benzoin carbonyl group and the lengthy synthesis. We have extended the group of photocleavable 3',5'-dimethoxybenzoin-based linkers by designing and synthesizing a linker in which the carbonyl group is protected as a dimethyl ketal. This protection is compatible with commonly used esterification and amide bond formation techniques, including the Fmoc/tBu strategy for solid phase peptide synthesis, is stable under mild acidic conditions, and can be quantitatively removed in <5 min by 3% TFA in dichloromethane. Irradiation of beads carrying peptides attached to the linker at 350 nm in aqueous or partially aqueous media affords >90% release after 30 min. The linker was synthesized from commercially available starting materials in five steps with an overall yield of 40% and without any column chromatography purification. Additionally, we developed a route to a dithiane-protected linker that requires only two steps and proceeds in 65% yield, a significant improvement over previous synthetic routes.

SIMULATION AND VISUALIZATION OF FLOW PATTERN IN MICROARRAYS FOR LIQUID PHASE OLIGONUCLEOTIDE AND PEPTIDE SYNTHESIS

PubMed Central

O-Charoen, Sirimon; Srivannavit, Onnop; Gulari, Erdogan

2008-01-01

Microfluidic microarrays have been developed for economical and rapid parallel synthesis of oligonucleotide and peptide libraries. For a synthesis system to be reproducible and uniform, it is crucial to have a uniform reagent delivery throughout the system. Computational fluid dynamics (CFD) is used to model and simulate the microfluidic microarrays to study geometrical effects on flow patterns. By proper design geometry, flow uniformity could be obtained in every microreactor in the microarrays. PMID:17480053

Solid-State NMR Studies of Amyloid Materials: A Protocol to Define an Atomic Model of Aβ(1-42) in Amyloid Fibrils.

PubMed

Xiao, Yiling; McElheny, Dan; Hoshi, Minako; Ishii, Yoshitaka

2018-01-01

Intense efforts have been made to understand the molecular structures of misfolded amyloid β (Aβ) in order to gain insight into the pathological mechanism of Alzheimer's disease. Solid-state NMR spectroscopy (SSNMR) is considered a primary tool for elucidating the structures of insoluble and noncrystalline amyloid fibrils and other amyloid assemblies. In this chapter, we describe a detailed protocol to obtain the first atomic model of the 42-residue human Aβ peptide Aβ(1-42) in structurally homogeneous amyloid fibrils from our recent SSNMR study (Nat Struct Mol Biol 22:499-505, 2015). Despite great biological and clinical interest in Aβ(1-42) fibrils, their structural details have been long-elusive until this study. The protocol is divided into four sections. First, the solid-phase peptide synthesis (SPPS) and purification of monomeric Aβ(1-42) is described. We illustrate a controlled incubation method to prompt misfolding of Aβ(1-42) into homogeneous amyloid fibrils in an aqueous solution with fragmented Aβ(1-42) fibrils as seeds. Next, we detail analysis of Aβ(1-42) fibrils by SSNMR to obtain structural restraints. Finally, we describe methods to construct atomic models of Aβ(1-42) fibrils based on SSNMR results through two-stage molecular dynamics calculations.

Citropin 1.1 Trifluoroacetate to Chloride Counter-Ion Exchange in HCl-Saturated Organic Solutions: An Alternative Approach.

PubMed

Sikora, Karol; Neubauer, Damian; Jaśkiewicz, Maciej; Kamysz, Wojciech

2018-01-01

In view of the increasing interest in peptides in various market sectors, a stronger emphasis on topics related to their production has been seen. Fmoc-based solid phase peptide synthesis, although being fast and efficient, provides final products with significant amounts of trifluoroacetate ions in the form of either a counter-ion or an unbound impurity. Because of the proven toxicity towards cells and peptide activity inhibition, ion exchange to more biocompatible one is purposeful. Additionally, as most of the currently used counter-ion exchange techniques are time-consuming and burdened by peptide yield reduction risk, development of a new approach is still a sensible solution. In this study, we examined the potential of peptide counter-ion exchange using non-aqueous organic solvents saturated with HCl. Counter-ion exchange of a model peptide, citropin 1.1 (GLFDVIKKVASVIGGL-NH 2 ), for each solvent was conducted through incubation with subsequent evaporation under reduced pressure, dissolution in water and lyophilization. Each exchange was performed four times and compared to a reference method-lyophilization of the peptide from an 0.1 M HCl solution. The results showed superior counter-ion exchange efficiency for most of the organic solutions in relation to the reference method. Moreover, HCl-saturated acetonitrile and tert -butanol provided a satisfying exchange level after just one repetition. Thus, those two organic solvents can be potentially introduced into routine peptide counter-ion exchange.

Biomolecule conjugation strategy using novel water-soluble phosphine-based chelating agents

DOEpatents

Katti, Kattesh V.; Gali, Hariprasad; Volkert, Wynn A.

2004-08-24

This invention describes a novel strategy to produce phosphine-functionalized biomolecules (e.g. peptides or proteins) for potential use in the design and development of site-specific radiopharmaceuticals for diagnosis or therapy of specific cancers. Hydrophilic alkyl phosphines, in general, tend to be oxidatively unstable. Therefore, incorporation of such phosphine functionalities on peptide (and other biomolecule) backbones, without oxidizing the P.sup.III centers, is difficult. In this context this discovery reports on a new technology by which phosphines, in the form of bifunctional chelating agents, can be directly incorporated on biomolecular backbones using manual synthetic or solid phase peptide synthesis methodologies. The superior ligating abilities of phosphine ligands, with various diagnostically (e.g. TC-99m) or therapeutically (e.g. Re186/188, Rh-105, Au-199) useful radiometals, coupled with the findings that the resulting complexes demonstrate high in vivo stability makes this approach useful in the development of radiolabeled biomolecules for applications in the design of tumor-specific radiopharmaceuticals.

Synthetic Covalently Linked Dimeric Form of H2 Relaxin Retains Native RXFP1 Activity and Has Improved In Vitro Serum Stability

PubMed Central

Nair, Vinojini B.; Bathgate, Ross A. D.; Separovic, Frances; Samuel, Chrishan S.; Hossain, Mohammed Akhter; Wade, John D.

2015-01-01

Human (H2) relaxin is a two-chain peptide member of the insulin superfamily and possesses potent pleiotropic roles including regulation of connective tissue remodeling and systemic and renal vasodilation. These effects are mediated through interaction with its cognate G-protein-coupled receptor, RXFP1. H2 relaxin recently passed Phase III clinical trials for the treatment of congestive heart failure. However, its in vivo half-life is short due to its susceptibility to proteolytic degradation and renal clearance. To increase its residence time, a covalent dimer of H2 relaxin was designed and assembled through solid phase synthesis of the two chains, including a judiciously monoalkyne sited B-chain, followed by their combination through regioselective disulfide bond formation. Use of a bisazido PEG7 linker and “click” chemistry afforded a dimeric H2 relaxin with its active site structurally unhindered. The resulting peptide possessed a similar secondary structure to the native monomeric H2 relaxin and bound to and activated RXFP1 equally well. It had fewer propensities to activate RXFP2, the receptor for the related insulin-like peptide 3. In human serum, the dimer had a modestly increased half-life compared to the monomeric H2 relaxin suggesting that additional oligomerization may be a viable strategy for producing longer acting variants of H2 relaxin. PMID:25685807

Total chemical synthesis of human matrix Gla protein

PubMed Central

Hackeng, Tilman M.; Rosing, Jan; Spronk, Henri M.H.; Vermeer, Cees

2001-01-01

Human matrix Gla protein (MGP) is a vitamin K–dependent extracellular matrix protein that binds Ca2+ ions and that is involved in the prevention of vascular calcification. MGP is a 10.6-kD protein (84 amino acids) containing five γ-carboxyglutamic acid (Gla) residues and one disulfide bond. Studies of the mechanism by which MGP prevents calcification of the arterial media are hampered by the low solubility of the protein (<10 μg/mL). Because of solubility problems, processing of a recombinantly expressed MGP-fusion protein chimera to obtain MGP was unsuccessful. Here we describe the total chemical synthesis of MGP by tBoc solid-phase peptide synthesis (SPPS) and native chemical ligation. Peptide Tyr1-Ala53 was synthesized on a derivatized resin yielding a C-terminal thioester group. Peptide Cys54-Lys84 was synthesized on Lys-PAM resin yielding a C-terminal carboxylic acid. Subsequent native chemical ligation of the two peptides resulted in the formation of a native peptide bond between Ala53 and Cys54. Folding of the 1–84-polypeptide chain in 3 M guanidine (pH 8) resulted in a decrease of molecular mass from 10,605 to 10,603 (ESI-MS), representing the loss of two protons because of the formation of the Cys54-Cys60 internal disulfide bond. Like native MGP, synthetic MGP had the same low solubility when brought into aqueous buffer solutions with physiological salt concentrations, confirming its native like structure. However, the solubility of MGP markedly increased in borate buffer at pH 7.4 in the absence of sodium chloride. Ca2+-binding to MGP was confirmed by analytical HPLC, on which the retention time of MGP was reduced in the presence of CaCl2. Circular dichroism studies revealed a sharp increase in α-helicity at 0.2 mM CaCl2 that may explain the Ca2+-dependent shift in high-pressure liquid chromatography (HPLC)-retention time of MGP. In conclusion, facile and efficient chemical synthesis in combination with native chemical ligation yielded MGP preparations that can aid in unraveling the mechanism by which MGP prevents vascular calcification. PMID:11274477

Antagonistic Activity of Lactobacillus plantarum C11: Two New Two-Peptide Bacteriocins, Plantaricins EF and JK, and the Induction Factor Plantaricin A

PubMed Central

Anderssen, Erlend L.; Diep, Dzung Bao; Nes, Ingolf F.; Eijsink, Vincent G. H.; Nissen-Meyer, Jon

1998-01-01

Six bacteriocinlike peptides (plantaricin A [PlnA], PlnE, PlnF, PlnJ, PlnK, and PlnN) produced by Lactobacillus plantarum C11 were detected by amino acid sequencing and mass spectrometry. Since purification to homogeneity was problematic, all six peptides were obtained by solid-phase peptide synthesis and were tested for bacteriocin activity. It was found that L. plantarum C11 produces two two-peptide bacteriocins (PlnEF and PlnJK); a strain-specific antagonistic activity was detected at nanomolar concentrations when PlnE and PlnF were combined and when PlnJ and PlnK were combined. Complementary peptides were at least 103 times more active when they were combined than when they were present individually, and optimal activity was obtained when the complementary peptides were present in approximately equal amounts. The interaction between complementary peptides was specific, since neither PlnE nor PlnF could complement PlnJ or PlnK, and none of these peptides could complement the peptides constituting the two-peptide bacteriocin lactococcin G. Interestingly, PlnA, which acts as an extracellular signal (pheromone) that triggers bacteriocin production, also possessed a strain-specific antagonistic activity. No bacteriocin activity could be detected for PlnN. PMID:9603847

Silencing of BCR/ABL Chimeric Gene in Human Chronic Myelogenous Leukemia Cell Line K562 by siRNA-Nuclear Export Signal Peptide Conjugates.

PubMed

Shinkai, Yasuhiro; Kashihara, Shinichi; Minematsu, Go; Fujii, Hirofumi; Naemura, Madoka; Kotake, Yojiro; Morita, Yasutaka; Ohnuki, Koichiro; Fokina, Alesya A; Stetsenko, Dmitry A; Filichev, Vyacheslav V; Fujii, Masayuki

2017-06-01

Herein we described the synthesis of siRNA-NES (nuclear export signal) peptide conjugates by solid phase fragment coupling and the application of them to silencing of bcr/abl chimeric gene in human chronic myelogenous leukemia cell line K562. Two types of siRNA-NES conjugates were prepared, and both sense strands at 5' ends were covalently linked to a NES peptide derived from TFIIIA and HIV-1 REV, respectively. Significant enhancement of silencing efficiency was observed for both of them. siRNA-TFIIIA NES conjugate suppressed the expression of BCR/ABL gene to 8.3% at 200 nM and 11.6% at 50 nM, and siRNA-HIV-1REV NES conjugate suppressed to 4.0% at 200 nM and 6.3% at 50 nM, whereas native siRNA suppressed to 36.3% at 200 nM and 30.2% at 50 nM. We could also show complex of siRNA-NES conjugate and designed amphiphilic peptide peptideβ7 could be taken up into cells with no cytotoxicity and showed excellent silencing efficiency. We believe that the complex siRNA-NES conjugate and peptideβ7 is a promising candidate for in vivo use and therapeutic applications.

The assembly and properties of protobiological structures - The beginnings of cellular peptide synthesis

NASA Technical Reports Server (NTRS)

Fox, S. W.; Nakashima, T.

1980-01-01

New data indicate that lysine-rich proteinoids have the ability to catalyze the synthesis of peptide bonds from a variety of amino acids and ATP. This capacity is evident in aqueous solution, in suspension of phase-separated complexes of lysine-rich proteinoid with acidic proteinoids, and in suspension of phase-separated particles composed of lysine-rich proteinoids with polynucleotides. Since the proteinoid complexes can contain other catalytic activities, including ability to catalyze internucleotide bond formation, it is inferred that the first protocells on earth already had a number of biological types of activity.

Expression and purification of isotopically labeled peptide inhibitors and substrates of cAMP-dependant protein kinase A for NMR analysis.

PubMed

Masterson, Larry R; Bortone, Nadia; Yu, Tao; Ha, Kim N; Gaffarogullari, Ece C; Nguyen, Oanh; Veglia, Gianluigi

2009-04-01

Extensive X-ray crystallographic studies carried out on the catalytic-subunit of protein kinase A (PKA-C) enabled the atomic characterization of inhibitor and/or substrate peptide analogues trapped at its active site. Yet, the structural and dynamic transitions of these peptides from the free to the bound state are missing. These conformational transitions are central to understanding molecular recognition and the enzymatic cycle. NMR spectroscopy allows one to study these phenomena under functionally relevant conditions. However, the amounts of isotopically labeled peptides required for this technique present prohibitive costs for solid-phase peptide synthesis. To enable NMR studies, we have optimized both expression and purification of isotopically enriched substrate/inhibitor peptides using a recombinant fusion protein system. Three of these peptides correspond to the cytoplasmic regions of the wild-type and lethal mutants of the membrane protein phospholamban, while the fourth peptide correspond to the binding epitope of the heat-stable protein kinase inhibitor (PKI(5-24)). The target peptides were fused to the maltose binding protein (MBP), which is further purified using a His(6) tag approach. This convenient protocol allows for the purification of milligram amounts of peptides necessary for NMR analysis.

Facile chemical synthesis and equilibrium unfolding properties of CopG

PubMed Central

Wales, Thomas E.; Richardson, Jane S.; Fitzgerald, Michael C.

2004-01-01

The 45-amino acid polypeptide chain of the homodimeric transcriptional repressor, CopG, was chemically synthesized by stepwise solid phase peptide synthesis (SPPS) using a protocol based on Boc-chemistry. The product obtained from the synthesis was readily purified by reversed-phase HPLC to give a good overall yield (21% by weight). Moreover, the synthetic CopG constructs prepared in this work folded into three-dimensional structures similar to the wild-type protein prepared using conventional recombinant methods as judged by far UV-CD spectroscopy. A fluorescent CopG analog, (Y39W)CopG, was also designed and chemically synthesized to facilitate biophysical studies of CopG’s protein folding and assembly reaction. The guanidinium chloride-induced equilibrium unfolding properties of the wild-type CopG and (Y39W)CopG constructs in this work were characterized and used to develop a model for CopG’s equilibrium unfolding reaction. Our results indicate that CopG’s folding and assembly reaction is well modeled by a two-state process involving folded dimer and unfolded monomer. Using this model, ΔGf and m-values of −13.42 ± 0.04 kcal/mole dimer and 1.92 ± 0.01 kcal/(mole M) were calculated for CopG. PMID:15169951

Recent Approaches Toward Solid Phase Synthesis of β-Lactams

NASA Astrophysics Data System (ADS)

Mandal, Bablee; Ghosh, Pranab; Basu, Basudeb

Since the discovery of penicillin in 1929, β-lactam antibiotics have been recognized as potentially chemotherapeutic drugs of incomparable effectiveness, conjugating a broad spectrum of activity with very low toxicity. The primary motif azetidin-2-one ring (β-lactam) has been considered as specific pharmacophores and scaffolds. With the advent of combinatorial chemistry and automated parallel synthesis coupled with ample interests from the pharmaceutical industries, recent trends have been driven mostly by adopting solid phase techniques and polymer-supported synthesis of β-lactams. The present survey will present an overview of the developments on the polymer-supported and solid phase techniques for the preparation of β-lactam ring or β-lactam containing antibiotics published over the last decade. Both unsubstituted and substitutions with different functional groups at various positions of β-lactams have been synthesized using solid phase technology. However, Wang resin and application of Staudinger [2+2] cycloaddition reaction have remained hitherto the major choice. It may be expected that other solid phase approaches involving different resins would be developed in the coming years.

Femtomolar Ln(III) affinity in peptide-based ligands containing unnatural chelating amino acids.

PubMed

Niedźwiecka, Agnieszka; Cisnetti, Federico; Lebrun, Colette; Delangle, Pascale

2012-05-07

The incorporation of unnatural chelating amino acids in short peptide sequences leads to lanthanide-binding peptides with a higher stability than sequences built exclusively from natural residues. In particular, the hexadentate peptide P(22), which incorporates two unnatural amino acids Ada(2) with aminodiacetate chelating arms, showed picomolar affinity for Tb(3+). To design peptides with higher denticity, expected to show higher affinity for Ln(3+), we synthesized the novel unnatural amino acid Ed3a(2) which carries an ethylenediamine triacetate side-chain and affords a pentadentate coordination site. The synthesis of the derivative Fmoc-Ed3a(2)(tBu)(3)-OH, with appropriate protecting groups for direct use in the solid phase peptide synthesis (Fmoc strategy), is described. The two high denticity peptides P(HD2) (Ac-Trp-Ed3a(2)-Pro-Gly-Ada(2)-Gly-NH(2)) and P(HD5) (Ac-Trp-Ada(2)-Pro-Gly-Ed3a(2)-Gly-NH(2)) led to octadentate Tb(3+) complexes with femtomolar stability in water. The position of the high denticity amino acid Ed3a(2) in the hexapeptide sequence appears to be critical for the control of the metal complex speciation. Whereas P(HD5) promotes the formation of polymetallic species in excess of Ln(3+), P(HD2) forms exclusively the mononuclear complex. The octadentate coordination of Tb(3+) by both P(HD) leads to total dehydration of the metal ion in the mononuclear complexes with long luminescence lifetimes (>2 ms). Hence, we demonstrated that unnatural amino acids carrying polyaminocarboxylate side-chains are interesting building blocks to design high affinity Ln-binding peptides. In particular the novel peptide P(HD2) forms a unique octadentate Tb(3+) complex with femtomolar stability in water and an improvement of the luminescence properties with respect to the trisaquo TbP(22) complex by a factor of 4.

Microwave-assisted solid phase conversion study of Meldrum's acid to ethylenetetracarboxylic dianhydride (C 6O 6)

NASA Astrophysics Data System (ADS)

Taherpour, Avat (Arman)

2010-01-01

Utilization of microwave irradiation provides an effective method for fast synthesizing of some important compounds. Microwave-assisted solid phase is an especial class in chemical synthesis. By the use of MW-irradiation on chemicals, sometimes interesting results can be seen. The synthesis of the interesting molecule ethylenetetracarboxylic dianhydride (C 6O 6) was attempted with a few different methods. In this study, the microwave-assisted solid phase conversion of Meldrum's acid to ethylenetetracarboxylic dianhydride was reported. This conversion was characterized by FT-IR, GC/MS and NMR spectroscopy results.

Non-nucleoside building blocks for copper-assisted and copper-free click chemistry for the efficient synthesis of RNA conjugates.

PubMed

Jayaprakash, K N; Peng, Chang Geng; Butler, David; Varghese, Jos P; Maier, Martin A; Rajeev, Kallanthottathil G; Manoharan, Muthiah

2010-12-03

Novel non-nucleoside alkyne monomers compatible with oligonucleotide synthesis were designed, synthesized, and efficiently incorporated into RNA and RNA analogues during solid-phase synthesis. These modifications allowed site-specific conjugation of ligands to the RNA oligonucleotides through copper-assisted (CuAAC) and copper-free strain-promoted azide-alkyne cycloaddition (SPAAC) reactions. The SPAAC click reactions of cyclooctyne-oligonucleotides with various classes of azido-functionalized ligands in solution phase and on solid phase were efficient and quantitative and occurred under mild reaction conditions. The SPAAC reaction provides a method for the synthesis of oligonucleotide-ligand conjugates uncontaminated with copper ions.

Design and synthesis of multifunctional gold nanoparticles bearing tumor-associated glycopeptide antigens as potential cancer vaccines.

PubMed

Brinãs, Raymond P; Sundgren, Andreas; Sahoo, Padmini; Morey, Susan; Rittenhouse-Olson, Kate; Wilding, Greg E; Deng, Wei; Barchi, Joseph J

2012-08-15

The development of vaccines against specific types of cancers will offer new modalities for therapeutic intervention. Here, we describe the synthesis of a novel vaccine construction prepared from spherical gold nanoparticles of 3-5 nm core diameters. The particles were coated with both the tumor-associated glycopeptides antigens containing the cell-surface mucin MUC4 with Thomsen Friedenreich (TF) antigen attached at different sites and a 28-residue peptide from the complement derived protein C3d to act as a B-cell activating "molecular adjuvant". The synthesis entailed solid-phase glycopeptide synthesis, design of appropriate linkers, and attachment chemistry of the various molecules to the particles. Attachment to the gold surface was mediated by a novel thiol-containing 33 atom linker which was further modified to be included as a third "spacer" component in the synthesis of several three-component vaccine platforms. Groups of mice were vaccinated either with one of the nanoplatform constructs or with control particles without antigen coating. Evaluation of sera from the immunized animals in enzyme immunoassays (EIA) against each glycopeptide antigen showed a small but statistically significant immune response with production of both IgM and IgG isotypes. Vaccines with one carbohydrate antigen (B, C, and E) gave more robust responses than the one with two contiguous disaccharides (D), and vaccine E with a TF antigen attached to threonine at the 10th position of the peptide was selected for IgG over IgM suggesting isotype switching. The data suggested that this platform may be a viable delivery system for tumor-associated glycopeptide antigens.

Concept of internal structural controls for evaluation of inactive synthetic peptide analogs: synthesis of [Orn13,14]apamin and its guanidination to an apamin derivative with full neurotoxic activity.

PubMed Central

Cosand, W L; Merrifield, R B

1977-01-01

The importance of arginine residues 13 and 14 in the bee venom neurotoxin, apamin, was teste by the synthesis of replacement analogs. [13,14-di-Ndelta-trifluoroacetylornithine]Apamin was synthesized by the solid phase method on a benzhydrylamine resin. It was deprotected to [13,14-diornithine]apamin, which was then guanidinated to produce the 4-homoarginine-13,14-diarginine analog, [Har4]apamin. Neither the trifluoroacetylornithine analog nor the ornithine analog produced any detectable symptoms when injected intravenously into mice. However, the synthetic [Har4]apamin exhibited the full neurotoxic activity of native apamin and of [Har4]apamin derived from the natural toxin. This provided an internal structural control for the correctness of the primary structure of the inactive synthetic analogs and strengthened the conclusion that one, or both, of the arginine residues plays an important role in the action of apamin. Images PMID:268626

Antifreeze glycopeptide analogues: microwave-enhanced synthesis and functional studies.

PubMed

Heggemann, Carolin; Budke, Carsten; Schomburg, Benjamin; Majer, Zsuzsa; Wissbrock, Marco; Koop, Thomas; Sewald, Norbert

2010-01-01

Antifreeze glycoproteins enable life at temperatures below the freezing point of physiological solutions. They usually consist of the repetitive tripeptide unit (-Ala-Ala-Thr-) with the disaccharide alpha-D-galactosyl-(1-3)-beta-N-acetyl-D-galactosamine attached to each hydroxyl group of threonine. Monoglycosylated analogues have been synthesized from the corresponding monoglycosylated threonine building block by microwave-assisted solid phase peptide synthesis. This method allows the preparation of analogues containing sequence variations which are not accessible by other synthetic methods. As antifreeze glycoproteins consist of numerous isoforms they are difficult to obtain in pure form from natural sources. The synthetic peptides have been structurally analyzed by CD and NMR spectroscopy in proton exchange experiments revealing a structure as flexible as reported for the native peptides. Microphysical recrystallization tests show an ice structuring influence and ice growth inhibition depending on the concentration, chain length and sequence of the peptides.

Synthesis of a Bifunctional Peptide Inhibitor-IgG1 Fc Fusion That Suppresses Experimental Autoimmune Encephalomyelitis.

PubMed

White, Derek R; Khedri, Zahra; Kiptoo, Paul; Siahaan, Teruna J; Tolbert, Thomas J

2017-07-19

Multiple sclerosis (MS) is a neurodegenerative disease that is estimated to affect over 2.3 million people worldwide. The exact cause for this disease is unknown but involves immune system attack and destruction of the myelin protein surrounding the neurons in the central nervous system. One promising class of compounds that selectively prevent the activation of immune cells involved in the pathway leading to myelin destruction are bifunctional peptide inhibitors (BPIs). Treatment with BPIs reduces neurodegenerative symptoms in experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. In this work, as an effort to further improve the bioactivity of BPIs, BPI peptides were conjugated to the N- and C-termini of the fragment crystallizable (Fc) region of the human IgG1 antibody. Initially, the two peptides were conjugated to IgG1 Fc using recombinant DNA technology. However, expression in yeast resulted in low yields and one of the peptides being heavily proteolyzed. To circumvent this problem, the poorly expressed peptide was instead produced by solid phase peptide synthesis and conjugated enzymatically using a sortase-mediated ligation. The sortase-mediated method showed near-complete conjugation yield as observed by SDS-PAGE and mass spectrometry in small-scale reactions. This method was scaled up to obtain sufficient quantities for testing the BPI-Fc fusion in mice induced with EAE. Compared to the PBS-treated control, mice treated with the BPI-Fc fusion showed significantly reduced disease symptoms, did not experience weight loss, and showed reduced de-myelination. These results demonstrate that the BPI peptides were highly active at suppressing EAE when conjugated to the large Fc scaffold in this manner.

Microwave-assisted synthesis of triple-helical, collagen-mimetic lipopeptides

PubMed Central

Banerjee, Jayati; Hanson, Andrea J; Muhonen, Wallace W; Shabb, John B; Mallik, Sanku

2018-01-01

Collagen-mimetic peptides and lipopeptides are widely used as substrates for matrix degrading enzymes, as new biomaterials for tissue engineering, as drug delivery systems and so on. However, the preparation and subsequent purification of these peptides and their fatty-acid conjugates are really challenging. Herein, we report a rapid microwave-assisted, solid-phase synthetic protocol to prepare the fatty-acid conjugated, triple-helical peptides containing the cleavage site for the enzyme matrix metalloproteinase-9 (MMP-9). We employed a PEG-based resin as the solid support and the amino acids were protected with Fmoc- and tert-butyl groups. The amino acids were coupled at 50 °C (25 W of microwave power) for 5 min. The deprotection reactions were carried out at 75 °C (35 W of microwave power) for 3 min. Using this protocol, a peptide containing 23 amino acids was synthesized and then conjugated to stearic acid in 14 h. PMID:20057380

Synthesis of Triamino Acid Building Blocks with Different Lipophilicities

PubMed Central

Maity, Jyotirmoy; Honcharenko, Dmytro; Strömberg, Roger

2015-01-01

To obtain different amino acids with varying lipophilicity and that can carry up to three positive charges we have developed a number of new triamino acid building blocks. One set of building blocks was achieved by aminoethyl extension, via reductive amination, of the side chain of ortnithine, diaminopropanoic and diaminobutanoic acid. A second set of triamino acids with the aminoethyl extension having hydrocarbon side chains was synthesized from diaminobutanoic acid. The aldehydes needed for the extension by reductive amination were synthesized from the corresponding Fmoc-L-2-amino fatty acids in two steps. Reductive amination of these compounds with Boc-L-Dab-OH gave the C4-C8 alkyl-branched triamino acids. All triamino acids were subsequently Boc-protected at the formed secondary amine to make the monomers appropriate for the N-terminus position when performing Fmoc-based solid-phase peptide synthesis. PMID:25876040

[68Ga]FSC-(RGD)3 a trimeric RGD peptide for imaging αvβ3 integrin expression based on a novel siderophore derived chelating scaffold—synthesis and evaluation

PubMed Central

Knetsch, Peter A.; Zhai, Chuangyan; Rangger, Christine; Blatzer, Michael; Haas, Hubertus; Kaeopookum, Piriya; Haubner, Roland; Decristoforo, Clemens

2015-01-01

Over the last years Gallium-68 (68Ga) has received tremendous attention for labeling of radiopharmaceuticals for positron emission tomography (PET). 68Ga labeling of biomolecules is currently based on bifunctional chelators containing aminocarboxylates (mainly DOTA and NOTA). We have recently shown that cyclic peptide siderophores have very good complexing properties for 68Ga resulting in high specific activities and excellent metabolic stabilities, in particular triacetylfusarinine-C (TAFC). We postulated, that, starting from its deacetylated form (Fusarinine-C (FSC)) trimeric bioconjugates are directly accessible to develop novel targeting peptide based 68Ga labeled radiopharmaceuticals. As proof of principle we report on the synthesis and 68Ga-radiolabeling of a trimeric FSC-RGD conjugate, [68Ga]FSC-(RGD)3, targeting αvβ3 integrin, which is highly expressed during tumor-induced angiogenesis. Synthesis of the RGD peptide was carried out applying solid phase peptide synthesis (SPPS), followed by the coupling to the siderophore [Fe]FSC via in situ activation using HATU/HOAt and DIPEA. Subsequent demetalation allowed radiolabeling of FSC-(RGD)3 with 68Ga. The radiolabeling procedure was optimized regarding peptide amount, reaction time, temperature as well buffer systems. For in vitro evaluation partition coefficient, protein binding, serum stability, αvβ3 integrin binding affinity, and tumor cell uptake were determined. For in vitro tests as well as for the biodistribution studies αvβ3 positive human melanoma M21 and αvβ3 negative M21-L cells were used. [68Ga]FSC-(RGD)3 was prepared with high radiochemical yield (> 98%). Distribution coefficient was − 3.6 revealing a hydrophilic character, and an IC50 value of 1.8 ± 0.6 nM was determined indicating a high binding affinity for αvβ3 integrin. [68Ga]FSC-(RGD)3 was stable in PBS (pH 7.4), FeCl3- and DTPA-solution as well as in fresh human serum at 37 °C for 2 hours. Biodistribution assay confirmed the receptor specific uptake found in vitro. Uptake in the αvβ3 positive tumor was 4.3% ID/g 60 min p.i. which was 3-fold higher than the monomeric [68Ga]NODAGA-RGD. Tumor to blood ratio of approx. 8 and tumor to muscle ratio of approx. 7 were observed. [68Ga]FSC-(RGD)3 serves as an example for the feasibility of a novel class of bifunctional chelators based on cyclic peptide siderophores and shows excellent targeting properties for αvβ3 integrin in vivo for imaging tumor-induced neovascularization. PMID:25459110

Structure and biological activities of eumenine mastoparan-AF (EMP-AF), a new mast cell degranulating peptide in the venom of the solitary wasp (Anterhynchium flavomarginatum micado).

PubMed

Konno, K; Hisada, M; Naoki, H; Itagaki, Y; Kawai, N; Miwa, A; Yasuhara, T; Morimoto, Y; Nakata, Y

2000-11-01

A new mast cell degranulating peptide, eumenine mastoparan-AF (EMP-AF), was isolated from the venom of the solitary wasp Anterhynchium flavomarginatum micado, the most common eumenine wasp found in Japan. The structure was analyzed by FAB-MS/MS together with Edman degradation, which was corroborated by solid-phase synthesis. The sequence of EMP-AF, Ile-Asn-Leu-Leu-Lys-Ile-Ala-Lys-Gly-Ile-Ile-Lys-Ser-Leu-NH(2), was similar to that of mastoparan, a mast cell degranulating peptide from a hornet venom; tetradecapeptide with C-terminus amidated and rich in hydrophobic and basic amino acids. In fact, EMP-AF exhibited similar activity to mastoparan in stimulating degranulation from rat peritoneal mast cells and RBL-2H3 cells. It also showed significant hemolytic activity in human erythrocytes. Therefore, this is the first example that a mast cell degranulating peptide is found in the solitary wasp venom. Besides the degranulation and hemolytic activity, EMP-AF also affects on neuromuscular transmission in the lobster walking leg preparation. Three analogs EMP-AF-1 approximately 3 were snythesized and biologically tested together with EMP-AF, resulting in the importance of the C-terminal amide structure for biological activities.

Automatic reactor for solid-phase synthesis of molecularly imprinted polymeric nanoparticles (MIP NPs) in water.

PubMed

Poma, Alessandro; Guerreiro, Antonio; Caygill, Sarah; Moczko, Ewa; Piletsky, Sergey

We report the development of an automated chemical reactor for solid-phase synthesis of MIP NPs in water. Operational parameters are under computer control, requiring minimal operator intervention. In this study, "ready for use" MIP NPs with sub-nanomolar affinity are prepared against pepsin A, trypsin and α-amylase in only 4 hours.

Single hydration of the peptide bond: the case of the Vince lactam.

PubMed

Écija, Patricia; Basterretxea, Francisco J; Lesarri, Alberto; Millán, Judith; Castaño, Fernando; Cocinero, Emilio J

2012-10-18

2-Azabicyclo[2.2.1]hept-5-en-3-one (ABH or Vince lactam) and its monohydrated complex (ABH···H(2)O) have been observed in a supersonic jet by Fourier transform microwave spectroscopy. ABH is broadly used in the synthesis of therapeutic drugs, whereas the ABH···H(2)O system offers a simple model to explain the conformational preferences of water linked to a constrained peptidic bond. A single predominant form of the Vince lactam and its singly hydrated complex have been detected, determining the rotational constants, centrifugal distortion constants, and nuclear quadrupole coupling tensor. The monohydrated complex is stabilized by two hydrogen bonds (C═O···H-O and N-H···O) closing a six-membered ring. The complexation energy has been estimated to be ∼10 kJ mol(-1) from experimental results. In addition, the observed structure in the gas phase has been compared with solid-phase diffraction data. The structural parameters and binding energies of ABH···H(2)O have also been compared with similar molecules containing peptide bonds. Ab initio (MP2) and density functional (M06-2X and B3LYP) methods have supported the experimental work, describing the rotational parameters and conformational landscape of the title compound and its singly hydrated complex.

Development of a (68)Ga-peptide tracer for PET GnRH1-imaging.

PubMed

Zoghi, Masoumeh; Jalilian, Amir R; Niazi, Ali; Johari-Daha, Fariba; Alirezapour, Behrouz; Ramezanpour, Saeed

2016-07-01

Total synthesis, quality control and preclinical evaluation of [(68)Ga]-DOTA-triptorelin ([(68)Ga]-DOTA-TRP) is reported as a possible PET radiotracer for GnRH receptor imaging. DOTA-TRP was totally synthesized in two steps and after characterization went through radiolabelling optimization studies followed by tracer stability. The biodistribution of the tracer in normal male rats and 4T1 tumour-bearing mice was performed in 120 min after i.v. injection. The peptide and the conjugates were synthesized with >95 % chemical purity. [(68)Ga]-DOTA-TRP complex was prepared in high radiochemical purity (>99 %, ITLC, HPLC) and specific activity of 1400-2100 MBq/nM at 95 °C using 40-60 μg of the peptide in 5-7 min followed by solid phase purification. The IC50 [nM] DOTA-TRP was comparable to the intact peptide, 0.11 ± 0.01 and 0.22 ± 0.05, respectively. The biodistribution of the tracer demonstrated kidney, stomach, and testes significant uptake, all in accordance with GnRH receptor ligands. Significant tumour uptake was observed in 4T1 tumour-bearing female mice 30-120 min post-injection with tumour:blood and tumour:muscle ratios of 28 and >50 in 60 min, respectively. Kidney is rapidly washed from the tracer. [(68)Ga]-DOTA-TRP can be proposed as a possible tracer for GnRH-R imaging studies.

Automatic reactor for solid-phase synthesis of molecularly imprinted polymeric nanoparticles (MIP NPs) in water

PubMed Central

Poma, Alessandro; Guerreiro, Antonio; Caygill, Sarah; Moczko, Ewa; Piletsky, Sergey

2015-01-01

We report the development of an automated chemical reactor for solid-phase synthesis of MIP NPs in water. Operational parameters are under computer control, requiring minimal operator intervention. In this study, “ready for use” MIP NPs with sub-nanomolar affinity are prepared against pepsin A, trypsin and α-amylase in only 4 hours. PMID:26722622

Semisynthetic protein nanoreactor for single-molecule chemistry

PubMed Central

Lee, Joongoo; Bayley, Hagan

2015-01-01

The covalent chemistry of individual reactants bound within a protein pore can be monitored by observing the ionic current flow through the pore, which acts as a nanoreactor responding to bond-making and bond-breaking events. In the present work, we incorporated an unnatural amino acid into the α-hemolysin (αHL) pore by using solid-phase peptide synthesis to make the central segment of the polypeptide chain, which forms the transmembrane β-barrel of the assembled heptamer. The full-length αHL monomer was obtained by native chemical ligation of the central synthetic peptide to flanking recombinant polypeptides. αHL pores with one semisynthetic subunit were then used as nanoreactors for single-molecule chemistry. By introducing an amino acid with a terminal alkyne group, we were able to visualize click chemistry at the single-molecule level, which revealed a long-lived (4.5-s) reaction intermediate. Additional side chains might be introduced in a similar fashion, thereby greatly expanding the range of single-molecule covalent chemistry that can be investigated by the nanoreactor approach. PMID:26504203

Preparation of a Trp-BODIPY fluorogenic amino acid to label peptides for enhanced live-cell fluorescence imaging.

PubMed

Mendive-Tapia, Lorena; Subiros-Funosas, Ramon; Zhao, Can; Albericio, Fernando; Read, Nick D; Lavilla, Rodolfo; Vendrell, Marc

2017-08-01

Fluorescent peptides are valuable tools for live-cell imaging because of the high specificity of peptide sequences for their biomolecular targets. When preparing fluorescent versions of peptides, labels must be introduced at appropriate positions in the sequences to provide suitable reporters while avoiding any impairment of the molecular recognition properties of the peptides. This protocol describes the preparation of the tryptophan (Trp)-based fluorogenic amino acid Fmoc-Trp(C 2 -BODIPY)-OH and its incorporation into peptides for live-cell fluorescence imaging-an approach that is applicable to most peptide sequences. Fmoc-Trp(C 2 -BODIPY)-OH contains a BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) fluorogenic core, which works as an environmentally sensitive fluorophore, showing high fluorescence in lipophilic conditions. It is attached to Trp via a spacer-free C-C linkage, resulting in a labeled amino acid that can mimic the molecular interactions of Trp, enabling wash-free imaging. This protocol covers the chemical synthesis of the fluorogenic amino acid Fmoc-Trp(C 2 -BODIPY)-OH (3-4 d), the preparation of the labeled antimicrobial peptide BODIPY-cPAF26 by solid-phase synthesis (6-7 d) and its spectral and biological characterization as a live-cell imaging probe for different fungal pathogens. As an example, we include a procedure for using BODIPY-cPAF26 for wash-free imaging of fungal pathogens, including real-time visualization of Aspergillus fumigatus (5 d for culturing, 1-2 d for imaging).

Polymer-Supported Reagents and [to the first power]H-[to the nineteenth power]F NMR Couplings: The Synthesis of 2-Fluoroacetophenone

ERIC Educational Resources Information Center

Pohl, Nicola; Schwarz, Kimberly

2008-01-01

We describe an experiment for the undergraduate organic laboratory curriculum in which 2-bromoacetophenone is converted to 2-fluoroacetophenone using a solid-phase nucleophilic fluorine source. The experiment introduces students to the utility of solid-phase reagents in organic synthesis, to NMR-active nuclei other than [to the first power]H…

Solid phase synthesis of phosphorothioate oligonucleotides utilizing diethyldithiocarbonate disulfide (DDD) as an efficient sulfur transfer reagent.

PubMed

Cheruvallath, Zacharia S; Kumar, R Krishna; Rentel, Claus; Cole, Douglas L; Ravikumar, Vasulinga T

2003-04-01

Diethyldithiodicarbonate (DDD), a cheap and easily prepared compound, is found to be a rapid and efficient sulfurizing reagent in solid phase synthesis of phosphorothioate oligodeoxyribonucleotides via the phosphoramidite approach. Product yield and quality based on IP-LC-MS compares well with high quality oligonucleotides synthesized using phenylacetyl disulfide (PADS) which is being used for manufacture of our antisense drugs.

Rare earth indates (RE: La-Yb): influence of the synthesis route and heat treatment on the crystal structure.

PubMed

Shukla, Rakesh; Grover, Vinita; Srinivasu, Kancharlapalli; Paul, Barnita; Roy, Anushree; Gupta, Ruma; Tyagi, Avesh Kumar

2018-05-15

Rare earth indates are an interesting class of compounds with rich crystallography. The present study explores the crystallographic phases observed in REInO3 (RE: La-Yb) systems and their dependence on synthesis routes and annealing temperature. All REInO3 compositions were synthesized by a solid state route as well as gel-combustion synthesis (GC) followed by annealing at different temperatures. The systems were well characterized by powder XRD studies and were analysed by Rietveld refinement for the structural parameters. The cell parameters were observed to decrease in accordance with the trend in ionic radii on proceeding from lighter to heavier rare earth ions. Interestingly, the synthesis route and the annealing temperature had a profound bearing on the phase relationships observed in the REInO3 series. The solid state synthesized samples depicted an orthorhombic phase (Pbnm) field for LaInO3 to SmInO3, followed by a hexagonal-type phase (P63cm) for GdInO3 to DyInO3. However, the phase field distribution was greatly influenced upon employing gel-combustion (GC) wherein both single-phasic hexagonal and orthorhombic phase fields were found to shrink. Annealing the GC-synthesized compositions to still higher temperatures (1250 °C) further evolved the phase boundaries. An important outcome of the study is observance of polymorphism in SmInO3 which crystallized in the hexagonal phase when synthesized by GC and orthorhombic phase by solid state synthesis. This reveals the all-important role played by synthesis conditions. The existence and energetics of the two polymorphs have been elucidated and discussed with the aid of theoretical studies.

Synthesis of disulfated peptides corresponding to the N-terminus of chemokines receptors CXCR6 (CXCR6 1-20) and DARC (DARC 8-42) using a sulfate-protecting group strategy.

PubMed

Ali, Ahmed M; Taylor, Scott D

2010-04-01

Tyrosine sulfation is a post translational modification that occurs on integral membrane and secreted proteins, and is required for mediating crucial biological processes. Until recently the synthesis of sTyr peptides, especially those containing multiple sTyr residues, were among the most challenging peptides to prepare. We recently described an efficient strategy for Fmoc-based solid phase synthesis of sTyr peptides in which the sulfate group in the sTyr residue(s) is protected with a DCV group (FmocTyr(SO(3)DCV)OH, 1). After cleavage of the peptide from the support the DCV group is removed by hydrogenolysis. Here we demonstrate that sTyr peptides containing Met or Trp residues can be prepared using our sulfate-protecting group strategy by preparing peptides corresponding to residues 1-20 of chemokine receptor CXCR6 and 8-42 of chemokine receptor DARC. Removing the DCV groups at the end of the syntheses was readily achieved, without any reduction of the indole ring in Trp, by performing the hydrogenolysis in the presence of triethylamine. These conditions were found to be particularly efficient for removing the DCV group and superiour to our original conditions using H(2), ammonium formate, Pd/C. The presence of Met was found not to interfere with the removal of the DCV group. The use of pseudoproline dipeptides and N-backbone protection with the 2,4-dimethoxybenzyl group were found to be very effective tactics for preventing aggregation and aspartimide formation during the synthesis of these peptides. We also report an alternative and more cost effective synthesis of amino acid 1. Copyright (c) 2010 European Peptide Society and John Wiley & Sons, Ltd.

Directing the phase behavior of polyelectrolyte complexes using chiral patterned peptides

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pacalin, Naomi M.; Leon, Lorraine; Tirrell, Matthew

Polyelectrolyte complexes (PECs) have a broad range of promising applications as soft materials due to their self-assembly and diversity of structure and chemical composition. Peptide polymer PECs are highly biocompatible and biodegradable, making them particularly useful for encapsulation of food additives and flavors, micellar drug delivery, medical and underwater adhesives, fetal membrane patches, and scaffolds for cell growth in tissue engineering. While parameters affecting PEC formation and stability in regards to charge effects are well researched, little is known about the effects of van der Waals interactions, hydrogen bonding, and secondary structure in these materials. Peptide chirality provides a uniquemore » opportunity to manipulate PEC phase to modulate the amount of solid-like (precipitate) or liquid-like (coacervate) character by influencing hydrogen bonding interactions among peptide chains. In previous work, we showed that chiral peptides form solid complexes, while complexes with even one racemic peptide were fluid. This raised the interesting question of how long a homochiral sequence must be to result in solid phase formation. In this work, we designed chiral patterned peptides of polyglutamic acid and polylysine ranging from 50 to 90% L-chiral residues with increasing numbers of sequential L-chiral residues before a chirality change. These polymers were mixed together to form PECs. We observed that 8 or more sequential L-chiral residues are necessary to achieve both the appearance of a precipitate phase and sustained beta-sheets in the complex, as determined by optical imaging and FTIR Spectroscopy. Less homochiral content results in formation of a coacervate phase. Thus, we show that chiral sequence can be used to control the phase transition of PECs. Understanding how to manipulate PEC phase using chiral sequence as presented here may enable tuning of the material properties to achieve the desired mechanical strength for coatings and polymer brushes, or the most effective molecular release kinetics for drug delivery applications, for example.« less

Directing the phase behavior of polyelectrolyte complexes using chiral patterned peptides

NASA Astrophysics Data System (ADS)

Pacalin, Naomi M.; Leon, Lorraine; Tirrell, Matthew

2016-10-01

Polyelectrolyte complexes (PECs) have a broad range of promising applications as soft materials due to their self-assembly and diversity of structure and chemical composition. Peptide polymer PECs are highly biocompatible and biodegradable, making them particularly useful for encapsulation of food additives and flavors, micellar drug delivery, medical and underwater adhesives, fetal membrane patches, and scaffolds for cell growth in tissue engineering. While parameters affecting PEC formation and stability in regards to charge effects are well researched, little is known about the effects of van der Waals interactions, hydrogen bonding, and secondary structure in these materials. Peptide chirality provides a unique opportunity to manipulate PEC phase to modulate the amount of solid-like (precipitate) or liquid-like (coacervate) character by influencing hydrogen bonding interactions among peptide chains. In previous work, we showed that chiral peptides form solid complexes, while complexes with even one racemic peptide were fluid. This raised the interesting question of how long a homochiral sequence must be to result in solid phase formation. In this work, we designed chiral patterned peptides of polyglutamic acid and polylysine ranging from 50 to 90% L-chiral residues with increasing numbers of sequential L-chiral residues before a chirality change. These polymers were mixed together to form PECs. We observed that 8 or more sequential L-chiral residues are necessary to achieve both the appearance of a precipitate phase and sustained β-sheets in the complex, as determined by optical imaging and FTIR Spectroscopy. Less homochiral content results in formation of a coacervate phase. Thus, we show that chiral sequence can be used to control the phase transition of PECs. Understanding how to manipulate PEC phase using chiral sequence as presented here may enable tuning of the material properties to achieve the desired mechanical strength for coatings and polymer brushes, or the most effective molecular release kinetics for drug delivery applications, for example.

Simple method to assess stability of immobilized peptide ligands against proteases.

PubMed

Giudicessi, Silvana L; Salum, María L; Saavedra, Soledad L; Martínez-Ceron, María C; Cascone, Osvaldo; Erra-Balsells, Rosa; Camperi, Silvia A

2017-09-01

Although peptides are used as affinity chromatography ligands, they could be digested by proteases. Usually, peptide stability is evaluated in solution, which differs from the resin-bounded peptide behavior. Furthermore, the study of the degradation products requires purification steps before analysis. Here, we describe an easy method to assess immobilized peptide stability. Sample peptides were synthesized on hydroxymethylbenzamide-ChemMatrix resin. Peptidyl-resin beads were then incubated with solutions containing proteases. Peptides were detached from the solid support with ammonia vapor and analyzed by matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry, allowing the detection of the whole peptides as well as their C-terminal degradation products. The method allowed a fast evaluation of peptide ligand stability in solid phase towards proteases that may be present in the crude sample before their use as ligands in affinity chromatography. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

An optimized chemical synthesis of human relaxin-2.

PubMed

Barlos, Kostas K; Gatos, Dimitrios; Vasileiou, Zoe; Barlos, Kleomenis

2010-04-01

Human gene 2 relaxin (RLX) is a member of the insulin superfamily and is a multi-functional factor playing a vital role in pregnancy, aging, fibrosis, cardioprotection, vasodilation, inflammation, and angiogenesis. RLX is currently applied in clinical trials to cure among others acute heart failure, fibrosis, and preeclampsia. The synthesis of RLX by chemical methods is difficult because of the insolubility of its B-chain and the required laborious and low yielding site-directed combination of its A (RLXA) and B (RLXB) chains. We report here that oxidation of the Met(25) residue of RLXB improves its solubility, allowing its effective solid-phase synthesis and application in random interchain combination reactions with RLXA. Linear Met(O)(25)-RLX B-chain (RLXBO) reacts with a mixture of isomers of bicyclic A-chain (bcRLXA) giving exclusively the native interchain combination. Applying this method Met(O)(25)-RLX (RLXO) was obtained in 62% yield and was easily converted to RLX in 78% yield, by reduction with ammonium iodide. Copyright (c) 2010 European Peptide Society and John Wiley & Sons, Ltd.

High-flexibility combinatorial peptide synthesis with laser-based transfer of monomers in solid matrix material.

PubMed

Loeffler, Felix F; Foertsch, Tobias C; Popov, Roman; Mattes, Daniela S; Schlageter, Martin; Sedlmayr, Martyna; Ridder, Barbara; Dang, Florian-Xuan; von Bojničić-Kninski, Clemens; Weber, Laura K; Fischer, Andrea; Greifenstein, Juliane; Bykovskaya, Valentina; Buliev, Ivan; Bischoff, F Ralf; Hahn, Lothar; Meier, Michael A R; Bräse, Stefan; Powell, Annie K; Balaban, Teodor Silviu; Breitling, Frank; Nesterov-Mueller, Alexander

2016-06-14

Laser writing is used to structure surfaces in many different ways in materials and life sciences. However, combinatorial patterning applications are still limited. Here we present a method for cost-efficient combinatorial synthesis of very-high-density peptide arrays with natural and synthetic monomers. A laser automatically transfers nanometre-thin solid material spots from different donor slides to an acceptor. Each donor bears a thin polymer film, embedding one type of monomer. Coupling occurs in a separate heating step, where the matrix becomes viscous and building blocks diffuse and couple to the acceptor surface. Furthermore, we can consecutively deposit two material layers of activation reagents and amino acids. Subsequent heat-induced mixing facilitates an in situ activation and coupling of the monomers. This allows us to incorporate building blocks with click chemistry compatibility or a large variety of commercially available non-activated, for example, posttranslationally modified building blocks into the array's peptides with >17,000 spots per cm(2).

Naturally Inspired Peptide Leads: Alanine Scanning Reveals an Actin‐Targeting Thiazole Analogue of Bisebromoamide

PubMed Central

Johnston, Heather J.; Boys, Sarah K.; Makda, Ashraff; Carragher, Neil O.

2016-01-01

Abstract Systematic alanine scanning of the linear peptide bisebromoamide (BBA), isolated from a marine cyanobacterium, was enabled by solid‐phase peptide synthesis of thiazole analogues. The analogues have comparable cytotoxicity (nanomolar) to that of BBA, and cellular morphology assays indicated that they target the actin cytoskeleton. Pathway inhibition in human colon tumour (HCT116) cells was explored by reverse phase protein array (RPPA) analysis, which showed a dose‐dependent response in IRS‐1 expression. Alanine scanning reveals a structural dependence to the cytotoxicity, actin targeting and pathway inhibition, and allows a new readily synthesised lead to be proposed. PMID:27304907

Pyrrole-Imidazole Polyamides: Manual Solid-Phase Synthesis.

PubMed

Pauff, Steven M; Fallows, Andrew J; Mackay, Simon P; Su, Wu; Cullis, Paul M; Burley, Glenn A

2015-12-01

Pyrrole-imidazole polyamides (PAs) are a family of DNA-binding peptides that bind in the minor groove of double-stranded DNA (dsDNA) in a sequence-selective, programmable fashion. This protocol describes a detailed manual procedure for the solid-phase synthesis of this family of compounds. The protocol entails solution-phase synthesis of the Boc-protected pyrrole (Py) and imidazole (Im) carboxylic acid building blocks. This unit also describes the importance of choosing the appropriate condensing agent to form the amide linkages between each building block. Finally, a monomeric coupling protocol and a fragment-based approach are described that delivers PAs in 13% to 30% yield in 8 days. Copyright © 2015 John Wiley & Sons, Inc.

[Cell-penetrating chimeric apoptotic peptide AVPI-LMWP/DNA co-delivery system for cancer therapy].

PubMed

Tan, Jiao; Wang, Ya-Ping; Wang, Hui-Xin; Liang, Jian-Ming; Zhang, Meng; Sun, Xun; Huang, Yong-Zhuo

2014-12-01

To develop a cell-penetrating chimeric apoptotic peptide AVPI-LMWP/DNA co-delivery system for cancer therapy, we prepared the AVPI-LMWP/pTRAIL self-assembled complexes containing a therapeutic combination of peptide drug AVPI and DNA drug TRAIL. The chimeric apoptotic peptide AVPI-LMWP was synthesized using the standard solid-phase synthesis. The cationic AVPI-LMWP could condense pTRAIL by electrostatic interaction. The physical-chemical properties of the AVPI-LMWP/pTRAIL complexes were characterized. The cellular uptake efficiency and the inhibitory activity of the AVPI-LMWP/pTRAIL complexes on tumor cell were also performed. The results showed that the AVPI-LMWP/pTRAIL complexes were successfully prepared by co-incubation. With the increase of mass ratio (AVPI-LMWP/DNA), the particle size was decreased and the zeta potential had few change. Agarose gel electrophoresis showed that AVPI-LMWP could fully bind and condense pTRAIL at a mass ratio above 15:1. Cellular uptake efficiency was improved along with the increased ratio of W(AVPI-LMWP)/WpTRAIL. The in vitro cytotoxicity experiments demonstrated that the AVPI-LMWP/pTRAIL (W:W = 20:1) complexes was significantly more effective than the pTRAIL, AVPI-LMWP alone or LMWP/pTRAIL complexes on inhibition of HeLa cell growth. Our studies indicated that the AVPI-LMWP/pTRAIL co-delivery system could deliver plasmid into HeLa cell and induce tumor cell apoptosis efficiently, which showed its potential in cancer therapy using combination of apoptoic peptide and gene drugs.

Rational design and synthesis of altered peptide ligands based on human myelin oligodendrocyte glycoprotein 35-55 epitope: inhibition of chronic experimental autoimmune encephalomyelitis in mice.

PubMed

Tselios, Theodore; Aggelidakis, Mihalis; Tapeinou, Anthi; Tseveleki, Vivian; Kanistras, Ioannis; Gatos, Dimitrios; Matsoukas, John

2014-11-04

Experimental autoimmune encephalomyelitis (EAE) is a demyelinating disease of the central nervous system and is an animal model of multiple sclerosis (MS). Although the etiology of MS remains unclear, there is evidence T-cell recognition of immunodominant epitopes of myelin proteins, such as the 35-55 epitope of myelin oligodendrocyte glycoprotein (MOG), plays a pathogenic role in the induction of chronic EAE. Cyclization of peptides is of great interest since the limited stability of linear peptides restricts their potential use as therapeutic agents. Herein, we have designed and synthesized a number of linear and cyclic peptides by mutating crucial T cell receptor (TCR) contact residues of the human MOG35-55 epitope. In particular, we have designed and synthesized cyclic altered peptide ligands (APLs) by mutating Arg41 with Ala or Arg41 and Arg46 with Ala. The peptides were synthesized in solid phase on 2-chlorotrityl chloride resin (CLTR-Cl) using the Fmoc/t-Bu methodology. The purity of final products was verified by RP-HPLC and their identification was achieved by ESI-MS. It was found that the substitutions of Arg at positions 41 and 46 with Ala results in peptide analogues that reduce the severity of MOG-induced EAE clinical symptoms in C57BL/6 mice when co-administered with mouse MOG35-55 peptide at the time of immunization.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ramkumar, Shwetha; Fan, Liang-Shih

A process for producing hydrogen comprising the steps of: (i) gasifying a fuel into a raw synthesis gas comprising CO, hydrogen, steam, sulfur and halide contaminants in the form of H.sub.2S, COS, and HX, wherein X is a halide; (ii) passing the raw synthesis gas through a water gas shift reactor (WGSR) into which CaO and steam are injected, the CaO reacting with the shifted gas to remove CO.sub.2, sulfur and halides in a solid-phase calcium-containing product comprising CaCO.sub.3, CaS and CaX.sub.2; (iii) separating the solid-phase calcium-containing product from an enriched gaseous hydrogen product; and (iv) regenerating the CaO bymore » calcining the solid-phase calcium-containing product at a condition selected from the group consisting of: in the presence of steam, in the presence of CO.sub.2, in the presence of synthesis gas, in the presence of H.sub.2 and O.sub.2, under partial vacuum, and combinations thereof.« less

Rapid Analysis of Protein Farnesyltransferase Substrate Specificity Using Peptide Libraries and Isoprenoid Diphosphate Analogues

PubMed Central

2015-01-01

Protein farnesytransferase (PFTase) catalyzes the farnesylation of proteins with a carboxy-terminal tetrapeptide sequence denoted as a Ca1a2X box. To explore the specificity of this enzyme, an important therapeutic target, solid-phase peptide synthesis in concert with a peptide inversion strategy was used to prepare two libraries, each containing 380 peptides. The libraries were screened using an alkyne-containing isoprenoid analogue followed by click chemistry with biotin azide and subsequent visualization with streptavidin-AP. Screening of the CVa2X and CCa2X libraries with Rattus norvegicus PFTase revealed reaction by many known recognition sequences as well as numerous unknown ones. Some of the latter occur in the genomes of bacteria and viruses and may be important for pathogenesis, suggesting new targets for therapeutic intervention. Screening of the CVa2X library with alkyne-functionalized isoprenoid substrates showed that those prepared from C10 or C15 precursors gave similar results, whereas the analogue synthesized from a C5 unit gave a different pattern of reactivity. Lastly, the substrate specificities of PFTases from three organisms (R. norvegicus, Saccharomyces cerevisiae, and Candida albicans) were compared using CVa2X libraries. R. norvegicus PFTase was found to share more peptide substrates with S. cerevisiae PFTase than with C. albicans PFTase. In general, this method is a highly efficient strategy for rapidly probing the specificity of this important enzyme. PMID:24841702

Synthesis and evaluation of Tc-99m and fluorescence-labeled elastin-derived peptide, VAPG for multimodal tumor imaging in murine tumor model.

PubMed

Kim, Myoung Hyoun; Kim, Chang Guhn; Kim, Seul-Gi; Kim, Dae-Weung

2017-12-01

We developed a Tc-99m and fluorescence-labeled peptide, Tc-99m TAMRA-GHEG-ECG-VAPG to target tumor cells and evaluated the diagnostic performance as a dual-modality imaging agent for tumor in a murine model. TAMRA-GHEG-ECG-VAPG was synthesized by using Fmoc solid-phase peptide synthesis. Radiolabeling of TAMRA-GHEG-ECG-VAPG with Tc-99m was done by using ligand exchange via tartrate. Binding affinity and in vitro cellular uptake studies were performed. Gamma camera imaging, biodistribution, and ex vivo imaging studies were performed in murine models with SW620 tumors. Tumor tissue slides were prepared and analyzed with immunohistochemistry by using confocal microscopy. After radiolabeling procedures with Tc-99m, Tc-99m TAMRA-GHEG-ECG-VAPG complexes were prepared in high yield (>96%). The K d of Tc-99m TAMRA-GHEG-ECG-VAPG determined by saturation binding was 16.8 ± 3.6 nM. Confocal microscopy images of SW620 cells incubated with TAMRA-GHEG-ECG-VAPG showed strong fluorescence in the cytoplasm. Gamma camera imaging revealed substantial uptake of Tc-99m TAMRA-GHEG-ECG-VAPG in tumors. Tumor uptake was effectively blocked by the coinjection of an excess concentration of VAPG. Specific uptake of Tc-99m TAMRA-GHEG-ECG-VAPG was confirmed by biodistribution, ex vivo imaging, and immunohistochemistry stain studies. In vivo and in vitro studies revealed substantial uptake of Tc-99m TAMRA-GHEG-ECG-VAPG in tumor cells. Tc-99m TAMRA-GHEG-ECG-VAPG has potential as a dual-modality tumor imaging agent. Copyright © 2017 John Wiley & Sons, Ltd.

Effect of antimicrobial peptide on dynamics of phosphocholine membrane: role of cholesterol and physical state of bilayer

DOE PAGES

Sharma, Veerendra K.; Mamontov, Eugene; Anunciado, Divina B.; ...

2015-06-24

Antimicrobial peptides are universal in all forms of life and are well known for their strong interaction with the cell membrane. This makes them a popular target for investigation of peptide-lipid interactions. Here we report the effect of melittin, an important antimicrobial peptide, on the dynamics of membranes based on 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipid in both the solid gel and fluid phases. To probe the phase transition, elastic neutron intensity temperature scans have been carried out on DMPC-based unilamellar vesicles (ULV) with and without melittin. We have found that addition of a small amount (0.2 mol%) melittin eliminates the steep fallmore » in the elastic intensity at 296 K associated with the solid gel to fluid phase transition, which is observed for pure DMPC vesicles. Quasielastic neutron scattering (QENS) experiments have been carried out on DMPC ULV in the solid gel and fluid phases with and without 0.2 mol % melittin. The data analysis invariably shows the presence of lateral and internal motions of the DMPC molecule. We found that melittin does have a profound effect on the dynamics of lipid molecules, especially on the lateral motion, and affects it in a different way, depending on the phase of the bilayers. In the solid gel phase, it acts as a plasticizer, enhancing the lateral motion of DMPC. However, in the fluid phase it acts as a stiffening agent, restricting the lateral motion of the lipid molecules. These observations are consistent with the mean squared displacements extracted from the elastic intensity temperature scans. Cholesterol is a vital component of eukaryotic membrane, which is a natural target for melittin. To investigate the effect of melittin on vesicles supplemented with cholesterol, QENS experiments have also been carried out on DMPC ULV with 20 mol% cholesterol in the presence and absence of 0.2 mol% melittin. Remarkably, the effects of melittin on the membrane dynamics disappear in the presence of 20 mol % cholesterol. Thus, our measurements indicate that the destabilizing effect of the peptide melittin on membranes can be mitigated by the presence of cholesterol.« less

Effect of antimicrobial peptide on dynamics of phosphocholine membrane: role of cholesterol and physical state of bilayer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sharma, Veerendra K.; Mamontov, Eugene; Anunciado, Divina B.

Antimicrobial peptides are universal in all forms of life and are well known for their strong interaction with the cell membrane. This makes them a popular target for investigation of peptide-lipid interactions. Here we report the effect of melittin, an important antimicrobial peptide, on the dynamics of membranes based on 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipid in both the solid gel and fluid phases. To probe the phase transition, elastic neutron intensity temperature scans have been carried out on DMPC-based unilamellar vesicles (ULV) with and without melittin. We have found that addition of a small amount (0.2 mol%) melittin eliminates the steep fallmore » in the elastic intensity at 296 K associated with the solid gel to fluid phase transition, which is observed for pure DMPC vesicles. Quasielastic neutron scattering (QENS) experiments have been carried out on DMPC ULV in the solid gel and fluid phases with and without 0.2 mol % melittin. The data analysis invariably shows the presence of lateral and internal motions of the DMPC molecule. We found that melittin does have a profound effect on the dynamics of lipid molecules, especially on the lateral motion, and affects it in a different way, depending on the phase of the bilayers. In the solid gel phase, it acts as a plasticizer, enhancing the lateral motion of DMPC. However, in the fluid phase it acts as a stiffening agent, restricting the lateral motion of the lipid molecules. These observations are consistent with the mean squared displacements extracted from the elastic intensity temperature scans. Cholesterol is a vital component of eukaryotic membrane, which is a natural target for melittin. To investigate the effect of melittin on vesicles supplemented with cholesterol, QENS experiments have also been carried out on DMPC ULV with 20 mol% cholesterol in the presence and absence of 0.2 mol% melittin. Remarkably, the effects of melittin on the membrane dynamics disappear in the presence of 20 mol % cholesterol. Thus, our measurements indicate that the destabilizing effect of the peptide melittin on membranes can be mitigated by the presence of cholesterol.« less

Antimicrobial Peptides: A Promising Therapeutic Strategy in Tackling Antimicrobial Resistance.

PubMed

Nuti, Ramya; Goud, Nerella S; Saraswati, A Prasanth; Alvala, Ravi; Alvala, Mallika

2017-01-01

Antimicrobial resistance (AMR) has posed a serious threat to global public health and it requires immediate action, preferably long term. Current drug therapies have failed to curb this menace due to the ability of microbes to circumvent the mechanisms through which the drugs act. From the drug discovery point of view, the majority of drugs currently employed for antimicrobial therapy are small molecules. Recent trends reveal a surge in the use of peptides as drug candidates as they offer remarkable advantages over small molecules. Newer synthetic strategies like organometalic complexes, Peptide-polymer conjugates, solid phase, liquid phase and recombinant DNA technology encouraging the use of peptides as therapeutic agents with a host of chemical functions, and tailored for specific applications. In the last decade, many peptide based drugs have been successfully approved by the Food and Drug Administration (FDA). This success can be attributed to their high specificity, selectivity and efficacy, high penetrability into the tissues, less immunogenicity and less tissue accumulation. Considering the enormity of AMR, the use of Antimicrobial Peptides (AMPs) can be a viable alternative to current therapeutics strategies. AMPs are naturally abundant allowing synthetic chemists to develop semi-synthetics peptide molecules. AMPs have a broad spectrum of activity towards microbes and they possess the ability to bypass the resistance induction mechanisms of microbes. The present review focuses on the potential applications of AMPs against various microbial disorders and their future prospects. Several resistance mechanisms and their strategies have also been discussed to highlight the importance in the current scenario. Breakthroughs in AMP designing, peptide synthesis and biotechnology have shown promise in tackling this challenge and has revived the interest of using AMPs as an important weapon in fighting AMR. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Use of unnatural amino acids to probe structure-activity relationships and mode-of-action of antimicrobial peptides.

PubMed

Tossi, Alessandro; Scocchi, Marco; Zahariev, Sotir; Gennaro, Renato

2012-01-01

Endogenous antimicrobial peptides (AMPs) can have multimodal mechanisms of bacterial inactivation, such as membrane lysis, interference with cell wall biosynthesis or membrane-based protein machineries, or translocation through the membrane to intracellular targets. The controlled variation of side-chain characteristics in their amino acid residues can provide much useful information on structure-activity relationships and mode-of-action, and also lead to improved activities. The small size and relatively low complexity of AMPs make them amenable to solid-phase peptide synthesis, facilitating the use of nonproteinogenic amino acids and vastly increasing the accessible molecular diversity of side chains. Here, we describe how such residues can be used to modulate such key parameters as cationicity, hydrophobicity, steric factors conformational stability, and H-bonding.

Tiered analytics for purity assessment of macrocyclic peptides in drug discovery: Analytical consideration and method development.

PubMed

Qian Cutrone, Jingfang Jenny; Huang, Xiaohua Stella; Kozlowski, Edward S; Bao, Ye; Wang, Yingzi; Poronsky, Christopher S; Drexler, Dieter M; Tymiak, Adrienne A

2017-05-10

Synthetic macrocyclic peptides with natural and unnatural amino acids have gained considerable attention from a number of pharmaceutical/biopharmaceutical companies in recent years as a promising approach to drug discovery, particularly for targets involving protein-protein or protein-peptide interactions. Analytical scientists charged with characterizing these leads face multiple challenges including dealing with a class of complex molecules with the potential for multiple isomers and variable charge states and no established standards for acceptable analytical characterization of materials used in drug discovery. In addition, due to the lack of intermediate purification during solid phase peptide synthesis, the final products usually contain a complex profile of impurities. In this paper, practical analytical strategies and methodologies were developed to address these challenges, including a tiered approach to assessing the purity of macrocyclic peptides at different stages of drug discovery. Our results also showed that successful progression and characterization of a new drug discovery modality benefited from active analytical engagement, focusing on fit-for-purpose analyses and leveraging a broad palette of analytical technologies and resources. Copyright © 2017. Published by Elsevier B.V.

Effect of grafted oligopeptides on friction.

PubMed

Iarikov, Dmitri D; Ducker, William A

2013-05-14

Frictional and normal forces in aqueous solution at 25 °C were measured between a glass particle and oligopeptide films grafted from a glass plate. Homopeptide molecules consisting of 11 monomers of either glutamine, leucine, glutamic acid, lysine, or phenylalanine and one heteropolymer were each "grafted from" an oxidized silicon wafer using microwave-assisted solid-phase peptide synthesis. The peptide films were characterized using X-ray photoelectron spectroscopy and secondary ion mass spectrometry. Frictional force measurements showed that the oligopeptides increased the magnitude of friction compared to that on a bare hydrophilic silicon wafer but that the friction was a strong function of the nature of the monomer unit. Overall we find that the friction is lower for more hydrophilic films. For example, the most hydrophobic monomer, leucine, exhibited the highest friction whereas the hydrophilic monomer, polyglutamic acid, exhibited the lowest friction at zero load. When the two surfaces had opposite charges, there was a strong attraction, adhesion, and high friction between the surfaces. Friction for all polymers was lower in phosphate-buffered saline than in pure water, which was attributed to lubrication via hydrated salt ions.

Introducing cymantrene labels into scattering scanning near-field infrared microscopy.

PubMed

Kopf, Ilona; N'Dongo, Harmel W Peindy; Ballout, Fouad; Schatzschneider, Ulrich; Bründermann, Erik; Havenith, Martina

2012-11-07

In this paper we investigate metal-organic compounds as infrared (IR) active labels by scattering scanning near-field infrared microscopy (IR s-SNOM, often also abbreviated as s-SNIM) with a lateral resolution of 90 × 90 nm(2). Tailor-made IR spectroscopic probes based on cymantrene (CpMn(CO)(3) with Cp = η(5)-C(5)H(5)) conjugated to a cysteine-modified pseudoneurotensin (pNT-Cys-OH) peptide were prepared by automated microwave-assisted solid phase peptide synthesis (SPPS) and characterized by HPLC, ESI-MS and IR. Well-defined patterned self-assembled monolayers on a gold surface were prepared by microcontact printing of 1-octadecanethiol (ODT) followed by additional incubation in ethanolic solution of the cymantrene-peptide derivative. The self-assembled monolayers have been evidenced by infrared reflection absorption spectroscopy (IRRAS) and AFM. CO laser source radiation was tuned (1944, 1900, 1798 and 1658 cm(-1)) for imaging contrast with good matching correlation between spectroscopic and topographic patterns at specific characteristic metal carbonyl and amide bands (1944 cm(-1) (λ = 5.14 μm) and 1658 cm(-1) (λ = 6.03 μm)). Cymantrene probes provide an attractive method to tag a unique spectroscopic feature on any bio(macro)molecule. Introducing such probes into super-resolution IR s-SNOM will enable molecular tracking and distribution studies even in complex biological systems.

Solid-phase synthesis of the nucleopeptide fragment H-Asp-Ser[pAAAGTAAGCC]-Glu-OH from the nucleoprotein of Bacillus subtilis phage phi 29.

PubMed Central

Dreef-Tromp, C M; van der Maarel, J C; van den Elst, H; van der Marel, G A; van Boom, J H

1992-01-01

The naturally occurring DNA-nucleopeptide H-Asp-Ser[5'-pAAAGTAAGCC-3']-Glu-OH was prepared via a solid-phase phosphite triester approach using N-2-(tert-butyldiphenylsilyloxymethyl)benzoyl protected nucleosides. The oligonucleotide was linked via the extremely base-labile oxalyl ester anchor to the solid support. PMID:1508685

Automated synthesis of arabinoxylan-oligosaccharides enables characterization of antibodies that recognize plant cell wall glycans.

PubMed

Schmidt, Deborah; Schuhmacher, Frank; Geissner, Andreas; Seeberger, Peter H; Pfrengle, Fabian

2015-04-07

Monoclonal antibodies that recognize plant cell wall glycans are used for high-resolution imaging, providing important information about the structure and function of cell wall polysaccharides. To characterize the binding epitopes of these powerful molecular probes a library of eleven plant arabinoxylan oligosaccharides was produced by automated solid-phase synthesis. Modular assembly of oligoarabinoxylans from few building blocks was enabled by adding (2-naphthyl)methyl (Nap) to the toolbox of orthogonal protecting groups for solid-phase synthesis. Conjugation-ready oligosaccharides were obtained and the binding specificities of xylan-directed antibodies were determined on microarrays. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Solid-phase synthesis of smac peptidomimetics incorporating triazoloprolines and biarylalanines.

PubMed

Le Quement, Sebastian T; Ishoey, Mette; Petersen, Mette T; Thastrup, Jacob; Hagel, Grith; Nielsen, Thomas E

2011-11-14

Apoptotic induction mechanisms are of crucial importance for the general homeostasis of multicellular organisms. In cancer the apoptotic pathways are downregulated, which, at least partly, is due to an abundance of inhibitors of apoptosis proteins (IAPs) that block the apoptotic cascade by deactivating proteolytic caspases. The Smac protein has an antagonistic effect on IAPs, thus providing structural clues for the synthesis of new pro-apoptotic compounds. Herein, we report a solid-phase approach for the synthesis of Smac-derived tetrapeptide libraries. On the basis of a common (N-Me)AVPF sequence, peptides incorporating triazoloprolines and biarylalanines were synthesized by means of Cu(I)-catalyzed azide-alkyne cycloaddition and Pd-catalyzed Suzuki cross-coupling reactions. Solid-phase procedures were optimized to high efficiency, thus accessing all products in excellent crude purities and yields (both typically above 90%). The peptides were subjected to biological evaluation in a live/dead cellular assay which revealed that structural decorations on the AVPF sequence indeed are highly important for cytotoxicity toward HeLa cells.

Calcium looping process for high purity hydrogen production integrated with capture of carbon dioxide, sulfur and halides

DOEpatents

Ramkumar, Shwetha; Fan, Liang-Shih

2013-07-30

A process for producing hydrogen comprising the steps of: (i) gasifying a fuel into a raw synthesis gas comprising CO, hydrogen, steam, sulfur and halide contaminants in the form of H.sub.2S, COS, and HX, wherein X is a halide; (ii) passing the raw synthesis gas through a water gas shift reactor (WGSR) into which CaO and steam are injected, the CaO reacting with the shifted gas to remove CO.sub.2, sulfur and halides in a solid-phase calcium-containing product comprising CaCO.sub.3, CaS and CaX.sub.2; (iii) separating the solid-phase calcium-containing product from an enriched gaseous hydrogen product; and (iv) regenerating the CaO by calcining the solid-phase calcium-containing product at a condition selected from the group consisting of: in the presence of steam, in the presence of CO.sub.2, in the presence of synthesis gas, in the presence of H.sub.2 and O.sub.2, under partial vacuum, and combinations thereof.

Formation of sodium bismuth titanate-barium titanate during solid-state synthesis

DOE PAGES

Hou, Dong; Aksel, Elena; Fancher, Chris M.; ...

2017-01-12

Phase formation of sodium bismuth titanate (Na 0.5Bi 0.5TiO 3 or NBT) and its solid solution with barium titanate (BaTiO 3 or BT) during the calcination process is studied using in situ high-temperature diffraction. The reactant powders were mixed and heated to 1000°C, while X-ray diffraction patterns were recorded continuously. Phase evolutions from starting materials to final perovskite products are observed, and different transient phases are identified. The formation mechanism of NBT and NBT–xBT perovskite structures is discussed, and a reaction sequence is suggested based on the observations. The in situ study leads to a new processing approach, which ismore » the use of nano-TiO 2, and gives insights to the particle size effect for solid-state synthesis products. Lastly, it was found that the use of nano-TiO 2 as reactant powder accelerates the synthesis process, decreases the formation of transient phases, and helps to obtain phase-pure products using a lower thermal budget.« less

Photoinactivation of Gram positive and Gram negative bacteria with the antimicrobial peptide (KLAKLAK)(2) conjugated to the hydrophilic photosensitizer eosin Y.

PubMed

Johnson, Gregory A; Muthukrishnan, Nandhini; Pellois, Jean-Philippe

2013-01-16

We test the hypothesis that the antimicrobial peptide (KLAKLAK)(2) enhances the photodynamic activity of the photosensitizer eosin Y upon conjugation. The conjugate eosin-(KLAKLAK)(2) was obtained by solid-phase peptide synthesis. Photoinactivation assays were performed against the Gram-negative bacteria Escherichia coli , Pseudomonas aeruginosa , and multidrug resistant Acinetobacter baumannii AYE, as well as the Gram-positive bacteria Staphylococcus aureus , and Staphylococcus epidermidis . Partitioning assays were performed with E. coli and S. aureus . Photohemolysis and photokilling assays were also performed to assess the photodynamic activity of the conjugate toward mammalian cells. Eosin-(KLAKLAK)(2) photoinactivates 99.999% of 10(8) CFU/mL of most bacteria tested at a concentration of 1 μM or below. In contrast, neither eosin Y nor (KLAKLAK)(2) cause any significant photoinactivation under similar conditions. The increase in photodynamic activity of the photosensitizer conferred by the antimicrobial peptide is in part due to the fact that (KLAKLAK)(2) promotes the association of eosin Y to bacteria. Eosin-(KLAKLAK)(2) does not significantly associate with red blood cells or the cultured mammalian cell lines HaCaT, COS-7, and COLO 316. Consequently, little photodamage or photokilling is observed with these cells under conditions for which bacterial photoinactivation is achieved. The peptide (KLAKLAK)(2) therefore significantly enhances the photodynamic activity of eosin Y toward both Gram-positive and Gram-negative bacteria while interacting minimally with human cells. Overall, our results suggest that antimicrobial peptides such as (KLAKLAK)(2) might serve as attractive agents that can target photosensitizers to bacteria specifically.

A Cyclic Altered Peptide Analogue Based on Myelin Basic Protein 87-99 Provides Lasting Prophylactic and Therapeutic Protection Against Acute Experimental Autoimmune Encephalomyelitis.

PubMed

Emmanouil, Mary; Tseveleki, Vivian; Triantafyllakou, Iro; Nteli, Agathi; Tselios, Theodore; Probert, Lesley

2018-01-31

In this report, amide-linked cyclic peptide analogues of the 87-99 myelin basic protein (MBP) epitope, a candidate autoantigen in multiple sclerosis (MS), are tested for therapeutic efficacy in experimental autoimmune encephalomyelitis (EAE). Cyclic altered peptide analogues of MBP 87-99 with substitutions at positions 91 and/or 96 were tested for protective effects when administered using prophylactic or early therapeutic protocols in MBP 72-85 -induced EAE in Lewis rats. The Lys 91 and Pro 96 of MBP 87-99 are crucial T-cell receptor (TCR) anchors and participate in the formation of trimolecular complex between the TCR-antigen (peptide)-MHC (major histocompability complex) for the stimulation of encephalitogenic T cells that are necessary for EAE induction and are implicated in MS. The cyclic peptides were synthesized using Solid Phase Peptide Synthesis (SPPS) applied on the 9-fluorenylmethyloxycarboxyl/tert-butyl Fmoc/tBu methodology and combined with the 2-chlorotrityl chloride resin (CLTR-Cl). Cyclo(91-99)[Ala 96 ]MBP 87-99 , cyclo(87-99)[Ala 91,96 ]MBP 87-99 and cyclo(87-99)[Arg 91 , Ala 96 ]MBP 87-99 , but not wild-type linear MBP 87-99 , strongly inhibited MBP 72-85 -induced EAE in Lewis rats when administered using prophylactic and early therapeutic vaccination protocols. In particular, cyclo(87-99)[Arg 91 , Ala 96 ]MBP 87-99 was highly effective in preventing the onset and development of clinical symptoms and spinal cord pathology and providing lasting protection against EAE induction.

Substitution determination of Fmoc-substituted resins at different wavelengths.

PubMed

Eissler, Stefan; Kley, Markus; Bächle, Dirk; Loidl, Günther; Meier, Thomas; Samson, Daniel

2017-10-01

In solid-phase peptide synthesis, the nominal batch size is calculated using the starting resin substitution and the mass of the starting resin. The starting resin substitution constitutes the basis for the calculation of a whole set of important process parameters, such as the number of amino acid derivative equivalents. For Fmoc-substituted resins, substitution determination is often performed by suspending the Fmoc-protected starting resin in 20% (v/v) piperidine in DMF to generate the dibenzofulvene-piperidine adduct that is quantified by ultraviolet-visible spectroscopy. The spectrometric measurement is performed at the maximum absorption wavelength of the dibenzofulvene-piperidine adduct, that is, at 301.0 nm. The recorded absorption value, the resin weight and the volume are entered into an equation derived from Lambert-Beer's law, together with the substance-specific molar absorption coefficient at 301.0 nm, in order to calculate the nominal substitution. To our knowledge, molar absorption coefficients between 7100 l mol -1  cm -1 and 8100 l mol -1  cm -1 have been reported for the dibenzofulvene-piperidine adduct at 301.0 nm. Depending on the applied value, the nominal batch size may differ up to 14%. In this publication, a determination of the molar absorption coefficients at 301.0 and 289.8 nm is reported. Furthermore, proof is given that by measuring the absorption at 289.8 nm the impact of wavelength accuracy is reduced. © 2017 The Authors Journal of Peptide Science published by European Peptide Society and John Wiley & Sons Ltd. © 2017 The Authors Journal of Peptide Science published by European Peptide Society and John Wiley & Sons Ltd.

Quantification and application of a liquid chromatography-tandem mass spectrometric method for the determination of WKYMVm peptide in rat using solid-phase extraction.

PubMed

Lee, Byeong Ill; Park, Min-Ho; Heo, Soon Chul; Park, Yuri; Shin, Seok-Ho; Byeon, Jin-Ju; Kim, Jae Ho; Shin, Young G

2018-03-01

A liquid chromatographic-electrospray ionization-time-of-flight/mass spectrometric (LC-ESI-TOF/MS) method was developed and applied for the determination of WKYMVm peptide in rat plasma to support preclinical pharmacokinetics studies. The method consisted of micro-elution solid-phase extraction (SPE) for sample preparation and LC-ESI-TOF/MS in the positive ion mode for analysis. Phenanthroline (10 mg/mL) was added to rat blood immediately for plasma preparation followed by addition of trace amount of 2 m hydrogen chloride to plasma before SPE for stability of WKYMVm peptide. Then sample preparation using micro-elution SPE was performed with verapamil as an internal standard. A quadratic regression (weighted 1/concentration 2 ), with the equation y = ax 2  + bx + c was used to fit calibration curves over the concentration range of 3.02-2200 ng/mL for WKYMVm peptide. The quantification run met the acceptance criteria of ±25% accuracy and precision values. For quality control samples at 15, 165 and 1820 ng/mL from the quantification experiment, the within-run and the between-run accuracy ranged from 92.5 to 123.4% with precision values ≤15.1% for WKYMVm peptide from the nominal values. This novel LC-ESI-TOF/MS method was successfully applied to evaluate the pharmacokinetics of WKYMVm peptide in rat plasma. Copyright © 2017 John Wiley & Sons, Ltd.

The use of solid supports to generate nucleic acid carriers.

PubMed

Unciti-Broceta, Asier; Díaz-Mochón, Juan José; Sánchez-Martín, Rosario M; Bradley, Mark

2012-07-17

Nucleic acids are the foundation stone of all cellular processes. Consequently, the use of DNA or RNA to treat genetic and acquired disorders (so called gene therapy) offers enormous potential benefits. The restitution of defective genes or the suppression of malignant genes could target a range of diseases, including cancers, inherited diseases (cystic fibrosis, muscular dystrophy, etc.), and viral infections. However, this strategy has a major barrier: the size and charge of nucleic acids largely restricts their transit into eukaryotic cells. Potential strategies to solve this problem include the use of a variety of natural and synthetic nucleic acid carriers. Driven by the aim and ambition of translating this promising therapeutic approach into the clinic, researchers have been actively developing advanced delivery systems for nucleic acids for more than 20 years. A decade ago we began our investigations of solid-phase techniques to construct families of novel nucleic acid carriers for transfection. We envisaged that the solid-phase synthesis of polycationic dendrimers and derivatized polyamimes would offer distinct advantages over solution phase techniques. Notably in solid phase synthesis we could take advantage of mass action and streamlined purification procedures, while simplifying the handling of compounds with high polarities and plurality of functional groups. Parallel synthesis methods would also allow rapid access to libraries of compounds with improved purities and yields over comparable solution methodologies and facilitate the development of structure activity relationships. We also twisted the concept of the solid-phase support on its head: we devised miniaturized solid supports that provided an innovative cell delivery vehicle in their own right, carrying covalently conjugated cargos (biomolecules) into cells. In this Account, we summarize the main outcomes of this series of chemically related projects.

Synthesis and evaluation of Tc-99m-labeled RRL-containing peptide as a non-invasive tumor imaging agent in a mouse fibrosarcoma model.

PubMed

Kim, Dae-Weung; Kim, Woo Hyoung; Kim, Myoung Hyoun; Kim, Chang Guhn

2015-11-01

Arginine-arginine-leucine (RRL) is considered a tumor endothelial cell-specific binding sequence. RRL-containing peptide targeting tumor vessels is an excellent candidate for tumor imaging. In this study, we developed RRL-containing hexapeptides and evaluated their feasibility as a tumor imaging agent in a HT-1080 fibrosarcoma-bearing murine model. The hexapeptide, glutamic acid-cysteine-glycine (ECG)-RRL was synthesized using Fmoc solid-phase peptide synthesis. Radiolabeling efficiency was evaluated using instant thin-layer chromatography. Uptake of Tc-99m ECG-RRL within HT-1080 cells was evaluated in vitro by confocal microscopy and cellular binding affinity was calculated. Gamma images were acquired In HT-1080 fibrosarcoma tumor-bearing mice, and the tumor-to-muscle uptake ratio was calculated. The inflammatory-to-normal muscle uptake ratio was also calculated in an inflammation mouse model. A biodistribution study was performed to calculate %ID/g. A high yield of Tc-99m ECG-RRL complexes was prepared after Tc-99m radiolabeling. Binding of Tc-99m ECG-RRL to tumor cells had was confirmed by in vitro studies. Gamma camera imaging in the murine model showed that Tc-99m ECG-RRL accumulated substantially in the subcutaneously engrafted tumor and that tumoral uptake was blocked by co-injecting excess RRL. Moreover, Tc-99m ECG-RRL accumulated minimally in inflammatory lesions. We successfully developed Tc-99m ECG-RRL as a new tumor imaging candidate. Specific tumoral uptake of Tc-99m ECG-RRL was evaluated both in vitro and in vivo, and it was determined to be a good tumor imaging candidate. Additionally, Tc-99m ECG-RRL effectively distinguished between cancerous tissue and inflammatory lesions.

The C-terminus of the B-chain of human insulin-like peptide 5 is critical for cognate RXFP4 receptor activity.

PubMed

Patil, Nitin A; Bathgate, Ross A D; Kocan, Martina; Ang, Sheng Yu; Tailhades, Julien; Separovic, Frances; Summers, Roger; Grosse, Johannes; Hughes, Richard A; Wade, John D; Hossain, Mohammed Akhter

2016-04-01

Insulin-like peptide 5 (INSL5) is an orexigenic peptide hormone belonging to the relaxin family of peptides. It is expressed primarily in the L-cells of the colon and has a postulated key role in regulating food intake. Its G protein-coupled receptor, RXFP4, is a potential drug target for treating obesity and anorexia. We studied the effect of modification of the C-terminus of the A and B-chains of human INSL5 on RXFP4 binding and activation. Three variants of human INSL5 were prepared using solid phase peptide synthesis and subsequent sequential regioselective disulfide bond formation. The peptides were synthesized as C-terminal acids (both A- and B-chains with free C-termini, i.e., the native form), amides (both chains as the C-terminal amide) and one analog with the C-terminus of its A-chain as the amide and the C-terminus of the B-chain as the acid. The results showed that C-terminus of the B-chain is more important than that of the A-chain for RXFP4 binding and activity. Amidation of the A-chain C-terminus does not have any effect on the INSL5 activity. The difference in RXFP4 binding and activation between the three peptides is believed to be due to electrostatic interaction of the free carboxylate of INSL5 with a positively charged residue (s), either situated within the INSL5 molecule itself or in the receptor extracellular loops.

Combinatorial synthesis of phosphors using arc-imaging furnace

NASA Astrophysics Data System (ADS)

Ishigaki, Tadashi; Toda, Kenji; Yoshimura, Masahiro; Uematsu, Kazuyoshi; Sato, Mineo

2011-10-01

We have applied a novel 'melt synthesis technique' rather than a conventional solid-state reaction to rapidly synthesize phosphor materials. During a synthesis, the mixture of oxides or their precursors is melted by light pulses (10-60 s) in an arc-imaging furnace on a water-cooled copper hearth to form a globule of 1-5 mm diameter, which is then rapidly cooled by turning off the light. Using this method, we synthesized several phosphor compounds including Y3Al5O12:Ce(YAG) and SrAl2O4:Eu,Dy. Complex phosphor oxides are difficult to produce by conventional solid-state reaction techniques because of the slow reaction rates among solid oxides; as a result, the oxides form homogeneous compounds or solid solutions. On the other hand, melt reactions are very fast (10-60 s) and result in homogeneous compounds owing to rapid diffusion and mixing in the liquid phase. Therefore, melt synthesis techniques are suitable for preparing multi component homogeneous compounds and solid solutions.

Confocal Raman microscopy for monitoring chemical reactions on single optically trapped, solid-phase support particles.

PubMed

Houlne, Michael P; Sjostrom, Christopher M; Uibel, Rory H; Kleimeyer, James A; Harris, Joel M

2002-09-01

Optical trapping of small structures is a powerful tool for the manipulation and investigation of colloidal and particulate materials. The tight focus excitation requirements of optical trapping are well suited to confocal Raman microscopy. In this work, an inverted confocal Raman microscope is developed for studies of chemical reactions on single, optically trapped particles and applied to reactions used in solid-phase peptide synthesis. Optical trapping and levitation allow a particle to be moved away from the coverslip and into solution, avoiding fluorescence interference from the coverslip. More importantly, diffusion of reagents into the particle is not inhibited by a surface, so that reaction conditions mimic those of particles dispersed in solution. Optical trapping and levitation also maintain optical alignment, since the particle is centered laterally along the optical axis and within the focal plane of the objective, where both optical forces and light collection are maximized. Hour-long observations of chemical reactions on individual, trapped silica particles are reported. Using two-dimensional least-squares analysis methods, the Raman spectra collected during the course of a reaction can be resolved into component contributions. The resolved spectra of the time-varying species can be observed, as they bind to or cleave from the particle surface.

[Synthesis and biological activity of substance P analogs].

PubMed

Liu, S Y; Zhong, M N; Dong, Y X; Li, Y L; Yuan, X M

1997-12-01

Substance P (SP) and its three analogs were synthesized through solid-phase procedure. The fully-protected peptide-resin was cleaved with anhydrous HF.--SH of Cys in [Cys5,9] SP(4-11) was protected with Acm, then deprotected through Iodine oxidation method, the disulfide bridge cyclization was formed subsequently. The crude product was purified on C18 RP-HPLC. The results of guinea-pig isolated ileum test (GPI) showed that the potency of the analogs was as follows: [AcGln5, Pro9] SP(5-11) > SP > SP(4-11) > [Cys5,9] SP(4-11). They induced GPI to contract very fast and showed the characteristics of tachykinin. On the writing test in mice, the results showed: [Cys5,9] SP(4-11) > SP > SP(4-11) > [AcGln5, Pro9] SP(5-11). The biological tests showed that the SP analogs have some extent of selectivity.

Convergent solid-phase synthesis of hirudin.

PubMed

Goulas, Spyros; Gatos, Dimitrios; Barlos, Kleomenis

2006-02-01

Hirudin variant 1 (HV1), a small protein consisting of 65 amino acids and three disulfide bonds, was synthesized by using Fmoc-based convergent methods on 2-chlorotrityl resin (CLTR). The linear sequence was assembled by the sequential condensation of 7 protected fragments, on the resin-bound 55-65 fragment. The conditions of fragment assembly were carefully studied to determine the most efficient synthetic protocol. Crude reduced [Cys(16, 28)(Acm)]-HV1 thus obtained was easily purified to homogeneity by RP-HPLC. Disulfide bridges were successfully formed by a two-step procedure, involving an oxidative folding step to form Cys(6)-Cys(14) and Cys(22)-Cys(39) linkages, followed by iodine oxidation to form the Cys(16)-Cys(28) bond. The correct disulfide bond alignment was established by peptide mapping using Staphylococcus aureus V8 protease at pH 4.5.

Synthesis of 3'-, or 5'-, or internal methacrylamido-modified oligonucleotides

DOEpatents

Golova, Julia B.; Chernov, Boris K.

2010-04-27

New modifiers were synthesized for incorporation of a methacrylic function in 3'-, 5'- and internal positions of oligonucleotides during solid phase synthesis. A modifier was used for synthesis of 5'-methacrylated oligonucleotides for preparation of microarrays by a co-polymerization method.

Persistent dopants and phase segregation in organolead mixed-halide perovskites

DOE PAGES

Rosales, Bryan A.; Men, Long; Cady, Sarah D.; ...

2016-07-25

Organolead mixed-halide perovskites such as CH 3NH 3PbX 3–aX' a (X, X' = I, Br, Cl) are interesting semiconductors because of their low cost, high photovoltaic power conversion efficiencies, enhanced moisture stability, and band gap tunability. Using a combination of optical absorption spectroscopy, powder X-ray diffraction (XRD), and, for the first time, 207Pb solid state nuclear magnetic resonance (ssNMR), we probe the extent of alloying and phase segregation in these materials. Because 207Pb ssNMR chemical shifts are highly sensitive to local coordination and electronic structure, and vary linearly with halogen electronegativity and band gap, this technique can provide the truemore » chemical speciation and composition of organolead mixed-halide perovskites. We specifically investigate samples made by three different preparative methods: solution phase synthesis, thermal annealing, and solid phase synthesis. 207Pb ssNMR reveals that nonstoichiometric dopants and semicrystalline phases are prevalent in samples made by solution phase synthesis. We show that these nanodomains are persistent after thermal annealing up to 200 °C. Further, a novel solid phase synthesis that starts from the parent, single-halide perovskites can suppress phase segregation but not the formation of dopants. Our observations are consistent with the presence of miscibility gaps and spontaneous spinodal decomposition of the mixed-halide perovskites at room temperature. This underscores how strongly different synthetic procedures impact the nanostructuring and composition of organolead halide perovskites. In conclusion, better optoelectronic properties and improved device stability and performance may be achieved through careful manipulation of the different phases and nanodomains present in these materials.« less

Unusual Reactivity of a Silver Mineralizing Peptide

PubMed Central

Carter, Carly Jo; Ackerson, Christopher J.; Feldheim, Daniel L.

2010-01-01

The ability of peptides selected via phage display to mediate the formation of inorganic nanoparticles is now well established. The atomic-level interactions between the selected peptides and the metal ion precursors are in most instances, however, largely obscure. We identified a new peptide sequence that is capable of mediating the formation of Ag nanoparticles. Surprisingly, nanoparticle formation requires the presence of peptide, HEPES buffer, and light; the absence of any one of these compromises nanoparticle formation. Electrochemical experiments revealed that the peptide binds Ag+ in a 3 Ag+:1 peptide ratio and significantly alters the Ag+ reduction potential. Alanine replacement studies yielded insight into the sequence-function relationships of Ag nanoparticle formation, including the Ag+ coordination sites and the residues necessary for Ag synthesis. In addition, the peptide was found to function when immobilized onto surfaces, and the specific immobilizing concentration could be adjusted to yield either spherical Ag nanoparticles or high aspect ratio nanowires. These studies further illustrate the range of interesting new solid-state chemistries possible using biomolecules. PMID:20552994

Preparation and evaluation of the tumor-specific antigen-derived synthetic mucin 1 peptide: A potential candidate for the targeting of breast carcinoma.

PubMed

Okarvi, Subhani M; Al Jammaz, Ibrahim

2016-07-01

The goal of this study was to prepare a synthetic peptide derived from breast tumor associated antigen and to evaluate its potential as a breast cancer imaging agent. A mucin 1 derived peptide was synthesized by solid-phase peptide synthesis and examined for its radiochemical and metabolic stability. The tumor cell binding affinity of (99m)Tc-MUC1 peptide was investigated on MUC1-positive T47D and MCF7 breast cancer cell lines. In vivo biodistribution was studied in normal Balb/c mice and in vivo tumor targeting and imaging in MCF7 and T47D tumor-bearing nude mice. The synthesized MUC1-derived peptide displayed high radiochemical and metabolic stability. In vitro tumor cell-binding on T47D and MCF7 cell lines demonstrated high affinity of (99m)Tc-MUC1 peptide towards human breast cancer cells (binding affinities in nanomolar range). Pharmacokinetic studies performed on Balb/c mice are characterized by an efficient clearance from the blood and excretion predominantly through the urinary system. In vivo tumor uptake in nude mice with MCF7 tumor xenografts was 2.77±0.63% ID/g as early as 1h p.i. whereas in nude mice with T47D human ductal breast epithelial cancer cells, the accumulation in the tumor was found to be 2.65±0.54% ID/g at 1h p.i. Also tumor lesion was detectable in γ-camera imaging. The tumor uptake values were always higher than the blood and muscle uptake, with good tumor retention and good tumor-to-blood and tumor-to-muscle ratios. A low to moderate (<5% ID/g) accumulation and retention of (99m)Tc-MUC1 was found in the major organs (i.e., lungs, stomach, liver, intestines, kidneys, etc.) in both normal and tumor-bearing mice. This study suggests that (99m)Tc-MUC1 tumor-antigen peptide may be a potential candidate for the targeted imaging of MUC1-positive human tumors and warrants further investigation. Copyright © 2016 Elsevier Inc. All rights reserved.

Unusual reactivity of a silver mineralizing peptide.

PubMed

Carter, Carly Jo; Ackerson, Christopher J; Feldheim, Daniel L

2010-07-27

The ability of peptides selected via phage display to mediate the formation of inorganic nanoparticles is now well established. The atomic-level interactions between the selected peptides and the metal ion precursors are in most instances, however, largely obscure. We identified a new peptide sequence that is capable of mediating the formation of Ag nanoparticles. Surprisingly, nanoparticle formation requires the presence of peptide, HEPES buffer, and light; the absence of any one of these compromises nanoparticle formation. Electrochemical experiments revealed that the peptide binds Ag+ in a 3 Ag+:1 peptide ratio and significantly alters the Ag+ reduction potential. Alanine replacement studies yielded insight into the sequence-function relationships of Ag nanoparticle formation, including the Ag+ coordination sites and the residues necessary for Ag synthesis. In addition, the peptide was found to function when immobilized onto surfaces, and the specific immobilizing concentration could be adjusted to yield either spherical Ag nanoparticles or high aspect ratio nanowires. These studies further illustrate the range of interesting new solid-state chemistries possible using biomolecules.

Solid-phase organic synthesis of difluoroalkyl entities using a novel fluorinating cleavage strategy: part 2. Synthesis of three small gem-difluorinated compound libraries using a dithiane linker.

PubMed

Wiehn, Matthias S; Fürniss, Daniel; Bräse, Stefan

2009-01-01

Three small compound biaryl libraries featuring a novel fluorinating cleavage strategy for preparation of a difluoromethyl group were assembled on solid supports. The average reaction yield per step was up to 96% in a synthetic sequence over five to six steps. Key features were Suzuki coupling reactions, transesterification with potassium cyanide and amidation reaction with trimethyl aluminum on solid supports.

Silicon nanowire synthesis by a vapor-liquid-solid approach

NASA Technical Reports Server (NTRS)

Mao, Aaron; Ng, H. T.; Nguyen, Pho; McNeil, Melanie; Meyyappan, M.

2005-01-01

Synthesis of silicon nanowires is studied by using a vapor-liquid-solid growth technique. Silicon tetrachloride reduction with hydrogen in the gas phase is used with gold serving as catalyst to facilitate growth. Only a narrow set of conditions of SiCl4 concentration and temperature yield straight nanowires. High concentrations and temperatures generally result in particulates, catalyst coverage and deactivation, and coatinglike materials.

Persistent dopants and phase segregation in organolead mixed-halide perovskites

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rosales, Bryan A.; Men, Long; Cady, Sarah D.

Organolead mixed-halide perovskites such as CH 3NH 3PbX 3–aX' a (X, X' = I, Br, Cl) are interesting semiconductors because of their low cost, high photovoltaic power conversion efficiencies, enhanced moisture stability, and band gap tunability. Using a combination of optical absorption spectroscopy, powder X-ray diffraction (XRD), and, for the first time, 207Pb solid state nuclear magnetic resonance (ssNMR), we probe the extent of alloying and phase segregation in these materials. Because 207Pb ssNMR chemical shifts are highly sensitive to local coordination and electronic structure, and vary linearly with halogen electronegativity and band gap, this technique can provide the truemore » chemical speciation and composition of organolead mixed-halide perovskites. We specifically investigate samples made by three different preparative methods: solution phase synthesis, thermal annealing, and solid phase synthesis. 207Pb ssNMR reveals that nonstoichiometric dopants and semicrystalline phases are prevalent in samples made by solution phase synthesis. We show that these nanodomains are persistent after thermal annealing up to 200 °C. Further, a novel solid phase synthesis that starts from the parent, single-halide perovskites can suppress phase segregation but not the formation of dopants. Our observations are consistent with the presence of miscibility gaps and spontaneous spinodal decomposition of the mixed-halide perovskites at room temperature. This underscores how strongly different synthetic procedures impact the nanostructuring and composition of organolead halide perovskites. In conclusion, better optoelectronic properties and improved device stability and performance may be achieved through careful manipulation of the different phases and nanodomains present in these materials.« less

DNA-Encoded Solid-Phase Synthesis: Encoding Language Design and Complex Oligomer Library Synthesis.

PubMed

MacConnell, Andrew B; McEnaney, Patrick J; Cavett, Valerie J; Paegel, Brian M

2015-09-14

The promise of exploiting combinatorial synthesis for small molecule discovery remains unfulfilled due primarily to the "structure elucidation problem": the back-end mass spectrometric analysis that significantly restricts one-bead-one-compound (OBOC) library complexity. The very molecular features that confer binding potency and specificity, such as stereochemistry, regiochemistry, and scaffold rigidity, are conspicuously absent from most libraries because isomerism introduces mass redundancy and diverse scaffolds yield uninterpretable MS fragmentation. Here we present DNA-encoded solid-phase synthesis (DESPS), comprising parallel compound synthesis in organic solvent and aqueous enzymatic ligation of unprotected encoding dsDNA oligonucleotides. Computational encoding language design yielded 148 thermodynamically optimized sequences with Hamming string distance ≥ 3 and total read length <100 bases for facile sequencing. Ligation is efficient (70% yield), specific, and directional over 6 encoding positions. A series of isomers served as a testbed for DESPS's utility in split-and-pool diversification. Single-bead quantitative PCR detected 9 × 10(4) molecules/bead and sequencing allowed for elucidation of each compound's synthetic history. We applied DESPS to the combinatorial synthesis of a 75,645-member OBOC library containing scaffold, stereochemical and regiochemical diversity using mixed-scale resin (160-μm quality control beads and 10-μm screening beads). Tandem DNA sequencing/MALDI-TOF MS analysis of 19 quality control beads showed excellent agreement (<1 ppt) between DNA sequence-predicted mass and the observed mass. DESPS synergistically unites the advantages of solid-phase synthesis and DNA encoding, enabling single-bead structural elucidation of complex compounds and synthesis using reactions normally considered incompatible with unprotected DNA. The widespread availability of inexpensive oligonucleotide synthesis, enzymes, DNA sequencing, and PCR make implementation of DESPS straightforward, and may prompt the chemistry community to revisit the synthesis of more complex and diverse libraries.

Silicon nanowire synthesis by a vapor-liquid-solid approach.

PubMed

Mao, Aaron; Ng, H T; Nguyen, Pho; McNeil, Melanie; Meyyappan, M

2005-05-01

Synthesis of silicon nanowires is studied by using a vapor-liquid-solid growth technique. Silicon tetrachloride reduction with hydrogen in the gas phase is used with gold serving as catalyst to facilitate growth. Only a narrow set of conditions of SiCl4 concentration and temperature yield straight nanowires. High concentrations and temperatures generally result in particulates, catalyst coverage and deactivation, and coatinglike materials.

Piezoelectric Ceramics of the (1 − x)Bi0.50Na0.50TiO3–xBa0.90Ca0.10TiO3 Lead-Free Solid Solution: Chemical Shift of the Morphotropic Phase Boundary, a Case Study for x = 0.06

PubMed Central

Vivar-Ocampo, Rodrigo; Pardo, Lorena; Ávila, David; Morán, Emilio; González, Amador M.; Bucio, Lauro; Villafuerte-Castrejón, María-Elena

2017-01-01

Research and development of lead-free piezoelectric materials are still the hottest topics in the field of piezoelectricity. One of the most promising lead-free family of compounds to replace lead zirconate–titanate for actuators is that of Bi0.50Na0.50TiO3 (BNT) based solid solutions. The pseudo-binary (1 − x)Bi0.50Na0.50TiO3–xBa1 − yCayTiO3 system has been proposed for high temperature capacitors and not yet fully explored as piezoelectric material. In this work, the solid solution with x = 0.06 and y = 0.10 was obtained by two different synthesis routes: solid state and Pechini, aiming at using reduced temperatures, both in synthesis (<800 °C) and sintering (<1150 °C), while maintaining appropriated piezoelectric performance. Crystal structure, ceramic grain size, and morphology depend on the synthesis route and were analyzed by X-ray diffraction, together with scanning and transmission electron microscopy. The effects of processing and ceramic microstructure on the structural, dielectric, ferroelectric, and piezoelectric properties were discussed in terms of a shift of the Morphotropic Phase Boundary, chemically induced by the synthesis route. PMID:28773096

Plasma Proteome Dynamics: Analysis of Lipoproteins and Acute Phase Response Proteins with 2H2O Metabolic Labeling*

PubMed Central

Li, Ling; Willard, Belinda; Rachdaoui, Nadia; Kirwan, John P.; Sadygov, Rovshan G.; Stanley, William C.; Previs, Stephen; McCullough, Arthur J.; Kasumov, Takhar

2012-01-01

Understanding the pathologies related to the regulation of protein metabolism requires methods for studying the kinetics of individual proteins. We developed a 2H2O metabolic labeling technique and software for protein kinetic studies in free living organisms. This approach for proteome dynamic studies requires the measurement of total body water enrichments by GC-MS, isotopic distribution of the tryptic peptide by LC-MS/MS, and estimation of the asymptotical number of deuterium incorporated into a peptide by software. We applied this technique to measure the synthesis rates of several plasma lipoproteins and acute phase response proteins in rats. Samples were collected at different time points, and proteins were separated by a gradient gel electrophoresis. 2H labeling of tryptic peptides was analyzed by ion trap tandem mass spectrometry (LTQ MS/MS) for measurement of the fractional synthesis rates of plasma proteins. The high sensitivity of LTQ MS in zoom scan mode in combination with 2H label amplification in proteolytic peptides allows detection of the changes in plasma protein synthesis related to animal nutritional status. Our results demonstrate that fasting has divergent effects on the rate of synthesis of plasma proteins, increasing synthesis of ApoB 100 but decreasing formation of albumin and fibrinogen. We conclude that this technique can effectively measure the synthesis of plasma proteins and can be used to study the regulation of protein homeostasis under physiological and pathological conditions. PMID:22393261

Piezoelectric Ceramics of the (1 - x)Bi0.50Na0.50TiO₃-xBa0.90Ca0.10TiO₃ Lead-Free Solid Solution: Chemical Shift of the Morphotropic Phase Boundary, a Case Study for x = 0.06.

PubMed

Vivar-Ocampo, Rodrigo; Pardo, Lorena; Ávila, David; Morán, Emilio; González, Amador M; Bucio, Lauro; Villafuerte-Castrejón, María-Elena

2017-07-01

Research and development of lead-free piezoelectric materials are still the hottest topics in the field of piezoelectricity. One of the most promising lead-free family of compounds to replace lead zirconate-titanate for actuators is that of Bi 0.50 Na 0.50 TiO₃ (BNT) based solid solutions. The pseudo-binary (1 - x )Bi 0.50 Na 0.50 TiO₃- x Ba 1 - y Ca y TiO₃ system has been proposed for high temperature capacitors and not yet fully explored as piezoelectric material. In this work, the solid solution with x = 0.06 and y = 0.10 was obtained by two different synthesis routes: solid state and Pechini, aiming at using reduced temperatures, both in synthesis (<800 °C) and sintering (<1150 °C), while maintaining appropriated piezoelectric performance. Crystal structure, ceramic grain size, and morphology depend on the synthesis route and were analyzed by X-ray diffraction, together with scanning and transmission electron microscopy. The effects of processing and ceramic microstructure on the structural, dielectric, ferroelectric, and piezoelectric properties were discussed in terms of a shift of the Morphotropic Phase Boundary, chemically induced by the synthesis route.

Peptide dendrimer-conjugates of ketoprofen: Synthesis and ex vivo and in vivo evaluations of passive diffusion, sonophoresis and iontophoresis for skin delivery.

PubMed

Hegde, Aswathi R; Rewatkar, Prarthana V; Manikkath, Jyothsna; Tupally, Karnaker; Parekh, Harendra S; Mutalik, Srinivas

2017-05-01

The aim of this study was to evaluate skin delivery of ketoprofen when covalently tethered to mildly cationic (2 + or 4 + ) peptide dendrimers prepared wholly by solid phase peptide synthesis. The amino acids glycine, arginine and lysine formed the dendrimer with ketoprofen tethered either to the lysine side-arm (N ε ) or periphery of dendrimeric branches. Passive diffusion, sonophoresis- and iontophoresis-assisted permeation of each peptide dendrimer-drug conjugate (D1-D4) was studied across mouse skin, both in vitro and in vivo. In addition, skin toxicity of dendrimeric conjugates when trialed with iontophoresis or sonophoresis was also evaluated. All dendrimeric conjugates improved aqueous solubility at least 5-fold, compared to ketoprofen alone, while also exhibiting appreciable lipophilicity. In vitro passive diffusion studies revealed that ketoprofen in its native form was delivered to a greater extent, compared with a dendrimer-conjugated form at the end of 24h (Q 24h (μg/cm 2 ): ketoprofen (68.06±3.62)>D2 (49.62±2.92)>D4 (19.20±0.89)>D1 (6.45±0.40)>D3 (2.21±0.19). However, sonophoresis substantially increased the skin permeation of ketoprofen-dendrimer conjugates in 30min (Q 30min (μg/cm 2 ): D4 (122.19±7.14)>D2 (66.74±3.86)>D1 (52.10±3.22)>D3 (41.66±3.22)) although ketoprofen alone again proved superior (Q 30min : 167.99±9.11μg/cm 2 ). Next, application of iontophoresis was trialed and shown to considerably increase permeation of dendrimeric ketoprofen in 6h (Q 6h (μg/cm 2 ): D2 (711.49±39.14)>D4 (341.23±16.43)>D3 (89.50±4.99)>D1 (50.91±2.98), with a Q 6h value of 96.60±5.12μg/cm 2 for ketoprofen alone). In vivo studies indicated that therapeutically relevant concentrations of ketoprofen could be delivered transdermally when iontophoresis was paired with D2 (985.49±43.25ng/mL). Further, histopathological analysis showed that the dendrimeric approach was a safe mode as ketoprofen alone. The present study successfully demonstrates that peptide dendrimer conjugates of ketoprofen, when combined with non-invasive modalities, such as iontophoresis can enhance skin permeation with clinically relevant concentrations achieved transdermally. Copyright © 2017 Elsevier B.V. All rights reserved.

Solid phase synthesis and antiprotozoal evaluation of di- and trisubstituted 5'-carboxamidoadenosine analogues.

PubMed

Rodenko, Boris; Detz, Remko J; Pinas, Victorine A; Lambertucci, Catia; Brun, Reto; Wanner, Martin J; Koomen, Gerrit-Jan

2006-03-01

The rapid increase of resistance to drugs commonly used in the treatment of tropical diseases such as malaria and African sleeping sickness calls for the prompt development of new safe and efficacious drugs. The pathogenic protozoan parasites lack the capability of synthesising purines de novo and they take up preformed purines from their host through various transmembrane transporters. Adenosine derivatives constitute a class of potential therapeutics due to their selective internalisation by these transporters. Automated solid-phase synthesis can speed up the process of lead finding and we pursued the solid-phase synthesis of di- and trisubstituted 5'-carboxamidoadenosine derivatives by using a safety-catch approach. While efforts with Kenner's sulfonamide linker remained fruitless, successful application of the hydrazide safety-catch linker allowed the construction of two representative combinatorial libraries. Their antiprotozoal evaluation identified two compounds with promising activity: N(6)-benzyl-5'-N-phenylcarboxamidoadenosine with an IC(50) value of 0.91 microM against Trypanosoma brucei rhodesiense and N(6)-diphenylethyl-5'-phenylcarboxamidoadenosine with an IC(50) value of 1.8 microM against chloroquine resistant Plasmodium falciparum.

Combinatorial Solid-Phase Synthesis of Aromatic Oligoamides: A Research-Based Laboratory Module for Undergraduate Organic Chemistry

ERIC Educational Resources Information Center

Fuller, Amelia A.

2016-01-01

A five-week, research-based experiment suitable for second-semester introductory organic laboratory students is described. Each student designs, prepares, and analyzes a combinatorial array of six aromatic oligoamides. Molecules are prepared on solid phase via a six-step synthetic sequence, and purities and identities are determined by analysis of…

Solid-phase synthesis and structure-activity relationships of novel biarylethers as melanin-concentrating hormone receptor-1 antagonists.

PubMed

Ma, Vu; Bannon, Anthony W; Baumgartner, Jamie; Hale, Clarence; Hsieh, Faye; Hulme, Christopher; Rorrer, Kirk; Salon, John; van Staden, Carlo; Tempest, Paul

2006-10-01

Melanin-concentrating hormone (MCH) is a cyclic 19 amino acid orexigenic neuropeptide. The action of MCH on feeding is thought to involve the activation of its respective G protein-coupled receptor MCH-R1. Consequently, antagonists that block MCH regulated MCH-R1 activity may provide a viable approach to the treatment of diet-induced obesity. This communication reports the discovery of a novel MCH-R1 receptor antagonist, the biarylether 7, identified through high throughput screening. The solid-phase synthesis and structure-activity relationship of related analogs is described.

Solid-phase synthesis of oligo-2-pyrimidinone-2'-deoxyribonucleotides and oligo-2-pyrimidinone-2'-deoxyriboside methylphosphonates.

PubMed Central

Zhou, Y; Ts'o, P O

1996-01-01

A synthetic method was developed for the synthesis of oligodeoxyribonucleotides and oligodeoxyribonucleoside methylphosphonates comprised exclusively of the fluorescent 2-pyrimidinone base for the first time. The method utilized the solid-phase 2-cyanoethylphosphoramidite and methylphosphonamidite chemistry for internucleotide couplings and a baselabile oxalyl linkage to anchor the oligomers onto the CPG support. Cleavage of the oligomers from the support was effected by a short treatment of the support with 5% ammonium hydroxide in methanol at room temperature, without any degradation of the base-sensitive 2-pyrimidinone residues or the base-sensitive methylphosphonate backbone. PMID:8758991

Multistep synthesis on SU-8: combining microfabrication and solid-phase chemistry on a single material.

PubMed

Cavalli, Gabriel; Banu, Shahanara; Ranasinghe, Rohan T; Broder, Graham R; Martins, Hugo F P; Neylon, Cameron; Morgan, Hywel; Bradley, Mark; Roach, Peter L

2007-01-01

SU-8 is an epoxy-novolac resin and a well-established negative photoresist for microfabrication and microengineering. The photopolymerized resist is an extremely highly crosslinked polymer showing outstanding chemical and physical robustness with residual surface epoxy groups amenable for chemical functionalization. In this paper we describe, for the first time, the preparation and surface modification of SU-8 particles shaped as microbars, the attachment of appropriate linkers, and the successful application of these particles to multistep solid-phase synthesis leading to oligonucleotides and peptides attached in an unambiguous manner to the support surface.

Effects of Primary Processing Techniques and Significance of Hall-Petch Strengthening on the Mechanical Response of Magnesium Matrix Composites Containing TiO2 Nanoparticulates

PubMed Central

Meenashisundaram, Ganesh Kumar; Nai, Mui Hoon; Gupta, Manoj

2015-01-01

In the present study, Mg (1.98 and 2.5) vol % TiO2 nanocomposites are primarily synthesized utilizing solid-phase blend-press-sinter powder metallurgy (PM) technique and liquid-phase disintegrated melt deposition technique (DMD) followed by hot extrusion. Microstructural characterization of the synthesized Mg-TiO2 nanocomposites indicated significant grain refinement with DMD synthesized Mg nanocomposites exhibiting as high as ~47% for 2.5 vol % TiO2 NPs addition. X-ray diffraction studies indicated that texture randomization of pure Mg depends not only on the critical amount of TiO2 NPs added to the Mg matrix but also on the adopted synthesis methodology. Irrespective of the processing technique, theoretically predicted tensile yield strength of Mg-TiO2 nanocomposites was found to be primarily governed by Hall-Petch mechanism. Among the synthesized Mg materials, solid-phase synthesized Mg 1.98 vol % TiO2 nanocomposite exhibited a maximum tensile fracture strain of ~14.5%. Further, the liquid-phase synthesized Mg-TiO2 nanocomposites exhibited higher tensile and compression properties than those primarily processed by solid-phase synthesis. The tensile-compression asymmetry values of the synthesized Mg-TiO2 nanocomposite was found to be lower than that of pure Mg with solid-phase synthesized Mg 1.98 vol % TiO2 nanocomposite exhibiting as low as 1.06. PMID:28347063

Glycotriazole-peptides derived from the peptide HSP1: synergistic effect of triazole and saccharide rings on the antifungal activity.

PubMed

Junior, Eduardo F C; Guimarães, Carlos F R C; Franco, Lucas L; Alves, Ricardo J; Kato, Kelly C; Martins, Helen R; de Souza Filho, José D; Bemquerer, Marcelo P; Munhoz, Victor H O; Resende, Jarbas M; Verly, Rodrigo M

2017-08-01

This work proposes a strategy that uses solid-phase peptide synthesis associated with copper(I)-catalyzed azide alkyne cycloaddition reaction to promote the glycosylation of an antimicrobial peptide (HSP1) containing a carboxyamidated C-terminus (HSP1-NH 2 ). Two glycotriazole-peptides, namely [p-Glc-trz-G 1 ]HSP1-NH 2 and [p-GlcNAc-trz-G 1 ]HSP1-NH 2 , were prepared using per-O-acetylated azide derivatives of glucose and N-acetylglucosamine in the presence of copper(II) sulfate pentahydrate (CuSO 4 ·5H 2 O) and sodium ascorbate as a reducing agent. In order to investigate the synergistic action of the carbohydrate motif linked to the triazole-peptide structure, a triazole derivative [trz-G 1 ]HSP1-NH 2 was also prepared. A set of biophysical approaches such as DLS, Zeta Potential, SPR and carboxyfluorescein leakage from phospholipid vesicles confirmed higher membrane disruption and lytic activities as well as stronger peptide-LUVs interactions for the glycotriazole-peptides when compared to HSP1-NH 2 and to its triazole derivative, which is in accordance with the performed biological assays: whereas HSP1-NH 2 presents relatively low and [trz-G 1 ]HSP1-NH 2 just moderate fungicidal activity, the glycotriazole-peptides are significantly more effective antifungal agents. In addition, the glycotriazole-peptides and the triazole derivative present strong inhibition effects on ergosterol biosynthesis in Candida albicans, when compared to HSP1-NH 2 alone. In conclusion, the increased fungicidal activity of the glycotriazole-peptides seems to be the result of (A) more pronounced membrane-disruptive properties, which is related to the presence of a saccharide ring, together with (B) the inhibition of ergosterol biosynthesis, which seems to be related to the presence of both the monosaccharide and the triazole rings.

AJIPHASE®: A Highly Efficient Synthetic Method for One-Pot Peptide Elongation in the Solution Phase by an Fmoc Strategy.

PubMed

Takahashi, Daisuke; Inomata, Tatsuji; Fukui, Tatsuya

2017-06-26

We previously reported an efficient peptide synthesis method, AJIPHASE®, that comprises repeated reactions and isolations by precipitation. This method utilizes an anchor molecule with long-chain alkyl groups as a protecting group for the C-terminus. To further improve this method, we developed a one-pot synthesis of a peptide sequence wherein the synthetic intermediates were isolated by solvent extraction instead of precipitation. A branched-chain anchor molecule was used in the new process, significantly enhancing the solubility of long peptides and the operational efficiency compared with the previous method, which employed precipitation for isolation and a straight-chain aliphatic group. Another prerequisite for this solvent-extraction-based strategy was the use of thiomalic acid and DBU for Fmoc deprotection, which facilitates the removal of byproducts, such as the fulvene adduct. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Isolation, structure, synthesis, and activity of a new member of the calcitonin gene-related peptide family from frog skin and molecular cloning of its precursor.

PubMed

Seon, A A; Pierre, T N; Redeker, V; Lacombe, C; Delfour, A; Nicolas, P; Amiche, M

2000-02-25

Calcitonin gene-related peptide has been extracted from the skin exudate of a single living specimen of the frog Phyllomedusa bicolor and purified to homogeneity by a two-step protocol. A total volume of 250 microl of exudate yielded 380 microg of purified peptide. Mass spectrometric analysis and gas phase sequencing of the purified peptide as well as chemical synthesis and cDNA analysis were consistent with the structure SCDTSTCATQRLADFLSRSGGIGSPDFVPTDVSANSF amide and the presence of a disulfide bridge linking Cys(2) and Cys(7). The skin peptide, named skin calcitonin gene-related peptide, differs significantly from all other members of the calcitonin gene-related peptide family of peptides at nine positions but binds with high affinity to calcitonin gene-related peptide receptors in the rat brain and acts as an agonist in the rat vas deferens bioassay with potencies equal to those of human CGRP. Reverse transcriptase-polymerase chain reaction coupled with cDNA cloning and sequencing demonstrated that skin calcitonin gene-related peptide isolated in the skin is identical to that present in the frog's central and enteric nervous systems. These data, which indicate for the first time the existence of calcitonin gene-related peptide in the frog skin, add further support to the brain-skin-gut triangle hypothesis as a useful tool in the identification and/or isolation of mammalian peptides that are present in the brain and other tissues in only minute quantities.

Elucidation of the surface characteristics and electrochemistry of high-performance LiNiO 2

DOE PAGES

Xu, Jing; Lin, Feng; Nordlund, Dennis; ...

2016-02-25

Phase pure LiNiO 2 was prepared using a solid-state method and the optimal synthesis conditions led to a remarkably high capacity of 200 mA h g $-$1 with excellent retention. The combination of bulk and surface characterization elucidated an essential role of the excess Li in phase formation during synthesis and the subsequent electrochemical performance.

A novel Tc-99m and fluorescence-labeled arginine-arginine-leucine-containing peptide as a multimodal tumor imaging agent in a murine tumor model.

PubMed

Kim, Myoung Hyoun; Kim, Seul-Gi; Kim, Dae-Weung

2018-06-15

We developed a Tc-99m and TAMRA-labeled peptide, Tc-99m arginine-arginine-leucine (RRL) peptide (TAMRA-GHEG-ECG-RRL), to target tumor cells and evaluated the diagnostic performance of Tc-99m TAMRA-GHEG-ECG-RRL as a dual-modality imaging agent for tumor in a murine model. TAMRA-GHEG-ECG-RRL was synthesized using Fmoc solid-phase peptide synthesis. Binding affinity and in vitro cellular uptake studies were performed. Gamma camera imaging, biodistribution, and ex vivo imaging studies were performed in murine models with PC-3 tumors. Tumor tissue slides were prepared and analyzed with immunohistochemistry using confocal microscopy. After radiolabeling procedures with Tc-99m, Tc-99m TAMRA-GHEG-ECG-RRL complexes were prepared in high yield (>96%). The K d of Tc-99m TAMRA-GHEG-ECG-RRL determined by saturation binding was 41.7 ± 7.8 nM. Confocal microscopy images of PC-3 cells incubated with TAMRA-GHEG-ECG-RRL showed strong fluorescence in the cytoplasm. Gamma camera imaging revealed substantial uptake of Tc-99m TAMRA-GHEG-ECG-RRL in tumors. Tumor uptake was effectively blocked by the coinjection of an excess concentration of RRL. Specific uptake of Tc-99m TAMRA-GHEG-ECG-RRL was confirmed by biodistribution, ex vivo imaging, and immunohistochemistry stain studies. In conclusion, in vivo and in vitro studies revealed substantial uptake of Tc-99m TAMRA-GHEG-ECG-RRL in tumors. Tc-99m TAMRA-GHEG-ECG-RRL has potential as a dual-modality tumor imaging agent. Copyright © 2018 John Wiley & Sons, Ltd.

Design of peptide substrates for nanosecond time-resolved fluorescence assays of proteases: 2,3-diazabicyclo[2.2.2]oct-2-ene as a noninvasive fluorophore.

PubMed

Hennig, Andreas; Florea, Mara; Roth, Doris; Enderle, Thilo; Nau, Werner M

2007-01-15

Fluorescence protease assays were investigated with peptide substrates containing a 2,3-diazabicyclo[2.2.2]oct-2-ene-labeled asparagine (Dbo) as a fluorescent amino acid. The special characteristic of the fluorophore Dbo is its exceedingly long fluorescence lifetime (ca. 300 ns in water under air), which allows the use of nanosecond time-resolved fluorescence (Nano-TRF) detection to efficiently suppress shorter-lived background emission. In addition, the natural amino acids tryptophan and tyrosine can be employed as intramolecular fluorescence quenchers, which facilitates substrate design. Fourteen synthetic peptide substrates (composed of 2-19 amino acids) and five enzymes (trypsin, pepsin, carboxypeptidase A, leucine aminopeptidase, and chymotrypsin) were investigated and, in all 28 examined combinations, enzymatic activity was detected by monitoring the increase in steady state fluorescence with time and determining the reaction rates as kcat/Km values, which ranged from 0.2 to 80x10(6) M-1 min-1. The results suggest an excellent compatibility of the very small and hydrophilic fluorescent probe Dbo with solid-phase peptide synthesis and the investigated proteases. For all 14 peptides the fluorescence lifetimes before and after enzymatic cleavage were measured and Nano-TRF measurements were performed in 384-well microplates. The fluorescence lifetimes of the different peptides provide the basis for the rational design of Dbo-based fluorescent substrates for protease assays. Measurements in Nano-TRF mode revealed, in addition to efficient suppression of background fluorescence, an increased differentiation between cleaved and uncleaved substrate. The Dbo-based assays can be adapted for high-throughput screening.

Combinatorial synthesis of phosphors using arc-imaging furnace

PubMed Central

Ishigaki, Tadashi; Toda, Kenji; Yoshimura, Masahiro; Uematsu, Kazuyoshi; Sato, Mineo

2011-01-01

We have applied a novel ‘melt synthesis technique’ rather than a conventional solid-state reaction to rapidly synthesize phosphor materials. During a synthesis, the mixture of oxides or their precursors is melted by light pulses (10–60 s) in an arc-imaging furnace on a water-cooled copper hearth to form a globule of 1–5 mm diameter, which is then rapidly cooled by turning off the light. Using this method, we synthesized several phosphor compounds including Y3Al5O12:Ce(YAG) and SrAl2O4:Eu,Dy. Complex phosphor oxides are difficult to produce by conventional solid-state reaction techniques because of the slow reaction rates among solid oxides; as a result, the oxides form homogeneous compounds or solid solutions. On the other hand, melt reactions are very fast (10–60 s) and result in homogeneous compounds owing to rapid diffusion and mixing in the liquid phase. Therefore, melt synthesis techniques are suitable for preparing multi component homogeneous compounds and solid solutions. PMID:27877432

Molecular Design, Structural Analysis and Antifungal Activity of Derivatives of Peptide CGA-N46.

PubMed

Li, Rui-Fang; Lu, Zhi-Fang; Sun, Ya-Nan; Chen, Shi-Hua; Yi, Yan-Jie; Zhang, Hui-Ru; Yang, Shuo-Ye; Yu, Guang-Hai; Huang, Liang; Li, Chao-Nan

2016-09-01

Chromogranin A (CGA)-N46, a derived peptide of human chromogranin A, has antifungal activity. To further research the active domain of CGA-N46, a series of derivatives were designed by successively deleting amino acid from both terminus of CGA-N46, and the amino acid sequence of each derivative was analyzed by bioinformatic software. Based on the predicted physicochemical properties of the peptides, including half-life time in mammalian reticulocytes (in vitro), yeast (in vivo) and E. coli (in vivo), instability index, aliphatic index and grand average of hydropathicity (GRAVY), the secondary structure, net charge, the distribution of hydrophobic residues and hydrophilic residues, the final derivatives CGA-N15, CGA-N16, CGA-N12 and CGA-N8 were synthesized by solid-phase peptide synthesis. The results of bioinformatic analysis showed that CGA-N46 and its derivatives were α-helix, neutral or weak positive charge, hydrophilic, and CGA-N12 and CGA-N8 were more stable than the other derivatives. The results of circular dichroism confirmed that CGA-N46 and its derived peptides displayed α-helical structure in an aqueous solution and 30 mM sodium dodecylsulfate, but α-helical contents decreased in hydrophobic lipid vesicles. CGA-N15, CGA-N16, CGA-N12 and CGA-N8 had higher antifungal activities than their mother peptide CGA-N46. Among of the derived peptides, CGA-N12 showed the least hemolytic activity. In conclusion, we have successfully identified the active domain of CGA-N46 with strong antifungal activity and weak hemolytic activity, which provides the possibility to develop a new class of antibiotics.

Solid-phase synthesis of molecularly imprinted nanoparticles.

PubMed

Canfarotta, Francesco; Poma, Alessandro; Guerreiro, Antonio; Piletsky, Sergey

2016-03-01

Molecularly imprinted polymers (MIPs) are synthetic materials, generally based on acrylic or methacrylic monomers, that are polymerized in the presence of a specific target molecule called the 'template' and capable of rebinding selectively to this target molecule. They have the potential to be low-cost and robust alternatives to biomolecules such as antibodies and receptors. When prepared by traditional synthetic methods (i.e., with free template in solution), their usefulness has been limited by high binding site heterogeneity, the presence of residual template and the fact that the production methods are complex and difficult to standardize. To overcome some of these limitations, we developed a method for the synthesis of MIP nanoparticles (nanoMIPs) using an innovative solid-phase approach, which relies on the covalent immobilization of the template molecules onto the surface of a solid support (glass beads). The obtained nanoMIPs are virtually free of template and demonstrate high affinity for the target molecule (e.g., melamine and trypsin in our published work). Because of an affinity separation step performed on the solid phase after polymerization, poor binders and unproductive polymer are removed, so the final product has more uniform binding characteristics. The overall protocol, starting from the immobilization of the template onto the solid phase and including the purification and characterization of the nanoparticles, takes up to 1 week.

Hydrophilic interaction liquid chromatography-solid phase extraction directly combined with protein precipitation for the determination of triptorelin in plasma.

PubMed

Wang, Jixia; Kong, Song; Yan, Jingyu; Jin, Gaowa; Guo, Zhimou; Shen, Aijin; Xu, Junyan; Zhang, Xiuli; Zou, Lijuan; Liang, Xinmiao

2014-06-01

Peptide drugs play a critical role in therapeutic treatment. However, as the complexity of plasma, determination of peptide drugs using liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a daunting task. To solve this problem, hydrophilic interaction liquid chromatography-solid phase extraction (HILIC-SPE) directly combined with protein precipitation (PPT) was developed for the selective extraction of triptorelin from plasma. The extracts were analyzed by reversed-phase liquid chromatography (RPLC). Proteins, phospholipids and highly polar interferences could be removed from plasma by the efficient combination of PPT, HILIC-SPE and RPLC-MS/MS. This method was evaluated by matrix effect, recovery and process efficiency at different concentration levels (50, 500 and 5,000 ng/mL) of triptorelin. Furthermore, the performance of HILIC-SPE was compared with that of reversed-phase C18 SPE and hydrophilic lipophilic balance (Oasis HLB) SPE. Among them, HILIC-SPE provided the minimum matrix effect (ranging from 96.02% to 103.41%), the maximum recovery (ranging from 80.68% to 90.54%) and the satisfactory process efficiency (ranging from 82.83% to 92.95%). The validated method was successfully applied to determine triptorelin in rat plasma. Copyright © 2014 Elsevier B.V. All rights reserved.

Solid phase monofunctionalization of gold nanoparticles using ionic exchange resin as polymer support.

PubMed

Zou, Jianhua; Dai, Qiu; Wang, Jinhai; Liu, Xiong; Huo, Qun

2007-07-01

A solid phase modification method using anionic exchange resin as polymer support was developed for the synthesis of monofunctional gold nanoparticles. Based on a "catch and release" mechanism to control the number of functional groups attached to the nanoparticle surface, bifunctional thiol ligands with a carboxylic acid end group were first immobilized at a controlled density on anionic exchange resin through electrostatic interactions. Gold nanoparticles were then immobilized to the anionic exchange resin by a one-to-one place exchange reaction between resin-bound thiol ligands and butanethiol-protected gold nanoparticles in solution. After cleaving off from the resin under mild conditions, gold nanoparticles with a single carboxyl group attached to the surface were obtained as the major product. Experimental conditions such as the solvents used for ligand loading and solid phase place exchange reaction, and the loading density of the ligands, were found to play a critical role towards the successful synthesis of monofunctional nanoparticles. Overall, the noncovalent bond-based ligand immobilization technique reported here greatly simplified the process of solid phase monofunctionalization of nanoparticles compared to a previously reported covalent bond-based ligand immobilization technique.

Synthesis of 4-(5-iodo-3-methylpyrazolyl) phenylsulfonamide and its elaboration to a COX II inhibitor library by solution-phase suzuki coupling using Pd/C as a solid-supported catalyst.

PubMed

Organ, Michael G; Mayer, Stanislas

2003-01-01

An effective synthesis of 4-(5-iodo-3-methylpyrazolyl) phenylsulfonamide has been developed. This aromatic iodide template served as an efficient oxidative addition partner for the preparation of a solution-phase library of Celecoxib analogues via Suzuki coupling using Pd/C, a readily filterable catalyst.

Inhibition of ovarian cancer cell proliferation by a cell cycle inhibitory peptide fused to a thermally responsive polypeptide carrier.

PubMed

Massodi, Iqbal; Moktan, Shama; Rawat, Aruna; Bidwell, Gene L; Raucher, Drazen

2010-01-15

Current treatment of solid tumors is limited by normal tissue tolerance, resulting in a narrow therapeutic index. To increase drug specificity and efficacy and to reduce toxicity in normal tissues, we have developed a polypeptide carrier for a cell cycle inhibitory peptide, which has the potential to be thermally targeted to the tumor site. The design of this polypeptide is based on elastin-like polypeptide (ELP). The coding sequence of ELP was modified by the addition of the cell penetrating peptide Bac-7 at the N-terminus and a 23 amino acid peptide derived from p21 at the C-terminus (Bac-ELP1-p21). Bac-ELP1-p21 is soluble in aqueous solutions below physiological temperature (37 degrees C) but aggregates when the temperature is raised above 39 degrees C, making it a promising thermally responsive therapeutic carrier that may be actively targeted to solid tumors by application of focused hyperthermia. While Bac-ELP1-p21 at 37 degrees C did not have any effect on SKOV-3 cell proliferation, the use of hyperthermia increased the antiproliferative effect of Bac-ELP1-p21 compared with a thermally unresponsive control polypeptide. Bac-ELP1-p21 displayed both a cytoplasmic and nuclear distribution in the SKOV-3 cells, with nuclear-localized polypeptide enriched in the heated cells, as revealed by confocal microscopy. Using Western blotting, we show that Bac-ELP1-p21 caused a decrease in Rb phosphorylation levels in cells treated at 42 degrees C. The polypeptide also induced caspase activation, PARP cleavage, and cell cycle arrest in S-phase and G2/M-phase. These studies indicate that ELP is a promising macromolecular carrier for the delivery of cell cycle inhibitory peptides to solid tumors.

Synthesis of graphene nanoribbons from amyloid templates by gallium vapor-assisted solid-phase graphitization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murakami, Katsuhisa, E-mail: k.murakami@bk.tsukuba.ac.jp; Dong, Tianchen; Kajiwara, Yuya

2014-06-16

Single- and double-layer graphene nanoribbons (GNRs) with widths of around 10 nm were synthesized directly onto an insulating substrate by solid-phase graphitization using a gallium vapor catalyst and carbon templates made of amyloid fibrils. Subsequent investigation revealed that the crystallinity, conductivity, and carrier mobility were all improved by increasing the temperature of synthesis. The carrier mobility of the GNR synthesized at 1050 °C was 0.83 cm{sup 2}/V s, which is lower than that of mechanically exfoliated graphene. This is considered to be most likely due to electron scattering by the defects and edges of the GNRs.

Phase-assisted synthesis and DNA unpacking evaluation of biologically inspired metallo nanocomplexes using peptide as unique building block.

PubMed

Raman, N; Sudharsan, S

2011-12-01

The goal of nanomaterials' surface modification using a biomaterial is to preserve the materials' bulk properties while modifying only their surface to possess desired recognition and specificity. Here, we have developed a phase-assisted, modified Brust-Schiffrin methodological synthesis of metallo nanocomplexes anchored by a peptide, N,N'-(1,3-propylene)-bis-hippuricamide. The spectral, thermal and morphological characterizations assure the formation of nanocomplexes. Therapeutic behavior of all the nanocomplexes has been well sighted by evaluating their DNA unpacking skills. In addition, we demonstrate their biological inspiration by targeting few bacterial and fungal strains. The in vitro antimicrobial investigation reports that all the nanocomplexes disrupt microbial cell walls/membranes efficiently and inhibit the growth of microbes. These sorts of nanocomplexes synthesized in large quantities and at low cost, deliver versatile biomedical applications, and can be used to treat various diseases which may often cause high mortality. Copyright © 2011 Elsevier Inc. All rights reserved.

One Step Combustion Synthesis Of YAG:Ce Phosphor For Solid State Lighting

NASA Astrophysics Data System (ADS)

Yadav, Pooja; Gupta, K. Vijay Kumar; Muley, Aarti; Joshi, C. P.; Moharil, S. V.

2011-10-01

YAG:Ce is an important phosphor having applications in various fields ranging from solid state lighting to scintillation detectors. YAG phosphors doped with activators are mainly synthesized by solid state reaction techniques that require high sintering temperatures (above 1500°C) to eliminate YAM and YAP phases. Though several soft chemical routes have been explored for synthesis of YAG, most of these methods are complex and phase pure materials are not obtained in one step, but prolonged annealing at temperatures around 1000 C or above becomes necessary. One step combustion synthesis of YAG:Ce3+ and related phosphors carried out at 500 C furnace temperature is reported here. Activation with Ce3+ could be achieved during the synthesis without taking recourse to any post-combustion thermal treatment. LEDs prepared from the combustion synthesized YAG:Ce3+, exhibited properties comparable to those produced from the commercial phosphor.

Predicting the growth of S i3N4 nanowires by phase-equilibrium-dominated vapor-liquid-solid mechanism

NASA Astrophysics Data System (ADS)

Zhang, Yongliang; Cai, Jing; Yang, Lijun; Wu, Qiang; Wang, Xizhang; Hu, Zheng

2017-09-01

Nanomaterial synthesis is experiencing a profound evolution from empirical science ("cook-and-look") to prediction and design, which depends on the deep insight into the growth mechanism. Herein, we report a generalized prediction of the growth of S i3N4 nanowires by nitriding F e28S i72 alloy particles across different phase regions based on our finding of the phase-equilibrium-dominated vapor-liquid-solid (PED-VLS) mechanism. All the predictions about the growth of S i3N4 nanowires, and the associated evolutions of lattice parameters and geometries of the coexisting Fe -Si alloy phases, are experimentally confirmed quantitatively. This progress corroborates the general validity of the PED-VLS mechanism, which could be applied to the design and controllable synthesis of various one-dimensional nanomaterials.

Arynes and Heteroarynes in the Synthesis of Dibenzocinnolines, Diazaxanthyledenes, and Triptycenes

NASA Astrophysics Data System (ADS)

Suh, Sung-Eun

Arynes are known as useful synthons in organic synthesis. In particular, reactions accompanying multiple arynes have been employed for the construction of arenes and heteroarenes of complex molecules. Employing known reactivity modes of arynes such as cycloadditions, nucleophilic addition, bond insertion, Alder-ene, annulation, desaturation, and polymerization, a wide variety of transformation of reactive starting materials led to the development of novel fluorophores and energy materials, as well as the synthesis of natural products. Harnessing the highly reactive arynes, the triple aryne-tetrazine (TAT) reaction was disclosed as a novel metal-free synthetic method for the preparation of dibenzo[de,g]cinnoline derivatives in a single operation. Dibenzo[de,g]cinnolines have been shown as potential fluorescent probes in cells. For the mechanism, multiple mechanistic steps of the TAT reaction were scrutinized by isolation of intermediates and byproducts as well as a computational study on the transition states and the competitive reactions pathways. A facile two-step synthesis of the reported structure of xylopyridine A was developed from a pyridyne precursor and 2-fluorobenzoic acid utilizing a pyridyne insertion reaction followed by reductive coupling. Simple transformation of the reported xylopyridine A structure have given photoactivatable dyes and specific organelle staining probes in either live or fixed cells and tissues, exhibiting high quantum yields, photostability, cell permeability and low toxicity. On the basis of these results, the synthesis of multistage photoactivatable dyes was designed and studied. Utilization of arynes allowed access to the synthesis of 9-substituted triptycene derivatives which have been recognized as three-way junction binders. Accompanying solid-phase peptide synthesis, the rapid diversification of the triptycene scaffold was achieved for screening in a nucleic acid junction binding assay.

An HLA-B27 Homodimer Specific Antibody Recognizes a Discontinuous Mixed-Disulfide Epitope as Identified by Affinity-Mass Spectrometry

NASA Astrophysics Data System (ADS)

Iuraşcu, Marius-Ionuţ; Marroquin Belaunzanar, Osiris; Cozma, Claudia; Petrausch, Ulf; Renner, Christoph; Przybylski, Michael

2016-06-01

HLA-B27 homodimer formation is believed to be a hallmark of HLA-B27 associated spondyloarthritides. Recently, we have generated a homodimer-specific monoclonal antibody (HD6) and have demonstrated that HLA-B27 homodimer complexes are present on monocytes of healthy HLA-B27 gene carriers at low levels, with significantly increased levels at active disease. The capability of the HD6 antibody to discriminate between correctly formed HLA-B27 heterotrimers and pathology-associated homodimers is striking and cannot be explained by the primary structure of HLA-B27. We hypothesized that HD6 accesses a unique epitope and used affinity-mass spectrometry for its identification. The HD6 antibody was immobilized on an activated sepharose affinity column, and HLA-B27 homodimer characterized for affinity. The epitope was identified by proteolytic epitope excision and MALDI mass spectrometry, and shown to comprise a discontinuous Cys-203- 257-Cys mixed-disulfide peptide structure that is not accessible in HLA-B27 heterotrimers due to protection by noncovalently linked β2-microglobulin. The epitope peptides were synthesized by solid phase peptide synthesis, and the two monomeric peptide components, HLA-B27(203-219) and HLA-B27(257-273), as well as the homo- and hetero-dimeric disulfide linked combinations prepared. The affinity binding constants KD towards the antibodies were determined using a surface acoustic wave (SAW) biosensor, and showed the highest affinity with a KD of approximately 40 nM to the HD6 antibody for the (203-219)-SS-(257-273) mixed disulfide epitope.

Synthesis mechanism and preparation of LaMgAl11O19 powder for plasma spraying

NASA Astrophysics Data System (ADS)

He, Mingtao; Meng, Huimin; Wang, Yuchao; Ren, Pengwei

2018-06-01

Lanthanide magnesium hexaaluminate (LaMgAl11O19) powders were successfully synthesized by the solid-state reaction method. The objective of this study was to investigate the synthesis mechanism of LaMgAl11O19 and prepare LaMgAl11O19 powders suitable for plasma spraying. The results show that LaAlO3 reacts with MgAl2O4 and Al2O3 to form LaMgAl11O19 at approximately 1300 °C. Single-phase LaMgAl11O19 powders were prepared successfully by solid-state reaction at a synthesis temperature of 1600 °C for 6 h. Unlike the particles in the synthesized powders, those of the centrifugally spray-dried powders have a spherical shape with uniform granularity and good flowability, density, and particle size distribution, making them suitable for plasma spraying. The synthesized powders and centrifugally spray-dried powders remained as a single phase after heat treatment at 1300 °C for 100 h, indicating that LaMgAl11O19 has excellent high-temperature stability.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pennington, M.W.

We have determined that ShN I, a 48-residue type 2 sea anemone toxin, delays the inactivation of the Na channel in lobster olfactory somas. The receptor for ShN I was identified in vesicle preparations of neuronal tissues from both crustaceans and mammals; however, the K{sub D} values for the former is more than 1,000 fold lower for the later. The binding of ({sup 125}I)-ShN I to this receptor was determined to be unaffected by Anemonia sulcata II, depolarization of the membrane, or veratridine. ShN I was unable to displace ({sup 125}I)-Androctonus austrialis Hector II, whereas unlabeled AaH II and Asmore » II displaced the labeled scorpion toxin from rat brain synaptosomes. This is the first characterization of a new Na channel receptor site which specifically binds type 2 anemone toxins. To study the interactions that specific amino acid residues of ShN I have with this receptor, we developed a strategy using solid phase peptide synthesis. Prior to the synthesis of analogs to ShN I, we assembled the native ShN I sequence and reoxidized the three intramolecular disulfide bonds. Chemical, physical, and pharmacological characterization of the purified synthetic ShN I showed it to be indistinguishable from the natural toxin.« less

Stimuli-sensitive hydrogels: ideal carriers for chronobiology and chronotherapy.

PubMed

Peppas, Nicholas A; Leobandung, William

2004-01-01

The development of solid-phase peptide synthesis in the early 1960s and recombinant DNA technology in the early 1970s boosted the scientific interest of utilizing proteins and peptides as potential therapeutic agents to battle poorly controlled diseases. While there has been rapid progress in the development and synthesis of new proteins and peptides as potential therapeutic agents, the formulation and development of the associated delivery systems is lacking. The development of delivery systems is equally important due to the problems of stability, low bioavailability and short half-life of proteins and peptides. The main problem in this field is that low stability leads to low bioavailability. In this review we draw attention to chrono-pharmacological drug-delivery systems, which can be used to match the delivery of therapeutic agents with the biological rhythm. They are very important especially in endocrinology and in vaccine therapy. We show that the treatment of hypopituitary dwarfism by administration of human growth-hormone-releasing hormone (GHRH) is more effective when GHRH is administered in a pulsatile manner that exhibits a period characteristic of the patient's circadian rhythm. Here we examine how to design novel chrono-pharmacological drug-delivery systems that should be able to release the therapeutic agents at predetermined intervals.

Design, synthesis and structure-activity relationship study of wollamide B; a new potential anti TB agent

PubMed Central

Laqua, Katja; Walkowska, Anna Maria; Cunningham, Fraser; Martinez-Martinez, Maria Santos; Cuevas-Zurita, Juan Carlos; Ballell-Pages, Lluís; Imming, Peter

2017-01-01

Wollamide B is a cationic antimycobacterial cyclohexapeptide that exhibits activity against Mycobacterium bovis (M. bovis) (IC50 of 3.1 μM). Aiming to define its structural activity relationship (SAR), optimizing potency and pharmacokinetic properties, libraries of analogues were synthesized following a standard Fmoc-based solid phase peptide synthesis approach. The antimycobacterial activities of wollamide B and all the synthesized analogues were tested against Mycobacterium tuberculosis (Mtb) H37Rv. Parallely, in vitro drug metabolism and pharmacokinetic (ADME) profiling was done for the synthesized compounds to evaluate their drug likeness. Among the 25 synthesized wollamides five of them showed potent activities with MICs ≤ 3.1 μM and found to be nontoxic against human HepG2 cells up to 100 μM. The results of the in vitro ADME profiling revealed the remarkable plasma stability and very good aqueous solubility of the class in general while the metabolic stability was found to be moderate to low. Of particular note, compounds 7c (MIC = 1.1 μM) and 13c (0.6 μM) that exhibited good balance of antimycobacterial activity vs. optimal pharmacokinetic properties could be used as a new lead for further development. PMID:28423019

A novel DNA/histone H4 peptide complex detects autoantibodies in systemic lupus erythematosus sera.

PubMed

Panza, Filomena; Alcaro, Maria Claudia; Petrelli, Fiorella; Angelotti, Francesca; Pratesi, Federico; Rovero, Paolo; Migliorini, Paola

2016-10-04

The detection of anti-dsDNA antibodies is critical for the diagnosis and follow-up of systemic lupus erythematosus (SLE) patients. The presently available assays are characterized by a non-optimal specificity (solid phase assays) or sensitivity (Crithidia Luciliae immunofluorescence test (CLIFT)). To overcome the limits of CLIFT and solid phase chromatin assays, we explored the diagnostic potential of an assay based on plasmid DNA containing a highly bent fragment of 211 bp from Crithidia Luciliae minicircles, complexed with histone peptides. Electrically neutral complexes of PK201/CAT plasmid (PK) DNA and histone 4 (H4) peptides were evaluated by electromobility shift assay. Complexes of H4 peptides and PK were absorbed to the solid phase to detect specific immunoglobulin G (IgG) in sera. Sera from 109 SLE patients, 100 normal healthy subjects, and 169 disease controls were tested. H4(14-34) containing the consensus sequence for DNA binding interacts with PK, retarding its migration. H4(14-34)/PK complexes were used to test sera by ELISA. Anti-H4-PK antibodies were detected in 56 % of SLE sera (more frequently in patients with skin or joint involvement) versus 5.9 % in disease controls; inhibition assays show that sera react with epitopes present on DNA or on the complex, not on the peptide. Antibody titer is correlated with European Consensus Lupus Activity Measurement (ECLAM) score and anti-complement component 1q (C1q) antibodies, negatively with C3 levels. Anti-H4-PK antibodies compared with CLIFT and solid phase dsDNA assays display moderate concordance. The H4/PK assay is a simple and reliable test which is useful for the differential diagnosis and evaluation of disease activity in SLE patients.

Peptide synthesis on glass substrate using acoustic droplet ejector.

PubMed

Youngki Choe; Shih-Jui Chen; Eun Sok Kim

2014-03-01

This paper describes the synthesis of a 9-mers-long peptide ladder structure of glycine on a modified glass surface using a nanoliter droplet ejector. To synthesize peptide on a glass substrate, SPOT peptide synthesis protocol was followed with a nozzleless acoustic droplet ejector being used to eject about 300 droplets of preactivated amino acid solution to dispense 60 nL of the solution per mer. The coupling efficiency of each mer was measured with FITC fluorescent tag to be 96%, resulting in net 70% efficiency for the whole 9-mer-long peptide of glycine. Usage of a nanoliter droplet ejector for SPOT peptide synthesis increases the density of protein array on a chip.

LiOH - H2O2 - H2O trinary system study for the selection of optimal conditions of lithium peroxide synthesis

NASA Astrophysics Data System (ADS)

Nefedov, R. A.; Ferapontov, Yu A.; Kozlova, N. P.

2016-01-01

Using solubility method the decay kinetics of peroxide products contained in liquid phase of LiOH - H2O2 - H2O trinary system with 2 to 6% by wt hydrogen peroxide content in liquid phase in 21 to 33 °C temperature range has been studied. Conducted studies have allowed to determine temperature and concentration limits of solid phase existence of Li2O2·H2O content, distinctness of which has been confirmed using chemical and qualitative X- ray phase analysis. Stabilizing effect of solid phase of Li2O2·H2O content on hydrogen peroxide decay contained in liquid phase of LiOH - H2O2 - H2O trinary system under conditions of experiments conducted has been shown.

PH dependent adhesive peptides

DOEpatents

Tomich, John; Iwamoto, Takeo; Shen, Xinchun; Sun, Xiuzhi Susan

2010-06-29

A novel peptide adhesive motif is described that requires no receptor or cross-links to achieve maximal adhesive strength. Several peptides with different degrees of adhesive strength have been designed and synthesized using solid phase chemistries. All peptides contain a common hydrophobic core sequence flanked by positively or negatively charged amino acids sequences.

Separation and purification of thymopentin with molecular imprinting membrane by solid phase extraction disks.

PubMed

Wang, Chaoli; Hu, Xiaoling; Guan, Ping; Wu, Danfeng; Qian, Liwei; Li, Ji; Song, Renyuan

2015-01-01

The synthesis and performance of molecularly imprinted membranes (MIMs) as a solid phase extraction packing materials for the separation and purification of thymopentin from crude samples was described. In order to increase structural selectivity and imprinting efficiency, surface-initiated ATRP and ionic liquid (1-vinyl-3-ethyl acetate imidazolium chloride) were used to prepare molecularly imprinting membranes. The results demonstrated that solid phase extraction disks stuffed by MIMs with ionic liquids as functional monomer demonstrated high isolation and purification of performance to the thymopentin. The molecular recognition of thymopentin was analyzed by using molecular modeling software. Copyright © 2014 Elsevier B.V. All rights reserved.

Hydrothermal synthesis of barium strontium titanate and bismuth titanate materials

NASA Astrophysics Data System (ADS)

Xu, Huiwen

Hydrothermal processing facilitates the synthesis of crystalline ceramic materials of varying composition or complex crystal structure. The present work can be divided into two parts. First is to study the low temperature hydrothermal synthesis of bismuth titanate. Second is to study both thermodynamic and kinetic aspects of the hydrothermally synthesized barium strontium titanate. A chelating agent was used to form a Bi-Ti gel precursor. By hydrothermally treating the Bi-Ti gel, crystalline bismuth titanate has been synthesized at 160°C for the first time. Microstructural evolution during the low temperature synthesis of bismuth titanate can be divided into two stages, including condensation of Bi-Ti gel particles and crystallization of bismuth titanate. Crystallization of bismuth titanate occurred by an in situ transformation mechanism at an early stage followed by a dissolution-reprecipitation mechanism. Phase separation was observed in hydrothermally synthesized barium strontium titanate (BST). By hydrothermally treating BST powders between 250°C--300°C, an asymmetrical miscibility gap was found in the BaTiO3-SrTiO 3 system at low temperatures (T ≤ 320°C). A subregular solid solution model was applied to calculate the equilibrium compositions and the Gibbs free energy of formation of BST solid solution at low temperatures (T ≤ 320°C). The Gibbs free energy of formation of Sr-rich BST phase is larger than that of Ba-rich BST phase. Kinetic studies of single phase BST solid solution at 80°C show that, compared to the BaTiO3 or Ba-rich BST, SrTiO3 and Sr-rich BST powders form at lower reaction rates.

New immobilisation protocol for the template used in solid-phase synthesis of MIP nanoparticles

NASA Astrophysics Data System (ADS)

Chen, Lu; Muhammad, Turghun; Yakup, Burabiye; Piletsky, Sergey A.

2017-06-01

As a novel imprinting method, solid-phase synthesis has proven to be a promising approach to prepare polymer nanoparticles with specific recognition sites for a template molecule. In this method, imprinted polymer nanoparticles were synthesized using template immobilized on a solid support. Herein, preparation of immobilized templates on quartz chips through homogeneous route was reported as an efficient alternative strategy to heterogeneous one. The template molecule indole-3-butyric acid (IBA) was reacted with 3-aminopropyltriethoxysilane (APTES) to produce silylated template (IBA-APTES), and it was characterized by IR, 1H NMR and GC-MS. Then, the silylated template molecule was grafted onto the activated surfaces of quartz chip to prepare immobilized template (SiO2@IBA-APTES). The immobilization was confirmed by contact angle, XPS, UV and fluorescence measurement. Immobilization protocol has shown good reproducibility and stability of the immobilized template. MIP nanoparticles were prepared with high selectivity toward the molecule immobilized onto the solid surface. This provides a new approach for the development of molecularly imprinted nanoparticles.

Hydrothermal Synthesis of Dicalcium Silicate Based Cement

NASA Astrophysics Data System (ADS)

Dutta, N.; Chatterjee, A.

2017-06-01

It is imperative to develop low energy alternative binders considering the large amounts of energy consumed as well as carbon dioxide emissions involved in the manufacturing of ordinary Portland cement. This study is on the synthesis of a dicalcium silicate based binder using a low temperature hydrothermal route.The process consists of synthesizing an intermediate product consisting of a calcium silicate hydrate phase with a Ca:Si ratio of 2:1 and further thermal treatment to produce the β-Ca2SiO4 (C2S) phase.Effect of various synthesis parameters like water to solid ratio, dwell time and temperature on the formation of the desired calcium silicate hydrate phase is reported along with effect of heating conditions for formation of the β-C2S phase. Around 77.45% of β-C2S phase was synthesized by thermal treatment of the intermediate phase at 820°C.

Biotin-transfer from a trifunctional crosslinker for identification of cell surface receptors of soluble protein ligands

PubMed Central

Tremblay, Tammy-Lynn; Hill, Jennifer J.

2017-01-01

Here we describe a novel crosslinker and its application as a biotin-transfer reagent to identify cell surface receptors of soluble protein ligands on live cells. This crosslinker contains three functional groups: an aldehyde-reactive aminooxy group, a sulfhydryl, and a biotin (ASB). It is readily synthesized via a 3-step addition reaction using standard solid-phase peptide synthesis methods and commercially available intermediates, allowing access to laboratories without specialized synthetic chemistry capabilities. For the biotin-transfer method, ASB is linked to a protein ligand through the sulfhydryl group in a two-step process that allows the introduction of a disulfide bond between the ligand and the crosslinker. Incubation of the labelled ligand with oxidized live cells leads to the formation of crosslinks with aldehyde-containing glycans on the cell surface receptor. Subsequent reduction of the disulfide bond results in biotin transfer from the ligand to the cell surface receptor. Protein biotinylation that is mediated by ligand binding to its receptor is differentiated from background biotinylation events by comparison with a similarly labelled control protein using comparative proteomic mass spectrometry to quantify streptavidin-bound proteins. Using this method, we successfully identified the cell surface receptors of a peptide hormone, a monoclonal antibody, and a single-domain antibody-Fc fusion construct. PMID:28422167

Induction of hepatic protein synthesis by a peptide in blood plasma of patients with sepsis and trauma.

PubMed

Loda, M; Clowes, G H; Dinarello, C A; George, B C; Lane, B; Richardson, W

1984-08-01

Accelerated release of amino acids from muscle and their uptake for protein synthesis by liver and other visceral tissues are characteristic of trauma or sepsis. Experimentally, this response is induced by interleukin-1 (IL-1) generated by activated macrophages in vitro. However, IL-1 has not been demonstrated in human blood. A small 4000-dalton peptide recently isolated from plasma of patients with sepsis and trauma induces muscle proteolysis and is called "proteolysis-inducing factor" (PIF). To test whether this agent has the ability also to induce hepatic protein synthesis, a series of animal experiments and clinical observations were undertaken. The structural and secretory (acute-phase reactants) in vitro protein synthesis in livers of normal rats injected intraperitoneally with IL-1 or PIF was significantly greater than that of normal rats or those injected with Ringer's lactate (p less than 0.01). In patients with sepsis and trauma the central plasma clearance rate of amino acids, a measure of visceral (principally hepatic) amino acid uptake, was elevated and correlated with the rates of protein synthesis in incubated liver slices obtained by biopsy at operation from the same patients (p less than 0.05). Both in vivo measured central plasma clearance rate of amino acids and in vitro measured hepatic protein synthesis correlated with plasma levels of PIF in the same patients (p less than 0.01 and p less than 0.05, respectively). We conclude that since PIF, and not IL-1, is present in human plasma and both are produced by activated macrophages, PIF seems to be the stable circulating cleavage product of IL-1, which induces not only muscle proteolysis but also hepatic protein synthesis, principally in the form of acute-phase reactants during infection and other states in which inflammation is present.

Solid-phase synthesis and chemical space analysis of a 190-membered alkaloid/terpenoid-like library

PubMed Central

Moura-Letts, Gustavo; DiBlasi, Christine M.; Bauer, Renato A.; Tan, Derek S.

2011-01-01

Alkaloid and terpenoid natural products display an extensive array of chemical frameworks and biological activities. However such scaffolds remain underrepresented in current screening collections and are, thus, attractive targets for the synthesis of natural product-based libraries that access underexploited regions of chemical space. Recently, we reported a systematic approach to the stereoselective synthesis of multiple alkaloid/terpenoid-like scaffolds using transition metal-mediated cycloaddition and cyclization reactions of enyne and diyne substrates assembled on a tert-butylsulfinamide lynchpin. We report herein the synthesis of a 190-membered library of alkaloid/terpenoid-like molecules using this synthetic approach. Translation to solid-phase synthesis was facilitated by the use of a tert-butyldiarylsilyl (TBDAS) linker that closely mimics the tert-butyldiphenysilyl protecting group used in the original solution-phase route development work. Unexpected differences in stereoselectivity and regioselectivity were observed in some reactions when carried out on solid support. Further, the sulfinamide moiety could be hydrolyzed or oxidized efficiently without compromising the TBDAS linker to provide additional amine and sulfonamide functionalities. Principal component analysis of the structural and physicochemical properties of these molecules confirmed that they access regions of chemical space that overlap with bona fide natural products and are distinct from areas addressed by conventional synthetic drugs and drug-like molecules. The influences of scaffolds and substituents were also evaluated, with both found to have significant impacts on location in chemical space and three-dimensional shape. Broad biological evaluation of this library will provide valuable insights into the abilities of natural product-based libraries to access similarly underexploited regions of biological space. PMID:21451137

Tidbits for the synthesis of bis(2-sulfanylethyl)amido (SEA) polystyrene resin, SEA peptides and peptide thioesters.

PubMed

Ollivier, Nathalie; Raibaut, Laurent; Blanpain, Annick; Desmet, Rémi; Dheur, Julien; Mhidia, Reda; Boll, Emmanuelle; Drobecq, Hervé; Pira, Silvain L; Melnyk, Oleg

2014-02-01

Protein total chemical synthesis enables the atom-by-atom control of the protein structure and therefore has a great potential for studying protein function. Native chemical ligation of C-terminal peptide thioesters with N-terminal cysteinyl peptides and related methodologies are central to the field of protein total synthesis. Consequently, methods enabling the facile synthesis of peptide thioesters using Fmoc-SPPS are of great value. Herein, we provide a detailed protocol for the preparation of bis(2-sulfanylethyl)amino polystyrene resin as a starting point for the synthesis of C-terminal bis(2-sulfanylethyl)amido peptides and of peptide thioesters derived from 3-mercaptopropionic acid. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.

Modification of the N-Terminus of a Calcium Carbonate Precipitating Peptide Affects Calcium Carbonate Mineralization.

PubMed

Usui, Kenji; Yokota, Shin-Ichiro; Ozaki, Makoto; Sakashita, Shungo; Imai, Takahito; Tomizaki, Kin-Ya

2018-01-01

A core sequence (the 9 C-terminal residues) of calcification-associated peptide (CAP- 1) isolated from the exoskeleton of the red swamp crayfish was previously shown to control calcium carbonate precipitation with chitin. In addition, a modified core sequence in which the phosphorylated serine at the N terminus is replaced with serine exhibits was also previously shown to alter precipitation characteristics with chitin. We focused on calcium carbonate precipitation and attempted to elucidate aspects of the mechanism underlying mineralization. We attempted to evaluate in detail the effects of modifying the N-terminus in the core sequence on calcium carbonate mineralization without chitin. The peptide modifications included phosphorylation, dephosphorylation, and a free or acetylated Nterminus. The peptides were synthesized manually on Wang resin using the DIPCI-DMAP method for the first residue, and Fmoc solid phase peptide synthesis with HBTU-HOBt for the subsequent residues. Prior to calcium carbonate precipitation, calcium carbonate was suspended in MilliQ water. Carbon dioxide gas was bubbled into the stirred suspension, then the remaining solid CaCO3 was removed by filtration. The concentration of calcium ions in the solution was determined by standard titration with ethylenediaminetetraacetate. Calcium carbonate precipitation was conducted in a micro tube for 3 h at 37°C. We used the micro-scale techniques AFM (atomic force microscopy) and TEM (transmission electron microscopy), and the macro-scale techniques chelate titration, HPLC, gel filtration, CD (circular dichroism) and DLS (dynamic light scattering). We determined the morphologies of the calcium carbonate deposits using AFM and TEM. The pS peptide provided the best control of the shape and size of the calcium carbonate round particles. The acetylated peptides (Ac-S and Ac-pS) provided bigger particles with various shapes. S peptide provided a mixture of bigger particles and amorphous particles. We verified these findings using DLS. All the peptide samples produced nanostructures of the expected size in agreement with the AFM and TEM results. We estimated the abilities of these peptides to precipitate calcium carbonate by determining the residual calcium hydrogen carbonate concentration by standard titration with ethylenediaminetetraacetate after calcium carbonate precipitation. The Ac-pS peptide showed the lowest residual calcium hydrogen carbonate concentration whereas the S peptide showed the highest, suggesting that the precipitating activities of these peptides towards calcium carbonate correlated with peptide net charge. Then the gel filtration results showed a large oligomer peak and a small oligomer/monomer peak for all peptide samples in agreement with the AFM, TEM and DLS results. CD measurements showed that all the peptides formed random-coil-like structures. Thus, we used both macro- and micro-observation techniques such as chelate titration, DLS, AFM and TEM to show that the calcium carbonate precipitating activities of four derivatives of the core sequence of CAP-1 may correlate with the peptide net charge. These peptides mainly act as a catalyst rather than as a binder or component of the calcium carbonate deposits (as a template). On the other hand, the morphologies of the calcium carbonate deposits appeared to be dependent on the ability of the peptide to assemble and act as a template. Consequently, elucidating the relationship between peptide sequence and the ability of the peptide to assemble would be indispensable for controlling precipitate morphologies in the near future. This knowledge would provide important clues for elucidating the relationship between peptide sequence and mineralization ability, including deposit morphology and precipitating activity, for use in nanobiochemistry and materials chemistry research. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Peptide o-aminoanilides as crypto-thioesters for protein chemical synthesis.

PubMed

Wang, Jia-Xing; Fang, Ge-Min; He, Yao; Qu, Da-Liang; Yu, Min; Hong, Zhang-Yong; Liu, Lei

2015-02-09

Fully unprotected peptide o-aminoanilides can be efficiently activated by NaNO2 in aqueous solution to furnish peptide thioesters for use in native chemical ligation. This finding enables the convergent synthesis of proteins from readily synthesizable peptide o-aminoanilides as a new type of crypto-thioesters. The practicality of this approach is shown by the synthesis of histone H2B from five peptide segments. Purification or solubilization tags, which are sometimes needed to improve the efficiency of protein chemical synthesis, can be incorporated into the o-aminoanilide moiety, as demonstrated in the preparation of the cyclic protein lactocyclicin Q. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

SAR by Oxime-Containing Peptide Libraries: Application to Tsg101 Ligand Optimization

PubMed Central

Liu, Fa; Stephen, Andrew G.; Waheed, Abdul A.; Aman, M. Javad; Freed, Eric O.; Fisher, Robert J.; Burke, Terrence R.

2008-01-01

HIV-1 viral assembly requires a direct interaction between a Pro-Thr-Ala-Pro (“PTAP”) motif in the viral protein Gag-p6 and the cellular endosomal sorting factor Tsg101. In an effort to develop competitive inhibitors of this interaction, an SAR study was conducted based on the application of post solid-phase oxime formation involving the sequential insertion of aminooxy-containing residues within a nonamer parent peptide followed by reaction with libraries of aldehydes. Approximately 15–20-fold enhancement in binding affinity was achieved by this approach. PMID:18655064

Distributed Drug Discovery: Advancing Chemical Education through Contextualized Combinatorial Solid-Phase Organic Laboratories

ERIC Educational Resources Information Center

Scott, William L.; Denton, Ryan E.; Marrs, Kathleen A.; Durrant, Jacob D.; Samaritoni, J. Geno; Abraham, Milata M.; Brown, Stephen P.; Carnahan, Jon M.; Fischer, Lindsey G.; Glos, Courtney E.; Sempsrott, Peter J.; O'Donnell, Martin J.

2015-01-01

The Distributed Drug Discovery (D3) program trains students in three drug discovery disciplines (synthesis, computational analysis, and biological screening) while addressing the important challenge of discovering drug leads for neglected diseases. This article focuses on implementation of the synthesis component in the second-semester…

Modified Ion-Conducting Ceramics Based on Lanthanum Gallate: Synthesis, Structure, and Properties

NASA Astrophysics Data System (ADS)

Kaleva, G. M.; Politova, E. D.; Mosunov, A. V.; Sadovskaya, N. V.

2018-06-01

A review is presented of the synthesis and complex investigation of modified ion-conducting ceramics based on heterosubstituted lanthanum gallate as a promising electrolyte material for solid oxide fuel cells. The effect the composition of multicomponent complex oxides has on the structure, microstructure, and electrophysical properties of ceramics is examined. Samples of ceramics with new compositions are produced via solid-state synthesis and modified with lithium fluoride. A drop is observed in the sintering temperature of the ceramics, caused by the liquid phase mechanism of sintering as a result of the low-melting superstoichiometric quantities of the additive. The effect lithium fluoride has on the process of phase formation, microstructure, and conductivity of the ceramics is investigated. It is found that samples modified with lithium fluoride display high density, dense grain packing, and high values of electrical conductivity at high temperatures.

Surface-modified multifunctional MIP nanoparticles

NASA Astrophysics Data System (ADS)

Moczko, Ewa; Poma, Alessandro; Guerreiro, Antonio; Perez de Vargas Sansalvador, Isabel; Caygill, Sarah; Canfarotta, Francesco; Whitcombe, Michael J.; Piletsky, Sergey

2013-04-01

The synthesis of core-shell molecularly imprinted polymer nanoparticles (MIP NPs) has been performed using a novel solid-phase approach on immobilised templates. The same solid phase also acts as a protective functionality for high affinity binding sites during subsequent derivatisation/shell formation. This procedure allows for the rapid synthesis, controlled separation and purification of high-affinity materials, with each production cycle taking just 2 hours. The aim of this approach is to synthesise uniformly sized imprinted materials at the nanoscale which can be readily grafted with various polymers without affecting their affinity and specificity. For demonstration purposes we grafted anti-melamine MIP NPs with coatings which introduce the following surface characteristics: high polarity (PEG methacrylate); electro-activity (vinylferrocene); fluorescence (eosin acrylate); thiol groups (pentaerythritol tetrakis(3-mercaptopropionate)). The method has broad applicability and can be used to produce multifunctional imprinted nanoparticles with potential for further application in the biosensors, diagnostics and biomedical fields and as an alternative to natural receptors.The synthesis of core-shell molecularly imprinted polymer nanoparticles (MIP NPs) has been performed using a novel solid-phase approach on immobilised templates. The same solid phase also acts as a protective functionality for high affinity binding sites during subsequent derivatisation/shell formation. This procedure allows for the rapid synthesis, controlled separation and purification of high-affinity materials, with each production cycle taking just 2 hours. The aim of this approach is to synthesise uniformly sized imprinted materials at the nanoscale which can be readily grafted with various polymers without affecting their affinity and specificity. For demonstration purposes we grafted anti-melamine MIP NPs with coatings which introduce the following surface characteristics: high polarity (PEG methacrylate); electro-activity (vinylferrocene); fluorescence (eosin acrylate); thiol groups (pentaerythritol tetrakis(3-mercaptopropionate)). The method has broad applicability and can be used to produce multifunctional imprinted nanoparticles with potential for further application in the biosensors, diagnostics and biomedical fields and as an alternative to natural receptors. Electronic supplementary information (ESI) available: Details of the synthesis of eosin O-acrylate monomer and 1H-NMR spectrum of MIP NPs post-derivatised with PEG shell. See DOI: 10.1039/c3nr00354j

Radiohybridization PET imaging of KRAS G12D mRNA expression in human pancreas cancer xenografts with [(64)Cu]DO3A-peptide nucleic acid-peptide nanoparticles.

PubMed

Chakrabarti, A; Zhang, K; Aruva, M R; Cardi, C A; Opitz, A W; Wagner, N J; Thakur, M L; Wickstrom, E

2007-06-01

There is a compelling need to image pancreas cancer at an early stage. Human pancreas cancer cells display elevated levels of KRAS protein due to high copy numbers of KRAS mRNA, and elevated levels of insulin-like growth factor 1 receptor (IGF1R) due to overexpression of IGF1R mRNA. Therefore we hypothesized that pancreas cancer could be detected in vivo with a single probe that targets both KRAS mRNA and IGF1R. Because positron emission tomography (PET) is a sensitive imaging technique, we designed a probe incorporating the positron-emitting nuclide (64)Cu. The KRAS-specific hybridization probe consisted of 1,4,7-tris(carboxymethylaza)cyclododecane-10-aza-acetyl (DO3A) on the N-terminus of a peptide nucleic acid (PNA) hybridization sequence (GCCATCAGCTCC) linked to a cyclized IGF1 peptide analog (d-Cys-Ser-Lys-Cys) on the C-terminus, for IGF1R-mediated endocytosis. A series of such KRAS radiohybridization probes with 0, 1, 2 or 3 mismatches to KRAS G12D mRNA, including exact matches to wild type KRAS mRNA and KRAS G12V mRNA, along with a double d(Ala) replacement IGF1 peptide control, were assembled by continuous solid phase synthesis. To test the hypothesis that KRAS-IGF1 dual probes could specifically image KRAS mRNA expression noninvasively in human IGF1R-overexpressing AsPC1 pancreas cancer xenografts in immunocompromised mice, [(64)Cu]PNA radiohybridization probes and controls were administered by tail vein. The [(64)Cu]KRAS-IGF1 radiohybridization probe yielded strong tumor contrast in PET images, 8.6 +/- 1.4-fold more intense in the center of human pancreas cancer xenografts than in the contralateral muscle at 4 h post-injection. Control experiments with single base KRASmismatches, an IGF1 peptide mismatch, and a breast cancer xenograft lacking KRAS activation yielded weak tumor contrast images. These experiments are consistent with our hypothesis for noninvasive PET imaging of KRAS oncogene expression in pancreas cancer xenografts. Imaging oncogene mRNAs with radiolabel-PNA-peptide nanoparticles might provide specific genetic characterization of preinvasive and invasive pancreas cancers for staging and choice of therapy.

Laminin peptide YIGSR induces collagen synthesis in Hs27 human dermal fibroblasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yoon, Jong Hyuk; Kim, Jaeyoon; Lee, Hyeongjoo

Highlights: Black-Right-Pointing-Pointer We identify a function of the YIGSR peptide to enhance collagen synthesis in Hs27. Black-Right-Pointing-Pointer YIGSR peptide enhanced collagen type 1 synthesis both of gene and protein levels. Black-Right-Pointing-Pointer There were no changes in cell proliferation and MMP-1 level in YIGSR treatment. Black-Right-Pointing-Pointer The YIGSR effect on collagen synthesis mediated activation of FAK, pyk2 and ERK. Black-Right-Pointing-Pointer The YIGSR-induced FAK and ERK activation was modulated by FAK and MEK inhibitors. -- Abstract: The dermal ECM is synthesized from fibroblasts and is primarily compromised of fibrillar collagen and elastic fibers, which support the mechanical strength and resiliency of skin,more » respectively. Laminin, a major glycoprotein located in the basement membrane, promotes cell adhesion, cell growth, differentiation, and migration. The laminin tyrosine-isoleucine-glycine-serine-arginine (YIGSR) peptide, corresponding to the 929-933 sequence of the {beta}1 chain, is known to be a functional motif with effects on the inhibition of tumor metastasis, the regulation of sensory axonal response and the inhibition of angiogenesis through high affinity to the 67 kDa laminin receptor. In this study, we identified a novel function of the YIGSR peptide to enhance collagen synthesis in human dermal fibroblasts. To elucidate this novel function regarding collagen synthesis, we treated human dermal fibroblasts with YIGSR peptide in both a time- and dose-dependent manner. According to subsequent experiments, we found that the YIGSR peptide strongly enhanced collagen type 1 synthesis without changing cell proliferation or cellular MMP-1 level. This YIGSR peptide-mediated collagen type 1 synthesis was modulated by FAK inhibitor and MEK inhibitor. This study clearly reveals that YIGSR peptide plays a novel function on the collagen type 1 synthesis of dermal fibroblasts and also suggests that YIGSR is a strong candidate peptide for the treatment of skin aging and wrinkles.« less

A High-Throughput Process for the Solid-Phase Purification of Synthetic DNA Sequences

PubMed Central

Grajkowski, Andrzej; Cieślak, Jacek; Beaucage, Serge L.

2017-01-01

An efficient process for the purification of synthetic phosphorothioate and native DNA sequences is presented. The process is based on the use of an aminopropylated silica gel support functionalized with aminooxyalkyl functions to enable capture of DNA sequences through an oximation reaction with the keto function of a linker conjugated to the 5′-terminus of DNA sequences. Deoxyribonucleoside phosphoramidites carrying this linker, as a 5′-hydroxyl protecting group, have been synthesized for incorporation into DNA sequences during the last coupling step of a standard solid-phase synthesis protocol executed on a controlled pore glass (CPG) support. Solid-phase capture of the nucleobase- and phosphate-deprotected DNA sequences released from the CPG support is demonstrated to proceed near quantitatively. Shorter than full-length DNA sequences are first washed away from the capture support; the solid-phase purified DNA sequences are then released from this support upon reaction with tetra-n-butylammonium fluoride in dry dimethylsulfoxide (DMSO) and precipitated in tetrahydrofuran (THF). The purity of solid-phase-purified DNA sequences exceeds 98%. The simulated high-throughput and scalability features of the solid-phase purification process are demonstrated without sacrificing purity of the DNA sequences. PMID:28628204

Purification and antibacterial activity of recombinant warnericin RK expressed in Escherichia coli.

PubMed

Verdon, Julien; Girardin, Nicolas; Marchand, Adrienne; Héchard, Yann; Berjeaud, Jean-Marc

2013-06-01

Warnericin RK is a small cationic peptide produced by Staphylococcus warneri RK. This peptide has an antimicrobial spectrum of activity almost restricted to the Legionella genus. It is a membrane-active peptide with a proposed detergent-like mechanism of action at high concentration. Moreover, the fatty acids content of Legionella was shown to modulate the peptide activity. In order to decipher the mode of action in details using solid-state NMR spectroscopy, large amount of an isotopic labeled peptide is required. Since it is less expensive to obtain such a peptide biologically, we report here methods to express warnericin RK in Escherichia coli with or without a fusion partner and to purify resulting recombinant peptides. The cDNA fragment encoding warnericin RK was synthesized and ligated into three expression vectors. Two fusion peptides, carrying polyhistidine tag in N- or C-terminal and a native peptide, without tag, were expressed in E. coli cells. Fusion peptides were purified, with a yield of 3 mg/l, by affinity chromatography and reverse-phase HPLC. The recombinant native peptide was purified using a two-step purification method consisting of a hydrophobic chromatography followed by a reverse-phase HPLC step with a yield of 1.4 mg/l. However, the anti-Legionella activity was lower for both tagged peptide probably because of structural modifications. So, the native recombinant peptide was preferentially chosen for (15)N-labeling experiments. Our results suggest that the developed production and purification procedures will be useful in obtaining a large quantity of recombinant isotope-labeled warnericin RK for further studies.

Novel fluorescent contrast agents for optical imaging of in vivo tumors based on a receptor-targeted dye-peptide conjugate platform

NASA Astrophysics Data System (ADS)

Bugaj, Joseph E.; Achilefu, Samuel I.; Dorshow, Richard B.; Rajagopalan, Raghavan

2001-04-01

We have designed, synthesized, and evaluated the efficacy of novel dye-peptide conjugates that are receptor specific. Contrary to the traditional approach of conjugating dyes to large proteins and antibodies, we used small peptide-dye conjugates that target over-expressed receptors on tumors. Despite the fact that the peptide and the dye probe have similar molecular mass, our results demonstrate that the affinity of the peptide for its receptor and the dye fluorescence properties are both retained. The use of small peptides has several advantages over large biomolecules, including ease of synthesis of a variety of compounds for potential combinatorial screening of new targets, reproducibility of high purity compounds, diffusiveness to solid tumors, and the ability to incorporate a variety of functional groups that modify the pharmacokinetics of the peptide-dye conjugates. The efficacy of these new fluorescent optical contrast agents was evaluated in vivo in well-characterized rat tumor lines expressing somatostatin (sst2) and bombesin receptors. A simple continuous wave optical imaging system was employed. The resulting optical images clearly show that successful specific tumor targeting was achieved. Thus, we have demonstrated that small peptide- dye conjugates are effective as contrast agents for optical imaging of tumors.

One-Bead-Two-Compound Thioether Bridged Macrocyclic γ-AApeptide Screening Library against EphA2.

PubMed

Shi, Yan; Challa, Sridevi; Sang, Peng; She, Fengyu; Li, Chunpu; Gray, Geoffrey M; Nimmagadda, Alekhya; Teng, Peng; Odom, Timothy; Wang, Yan; van der Vaart, Arjan; Li, Qi; Cai, Jianfeng

2017-11-22

Identification of molecular ligands that recognize peptides or proteins is significant but poses a fundamental challenge in chemical biology and biomedical sciences. Development of cyclic peptidomimetic library is scarce, and thus discovery of cyclic peptidomimetic ligands for protein targets is rare. Herein we report the unprecedented one-bead-two-compound (OBTC) combinatorial library based on a novel class of the macrocyclic peptidomimetics γ-AApeptides. In the library, we utilized the coding peptide tags synthesized with Dde-protected α-amino acids, which were orthogonal to solid phase synthesis of γ-AApeptides. Employing the thioether linkage, the desired macrocyclic γ-AApeptides were found to be effective for ligand identification. Screening the library against the receptor tyrosine kinase EphA2 led to the discovery of one lead compound that tightly bound to EphA2 (K d = 81 nM) and potently antagonized EphA2-mediated signaling. This new approach of macrocyclic peptidomimetic library may lead to a novel platform for biomacromolecular surface recognition and function modulation.

Pre-clinical evaluation of eight DOTA coupled gastrin-releasing peptide receptor (GRP-R) ligands for in vivo targeting of receptor-expressing tumors.

PubMed

Accardo, Antonella; Galli, Filippo; Mansi, Rosalba; Del Pozzo, Luigi; Aurilio, Michela; Morisco, Anna; Ringhieri, Paola; Signore, Alberto; Morelli, Giancarlo; Aloj, Luigi

2016-12-01

Overexpression of the gastrin-releasing peptide receptor (GRP-R) has been documented in several human neoplasms such as breast, prostate, and ovarian cancer. There is growing interest in developing radiolabeled peptide-based ligands toward these receptors for the purpose of in vivo imaging and radionuclide therapy of GRP-R-overexpressing tumors. A number of different peptide sequences, isotopes, and labeling methods have been proposed for this purpose. The aim of this work is to perform a direct side-by-side comparison of different GRP-R binding peptides utilizing a single labeling strategy to identify the most suitable peptide sequence. Solid-phase synthesis of eight derivatives (BN1-8) designed based on literature analysis was carried out. Peptides were coupled to the DOTA chelator through a PEG4 spacer at the N-terminus. Derivatives were characterized for serum stability, binding affinity on PC-3 human prostate cancer cells, biodistribution in tumor-bearing mice, and gamma camera imaging at 1, 6, and 24 h after injection. Serum stability was quite variable among the different compounds with half-lives ranging from 16 to 400 min at 37 °C. All compounds tested showed K d values in the nanomolar range with the exception of BN3 that showed no binding. Biodistribution and imaging studies carried out for compounds BN1, BN4, BN7, and BN8 showed targeting of the GRP-R-positive tumors and the pancreas. The BN8 compound (DOTA-PEG-DPhe-Gln-Trp-Ala-Val-NMeGly-His-Sta-Leu-NH2) showed high affinity, the longest serum stability, and the highest target-to-background ratios in biodistribution and imaging experiments among the compounds tested. Our results indicate that the NMeGly for Gly substitution and the Sta-Leu substitution at the C-terminus confer high serum stability while maintaining high receptor affinity, resulting in biodistribution properties that outperform those of the other peptides.

Biological and surface-active properties of double-chain cationic amino acid-based surfactants.

PubMed

Greber, Katarzyna E; Dawgul, Małgorzata; Kamysz, Wojciech; Sawicki, Wiesław; Łukasiak, Jerzy

2014-08-01

Cationic amino acid-based surfactants were synthesized via solid phase peptide synthesis and terminal acylation of their α and ε positions with saturated fatty acids. Five new lipopeptides, N-α-acyl-N-ε-acyl lysine analogues, were obtained. Minimum inhibitory concentration and minimum bactericidal (fungicidal) concentration were determined on reference strains of bacteria and fungi to evaluate the antimicrobial activity of the lipopeptides. Toxicity to eukaryotic cells was examined via determination of the haemolytic activities. The surface-active properties of these compounds were evaluated by measuring the surface tension and formation of micelles as a function of concentration in aqueous solution. The cationic surfactants demonstrated diverse antibacterial activities dependent on the length of the fatty acid chain. Gram-negative bacteria and fungi showed a higher resistance than Gram-positive bacterial strains. It was found that the haemolytic activities were also chain length-dependent values. The surface-active properties showed a linear correlation between the alkyl chain length and the critical micelle concentration.

A study of room-temperature LixMn1.5Ni0.5O4 solid solutions

NASA Astrophysics Data System (ADS)

Saravanan, Kuppan; Jarry, Angelique; Kostecki, Robert; Chen, Guoying

2015-01-01

Understanding the kinetic implication of solid-solution vs. biphasic reaction pathways is critical for the development of advanced intercalation electrode materials. Yet this has been a long-standing challenge in materials science due to the elusive metastable nature of solid solution phases. The present study reports the synthesis, isolation, and characterization of room-temperature LixMn1.5Ni0.5O4 solid solutions. In situ XRD studies performed on pristine and chemically-delithiated, micron-sized single crystals reveal the thermal behavior of LixMn1.5Ni0.5O4 (0 <= x <= 1) cathode material consisting of three cubic phases: LiMn1.5Ni0.5O4 (Phase I), Li0.5Mn1.5Ni0.5O4 (Phase II) and Mn1.5Ni0.5O4 (Phase III). A phase diagram capturing the structural changes as functions of both temperature and Li content was established. The work not only demonstrates the possibility of synthesizing alternative electrode materials that are metastable in nature, but also enables in-depth evaluation on the physical, electrochemical and kinetic properties of transient intermediate phases and their role in battery electrode performance.

A study of room-temperature LixMn1.5Ni0.5O4 solid solutions

PubMed Central

Saravanan, Kuppan; Jarry, Angelique; Kostecki, Robert; Chen, Guoying

2015-01-01

Understanding the kinetic implication of solid-solution vs. biphasic reaction pathways is critical for the development of advanced intercalation electrode materials. Yet this has been a long-standing challenge in materials science due to the elusive metastable nature of solid solution phases. The present study reports the synthesis, isolation, and characterization of room-temperature LixMn1.5Ni0.5O4 solid solutions. In situ XRD studies performed on pristine and chemically-delithiated, micron-sized single crystals reveal the thermal behavior of LixMn1.5Ni0.5O4 (0 ≤ x ≤ 1) cathode material consisting of three cubic phases: LiMn1.5Ni0.5O4 (Phase I), Li0.5Mn1.5Ni0.5O4 (Phase II) and Mn1.5Ni0.5O4 (Phase III). A phase diagram capturing the structural changes as functions of both temperature and Li content was established. The work not only demonstrates the possibility of synthesizing alternative electrode materials that are metastable in nature, but also enables in-depth evaluation on the physical, electrochemical and kinetic properties of transient intermediate phases and their role in battery electrode performance. PMID:25619504

Acyl guanidine inhibitors of β-secretase (BACE-1): optimization of a micromolar hit to a nanomolar lead via iterative solid- and solution-phase library synthesis.

PubMed

Gerritz, Samuel W; Zhai, Weixu; Shi, Shuhao; Zhu, Shirong; Toyn, Jeremy H; Meredith, Jere E; Iben, Lawrence G; Burton, Catherine R; Albright, Charles F; Good, Andrew C; Tebben, Andrew J; Muckelbauer, Jodi K; Camac, Daniel M; Metzler, William; Cook, Lynda S; Padmanabha, Ramesh; Lentz, Kimberley A; Sofia, Michael J; Poss, Michael A; Macor, John E; Thompson, Lorin A

2012-11-08

This report describes the discovery and optimization of a BACE-1 inhibitor series containing an unusual acyl guanidine chemotype that was originally synthesized as part of a 6041-membered solid-phase library. The synthesis of multiple follow-up solid- and solution-phase libraries facilitated the optimization of the original micromolar hit into a single-digit nanomolar BACE-1 inhibitor in both radioligand binding and cell-based functional assay formats. The X-ray structure of representative inhibitors bound to BACE-1 revealed a number of key ligand:protein interactions, including a hydrogen bond between the side chain amide of flap residue Gln73 and the acyl guanidine carbonyl group, and a cation-π interaction between Arg235 and the isothiazole 4-methoxyphenyl substituent. Following subcutaneous administration in rats, an acyl guanidine inhibitor with single-digit nanomolar activity in cells afforded good plasma exposures and a dose-dependent reduction in plasma Aβ levels, but poor brain exposure was observed (likely due to Pgp-mediated efflux), and significant reductions in brain Aβ levels were not obtained.

Synthesis of oligonucleotides on a soluble support

PubMed Central

2017-01-01

Oligonucleotides are usually prepared in lab scale on a solid support with the aid of a fully automated synthesizer. Scaling up of the equipment has allowed industrial synthesis up to kilogram scale. In spite of this, solution-phase synthesis has received continuous interest, on one hand as a technique that could enable synthesis of even larger amounts and, on the other hand, as a gram scale laboratory synthesis without any special equipment. The synthesis on a soluble support has been regarded as an approach that could combine the advantageous features of both the solution and solid-phase syntheses. The critical step of this approach is the separation of the support-anchored oligonucleotide chain from the monomeric building block and other small molecular reagents and byproducts after each coupling, oxidation and deprotection step. The techniques applied so far include precipitation, extraction, chromatography and nanofiltration. As regards coupling, all conventional chemistries, viz. phosphoramidite, H-phosphonate and phosphotriester strategies, have been attempted. While P(III)-based phosphoramidite and H-phosphonate chemistries are almost exclusively used on a solid support, the “outdated” P(V)-based phosphotriester chemistry still offers one major advantage for the synthesis on a soluble support; the omission of the oxidation step simplifies the coupling cycle. Several of protocols developed for the soluble-supported synthesis allow the preparation of both DNA and RNA oligomers of limited length in gram scale without any special equipment, being evidently of interest for research groups that need oligonucleotides in large amounts for research purposes. However, none of them has really tested at such a scale that the feasibility of their industrial use could be critically judged. PMID:28781703

A simple solid phase, peptide-based fluorescent assay for the efficient and universal screening of HRV 3C protease inhibitors.

PubMed

Schünemann, Katrin; Connelly, Stephen; Kowalczyk, Renata; Sperry, Jonathan; Wilson, Ian A; Fraser, John D; Brimble, Margaret A

2012-08-01

With over a 100 different serotypes, the human rhinovirus (HRV) is the major aetiological agent for the common cold, for which only symptomatic treatment is available. HRV maturation and replication is entirely dependent on the activity of a virally encoded 3C protease that represents an attractive target for the development of therapeutics to treat the common cold. Although a variety of small molecules and peptidomimetics have been found to inhibit HRV 3C protease, no universally compatible assay exists to reliably quantify the activity of the enzyme in vitro. Herein we report the development of a universal and robust solid phase peptide assay that utilizes the full HRV-14 3C protease recognition sequence and the release of 5(6)-carboxyfluorescein to sensitively quantify protease activity. This novel assay overcomes several limitations of existing assays allowing for the simple and efficient analysis of HRV-14 3C protease activity facilitating both high-throughput screening and the accurate kinetic study of HRV-14 3C protease inhibitors. Copyright © 2012 Elsevier Ltd. All rights reserved.

Fluorous Parallel Synthesis of A Hydantoin/Thiohydantoin Library

PubMed Central

Lu, Yimin; Zhang, Wei

2007-01-01

Fluorous tagging strategy is applied to solution-phase parallel synthesis of a library containing hydantoin and thiohydantoin analogs. Two perfluoroalkyl (Rf)-tagged α-amino esters each react with 6 aromatic aldehydes under reductive amination conditions. Twelve amino esters then each react with 10 isocyanates and isothiocyanates in parallel. The resulting 120 ureas and thioureas undergo spontaneous cyclization to form the corresponding hydantoins and thiohydantoins. The intermediate and final product purifications are performed with solid-phase extraction (SPE) over FluoroFlash™ cartridges, no chromatography is required. Using standard instruments and straightforward SPE technique, one chemist accomplished the 120-member library synthesis in less than 5 working days, including starting material synthesis and product analysis. PMID:15789556

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, S.-S.; IGE Therapeutics, Inc., Cellular and Cancer Immunology, 6370 Lusk Boulevard, F109, San Diego, CA 92121; Yang Yongmin

GFP-C{kappa} fusion protein was previously shown selectable on ribosome display platform with solid phase antibodies against GFP determinant [Y.-M. Yang, T.J. Barankiewicz, M. He, M. Taussig, S.-S. Chen, Selection of antigenic markers on a GFP-C{kappa} fusion scaffold with high sensitivity by eukaryotic ribosome display, Biochem. Biophys. Res. Commun. 359 (2007) 251-257]. Herein, we show that members of aptameric peptide library constructed within the site 6 and site 8/9 loops of GFP of the ribosome display construct are selectable upon binding to the solid phase IgE antigen. An input of 1.0 {mu}g of the dual site aptameric GFP library exhibiting amore » diversity of 7.5 x 10{sup 11} was transcribed, translated and incubated with solid phase IgE. RT-PCR products were amplified from mRNA of the aptamer-ribosome-mRNA (ARM) complex captured on the solid phase IgE. Clones of aptameric GFP were prepared from RT-PCR product of ARM complex following repetitive selection. Recombinant aptameric GFP proteins from the selected clones bind IgE coated on the 96-well plate, and the binding was abrogated by incubation with soluble human IgE but not human IgG. Selected aptameric GFP proteins also exhibit binding to three different sources of human IgE (IgE PS, BED, and JW8) but not irrelevant proteins. These observations indicate that appropriately selected aptameric GFP on a solid phase ligand by ribosome display may serve as an affinity reagent for blocking reactivity of a biological ligand.« less

Detergent enhances binding of a secreted HLA-A2 molecule to solid phase peptides.

PubMed

Tussey, L G; Frelinger, J A

1991-11-01

We have constructed a secreted analogue (sA2) of the human class I molecule HLA-A2. sA2 was affinity purified both in the presence and absence of detergent and the effects of detergent on the magnitude and specificity of A2 binding to solid phase peptides tested. sA2 purified in the presence of detergent and detergent-solubilized A2 are shown to function comparably in the binding of the synthetic peptide M.Y + 57-68, a known T-cell epitope derived from the influenza A matrix protein. The molecules binding to M.Y + 57-68 typically represent 8% to 10% of the added protein. In contrast, less than 1% of sA2 protein purified in the absence of detergent binds M.Y + 57-68. This reduced binding is not due to a change in the affinity of sA2 for M.Y + 57-68. Addition of detergent at various stages of the purification and iodination procedures indicates that the longer the sA2 molecules are exposed to detergent the better they bind. However, the concentration of detergent during the actual binding assay does not appear to be critical. We also find that while the sA2-detergent and the sA2-no detergent molecules differ in the extent to which they bind various peptides, they do not differ in their patterns of binding. We conclude that detergent probably does not influence the specificity of class I/peptide binding but does increase the number of sA2 molecules that can participate in the binding of peptide either by generating and stabilizing "empty" sA2 molecules or by stabilizing a structure that is more amenable to binding peptide.

Design of Hybrid Solid Polymer Electrolytes: Structure and Properties

NASA Technical Reports Server (NTRS)

Bronstein, Lyudmila M.; Karlinsey, Robert L.; Ritter, Kyle; Joo, Chan Gyu; Stein, Barry; Zwanziger, Josef W.

2003-01-01

This paper reports synthesis, structure, and properties of novel hybrid solid polymer electrolytes (SPE's) consisting of organically modified aluminosilica (OM-ALSi), formed within a poly(ethylene oxide)-in-salt (Li triflate) phase. To alter the structure and properties we fused functionalized silanes containing poly(ethylene oxide) (PEO) tails or CN groups.

Solid-state reaction synthesis for mixed-phase Eu3+-doped bismuth molybdate and its luminescence properties

NASA Astrophysics Data System (ADS)

Liang, Danyang; Ding, Yu; Wang, Nan; Cai, Xiaomeng; Li, Jia; Han, Linyu; Wang, Shiqi; Han, Yuanyuan; Jia, Guang; Wang, Liyong

2017-09-01

A method for mixed-phase bismuth molybdate doped with Eu3+ ions was developed by solid-state reaction assisting with polyvinyl alcohol (PVA). The results of powder X-ray diffraction showed a mixed-phase structure and the microscopical characterization technology revealed the formation process with the addition of PVA. As a structure inducer, the PVA molecules played a vital role in the formation of phase structure. The as-obtained Eu3+-doped bismuth molybdates were also characterized by using different spectroscopic techniques including FTIR and photoluminescence (PL). The results show that doping concentration, PVA addition and calcination temperature affect photoluminescence properties remarkably.

Synthesis of high quality phosphorothioate oligonucleotides as antisense drugs. Use of I-linker in the elimination of 3'-terminal phosphorothioate monoesters.

PubMed

Ravikumar, Vasulinga T; Kumar, R Krishna; Capaldi, Daniel C; Cole, Douglas L

2003-01-01

Detritylation of a 5'-O-DMT-2'-deoxyadenosine moiety attached to solid support under acidic condition leads to depurination during oligonucleotide synthesis. Deprotection followed by reversed phase HPLC purification leads to desired oligonucleotide contaminated with significant levels of 3'-terminal phosphorothiaote (3'-TPT) monoester (n-1)-mer. However, it is demonstrated that attachment of dA nucleoside through its exocyclic amino group to solid support leads to substantial reduction of 3'-TPT formation thereby improving the quality of oligonucleotide synthesized.

Hydrocarbon synthesis catalyst and method of preparation

DOEpatents

Sapienza, Richard S.; Sansone, Michael J.; Slegeir, William A. R.

1983-08-02

A catalyst for the synthesis of hydrocarbons from carbon monoxide and hydrogen composed of palladium or platinum and cobalt supported on a solid phase is disclosed. The catalyst is prepared by heating a heterogeneous component of the palladium or platinum deposited on the solid support in a solution of cobalt carbonyl or precursors thereof. The catalyst exhibits excellent activity, stability in air, and produces highly desirable product fractions even with dilute gaseous reactants. The catalyst is preferably used in dilute slurry form, which is desirable from a heat transfer standpoint.

Production and Characterization of a New α-Glucosidase Inhibitory Peptide from Aspergillus oryzae N159-1

PubMed Central

Kang, Min-Gu; Yi, Sung-Hun

2013-01-01

An α-glucosidase inhibitor was developed from Aspergillus oryzae N159-1, which was screened from traditional fermented Korean foods. The intracellular concentration of the inhibitor reached its highest level when the fungus was cultured in tryptic soy broth medium at 27℃ for five days. The inhibitor was purified using a series of purification steps involving ultrafiltration, Sephadex G-25 gel permeation chromatography, strong cation exchange solid phase extraction, reverse-phase high performance liquid chromatography, and size exclusion chromatography. The final yield of the purification was 1.9%. Results of the liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis indicated that the purified α-glucosidase inhibitor was a tri-peptide, Pro-Phe-Pro, with the molecular weight of 360.1 Da. The IC50 value of the peptide against α-glucosidase activity was 3.1 mg/mL. Using Lineweaver-Burk plot analysis, the inhibition pattern indicated that the inhibitor acts as a mixed type inhibitor. PMID:24198670

Hydrocarbon synthesis catalyst and method of preparation

DOEpatents

Sapienza, R.S.; Sansone, M.J.; Slegeir, W.A.R.

1983-08-02

A catalyst for the synthesis of hydrocarbons from carbon monoxide and hydrogen composed of palladium or platinum and cobalt supported on a solid phase is disclosed. The catalyst is prepared by heating a heterogeneous component of the palladium or platinum deposited on the solid support in a solution of cobalt carbonyl or precursors thereof. The catalyst exhibits excellent activity, stability in air, and produces highly desirable product fractions even with dilute gaseous reactants. The catalyst is preferably used in dilute slurry form, which is desirable from a heat transfer standpoint. 9 figs.

Difference between beta1-adrenoceptor autoantibodies of human and animal origin-Limitations detecting beta1-adrenoceptor autoantibodies using peptide based ELISA technology.

PubMed

Wenzel, Katrin; Schulze-Rothe, Sarah; Müller, Johannes; Wallukat, Gerd; Haberland, Annekathrin

2018-01-01

Cell-based analytics for the detection of the beta1-adrenoceptor autoantibody (beta1-AAB) are functional, yet difficult to handle, and should be replaced by easily applicable, routine lab methods. Endeavors to develop solid-phase-based assays such as ELISA to exploit epitope moieties for trapping autoantibodies are ongoing. These solid-phase-based assays, however, are often unreliable when used with human patient material, in contrast to animal derived autoantibodies. We therefore tested an immunogen peptide-based ELISA for the detection of beta1-AAB, and compared commercially available goat antibodies against the 2nd extracellular loop of human beta1-adrenoceptor (ADRB1-AB) to autoantibodies enriched from patient material. The functionality of these autoantibodies was tested in a cell based assay for comparison and their structural appearance was investigated using 2D gel electrophoresis. The ELISA showed a limit of detection for ADRB1-AB of about 1.5 nmol antibody/L when spiked in human control serum and only about 25 nmol/L when spiked in species identical (goat) matrix material. When applied to samples of human origin, the ELISA failed to identify the specific beta1-AABs. A low concentration of beta1-AAB, together with structural inconsistency of the patient originated samples as seen from the 2D Gel appearance, might contribute to the failure of the peptide based ELISA technology to detect human beta1-AABs.

Preparation and characterization of mechanically alloyed AB3-type based material LaMg2Ni5Al4 and its solid-gaz hydrogen storage reaction

NASA Astrophysics Data System (ADS)

Jaafar, Hassen; Aymard, Luc; Dachraoui, Walid; Demortière, Arnaud; Abdellaoui, Mohieddine

2018-04-01

We developed in the present paper the synthesis of a new AB3-type compound LaMg2Ni5Al4 by mechanical alloying (MA) process. ​​X-ray diffraction analysis (XRD) was used to determine the structural properties and the phase evolution of the powder mixtures. Two different synthesis pathways have been investigated. The first starting from elemental metals and the second from a mixture of two binary compounds LaNi5 (CaCu5-type structure, P6/mmm space group) and Al(Mg) solid solution (cubic Fm-3 m space group). The results show multiphase alloys which contain LaMg2Ni5Al4 main phase with hexagonal PuNi3-type structure (R-3 m space group). Rietveld analysis shows that using a planetary ball mill, we obtain a good yield of LaMg2Ni5Al4 compound after 5 h of mechanical alloying for both synthesis pathways. TEM analysis confirmed XRD results. SEM-EDX analysis of the final product was in agreement with the nominal chemical formula. A setup of possible solid-gaz hydrogenation reaction will be described so far at the end of this work. Electrochemical results demonstrate evidence on hydrogen absorption in the AB3 material and the discharge capacity was equal to 5.9 H/f.u.

Direct on-chip DNA synthesis using electrochemically modified gold electrodes as solid support

NASA Astrophysics Data System (ADS)

Levrie, Karen; Jans, Karolien; Schepers, Guy; Vos, Rita; Van Dorpe, Pol; Lagae, Liesbet; Van Hoof, Chris; Van Aerschot, Arthur; Stakenborg, Tim

2018-04-01

DNA microarrays have propelled important advancements in the field of genomic research by enabling the monitoring of thousands of genes in parallel. The throughput can be increased even further by scaling down the microarray feature size. In this respect, microelectronics-based DNA arrays are promising as they can leverage semiconductor processing techniques with lithographic resolutions. We propose a method that enables the use of metal electrodes for de novo DNA synthesis without the need for an insulating support. By electrochemically functionalizing gold electrodes, these electrodes can act as solid support for phosphoramidite-based synthesis. The proposed method relies on the electrochemical reduction of diazonium salts, enabling site-specific incorporation of hydroxyl groups onto the metal electrodes. An automated DNA synthesizer was used to couple phosphoramidite moieties directly onto the OH-modified electrodes to obtain the desired oligonucleotide sequence. Characterization was done via cyclic voltammetry and fluorescence microscopy. Our results present a valuable proof-of-concept for the integration of solid-phase DNA synthesis with microelectronics.

Synthesis and Surface Activity of Cationic Amino Acid-Based Surfactants in Aqueous Solution.

PubMed

Greber, Katarzyna E

2017-01-01

I studied the possibility of using amino acid-based surfactants as emulsifiers at the same time as preservatives. Fourteen lipopeptides were synthesized employing a solid phase peptide synthesis procedure. All compounds were designed to be positively charged from +1 to +4 and acylated with fatty acid chain-palmitic and miristic. The surface activity of the obtained lipopeptides was tested using a semi-automatic tensiometer to calculate parameters describing the behavior of lipopeptides in the air/water interface. Such parameters as CMC, surface tension at the CMC point ( σ CMC ), effectiveness ( π CMC ), and efficiency (pC20) were measured. Emulsifying properties of all lipopeptides were also examined. The studies reveal that the surface active properties of synthesized compounds strongly depend on the length of alkyl chains as well as on the composition of amino acid polar heads. The critical micelle concentration decreases with increasing alkyl chain length of lipopeptides with the same polar head. The effectiveness and efficiency decrease when the number of amino acids in the polar head increases. All lipopeptides established a very weak emulsification power and created unstable water/Miglyol 812 and water/paraffin oil emulsions. Results suggest that lipopeptides cannot be used as emulsifiers; nonetheless, it is possible to use them as auxiliary surfactants with disinfectant properties in combination with more potent emulsifiers.

Prebiotic Peptide (Amide) Bond Synthesis Accelerated by Glycerol and Bicarbonate Under Neutral to Alkaline Dry-Down Conditions

NASA Technical Reports Server (NTRS)

Forsythe, J. G.; Weber, A. L.

2017-01-01

Past studies of prebiotic peptide bond synthesis have generally been carried out in the acidic to neutral pH range [1, 2]. Here we report a new process for peptide bond (amide) synthesis in the neutral to alkaline pH range that involves simple dry-down heating of amino acids in the presence of glycerol and bicarbonate. Glycerol was included in the reaction mixture as a solvent and to provide hydroxyl groups for possible formation of ester intermediates previously implicated in peptide bond synthesis under acidic to neutral conditions [1]. Bicarbonate was added to raise the reaction pH to 8-9.

Enzymatic triggered release of an HIV-1 entry inhibitor from prostate specific antigen degradable microparticles.

PubMed

Clark, Meredith R; Aliyar, Hyder A; Lee, Chang-won; Jay, Julie I; Gupta, Kavita M; Watson, Karen M; Stewart, Russell J; Buckheit, Robert W; Kiser, Patrick F

2011-07-15

This paper describes the design, construction and characterization of the first anti-HIV drug delivery system that is triggered to release its contents in the presence of human semen. Microgel particles were synthesized with a crosslinker containing a peptide substrate for the seminal serine protease prostate specific antigen (PSA) and were loaded with the HIV-1 entry inhibitor sodium poly(styrene-4-sulfonate) (pSS). The particles were composed of N-2-hydroxyproplymethacrylamide and bis-methacrylamide functionalized peptides based on the PSA substrates GISSFYSSK and GISSQYSSK. Exposure to human seminal plasma (HSP) degraded the microgel network and triggered the release of the entrapped antiviral polymer. Particles with the crosslinker composed of the substrate GISSFYSSK showed 17 times faster degradation in seminal plasma than that of the crosslinker composed of GISSQYSSK. The microgel particles containing 1 mol% GISSFYSSK peptide crosslinker showed complete degradation in 30 h in the presence of HSP at 37°C and pSS released from the microgels within 30 min reached a concentration of 10 μg/mL, equivalent to the published IC(90) for pSS. The released pSS inactivated HIV-1 in the presence of HSP. The solid phase synthesis of the crosslinkers, preparation of the particles by inverse microemulsion polymerization, HSP-triggered release of pSS and inactivation of HIV-1 studies are described. Copyright © 2011 Elsevier B.V. All rights reserved.

The critical role of peptide chemistry in the life sciences.

PubMed

Kent, Stephen B H

2015-03-01

Peptide chemistry plays a key role in the synthesis and study of protein molecules and their functions. Modern ligation methods enable the total synthesis of enzymes and the systematic dissection of the chemical basis of enzyme catalysis. Predicted developments in peptide science are described. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.

Two novel solvent system compositions for protected synthetic peptide purification by centrifugal partition chromatography.

PubMed

Amarouche, Nassima; Giraud, Matthieu; Forni, Luciano; Butte, Alessandro; Edwards, F; Borie, Nicolas; Renault, Jean-Hugues

2014-04-11

Protected synthetic peptide intermediates are often hydrophobic and not soluble in most common solvents. They are thus difficult to purify by preparative reversed-phase high-performance liquid chromatography (RP-HPLC), usually used for industrial production. It is then challenging to develop alternative chromatographic purification processes. Support-free liquid-liquid chromatographic techniques, including both hydrostatic (centrifugal partition chromatography or CPC) and hydrodynamic (counter-current chromatography or CCC) devices, are mainly involved in phytochemical studies but have also been applied to synthetic peptide purification. In this framework, two new biphasic solvent system compositions covering a wide range of polarity were developed to overcome solubility problems mentioned above. The new systems composed of heptane/tetrahydrofuran/acetonitrile/dimethylsulfoxide/water and heptane/methyl-tetrahydrofuran/N-methylpyrrolidone/water were efficiently used for the CPC purification of a 39-mer protected exenatide (Byetta®) and a 8-mer protected peptide intermediate of bivalirudin (Angiox®) synthesis. Phase compositions of the different biphasic solvent systems were determined by (1)H nuclear magnetic resonance. Physico-chemical properties including viscosity, density and interfacial tension of these biphasic systems are also described. Copyright © 2014 Elsevier B.V. All rights reserved.

Preparation and Evaluation at the Delta Opioid Receptor of a Series of Linear Leu-Enkephalin Analogues Obtained by Systematic Replacement of the Amides

PubMed Central

2013-01-01

Leu-enkephalin analogues, in which the amide bonds were sequentially and systematically replaced either by ester or N-methyl amide bonds, were prepared using classical organic chemistry as well as solid phase peptide synthesis (SPPS). The peptidomimetics were characterized using competition binding, ERK1/2 phosphorylation, receptor internalization, and contractility assays to evaluate their pharmacological profile over the delta opioid receptor (DOPr). The lipophilicity (LogD7.4) and plasma stability of the active analogues were also measured. Our results revealed that the last amide bond can be successfully replaced by either an ester or an N-methyl amide bond without significantly decreasing the biological activity of the corresponding analogues when compared to Leu-enkephalin. The peptidomimetics with an N-methyl amide function between residues Phe and Leu were found to be more lipophilic and more stable than Leu-enkephalin. Findings from the present study further revealed that the hydrogen-bond donor properties of the fourth amide of Leu-enkephalin are not important for its biological activity on DOPr. Our results show that the systematic replacement of amide bonds by isosteric functions represents an efficient way to design and synthesize novel peptide analogues with enhanced stability. Our findings further suggest that such a strategy can also be useful to study the biological roles of amide bonds. PMID:23650868

Construction of 1,3,4-Oxadiazole and 1,3,4-Thiadiazole Library with a High Level of Skeletal Diversity Based on Branching Diversity-Oriented Synthesis on Solid-Phase Supports.

PubMed

Ha, Ji-Eun; Yang, Seung-Ju; Gong, Young-Dae

2018-02-12

An efficient solid-phase synthetic route for the construction of 1,3,4-oxadiazole and 1,3,4-thiadiazole libraries based on branching diversity-oriented synthesis (DOS) has been developed in this study. The core skeleton resins, 1,3,4-oxadiazole and 1,3,4-thiadiazole, were obtained through desulfurative and dehydrative cyclizations of thiosemicarbazide resin, respectively. Various functional groups have been introduced to the core skeleton resins, such as aryl, amine, amide, urea, thiourea, and an amino acid. Most of the libraries were purified by simple trituration without extraction or column chromatography after cleavage of the products from the solid-supported resin. As a result, we obtained high yields of pure 1,3,4-oxadiazole and 1,3,4-thiadiazole derivatives (total numbers = 128). Finally, we confirmed the drug-like properties of our library by calculation of physicochemical properties, displays of the skeletal diversities of the library in 3D-space, and occupation of a broad range of areas by their functional groups.

Design of RGD-ATWLPPR peptide conjugates for the dual targeting of αVβ3 integrin and neuropilin-1.

PubMed

Thoreau, Fabien; Vanwonterghem, Laetitia; Henry, Maxime; Coll, Jean-Luc; Boturyn, Didier

2018-05-18

Targeting the tumour microenvironment is a promising strategy to detect and/or treat cancer. The design of selective compounds that co-target several receptors frequently overexpressed in solid tumours may allow a reliable and selective detection of tumours. Here we report the modular synthesis of compounds encompassing ligands of αVβ3 integrin and neuropilin-1 that are overexpressed in the tumour microenvironment. These compounds were then evaluated through cellular experiments and imaging of tumours in mice. We observed that the peptide that displays both ligands is more specifically accumulating in the tumours than in controls. Simultaneous interaction with αVβ3 integrin and NRP1 induces NRP1 stabilization at the cell membrane surface which is not observed with the co-injection of the controls.

Evaluating Ga-68 Peptide Conjugates for Targeting VPAC Receptors: Stability and Pharmacokinetics.

PubMed

Kumar, Pardeep; Tripathi, Sushil K; Chen, C P; Wickstrom, Eric; Thakur, Mathew L

2018-05-25

In recent years, considerable progress has been made in the use of gallium-68 labeled receptor-specific peptides for imaging oncologic diseases. The objective was to examine the stability and pharmacokinetics of [ 68 Ga]NODAGA and DOTA-peptide conjugate targeting VPAC [combined for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP)] receptors on tumor cells. A VPAC receptor-specific peptide was chosen as a model peptide and conjugated to NODAGA and DOTA via solid-phase synthesis. The conjugates were characterized by HPLC and MALDI-TOF. Following Ga-68 chelation, the radiochemical purity of Ga-68 labeled peptide conjugate was determined by radio-HPLC. The stability was tested against transmetallation using 100 nM Fe 3+ /Zn 2+ /Ca 2+ ionic solution and against transchelation using 200 μM DTPA solution. The ex vivo and in vivo stability of the Ga-68 labeled peptide conjugate was tested in mouse plasma and urine. Receptor specificity was determined ex vivo by cell binding assays using human breast cancer BT474 cells. Positron emission tomography (PET)/X-ray computed tomography (CT) imaging, tissue distribution, and blocking studies were performed in mice bearing BT474 xenografts. The chemical and radiochemical purity was greater than 95 % and both conjugates were stable against transchelation and transmetallation. Ex vivo stability at 60 min showed that the NODAGA-peptide-bound Ga-68 reduced to 42.1 ± 3.7 % (in plasma) and 37.4 ± 2.9 % (in urine), whereas the DOTA-peptide-bound Ga-68 was reduced to 1.2 ± 0.3 % (in plasma) and 4.2 ± 0.4 % (in urine) at 60 min. Similarly, the in vivo stability for [ 68 Ga]NODAGA-peptide was decreased to 2.1 ± 0.2 % (in plasma) and 2.2 ± 0.4 % (in urine). For [ 68 Ga]DOTA-peptide, it was decreased to 1.4 ± 0.3 % (in plasma) and 1.2 ± 0.4 % (in urine) at 60 min. The specific BT474 cell binding was 53.9 ± 0.8 % for [ 68 Ga]NODAGA-peptide, 25.8 ± 1.4 % for [ 68 Ga]-DOTA-peptide, and 18.8 ± 2.5 % for [ 68 Ga]GaCl 3 at 60 min. Inveon microPET/CT imaging at 1 h post-injection showed significantly (p < 0.05) higher tumor to muscle (T/M) ratio for [ 68 Ga]NODAGA-peptide (3.4 ± 0.3) as compared to [ 68 Ga]DOTA-peptide (1.8 ± 0.6). For [ 68 Ga]GaCl 3 and blocked mice, their ratios were 1.5 ± 0.6 and 1.5 ± 0.3 respectively. The tissue distributions data were similar to the PET imaging data. NODAGA is superior to DOTA in terms of radiolabeling kinetics. The method of radiolabeling was reproducible and yielded higher specific activity. Although both agents have relatively low in vivo stability, PET/CT imaging studies delineated BC tumors with [ 68 Ga]NODAGA-peptide, but not with [ 68 Ga]DOTA-peptide.

Investigation of SP94 Peptide as a Specific Probe for Hepatocellular Carcinoma Imaging and Therapy

PubMed Central

Li, Yanli; Hu, Yan; Xiao, Jie; Liu, Guobing; Li, Xiao; Zhao, Yanzhao; Tan, Hui; Shi, Hongcheng; Cheng, Dengfeng

2016-01-01

SP94 (SFSIIHTPILPL), a novel peptide, has shown specific binding to hepatocellular carcinoma (HCC) cells. We aimed to investigate the capability of SP94 as a targeting probe for HCC imaging and therapy following labeling with technetium-99m (99mTc) and rhenium-188 (188Re). HYNIC-SP94 was prepared by solid phase synthesis and then labeled with 99mTc. Cell competitive binding, internalization assay, in vitro and in vivo stability, biodistribution and micro-single photon emission computed tomography /computed tomography (SPECT/CT) imaging studies were performed to investigate the capability of 99mTc tricine-EDDA/HYNIC-SP94 as a specific HCC imaging probe. Initial promising targeting results inspired evaluation of its therapeutic effect when labeled by 188Re. HYNIC-SP94 was then labeled again with 188Re to perform cell apoptosis, microSPECT/CT imaging evaluation and immunohistochemistry. Huh-7 cells exhibited typical apoptotic changes after 188Re irradiation. According to 99mTc tricine-EDDA/HYNIC-SP94 microSPECT/CT imaging, tumor uptake was significantly decreased compared with that of pre-treatment with 188Re-HYNIC-SP94. The immunohistochemistry also displayed obvious necrosis and apoptosis as well as inhibition of proliferation in the 188Re-HYNIC-SP94 treatment group. The results supported that 99mTc tricine-EDDA/HYNIC-SP94 is able to target HCC cells and 188Re-HYNIC- SP94 holds potential as a therapeutic agent for HCC, making 99mTc/188Re-HYNIC-SP94 a promising targeting probe for HCC imaging and therapy. PMID:27649935

Enhanced solubility and targeted delivery of curcumin by lipopeptide micelles.

PubMed

Liang, Ju; Wu, Wenlan; Lai, Danyu; Li, Junbo; Fang, Cailin

2015-01-01

A lipopeptide (LP)-containing KKGRGDS as the hydrophilic heads and lauric acid (C12) as the hydrophobic tails has been designed and prepared by standard solid-phase peptide synthesis technique. LP can self-assemble into spherical micelles with the size of ~30 nm in PBS (phosphate buffer saline) (pH 7.4). Curcumin-loaded LP micelles were prepared in order to increase the water solubility, sustain the releasing rate, and improve the tumor targeted delivery of curcumin. Water solubility, cytotoxicity, in vitro release behavior, and intracellular uptake of curcumin-loaded LP micelles were investigated. The results showed that LP micelles can increase the water solubility of curcumin 1.1 × 10(3) times and sustain the release of curcumin in a low rate. Curcumin-loaded LP micelles showed much higher cell inhibition than free curcumin on human cervix carcinoma (HeLa) and HepG2 cells. When incubating these curcumin-loaded micelles with HeLa and COS7 cells, due to the over-expression of integrins on cancer cells, the micelles can efficiently use the tumor-targeting function of RGD (functionalized peptide sequences: Arg-Gly-Asp) sequence to deliver the drug into HeLa cells, and better efficiency of the self-assembled LP micelles for curcumin delivery than crude curcumin was also confirmed by LCSM (laser confocal scanning microscope) assays. Combined with the enhanced solubility and higher cell inhibition, LP micelles reported in this study may be promising in clinical application for targeted curcumin delivery.

Efficiency, error and yield in light-directed maskless synthesis of DNA microarrays

PubMed Central

2011-01-01

Background Light-directed in situ synthesis of DNA microarrays using computer-controlled projection from a digital micromirror device--maskless array synthesis (MAS)--has proved to be successful at both commercial and laboratory scales. The chemical synthetic cycle in MAS is quite similar to that of conventional solid-phase synthesis of oligonucleotides, but the complexity of microarrays and unique synthesis kinetics on the glass substrate require a careful tuning of parameters and unique modifications to the synthesis cycle to obtain optimal deprotection and phosphoramidite coupling. In addition, unintended deprotection due to scattering and diffraction introduce insertion errors that contribute significantly to the overall error rate. Results Stepwise phosphoramidite coupling yields have been greatly improved and are now comparable to those obtained in solid phase synthesis of oligonucleotides. Extended chemical exposure in the synthesis of complex, long oligonucleotide arrays result in lower--but still high--final average yields which approach 99%. The new synthesis chemistry includes elimination of the standard oxidation until the final step, and improved coupling and light deprotection. Coupling Insertions due to stray light are the limiting factor in sequence quality for oligonucleotide synthesis for gene assembly. Diffraction and local flare are by far the largest contributors to loss of optical contrast. Conclusions Maskless array synthesis is an efficient and versatile method for synthesizing high density arrays of long oligonucleotides for hybridization- and other molecular binding-based experiments. For applications requiring high sequence purity, such as gene assembly, diffraction and flare remain significant obstacles, but can be significantly reduced with straightforward experimental strategies. PMID:22152062

A direct method for the synthesis of orthogonally protected furyl- and thienyl- amino acids.

PubMed

Hudson, Alex S; Caron, Laurent; Colgin, Neil; Cobb, Steven L

2015-04-01

The synthesis of unnatural amino acids plays a key part in expanding the potential application of peptide-based drugs and in the total synthesis of peptide natural products. Herein, we report a direct method for the synthesis of orthogonally protected 5-membered heteroaromatic amino acids.

Multiple Locations of Peptides in the Hydrocarbon Core of Gel-Phase Membranes Revealed by Peptide 13C to Lipid 2H Rotational-Echo Double-Resonance Solid-State Nuclear Magnetic Resonance

PubMed Central

2015-01-01

Membrane locations of peptides and proteins are often critical to their functions. Solid-state rotational-echo double-resonance (REDOR) nuclear magnetic resonance is applied to probe the locations of two peptides via peptide 13CO to lipid 2H distance measurements. The peptides are KALP, an α-helical membrane-spanning peptide, and HFP, the β-sheet N-terminal fusion peptide of the HIV gp41 fusion protein that plays an important role in HIV–host cell membrane fusion. Both peptides are shown to have at least two distinct locations within the hydrocarbon core of gel-phase membranes. The multiple locations are attributed to snorkeling of lysine side chains for KALP and to the distribution of antiparallel β-sheet registries for HFP. The relative population of each location is also quantitated. To the best of our knowledge, this is the first clear experimental support of multiple peptide locations within the membrane hydrocarbon core. These data are for gel-phase membranes, but the approach should work for liquid-ordered membranes containing cholesterol and may be applicable to liquid-disordered membranes with appropriate additional analysis to take into account protein and lipid motion. This paper also describes the methodological development of 13CO–2H REDOR using the lyophilized I4 peptide that is α-helical and 13CO-labeled at A9 and 2Hα-labeled at A8. The I4 spins are well-approximated as an ensemble of isolated 13CO–2H spin pairs each separated by 5.0 Å with a 37 Hz dipolar coupling. A pulse sequence with rectangular 100 kHz 2H π pulses results in rapid and extensive buildup of REDOR (ΔS/S0) with a dephasing time (τ). The buildup is well-fit by a simple exponential function with a rate of 24 Hz and an extent close to 1. These parameter values reflect nonradiative transitions between the 2H spin states during the dephasing period. Each spin pair spends approximately two-thirds of its time in the 13CO–2H (m = ±1) states and approximately one-third of its time in the 13CO–2H (m = 0) state and contributes to the ΔS/S0 buildup during the former but not the latter time segments. PMID:25531389

Effect of solid surface charge on the binding behaviour of a metal-binding peptide

PubMed Central

Donatan, Senem; Sarikaya, Mehmet; Tamerler, Candan; Urgen, Mustafa

2012-01-01

Over the last decade, solid-binding peptides have been increasingly used as molecular building blocks coupling bio- and nanotechnology. Despite considerable research being invested in this field, the effects of many surface-related parameters that define the binding of peptide to solids are still unknown. In the quest to control biological molecules at solid interfaces and, thereby, tailoring the binding characteristics of the peptides, the use of surface charge of the solid surface may probably play an important role, which then can be used as a potential tuning parameter of peptide adsorption. Here, we report quantitative investigation on the viscoelastic properties and binding kinetics of an engineered gold-binding peptide, 3RGBP1, adsorbed onto the gold surface at different surface charge densities. The experiments were performed in aqueous solutions using an electrochemical dissipative quartz crystal microbalance system. Hydrodynamic mass, hydration state and surface coverage of the adsorbed peptide films were determined as a function of surface charge density of the gold metal substrate. Under each charged condition, binding of 3rGBP1 displayed quantitative differences in terms of adsorbed peptide amount, surface coverage ratio and hydration state. Based on the intrinsically disordered structure of the peptide, we propose a possible mechanism for binding of the peptide that can be used for tuning surface adsorption in further studies. Controlled alteration of peptide binding on solid surfaces, as shown here, may provide novel methods for surface functionalization used for bioenabled processing and fabrication of future micro- and nanodevices. PMID:22491974

Solid-Phase Synthesis of RNA Analogs Containing Phosphorodithioate Linkages.

PubMed

Yang, Xianbin

2017-09-18

The oligoribonucleotide phosphorodithioate (PS2-RNA) modification uses two sulfur atoms to replace two non-bridging oxygen atoms at an internucleotide phosphorodiester backbone linkage. Like a natural phosphodiester RNA backbone linkage, a PS2-modified backbone linkage is achiral at phosphorus. PS2-RNAs are highly stable to nucleases and several in vitro assays have demonstrated their biological activity. For example, PS2-RNAs silenced mRNA in vitro and bound to protein targets in the form of PS2-aptamers (thioaptamers). Thus, the interest in and promise of PS2-RNAs has drawn attention to synthesizing, isolating, and characterizing these compounds. RNA-thiophosphoramidite monomers are commercially available from AM Biotechnologies and this unit describes an effective methodology for solid-phase synthesis, deprotection, and purification of RNAs having PS2 internucleotide linkages. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Solid-phase synthesis of Cu2MoS4 nanoparticles for degradation of methyl blue under a halogen-tungsten lamp

NASA Astrophysics Data System (ADS)

Li, Shi-na; Ma, Rui-xin; Wang, Cheng-yan

2018-03-01

The Cu2MoS4 nanoparticles were prepared using a relatively simple and convenient solid-phase process, which was applied for the first time. The crystalline structure, morphology, and optical properties of Cu2MoS4 nanoparticles were characterized using X-ray diffraction, X-ray photoelectron spectroscopy, field emission scanning electron microscopy, and UV-vis spectrophotometry. Cu2MoS4 nanoparticles having a band gap of 1.66 eV exhibits good photocatalytic activity in the degradation of methylene blue, which indicates that this simple process may be critical to facilitate the cheap production of photocatalysts.

Tailor Made Synthesis of T-Shaped and π-STACKED Dimers in the Gas Phase: Concept for Efficient Drug Design and Material Synthesis

NASA Astrophysics Data System (ADS)

Kumar, Sumit; Das, Aloke

2013-06-01

Non-covalent interactions play a key role in governing the specific functional structures of biomolecules as well as materials. Thus molecular level understanding of these intermolecular interactions can help in efficient drug design and material synthesis. It has been found from X-ray crystallography that pure hydrocarbon solids (i.e. benzene, hexaflurobenzene) have mostly slanted T-shaped (herringbone) packing arrangement whereas mixed solid hydrocarbon crystals (i.e. solid formed from mixtures of benzene and hexafluorobenzene) exhibit preferentially parallel displaced (PD) π-stacked arrangement. Gas phase spectroscopy of the dimeric complexes of the building blocks of solid pure benzene and mixed benzene-hexafluorobenzene adducts exhibit similar structural motifs observed in the corresponding crystal strcutures. In this talk, I will discuss about the jet-cooled dimeric complexes of indole with hexafluorobenzene and p-xylene in the gas phase using Resonant two photon ionzation and IR-UV double resonance spectroscopy combined with quantum chemistry calculations. In stead of studying benzene...p-xylene and benzene...hexafluorobenzene dimers, we have studied corresponding indole complexes because N-H group is much more sensitive IR probe compared to C-H group. We have observed that indole...hexafluorobenzene dimer has parallel displaced (PD) π-stacked structure whereas indole...p-xylene has slanted T-shaped structure. We have shown here selective switching of dimeric structure from T-shaped to π-stacked by changing the substituent from electron donating (-CH3) to electron withdrawing group (fluorine) in one of the complexing partners. Thus, our results demonstrate that efficient engineering of the non-covalent interactions can lead to efficient drug design and material synthesis.

Structure Evolution and Reactivity of the Sc(2- x)V xO3+δ (0 ≤ x ≤ 2.0) System.

PubMed

Lussier, Joey A; Simon, Fabian J; Whitfield, Pamela S; Singh, Kalpana; Thangadurai, Venkataraman; Bieringer, Mario

2018-05-07

Solid oxide fuel cells (SOFCs) are solid-state electrochemical devices that directly convert chemical energy of fuels into electricity with high efficiency. Because of their fuel flexibility, low emissions, high conversion efficiency, no moving parts, and quiet operation, they are considered as a promising energy conversion technology for low carbon future needs. Solid-state oxide and proton conducting electrolytes play a crucial role in improving the performance and market acceptability of SOFCs. Defect fluorite phases are some of the most promising fast oxide ion conductors for use as electrolytes in SOFCs. We report the synthesis, structure, phase diagram, and high-temperature reactivity of the Sc (2- x) V x O 3+δ (0 ≤ x ≤ 2.00) oxide defect model system. For all Sc (2- x) V x O 3.0 phases with x ≤ 1.08 phase-pure bixbyite-type structures are found, whereas for x ≥ 1.68 phase-pure corundum structures are reported, with a miscibility gap found for 1.08 < x < 1.68. Structural details obtained from the simultaneous Rietveld refinements using powder neutron and X-ray diffraction data are reported for the bixbyite phases, demonstrating a slight V 3+ preference toward the 8b site. In situ X-ray diffraction experiments were used to explore the oxidation of the Sc (2- x) V x O 3.0 phases. In all cases ScVO 4 was found as a final product, accompanied by Sc 2 O 3 for x < 1.0 and V 2 O 5 when x > 1.0; however, the oxidative pathway varied greatly throughout the series. Comments are made on different synthesis strategies, including the effect on crystallinity, reaction times, rate-limiting steps, and reaction pathways. This work provides insight into the mechanisms of solid-state reactions and strategic guidelines for targeted materials synthesis.

The effect of B{sub 2}O{sub 3} flux on growth NLBCO superconductor by solid state reaction and wet-mixing methods

DOE Office of Scientific and Technical Information (OSTI.GOV)

Suharta, W. G., E-mail: wgsuharta@gmail.com; Wendri, N.; Ratini, N.

The synthesis of B{sub 2}O{sub 3} flux substituted NLBCO superconductor NdBa{sub 1.75}La{sub 0.25}Cu{sub 3}O{sub 7-∂} has been done using solid state reaction and wet-mixing methods in order to obtain homogeneous crystals and single phase. From DTA/TGA characteritations showed the synthesis process by wet-mixing requires a lower temperature than the solid state reaction in growing the superconductor NdBa{sub 1.75}La{sub 0.25}Cu{sub 3}O{sub 7-∂}. Therefore, in this research NdBa{sub 1.75}La{sub 0.25}Cu{sub 3}O{sub 7-∂} sample calcinated at 650°C for wet-mixing method and 820°C for solid state reaction methods. The all samples was sintered at 950°C for ten hours. Crystallinity of the sample was confirmedmore » using X-ray techniques and generally obtained sharp peaks that indicates the sample already well crystallized. Search match analyses for diffraction data gave weight fractions of impurity phase of the solid state reaction method higher than wet-mixing method. In this research showed decreasing the price of the lattice parameter about 1% with the addition of B{sub 2}O{sub 3} flux for the both synthesis process and 2% of wet mixing process for all samples. Characterization using scanning electron microscopy (SEM) showed the distribution of crystal zise for wet-mixing method more homogeneous than solid state reaction method, with he grain size of samples is around 150–250 nm. The results of vibrating sample magnetometer (VSM) showed the paramagnetic properties for all samples.« less

Entropic Anomaly Observed in Lipid Polymorphisms Induced by Surfactant Peptide SP-B(1-25).

PubMed

Tran, Nhi; Kurian, Justin; Bhatt, Avni; McKenna, Robert; Long, Joanna R

2017-10-05

The N-terminal 25 amino-acid residues of pulmonary surfactant protein B (SP-B 1-25 ) induces unusual lipid polymorphisms in a model lipid system, 4:1 DPPC/POPG, mirroring the lipid composition of native pulmonary surfactant. It is widely suggested that SP-B 1-25 -induced lipid polymorphisms within the alveolar aqueous subphase provide a structural platform for rapid lipid adsorption to the air-water interface. Here, we characterize in detail the phase behavior of DPPC and POPG in hydrated lipid assemblies containing therapeutic levels of SP-B 1-25 using 2 H and 31 P solid state NMR spectroscopy. The appearance of a previously observed isotropic lipid phase is found to be highly dependent on the thermal cycling of the samples. Slow heating of frozen samples leads to phase separation of DPPC into a lamellar phase whereas POPG lipids interact with the peptide to form an isotropic phase at physiologic temperature. Rapid heating of frozen samples to room temperature leads to strongly isotropic phase behavior for both DPPC and POPG lipids, with DPPC in exchange between isotropic and interdigitated phases. 31 P T 2 relaxation times confirm the isotropic phase to be consistent with a lipid cubic phase. The observed phases exhibit thermal stability up to physiologic temperature (37 °C) and are consistent with the formation of a ripple phase containing a large number of peptide-induced membrane structural defects enabling rapid transit of lipids between lipid lamellae. The coexistance of a lipid cubic phase with interdigitated lipids suggests a specific role for the highly conserved N-terminus of SP-B in stabilizing this unusual lipid polymorphism.

VQS (vapor-quasiliquid-solid, vapor-quasisolid-solid) mechanism presents a unified foundation for the syntheses of nanotubes, primarily carbon nanotubes

NASA Astrophysics Data System (ADS)

Mohammad, S. Noor

2017-09-01

Nanotubes are synthesized almost entirely by metal-catalyst-free and metal-catalyst-mediated non-eutectic mechanism(s). An investigation has been carried out to understand the basics of this mechanism. Various possible chemical and physical processes involved in nanotube synthesis have been researched. Various components and attributes of nanotube synthesis have been evaluated. Phase transitions, alloy formation, porosity, carrier transport and the fundamentals underlying them have been examined. Nanoparticle surfaces conducive to nanotube synthesis have been examined. The role of surface treatment, which includes oxidation, oxygenation, acid treatment, plasma treatment, water treatment, sputtering, etc in creating such surfaces, has been investigated. The role of surface treatment and phase transitions as functions of temperature, pressure, ambient, contaminants, surface amorphicity, etc in creating diffusion paths for the diffusion of growth species for supersaturation and nucleation has been explored. Interdiffusion of catalyst and source materials, and hence exchange of materials, on the nanoparticle surface, have been elucidated. This exchange of materials on catalyst surface appears to add a new dimension to the synthesis kinetics. Integrated together, they reveal a general mechanism for probably all metal-catalyst-free and metal-catalyst-mediated non-eutectic nanotube synthesis. Available experiments strongly support the proposed mechanism; they suggest that this mechanism has a broad appeal.

Composition and Morphology Control of Metal Dichalcogenides via Chemical Vapor Deposition for Photovoltaic and Nanoelectronic Applications

NASA Astrophysics Data System (ADS)

Samad, Leith L. J.

The body of work reviewed here encompasses a variety of metal dichalcogenides all synthesized using chemical vapor deposition (CVD) for solar and electronics applications. The first reported phase-pure CVD synthesis of iron pyrite thin films is presented with detailed structural and electrochemical analysis. The phase-pure thin film and improved crystal growth on a metallic backing material represents one of the best options for potential solar applications using iron pyrite. Large tin-sulfur-selenide solid solution plates with tunable bandgaps were also synthesized via CVD as single-crystals with a thin film geometry. Solid solution tin-sulfur-selenide plates were demonstrated to be a new material for solar cells with the first observed solar conversion efficiencies up to 3.1%. Finally, a low temperature molybdenum disulfide vertical heterostructure CVD synthesis with layered controlled growth was achieved with preferential growth enabled by Van der Waals epitaxy. Through recognition of additional reaction parameters, a fully regulated CVD synthesis enabled the controlled growth of 1-6 molybdenum disulfide monolayers for nanoelectronic applications. The improvements in synthesis and materials presented here were all enabled by the control afforded by CVD such that advances in phase purity, growth, and composition control of several metal dichalcogenides were achieved. Further work will be able to take full advantage of these advances for future solar and electronics technologies.

A novel endogenous inhibitor of phenoloxidase from Musca domestica has a cystine motif commonly found in snail and spider toxins.

PubMed

Daquinag, A C; Sato, T; Koda, H; Takao, T; Fukuda, M; Shimonishi, Y; Tsukamoto, T

1999-02-16

Phenoloxidase inhibitor (POI), found in the hemolymph of housefly pupae, is a novel dopa-containing and cystine-rich peptide that competitively inhibits phenoloxidase with a Ki in the nanomolar range. [Tyr32]POI is a potential precursor molecule also found in the hemolymph that may be posttranslationally oxidized to the dopa-containing peptide after creation of a rigid structure. By employing both a solid-phase peptide synthesis system based on a 9-fluorenylmethoxycarbonyl strategy and a specific air oxidation technique to ensure correct folding, we have been able to synthesize [Tyr32]POI. The synthetic [Tyr32]POI was confirmed to be identical to the native [Tyr32]POI by coelution high-performance liquid chromatography analysis and by enzymatic analysis using the phenoloxidase inhibition assay. To determine the disulfide pairings within the peptides, a series of enzyme hydrolyses and partial reduction/alkylation steps were performed. Three cystine pairs (Cys11-Cys25, Cys18-Cys29, and Cys24-Cys36) were determined by identification of the resulting peptides. The disulfide pairings of the two adjacent Cys residues (Cys11-Cys25 and Cys24-Cys36) were unambiguously assigned by comparing the derived fragments with the two possible isomers synthesized through a novel disulfide-linking technique. The arrangement of the disulfide bridges in POI was found to be topologically identical to those found for several peptides within the inhibitor cystine knot structural family. Although these peptides share a low primary sequence homology and display a diversity of biological functions, they nonetheless share similarities in their cystine motifs and tertiary structure. The tertiary structure model of POI, which was derived through molecular dynamics and energy minimization studies using restraints with determined disulfide connectivities, suggests that POI is a new class member of the inhibitor cystine-knot structural family.

Synthesis, properties, and application in peptide chemistry of a magnetically separable and reusable biocatalyst

NASA Astrophysics Data System (ADS)

Liria, Cleber W.; Ungaro, Vitor A.; Fernandes, Raphaella M.; Costa, Natália J. S.; Marana, Sandro R.; Rossi, Liane M.; Machini, M. Teresa

2014-11-01

Enzyme-catalyzed chemical processes are selective, very productive, and generate little waste. Nevertheless, they may be optimized using enzymes bound to solid supports, which are particularly important for protease-mediated reactions since proteases undergo fast autolysis in solution. Magnetic nanoparticles are suitable supports for this purpose owing to their high specific surface area and to be easily separated from reaction media. Here we describe the immobilization of bovine α-chymotrypsin (αCT) on silica-coated superparamagnetic nanoparticles (Fe3O4@silica) and the characterization of the enzyme-nanoparticle hybrid (Fe3O4@silica-αCT) in terms of protein content, properties, recovery from reaction media, application, and reuse in enzyme-catalyzed peptide synthesis. The results revealed that (i) full acid hydrolysis of the immobilized protease followed by amino acid analysis of the hydrolyzate is a reliable method to determine immobilization yield; (ii) despite showing lower amidase activity and a lower K cat/ K m value for a specific substrate than free αCT, the immobilized enzyme is chemically and thermally more stable, magnetically recoverable from reaction media, and can be consecutively reused for ten cycles to catalyze the amide bond hydrolysis and ester hydrolysis of the protected dipeptide Z-Ala-Phe-OMe. Altogether, these properties indicate the potential of Fe3O4@silica-αCT to act as an efficient, suitably stable, and reusable catalyst in amino acid, peptide, and protein chemistry as well as in proteomic studies.

Synthesis and structural studies on cerium substituted La0.4Ca0.6MnO3 as solid oxide fuel cell electrode material

NASA Astrophysics Data System (ADS)

Singh, Monika; Kumar, Dinesh; Singh, Akhilesh Kumar

2018-04-01

For solid oxide fuel cell electrode material, calcium doped lanthanum manganite La0.4Ca0.6MnO3 (LCMO) and cerium-incorporated on Ca-site with composition La0.40Ca0.55Ce0.05MnO3 (LCCMO) were synthesized using most feasible and efficient glycine-nitrate method. The formation of crystalline single phase was confirmed by x-ray diffraction (XRD). The Rietveld analysis reveals that both systems crystallize into orthorhombic crystal structure with Pnma space group. Additionally, 8 mole % Y2O3 stabilized ZrO2 (8YSZ) solid electrolyte was also synthesized using high energy ball mill to check the reaction with electrode materials. It was found that the substitution of Ce+4 cations in LCMO perovskite suppressed formation of undesired insulating CaZrO3 phase.

Glycerol derivatives of cutin and suberin monomers: synthesis and self-assembly.

PubMed

Douliez, Jean-Paul; Barrault, Joël; Jerome, François; Heredia, Antonio; Navailles, Laurence; Nallet, Frédéric

2005-01-01

Glycerol derivatives of cutin and suberin monomers were synthesized by acid catalysis. Their dispersion in an aqueous solution was examined by phase contrast microscopy, neutron scattering, and solid state NMR. It is shown that the phase behavior strongly depends on the nature of the derivatives forming either lumps of aggregated membranes or well dispersed membranes.

High-Pressure Synthesis: A New Frontier in the Search for Next-Generation Intermetallic Compounds.

PubMed

Walsh, James P S; Freedman, Danna E

2018-06-19

The application of high pressure adds an additional dimension to chemical phase space, opening up an unexplored expanse bearing tremendous potential for discovery. Our continuing mission is to explore this new frontier, to seek out new intermetallic compounds and new solid-state bonding. Simple binary elemental systems, in particular those composed of pairs of elements that do not form compounds under ambient pressures, can yield novel crystalline phases under compression. Thus, high-pressure synthesis can provide access to solid-state compounds that cannot be formed with traditional thermodynamic methods. An emerging approach for the rapid exploration of composition-pressure-temperature phase space is the use of hand-held high-pressure devices known as diamond anvil cells (DACs). These devices were originally developed by geologists as a way to study minerals under conditions relevant to the earth's interior, but they possess a host of capabilities that make them ideal for high-pressure solid-state synthesis. Of particular importance, they offer the capability for in situ spectroscopic and diffraction measurements, thereby enabling continuous reaction monitoring-a powerful capability for solid-state synthesis. In this Account, we provide an overview of this approach in the context of research we have performed in the pursuit of new intermetallic compounds. We start with a discussion of pressure as a fundamental experimental variable that enables the formation of intermetallic compounds that cannot be isolated under ambient conditions. We then introduce the DAC apparatus and explain how it can be repurposed for use as a synthetic vessel with which to explore this phase space, going to extremes of pressure where no chemist has gone before. The remainder of the Account is devoted to discussions of recent experiments we have performed with this approach that have led to the discovery of novel intermetallic compounds in the Fe-Bi, Cu-Bi, and Ni-Bi systems, with a focus on the cutting-edge methods that made these experiments possible. We review the use of in situ laser heating at high pressure, which led to the discovery of FeBi 2 , the first binary intermetallic compound in the Fe-Bi system. Our work in the Cu-Bi system is described in the context of in situ experiments carried out in the DAC to map its high-pressure phase space, which revealed two intermetallic phases (Cu 11 Bi 7 and CuBi). Finally, we review the discovery of β-NiBi, a novel high-pressure phase in the Ni-Bi system. We hope that this Account will inspire the next generation of solid-state chemists to boldly explore high-pressure phase space.

Boron nitride as desalting material in combination with phosphopeptide enrichment in shotgun proteomics.

PubMed

Furuhashi, Takeshi; Nukarinen, Ella; Ota, Shigenori; Weckwerth, Wolfram

2014-05-01

Hydrophilic peptides in shotgun proteomics have been shown to be problematic in conventional chromatography. Typically, C18 solid phase extraction or peptide traps are used for desalting the sample prior to mass spectrometry analysis, but the capacity to retain hydrophilic peptides is not very high, causing a bias toward more hydrophobic peptides. This is particularly problematic in phosphoproteomic studies. We tested the compatibility of commercially available boron nitride as a novel material for peptide desalting. Boron nitride can be used to recover a wide range of peptides with different physicochemical properties comparable to combined C18 and graphite carbon material. Copyright © 2014. Published by Elsevier Inc.

Alanine analogues of [D-Trp]CJ-15,208: novel opioid activity profiles and prevention of drug- and stress-induced reinstatement of cocaine-seeking behaviour.

PubMed

Aldrich, J V; Senadheera, S N; Ross, N C; Reilley, K A; Ganno, M L; Eans, S E; Murray, T F; McLaughlin, J P

2014-07-01

The novel macrocyclic peptide cyclo[Phe-D-Pro-Phe-D-Trp] ([D-Trp]CJ-15,208) exhibits κ opioid (KOP) receptor antagonist activity in both in vitro and in vivo assays. The four alanine analogues of this peptide were synthesized and characterized both in vitro and in vivo to assess the contribution of different amino acid residues to the activity of [D-Trp]CJ-15,208. The peptides were synthesized by a combination of solid phase peptide synthesis and cyclization in solution. The analogues were evaluated in vitro in receptor binding and functional assays, and in vivo with mice using a tail-withdrawal assay for antinociceptive and opioid antagonist activity. Mice demonstrating extinction of cocaine conditioned-place preference (CPP) were pretreated with selected analogues to evaluate prevention of stress or cocaine-induced reinstatement of CPP. The alanine analogues displayed pharmacological profiles in vivo distinctly different from [D-Trp]CJ-15,208. While the analogues exhibited varying opioid receptor affinities and κ and μ opioid receptor antagonist activity in vitro, they produced potent opioid receptor-mediated antinociception (ED50 = 0.28-4.19 nmol, i.c.v.) in vivo. Three of the analogues also displayed KOP receptor antagonist activity in vivo. Pretreatment with an analogue exhibiting both KOP receptor agonist and antagonist activity in vivo prevented both cocaine- and stress-induced reinstatement of cocaine-seeking behaviour in the CPP assay in a time-dependent manner. These unusual macrocyclic peptides exhibit in vivo opioid activity profiles different from the parent compound and represent novel compounds for potential development as therapeutics for drug abuse and possibly as analgesics. © 2014 The British Pharmacological Society.

Solid-Phase Extraction Strategies to Surmount Body Fluid Sample Complexity in High-Throughput Mass Spectrometry-Based Proteomics

PubMed Central

Bladergroen, Marco R.; van der Burgt, Yuri E. M.

2015-01-01

For large-scale and standardized applications in mass spectrometry- (MS-) based proteomics automation of each step is essential. Here we present high-throughput sample preparation solutions for balancing the speed of current MS-acquisitions and the time needed for analytical workup of body fluids. The discussed workflows reduce body fluid sample complexity and apply for both bottom-up proteomics experiments and top-down protein characterization approaches. Various sample preparation methods that involve solid-phase extraction (SPE) including affinity enrichment strategies have been automated. Obtained peptide and protein fractions can be mass analyzed by direct infusion into an electrospray ionization (ESI) source or by means of matrix-assisted laser desorption ionization (MALDI) without further need of time-consuming liquid chromatography (LC) separations. PMID:25692071

Alanine analogues of [D-Trp]CJ-15,208: novel opioid activity profiles and prevention of drug- and stress-induced reinstatement of cocaine-seeking behaviour

PubMed Central

Aldrich, J V; Senadheera, S N; Ross, N C; Reilley, K A; Ganno, M L; Eans, S E; Murray, T F; McLaughlin, J P

2014-01-01

BACKGROUND AND PURPOSE The novel macrocyclic peptide cyclo[Phe-D-Pro-Phe-D-Trp] ([D-Trp]CJ-15,208) exhibits κ opioid (KOP) receptor antagonist activity in both in vitro and in vivo assays. The four alanine analogues of this peptide were synthesized and characterized both in vitro and in vivo to assess the contribution of different amino acid residues to the activity of [D-Trp]CJ-15,208. EXPERIMENTAL APPROACH The peptides were synthesized by a combination of solid phase peptide synthesis and cyclization in solution. The analogues were evaluated in vitro in receptor binding and functional assays, and in vivo with mice using a tail-withdrawal assay for antinociceptive and opioid antagonist activity. Mice demonstrating extinction of cocaine conditioned-place preference (CPP) were pretreated with selected analogues to evaluate prevention of stress or cocaine-induced reinstatement of CPP. KEY RESULTS The alanine analogues displayed pharmacological profiles in vivo distinctly different from [D-Trp]CJ-15,208. While the analogues exhibited varying opioid receptor affinities and κ and μ opioid receptor antagonist activity in vitro, they produced potent opioid receptor-mediated antinociception (ED50 = 0.28–4.19 nmol, i.c.v.) in vivo. Three of the analogues also displayed KOP receptor antagonist activity in vivo. Pretreatment with an analogue exhibiting both KOP receptor agonist and antagonist activity in vivo prevented both cocaine- and stress-induced reinstatement of cocaine-seeking behaviour in the CPP assay in a time-dependent manner. CONCLUSIONS AND IMPLICATIONS These unusual macrocyclic peptides exhibit in vivo opioid activity profiles different from the parent compound and represent novel compounds for potential development as therapeutics for drug abuse and possibly as analgesics. PMID:24588614

Highly efficient solid phase synthesis of oligonucleotide analogs containing phosphorodithioate linkages

PubMed Central

Capaldi, Daniel C.; Cole, Douglas L.; Ravikumar, Vasulinga T.

2000-01-01

A triester method for the synthesis of deoxynucleoside phosphorodithioate dimers is described. The phosphorodithioate linkage is introduced using a new dithiophosphorylating reagent DPSE-SP(S)Cl2 where DPSE = 2-diphenylmethylsilylethyl. This group is removed quickly using tetra-butylammonium fluoride leading to the quantitative formation of phosphorodithioate diesters uncontaminated with the corresponding phosphorothioates. The utility of this group is demonstrated by the synthesis of a pentadecathymidylic acid, [T(PS2)T(PO2)]7T, which contains alternating phosphorodithioate/phosphate diester internucleotide linkages. PMID:10756207

A novel Tc-99 m and fluorescence labeled peptide as a multimodal imaging agent for targeting angiogenesis in a murine tumor model.

PubMed

Kim, Myoung Hyoun; Kim, Chang Guhn; Kim, Seul-Gi; Kim, Dae-Weung

2016-11-01

The serine-aspartic acid-valine (SDV) peptide binds specifically to integrin α V β 3 . In the present study, we successfully developed a TAMRA-GHEG-ECG-SDV peptide labeled with both Tc-99 m and TAMRA to target the integrin α V β 3 of tumor cells; furthermore, we evaluated the diagnostic performance of Tc-99 m TAMRA-GHEG-ECG-SDV as a dual-modality imaging agent for tumor of the murine model. TAMRA-GHEG-ECG-SDV was synthesized using Fmoc solid-phase peptide synthesis. Radiolabeling of TAMRA-GHEG-ECG-SDV with Tc-99 m was done using ligand exchange methods. Labeling stability and cytotoxicity studies were performed. Gamma camera imaging, biodistribution and ex vivo imaging studies were performed in murine models with HT-1080 and HT-29 tumors. A tumor tissue slide was prepared and analyzed using confocal microscopy. After radiolabeling procedures with Tc-99 m, the Tc-99 m TAMRA-GHEG-ECG-SDV complexes were prepared in high yield (>99%). In the gamma camera imaging study, a substantial uptake of Tc-99 m TAMRA-GHEG-ECG-SDV into HT-1080 tumor (integrin α V β 3 positive) and low uptake of Tc-99 m TAMRA-GHEG-ECG-SDV into HT-29 tumor (integrin α V β 3 negative) were demonstrated. A competition study revealed that HT-1080 tumor uptake was effectively blocked by the co-injection of an excess concentration of SDV. Specific uptake of Tc-99 m TAMRA-GHEG-ECG-SDV was confirmed by biodistribution, ex vivo imaging and confocal microscopy studies. Our in vivo and in vitro studies revealed substantial uptake of Tc-99 m TAMRA-GHEG-ECG-SDV in the integrin α V β 3 -positive tumor. Tc-99 m TAMRA-GHEG-ECG-SDV could be a good candidate for a dual-modality imaging agent targeting tumor angiogenesis. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Synthesis and Biological Evaluation of Copper-64 Radiolabeled [DUPA-6-Ahx-(NODAGA)-5-Ava-BBN(7-14)NH2], a Novel Bivalent Targeting Vector Having Affinity for Two Distinct Biomarkers (GRPr/PSMA) of Prostate Cancer

PubMed Central

Bandari, Rajendra Prasad; Jiang, Zongrun; Reynolds, Tamila Stott; Bernskoetter, Nicole E.; Szczodroski, Ashley F.; Bassuner, Kurt J.; Kirkpatrick, Daniel L.; Rold, Tammy L.; Sieckman, Gary L.; Hoffman, Timothy J.; Connors, James P.; Smith, Charles J.

2014-01-01

Gastrin-releasing peptide receptors (GRPr) and prostate-specific membrane antigen (PSMA) are two identifying biomarkers expressed in very high numbers on prostate cancer cells and could serve as a useful tool for molecular targeting and diagnosis of disease via positron-emission tomography (PET). The aim of this study was to produce the multipurpose, bivalent [DUPA-6-Ahx-(64Cu-NODAGA)-5-Ava-BBN(7-14)NH2] radioligand for prostate cancer imaging, where DUPA = 2-[3-(1,3-Bis-tertbutoxycarbonylpropyl)-ureido]pentanedioic acid, a small-molecule, PSMA-targeting probe, 6Ahx = 6-aminohexanoic acid, 5-Ava = 5-aminovaleric acid, NODAGA = [2-(4,7-biscarboxymethyl)-1,4,7-(triazonan-1-yl)pentanedioic acid] (a derivative of NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid)), and BBN(7-14)NH2 = bombesin or BBN, a GRPr-specific peptide targeting probe. Methods The PSMA/GRPr dual targeting ligand precursor [DUPA-6-Ahx-K-5-Ava-BBN(7-14)NH2], was synthesized by solid-phase and manual peptide synthesis, after which NODAGA was added via manual conjugation to the ε-amine of lysine (K). The new bivalent GRPr/PSMA targeting vector was purified by reversed-phase high performance liquid chromatography (RP-HPLC), characterized by electrospray-ionization mass spectrometry (ESI-MS), and metallated with 64CuCl2 and natCuCl2. The receptor binding affinity was evaluated in human, prostate, PC-3 (GRPr-positive) and LNCaP (PSMA-positive) cells and the tumor-targeting efficacy determined in severe combined immunodeficient (SCID) and athymic nude mice bearing PC-3 and LNCaP tumors. Whole-body maximum intensity microPET/CT images of PC-3/LNCaP tumor-bearing mice were obtained 18 h post-injection (p.i.). Results Competitive binding assays in PC-3 and LNCaP cells indicated high receptor binding affinity for the [DUPA-6-Ahx-(natCu-NODAGA)-5-Ava-BBN(7-14)NH2] conjugate. MicroPET scintigraphy in PC-3/LNCaP tumor-bearing mice indicated that xenografted tumors were visible at 18 h p.i. with collateral, background radiation also being observed in non-target tissue. Conclusions [DUPA-6-Ahx-(64Cu-NODAGA)-5-Ava-BBN(7-14)NH2] targeting vector, as described herein, is the first example of a dual GRPr-/PSMA-targeting radioligand for molecular imaging prostate tumors. Detailed in vitro studies and microPET molecular imaging investigations of [DUPA-6-Ahx-(64Cu-NODAGA)-5-Ava-BBN(7-14)NH2] in tumor-bearing mice indicates that further studies are necessary to optimize uptake and retention of tracer in GRPr- and PSMA-positive tissues. PMID:24508213

Synthesis and biological evaluation of copper-64 radiolabeled [DUPA-6-Ahx-(NODAGA)-5-Ava-BBN(7-14)NH2], a novel bivalent targeting vector having affinity for two distinct biomarkers (GRPr/PSMA) of prostate cancer.

PubMed

Bandari, Rajendra Prasad; Jiang, Zongrun; Reynolds, Tamila Stott; Bernskoetter, Nicole E; Szczodroski, Ashley F; Bassuner, Kurt J; Kirkpatrick, Daniel L; Rold, Tammy L; Sieckman, Gary L; Hoffman, Timothy J; Connors, James P; Smith, Charles J

2014-04-01

Gastrin-releasing peptide receptors (GRPr) and prostate-specific membrane antigen (PSMA) are two identifying biomarkers expressed in very high numbers on prostate cancer cells and could serve as a useful tool for molecular targeting and diagnosis of disease via positron-emission tomography (PET). The aim of this study was to produce the multipurpose, bivalent [DUPA-6-Ahx-((64)Cu-NODAGA)-5-Ava-BBN(7-14)NH2] radioligand for prostate cancer imaging, where DUPA = (2-[3-(1,3-dicarboxypropyl)-ureido]pentanedioic acid), a small-molecule, PSMA-targeting probe, 6Ahx = 6-aminohexanoic acid, 5-Ava = 5-aminovaleric acid, NODAGA = [2-(4,7-biscarboxymethyl)-1,4,7-(triazonan-1-yl)pentanedioic acid] (a derivative of NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid)), and BBN(7-14)NH2 = bombesin, a GRPr-specific peptide targeting probe. The PSMA/GRPr dual targeting ligand precursor [DUPA-6-Ahx-K-5-Ava-BBN(7-14)NH2], was synthesized by solid-phase and manual peptide synthesis, after which NODAGA was added via manual conjugation to the ε-amine of lysine (K). The new bivalent GRPr/PSMA targeting vector was purified by reversed-phase high performance liquid chromatography (RP-HPLC), characterized by electrospray-ionization mass spectrometry (ESI-MS), and metallated with (64)CuCl2 and (nat)CuCl2. The receptor binding affinity was evaluated in human, prostate, PC-3 (GRPr-positive) and LNCaP (PSMA-positive) cells and the tumor-targeting efficacy determined in severe combined immunodeficient (SCID) and athymic nude mice bearing PC-3 and LNCaP tumors. Whole-body maximum intensity microPET/CT images of PC-3/LNCaP tumor-bearing mice were obtained 18 h post-injection (p.i.). Competitive binding assays in PC-3 and LNCaP cells indicated high receptor binding affinity for the [DUPA-6-Ahx-((nat)Cu-NODAGA)-5-Ava-BBN(7-14)NH2] conjugate. MicroPET scintigraphy in PC-3/LNCaP tumor-bearing mice indicated that xenografted tumors were visible at 18h p.i. with collateral, background radiation also being observed in non-target tissue. DUPA-6-Ahx-((64)Cu-NODAGA)-5-Ava-BBN(7-14)NH2] targeting vector, as described herein, is the first example of a dual GRPr-/PSMA-targeting radioligand for molecular of imaging prostate tumors. Detailed in vitro studies and microPET molecular imaging investigations of [DUPA-6-Ahx-((64)Cu-NODAGA)-5-Ava-BBN(7-14)NH2 in tumor-bearing mice indicate that further studies are necessary to optimize uptake and retention of tracer in GRPr- and PSMA-positive tissues. Published by Elsevier Inc.

Parallel solid-phase synthesis and high-throughput 1H NMR evaluation of a 96-member 1,2,4-trisubstituted-pyrimidin-6-one-5-carboxylic acid library.

PubMed

Hamper, Bruce C; Kesselring, Allen S; Chott, Robert C; Yang, Shengtian

2009-01-01

A solid-phase organic synthesis method has been developed for the preparation of trisubstituted pyrimidin-6-one carboxylic acids 12, which allows elaboration to a 3-dimensional combinatorial library. Three substituents are introduced by initial Knoevenagel condensation of an aldehyde and malonate ester resin 7 to give resin bound 1. Cyclization of 1 with an N-substituted amidine 10, oxidation, and cleavage afforded pyrimidinone 12. The initial solid-phase reaction sequence was followed by gel-phase (19)FNMR and direct-cleavage (1)H NMR of intermediate resins to determine the optimal conditions. The scope of the method for library production was determined by investigation of a 3 x 4 pilot library of twelve compounds. Cyclocondensation of N-methylamidines and 7 followed by CAN oxidation gave mixtures of the resin bound pyrimidin-6-one 11 and the regioisomeric pyrimidin-4-one 15, which after cleavage from the resin afforded a nearly 1:1 mixture of pyrimidin-6-one and pyrimidin-4-one carboxylic acids 12 and 16, respectively. The regiochemical assignment was confirmed by ROESY1D and gHMBC NMR experiments. A library was prepared using 8 aldehydes, 3 nitriles, and 4 amines to give a full combinatorial set of 96 pyrimidinones 12. Confirmation of structural identity and purity was carried out by LCMS using coupled ELS detection and by high-throughput flow (1)H NMR.

Phase formation and UV luminescence of Gd3+ doped perovskite-type YScO3

NASA Astrophysics Data System (ADS)

Shimizu, Yuhei; Ueda, Kazushige

2016-10-01

Synthesis of pure and Gd3+doped perovskite-type YScO3 was attempted by a polymerized complex (PC) method and solid state reaction (SSR) method. Crystalline phases and UV luminescence of samples were examined with varying heating temperatures. The perovskite-type single phase was not simply formed in the SSR method, as reported in some literatures, and two cubic C-type phases of starting oxide materials remained forming slightly mixed solid solutions. UV luminescence of Gd3+ doped samples increased with an increase in heating temperatures and volume of the perovskite-type phase. In contrast, a non-crystalline precursor was crystallized to a single C-type phase at 800 °C in the PC method forming a completely mixed solid solution. Then, the phase of perovskite-type YScO3 formed at 1200 °C and its single phase was obtained at 1400 °C. It was revealed that high homogeneousness of cations was essential to generate the single perovskite-phase of YScO3. Because Gd3+ ions were also dissolved into the single C-type phase in Gd3+ doped samples, intense UV luminescence was observed above 800 °C in both C-type phase and perovskite-type phase.

Integrated electrokinetically driven microfluidic devices with pH-mediated solid-phase extraction coupled to microchip electrophoresis for preterm birth biomarkers.

PubMed

Sonker, Mukul; Knob, Radim; Sahore, Vishal; Woolley, Adam T

2017-07-01

Integration in microfluidics is important for achieving automation. Sample preconcentration integrated with separation in a microfluidic setup can have a substantial impact on rapid analysis of low-abundance disease biomarkers. Here, we have developed a microfluidic device that uses pH-mediated solid-phase extraction (SPE) for the enrichment and elution of preterm birth (PTB) biomarkers. Furthermore, this SPE module was integrated with microchip electrophoresis for combined enrichment and separation of multiple analytes, including a PTB peptide biomarker (P1). A reversed-phase octyl methacrylate monolith was polymerized as the SPE medium in polyethylene glycol diacrylate modified cyclic olefin copolymer microfluidic channels. Eluent for pH-mediated SPE of PTB biomarkers on the monolith was optimized using different pH values and ionic concentrations. Nearly 50-fold enrichment was observed in single channel SPE devices for a low nanomolar solution of P1, with great elution time reproducibility (<7% RSD). The monolith binding capacity was determined to be 400 pg (0.2 pmol). A mixture of a model peptide (FA) and a PTB biomarker (P1) was extracted, eluted, injected, and then separated by microchip electrophoresis in our integrated device with ∼15-fold enrichment. This device shows important progress towards an integrated electrokinetically operated platform for preconcentration and separation of biomarkers. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Highly efficient solid phase synthesis of oligonucleotide analogs containing phosphorodithioate linkages.

PubMed

Capaldi, D C; Cole, D L; Ravikumar, V T

2000-05-01

A triester method for the synthesis of deoxynucleoside phosphorodithioate dimers is described. The phosphorodithioate linkage is introduced using a new dithiophosphorylating reagent DPSE-SP(S)Cl(2)where DPSE = 2-diphenylmethylsilylethyl. This group is removed quickly using tetra-butylammonium fluoride leading to the quantitative formation of phosphorodithioate diesters uncontaminated with the corresponding phosphorothioates. The utility of this group is demonstrated by the synthesis of a penta-decathymidylic acid, [T(PS(2))T(PO(2))](7)T, which contains alternating phosphorodithioate/phosphate diester internucleotide linkages.

A Radiofluorinated Divalent Cystine Knot Peptide for Tumor PET Imaging

DOE PAGES

Jiang, Lei; Kimura, Richard H.; Ma, Xiaowei; ...

2014-04-09

A divalent knottin containing two separate integrin binding epitopes (RGD) in the adjacent loops, 3-4A, was recently developed and reported in our previous publication. In the current study, 3-4A was radiofluorinated with a 4-nitrophenyl 2- 18F-fluoropropinate ( 18F-NFP) group and the resulting divalent positron emission tomography (PET) probe, 18F-FP–3-4A, was evaluated as a novel imaging probe to detect integrin αvβ3 positive tumors in living animals. Knottin 3-4A was synthesized by solid phase peptide synthesis, folded, and site-specifically conjugated with 18/19F-NFP to produce the fluorinated peptide 18/19F-fluoropropinate-3-4A ( 18/19F-FP–3-4A). The stability of 18F-FP–3-4A was tested in both phosphate buffered saline (PBS)more » buffer and mouse serum. Cell uptake assays of the radiolabeled peptides were performed using U87MG cells. In addition, small animal PET imaging and biodistribution studies of 18F-FP–3-4A were performed in U87MG tumor-bearing mice. The receptor targeting specificity of the radiolabeled peptide was also verified by coinjecting the probe with a blocking peptide cyclo(RGDyK). Our study showed that 18F-FP–3-4A exhibited excellent stability in PBS buffer (pH 7.4) and mouse serum. Small animal PET imaging and biodistribution data revealed that 18F-FP–3-4A exhibited rapid and good tumor uptake (3.76 ± 0.59% ID/g and 2.22 ± 0.62% ID/g at 0.5 and 1 h, respectively). 18F-FP–3-4A was rapidly cleared from the normal tissues, resulting in excellent tumor-to-normal tissue contrasts. For example, liver uptake was only 0.39 ± 0.07% ID/g and the tumor to liver ratio was 5.69 at 1 h p.i. Furthermore, coinjection of cyclo(RGDyK) with 18F-FP–3-4A significantly inhibited tumor uptake (0.41 ± 0.12 vs 1.02 ± 0.19% ID/g at 2.5 h) in U87MG xenograft models, demonstrating specific accumulation of the probe in the tumor. In summary, the divalent probe 18F-FP–3-4A is characterized by rapid and high tumor uptake and excellent tumor-to-normal tissue ratios. 18F-FP–3-4A is a highly promising knottin based PET probe for translating into clinical imaging of tumor angiogenesis.« less

Salt-assistant combustion synthesis of nanocrystalline Nd{sub 2}(Zr{sub 1-x}Sn{sub x}){sub 2}O{sub 7} (0 {<=} x {<=} 1) solid solutions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tong Yuping, E-mail: huabeitong@yahoo.cn; Wang Yanping

2009-11-15

Nanocrystalline Nd{sub 2}(Zr{sub 1-x}Sn{sub x}){sub 2}O{sub 7} series solid solutions were prepared by a convenient salt-assisted combustion process using glycine as fuel. The samples were characterized by X-ray diffraction, Fourier transform infrared spectroscopy, Raman spectroscopy, transmission electron microscopy and high-resolution transmission electron microscopy. The results showed the Zr ion can be partially replaced by Sn ion. The partial substituted products were still single-phase solid solutions and the crystal form remained unchanged. TEM images reveal that the products are composed of well-dispersed square-shaped nanocrystals. The method provides a convenient and low-cost route for the synthesis of nanostructures of oxide materials.

Surface-modified multifunctional MIP nanoparticles.

PubMed

Moczko, Ewa; Poma, Alessandro; Guerreiro, Antonio; Perez de Vargas Sansalvador, Isabel; Caygill, Sarah; Canfarotta, Francesco; Whitcombe, Michael J; Piletsky, Sergey

2013-05-07

The synthesis of core-shell molecularly imprinted polymer nanoparticles (MIP NPs) has been performed using a novel solid-phase approach on immobilised templates. The same solid phase also acts as a protective functionality for high affinity binding sites during subsequent derivatisation/shell formation. This procedure allows for the rapid synthesis, controlled separation and purification of high-affinity materials, with each production cycle taking just 2 hours. The aim of this approach is to synthesise uniformly sized imprinted materials at the nanoscale which can be readily grafted with various polymers without affecting their affinity and specificity. For demonstration purposes we grafted anti-melamine MIP NPs with coatings which introduce the following surface characteristics: high polarity (PEG methacrylate); electro-activity (vinylferrocene); fluorescence (eosin acrylate); thiol groups (pentaerythritol tetrakis(3-mercaptopropionate)). The method has broad applicability and can be used to produce multifunctional imprinted nanoparticles with potential for further application in the biosensors, diagnostics and biomedical fields and as an alternative to natural receptors.

Two-dimensional tin selenide nanostructures for flexible all-solid-state supercapacitors.

PubMed

Zhang, Chunli; Yin, Huanhuan; Han, Min; Dai, Zhihui; Pang, Huan; Zheng, Yulin; Lan, Ya-Qian; Bao, Jianchun; Zhu, Jianmin

2014-04-22

Due to their unique electronic and optoelectronic properties, tin selenide nanostructures show great promise for applications in energy storage and photovoltaic devices. Despite the great progress that has been achieved, the phase-controlled synthesis of two-dimensional (2D) tin selenide nanostructures remains a challenge, and their use in supercapacitors has not been explored. In this paper, 2D tin selenide nanostructures, including pure SnSe2 nanodisks (NDs), mixed-phase SnSe-SnSe2 NDs, and pure SnSe nanosheets (NSs), have been synthesized by reacting SnCl2 and trioctylphosphine (TOP)-Se with borane-tert-butylamine complex (BTBC) and 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone. Utilizing the interplay of TOP and BTBC and changing only the amount of BTBC, the phase-controlled synthesis of 2D tin selenide nanostructures is realized for the first time. Phase-dependent pseudocapacitive behavior is observed for the resulting 2D nanostructures. The specific capacitances of pure SnSe2 NDs (168 F g(-1)) and SnSe NSs (228 F g(-1)) are much higher than those of other reported materials (e.g., graphene-Mn3O4 nanorods and TiN mesoporous spheres); thus, these tin selenide materials were used to fabricate flexible, all-solid-state supercapacitors. Devices fabricated with these two tin selenide materials exhibited high areal capacitances, good cycling stabilities, excellent flexibilities, and desirable mechanical stabilities, which were comparable to or better than those reported recently for other solid-state devices based on graphene and 3D GeSe2 nanostructures. Additionally, the rate capability of the SnSe2 NDs device was much better than that of the SnSe NS device, indicating that SnSe2 NDs are promising active materials for use in high-performance, flexible, all-solid-state supercapacitors.

Stabilization of Angiotensin-(1-7) by key substitution with a cyclic non-natural amino acid.

PubMed

Wester, Anita; Devocelle, Marc; Tallant, E Ann; Chappell, Mark C; Gallagher, Patricia E; Paradisi, Francesca

2017-10-01

Angiotensin-(1-7) [Ang-(1-7)], a heptapeptide hormone of the renin-angiotensin-aldosterone system, is a promising candidate as a treatment for cancer that reflects its anti-proliferative and anti-angiogenic properties. However, the peptide's therapeutic potential is limited by the short half-life and low bioavailability resulting from rapid enzymatic metabolism by peptidases including angiotensin-converting enzyme (ACE) and dipeptidyl peptidase 3 (DPP 3). We report the facile assembly of three novel Ang-(1-7) analogues by solid-phase peptide synthesis which incorporates the cyclic non-natural δ-amino acid ACCA. The analogues containing the ACCA substitution at the site of ACE cleavage exhibit complete resistance to human ACE, while substitution at the DDP 3 cleavage site provided stability against DPP 3 hydrolysis. Furthermore, the analogues retain the anti-proliferative properties of Ang-(1-7) against the 4T1 and HT-1080 cancer cell lines. These results suggest that ACCA-substituted Ang-(1-7) analogues which show resistance against proteolytic degradation by peptidases known to hydrolyze the native heptapeptide may be novel therapeutics in the treatment of cancer.

Defined presentation of carbohydrates on a duplex DNA scaffold.

PubMed

Schlegel, Mark K; Hütter, Julia; Eriksson, Magdalena; Lepenies, Bernd; Seeberger, Peter H

2011-12-16

A new method for the spatially defined alignment of carbohydrates on a duplex DNA scaffold is presented. The use of an N-hydroxysuccinimide (NHS)-ester phosphoramidite along with carbohydrates containing an alkylamine linker allows for on-column labeling during solid-phase oligonucleotide synthesis. This modification method during solid-phase synthesis only requires the use of minimal amounts of complex carbohydrates. The covalently attached carbohydrates are presented in the major groove of the B-form duplex DNA as potential substrates for murine type II C-type lectin receptors mMGL1 and mMGL2. CD spectroscopy and thermal melting revealed only minimal disturbance of the overall helical structure. Surface plasmon resonance and cellular uptake studies with bone-marrow-derived dendritic cells were used to assess the capability of these carbohydrate-modified duplexes to bind to mMGL receptors. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

One pot synthesis of magnetic graphene/carbon nanotube composites as magnetic dispersive solid-phase extraction adsorbent for rapid determination of oxytetracycline in sewage water.

PubMed

Sun, Yunyun; Tian, Jing; Wang, Lu; Yan, Hongyuan; Qiao, Fengxia; Qiao, Xiaoqiang

2015-11-27

A simple and time-saving one pot synthesis of magnetic graphene/carbon nanotube composites (M-G/CNTs) was developed that could avoid the tedious drying process of graphite oxide, and G/CNTs were modified by Fe3O4 nanoparticles in the reduction procedure. It contributed to a shorten duration of the synthesis process of M-G/CNTs. The obtained M-G/CNTs were characterized and the results indicated that CNTs and Fe3O4 nanoparticles were served as spacer distributing to the layers of graphene, which was beneficial for enlarging surface area and improving extraction efficiency. Moreover, M-G/CNTs showed good magnetic property and outstanding thermal stability. Then M-G/CNTs were applied as adsorbent of magnetic dispersive solid-phase extraction for rapid extraction and determination of oxytetracycline in sewage water. Under the optimum conditions, good linearity was obtained in the range of 20-800ngmL(-1) and the recoveries were ranged from 95.5% to 112.5% with relative standard deviations less than 5.8%. Copyright © 2015 Elsevier B.V. All rights reserved.

Natriuretic peptide system in the rat submaxillary gland.

PubMed

Jankowski, M; Petrone, C; Tremblay, J; Gutkowska, J

1996-04-09

Natriuretic peptides and their receptors were characterized in rat submaxillary glands (SGs). Reverse phase-high performance liquid chromatography (HPLC) of rat SGs extracts revealed the presence of the 28-amino-acid (AA) circulating peptide ANP (Ser99-Tyr126) and the 126-AA prohormone (Asn1-Tyr126). The presence of ANP prohormone indicated that SGs are a site of ANP synthesis. Indeed, ANP mRNAs were demonstrated. ANP mRNA was 10 times lower than in the lung and only about 7 times lower than in the hypothalamus. ANP content in SG was determined as 30 +/- 8 ng/mg of protein (n = 7). In addition the presence of another member of the natriuretic peptide family, C-type natriuretic peptide (CNP), was found in SG. The CNP level of 293 +/- 38 pg/mg protein was significantly higher than in the lungs (44 +/- 6 pg/mg protein, P < 0.001, n = 5), but about 15 times lower than in hypothalamus (4.5 +/- 0.6 ng/mg protein, P < 0.001, n = 6). Both guanylyl cyclase and clearance receptors were expressed in SG. The presence of natriuretic peptide transcripts and their receptors suggests a role in rat SG functions.

[Rhythm of protein synthesis in cultures of hepatocytes from rats of different ages. Norm and effect of the peptide livagen].

PubMed

Brodskiĭ, V Ia; Khavinson, V Kh; Zolotarev, Iu A; Nechaeva, N V; Malinin, V V; Novikova, T E; Gvazava, I G; Fateeva, V I

2001-01-01

The circumhoralian rhythm of protein synthesis was determined in a monolayer culture of hepatocytes from rats at the age of 1 to 24 months and weighing from 45 to 480 g, respectively. The peptide lyvagen (Lys-Glu-Asp-Ala) obtained by directed chemical synthesis on the basis of amino acid analysis of the liver polypeptide preparations increased the level of protein synthesis in the hepatocytes from rats of different ages; the highest effect was observed in the cells of old animals. In old rats, lyvagen increased the amplitude of protein synthesis fluctuations. The peptide epitalon (Ala-Glu-Asp-Gly) constructed on the basis of analysis of the epiphysis peptides did not change the intensity of protein synthesis in the cultured hepatocytes.

Exposing high-energy surfaces by rapid-anneal solid phase epitaxy

DOE PAGES

Wang, Y.; Song, Y.; Peng, R.; ...

2017-08-08

The functional design of nanoscale transition metal oxide heterostructures depends critically on the growth of atomically flat epitaxial thin films. Much of the time, improved functionality is expected for heterostructures and surfaces with orientations that do not have the lowest surface free energy. For example, crystal faces with a high surface free energy, such as rutile (001) planes, frequently exhibit higher catalytic activities but are correspondingly harder to synthesize due to energy-lowering faceting transitions. We propose a broadly applicable rapid-anneal solid phase epitaxial synthesis approach for the creation of atomically flat, high surface free energy oxide heterostructures. We also demonstratemore » its efficacy via the synthesis of atomically flat, epitaxial RuO 2(001) films with a superior oxygen evolution activity, quantified by their lower onset potential and higher current density, relative to that of more common RuO 2(110) films.« less

Automated synthesis of a 96 product-sized library of triazole derivatives using a solid phase supported copper catalyst.

PubMed

Jlalia, Ibtissem; Beauvineau, Claire; Beauvière, Sophie; Onen, Esra; Aufort, Marie; Beauvineau, Aymeric; Khaba, Eihab; Herscovici, Jean; Meganem, Faouzi; Girard, Christian

2010-04-28

This article deal with the parallel synthesis of a 96 product-sized library using a polymer-based copper catalyst that we developed which can be easily separated from the products by simple filtration. This gave us the opportunity to use this catalyst in an automated chemical synthesis station (Chemspeed ASW-2000). Studies and results about the preparation of the catalyst, its use in different solvent systems, its recycling capabilities and its scope and limitations in the synthesis of this library will be addressed. The synthesis of the triazole library and the very good results obtained will finally be discussed.

Synthesis, Structure, and Thermal Properties of Ca5Ga6O14

NASA Astrophysics Data System (ADS)

Tolkacheva, A. S.; Shkerin, S. N.; Kuzmin, A. V.; Plaksin, S. V.; Korzun, I. V.; Kochedykov, V. A.; Yaroslavtseva, T. V.; Vovkotrub, E. G.

2018-07-01

Calcium gallate Ca5Ga6O14 is synthesized by solid-phase means. Its melting point is 1325 ± 2°C. A phase transition of the second kind is observed in the temperature interval of 750-800°C. The temperature dependence of a thermal linear expansion coefficient within 200-900°C is given. Vibrational spectroscopy data confirm that Ca5Ga6O14 contains not only GaO4 tetrahedra but also GaO6 octahedra.

Nucleoside-O-Methyl-(H)-Phosphinates: Novel Monomers for the Synthesis of Methylphosphonate Oligonucleotides Using H-Phosphonate Chemistry.

PubMed

Kostov, Ondřej; Páv, Ondřej; Rosenberg, Ivan

2017-09-18

This unit comprises the straightforward synthesis of protected 2'-deoxyribonucleoside-O-methyl-(H)-phosphinates in both 3'- and 5'-series. These compounds represent a new class of monomers compatible with the solid-phase synthesis of oligonucleotides using H-phosphonate chemistry and are suitable for the preparation of both 3'- and 5'-O-methylphosphonate oligonucleotides. The synthesis of 4-toluenesulfonyloxymethyl-(H)-phosphinic acid as a new reagent for the preparation of O-methyl-(H)-phosphinic acid derivatives is described. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Microwave-assisted synthesis of medicinally relevant indoles.

PubMed

Patil, S A; Patil, R; Miller, D D

2011-01-01

Indoles represent an important structural class in medicinal chemistry with broad spectrum of biological activities. The synthesis of indoles, therefore, has attracted enormous attention from synthetic chemists. Microwave methods for the preparation of indole analogs have been developed to speed up the synthesis, therefore, microwave assisted organic synthesis (MAOS) in controlled conditions is an invaluable technique for medicinal chemistry. In this review, indole forming classical reactions such as Fischer, Madelung, Bischler-Mohlau, Batcho-Leimgruber, Hemetsberger-Knittel, Graebe-Ullmann, Diels-Alder and Wittig type reactions using microwave radiation has been summarized. In addition, metal mediated cyclizations along with solid phase synthesis of indoles have been discussed. © 2011 Bentham Science Publishers Ltd.

A study of room-temperature Li xMn 1.5Ni 0.5O 4 solid solutions

DOE PAGES

Saravanan, Kuppan; Jarry, Angelique; Kostecki, Robert; ...

2015-01-26

Understanding the kinetic implication of solid-solution vs. biphasic reaction pathways is critical for the development of advanced intercalation electrode materials. Yet this has been a long-standing challenge in materials science due to the elusive metastable nature of solid solution phases. The present study reports the synthesis, isolation, and characterization of room-temperature Li xMn 1.5Ni 0.5O 4 solid solutions. In situ XRD studies performed on pristine and chemically-delithiated, micron-sized single crystals reveal the thermal behavior of Li xMn 1.5Ni 0.5O 4 (0 ≤ x ≤ 1) cathode material consisting of three cubic phases: LiMn 1.5Ni 0.5O 4 (Phase I), Li 0.5Mnmore » 1.5Ni 0.5O 4 (Phase II) and Mn 1.5Ni 0.5O 4 (Phase III). A phase diagram capturing the structural changes as functions of both temperature and Li content was established. In conclusion, the work not only demonstrates the possibility of synthesizing alternative electrode materials that are metastable in nature, but also enables in-depth evaluation on the physical, electrochemical and kinetic properties of transient intermediate phases and their role in battery electrode performance.« less

Synthesis of Ca(BH4)2 from Synthetic Colemanite Used in Hydrogen Storage by Mechanochemical Reaction

NASA Astrophysics Data System (ADS)

Karabulut, Ahmet F.; Guru, Metin; Boynueğri, Tuğba A.; Aydin, Mustafa Yasir

2016-08-01

In this study, synthesis of Ca(BH4)2 has been carried out with a solid phase reaction in which synthetic colemanite has been used as a raw material. Three dimensional high energy spex collider was selected for this mechanochemical reaction. Calcium borohydride is one of the most valuable metal borohydrides. In order to produce calcium borohydride economically, anhydrous colemanite mineral has been used as reactant. Calcium borohydride has been directly manufactured from anhydrous colemanite in spex-type ball milling without the need for any intermediate product. Thus, the advantages of this method over wet chemical procedure (such as having no intermediate product, no azeotropic limitations and no need of regaining product from solution after production by using evaporation, crystallization and drying processes) have made it possible to achieve the desired economical gains. Parametric experiments were conducted to determine the best conditions for the highest yield of solid phase reaction in the spex-type ball milling. Best results have been determined by using areas of related peaks in spectra of Fourier transform infrared spectroscopy (FT-IR). In order to use peaks area for determining Ca(BH4)2 concentration, a calibration graph of FT-IR absorbance peak areas has been created by using samples with known different concentrations of commercial Ca(BH4)2. Optimum amounts of calcium hydride and synthesis reaction time were found to be 2.1 times the stoichiometric ratio and 2500 min, respectively. As a result of these optimizations, the maximum yield of the solid phase reaction carried out by the spex-type ball milling has been determined as 93%.

Solid state synthesis of poly(dichlorophosphazene)

DOEpatents

Allen, Christopher W.; Hneihen, Azzam S.; Peterson, Eric S.

2001-01-01

A method for making poly(dichlorophosphazene) using solid state reactants is disclosed and described. The present invention improves upon previous methods by removing the need for chlorinated hydrocarbon solvents, eliminating complicated equipment and simplifying the overall process by providing a "single pot" two step reaction sequence. This may be accomplished by the condensation reaction of raw materials in the melt phase of the reactants and in the absence of an environmentally damaging solvent.

Library design practices for success in lead generation with small molecule libraries.

PubMed

Goodnow, R A; Guba, W; Haap, W

2003-11-01

The generation of novel structures amenable to rapid and efficient lead optimization comprises an emerging strategy for success in modern drug discovery. Small molecule libraries of sufficient size and diversity to increase the chances of discovery of novel structures make the high throughput synthesis approach the method of choice for lead generation. Despite an industry trend for smaller, more focused libraries, the need to generate novel lead structures makes larger libraries a necessary strategy. For libraries of a several thousand or more members, solid phase synthesis approaches are the most suitable. While the technology and chemistry necessary for small molecule library synthesis continue to advance, success in lead generation requires rigorous consideration in the library design process to ensure the synthesis of molecules possessing the proper characteristics for subsequent lead optimization. Without proper selection of library templates and building blocks, solid phase synthesis methods often generate molecules which are too heavy, too lipophilic and too complex to be useful for lead optimization. The appropriate filtering of virtual library designs with multiple computational tools allows the generation of information-rich libraries within a drug-like molecular property space. An understanding of the hit-to-lead process provides a practical guide to molecular design characteristics. Examples of leads generated from library approaches also provide a benchmarking of successes as well as aspects for continued development of library design practices.

Preparation and biological evaluation of [(99m)Tc/EDDA/Tricine/HYNIC(0), BzThi(3)]-octreotide for somatostatin receptor-positive tumor imaging.

PubMed

Erfani, Mostafa; Shafiei, Mohammad; Mazidi, Mohammad; Goudarzi, Mostafa

2013-04-01

Somatostatin-derived analogues play an important role in the diagnosis and treatment of neuroendocrine tumors. The aim of this study was to evaluate a new somatostatin analogue designed for labeling with (99m)Tc: [6-hydrazinopyridine-3-carboxylic acid (HYNIC(0)), β-(3-benzothienyl)-Ala (BzThi(3))]-octreotide ([HYNIC]-BOC), using ethylenediamine-N,N'-diacetic acid (EDDA) and tricine as coligands. Synthesis was performed on a solid phase using a standard Fmoc strategy. The HYNIC-peptide conjugate was radiolabeled with (99m)Tc and characterized by ITLC and high-performance liquid chromatography (HPLC). In vitro studies were carried out in sstr2 expressing AR4-2J cell lines. In vivo distribution studies were performed in rats bearing the AR4-2J tumor. The radiolabeled complex could be prepared at high-specific activities and >95% radiochemical yield as determined by HPLC. The peptide conjugate showed high-affinity binding for sstr2. The radioligand showed high and specific internalization into AR4-2J cells (18.19%±0.21% at 4 hours). In vivo distribution studies in rats bearing tumor have shown a receptor-specific uptake of radioactivity in somatostatin receptor-positive organs. After 4 hours, uptake in the AR4-2J tumor was 1.71%±0.36% injected dose per gram tissue (%ID/g). These data show that [(99m)Tc/EDDA/Tricine/HYNIC(0), BzThi(3)]-octreotide is a specific radioligand for the somatostatin receptor-positive tumors and is a suitable candidate for clinical studies.

Extendable nickel complex tapes that reach NIR absorptions.

PubMed

Audi, Hassib; Chen, Zhongrui; Charaf-Eddin, Azzam; D'Aléo, Anthony; Canard, Gabriel; Jacquemin, Denis; Siri, Olivier

2014-12-14

Stepwise synthesis of linear nickel complex oligomer tapes with no need for solid-phase support has been achieved. The control of the length in flat arrays allows a fine-tuning of the absorption properties from the UV to the NIR region.

Targeting hypoxia in tumors using 2-nitroimidazoles with peptidic chelators for technetium-99m: effect of lipophilicity.

PubMed

Zhang, X; Su, Z F; Ballinger, J R; Rauth, A M; Pollak, A; Thornback, J R

2000-01-01

Tumor hypoxia is an important prognostic factor for response to therapy. Radiolabeled 2-nitroimidazoles have been used for imaging hypoxia, and the octanol/water partition coefficient (P) of these compounds appears to play a crucial role in their suitability for imaging. A series of 11 2-nitroimidazoles coupled to peptidic chelators for (99m)Tc with divergent P was developed and evaluated in an in vitro system. Two classes of N(3)S chelators were used: dialkyl-Gly-Ser-Cys-linker-2-nitroimidazole (Class I) and dialkyl-Gly-Lys(2-nitroimidazole)-Cys (Class II). The chelators were prepared by automated solid-phase peptide synthesis. Xanthine oxidase was able to reduce the 2-nitroimidiazole moiety on the ligands, but the rate of reduction varied 5-fold among the different chelators. The chelators were labeled by transchelation from [(99m)Tc]gluconate at temperatures between 22 and 100 degrees C. The reaction mixtures were analyzed by HPLC and their P values determined. The accumulation of each complex in suspension cultures of Chinese hamster ovary cells incubated under aerobic or extremely hypoxic conditions was determined. Radiochemical yields ranged from 5 to 80% for the 11 compounds. HPLC showed that some of the compounds formed two complexes with (99m)Tc, possibly syn and anti conformations with respect to the Tc=O bond. In general, the Class I chelators labeled more readily than the class II chelators. The P values of the (99m)Tc complexes varied from 0.0002 to 5 and were generally in accordance with predictions based on structure. There were also differences in P as a function of pH; the free acids had a lower P at pH 7.4 than at pH 2.0 due to ionization, whereas the amides did not show this effect. Accumulation levels in aerobic cells were related to P but varied over a narrow range. Four of the 11 compounds showed selective accumulation in hypoxic cells. The peptidic class of 2-nitroimidazoles, with flexible design and convenient solid-phase synthesis, deserves further study as agents for imaging hypoxia in tumors.

Synthesis of refractory organic matter in the ionized gas phase of the solar nebula

PubMed Central

Kuga, Maïa; Marty, Bernard; Marrocchi, Yves; Tissandier, Laurent

2015-01-01

In the nascent solar system, primitive organic matter was a major contributor of volatile elements to planetary bodies, and could have played a key role in the development of the biosphere. However, the origin of primitive organics is poorly understood. Most scenarios advocate cold synthesis in the interstellar medium or in the outer solar system. Here, we report the synthesis of solid organics under ionizing conditions in a plasma setup from gas mixtures (H2(O)−CO−N2−noble gases) reminiscent of the protosolar nebula composition. Ionization of the gas phase was achieved at temperatures up to 1,000 K. Synthesized solid compounds share chemical and structural features with chondritic organics, and noble gases trapped during the experiments reproduce the elemental and isotopic fractionations observed in primitive organics. These results strongly suggest that both the formation of chondritic refractory organics and the trapping of noble gases took place simultaneously in the ionized areas of the protoplanetary disk, via photon- and/or electron-driven reactions and processing. Thus, synthesis of primitive organics might not have required a cold environment and could have occurred anywhere the disk is ionized, including in its warm regions. This scenario also supports N2 photodissociation as the cause of the large nitrogen isotopic range in the solar system. PMID:26039983

Preparation of Conjugates of Cytotoxic Lupane Triterpenes with Biotin.

PubMed

Soural, Miroslav; Hodon, Jiri; Dickinson, Niall J; Sidova, Veronika; Gurska, Sona; Dzubak, Petr; Hajduch, Marian; Sarek, Jan; Urban, Milan

2015-12-16

To better understand the mechanism of action of antitumor triterpenes, we are developing methods to identify their molecular targets. A promising method is based on combination of quantitative proteomics with SILAC and uses active compounds anchored to magnetic beads via biotin-streptavidin interaction. We developed a simple and fast solid-phase synthetic technique to connect terpenes to biotin through a linker. Betulinic acid was biotinylated from three different conjugation sites for use as a standard validation tool since many molecular targets of this triterpene are already known. Then, a set of four other cytotoxic triterpenoids was biotinylated. Biotinylated terpenes were similarly cytotoxic to their nonbiotinylated parents, which suggests that the target identification should not be influenced by linker or biotin. The developed solid-phase synthetic approach is the first attempt to use solid-phase synthesis to connect active triterpenes to biotin and is applicable as a general procedure for routine conjugation of triterpenes with other molecules of choice.

A novel method for the synthesis of zirconia powder

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bohe, A.E.; Pasquevich, D.M.

A novel method for the synthesis of zirconia powder is presented in this paper. The formation of fine particles of zirconia takes place when metallic zirconium and hematite are heated in the presence of gaseous chlorine. The overall process, which can be described by the following reaction: 3 Zr(s) + 2 Fe{sub 2}O{sub 3}(s) {r_arrow} 3 ZrO{sub 2}(s) + 4 Fe(s), occurs by a mass-transport mechanism through the vapor phase between 723 and 1223 K. The vapor-mass transport among the solid species takes place by means of zirconium and iron chlorides. The fundamentals of synthesis are discussed on the basismore » of a detailed thermodynamic analysis of reactions involved in the process, as well as by a characterization of the solid phases formed at various temperatures at XRD and SEM examinations.« less

Synthesis and profiling of a diverse collection of azetidine-based scaffolds for the development of CNS-focused lead-like libraries.

PubMed

Lowe, Jason T; Lee, Maurice D; Akella, Lakshmi B; Davoine, Emeline; Donckele, Etienne J; Durak, Landon; Duvall, Jeremy R; Gerard, Baudouin; Holson, Edward B; Joliton, Adrien; Kesavan, Sarathy; Lemercier, Berenice C; Liu, Haibo; Marié, Jean-Charles; Mulrooney, Carol A; Muncipinto, Giovanni; Welzel-O'Shea, Morgan; Panko, Laura M; Rowley, Ann; Suh, Byung-Chul; Thomas, Meryl; Wagner, Florence F; Wei, Jingqiang; Foley, Michael A; Marcaurelle, Lisa A

2012-09-07

The synthesis and diversification of a densely functionalized azetidine ring system to gain access to a wide variety of fused, bridged, and spirocyclic ring systems is described. The in vitro physicochemical and pharmacokinetic properties of representative library members are measured in order to evaluate the use of these scaffolds for the generation of lead-like molecules to be used in targeting the central nervous system. The solid-phase synthesis of a 1976-membered library of spirocyclic azetidines is also described.

Synthesis of sub-10 nm solid lipid nanoparticles for topical and biomarker detection applications

NASA Astrophysics Data System (ADS)

Calderón-Colón, Xiomara; Patchan, Marcia W.; Theodore, Mellisa L.; Le, Huong T.; Sample, Jennifer L.; Benkoski, Jason J.; Patrone, Julia B.

2014-02-01

Solid lipid nanoparticles (SLNs) are a promising platform for sensing in vivo biomarkers due to their biocompatibility, stability, and their ability to carry a wide range of active ingredients. The skin is a prominent target organ for numerous inflammatory and stress-related biomarkers, making it an excellent site for early detection of physiological imbalance and application of sensory nanoparticles. Though smaller particle size has generally been correlated with increased penetration of skin models, there has been little attention paid to the significance of other nanoparticle synthesis parameters with respect to their physical properties. In this study, we demonstrate the synthesis of sub-10 nm SLNs by the phase inversion temperature (PIT) method. These particles were specifically designed for topical delivery of hydrogen peroxide-detecting chemiluminescent dyes. A systematic design of experiments approach was used to investigate the role of the processing variables on SLN form and properties. The processing variables were correlated with the SLN properties (e.g., dye solubility, phase inversion temperature, particle size, polydispersity, melting point, and latent heat of melting). Statistical analysis revealed that the PIT method, while allowing total control over the thermal properties, resulted in well-controlled synthesis of ultra-small particles, while allowing great flexibility in the processing conditions and incorporated compounds.

LiMn2O4–yBryNanoparticles Synthesized by a Room Temperature Solid-State Coordination Method

PubMed Central

2009-01-01

LiMn2O4–yBrynanoparticles were synthesized successfully for the first time by a room temperature solid-state coordination method. X-ray diffractometry patterns indicated that the LiMn2O4–yBrypowders were well-crystallized pure spinel phase. Transmission electron microscopy images showed that the LiMn2O4–yBrypowders consisted of small and uniform nanosized particles. Synthesis conditions such as the calcination temperature and the content of Br−were investigated to optimize the ideal condition for preparing LiMn2O4–yBrywith the best electrochemical performances. The optimized synthesis condition was found in this work; the calcination temperature is 800 °C and the content of Br−is 0.05. The initial discharge capacity of LiMn2O3.95Br0.05obtained from the optimized synthesis condition was 134 mAh/g, which is far higher than that of pure LiMn2O4, indicating introduction of Br−in LiMn2O4is quite effective in improving the initial discharge capacity. PMID:20628635

Room temperature synthesis of copper indium diselenide in non-aqueous solution using an organoindium reagent

NASA Technical Reports Server (NTRS)

Hepp, Aloysius F.; Andras, Maria T.; Bailey, Sheila G.; Duraj, Stan A.

1992-01-01

A novel two-phase synthesis of CuInSe2 at 25 C from Cu2Se and Cp3In in 4-methylpyridine has been discovered. Characterization of the material produced shows it to be platelet-shaped crystallites with an average particle size of 10 microns, less than 2 percent C and H, with a small amount of unidentified crystalline impurity. The results demonstrate that it is possible to produce from solution a material that is ordinarily synthesized in bulk or films at much higher temperatures or using extraneous reagents and/or electrons. The use of a solid-state reagent as a starting material which is converted to another solid-state compound by an organometallic reagent has tremendous potential to produce precursors for a wide range of solid-state materials of interest to the electronics, defense, and aerospace communities.

Scope & Limitations of Fmoc Chemistry SPPS-Based Approaches to the Total Synthesis of Insulin Lispro via Ester Insulin

PubMed Central

Dhayalan, Balamurugan; Mandal, Kalyaneswar; Rege, Nischay; Weiss, Michael A.; Eitel, Simon H.; Meier, Thomas; Schoenleber, Ralph O.; Kent, Stephen B.H.

2017-01-01

We have systematically explored three approaches based on Fmoc chemistry SPPS for the total chemical synthesis of the key depsipeptide intermediate for the efficient total chemical synthesis of insulin. The approaches used were: stepwise Fmoc chemistry SPPS; the ‘hybrid method’, in which maximally-protected peptide segments made by Fmoc chemistry SPPS are condensed in solution; and, native chemical ligation using peptide-thioester segments generated by Fmoc chemistry SPPS. A key building block in all three approaches was a Glu[Oβ(Thr)] ester-linked dipeptide equipped with a set of orthogonal protecting groups compatible with Fmoc chemistry SPPS. The most effective method for the preparation of the 51 residue ester-linked polypeptide chain of ester insulin was the use of unprotected peptide-thioester segments, prepared from peptide-hydrazides synthesized by Fmoc chemistry SPPS, and condensed by native chemical ligation. High resolution X-ray crystallography confirmed the disulfide pairings and three-dimensional structure of synthetic insulin lispro prepared from ester insulin lispro by this route. Further optimization of these pilot studies should yield an effective total chemical synthesis of insulin lispro (Humalog) based on peptide synthesis by Fmoc chemistry SPPS. PMID:27905149

Programmable polyproteams built using twin peptide superglues

PubMed Central

Veggiani, Gianluca; Nakamura, Tomohiko; Brenner, Michael D.; Yan, Jun; Robinson, Carol V.; Howarth, Mark

2016-01-01

Programmed connection of amino acids or nucleotides into chains introduced a revolution in control of biological function. Reacting proteins together is more complex because of the number of reactive groups and delicate stability. Here we achieved sequence-programmed irreversible connection of protein units, forming polyprotein teams by sequential amidation and transamidation. SpyTag peptide is engineered to spontaneously form an isopeptide bond with SpyCatcher protein. By engineering the adhesin RrgA from Streptococcus pneumoniae, we developed the peptide SnoopTag, which formed a spontaneous isopeptide bond to its protein partner SnoopCatcher with >99% yield and no cross-reaction to SpyTag/SpyCatcher. Solid-phase attachment followed by sequential SpyTag or SnoopTag reaction between building-blocks enabled iterative extension. Linear, branched, and combinatorial polyproteins were synthesized, identifying optimal combinations of ligands against death receptors and growth factor receptors for cancer cell death signal activation. This simple and modular route to programmable “polyproteams” should enable exploration of a new area of biological space. PMID:26787909

A multiphase transitioning peptide hydrogel for suturing ultrasmall vessels

NASA Astrophysics Data System (ADS)

Smith, Daniel J.; Brat, Gabriel A.; Medina, Scott H.; Tong, Dedi; Huang, Yong; Grahammer, Johanna; Furtmüller, Georg J.; Oh, Byoung Chol; Nagy-Smith, Katelyn J.; Walczak, Piotr; Brandacher, Gerald; Schneider, Joel P.

2016-01-01

Many surgeries are complicated by the need to anastomose, or reconnect, micrometre-scale vessels. Although suturing remains the gold standard for anastomosing vessels, it is difficult to place sutures correctly through collapsed lumen, making the procedure prone to failure. Here, we report a multiphase transitioning peptide hydrogel that can be injected into the lumen of vessels to facilitate suturing. The peptide, which contains a photocaged glutamic acid, forms a solid-like gel in a syringe and can be shear-thin delivered to the lumen of collapsed vessels (where it distends the vessel) and the space between two vessels (where it is used to approximate the vessel ends). Suturing is performed directly through the gel. Light is used to initiate the final gel-sol phase transition that disrupts the hydrogel network, allowing the gel to be removed and blood flow to resume. This gel adds a new tool to the armamentarium for micro- and supermicrosurgical procedures.

Synthesis of refractory organic matter in the ionized gas phase of the solar nebula.

PubMed

Kuga, Maïa; Marty, Bernard; Marrocchi, Yves; Tissandier, Laurent

2015-06-09

In the nascent solar system, primitive organic matter was a major contributor of volatile elements to planetary bodies, and could have played a key role in the development of the biosphere. However, the origin of primitive organics is poorly understood. Most scenarios advocate cold synthesis in the interstellar medium or in the outer solar system. Here, we report the synthesis of solid organics under ionizing conditions in a plasma setup from gas mixtures (H2(O)-CO-N2-noble gases) reminiscent of the protosolar nebula composition. Ionization of the gas phase was achieved at temperatures up to 1,000 K. Synthesized solid compounds share chemical and structural features with chondritic organics, and noble gases trapped during the experiments reproduce the elemental and isotopic fractionations observed in primitive organics. These results strongly suggest that both the formation of chondritic refractory organics and the trapping of noble gases took place simultaneously in the ionized areas of the protoplanetary disk, via photon- and/or electron-driven reactions and processing. Thus, synthesis of primitive organics might not have required a cold environment and could have occurred anywhere the disk is ionized, including in its warm regions. This scenario also supports N2 photodissociation as the cause of the large nitrogen isotopic range in the solar system.

Synthesis and superconductivity of highly underdoped HgBa2CuO4+δ

NASA Astrophysics Data System (ADS)

Edwards, P. P.; Gameson, I.; Fletcher, A.; Peacock, G. B.

1998-05-01

The highest transition temperature superconductors are found within the complex homologous series HgBa2Can-1CunO2n+2+δ (n=1-7), with the third member, HgBa2Ca2Cu3O8+δ possessing the record-high transition temperature (Tc) of 135 K at room pressure. The first member of this family, HgBa2CuO4+δ having a Tc of up to 97 K, displays the highest transition temperature for any analogous compounds with a single copper-layer. The chemical reaction for the formation of this material is intrinsically complex due to the natural high volatility of mercury-bearing compounds; chemical synthesis has been postulated to proceed via a solid-vapour reaction. With this in mind, we have developed a mixed solid/vapour phase synthesis for HgBa2CuO4+δ using what one might term a `remote' source of mercury, in this case elemental Hg itself. Interestingly, because of the zero oxidation state of elemental mercury in the reagent mixture, the synthesis reaction proceeds under reducing conditions. By this route, a highly underdoped state (Tc<=35 K) of the superconducting phase HgBa2CuO4+δ is readily obtained. This level of underdoping is extremely difficult to achieve by more conventional synthetic routes. We comment on the unusually high oxygen affinity of the resulting underdoped compound, in relation to other cuprate superconductors, and the implied mobility of oxygen defects within the crystal structure.

Conformational Plasticity of the Cell-Penetrating Peptide SAP As Revealed by Solid-State 19F-NMR and Circular Dichroism Spectroscopies.

PubMed

Afonin, Sergii; Kubyshkin, Vladimir; Mykhailiuk, Pavel K; Komarov, Igor V; Ulrich, Anne S

2017-07-13

The cell-penetrating peptide SAP, which was designed as an amphipathic poly-l-proline helix II (PPII), was suggested to self-assemble into regular fibrils that are relevant for its internalization. Herein we have analyzed the structure of SAP in the membrane-bound state by solid-state 19 F-NMR, which revealed other structural states, in addition to the expected surface-aligned PPII. Trifluoromethyl-bicyclopentyl-glycine (CF 3 -Bpg) and two rigid isomers of trifluoromethyl-4,5-methanoprolines (CF 3 -MePro) were used as labels for 19 F-NMR analysis. The equilibria between different conformations of SAP were studied and were found to be shifted by the substituents at Pro-11. Synchrotron-CD results suggested that substituting Pro-11 by CF 3 -MePro governed the coil-to-PPII equilibrium in solution and in the presence of a lipid bilayer. Using CD and 19 F-NMR, we examined the slow kinetics of the association of SAP with membranes and the dependence of the SAP conformational dynamics on the lipid composition. The peptide did not bind to lipids in the solid ordered phase and aggregated only in the liquid ordered "raft"-like bilayers. Self-association could not be detected in solution or in the presence of liquid disordered membranes. Surface-bound amphipathic SAP in a nonaggregated state was structured as a mixture of nonideal extended conformations reflecting the equilibrium already present in solution, i.e., before binding to the membrane.

First principles materials design of novel functional oxides

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cooper, Valentino R.; Voas, Brian K.; Bridges, Craig A.

2016-05-31

We review our efforts to develop and implement robust computational approaches for exploring phase stability to facilitate the prediction-to-synthesis process of novel functional oxides. These efforts focus on a synergy between (i) electronic structure calculations for properties predictions, (ii) phenomenological/empirical methods for examining phase stability as related to both phase segregation and temperature-dependent transitions and (iii) experimental validation through synthesis and characterization. We illustrate this philosophy by examining an inaugural study that seeks to discover novel functional oxides with high piezoelectric responses. Lastly, our results show progress towards developing a framework through which solid solutions can be studied to predictmore » materials with enhanced properties that can be synthesized and remain active under device relevant conditions.« less

The Synthesis of Beta-Peptides Containing Guanidino Groups

NASA Technical Reports Server (NTRS)

Wen, Ke; Han, Hyunsoo; Hoffman, Timothy Z.; Janda, Kim D.; Orgel, Leslie E.

2003-01-01

The synthesis of the beta-peptide 1 by the postsynthetic modification of the corresponding amino-containing peptide 3 is described. The potential of 1 to act as a template for the ligation of complementary negatively-charged peptides is discussed.

Immobilization and functional reconstitution of antibody Fab fragment by solid-phase refolding.

PubMed

Kumada, Yoichi; Hamasaki, Kyoto; Nakagawa, Aya; Sasaki, Eiju; Shirai, Tatsunori; Okumura, Masahiro; Inoue, Manami; Kishimoto, Michimasa

2013-12-31

In this study, we demonstrated the successful preparation of a Fab antibody-immobilized hydrophilic polystyrene (phi-PS) plate via one- and two-step solid-phase refolding methods. Both polystyrene-binding peptide (PS-tag)-fused Fd fragment of heavy chain (Fab H-PS) and full-length of light-chain (Fab L-PS) were individually produced in insoluble fractions of Escherichia coli cells, and they were highly purified in the presence of 8M of urea. Antigen-binding activities of Fab antibody immobilized were correctly recovered by the one-step solid-phase refolding method that a mixture of Fab H-PS and Fab L-PS was immobilized in the presence of 0.5-2M urea, followed by surface washing of the phi-PS plate with PBST. These results indicate that by genetic fusion of a PS-tag, a complex between Fab H and Fab L was efficiently immobilized on the surface of a phi-PS plate even in the presence of a low concentration of urea, and was then correctly refolded to retain its high antigen-binding activity via removal of the urea. A two-step solid-phase refolding method whereby Fab H-PS and Fab L-PS were successively refolded on the surface of a phi-PS plate also resulted in Fab antibody formation on the plate. Furthermore, both the binding affinity and the specificity of the Fab antibody produced by the two-step method were highly maintained, according to the results of sandwich ELISA and competitive ELISA using Fab antibody-immobilized plate via two-step solid-phase refolding. Thus, the solid-phase refolding method demonstrated in this study should be quite useful for the preparation of a Fab antibody-immobilized PS surface with high efficiency from individually produced Fab H-PS and Fab L-PS. This method will be applicable to the preparation of a large Fab antibody library on the surface of a PS plate for use in antibody screening. © 2013. Published by Elsevier B.V. All rights reserved.

Programmable polyproteams built using twin peptide superglues.

PubMed

Veggiani, Gianluca; Nakamura, Tomohiko; Brenner, Michael D; Gayet, Raphaël V; Yan, Jun; Robinson, Carol V; Howarth, Mark

2016-02-02

Programmed connection of amino acids or nucleotides into chains introduced a revolution in control of biological function. Reacting proteins together is more complex because of the number of reactive groups and delicate stability. Here we achieved sequence-programmed irreversible connection of protein units, forming polyprotein teams by sequential amidation and transamidation. SpyTag peptide is engineered to spontaneously form an isopeptide bond with SpyCatcher protein. By engineering the adhesin RrgA from Streptococcus pneumoniae, we developed the peptide SnoopTag, which formed a spontaneous isopeptide bond to its protein partner SnoopCatcher with >99% yield and no cross-reaction to SpyTag/SpyCatcher. Solid-phase attachment followed by sequential SpyTag or SnoopTag reaction between building-blocks enabled iterative extension. Linear, branched, and combinatorial polyproteins were synthesized, identifying optimal combinations of ligands against death receptors and growth factor receptors for cancer cell death signal activation. This simple and modular route to programmable "polyproteams" should enable exploration of a new area of biological space.

Semisynthetic Enzymes by Protein-Peptide Site-Directed Covalent Conjugation: Methods and Applications.

PubMed

Palomo, Jose M

2017-01-01

This chapter describes the rational design and synthesis of semisynthetic lipases by site-directed incorporation of tailor-made peptides on the lipase-lid site to improve its activity, specificity, and enantioselectivity in specific biotransformations. Cysteine was genetically introduced at a particular point of the oligopeptide lid of the enzyme, and cysteine-containing peptides, complementary to the amino acid sequence on the lid site of Geobacillus thermocatenulatus lipase (BTL), were covalently attached on the lid of two different cysteine-BTL variants based on a fast thiol-disulfide exchange ligation followed by desulfurization. The BTL variants were initially immobilized on solid support to introduce the advantages of solid-state chemistry, such as quantitative transformations, easy purification, and recyclability. In the two different immobilized variants BTL-A193C and BTL-L230C, the cysteine was then activated with 2-dipyridyldisulfide to help the disulfide exchange with the peptide, generating the semisynthetic enzyme in high yield. Excellent results of improvement of activity and selectivity were obtained. For example, the peptide-BTL conjugate (at position 193) was 40-fold more active than the corresponding unmodified enzyme for the hydrolysis of per-acetylated thymidine at pH 5, or fourfold in the desymmetrization of dimethyl-3-phenylglutarate at pH 7. The new enzyme also exhibited excellent enantioselectivity in the desymmetrization reaction with enantiomeric excess (ee) of >99% when compared to that of the unmodified enzyme (ee=78%). © 2017 Elsevier Inc. All rights reserved.

Molten salt synthesis of nanocrystalline phase of high dielectric constant material CaCu3Ti4O12.

PubMed

Prakash, B Shri; Varma, K B R

2008-11-01

Nanocrystalline powders of giant dielectric constant material, CaCu3Ti4O12 (CCTO), have been prepared successfully by the molten salt synthesis (MSS) using KCl at 750 degrees C/10 h, which is significantly lower than the calcination temperature (approximately 1000 degrees C) that is employed to obtain phase pure CCTO in the conventional solid-state reaction route. The water washed molten salt synthesized powder, characterized by X-ray powder diffraction (XRD), Scanning electron microscopy (SEM), and Transmission electron microscopy (TEM) confirmed to be a phase pure CCTO associated with approximately 150 nm sized crystallites of nearly spherical shape. The decrease in the formation temperature/duration of CCTO in MSS method was attributed to an increase in the diffusion rate or a decrease in the diffusion length of reacting ions in the molten salt medium. As a consequence of liquid phase sintering, pellets of as-synthesized KCl containing CCTO powder exhibited higher sinterability and grain size than that of KCl free CCTO samples prepared by both MSS method and conventional solid-state reaction route. The grain size and the dielectric constant of KCl containing CCTO ceramics increased with increasing sintering temperature (900 degrees C-1050 degrees C). Indeed the dielectric constants of these ceramics were higher than that of KCl free CCTO samples prepared by both MSS method and those obtained via the solid-state reaction route and sintered at the same temperature. Internal barrier layer capacitance (IBLC) model was invoked to correlate the observed dielectric constant with the grain size in these samples.

Urinary C-peptide as a method for monitoring body mass changes in captive bonobos (Pan paniscus).

PubMed

Deschner, Tobias; Kratzsch, Jürgen; Hohmann, Gottfried

2008-11-01

In recent years methodological improvements have allowed for more precise estimates of nutrient intake in wild primates. However, estimates of energetic condition have remained relatively imprecise due to the difficulties of estimating digestive efficiency and energy expenditure in these animals. In the absence of a reliable intake-expenditure calculation, a method is needed that directly links changes in energetic condition, such as body mass, to physiological changes that can be detected via markers in body excretions such as urine or feces. One promising marker is C-peptide, a metabolic byproduct of insulin synthesis. Here we present the results of a food restriction experiment carried out in a group of captive bonobos (Pan paniscus). We measured changes in food availability and body mass and determined urinary C-peptide levels with the help of a time-resolved fluoroimmunoassay routinely used for measuring C-peptide in human blood. Urinary C-peptide levels decreased during a period of food restriction and increased again when food availability was continuously increased. During this refeeding phase an increase in body mass was significantly correlated with an increase in urinary C-peptide levels. Our results suggest that urinary C-peptide levels are an accurate indicator of individual energy balance. In conclusion, measuring C-peptide in urine is a promising method to quantify the energetic condition of wild apes.

Variable dimensionality in the uranium fluoride/2-methyl-piperazine system: Synthesis and structures of UFO-5, -6, and -7; Zero-, one-, and two-dimensional materials with unprecedented topologies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Francis, R.J.; Halasyamani, P.S.; Bee, J.S.

Recently, low temperature (T < 300 C) hydrothermal reactions of inorganic precursors in the presence of organic cations have proven highly productive for the synthesis of novel solid-state materials. Interest in these materials is driven by the astonishingly diverse range of structures produced, as well as by their many potential materials chemistry applications. This report describes the high yield, phase pure hydrothermal syntheses of three new uranium fluoride phases with unprecedented structure types. Through the systematic control of the synthesis conditions the authors have successfully controlled the architecture and dimensionality of the phase formed and selectively synthesized novel zero-, one-,more » and two-dimensional materials.« less

Monoclinic β-Li2TiO3 nanocrystalline particles employing novel urea assisted solid state route: Synthesis, characterization and sintering behavior

NASA Astrophysics Data System (ADS)

Tripathi, Biranchi M.; Mohanty, Trupti; Prakash, Deep; Tyagi, A. K.; Sinha, P. K.

2017-07-01

Pure phase monoclinic nano-crystalline Li2TiO3 powder was synthesized by a novel urea assisted solid state synthesis method using readily available and economical precursors. A single phase and well crystalline Li2TiO3 powder has been obtained at slightly lower temperature (600-700 °C) and shorter duration (2 h) as compared to the conventional solid state method. The proposed method has significant advantages in comparison to other viable methods mainly in terms of phase purity, powder properties and sinterability. Analysis of chemical composition using inductively coupled plasma atomic emission spectroscopy (ICP-AES) shows no loss of lithium from Li2TiO3 in the proposed method. The emergence of monoclinic Li2TiO3 phase was confirmed by X-ray diffraction (XRD) pattern of as-synthesized powder. The crystallite size of Li2TiO3 powder was calculated to be in the range of 15-80 nm, which varied as a function of urea composition and temperature. The morphology of as-prepared Li2TiO3 powders was examined by scanning electron microscope (SEM). The effect of urea composition on phase and morphology was investigated so as to delineate the role of urea. Upon sintering at < 1000 °C temperature, the Li2TiO3 powder compact attained about 98% of the theoretical density with fine grained (grain size: 2-3 μm) microstructure. It indicates excellent sinter-ability of Li2TiO3 powder synthesized by the proposed method. The fine grained structure is desirable for better tritium breeding performance of Li2TiO3. Electrochemical impedance spectroscopy at variable temperature showed good electrical properties of Li2TiO3. The proposed method is simple, anticipated to be cost effective and convenient to realise for large scale production of phase pure nanocrystalline and having significantly enhanced sinter-ability Li2TiO3 powder.

Synthesis of gold structures by gold-binding peptide governed by concentration of gold ion and peptide.

PubMed

Kim, Jungok; Kim, Dong-Hun; Lee, Sylvia J; Rheem, Youngwoo; Myung, Nosang V; Hur, Hor-Gil

2016-08-01

Although biological synthesis methods for the production of gold structures by microorganisms, plant extracts, proteins, and peptide have recently been introduced, there have been few reports pertaining to controlling their size and morphology. The gold ion and peptide concentrations affected on the size and uniformity of gold plates by a gold-binding peptide Midas-11. The higher concentration of gold ions produced a larger size of gold structures reached 125.5 μm, but an increased amount of Midas-11 produced a smaller size of gold platelets and increased the yield percentage of polygonal gold particles rather than platelets. The mechanisms governing factors controlling the production of gold structures were primarily related to nucleation and growth. These results indicate that the synthesis of gold architectures can be controlled by newly isolated and substituted peptides under different reaction conditions.

Uncovering the design rules for peptide synthesis of metal nanoparticles.

PubMed

Tan, Yen Nee; Lee, Jim Yang; Wang, Daniel I C

2010-04-28

Peptides are multifunctional reagents (reducing and capping agents) that can be used for the synthesis of biocompatible metal nanoparticles under relatively mild conditions. However, the progress in peptide synthesis of metal nanoparticles has been slow due to the lack of peptide design rules. It is difficult to establish sequence-reactivity relationships from peptides isolated from biological sources (e.g., biomineralizing organisms) or selected by combinatorial display libraries because of their widely varying compositions and structures. The abundance of random and inactive amino acid sequences in the peptides also increases the difficulty in knowledge extraction. In this study, a "bottom-up" approach was used to formulate a set of rudimentary rules for the size- and shape-controlled peptide synthesis of gold nanoparticles from the properties of the 20 natural alpha-amino acids for AuCl(4)(-) reduction and binding to Au(0). It was discovered that the reduction capability of a peptide depends on the presence of certain reducing amino acid residues, whose activity may be regulated by neighboring residues with different Au(0) binding strengths. Another finding is the effect of peptide net charge on the nucleation and growth of the Au nanoparticles. On the basis of these understandings, several multifunctional peptides were designed to synthesize gold nanoparticles in different morphologies (nanospheres and nanoplates) and with sizes tunable by the strategic placement of selected amino acid residues in the peptide sequence. The methodology presented here and the findings are useful for establishing the scientific basis for the rational design of peptides for the synthesis of metal nanostructures.

Mycoestrogen determination in cow milk: Magnetic solid-phase extraction followed by liquid chromatography and tandem mass spectrometry analysis.

PubMed

Capriotti, Anna Laura; Cavaliere, Chiara; Foglia, Patrizia; La Barbera, Giorgia; Samperi, Roberto; Ventura, Salvatore; Laganà, Aldo

2016-12-01

Recently, magnetic solid-phase extraction has gained interest because it presents various operational advantages over classical solid-phase extraction. Furthermore, magnetic nanoparticles are easy to prepare, and various materials can be used in their synthesis. In the literature, there are only few studies on the determination of mycoestrogens in milk, although their carryover in milk has occurred. In this work, we wanted to develop the first (to the best of our knowledge) magnetic solid-phase extraction protocol for six mycoestrogens from milk, followed by liquid chromatography and tandem mass spectrometry analysis. Magnetic graphitized carbon black was chosen as the adsorbent, as this carbonaceous material, which is very different from the most diffuse graphene and carbon nanotubes, had already shown selectivity towards estrogenic compounds in milk. The graphitized carbon black was decorated with Fe 3 O 4 , which was confirmed by the characterization analyses. A milk deproteinization step was avoided, using only a suitable dilution in phosphate buffer as sample pretreatment. The overall process efficiency ranged between 52 and 102%, whereas the matrix effect considered as signal suppression was below 33% for all the analytes even at the lowest spiking level. The obtained method limits of quantification were below those of other published methods that employ classical solid-phase extraction protocols. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Electrochemical Control of Peptide Self-Organization on Atomically Flat Solid Surfaces: A Case Study with Graphite.

PubMed

Seki, Takakazu; So, Christopher R; Page, Tamon R; Starkebaum, David; Hayamizu, Yuhei; Sarikaya, Mehmet

2018-02-06

The nanoscale self-organization of biomolecules, such as proteins and peptides, on solid surfaces under controlled conditions is an important issue in establishing functional bio/solid soft interfaces for bioassays, biosensors, and biofuel cells. Electrostatic interaction between proteins and surfaces is one of the most essential parameters in the adsorption and self-assembly of proteins on solid surfaces. Although the adsorption of proteins has been studied with respect to the electrochemical surface potential, the self-assembly of proteins or peptides forming well-organized nanostructures templated by lattice structure of the solid surfaces has not been studied in the relation to the surface potential. In this work, we utilize graphite-binding peptides (GrBPs) selected by the phage display method to investigate the relationship between the electrochemical potential of the highly ordered pyrolytic graphite (HOPG) and peptide self-organization forming long-range-ordered structures. Under modulated electrical bias, graphite-binding peptides form various ordered structures, such as well-ordered nanowires, dendritic structures, wavy wires, amorphous (disordered) structures, and islands. A systematic investigation of the correlation between peptide sequence and self-organizational characteristics reveals that the presence of the bias-sensitive amino acid modules in the peptide sequence has a significant effect on not only surface coverage but also on the morphological features of self-assembled structures. Our results show a new method to control peptide self-assembly by means of applied electrochemical bias as well as peptide design-rules for the construction of functional soft bio/solid interfaces that could be integrated in a wide range of practical implementations.

Perovskite solid solutions with multiferroic morphotropic phase boundaries and property enhancement

NASA Astrophysics Data System (ADS)

Algueró, M.; Amorín, H.; Fernández-Posada, C. M.; Peña, O.; Ramos, P.; Vila, E.; Castro, A.

2016-05-01

Recently, large phase-change magnetoelectric response has been anticipated by a first-principles investigation of phases in the BiFeO3-BiCoO3 perovskite binary system, associated with the existence of a discontinuous morphotropic phase boundary (MPB) between multiferroic polymorphs of rhombohedral and tetragonal symmetries. This might be a general property of multiferroic phase instabilities, and a novel promising approach for room temperature magnetoelectricity. We review here our current investigations on the identification and study of additional material systems, alternative to BiFeO3-BiCoO3 that has only been obtained by high pressure synthesis. Three systems, whose phase diagrams were, in principle, liable to show multiferroic MPBs have been addressed: the BiMnO3-PbTiO3 and BiFeO3-PbTiO3 binary systems, and the BiFeO3-BiMnO3-PbTiO3 ternary one. A comprehensive study of multiferroism across different solid solutions was carried out based on electrical and magnetic characterizations, complemented with mechanical and electromechanical measurements. An in-depth structural analysis was also accomplished when necessary.

Synthesis, structural and semiconducting properties of Ba(Cu1/3 Sb2/3)O3-PbTiO3 solid solutions

NASA Astrophysics Data System (ADS)

Singh, Chandra Bhal; Kumar, Dinesh; Prashant, Verma, Narendra Kumar; Singh, Akhilesh Kumar

2018-05-01

We report the synthesis and properties of a new solid solution 0.05Ba(Cu1/3Sb2/3)O3-0.95PbTiO3 (BCS-PT) which shows the semiconducting properties. In this study, we have designed new perovskite-type (ABO3) solid solution of BCS-PT that have tunable optical band gap. BCS-PT compounds were prepared by conventional solid-state reaction method and their structural, micro-structural and optical properties were analyzed. The calcination temperature for BCS-PT solid solutions has been optimized to obtain a phase pure system. The Reitveld analysis of X-ray data show that all samples crystallize in tetragonal crystal structure with space group P4mm. X-ray investigation revealed that increase in calcination temperature led to increase of lattice parameter `a' while `c' parameter value lowered. The band gap of PbTiO3 is reduced from 3.2 eV to 2.8 eV with BCS doping and with increasing calcination temperature it further reduces to 2.56 eV. The reduced band gap indicated that the compounds are semiconducting and can be used for photovoltaic device applications.

The 3-5 semiconductor solid solution single crystal growth. [low gravity float zone growth experiments using gallium indium antimonides and cadmium tellurides

NASA Technical Reports Server (NTRS)

Gertner, E. R.

1980-01-01

Techniques used for liquid and vapor phase epitaxy of gallium indium arsenide are described and the difficulties encountered are examined. Results show that the growth of bulk III-V solid solution single crystals in a low gravity environment will not have a major technological impact. The float zone technique in a low gravity environment is demonstrated using cadmium telluride. It is shown that this approach can result in the synthesis of a class of semiconductors that can not be grown in normal gravity because of growth problems rooted in the nature of their phase diagrams.

Evidence for Novel β-Sheet Structures in Iowa Mutant β-Amyloid Fibrils†

PubMed Central

Tycko, Robert; Sciarretta, Kimberly L.; Orgel, Joseph P. R. O.; Meredith, Stephen C.

2009-01-01

Asp23-to-Asn mutation within the coding sequence of β-amyloid, called the Iowa mutation, is associated with early onset, familial Alzheimer’s disease and cerebral amyloid angiopathy, in which patients develop neuritic plaques and massive vascular deposition predominantly of the mutant peptide. We examined the mutant peptide, D23N-Aβ40, by electron microscopy, X-ray diffraction, and solid-state NMR spectroscopy. D23N-Aβ40 forms fibrils considerably faster than the wild-type peptide (k = 3.77 × 10-3 min-1 and 1.07 × 10-4 min-1 for D23N-Aβ40 and the wild-type peptide WT-Aβ40, respectively) and without a lag phase. Electron microscopy shows that D23N-Aβ40 forms fibrils with multiple morphologies. X-ray fiber diffraction shows a cross-β pattern, with a sharp reflection at 4.7 Å and a broad reflection at 9.4 Å, which is notably smaller than the value for WT-Aβ40 fibrils (10.4 Å). Solid-state NMR measurements indicate molecular level polymorphism of the fibrils, with only a minority of D23N-Aβ40 fibrils containing the in-register, parallel β-sheet structure commonly found in WT-Aβ40 fibrils and most other amyloid fibrils. Antiparallel β-sheet structures in the majority of fibrils are indicated by measurements of intermolecular distances through 13C-13C and 15N-13C dipole-dipole couplings. An intriguing possibility exists that there is a relationship between the aberrant structure of D23N-Aβ40 fibrils and the unusual vasculotropic clinical picture in these patients. PMID:19358576

Evidence for novel beta-sheet structures in Iowa mutant beta-amyloid fibrils.

PubMed

Tycko, Robert; Sciarretta, Kimberly L; Orgel, Joseph P R O; Meredith, Stephen C

2009-07-07

Asp23-to-Asn mutation within the coding sequence of beta-amyloid, called the Iowa mutation, is associated with early onset, familial Alzheimer's disease and cerebral amyloid angiopathy, in which patients develop neuritic plaques and massive vascular deposition predominantly of the mutant peptide. We examined the mutant peptide, D23N-Abeta40, by electron microscopy, X-ray diffraction, and solid-state NMR spectroscopy. D23N-Abeta40 forms fibrils considerably faster than the wild-type peptide (k = 3.77 x 10(-3) min(-1) and 1.07 x 10(-4) min(-1) for D23N-Abeta40 and the wild-type peptide WT-Abeta40, respectively) and without a lag phase. Electron microscopy shows that D23N-Abeta40 forms fibrils with multiple morphologies. X-ray fiber diffraction shows a cross-beta pattern, with a sharp reflection at 4.7 A and a broad reflection at 9.4 A, which is notably smaller than the value for WT-Abeta40 fibrils (10.4 A). Solid-state NMR measurements indicate molecular level polymorphism of the fibrils, with only a minority of D23N-Abeta40 fibrils containing the in-register, parallel beta-sheet structure commonly found in WT-Abeta40 fibrils and most other amyloid fibrils. Antiparallel beta-sheet structures in the majority of fibrils are indicated by measurements of intermolecular distances through (13)C-(13)C and (15)N-(13)C dipole-dipole couplings. An intriguing possibility exists that there is a relationship between the aberrant structure of D23N-Abeta40 fibrils and the unusual vasculotropic clinical picture in these patients.

Microwave-assisted solid-phase synthesis of highly fluorescent carbon nanoparticles and its application in intracellular pH sensing.

PubMed

Yang, Shenghong; Chen, Xiao; Liu, Shuqin; Wang, Fuxin; Ouyang, Gangfeng

2018-08-15

Fluorescent carbon nanoparticles (FCNPs) have been deeply researched and widely applied in recent years due to their good optics performance, chemical stability and biocompatibility. Herein, a green and rapid microwave-assisted solid-phase synthesis (solvent-free) approach was proposed for the fabrication of highly FCNPs in a very short period of time, 4 min. The as-prepared FCNPs can emit a blue emission with quantum yield of up to 63.2% in water solution and show yellow fluorescence in the solid state. The FCNPs also exhibit special solvent effect that the fluorescence emission can be adjusted by controlling the solvent ratio of ethanol and water. Most importantly, the FCNPs possess a narrow-range pH response. The probe responds linearly and rapidly to minor pH fluctuations within the range of 3.47-5.10 and the correlation coefficient is above 0.99. The proposed FCNPs also exhibit high photostability and reusability. As expected, the cell imaging and intracellular pH monitoring was achieved successfully in living SMMC 7721 hepatoma cells by this probe. The FCNPs is promising as a convenient and general fluorescent pH sensor for bioimaging applications. Copyright © 2018. Published by Elsevier B.V.

Subeutectic Synthesis of Epitaxial Si-NWs with Diverse Catalysts Using a Novel Si Precursor

PubMed Central

2010-01-01

The applicability of a novel silicon precursor with respect to reasonable nanowire (NW) growth rates, feasibility of epitaxial NW growth and versatility with respect to diverse catalysts was investigated. Epitaxial growth of Si-NWs was achieved using octochlorotrisilane (OCTS) as Si precursor and Au as catalyst. In contrast to the synthesis approach with SiCl4 as precursor, OCTS provides Si without the addition of H2. By optimizing the growth conditions, effective NW synthesis is shown for alternative catalysts, in particular, Cu, Ag, Ni, and Pt with the latter two being compatible to complementary metal-oxide-semiconductor technology. As for these catalysts, the growth temperatures are lower than the lowest liquid eutectic; we suggest that the catalyst particle is in the solid state during NW growth and that a solid-phase diffusion process, either in the bulk, on the surface, or both, must be responsible for NW nucleation. PMID:20843058

Optimisation of the synthesis of vancomycin-selective molecularly imprinted polymer nanoparticles using automatic photoreactor.

PubMed

Muzyka, Kateryna; Karim, Khalku; Guerreiro, Antonio; Poma, Alessandro; Piletsky, Sergey

2014-03-31

A novel optimized protocol for solid-state synthesis of molecularly imprinted polymer nanoparticles (nanoMIPs) with specificity for antibiotic vancomycin is described. The experimental objective was optimization of the synthesis parameters (factors) affecting the yield of obtained nanoparticles which have been synthesized using the first prototype of an automated solid-phase synthesizer. Applications of experimental design (or design of experiments) in optimization of nanoMIP yield were carried out using MODDE 9.0 software. The factors chosen in the model were the amount of functional monomers in the polymerization mixture, irradiation time, temperature during polymerization, and elution temperature. In general, it could be concluded that the irradiation time is the most important and the temperature was the least important factor which influences the yield of nanoparticles. Overall, the response surface methodology proved to be an effective tool in reducing time required for optimization of complex experimental conditions.

Optimisation of the synthesis of vancomycin-selective molecularly imprinted polymer nanoparticles using automatic photoreactor

PubMed Central

2014-01-01

A novel optimized protocol for solid-state synthesis of molecularly imprinted polymer nanoparticles (nanoMIPs) with specificity for antibiotic vancomycin is described. The experimental objective was optimization of the synthesis parameters (factors) affecting the yield of obtained nanoparticles which have been synthesized using the first prototype of an automated solid-phase synthesizer. Applications of experimental design (or design of experiments) in optimization of nanoMIP yield were carried out using MODDE 9.0 software. The factors chosen in the model were the amount of functional monomers in the polymerization mixture, irradiation time, temperature during polymerization, and elution temperature. In general, it could be concluded that the irradiation time is the most important and the temperature was the least important factor which influences the yield of nanoparticles. Overall, the response surface methodology proved to be an effective tool in reducing time required for optimization of complex experimental conditions. PMID:24685151

Homogeneous synthesis of cellulose acrylate-g-poly (n-alkyl acrylate) solid-solid phase change materials via free radical polymerization.

PubMed

Qian, Yong-Qiang; Han, Na; Bo, Yi-Wen; Tan, Lin-Li; Zhang, Long-Fei; Zhang, Xing-Xiang

2018-08-01

A novel solid-solid phase change materials, namely, cellulose acrylate-g-poly (n-alkyl acrylate) (CA-g-PAn) (n = 14, 16 and 18) were successfully synthesized by free radical polymerization in N, N-dimethylacetamide (DMAc). The successful grafting was confirmed by fourier transform infrared spectra (FT-IR) and nuclear magnetic resonance (NMR). The properties of the CA-g-PAn copolymers were investigated by differential scanning calorimetry (DSC), thermogravimetric analysis (TGA). The phase change temperatures and the melting enthalpies of CA-g-PAn copolymers are in the range of 10.1-53.2 °C and 15-95 J/g, respectively. It can be adjusted by the contents of poly (n-alkyl acrylate) and the length of alkyl side-chain. The thermal resistant temperatures of CA-g-PA14, 16 and 18 copolymers are 308 °C, 292 °C and 273 °C, respectively. It show that all of grafting materials exhibit good thermal stability and shape stability. Therefore, it is expected to be applied in the cellulose-based thermos-regulating field. Copyright © 2018 Elsevier Ltd. All rights reserved.

Preparation, characterization, and application of poly(vinyl alcohol)-graft-poly(ethylene glycol) resins: novel polymer matrices for solid-phase synthesis.

PubMed

Luo, Juntao; Pardin, Christophe; Zhu, X X; Lubell, William D

2007-01-01

Spherical crosslinked poly(vinyl alcohol) (PVA) beads with good mechanical stability were prepared by reverse-suspension polymerization, using dimethyl sulfoxide (DMSO) as a cosolvent in an aqueous phase. Poly(ethylene glycol)s with varying chain lengths were grafted onto the PVA beads by anionic polymerization of ethylene oxide. The thermal behavior, morphology, and swelling were evaluated for each of the new polymer matrices. High loading and good swelling in water and organic solvents were characteristic of the PEG-grafted PVA beads. The polymer beads also exhibited good mechanical and chemical stability and were unaffected by treatment with 6 N HCl and with 6 N NaOH. The hydroxyl groups of the PVA-PEG beads were converted into aldehyde, carboxylic acid, and isocyanate functions to provide scavenger resins and were extended by way of a benzyl alcohol in a Wang linker. The transglutaminase substrates dipeptides (Z-Gln-Gly) and heptapeptides (Pro-Asn-Pro-Gln-Leu-Pro-Phe) were synthesized on PVA-PEG_5, PVA-PEG_20, and the Wang linker-derivatized PVA-PEG resins. The cleavage of the peptides from the resins using MeOH/NH3 mixture at different temperatures (0 degrees C and room temp) and 50% TFA/DCM provided, respectively, peptide methyl esters, amides, and acids in good yields and purity as assessed by LC-MS analysis.

Low incidence of anti-drug antibodies in patients with type 2 diabetes treated with once-weekly glucagon-like peptide-1 receptor agonist dulaglutide.

PubMed

Milicevic, Z; Anglin, G; Harper, K; Konrad, R J; Skrivanek, Z; Glaesner, W; Karanikas, C A; Mace, K

2016-05-01

Therapeutic administration of peptides may result in anti-drug antibody (ADA) formation, hypersensitivity adverse events (AEs) and reduced efficacy. As a large peptide, the immunogenicity of once-weekly glucagon-like peptide-1 (GLP-1) receptor agonist dulaglutide is of considerable interest. The present study assessed the incidence of treatment-emergent dulaglutide ADAs, hypersensitivity AEs, injection site reactions (ISRs), and glycaemic control in ADA-positive patients in nine phase II and phase III trials (dulaglutide, N = 4006; exenatide, N = 276; non-GLP-1 comparators, N = 1141). Treatment-emergent dulaglutide ADAs were detected using a solid-phase extraction acid dissociation binding assay. Neutralizing ADAs were detected using a cell-based assay derived from human endothelial kidney cells (HEK293). A total of 64 dulaglutide-treated patients (1.6% of the population) tested ADA-positive versus eight (0.7%) from the non-GLP-1 comparator group. Of these 64 patients, 34 (0.9%) had dulaglutide-neutralizing ADAs, 36 (0.9%) had native-sequence GLP-1 (nsGLP-1) cross-reactive ADAs and four (0.1%) had nsGLP-1 neutralization ADAs. The incidence of hypersensitivity AEs and ISRs was similar in the dulaglutide versus placebo groups. No dulaglutide ADA-positive patient reported hypersensitivity AEs. Because of the low incidence of ADAs, it was not possible to establish their effect on glycaemic control. © 2016 John Wiley & Sons Ltd.

A Synthesis and Crystal Chemical Study of the Fast Ion Conductor Li7–3xGaxLa3 Zr2O12 with x = 0.08 to 0.84

PubMed Central

2014-01-01

Fast-conducting phase-pure cubic Ga-bearing Li7La3Zr2O12 was obtained using solid-state synthesis methods with 0.08 to 0.52 Ga3+ pfu in the garnet. An upper limit of 0.72 Ga3+ pfu in garnet was obtained, but the synthesis was accompanied by small amounts of La2Zr2O12 and LiGaO3. The synthetic products were characterized by X-ray powder diffraction, electron microprobe and SEM analyses, ICP-OES measurements, and 71Ga MAS NMR spectroscopy. The unit-cell parameter, a0, of the various garnets does not vary significantly as a function of Ga3+ content, with a value of about 12.984(4) Å. Full chemical analyses for the solid solutions were obtained giving: Li7.08Ga0.06La2.93Zr2.02O12, Li6.50Ga0.15La2.96Zr2.05O12, Li6.48Ga0.23La2.93Zr2.04O12, Li5.93Ga0.36La2.94Zr2.01O12, Li5.38Ga0.53La2.96Zr1.99O12, Li4.82Ga0.60La2.96Zr2.00O12, and Li4.53Ga0.72La2.94Zr1.98O12. The NMR spectra are interpreted as indicating that Ga3+ mainly occurs in a distorted 4-fold coordinated environment that probably corresponds to the general 96h crystallographic site of garnet. PMID:24874559

Nanostructures by ion beams

NASA Astrophysics Data System (ADS)

Schmidt, B.

Ion beam techniques, including conventional broad beam ion implantation, ion beam synthesis and ion irradiation of thin layers, as well as local ion implantation with fine-focused ion beams have been applied in different fields of micro- and nanotechnology. The ion beam synthesis of nanoparticles in high-dose ion-implanted solids is explained as phase separation of nanostructures from a super-saturated solid state through precipitation and Ostwald ripening during subsequent thermal treatment of the ion-implanted samples. A special topic will be addressed to self-organization processes of nanoparticles during ion irradiation of flat and curved solid-state interfaces. As an example of silicon nanocrystal application, the fabrication of silicon nanocrystal non-volatile memories will be described. Finally, the fabrication possibilities of nanostructures, such as nanowires and chains of nanoparticles (e.g. CoSi2), by ion beam synthesis using a focused Co+ ion beam will be demonstrated and possible applications will be mentioned.

Optimisation of the synthesis of vancomycin-selective molecularly imprinted polymer nanoparticles using automatic photoreactor

NASA Astrophysics Data System (ADS)

Muzyka, Kateryna; Karim, Khalku; Guerreiro, Antonio; Poma, Alessandro; Piletsky, Sergey

2014-03-01

A novel optimized protocol for solid-state synthesis of molecularly imprinted polymer nanoparticles (nanoMIPs) with specificity for antibiotic vancomycin is described. The experimental objective was optimization of the synthesis parameters (factors) affecting the yield of obtained nanoparticles which have been synthesized using the first prototype of an automated solid-phase synthesizer. Applications of experimental design (or design of experiments) in optimization of nanoMIP yield were carried out using MODDE 9.0 software. The factors chosen in the model were the amount of functional monomers in the polymerization mixture, irradiation time, temperature during polymerization, and elution temperature. In general, it could be concluded that the irradiation time is the most important and the temperature was the least important factor which influences the yield of nanoparticles. Overall, the response surface methodology proved to be an effective tool in reducing time required for optimization of complex experimental conditions.

Fabrication of Cu2ZnSn(S,Se)4 (CZTSSe) absorber films based on solid-phase synthesis and blade coating processes

NASA Astrophysics Data System (ADS)

Ma, Ruixin; Yang, Fan; Li, Shina; Zhang, Xiaoyong; Li, Xiang; Cheng, Shiyao; Liu, Zilin

2016-04-01

CZTSSe is an important earth abundant collection of materials for the development of low cost and high efficiency thin film solar cells. This work developed a simple non-vacuum-based route to fabricate CZTSSe absorber films. This was demonstrated by first synthesizing Cu2ZnSnS4 (CZTS) nano-crystalline based on solid-phase synthesis. Then a stable colloidal ink composed of CZTS nano-crystalline was blade coated on Mo-coated substrates followed by an annealing process under Ar atmosphere. After CZTS films formation, the films were sintered into CZTSSe absorber films by exposing them under Selenium vapor. The formation of a kesterite type CZTS was confirmed using X-ray diffraction and Raman scattering measurements. The band gap of CZTSSe absorber films was determined to be 1.26 eV, which was appropriate for use as an absorber layer in thin film solar cells. The CZTSSe absorber films showed a good photovoltatic performance, demonstrating this simple approach had great potential for CZTSSe solar cell production.

Applications of reversible covalent chemistry in analytical sample preparation.

PubMed

Siegel, David

2012-12-07

Reversible covalent chemistry (RCC) adds another dimension to commonly used sample preparation techniques like solid-phase extraction (SPE), solid-phase microextraction (SPME), molecular imprinted polymers (MIPs) or immuno-affinity cleanup (IAC): chemical selectivity. By selecting analytes according to their covalent reactivity, sample complexity can be reduced significantly, resulting in enhanced analytical performance for low-abundance target analytes. This review gives a comprehensive overview of the applications of RCC in analytical sample preparation. The major reactions covered include reversible boronic ester formation, thiol-disulfide exchange and reversible hydrazone formation, targeting analyte groups like diols (sugars, glycoproteins and glycopeptides, catechols), thiols (cysteinyl-proteins and cysteinyl-peptides) and carbonyls (carbonylated proteins, mycotoxins). Their applications range from low abundance proteomics to reversible protein/peptide labelling to antibody chromatography to quantitative and qualitative food analysis. In discussing the potential of RCC, a special focus is on the conditions and restrictions of the utilized reaction chemistry.

Biochemical characterization of a new maize (Zea mays L.) peptide growth factor.

PubMed

Rodríguez-López, Cesar David; Rodríguez-Romero, Adela; Aguilar, Raúl; de Jiménez, Estela Sánchez

2011-01-01

Coordination of cell growth and cell division is very important for living organisms in order for these to develop harmonically. The present research is concerned with the purification and characterization of a new peptide hormone, namely ZmIGF (Zea mays insulin-like growth factor), which regulates growth and cell division in maize tissues. ZmIGF is a peptide of 5.7 kDa, as determined by mass spectroscopy. It was isolated either from maize embryonic axes of 48-h germinated seeds or from embryogenic callus and purified through several chromatographic procedures to obtain a single peak as shown by Reverse Phase High-Performance Liquid Chromatography (RP-HPLC). This peptide exhibits a well defined α-helix structure by circular dichroism analysis, similar to that reported for Insulin or for Insulin-like growth factor (IGF-1). Further, ZmIGF seems to perform, in maize, a similar function to that reported for insulin or peptides from the IGF family in animals. Indeed, maize tissues stimulated either by ZmIGF or insulin showed to induce selective synthesis of ribosomal proteins as well as of DNA. Taken together, the previously mentioned data strongly suggest that plants contain a peptide hormone of the IGF family, highly conserved through evolution that regulates growth and development.

Mitotic MELK-eIF4B signaling controls protein synthesis and tumor cell survival

PubMed Central

Wang, Yubao; Begley, Michael; Li, Qing; Huang, Hai-Tsang; Lako, Ana; Eck, Michael J.; Gray, Nathanael S.; Mitchison, Timothy J.; Cantley, Lewis C.; Zhao, Jean J.

2016-01-01

The protein kinase maternal and embryonic leucine zipper kinase (MELK) is critical for mitotic progression of cancer cells; however, its mechanisms of action remain largely unknown. By combined approaches of immunoprecipitation/mass spectrometry and peptide library profiling, we identified the eukaryotic translation initiation factor 4B (eIF4B) as a MELK-interacting protein during mitosis and a bona fide substrate of MELK. MELK phosphorylates eIF4B at Ser406, a modification found to be most robust in the mitotic phase of the cell cycle. We further show that the MELK–eIF4B signaling axis regulates protein synthesis during mitosis. Specifically, synthesis of myeloid cell leukemia 1 (MCL1), an antiapoptotic protein known to play a role in cancer cell survival during cell division, depends on the function of MELK-elF4B. Inactivation of MELK or eIF4B results in reduced protein synthesis of MCL1, which, in turn, induces apoptotic cell death of cancer cells. Our study thus defines a MELK–eIF4B signaling axis that regulates protein synthesis during mitosis, and consequently influences cancer cell survival. PMID:27528663

Surface-modified multifunctional MIP nanoparticles

PubMed Central

Moczko, Ewa; Poma, Alessandro; Guerreiro, Antonio; de Vargas Sansalvador, Isabel Perez; Caygill, Sarah; Canfarotta, Francesco; Whitcombe, Michael J.; Piletsky, Sergey

2015-01-01

The synthesis of core-shell molecularly imprinted polymer nanoparticles (MIP NPs) has been performed using a novel solid-phase approach on immobilised templates. The same solid phase also acts as protective functionality for high affinity binding sites during subsequent derivatisation/shell formation. This procedure allows for the rapid synthesis, controlled separation and purification of high-affinity materials, with each production cycle taking just 2 hours. The aim of this approach is to synthesise uniformly-sized imprinted materials at the nanoscale which can be readily grafted with various polymers without affecting their affinity and specificity. For demonstration purposes we grafted anti-melamine MIP NPs with coatings which introduce the following surface characteristics: high polarity (PEG methacrylate); electro-activity (vinyl ferrocene); fluorescence (eosin acrylate); thiol groups (pentaerythritol tetrakis(3-mercaptopropionate)). The method has broad applicability and can be used to produce multifunctional imprinted nanoparticles with potential for further application in the biosensors, diagnostics and biomedical fields and as an alternative to natural receptors. PMID:23503559

Synthesis of high-oxidation Y-Ba-Cu-O phases in superoxygenated thin films

NASA Astrophysics Data System (ADS)

Zhang, H.; Gauquelin, N.; McMahon, C.; Hawthorn, D. G.; Botton, G. A.; Wei, J. Y. T.

2018-03-01

It is known that solid-state reaction in high-pressure oxygen can stabilize high-oxidation phases of Y-Ba-Cu-O superconductors in powder form. We extend this superoxygenation concept of synthesis to thin films which, due to their large surface-to-volume ratio, are more reactive thermodynamically. Epitaxial thin films of YBa2Cu3O7 -δ grown by pulsed laser deposition are annealed at up to 700 atm O2 and 900 ∘C , in conjunction with Cu enrichment by solid-state diffusion. The films show the clear formation of Y2Ba4Cu7O15 -δ and Y2Ba4Cu8O16 as well as regions of YBa2Cu5O9 -δ and YBa2Cu6O10 -δ phases, according to scanning transmission electron microscopy, x-ray diffraction, and x-ray absorption spectroscopy. Similarly annealed YBa2Cu3O7 -δ powders show no phase conversion. Our results demonstrate a route of synthesis towards discovering more complex phases of cuprates and other superconducting oxides.

Novel 64Cu Labeled RGD2-BBN Heterotrimers for PET Imaging of Prostate Cancer.

PubMed

Lucente, Ermelinda; Liu, Hongguang; Liu, Yang; Hu, Xiang; Lacivita, Enza; Leopoldo, Marcello; Cheng, Zhen

2018-05-16

Bombesin receptor 2 (BB 2 ) and integrin α v β 3 receptor are privileged targets for molecular imaging of cancer because of their overexpression in a number of tumor tissues. The most recent developments in heterodimer-based radiopharmaceuticals concern BB 2 - and integrin α v β 3 -targeting compounds, consisting of bombesin (BBN) and cyclic arginine-glycine-aspartic acid peptides (RGD), connected through short length linkers. Molecular imaging probes based on RGD-BBN heterodimer design exhibit improved tumor targeting efficacy compared to the single-receptor targeting peptide monomers. However, their application in clinical study is restricted because of inefficient synthesis or unfavorable in vivo properties, which could depend on the short linker nature. Thus, the aim of the present study was to develop a RGD 2 -BBN heterotrimer, composed of (7-14)BBN-NH 2 peptide (BBN) linked to the E[ c(RGDyK)] 2 dimer peptide (RGD 2 ), bearing the new linker type [Pro-Gly] 12 . The heterodimer E[c(RGDyK)] 2 -PEG 3 -Glu-(Pro-Gly) 12 -BBN(7-14)-NH 2 (RGD 2 -PG 12 -BBN) was prepared through conventional solid phase synthesis, then conjugated with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or 1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid (NODA-GA). In 64 Cu labeling, the NODA-GA chelator showed superior radiochemical characteristics compared to DOTA (70% vs 40% yield, respectively). Both conjugates displayed dual targeting ability, showing good α v β 3 affinities and high BB 2 receptor affinities which, in the case of the NODA-GA conjugate, were in the same range as the best RGD-BBN heterodimer ligands reported to date ( K i = 24 nM). 64 Cu-DOTA and 64 Cu-NODA-GA probes were also found to be stable after 1 h incubation in mouse serum (>90%). In a microPET study in prostate cancer PC-3 xenograft mice, both probes showed low tumor uptake, probably due to poor pharmacokinetic properties in vivo. Overall, our study demonstrates that novel RGD-BBN heterodimer with long linker can be prepared and they preserve high binding affinities to BB 2 and integrin α v β 3 receptor binding ability. The present study represents a step forward in the design of effective heterodimer or heterotrimer probes for dual targeting.

Two-dimensional parallel array technology as a new approach to automated combinatorial solid-phase organic synthesis

PubMed

Brennan; Biddison; Frauendorf; Schwarcz; Keen; Ecker; Davis; Tinder; Swayze

1998-01-01

An automated, 96-well parallel array synthesizer for solid-phase organic synthesis has been designed and constructed. The instrument employs a unique reagent array delivery format, in which each reagent utilized has a dedicated plumbing system. An inert atmosphere is maintained during all phases of a synthesis, and temperature can be controlled via a thermal transfer plate which holds the injection molded reaction block. The reaction plate assembly slides in the X-axis direction, while eight nozzle blocks holding the reagent lines slide in the Y-axis direction, allowing for the extremely rapid delivery of any of 64 reagents to 96 wells. In addition, there are six banks of fixed nozzle blocks, which deliver the same reagent or solvent to eight wells at once, for a total of 72 possible reagents. The instrument is controlled by software which allows the straightforward programming of the synthesis of a larger number of compounds. This is accomplished by supplying a general synthetic procedure in the form of a command file, which calls upon certain reagents to be added to specific wells via lookup in a sequence file. The bottle position, flow rate, and concentration of each reagent is stored in a separate reagent table file. To demonstrate the utility of the parallel array synthesizer, a small combinatorial library of hydroxamic acids was prepared in high throughput mode for biological screening. Approximately 1300 compounds were prepared on a 10 μmole scale (3-5 mg) in a few weeks. The resulting crude compounds were generally >80% pure, and were utilized directly for high throughput screening in antibacterial assays. Several active wells were found, and the activity was verified by solution-phase synthesis of analytically pure material, indicating that the system described herein is an efficient means for the parallel synthesis of compounds for lead discovery. Copyright 1998 John Wiley & Sons, Inc.

Carbothermal shock synthesis of high-entropy-alloy nanoparticles

NASA Astrophysics Data System (ADS)

Yao, Yonggang; Huang, Zhennan; Xie, Pengfei; Lacey, Steven D.; Jacob, Rohit Jiji; Xie, Hua; Chen, Fengjuan; Nie, Anmin; Pu, Tiancheng; Rehwoldt, Miles; Yu, Daiwei; Zachariah, Michael R.; Wang, Chao; Shahbazian-Yassar, Reza; Li, Ju; Hu, Liangbing

2018-03-01

The controllable incorporation of multiple immiscible elements into a single nanoparticle merits untold scientific and technological potential, yet remains a challenge using conventional synthetic techniques. We present a general route for alloying up to eight dissimilar elements into single-phase solid-solution nanoparticles, referred to as high-entropy-alloy nanoparticles (HEA-NPs), by thermally shocking precursor metal salt mixtures loaded onto carbon supports [temperature ~2000 kelvin (K), 55-millisecond duration, rate of ~105 K per second]. We synthesized a wide range of multicomponent nanoparticles with a desired chemistry (composition), size, and phase (solid solution, phase-separated) by controlling the carbothermal shock (CTS) parameters (substrate, temperature, shock duration, and heating/cooling rate). To prove utility, we synthesized quinary HEA-NPs as ammonia oxidation catalysts with ~100% conversion and >99% nitrogen oxide selectivity over prolonged operations.

Catalytic RNA and synthesis of the peptide bond

NASA Technical Reports Server (NTRS)

Usher, D. A.; Kozlowski, M.; Zou, X.

1991-01-01

We are studying whether the L-19 IVS ribozyme from Tetrahymena thermophila can catalyze the formation of the peptide bond when it is supplied with synthetic aminoacyl oligonucleotides. If this reaction works, it could give us some insight into the mechanism of peptide bond formation and the origin of coded protein synthesis. Two short oligoribonucleotides, CCCCC and a protected form of CCCCU were prepared; the former was made by the controlled hydrolysis of Poly(C), and the later by multistep chemical synthesis from the protected monomers. The homopentamer was then aminocylated using C-14 labelled Boc-protected glycine imidazolide. This aminoacylated oligo-nucleotide has now been shown to enter the active site of the L-19 IVS, and aminoacyl transfer, and peptide bond formation reactions are being sought. Our synthesis of CCCCU made us aware of the inadequacy of many of the 2'- hydroxyl protecting groups that are in use today and we therefore designed a new 2'- protecting group that is presently being tested.

Design, Synthesis and Affinity Properties of Biologically Active Peptide and Protein Conjugates of Cotton Cellulose

DOE Office of Scientific and Technical Information (OSTI.GOV)

Edwards, J. V.; Goheen, Steven C.

The formation of peptide and protein conjugates of cellulose on cotton fabrics provides promising leads for the development of wound healing, antibacterial, and decontaminating textiles. An approach to the design, synthesis, and analysis of bioconjugates containing cellulose peptide and protein conjugates includes: 1) computer graphic modeling for a rationally designed structure; 2) attachment of the peptide or protein to cotton cellulose through a linker amino acid, and 3) characterization of the resulting bioconjugate. Computer graphic simulation of protein and peptide cellulose conjugates gives a rationally designed biopolymer to target synthetic modifications to the cotton cellulose. Techniques for preparing these typesmore » of conjugates involve both sequential assembly of the peptide on the fabric and direct crosslinking of the peptide or protein as cellulose bound esters or carboxymethylcellulose amides.« less

Peptide protected gold clusters: chemical synthesis and biomedical applications

NASA Astrophysics Data System (ADS)

Yuan, Qing; Wang, Yaling; Zhao, Lina; Liu, Ru; Gao, Fuping; Gao, Liang; Gao, Xueyun

2016-06-01

Bridging the gap between atoms and nanoparticles, noble metal clusters with atomic precision continue to attract considerable attention due to their important applications in catalysis, energy transformation, biosensing and biomedicine. Greatly different to common chemical synthesis, a one-step biomimetic synthesis of peptide-conjugated metal clusters has been developed to meet the demand of emerging bioapplications. Under mild conditions, multifunctional peptides containing metal capturing, reactive and targeting groups are rationally designed and elaborately synthesized to fabricate atomically precise peptide protected metal clusters. Among them, peptide-protected Au Cs (peptide-Au Cs) possess a great deal of exceptional advantages such as nanometer dimensions, high photostability, good biocompatibility, accurate chemical formula and specific protein targeting capacity. In this review article, we focus on the recent advances in potential theranostic fields by introducing the rising progress of peptide-Au Cs for biological imaging, biological analysis and therapeutic applications. The interactions between Au Cs and biological systems as well as potential mechanisms are also our concerned theme. We expect that the rapidly growing interest in Au Cs-based theranostic applications will attract broader concerns across various disciplines.

Phase transitions of sodium niobate powder and ceramics, prepared by solid state synthesis

NASA Astrophysics Data System (ADS)

Koruza, J.; Tellier, J.; Malič, B.; Bobnar, V.; Kosec, M.

2010-12-01

Phase transitions of sodium niobate, prepared by the solid state synthesis method, were examined using dielectric measurements, differential scanning calorimetry, and high temperature x-ray diffraction, in order to contribute to the clarification of its structural behavior below 400 °C. Four phase transitions were detected in the ceramic sample using dielectric measurements and differential scanning calorimetry and the obtained temperatures were in a good agreement with previous reports for the transitions of the P polymorph. The anomaly observed by dielectric measurements in the vicinity of 150 °C was frequency dependent and could be related to the dynamics of the ferroelectric nanoregions. The phase transitions of the as-synthesized NaNbO3 powder were investigated using differential scanning calorimetry and high temperature x-ray diffraction. The results show the existence of the Q polymorph at room temperature, not previously reported for the powder, which undergoes a transition to the R polymorph upon heating through a temperature region between 265 and 326.5 °C. This transition is mainly related to the displacement of Na into a more symmetric position and a minor change in the tilting system. The structures at room temperature, 250, 300, and 420 °C were refined by the Rietveld method and the evolution of the tilting system of the octahedral network and cationic displacement are reported.

C-Terminal Protein Characterization by Mass Spectrometry: Isolation of C-Terminal Fragments from Cyanogen Bromide-Cleaved Protein

PubMed Central

Nika, Heinz; Hawke, David H.; Angeletti, Ruth Hogue

2014-01-01

A sample preparation method for protein C-terminal peptide isolation from cyanogen bromide (CNBr) digests has been developed. In this strategy, the analyte was reduced and carboxyamidomethylated, followed by CNBr cleavage in a one-pot reaction scheme. The digest was then adsorbed on ZipTipC18 pipette tips for conjugation of the homoserine lactone-terminated peptides with 2,2′-dithiobis (ethylamine) dihydrochloride, followed by reductive release of 2-aminoethanethiol from the derivatives. The thiol-functionalized internal and N-terminal peptides were scavenged on activated thiol sepharose, leaving the C-terminal peptide in the flow-through fraction. The use of reversed-phase supports as a venue for peptide derivatization enabled facile optimization of the individual reaction steps for throughput and completeness of reaction. Reagents were replaced directly on the support, allowing the reactions to proceed at minimal sample loss. By this sequence of solid-phase reactions, the C-terminal peptide could be recognized uniquely in mass spectra of unfractionated digests by its unaltered mass signature. The use of the sample preparation method was demonstrated with low-level amounts of a whole, intact model protein. The C-terminal fragments were retrieved selectively and efficiently from the affinity support. The use of covalent chromatography for C-terminal peptide purification enabled recovery of the depleted material for further chemical and/or enzymatic manipulation. The sample preparation method provides for robustness and simplicity of operation and is anticipated to be expanded to gel-separated proteins and in a scaled-up format to high-throughput protein profiling in complex biological mixtures. PMID:24688319

Solid-phase synthesis and insights into structure-activity relationships of safinamide analogues as potent and selective inhibitors of type B monoamine oxidase.

PubMed

Leonetti, Francesco; Capaldi, Carmelida; Pisani, Leonardo; Nicolotti, Orazio; Muncipinto, Giovanni; Stefanachi, Angela; Cellamare, Saverio; Caccia, Carla; Carotti, Angelo

2007-10-04

Safinamide, (S)-N2-{4-[(3-fluorobenzyl)oxy]benzyl}alaninamide methanesulfonate, which is in phase III clinical trials as an anti-Parkinson drug, and a library of alkanamidic analogues were prepared through an expeditious solid-phase synthesis and evaluated for their monoamine oxidase B (MAO-B) and monoamine oxidase A (MAO-A) inhibitory activity and selectivity. (S)-3-Chlorobenzyloxyalaninamide (8) and (S)-3-chlorobenzyloxyserinamide (13) derivatives proved to be more potent MAO-B inhibitors than safinamide (IC50 = 33 and 43 nM, respectively, vs 98 nM) but with a lower MAO-B selectivity (SI = 3455 and 1967, respectively, vs 5918). The highest MAO-B inhibitory potency (IC50 = 17 nM) and a good selectivity (SI = 2941) were displayed by (R)-21, a tetrahydroisoquinoline analogue of safinamide. Structure-affinity relationships and docking simulations pointed out strong negative steric effects of alpha-aminoamide side chains and para substituents of the benzyloxy groups and favorable hydrophobic interactions of meta substituents. The significantly diverse MAO-B affinities of a number of R and S alpha-aminoamide enantiomers, including the two rigid analogues (21) of safinamide, indicated likely enantioselective interactions at the enzymatic binding sites.

Current Trends in Sensors Based on Conducting Polymer Nanomaterials

PubMed Central

Yoon, Hyeonseok

2013-01-01

Conducting polymers represent an important class of functional organic materials for next-generation electronic and optical devices. Advances in nanotechnology allow for the fabrication of various conducting polymer nanomaterials through synthesis methods such as solid-phase template synthesis, molecular template synthesis, and template-free synthesis. Nanostructured conducting polymers featuring high surface area, small dimensions, and unique physical properties have been widely used to build various sensor devices. Many remarkable examples have been reported over the past decade. The enhanced sensitivity of conducting polymer nanomaterials toward various chemical/biological species and external stimuli has made them ideal candidates for incorporation into the design of sensors. However, the selectivity and stability still leave room for improvement. PMID:28348348

Solid-Phase Enrichment and Analysis of Azide-Labeled Natural Products: Fishing Downstream of Biochemical Pathways.

PubMed

Pérez, Alexander J; Wesche, Frank; Adihou, Hélène; Bode, Helge B

2016-01-11

Many methods have been devised over the decades to trace precursors of specific molecules in cellular environments as, for example, in biosynthesis studies. The advent of click chemistry has facilitated the powerful combination of tracing and at the same time sieving the highly complex metabolome for compounds derived from simple or complex starting materials, especially when the click reaction takes place on a solid support. While the principle of solid-phase click reactions has already been successfully applied for selective protein and peptide enrichment, the successful enrichment of much smaller primary and secondary metabolites, showing great structural diversity and undergoing many different biosynthetic steps, has seen only little development. For bacterial secondary metabolism, a far broader tolerance for "clickable" precursors was observed than in ribosomal proteinogenesis, thus making this method a surprisingly valuable tool for the tracking and discovery of compounds within the cellular biochemical network. The implementation of this method has led to the identification of several new compounds from the bacterial genera Photorhabdus and Xenorhabdus, clearly proving its power. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Prebiotic Peptide (Amide) Bond Synthesis Accelerated by Glycerol and Bicarbonate Under Neutral to Alkaline Dry-Down Conditions

NASA Astrophysics Data System (ADS)

Forsythe, J. G.; Weber, A. L.

2017-07-01

We report a new process for robust peptide bond synthesis in the pH 6–10 range that involves dry-down heating of amino acids in the presence of glycerol and bicarbonate (substrates: L-alanine, L-2-aminobutyric acid, β-alanine, isoserine).

Efficient methods for attaching non-radioactive labels to the 5' ends of synthetic oligodeoxyribonucleotides.

PubMed Central

Agrawal, S; Christodoulou, C; Gait, M J

1986-01-01

The syntheses are described of two types of linker molecule useful for the specific attachment of non-radioactive labels such as biotin and fluorophores to the 5' terminus of synthetic oligodeoxyribonucleotides. The linkers are designed such that they can be coupled to the oligonucleotide as a final step in solid-phase synthesis using commercial DNA synthesis machines. Increased sensitivity of biotin detection was possible using an anti-biotin hybridoma/peroxidase detection system. PMID:3748808

Generic Biocombinatorial Strategy to Select Tailor-Made Stabilizers for Sol-Gel Nanoparticle Synthesis.

PubMed

Hanßke, Felix; Kemnitz, Erhard; Börner, Hans G

2015-09-09

A generic route for the selection of nanoparticle stabilizers via biocombinatorial means of phage display peptide screening is presented, providing magnesium fluoride nanoparticle synthesis as example. Selected sequence-specific MgF2 binders are evaluated for their adsorption behavior. Peptide-polymer conjugates derived from the best binding peptide are used for the stabilization of MgF2 sol nanoparticles, yielding fully redispersable dry states and improoving processability significantly. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Antifreeze glycopeptide diastereomers.

PubMed

Nagel, Lilly; Budke, Carsten; Dreyer, Axel; Koop, Thomas; Sewald, Norbert

2012-01-01

Antifreeze glycopeptides (AFGPs) are a special class of biological antifreeze agents, which possess the property to inhibit ice growth in the body fluids of arctic and antarctic fish and, thus, enable life under these harsh conditions. AFGPs are composed of 4-55 tripeptide units -Ala-Ala-Thr- glycosylated at the threonine side chains. Despite the structural homology among all the fish species, divergence regarding the composition of the amino acids occurs in peptides from natural sources. Although AFGPs were discovered in the early 1960s, the adsorption mechanism of these macromolecules to the surface of the ice crystals has not yet been fully elucidated. Two AFGP diastereomers containing different amino acid configurations were synthesized to study the influence of amino acid stereochemistry on conformation and antifreeze activity. For this purpose, peptides containing monosaccharide-substituted allo-L- and D-threonine building blocks were assembled by solid-phase peptide synthesis (SPPS). The retro-inverso AFGP analogue contained all amino acids in D-configuration, while the allo-L-diastereomer was composed of L-amino acids, like native AFGPs, with replacement of L-threonine by its allo-L-diastereomer. Both glycopeptides were analyzed regarding their conformational properties, by circular dichroism (CD), and their ability to inhibit ice recrystallization in microphysical experiments.

Peptide synthesis in early earth hydrothermal systems

USGS Publications Warehouse

Lemke, K.H.; Rosenbauer, R.J.; Bird, D.K.

2009-01-01

We report here results from experiments and thermodynamic calculations that demonstrate a rapid, temperature-enhanced synthesis of oligopeptides from the condensation of aqueous glycine. Experiments were conducted in custom-made hydrothermal reactors, and organic compounds were characterized with ultraviolet-visible procedures. A comparison of peptide yields at 260??C with those obtained at more moderate temperatures (160??C) gives evidence of a significant (13 kJ ?? mol-1) exergonic shift. In contrast to previous hydrothermal studies, we demonstrate that peptide synthesis is favored in hydrothermal fluids and that rates of peptide hydrolysis are controlled by the stability of the parent amino acid, with a critical dependence on reactor surface composition. From our study, we predict that rapid recycling of product peptides from cool into near-supercritical fluids in mid-ocean ridge hydrothermal systems will enhance peptide chain elongation. It is anticipated that the abundant hydrothermal systems on early Earth could have provided a substantial source of biomolecules required for the origin of life. Astrobiology 9, 141-146. ?? 2009 Mary Ann Liebert, Inc. 2009.

One-Pot Isomerization–Cross Metathesis–Reduction (ICMR) Synthesis of Lipophilic Tetrapeptides

PubMed Central

2015-01-01

An efficient, versatile and rapid method toward homologue series of lipophilic tetrapeptide derivatives (herein, the opioid peptides H-TIPP-OH and H-DIPP-OH) is reported. High atom economy and a minimal number of synthetic steps resulted from a one-pot tandem isomerization-cross metathesis-reduction sequence (ICMR), applicable both in solution and solid phase methodology. The broadly applicable synthesis proceeds with short reaction times and simple work-up, as illustrated in this work for alkylated opioid tetrapeptides. PMID:24906051

Synthesis of ion-exchange resin for selective thorium and uranyl ions sorption

NASA Astrophysics Data System (ADS)

Konovalov, Konstantin; Sachkov, Victor

2017-11-01

In this work, the method of ion-exchange resin synthesis selective to radionuclides (uranium and thorium) is presented. The method includes synthesis of polymeric styrene-divinylbenzene macroporous matrix with size of 0.1-0.2 mm, and its subsequent transformation by nitration and then reduction by tin (II) chloride. For passivation of active primary amines partially oxidation by oxygen from air is used. Obtained ion-exchange resin has ratio of sorption sum U+Th to sorption sum of other total rare-earth elements as 1:1.88 at ratio of solid to liquid phase 1:200. The proposed method of ion-exchange resin synthesis is scaled-up for laboratory reactors with volume of 5 and 50 liters.

Design, synthesis of allosteric peptide activator for human SIRT1 and its biological evaluation in cellular model of Alzheimer's disease.

PubMed

Kumar, Rahul; Nigam, Lokesh; Singh, Amrendra Pratap; Singh, Kusum; Subbarao, Naidu; Dey, Sharmistha

2017-02-15

Sirtuin 1 (SIRT1) is one of the member of the mammalian proteins of the Sirtuin family of NAD + dependent deacetylases, has recently been shown to attenuate amyloidogenic processing of amyloid protein precursor (APP) in in-vitro cell culture studies and transgenic mouse models of Alzheimer's disease (AD). SIRT1 has been shown to have a protective role against (AD). It has been reported earlier that increasing SIRT1 activity can prevent AD in mice model. Tripeptide as an activator of SIRT1 were screened on the basis of structural information by molecular docking and synthesized by solid phase method. The enhancement of biochemical activity of pure recombinant SIRT1 as well as SIRT1 in serum of AD patients in presence of tripeptide was done by Fluorescent Activity Assay. The activity of SIRT1 by peptide was assessed in IMR-32 cell line by measuring acetylated p53 level. Further the protective effect of SIRT1 activator in cellular model of AD was analyzed by MTT assay. We find CWR tripeptide as a SIRT1 activator by molecular docking, enhanced the activity of SIRT1 protein by lowering the Michaelis constant, Km by allosteric mechanism. The activity of serum SIRT1 of AD was also increases by CWR. It also decreased the acetylation of p53 in IMR32 neuroblastoma cells and protected the cell death caused by Aβ amyloid fragments in cell line model of AD. Thus, it can be concluded that CWR may serve as platform to elucidate further small molecule activator as a therapeutic agent for AD targeting SIRT1. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Preclinical evaluation of [99mTc/EDDA/tricine/HYNIC0, 1-Nal3, Thr8]-octreotide as a new analogue in the detection of somatostatin-receptor-positive tumors.

PubMed

Gandomkar, Mostafa; Najafi, Reza; Shafiei, Mohammad; Mazidi, Mohammad; Ebrahimi, Sayed Esmaeil Sadat

2007-08-01

Radiolabeled somatostatin analogues are important tools for the in vivo localization and targeted radionuclide therapy of somatostatin-receptor-positive tumors. The aim of this study was to evaluate a new somatostatin analogue designed for the labeling with (99m)Tc: [6-hydrazinopyridine-3-carboxylic acid (HYNIC(0)), 1-Nal(3), Thr(8)]-octreotide ([HYNIC]-NATE), using ethylenediamine-N,N'-diacetic acid (EDDA) and tricine as coligands. Synthesis was preformed on a solid phase using a standard Fmoc strategy. Labeling with (99m)Tc was performed at 100 degrees C for 10 min using SnCl(2) as a reductant. Radiochemical analysis involved ITLC and high-performance liquid chromatography methods. Peptide conjugate affinity was determined in AR4-2J cell membranes. The internalization and externalization rates were studied in sstr(2)-expressing AR4-2J cells. Biodistribution of radiopeptide was studied in rats bearing the AR4-2J tumor. Radiolabeling was performed at high specific activities, and radiochemical purity was >95%. Peptide conjugate showed high affinity binding for sstr(2). The radioligand showed a moderate and specific internalization into AR4-2J cells (14.13+/-0.61% at 4 h). In animal biodistribution studies, a receptor-specific uptake of radioactivity was observed in somatostatin-receptor-positive organs. After 4 h, uptake in the AR4-2J tumor was 1.33+/-0.23%ID/g (percentage of injected dose per gram of tissue). These data show that [(99m)Tc/EDDA/tricine/HYNIC]-NATE is a specific radioligand for the somatostatin-receptor-positive tumors and is a suitable candidate for clinical studies.

Identification of Dmt-D-Lys-Phe-Phe-OH as a highly antinociceptive tetrapeptide metabolite of the opioid-neurotensin hybrid peptide PK20.

PubMed

Kleczkowska, Patrycja; Bojnik, Engin; Leśniak, Anna; Kosson, Piotr; Van den Eynde, Isabelle; Ballet, Steven; Benyhe, Sandor; Tourwé, Dirk; Lipkowski, Andrzej W

2013-01-01

Recently, we presented a novel compound (PK20, Dmt-D-Lys-Phe-Phe-Lys-Lys-Pro-Phe-Tle-Leu-OH) that targets single entity opioid and neurotensin pharmacophores. This endomorphin-2-like opioid peptide was introduced as a highly active analgesic because it elicited a strong dose- and time-dependent antinociceptive response when administered centrally and peripherally. Its pain-relieving activity was observed as rapidly as 5 min after drug injection. Such promising results led us to perform further studies, such as determining the resistance to enzymatic degradation, which resulted in obtaining a very stable opioid pharmacore PK20 metabolite. The synthesis of PK20 and its N-terminal tetrapeptide fragment has been accomplished using solid phase peptide chemistry. The biological stability of peptides has been measured in human serum and analyzed by HPLC/MS. Peptides were pharmacologically characterized in in vitro MOP and DOP receptor binding as well as [(35)S]GTPγS receptor binding assays. Antinociceptive properties of compounds were measured by in vivo assays in C57Bl6 mice after intravenous or intrathecal applications. Dmt-D-Lys-Phe-Phe-OH (PK20M), an N-terminal tetrapeptide metabolite of the opioid-neurotensin hybrid peptide PK20, is characterized by a long duration of action, as demonstrated by a preserved, long-lasting analgesic effect even 2 h post-injection (average % MPE = 69.33). In rat brain membranes, PK20M efficiently displaced both the MOP and DOP receptor selective radioprobes [(3)H]DAMGO and [(3)H]DIDI (pKi of 9.52 and 7.86, respectively) and potently stimulated [(35)S]GTPγS binding, proving full agonism at both receptor types. In the [(35)S]GTPγS assay, which measured the agonist-mediated G protein activation, PK20M together with PK20 and Met-enkephalin were potent stimulators of the regulatory G proteins. The relative affinities of PK20M for the μ and δ receptor subtypes revealed μ-receptor selectivity. The novel MOP receptor selective metabolite has been shown to possess opioid subtype receptor selectivity, high potency, and effective analgesic activities as measured in various bioassays.

Site-Specific Antibody Labeling by Covalent Photoconjugation of Z Domains Functionalized for Alkyne-Azide Cycloaddition Reactions.

PubMed

Perols, Anna; Arcos Famme, Melina; Eriksson Karlström, Amelie

2015-11-01

Antibodies are extensively used in research, diagnostics, and therapy, and for many applications the antibodies need to be labeled. Labeling is typically performed by using amine-reactive probes that target surface-exposed lysine residues, resulting in heterogeneously labeled antibodies. An alternative labeling strategy is based on the immunoglobulin G (IgG)-binding protein domain Z, which binds to the Fc region of IgG. Introducing the photoactivable amino acid benzoylphenylalanine (BPA) into the Z domain makes it possible for a covalent bond to be be formed between the Z domain and the antibody on UV irradiation, to produce a site-specifically labeled product. Z32 BPA was synthesized by solid-phase peptide synthesis and further functionalized to give alkyne-Z32 BPA and azide-Z32 BPA for Cu(I) -catalyzed cycloaddition, as well as DBCO-Z32 BPA for Cu-free strain-promoted cycloaddition. The Z32 BPA variants were conjugated to the human IgG1 antibody trastuzumab and site-specifically labeled with biotin or fluorescein. The fluorescently labeled trastuzumab showed specific staining of the membranes of HER2-expressing cells in immunofluorescence microscopy. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

N-terminal galanin-(1-16) fragment is an agonist at the hippocampal galanin receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fisone, G.; Berthold, M.; Bedecs, K.

1989-12-01

The galanin N-terminal fragment (galanin-(1-16)) has been prepared by solid-phase synthesis and by enzymic cleavage of galanin by endoproteinase Asp-N. This peptide fragment displaced {sup 125}I-labeled galanin in receptor autoradiography experiments on rat forebrain and spinal cord and in equilibrium binding experiments from high-affinity binding sites in the ventral hippocampus with an IC50 of approximately 3 nM. In tissue slices of the same brain area, galanin-(1-16), similarly to galanin, inhibited the muscarinic agonist-stimulated breakdown of inositol phospholipids. Upon intracerebroventricular administration, galanin-(1-16) (10 micrograms/15 microliters) also inhibited the scopolamine (0.3 mg/kg, s.c.)-evoked release of acetylcholine, as studied in vivo by microdialysis.more » Substitution of (L-Trp2) for (D-Trp2) resulted in a 500-fold loss in affinity as compared with galanin-(1-16). It is concluded that, in the ventral hippocampus, the N-terminal galanin fragment (galanin-(1-16)) is recognized by the galanin receptors controlling acetylcholine release and muscarinic agonist-stimulated inositol phospholipid breakdown as a high-affinity agonist and that amino acid residue (Trp2) plays an important role in the receptor-ligand interactions.« less

Antifreeze glycopeptide diastereomers

PubMed Central

Nagel, Lilly; Budke, Carsten; Dreyer, Axel; Koop, Thomas

2012-01-01

Summary Antifreeze glycopeptides (AFGPs) are a special class of biological antifreeze agents, which possess the property to inhibit ice growth in the body fluids of arctic and antarctic fish and, thus, enable life under these harsh conditions. AFGPs are composed of 4–55 tripeptide units -Ala-Ala-Thr- glycosylated at the threonine side chains. Despite the structural homology among all the fish species, divergence regarding the composition of the amino acids occurs in peptides from natural sources. Although AFGPs were discovered in the early 1960s, the adsorption mechanism of these macromolecules to the surface of the ice crystals has not yet been fully elucidated. Two AFGP diastereomers containing different amino acid configurations were synthesized to study the influence of amino acid stereochemistry on conformation and antifreeze activity. For this purpose, peptides containing monosaccharide-substituted allo-L- and D-threonine building blocks were assembled by solid-phase peptide synthesis (SPPS). The retro-inverso AFGP analogue contained all amino acids in D-configuration, while the allo-L-diastereomer was composed of L-amino acids, like native AFGPs, with replacement of L-threonine by its allo-L-diastereomer. Both glycopeptides were analyzed regarding their conformational properties, by circular dichroism (CD), and their ability to inhibit ice recrystallization in microphysical experiments. PMID:23209499

Thermodynamic approach to the paradox of diamond formation with simultaneous graphite etching in the low pressure synthesis of diamond

NASA Astrophysics Data System (ADS)

Hwang, Nong M.; Yoon, Duk Y.

1996-03-01

In spite of the critical handicap from the thermodynamic point of view, the atomic hydrogen hypothesis is strongly supported by experimental observations of diamond deposition with simultaneous graphite etching. Thermodynamic analysis of the CH system showed that at ˜ 1500 K, carbon solubility in the gas phase is minimal and thus, the equilibrium fraction of solid carbon is maximal. Depending on whether gas phase nucleation takes place or not, the driving force is for deposition or for etching of solid carbon below ˜ 1500 K for the input gas of the typical mixture of 1% CH 499% H 2. The previous observation of etching of the graphite substrate is not expected unless solid carbon precipitated in the gas phase. By rigorous thermodynamic analysis of the previous experimental observations of diamond deposition with simultaneous graphite etching, we suggested that the previous implicit assumption that diamond deposits by an atomic unit should be the weakest point leading to the thermodynamic paradox. The experimental observations could be successfully explained without violating thermodynamics by assuming that the diamond phase had nucleated in the gas phase as fine clusters.

Size-controlled synthesis of Pd nanocrystals using a specific multifunctional peptide

NASA Astrophysics Data System (ADS)

Chiu, Chin-Yi; Li, Yujing; Huang, Yu

2010-06-01

Here we report a peptide-mediated synthesis of Pd NCs in aqueous solution with controllable size in the sub-10 nanometre regime. The specific multifunctional peptide Q7 selected using the phage display technique can bind to the Pd NC surface and act as a stabilizer to mediate Pd crystal nucleation and growth. At the nucleation stage, Q7 bound to and helped stabilize the different-sized small Pd NC nuclei achieved using different concentrations of the external reducing agent, NaBH4. At the growth stage, Q7 played the dual role of binding to and reducing the precursor onto the existing nuclei, which led to the further controllable growth of the Pd NCs. By using the variable sizes of nuclei as seeds, and by introducing different amounts of precursors Pd NCs with tunable sizes from 2.6 to 6.6 nm were achieved with good size distribution.Here we report a peptide-mediated synthesis of Pd NCs in aqueous solution with controllable size in the sub-10 nanometre regime. The specific multifunctional peptide Q7 selected using the phage display technique can bind to the Pd NC surface and act as a stabilizer to mediate Pd crystal nucleation and growth. At the nucleation stage, Q7 bound to and helped stabilize the different-sized small Pd NC nuclei achieved using different concentrations of the external reducing agent, NaBH4. At the growth stage, Q7 played the dual role of binding to and reducing the precursor onto the existing nuclei, which led to the further controllable growth of the Pd NCs. By using the variable sizes of nuclei as seeds, and by introducing different amounts of precursors Pd NCs with tunable sizes from 2.6 to 6.6 nm were achieved with good size distribution. Electronic Supplementary Information (ESI) available. Experimental details for peptide selection, peptide synthesis and Pd NCs synthesis; Q7 peptide sequence molecular structure and characterization; TEM images of Pd NCs. See DOI: 10.1039/c0nr00194e/

Biomimetic and Aggregation-Driven Crystallization Route for Room-Temperature Material Synthesis: Growth of β-Ga2O3 Nanoparticles Using Peptide Assemblies as Nanoreactors

PubMed Central

Lee, Sang-Yup; Gao, Xueyun; Matsui, Hiroshi

2008-01-01

The room temperature synthesis of β-Ga2O3 nanocrystal was examined by coupling two biomimetic crystallization techniques, the enzymatic peptide nano-assembly templating and the aggregation-driven crystallization. The catalytic template of peptide assembly nucleated and mineralized primary β-Ga2O3 crystals, and then fused them to grow single-crystalline and monodisperse nanoparticles in the cavity of the peptide assembly at room temperature. In this work, the peptide assembly was exploited as a nano-reactor with an enzymatic functionality catalyzing the hydrolysis of gallium precursors. In addition, the characteristic ring-structure of peptide assembly is expected to provide an efficient dehydration pathway and the crystallization control over the surface tension, which are advantageous for the β-Ga2O3 crystal growth. This multifunctional peptide assembly could be applied for syntheses of a variety of nanomaterials that are kinetically difficult to grow at room temperature. PMID:17302413

Molten salt synthesis of La0.8Sr0.2MnO3 powders for SOFC cathode electrode

NASA Astrophysics Data System (ADS)

Gu, Sin-il; Shin, Hyo-soon; Hong, Youn-woo; Yeo, Dong-hun; Kim, Jong-hee; Nahm, Sahn; Yoon, Sang-ok

2012-08-01

For La0.8Sr0.2MnO3 (LSM) perovskite, used as the cathode material for solid oxide fuel cells (SOFC), it is known that the formation of a triple-phase-boundary is restrained due to the formation of a second phase at the YSZ/electrode interface at high temperature. To decrease the 2nd phase, lowering the sintering temperature has been used. LSM powder was synthesized by molten salt synthesis method to control its particle size, shape, and agglomeration. We have characterized the phase formation, particle size, shape, and sintering behavior of LSM in the synthesis using the variation of KCl, LiCl, KF and its mixed salts as raw materials. In the case of KCl and KCl-KF salts, the particle size and shape of the LSM was well controlled and synthesized. However, in the case of LiCl and KCl-LiCl salts, LiMnOx as 2nd phase and LSM were synthesized simultaneously. In the case of the mixed salt of KCl-KF, the growth mechanism of the LSM particle was changed from `diffusion-controlled' to `reaction-controlled' according to the amount of mixed salt. The sintering temperature can be decreased below 1000 °C by using the synthesized LSM powder.

Investigation on phase transitions of 1-decylammonium hydrochloride as the potential thermal energy storage material

NASA Astrophysics Data System (ADS)

Dan, Wen-Yan; Di, You-Ying; He, Dong-Hua; Liu, Yu-Pu

2011-02-01

1-Decylammonium hydrochloride was synthesized by the method of liquid phase synthesis. Chemical analysis, elemental analysis, and X-ray single crystal diffraction techniques were applied to characterize its composition and structure. Low-temperature heat capacities of the compounds were measured with a precision automated adiabatic calorimeter over the temperature range from 78 to 380 K. Three solid-solid phase transitions have been observed at the peak temperatures of 307.52 ± 0.13, 325.02 ± 0.19, and 327.26 ± 0.07 K. The molar enthalpies and entropies of three phase transitions were determined based on the analysis of heat capacity curves. Experimental molar heat capacities were fitted to two polynomial equations of the heat capacities as a function of temperature by least square method. Smoothed heat capacities and thermodynamic functions of the compound relative to the standard reference temperature 298.15 K were calculated and tabulated at intervals of 5 K based on the fitted polynomials.

Expression, stabilization and purification of membrane proteins via diverse protein synthesis systems and detergents involving cell-free associated with self-assembly peptide surfactants.

PubMed

Zheng, Xuan; Dong, Shuangshuang; Zheng, Jie; Li, Duanhua; Li, Feng; Luo, Zhongli

2014-01-01

G-protein coupled receptors (GPCRs) are involved in regulating most of physiological actions and metabolism in the bodies, which have become most frequently addressed therapeutic targets for various disorders and diseases. Purified GPCR-based drug discoveries have become routine that approaches to structural study, novel biophysical and biochemical function analyses. However, several bottlenecks that GPCR-directed drugs need to conquer the problems including overexpression, solubilization, and purification as well as stabilization. The breakthroughs are to obtain efficient protein yield and stabilize their functional conformation which are both urgently requiring of effective protein synthesis system methods and optimal surfactants. Cell-free protein synthesis system is superior to the high yields and post-translation modifications, and early signs of self-assembly peptide detergents also emerged to superiority in purification of membrane proteins. We herein focus several predominant protein synthesis systems and surfactants involving the novel peptide detergents, and uncover the advantages of cell-free protein synthesis system with self-assembling peptide detergents in purification of functional GPCRs. This review is useful to further study in membrane proteins as well as the new drug exploration. Copyright © 2014 Elsevier Inc. All rights reserved.

Ocellatin peptides from the skin secretion of the South American frog Leptodactylus labyrinthicus (Leptodactylidae): characterization, antimicrobial activities and membrane interactions.

PubMed

Gusmão, Karla A G; Dos Santos, Daniel M; Santos, Virgílio M; Cortés, María Esperanza; Reis, Pablo V M; Santos, Vera L; Piló-Veloso, Dorila; Verly, Rodrigo M; de Lima, Maria Elena; Resende, Jarbas M

2017-01-01

The availability of antimicrobial peptides from several different natural sources has opened an avenue for the discovery of new biologically active molecules. To the best of our knowledge, only two peptides isolated from the frog Leptodactylus labyrinthicus , namely pentadactylin and ocellatin-F1, have shown antimicrobial activities. Therefore, in order to explore the antimicrobial potential of this species, we have investigated the biological activities and membrane interactions of three peptides isolated from the anuran skin secretion. Three peptide primary structures were determined by automated Edman degradation. These sequences were prepared by solid-phase synthesis and submitted to activity assays against gram-positive and gram-negative bacteria and against two fungal strains. The hemolytic properties of the peptides were also investigated in assays with rabbit blood erythrocytes. The conformational preferences of the peptides and their membrane interactions have been investigated by circular dichroism spectroscopy and liposome dye release assays. The amino acid compositions of three ocellatins were determined and the sequences exhibit 100% homology for the first 22 residues (ocellatin-LB1 sequence). Ocellatin-LB2 carries an extra Asn residue and ocellatin-F1 extra Asn-Lys-Leu residues at C-terminus. Ocellatin-F1 presents a stronger antibiotic potential and a broader spectrum of activities compared to the other peptides. The membrane interactions and pore formation capacities of the peptides correlate directly with their antimicrobial activities, i.e., ocellatin-F1 > ocellatin-LB1 > ocellatin-LB2. All peptides acquire high helical contents in membrane environments. However, ocellatin-F1 shows in average stronger helical propensities. The obtained results indicate that the three extra amino acid residues at the ocellatin-F1 C-terminus play an important role in promoting stronger peptide-membrane interactions and antimicrobial properties. The extra Asn-23 residue present in ocellatin-LB2 sequence seems to decrease its antimicrobial potential and the strength of the peptide-membrane interactions.

13C-13C rotational resonance in a transmembrane peptide: A comparison of the fluid and gel phases

NASA Astrophysics Data System (ADS)

Langlais, Denis B.; Hodges, Robert S.; Davis, James H.

1999-05-01

A comparative study of two doubly 13C labeled amphiphilic transmembrane peptides was undertaken to determine the potential of rotational resonance for measuring internuclear distances through the direct dipolar coupling in the presence of motion. The two peptides, having the sequence acetyl-K2-G-L16-K2-A-amide, differed only in the position of 13C labels. The first peptide, [1-13C]leu11:[α-13C]leu12, had labels on adjacent residues, at the carbonyl of leu11 and the α carbon of leu12. The second, [1-13C]leu8:[α-13\\|C]leu11, was labeled on consecutive turns of the α-helical peptide. The internuclear distance between labeled positions of the first peptide, which for an ideal α helix has a value of 2.48 Å, is relatively independent of internal flexibility or peptide conformational change. The dipolar coupling between these two nuclei is sensitive to motional averaging by molecular reorientation, however, making this peptide ideal for investigating these motions. The internuclear distance between labels on the second peptide has an expected static ideal α-helix value of 4.6 Å, but this is sensitive to internal flexibility. In addition, the dipolar coupling between these two nuclei is much weaker because of their larger separation, making this peptide a much more difficult test of the rotational resonance technique. The dipolar couplings between the labeled nuclei of these two peptides were measured by rotational resonance in the dry peptide powders and in multilamellar dispersions with dimyristoylphosphatidylcholine in the gel phase, at -10 °C, and in the fluid phase, at 40 °C. The results for the peptide having adjacent labels can be readily interpreted in terms of a simple model for the peptide motion. The results for the second peptide show that, in the fluid phase, the motionally averaged dipolar coupling is too small to be measured by rotational resonance. Rotational resonance, rotational echo double resonance, and related techniques can be used to obtain reliable and valuable dipolar couplings in static solid and membrane systems. The interpretation of these couplings in terms of internuclear distances is straightforward in the absence of molecular motion. These techniques hold considerable promise for membrane protein structural studies under conditions, such as at low temperatures, where molecular motion does not modulate the dipolar couplings. However, a typical membrane at physiological temperatures exhibits complex molecular motions. In the absence of an accurate and detailed description of both internal and whole body molecular motions, it is unlikely that techniques of this type, which are based on extracting distances from direct internuclear dipolar couplings, can be used to study molecular structure under these conditions. Furthermore, the reduction in the strengths of the dipolar couplings by these motions dramatically reduces the useful range of distances which can be measured.

Alanine scan of the peptide antibiotic feglymycin: assessment of amino acid side chains contributing to antimicrobial activity.

PubMed

Hänchen, Anne; Rausch, Saskia; Landmann, Benjamin; Toti, Luigi; Nusser, Antje; Süssmuth, Roderich D

2013-03-18

The antibiotic feglymycin is a linear 13-mer peptide synthesized by the bacterium Streptomyces sp. DSM 11171. It mainly consists of the nonproteinogenic amino acids 4-hydroxyphenylglycine and 3,5-dihydroxyphenylglycine. An alanine scan of feglymycin was performed by solution-phase peptide synthesis in order to assess the significance of individual amino acid side chains for biological activity. Hence, 13 peptides were synthesized from di- and tripeptide building blocks, and subsequently tested for antibacterial activity against Staphylococcus aureus strains. Furthermore we tested the inhibition of peptidoglycan biosynthesis enzymes MurA and MurC, which are inhibited by feglymycin. Whereas the antibacterial activity is significantly based on the three amino acids D-Hpg1, L-Hpg5, and L-Phe12, the inhibitory activity against MurA and MurC depends mainly on L-Asp13. The difference in the position dependence for antibacterial activity and enzyme inhibition suggests multiple molecular targets in the modes of action of feglymycin. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Effect of polynucleotides on the dimerization of glycine. [abiological protein synthesis in primitive earth conditions

NASA Technical Reports Server (NTRS)

Mizutani, H.; Ponnamperuma, C.

1981-01-01

Results from experiments to determine the effect of polynucleotides on abiological formation of peptide bonds are reported. The reaction between glycine molecules in an aqueous phase in the presence of a condensing agent was chosen as a model, with polyphosphates being selected as the condensing agent for biologically relevant peptide formation. Four types of polynucleotides were used: polygluanic acid (G), polyuridic acid (U), polyadenylic acid (A), and polycytidylic acid (C); the effects of small anions, acetate, chloride, and phosphate, were also studied. Procedures are given, including concentrations, pH, and incubation time, and the type of amino acid analyzer. The diglycine yields were, in order of most to least: G, C, A, U, and are diagrammed as a function of time; rate of formation followed the same order of magnitude as the final yields. Anion presence displayed no discernible effect. The results are taken to indicate that polynucleotides do have an effect on the formation of peptide bonds, an effect significant in the understanding of chemical evolution.

Solution based synthesis of mixed-phase materials in the Li2TiO3-Li4SiO4 system

NASA Astrophysics Data System (ADS)

Hanaor, Dorian A. H.; Kolb, Matthias H. H.; Gan, Yixiang; Kamlah, Marc; Knitter, Regina

2015-01-01

As candidate tritium breeder materials for use in the ITER helium cooled pebble bed, ceramic multiphasic compounds lying in the region of the quasi-binary lithium metatitanate-lithium orthosilicate system may exhibit mechanical and physical advantages relative to single phase materials. Here we present an organometallic solution-based synthesis procedure for the low-temperature fabrication of compounds in the Li2TiO3-Li4SiO4 region and investigate phase stability and transformations through temperature varied X-ray diffraction and scanning calorimetry. Results demonstrate that the metatitanate and metasilicate phases Li2TiO3 and Li2SiO3 readily crystallise in nanocrystalline form at temperatures below 180 °C. Lithium deficiency in the region of 5% results from Li sublimation from Li4SiO4 and/or from excess Li incorporation in the metatitanate phase and brings about a stoichiometry shift, with product compounds exhibiting mixed lithium orthosilicate/metasilicate content towards the Si rich region and predominantly Li2TiO3 content towards the Ti rich region. Above 1150 °C the transformation of monoclinic to cubic γ-Li2TiO3 disordered solid-solution occurs while the melting of silicate phases indicates a likely monotectic type system with a solidus line in the region 1050-1100 °C. Synthesis procedures involving a lithium chloride precursor are not likely to be a viable option for breeder pebble synthesis as this route was found to yield materials with a more significant Li-deficiency exhibiting the crystallisation of the Li2TiSiO5 phase at intermediate compositions.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murgia, Fabrizio; Antitomaso, Philippe; Stievano, Lorenzo

The ternary Chevrel phase Cu{sub 2}Mo{sub 6}S{sub 8} was successfully synthetized using a simple and cost-effective solid-state microwave-assisted reaction. While solid-state routes require days of high-temperature treatment under inert atmosphere, highly pure and crystalline Cu{sub 2}Mo{sub 6}S{sub 8} could be obtained in only 400 s from this precursor, the Chevrel binary phase Mo{sub 6}S{sub 8} was then obtained by copper removal through acidic leaching, and was evaluated as a positive electrode material for Mg-battery. The electrochemical performance in half-cell configuration shows reversible capacity exceeding 80 mAh/g, which is comparable to previous works carried out with materials synthesized by conventional high-temperaturemore » solid-state routes. - Graphical abstract: Ultrafast micro-wave synthesis of Chevrel phase Cu{sub 2}Mo{sub 6}S{sub 8} towards Mo{sub 6}S{sub 8} as positive electrode of Mg-battery. - Highlights: • Chevrel phase Cu{sub 2}Mo{sub 6}S{sub 8} is synthesized by fast microwave-assisted solid-state reaction. • Highly-pure and well-crystalline Cu{sub 2}Mo{sub 6}S{sub 8} is obtained. • Mo{sub 6}S{sub 8} obtained from leaching is tested as a positive electrode for Mg batteries.« less

Drug forecast - the peptide deformylase inhibitors as antibacterial agents.

PubMed

Guay, David R P

2007-08-01

The relatively rapid development of microbial resistance after the entry of every new antimicrobial into the marketplace necessitates a constant supply of new agents to maintain effective pharmacotherapy. Despite extensive efforts to identify novel lead compounds from molecular targets, only the peptide deformylase inhibitors (PDIs) have shown any real promise, with some advancing to phase I human trials. Bacterial peptide deformylase, which catalyzes the removal of the N-formyl group from N-terminal methionine following translation, is essential for bacterial protein synthesis, growth, and survival. The majority of PDIs are pseudopeptide hydroxamic acids and two of these (IV BB-83698 and oral NVP LBM-415) entered phase I human trials. However, agents to the present have suffered from major potential liabilities. Their in vitro activity has been limited to gram-positive aerobes and some anaerobes and has been quite modest against the majority of such species (MIC(90) values ranging from 1-8 mg/L). They have exerted bacteriostatic, not bacteriocidal, activity, thus reducing their potential usefulness in the management of serious infections in the immunocompromised. The relative ease with which microorganisms have been able to develop resistance and the multiple available mechanisms of resistance (mutations in fmt, defB, folD genes; AcrAB/TolC efflux pump; overexpression of peptide deformylase) are worrisome. These could portend a short timespan of efficacy after marketing. Despite these current liabilities, further pursuit of more potent and broader spectrum PDIs which are less susceptible to bacterial mechanisms of resistance is still warranted.

The utility of tumor-specifically internalizing peptides for targeted siRNA delivery into human solid tumors.

PubMed

Un, Frank; Zhou, Bingsen; Yen, Yun

2012-11-01

Ribonucleotide reductase composed of the hRRM1 and hRRM2 subunits catalyzes the conversion of ribonucleotides to their corresponding deoxy forms for DNA replication. Anti-hRRM2 siRNA degrades hRRM2's mRNA and suppresses tumorigenesis. A Phase I clinical trial demonstrated its therapy potential. HN-1 represents a tumor-specifically internalizing peptide for targeted-drug delivery into human head and neck squamous cell carcinoma. Internalization of peptide was monitored by fluorescence microscopy. The peptide-siRNA conjugate was chemically synthesized. The hRRM2 expression was monitored by western blot analysis. HN-1(TYR) (HN-1 with two N-terminally added tyrosines) was internalized by human head and neck or breast cancer cells. Anti-hRRM2 siRNA(R) (resistant to RNase degradation) was conjugated to HN-1(TYR) without compromising their properties. The treatment with HN-1(TYR)-anti-hRRM2 siRNA(R) partly suppressed the endogenously expressed hRRM2 in human breast cancer cells. Our results establish the utility of tumor-specifically internalizing peptides for targeted siRNA delivery into human cancer cells.

Shifting Native Chemical Ligation into Reverse through N→S Acyl Transfer

PubMed Central

Macmillan, Derek; Adams, Anna; Premdjee, Bhavesh

2011-01-01

Peptide thioester synthesis by N→S acyl transfer is being intensively explored by many research groups the world over. Reasons for this likely include the often straightforward method of precursor assembly using Fmoc-based chemistry and the fundamentally interesting acyl migration process. In this review we introduce recent advances in this exciting area and discuss, in more detail, our own efforts towards the synthesis of peptide thioesters through N→S acyl transfer in native peptide sequences. We have found that several peptide thioesters can be readily prepared and, what’s more, there appears to be ample opportunity for further development and discovery. PMID:22347724

Peptide/protein-polymer conjugates: synthetic strategies and design concepts.

PubMed

Gauthier, Marc A; Klok, Harm-Anton

2008-06-21

This feature article provides a compilation of tools available for preparing well-defined peptide/protein-polymer conjugates, which are defined as hybrid constructs combining (i) a defined number of peptide/protein segments with uniform chain lengths and defined monomer sequences (primary structure) with (ii) a defined number of synthetic polymer chains. The first section describes methods for post-translational, or direct, introduction of chemoselective handles onto natural or synthetic peptides/proteins. Addressed topics include the residue- and/or site-specific modification of peptides/proteins at Arg, Asp, Cys, Gln, Glu, Gly, His, Lys, Met, Phe, Ser, Thr, Trp, Tyr and Val residues and methods for producing peptides/proteins containing non-canonical amino acids by peptide synthesis and protein engineering. In the second section, methods for introducing chemoselective groups onto the side-chain or chain-end of synthetic polymers produced by radical, anionic, cationic, metathesis and ring-opening polymerization are described. The final section discusses convergent and divergent strategies for covalently assembling polymers and peptides/proteins. An overview of the use of chemoselective reactions such as Heck, Sonogashira and Suzuki coupling, Diels-Alder cycloaddition, Click chemistry, Staudinger ligation, Michael's addition, reductive alkylation and oxime/hydrazone chemistry for the convergent synthesis of peptide/protein-polymer conjugates is given. Divergent approaches for preparing peptide/protein-polymer conjugates which are discussed include peptide synthesis from synthetic polymer supports, polymerization from peptide/protein macroinitiators or chain transfer agents and the polymerization of peptide side-chain monomers.

Chemical synthesis, NMR analysis and evaluation on a cancer xenograft model (HL-60) of the aminosteroid derivative RM-133.

PubMed

Maltais, René; Hospital, Audrey; Delhomme, Audrey; Roy, Jenny; Poirier, Donald

2014-04-01

The aminosteroid derivative RM-133 has been reported to be a promising pro-apoptotic agent showing activity on various cancer cell lines. Following the development of solid-phase synthesis that generated a series of libraries of aminosteroid derivatives, we now report the development of a convenient liquid phase chemical synthesis of RM-133, the most promising candidate, in order to obtain sufficient quantities to proceed with the first preclinical assays. A simple and convergent six-step synthesis was designed and allowed the preparation of a gram-quantity scale of RM-133. This aminosteroid derivative was also fully characterized by NMR experiments which revealed an interesting mixture of conformers. Finally, the in vivo potency of RM-133 was evaluated on a xenograft model in nude mice with HL-60 tumors, which has resulted in the blocking of tumor progression by 57%. Copyright © 2014 Elsevier Inc. All rights reserved.

DNA - peptide polyelectrolyte complexes: Phase control by hybridization

NASA Astrophysics Data System (ADS)

Vieregg, Jeffrey; Lueckheide, Michael; Marciel, Amanda; Leon, Lorraine; Tirrell, Matthew

DNA is one of the most highly-charged molecules known, and interacts strongly with charged molecules in the cell. Condensation of long double-stranded DNA is one of the classic problems of biophysics, but the polyelectrolyte behavior of short and/or single-stranded nucleic acids has attracted far less study despite its importance for both biological and engineered systems. We report here studies of DNA oligonucleotides complexed with cationic peptides and polyamines. As seen previously for longer sequences, double-stranded oligonucleotides form solid precipitates, but single-stranded oligonucleotides instead undergo liquid-liquid phase separation to form coacervate droplets. Complexed oligonucleotides remain competent for hybridization, and display sequence-dependent environmental response. We observe similar behavior for RNA oligonucleotides, and methylphosphonate substitution of the DNA backbone indicates that nucleic acid charge density controls whether liquid or solid complexes are formed. Liquid-liquid phase separations of this type have been implicated in formation of membraneless organelles in vivo, and have been suggested as protocells in early life scenarios; oligonucleotides offer an excellent method to probe the physics controlling these phenomena.

Off-Line Multidimensional Liquid Chromatography and Auto Sampling Result in Sample Loss in LC/LC–MS/MS

PubMed Central

2015-01-01

Large-scale proteomics often employs two orthogonal separation methods to fractionate complex peptide mixtures. Fractionation can involve ion exchange separation coupled to reversed-phase separation or, more recently, two reversed-phase separations performed at different pH values. When multidimensional separations are combined with tandem mass spectrometry for protein identification, the strategy is often referred to as multidimensional protein identification technology (MudPIT). MudPIT has been used in either an automated (online) or manual (offline) format. In this study, we evaluated the performance of different MudPIT strategies by both label-free and tandem mass tag (TMT) isobaric tagging. Our findings revealed that online MudPIT provided more peptide/protein identifications and higher sequence coverage than offline platforms. When employing an off-line fractionation method with direct loading of samples onto the column from an eppendorf tube via a high-pressure device, a 5.3% loss in protein identifications is observed. When off-line fractionated samples are loaded via an autosampler, a 44.5% loss in protein identifications is observed compared with direct loading of samples onto a triphasic capillary column. Moreover, peptide recovery was significantly lower after offline fractionation than in online fractionation. Signal-to-noise (S/N) ratio, however, was not significantly altered between experimental groups. It is likely that offline sample collection results in stochastic peptide loss due to noncovalent adsorption to solid surfaces. Therefore, the use of the offline approaches should be considered carefully when processing minute quantities of valuable samples. PMID:25040086

A green synthesis of a layered titanate, potassium lithium titanate; lower temperature solid-state reaction and improved materials performance

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ogawa, Makoto, E-mail: waseda.ogawa@gmail.com; Department of Earth Sciences, Waseda University, 1-6-1 Nishiwaseda, Shinjuku-ku, Tokyo 169-8050; Morita, Masashi, E-mail: m-masashi@y.akane.waseda.jp

2013-10-15

A layered titanate, potassium lithium titanate, with the size range from 0.1 to 30 µm was prepared to show the effects of the particle size on the materials performance. The potassium lithium titanate was prepared by solid-state reaction as reported previously, where the reaction temperature was varied. The reported temperature for the titanate preparation was higher than 800 °C, though 600 °C is good enough to obtain single-phase potassium lithium titanate. The lower temperature synthesis is cost effective and the product exhibit better performance as photocatalysts due to surface reactivity. - Graphical abstract: Finite particle of a layered titanate, potassiummore » lithium titanate, was prepared by solid-state reaction at lower temperature to show modified materials performance. Display Omitted - Highlights: • Potassium lithium titanate was prepared by solid-state reaction. • Lower temperature reaction resulted in smaller sized particles of titanate. • 600 °C was good enough to obtain single phased potassium lithium titanate. • The product exhibited better performance as photocatalyst.« less

Direct synthesis of nitrogen-doped graphene on platinum wire as a new fiber coating method for the solid-phase microextraction of BXes in water samples: Comparison of headspace and cold-fiber headspace modes.

PubMed

Memarian, Elham; Hosseiny Davarani, Saied Saeed; Nojavan, Saeed; Movahed, Siyavash Kazemi

2016-09-07

In this work, a new solid-phase microextraction fiber was prepared based on nitrogen-doped graphene (N-doped G). Moreover, a new strategy was proposed to solve problems dealt in direct coating of N-doped G. For this purpose, first, Graphene oxide (GO) was coated on Pt wire by electrophoretic deposition method. Then, chemical reduction of coated GO to N-doped G was accomplished by hydrazine and NH3. The prepared fiber showed good mechanical and thermal stabilities. The obtained fiber was used in two different modes (conventional headspace solid-phase microextraction and cold-fiber headspace solid-phase microextraction (CF-HS-SPME)). Both modes were optimized and applied for the extraction of benzene and xylenes from different aqueous samples. All effective parameters including extraction time, salt content, stirring rate, and desorption time were optimized. The optimized CF-HS-SPME combined with GC-FID showed good limit of detections (LODs) (0.3-2.3 μg/L), limit of quantifications (LOQs) (1.0-7.0 μg/L) and linear ranges (1.0-5000 μg/L). The developed method was applied for the analysis of benzene and xylenes in rainwater and some wastewater samples. Copyright © 2016 Elsevier B.V. All rights reserved.

Protocol for the Solid-phase Synthesis of Oligomers of RNA Containing a 2'-O-thiophenylmethyl Modification and Characterization via Circular Dichroism.

PubMed

Francis, Andrew J; Resendiz, Marino J E

2017-07-28

Solid-phase synthesis has been used to obtain canonical and modified polymers of nucleic acids, specifically of DNA or RNA, which has made it a popular methodology for applications in various fields and for different research purposes. The procedure described herein focuses on the synthesis, purification, and characterization of dodecamers of RNA 5'-[CUA CGG AAU CAU]-3' containing zero, one, or two modifications located at the C2'-O-position. The probes are based on 2-thiophenylmethyl groups, incorporated into RNA nucleotides via standard organic synthesis and introduced into the corresponding oligonucleotides via their respective phosphoramidites. This report makes use of phosphoramidite chemistry via the four canonical nucleobases (Uridine (U), Cytosine (C), Guanosine (G), Adenosine (A)), as well as 2-thiophenylmethyl functionalized nucleotides modified at the 2'-O-position; however, the methodology is amenable for a large variety of modifications that have been developed over the years. The oligonucleotides were synthesized on a controlled-pore glass (CPG) support followed by cleavage from the resin and deprotection under standard conditions, i.e., a mixture of ammonia and methylamine (AMA) followed by hydrogen fluoride/triethylamine/N-methylpyrrolidinone. The corresponding oligonucleotides were purified via polyacrylamide electrophoresis (20% denaturing) followed by elution, desalting, and isolation via reversed-phase chromatography (Sep-pak, C18-column). Quantification and structural parameters were assessed via ultraviolet-visible (UV-vis) and circular dichroism (CD) photometric analysis, respectively. This report aims to serve as a resource and guide for beginner and expert researchers interested in embarking in this field. It is expected to serve as a work-in-progress as new technologies and methodologies are developed. The description of the methodologies and techniques within this document correspond to a DNA/RNA synthesizer (refurbished and purchased in 2013) that uses phosphoramidite chemistry.

Hybrid polyurea elastomers with enzymatic degradation and tunable mechanical properties

PubMed Central

Sears, Nicholas A; Pena-Galea, Geraldine; Cereceres, Stacy N; Cosgriff-Hernandez, Elizabeth

2016-01-01

Herein, we report on the synthesis and characterization of enzymatically labile polyureas for use as a tissue-engineered ligament scaffold. Polyureas were selected due to their excellent tensile properties, fatigue resistance, and highly tunable nature. Incorporation of a collagenase-sensitive peptide into the backbone of the polyurea provided a means to confer cell-responsive degradation to the synthetic polymer. Chemical, morphological, and mechanical testing were used to confirm incorporation of the peptide and characterize polyurea films. Notably, the incorporation of the peptide resulted in an increase in modulus, elongation, and tensile strength. This was attributed to an increase in phase mixing and an increase in hydrogen bonding between the hard and soft segments. Candidate polyureas with varying levels of collagen-mimetic peptide (0%, 10%, 20%) were then subjected to degradation in collagenase media or buffer at 37°C over 4 weeks. Statistically significant decreases in strength and elongation were observed in polyureas with 20% peptide content after collagenase treatment, whereas specimens in phosphate-buffered saline showed no statistically significant difference. These observations confirmed that enzyme-specific degradation was conferred to the polyurea. Overall, these polyureas hold great promise as a material for ligament reconstruction due to the promising mechanical properties and potential for cell-mediated degradation. PMID:27994846

Enzymatic Glycosylation by Transferases

NASA Astrophysics Data System (ADS)

Blixt, Ola; Razi, Nahid

Glycosyltransferases are important biological catalysts in cellular systems generating complex cell surface glycans involved in adhesion and signaling processes. Recent advances in glycoscience have increased the demands to access significant amount of glycans representing the glycome. Glycosyltransferases are now playing a key role for in vitro synthesis of oligosaccharides and the bacterial genome are increasingly utilized for cloning and over expression of active transferases in glycosylation reactions. This chapter highlights the recent progress towards preparative synthesis of oligosaccharides representing terminal sequences of glycoproteins and glycolipids using recombinant transferases. Transferases are also being explored in the context of solid-phase synthesis, immobilized on resins and over expression in vivo by engineered bacteria.

Ultrasound assisted combustion synthesis of TiC in Al-Ti-C system.

PubMed

Liu, Zhiwei; Rakita, Milan; Xu, Wilson; Wang, Xiaoming; Han, Qingyou

2015-11-01

This research investigated the effects of high-intensity ultrasound on the combustion synthesis of TiC particles in Al-Ti-C system. The process involved that high-intensity ultrasound was applied on the surface of a compacted Al-Ti-C pellet directly through a Nb probe during the thermal explosion reaction. By comparing with the sample without ultrasonic treatment, it was found that the thermal explosion reaction for synthesizing TiC phase could take place thoroughly in the ultrasonically treated sample. During the process of synthesizing TiC phase, the dissolution of solid graphite particles into the Al-Ti melt, as well as the nucleation and growth of TiC particles could be promoted effectively due to the effects of ultrasound, leading to an enhancement of the formation of TiC particles. Ultrasound assisted combustion synthesis as a simple and effective approach was proposed for synthesizing materials in this research. Copyright © 2015 Elsevier B.V. All rights reserved.

A simple method for the small scale synthesis and solid-phase extraction purification of steroid sulfates.

PubMed

Waller, Christopher C; McLeod, Malcolm D

2014-12-01

Steroid sulfates are a major class of steroid metabolite that are of growing importance in fields such as anti-doping analysis, the detection of residues in agricultural produce or medicine. Despite this, many steroid sulfate reference materials may have limited or no availability hampering the development of analytical methods. We report simple protocols for the rapid synthesis and purification of steroid sulfates that are suitable for adoption by analytical laboratories. Central to this approach is the use of solid-phase extraction (SPE) for purification, a technique routinely used for sample preparation in analytical laboratories around the world. The sulfate conjugates of sixteen steroid compounds encompassing a wide range of steroid substitution patterns and configurations are prepared, including the previously unreported sulfate conjugates of the designer steroids furazadrol (17β-hydroxyandrostan[2,3-d]isoxazole), isofurazadrol (17β-hydroxyandrostan[3,2-c]isoxazole) and trenazone (17β-hydroxyestra-4,9-dien-3-one). Structural characterization data, together with NMR and mass spectra are reported for all steroid sulfates, often for the first time. The scope of this approach for small scale synthesis is highlighted by the sulfation of 1μg of testosterone (17β-hydroxyandrost-4-en-3-one) as monitored by liquid chromatography-mass spectrometry (LCMS). Copyright © 2014 Elsevier Inc. All rights reserved.

Synthesis of peptide thioacids at neutral pH using bis(2-sulfanylethyl)amido peptide precursors.

PubMed

Pira, Silvain L; Boll, Emmanuelle; Melnyk, Oleg

2013-10-18

Reaction of bis(2-sulfanylethyl)amido (SEA) peptides with triisopropylsilylthiol in water at neutral pH yields peptide thiocarboxylates. An alkylthioester derived from β-alanine was used to trap the released bis(2-sulfanylethyl)amine and displace the equilibrium toward the peptide thiocarboxylate.

Vibrational circular dichroism of a 2,5-diketopiperazine (DKP) peptide: Evidence for dimer formation in cyclo LL or LD diphenylalanine in the solid state.

PubMed

Pérez-Mellor, Ariel; Zehnacker, Anne

2017-02-01

The diastereomer diketopiperazine (DKP) peptides built on phenylalanine, namely, cyclo diphenylalanine LPhe-LPhe and LPhe-DPhe, were studied in the solid phase by vibrational circular dichroism (VCD) coupled to quantum chemical calculations. The unit structure of cyclo LPhe-LPhe in KBr pellets is a dimer bridged by two strong NH…O hydrogen bonds. The intense bisignate signature in the CO stretch region is interpreted in terms of two contributions arising from the free COs of the dimer and the antisymmetrical combination of the bound COs. In contrast, cyclo LPhe-DPhe shows no VCD signal in relation to its symmetric nature. © 2017 Wiley Periodicals, Inc.

Synthesis of the biologically active natural product cyclodepsipeptides apratoxin A and its analogues.

PubMed

Doi, Takayuki

2014-01-01

This paper describes the synthetic studies conducted on a marine natural product, cyclodepsipeptide apratoxin A. Total synthesis of the oxazoline analogue of apratoxin A was achieved. The conversion of oxazoline to thioamide, as well as thioamide formation from a serine-derived compound, were both unsuccessful. However, thiazoline formation from a cysteine-derived compound led to the total synthesis of apratoxin A. An in vivo study on synthetic apratoxin A revealed that it has potent antitumor activity, but with significant toxicity. Solid-phase synthesis of apratoxin A was accomplished using a preformed thiazoline derivative as a coupling unit. This method was used to synthesize several azido-containing analogues as precursors of molecular probes, and these analogues exhibited potent biological activity.

Facile purification and click labeling with 2-[ 18F]fluoroethyl azide using solid phase extraction cartridges

DOE PAGES

Zhou, Dong; Chu, Wenhua; Peng, Xin; ...

2014-11-04

In this paper, a facile method was developed to purify 2-[ 18F]fluoroethyl azide ([ 18F]FEA) using a C18 cartridge and an Oasis® HLB cartridge in series, in which [18F]FEA was exclusively trapped on the HLB cartridge. [ 18F]FEA can be eluted for reactions in solution; alternatively click labeling can be carried out on the HLB cartridge itself by loading an alkyne substrate and copper (I) catalyst dissolved in DMF onto the cartridge. Finally, this solid phase extraction methodology for purification and click labeling with [ 18F]FEA, either in solution or on the cartridge, is safe, simple, reproducible in high yield,more » and compatible with automated synthesis of 18F-labeled PET tracers.« less

Solution- and solid-phase parallel synthesis of 4-alkoxy-substituted pyrimidines with high molecular diversity.

PubMed

Font, David; Heras, Montserrat; Villalgordo, José M

2003-01-01

A simple and straightforward methodology toward the synthesis of novel 2,6-disubstituted-4-alkoxypyrimidine derivatives of type 16 and 19 has been developed. This methodology, initially developed in solution, can be perfectly adapted to the solid support under analogous conditions, taking full advantage of automated parallel synthesis systems. This successful methodology benefits from the key role played by the thioether linkage placed at the 2-position in 3, 9, or 13 in a double manner: on one side, the steric effect exerted by the thioether linkage is likely to be responsible for the very high observed selectivity toward the formation of the O-alkylation products. On the other side, this sulfur linkage can serve not only as a robust point of attachment for the heterocycle, stable to a number of reaction conditions, but also as a means of introducing a new element of diversity through activation to the corresponding sulfone (safety-catch linker concept) and subsequent ipso-substitution reaction with a variety of different N-nucleophiles.

Ab initio computational study of reaction mechanism of peptide bond formation on HF/6-31G(d,p) level

NASA Astrophysics Data System (ADS)

Siahaan, P.; Lalita, M. N. T.; Cahyono, B.; Laksitorini, M. D.; Hildayani, S. Z.

2017-02-01

Peptide plays an important role in modulation of various cell functions. Therefore, formation reaction of the peptide is important for chemical reactions. One way to probe the reaction of peptide synthesis is a computational method. The purpose of this research is to determine the reaction mechanism for peptide bond formation on Ac-PV-NH2 and Ac-VP-NH2 synthesis from amino acid proline and valine by ab initio computational approach. The calculations were carried out by theory and basis set HF/6-31G(d,p) for four mechanisms (path 1 to 4) that proposed in this research. The results show that the highest of the rate determining step between reactant and transition state (TS) for path 1, 2, 3, and 4 are 163.06 kJ.mol-1, 1868 kJ.mol-1, 5685 kJ.mol-1, and 1837 kJ.mol-1. The calculation shows that the most preferred reaction of Ac-PV-NH2 and Ac-VP-NH2 synthesis from amino acid proline and valine are on the path 1 (initiated with the termination of H+ in proline amino acid) that produce Ac-PV-NH2.

Protein chemical synthesis by α-ketoacid-hydroxylamine ligation.

PubMed

Harmand, Thibault J; Murar, Claudia E; Bode, Jeffrey W

2016-06-01

Total chemical synthesis of proteins allows researchers to custom design proteins without the complex molecular biology that is required to insert non-natural amino acids or the biocontamination that arises from methods relying on overexpression in cells. We describe a detailed procedure for the chemical synthesis of proteins with the α-ketoacid-hydroxylamine (KAHA ligation), using (S)-5-oxaproline (Opr) as a key building block. This protocol comprises two main parts: (i) the synthesis of peptide fragments by standard fluorenylmethoxycarbonyl (Fmoc) chemistry and (ii) the KAHA ligation between fragments containing Opr and a C-terminal peptide α-ketoacid. This procedure provides an alternative to native chemical ligation (NCL) that could be valuable for the synthesis of proteins, particularly targets that do not contain cysteine residues. The ligation conditions-acidic DMSO/H2O or N-methyl-2-pyrrolidinone (NMP)/H2O-are ideally suited for solubilizing peptide segments, including many hydrophobic examples. The utility and efficiency of the protocol is demonstrated by the total chemical synthesis of the mature betatrophin (also called ANGPTL8), a 177-residue protein that contains no cysteine residues. With this protocol, the total synthesis of the betatrophin protein has been achieved in around 35 working days on a multimilligram scale.

Alpha-V Integrin Targeted PET Imagining of Breast Cancer Angiogenesis and Lose-Dose Metronomic Anti-Angiogenic Chemotherapy Efficacy

DTIC Science & Technology

2005-08-01

Chen X, Plasencia C, Hou Y, Neamati N. Synthesis and Biological Evaluation of Dimeric RGD Peptide-Paclitaxel Conjugate as Model for Integrin Targeted...Plasencia C, Hou Y, Neamati N. Synthesis and biological evaluation of dimeric RGD peptide-paclitaxel conjugate as a model for integrin-targeted drug...Targeted Imaging of Lung Cancer. Neoplasia 2005;7:271-279. 6. Chen X, Plasencia C, Hou Y, Neamati N. Synthesis and Biological Evaluation of Dimeric RGD

Low Temperature Synthesis, Chemical and Electrochemical Characterization of LiNi(x)Co(1-x)O2 (0 less than x less than 1)

NASA Technical Reports Server (NTRS)

Nanjundaswamy, K. S.; Standlee, D.; Kelly, C. O.; Whiteley, R. V., Jr.

1997-01-01

A new method of synthesis for the solid solution cathode materials LiNi(x)Co(1-x)O2 (0 less than x less than 1) involving enhanced reactions at temperatures less than or equal to 700 deg. C, between metal oxy-hydroxide precursors MOOH (M = Ni, Co) and Li-salts (Li2CO3, LiOH, and LiNO3) has been investigated. The effects of synthesis conditions and sources of Li, on phase purity, microstructure, and theoretical electrochemical capacity (total M(3+) content) are characterized by powder X-ray diffraction analysis, scanning electron microscopy, chemical analysis and room temperature magnetic susceptibility. An attempt has been made to correlate the electrochemical properties with the synthesis conditions and microstructure.

Low-temperature synthesis of homogeneous solid solutions of scheelite-structured Ca 1-xSr xWO 4 and Sr 1-xBa xWO 4 nanocrystals

DOE PAGES

Culver, Sean P.; Greaney, Matthew J.; Tinoco, Antonio; ...

2015-07-24

Here, a series of compositionally complex scheelite-structured nanocrystals of the formula A 1-xA’ xWO 4 (A = Ca, Sr, Ba) have been prepared under benign synthesis conditions using the vapor diffusion sol–gel method. Discrete nanocrystals with sub-20 nm mean diameters were obtained after kinetically controlled hydro- lysis and polycondensation at room temperature, followed by composition-dependent thermal aging at or below 60 °C. Rietveld analysis of X-ray diffraction data and Raman spectroscopy verified the synthesis of continuous and phase-pure nanocrystal solid solutions across the entire composition space for A 1-xA’ xWO 4, where 0 ≤ x ≤ 1. Elemental analysis bymore » X-ray photoelectron and inductively coupled plasma- atomic emission spectroscopies demonstrated excellent agreement between the nominal and experi- mentally determined elemental stoichiometries, while energy dispersive X-ray spectroscopy illustrated good spatial elemental homogeneity within these nanocrystals synthesized under benign conditions.« less

Solid solubility, phase transitions, thermal expansion, and compressibility in Sc{sub 1−x}Al{sub x}F{sub 3}

DOE Office of Scientific and Technical Information (OSTI.GOV)

Morelock, Cody R.; Gallington, Leighanne C.; Wilkinson, Angus P., E-mail: angus.wilkinson@chemistry.gatech.edu

2015-02-15

With the goal of thermal expansion control, the synthesis and properties of Sc{sub 1−x}Al{sub x}F{sub 3} were investigated. The solubility limit of AlF{sub 3} in ScF{sub 3} at ∼1340 K is ∼50%. Solid solutions (x≤0.50) were characterized by synchrotron powder diffraction at ambient pressure between 100 and 900 K and at pressures <0.414 GPa while heating from 298 to 523 K. A phase transition from cubic to rhombohedral is observed. The transition temperature increases smoothly with Al{sup 3+} content, approaching 500 K at the solid solubility limit, and also upon compression at fixed Al{sup 3+} content. The slope of themore » pressure–temperature phase boundary is ∼0.5 K MPa{sup −1}, which is steep relative to that for most symmetry-lowering phase transitions in perovskites. The volume coefficient of thermal expansion (CTE) for the rhombohedral phase is strongly positive, but the cubic-phase CTE varies from negative (x<0.15) to near-zero (x=0.15) to positive (x>0.20) between ∼600 and 800 K. The cubic solid solutions elastically stiffen on heating, while Al{sup 3+} substitution causes softening at a given temperature. - Graphical abstract: The cubic-phase coefficient of thermal expansion for Sc{sub 1−x}Al{sub x}F{sub 3}(solubility limit ∼50% at ∼1340 K) becomes more positive with increased Al{sup 3+} substitution, but the average isothermal bulk modulus decreases (elastic softening). - Highlights: • The solubility limit of AlF{sub 3} in ScF{sub 3} at ∼1340 K is ∼50%. • The phase transition temperature of Sc{sub 1−x}Al{sub x}F{sub 3} increases smoothly with x. • The cubic-phase volume CTE varies from negative to positive with increasing x. • The cubic solid solutions elastically stiffen on heating. • Al{sup 3+} substitution causes softening at a given temperature.« less

Magnetic nanoparticles-nylon 6 composite for the dispersive micro solid phase extraction of selected polycyclic aromatic hydrocarbons from water samples.

PubMed

Reyes-Gallardo, Emilia M; Lucena, R; Cárdenas, S; Valcárcel, M

2014-06-06

In this article, the easy synthesis of magnetic nanoparticles-nylon 6 composite is presented, characterized and applied in the microextraction field. The one-step synthesis of the composite is performed by a solvent changeover playing with the different solubility of the polymeric network in formic acid and water. The new material has been characterized by different techniques including infrared spectroscopy, transmission and scanning microscopy. The extraction performance of the composite under a dispersive micro solid phase extraction format has been evaluated by determining four polycyclic aromatic hydrocarbons (benzo[b]fluoranthene, fluoranthene, indeno[1,2,3-cd]pyrene and phenanthrene) in water using ultra performance liquid chromatography (UPLC) combined with photo diode array detection. The developed methodology allows the determination of the analytes with limits of detection in the range from 0.05 μg/L (benzo[b]fluoranthene) to 0.58 μg/L (phenanthrene). The repeatability of the method was better than 6.9% at the limit of quantification level. The relative recoveries varied in the interval 80-111%. Copyright © 2014 Elsevier B.V. All rights reserved.

Effects of 8-mer acidic peptide concentration on the morphology and photoluminescence of synthesized ZnO nanomaterials

NASA Astrophysics Data System (ADS)

Moon, Chung Hee; Tousi, Marzieh; Cheeney, Joseph; Ngo-Duc, Tam-Triet; Zuo, Zheng; Liu, Jianlin; Haberer, Elaine D.

2015-11-01

An 8-mer ZnO-binding peptide, VPGAAEHT, was identified using a M13 pVIII phage display library and employed as an additive during aqueous-based ZnO synthesis at 65 °C. Unlike most other well-studied ZnO-binding sequences which are strongly basic (pI > pH 7), the 8-mer peptide was overall acidic (pI < pH 7) in character, including only a single basic residue. The selected peptide strongly influenced ZnO nanostructure formation. Morphology and optical emission properties were found to be dependent on the concentration of peptide additive. Using lower peptide concentrations (<0.1 mM), single crystal hexagonal rods and platelets were produced, and using higher peptide concentrations (≥0.1 mM), polycrystalline layered platelets, yarn-like structures, and microspheres were assembled. Photoluminescence analysis revealed a characteristic ZnO band-edge peak, as well as sub-bandgap emission peaks. Defect-related green emission, typically associated with surface-related oxygen and zinc vacancies, was significantly reduced by the peptide additive, while blue emission, attributable to oxygen and zinc interstitials, emerged with increased peptide concentrations. Peptide-directed synthesis of ZnO materials may be useful for gas sensing and photocatalytic applications in which properly engineered morphology and defect levels have demonstrated enhanced performance.

Parallel Solid-Phase Synthesis Using a New Diethylsilylacetylenic Linker and Leading to Mestranol Derivatives with Potent Antiproliferative Activities on Multiple Cancer Cell Lines.

PubMed

Dutour, Raphael; Maltais, Rene; Perreault, Martin; Roy, Jenny; Poirier, Donald

2018-03-07

RM-133 belongs to a new family of aminosteroid derivatives demonstrating interesting anticancer properties, as confirmed in vivo in four mouse cancer xenograft models. However, the metabolic stability of RM-133 needs to be improved. After investigation, the replacement of its androstane scaffold by a more stable estrane scaffold led to the development of the mestranol derivative RM-581. Using solid-phase strategy involving five steps, we quickly synthesized a series of RM-581 analogs using the recently-developed diethylsilyl acetylenic linker. To establish structure-activity relationships, we then investigated their antiproliferative potency on a panel of cancer cell lines from various cancers (breast, prostate, ovarian and pancreatic). Some of the mestranol derivatives have shown in vitro anticancer activities that are close to, or better than those observed for RM-581. Compound 23, a mestranol derivative having a ((3,5-dimethylbenzoyl)-L-prolyl)piperazine side chain at position C2, was found to be active as an antiproliferative agent (IC50 = 0.38 ± 0.34 to 3.17 ± 0.10 µM) and to be twice as active as RM-581 on LNCaP, PC-3, MCF-7, PANC-1 and OVCAR-3 cancer cells (IC50 = 0.56 ± 0.30, 0.89 ± 0.63, 1.36 ± 0.31, 2.47 ± 0.91 and 3.17 ± 0.10 µM, respectively). Easily synthesized in good yields by both solid-phase organic synthesis and classic solution-phase chemistry, this promising candidate could be used as an antiproliferative agent on a variety of cancers, notably pancreatic and ovarian cancers, both having very bad prognoses. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Functional analysis of Pacific oyster (Crassostrea gigas) β-thymosin: Focus on antimicrobial activity.

PubMed

Nam, Bo-Hye; Seo, Jung-Kil; Lee, Min Jeong; Kim, Young-Ok; Kim, Dong-Gyun; An, Cheul Min; Park, Nam Gyu

2015-07-01

An antimicrobial peptide, ∼5 kDa in size, was isolated and purified in its active form from the mantle of the Pacific oyster Crassostrea gigas by C18 reversed-phase high-performance liquid chromatography. Matrix-assisted laser desorption ionisation time-of-flight analysis revealed 4656.4 Da of the purified and unreduced peptide. A comparison of the N-terminal amino acid sequence of oyster antimicrobial peptide with deduced amino acid sequences in our local expressed sequence tag (EST) database of C. gigas (unpublished data) revealed that the oyster antimicrobial peptide sequence entirely matched the deduced amino acid sequence of an EST clone (HM-8_A04), which was highly homologous with the β-thymosin of other species. The cDNA possessed a 126-bp open reading frame that encoded a protein of 41 amino acids. To confirm the antimicrobial activity of C. gigas β-thymosin, we overexpressed a recombinant β-thymosin (rcgTβ) using a pET22 expression plasmid in an Escherichia coli system. The antimicrobial activity of rcgTβ was evaluated and demonstrated using a bacterial growth inhibition test in both liquid and solid cultures. Copyright © 2015 Elsevier Ltd. All rights reserved.

Development of nanostructured EuAl2O4 phosphors with strong long-UV excitation.

PubMed

Hirata, Gustavo A; Bosze, Eric J; McKittrick, Joanna

2008-12-01

Fueled by the need to develop novel materials for applications in solid state white-emitting lamps we have improved a new low-cost, clean and efficient technique to produce high luminescence phosphors with strong excitation in the long-UV range (350-400 nm) which makes them useful for applications in GaN-based solid state lamps. In this work, pressurized combustion synthesis has been successfully used to develop EuAl2O4 (europium aluminate), a new green photoluminescent material with monoclinic structure. The combustion synthesis reaction conditions can be adjusted to produce either the AlEuO3 orthorhombic phase at low pressures (0.1 MPa), or the new monoclinic EuAl2O4 phase, which is apparently more thermodynamically favorable at higher combustion reaction pressures (1.4 MPa). The luminescent material is a high surface area powder (approximately 50 m2/g) composed mainly of nanostructured needles and plates with 5-10 nm in diameter and 100-150 nm in length. A broad emission peak centered at 530 nm with a decay time of 1.5 approximately 2 ms is obtained at the maximum excitation wavelength lambda(exc) = 370 nm.

Facile synthesis of poly(ionic liquid)-bonded magnetic nanospheres as a high-performance sorbent for the pretreatment and determination of phenolic compounds in water samples.

PubMed

Bi, Wentao; Wang, Man; Yang, Xiaodi; Row, Kyung Ho

2014-07-01

Poly(ionic liquid)-bonded magnetic nanospheres were easily synthesized and applied to the pretreatment and determination of phenolic compounds in water samples, which have detrimental effects on water quality and the health of living beings. The high affinity of poly(ionic liquid)s toward the target compounds as well as the magnetic behavior of Fe3 O4 were combined in this material to provide an efficient and simple magnetic solid-phase extraction approach. The adsorption behavior of the poly(ionic liquid)-bonded magnetic nanospheres was examined to optimize the synthesis. Different parameters affecting the magnetic solid-phase extraction of phenolic compounds were assessed in terms of adsorption and recovery. Under the optimal conditions, the proposed method showed excellent detection sensitivity with limits of detection in the range of 0.3-0.8 ng/mL and precision in the range of 1.2-3.3%. This method was also applied successfully to the analysis of real water samples; good spiked recoveries over the range of 82.5-99.2% were obtained. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Resin comparison and fast automated stepwise conventional synthesis of human SDF-1alpha.

PubMed

Patel, Hirendra; Chantell, Christina A; Fuentes, German; Menakuru, Mahendra; Park, Jae H

2008-12-01

Human SDF-1alpha contains 68 amino acids and is a member of the chemokine family of peptides. This long peptide was synthesized stepwise using classical conditions in 101 h. The reaction times were then reduced to deprotection times of 2 x 2 min and coupling times of 2 x 2.5 min, resulting in a total synthesis time of 22 h. The effect of different resins, resin substitutions and deprotection reagents on the crude peptide purities was compared. A small portion of crude peptide was purified using an RP-HPLC column and the mass of the final product was confirmed with MALDI-TOF mass spectrometry. Copyright 2008 European Peptide Society and John Wiley & Sons, Ltd.

Fluorine-ion conductivity of different technological forms of solid electrolytes R{sub 1–y}M{sub y}F{sub 3–y} (LaF{sub 3} Type ) (M = Ca, Sr, Ba; R Are Rare Earth Elements)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sorokin, N. I., E-mail: nsorokin1@yandex.ru; Sobolev, B. P.

We have investigated the conductivity of some representatives of different technological forms of fluoride-conducting solid electrolytes R{sub 1–y}M{sub y}F{sub 3–y} (M = Ca, Sr, Ba; R are rare earth elements) with an LaF{sub 3} structure: single crystals, cold- and hot-pressing ceramics based on a charge prepared in different ways (mechanochemical synthesis, solid-phase synthesis, and fragmentation of single crystals), polycrystalline alloys, etc. It is shown (by impedance spectroscopy), that different technological forms of identical chemical composition (R, M, y) exhibit different electrical characteristics. The maximum conductivity is observed for the single-crystal form of R{sub 1–y}M{sub y}F{sub 3–y} tysonite phases, which providesmore » (in contrast to other technological forms) the formation of true volume ion-conducting characteristics.« less

Characterization of low-abundance species in the active pharmaceutical ingredient of CIGB-300: A clinical-grade anticancer synthetic peptide.

PubMed

Garay, Hilda; Espinosa, Luis Ariel; Perera, Yasser; Sánchez, Aniel; Diago, David; Perea, Silvio E; Besada, Vladimir; Reyes, Osvaldo; González, Luis Javier

2018-04-20

CIGB-300 is a first-in-class synthetic peptide-based drug of 25 amino acids currently undergoing clinical trials in cancer patients. It contains an amidated disulfide cyclic undecapeptide fused to the TAT cell-penetrating peptide through a beta-alanine spacer. CIGB-300 inhibits the CK2-mediated phosphorylation leading to apoptosis of tumor cells in vitro, and in vivo in cancer patients. Despite the clinical development of CIGB-300, the characterization of peptide-related impurities present in the active pharmaceutical ingredient has not been reported earlier. In the decision tree of ICHQ3A(R2) guidelines, the daily doses intake, the abundance, and the identity of the peptide-related species are pivotal nodes that define actions to be taken (reporting, identification, and qualification). For this, purity was first assessed by reverse-phase chromatography (>97%) and low-abundance impurities (≤0.27%) were collected and identified by mass spectrometry. Most of the impurities were generated during peptide synthesis, the spontaneous air oxidation of the reduced peptide, and the lyophilization step. The most abundant impurity, with no biological activity, was the full-length peptide containing Met 17 transformed into a sulfoxide residue. Interestingly, parallel and antiparallel dimers of CIGB-300 linked by 2 intermolecular disulfide bonds exhibited a higher antiproliferative activity than the CIGB-300 monomer. Likewise, very low abundance trimers and tetramers of CIGB-300 linked by disulfide bonds (≤0.01%) were also detected. Here we describe for the first time the presence of active dimeric species whose feasibility as novel CIGB-300 derived entities merits further investigation. Copyright © 2018 European Peptide Society and John Wiley & Sons, Ltd.

From "Frontiers of Astronomy" to Astrobiology

NASA Astrophysics Data System (ADS)

Kwok, Sun

2011-10-01

In his book Frontiers of Astronomy, Fred Hoyle outlined a number of ideas on the stellar synthesis of solid-state materials and their ejection into the interstellar medium. He also considered the possibility of interstellar organics being integrated into the early Earth during the accretion phase of planetary formation. These organics may have played a role in the origin of life and the creation of fossil fuels. In this paper, we assess these ideas with modern observational evidence, in particular on the evidence of stellar synthesis of complex organics and their delivery to the early Solar System.

C-peptide prevents SMAD3 binding to alpha promoters to inhibit collagen type IV synthesis.

PubMed

Li, Yanning; Zhong, Yan; Gong, Wenjian; Gao, Xuehan; Qi, Huanli; Liu, Kun; Qi, Jinsheng

2018-07-01

Activation of transforming growth factor β1 (TGFB1)/SMAD3 signaling may lead to additional synthesis of collagen type IV (COL4), which is a major contributor to extracellular matrix (ECM) accumulation in diabetic nephropathy (DN). C-peptide can attenuate fibrosis to have unique beneficial effects in DN. However, whether and how C-peptide affects TGFB1/SMAD3-activated COL4 synthesis is unclear. In this study, pathological changes, expression of COL4 a1-a5 chains ( Col4a1-a5 ), COL4 distribution and protein and TGFB1 and SMAD3 protein were first assessed in a rat model of diabetes. Then, rat mesangial cells were treated with high glucose (HG) and/or C-peptide to investigate the underlying mechanism. Col4a1-a5 expression, COL4 protein and secretion, TGFB1 protein, SMAD3 nuclear translocation and binding of SMAD3 to its cognate sites in the promoters of Col4a1a2 , Col4a3a4 and Col4a5 were measured. It was found that C-peptide attenuated glomerular pathological changes and suppressed renal Col4a1 -a5 mRNA expression, COL4 protein content and TGFB1 protein content. C-peptide had a dose-dependent effect to inhibit Col4a1-a5 mRNA expression, COL4 protein content and secretion, in HG-stimulated mesangial cells. In addition, the HG-induced increase in TGFB1 protein content was significantly reduced by C-peptide. Although not apparently affecting SMAD3 nuclear translocation, C-peptide prevented SMAD3 from binding to its sites in the Col4a1a2 , Col4a3a4 and Col4a5 promoters in HG-stimulated mesangial cells. In conclusion, C-peptide could prevent SMAD3 from binding to its sites in the Col4a1a2 , Col4a3a4 and Col4a5 promoters, to inhibit COL4 generation. These results may provide a mechanism for the alleviation of fibrosis in DN by C-peptide. © 2018 Society for Endocrinology.

Template-directed synthesis of silica nanotubes for explosive detection.

PubMed

Yildirim, Adem; Acar, Handan; Erkal, Turan S; Bayindir, Mehmet; Guler, Mustafa O

2011-10-01

Fluorescent porous organic-inorganic thin films are of interest of explosive detection because of their vapor phase fluorescence quenching property. In this work, we synthesized fluorescent silica nanotubes using a biomineralization process through self-assembled peptidic nanostructures. We designed and synthesized an amyloid-like peptide self-assembling into nanofibers to be used as a template for silica nanotube formation. The amine groups on the peptide nanofibrous system were used for nucleation of silica nanostructures. Silica nanotubes were used to prepare highly porous surfaces, and they were doped with a fluorescent dye by physical adsorption for explosive sensing. These porous surfaces exhibited fast, sensitive, and highly selective fluorescence quenching against nitro-explosive vapors. The materials developed in this work have vast potential in sensing applications due to enhanced surface area. © 2011 American Chemical Society

Peptide Bond Synthesis by a Mechanism Involving an Enzymatic Reaction and a Subsequent Chemical Reaction*

PubMed Central

Abe, Tomoko; Hashimoto, Yoshiteru; Zhuang, Ye; Ge, Yin; Kumano, Takuto; Kobayashi, Michihiko

2016-01-01

We recently reported that an amide bond is unexpectedly formed by an acyl-CoA synthetase (which catalyzes the formation of a carbon-sulfur bond) when a suitable acid and l-cysteine are used as substrates. DltA, which is homologous to the adenylation domain of nonribosomal peptide synthetase, belongs to the same superfamily of adenylate-forming enzymes, which includes many kinds of enzymes, including the acyl-CoA synthetases. Here, we demonstrate that DltA synthesizes not only N-(d-alanyl)-l-cysteine (a dipeptide) but also various oligopeptides. We propose that this enzyme catalyzes peptide synthesis by the following unprecedented mechanism: (i) the formation of S-acyl-l-cysteine as an intermediate via its “enzymatic activity” and (ii) subsequent “chemical” S → N acyl transfer in the intermediate, resulting in peptide formation. Step ii is identical to the corresponding reaction in native chemical ligation, a method of chemical peptide synthesis, whereas step i is not. To the best of our knowledge, our discovery of this peptide synthesis mechanism involving an enzymatic reaction and a subsequent chemical reaction is the first such one to be reported. This new process yields peptides without the use of a thioesterified fragment, which is required in native chemical ligation. Together with these findings, the same mechanism-dependent formation of N-acyl compounds by other members of the above-mentioned superfamily demonstrated that all members most likely form peptide/amide compounds by using this novel mechanism. Each member enzyme acts on a specific substrate; thus, not only the corresponding peptides but also new types of amide compounds can be formed. PMID:26586916

Environmental information volume: Liquid Phase Methanol (LPMEOH{trademark}) project

DOE Office of Scientific and Technical Information (OSTI.GOV)

NONE

1996-05-01

The purpose of this project is to demonstrate the commercial viability of the Liquid Phase Methanol Process using coal-derived synthesis gas, a mixture of hydrogen and carbon monoxide. This report describes the proposed actions, alternative to the proposed action, the existing environment at the coal gasification plant at Kingsport, Tennessee, environmental impacts, regulatory requirements, offsite fuel testing, and DME addition to methanol production. Appendices include the air permit application, solid waste permits, water permit, existing air permits, agency correspondence, and Eastman and Air Products literature.

Peptide array-based interaction assay of solid-bound peptides and anchorage-dependant cells and its effectiveness in cell-adhesive peptide design.

PubMed

Kato, Ryuji; Kaga, Chiaki; Kunimatsu, Mitoshi; Kobayashi, Takeshi; Honda, Hiroyuki

2006-06-01

Peptide array, the designable peptide library covalently synthesized on cellulose support, was applied to assay peptide-cell interaction, between solid-bound peptides and anchorage-dependant cells, to study objective peptide design. As a model case, cell-adhesive peptides that could enhance cell growth as tissue engineering scaffold material, was studied. On the peptide array, the relative cell-adhesion ratio of NIH/3T3 cells was 2.5-fold higher on the RGDS (Arg-Gly-Asp-Ser) peptide spot as compared to the spot with no peptide, thus indicating integrin-mediated peptide-cell interaction. Such strong cell adhesion mediated by the RGDS peptide was easily disrupted by single residue substitution on the peptide array, thus indicating that the sequence recognition accuracy of cells was strictly conserved in our optimized scheme. The observed cellular morphological extension with active actin stress-fiber on the RGD motif-containing peptide supported our strategy that peptide array-based interaction assay of solid-bound peptide and anchorage-dependant cells (PIASPAC) could provide quantitative data on biological peptide-cell interaction. The analysis of 180 peptides obtained from fibronectin type III domain (no. 1447-1629) yielded 18 novel cell-adhesive peptides without the RGD motif. Taken together with the novel candidates, representative rules of ineffective amino acid usage were obtained from non-effective candidate sequences for the effective designing of cell-adhesive peptides. On comparing the amino acid usage of the top 20 and last 20 peptides from the 180 peptides, the following four brief design rules were indicated: (i) Arg or Lys of positively charged amino acids (except His) could enhance cell adhesion, (ii) small hydrophilic amino acids are favored in cell-adhesion peptides, (iii) negatively charged amino acids and small amino acids (except Gly) could reduce cell adhesion, and (iv) Cys and Met could be excluded from the sequence combination since they have less influence on the peptide design. Such rules that are indicative of the nature of the functional peptide sequence can be obtained only by the mass comparison analysis of PIASPAC using peptide array. By following such indicative rules, numerous amino acid combinations can be effectively screened for further examination of novel peptide design.

A versatile expression vector for the growth and amplification of unmodified phage display polypeptides.

PubMed

Winton, Alexander J; Baptiste, Janae L; Allen, Mark A

2018-09-01

Proteins and polypeptides represent nature's most complex and versatile polymer. They provide complicated shapes, diverse chemical functionalities, and tightly regulated and controlled sizes. Several disease states are related to the misfolding or overproduction of polypeptides and yet polypeptides are present in several therapeutic molecules. In addition to biological roles; short chain polypeptides have been shown to interact with and drive the bio-inspired synthesis or modification of inorganic materials. This paper outlines the development of a versatile cloning vector which allows for the expression of a short polypeptide by controlling the incorporation of a desired DNA coding insert. As a demonstration of the efficacy of the expression system, a solid binding polypeptide identified from M13 phage display was expressed and purified. The solid binding polypeptide was expressed as a soluble 6xHis-SUMO tagged construct. Expression was performed in E. coli using auto-induction followed by Ni-NTA affinity chromatography and ULP1 protease cleavage. Methodology demonstrates the production of greater than 8 mg of purified polypeptide per liter of E. coli culture. Isotopic labeling of the peptide is also demonstrated. The versatility of the designed cloning vector, use of the 6xHis-SUMO solubility partner, bacterial expression in auto-inducing media and the purification methodology make this expressionun vector a readily scalable and user-friendly system for the creation of desired peptide domains. Copyright © 2018. Published by Elsevier Inc.

Synthesis, microstructure and dielectric properties of zirconium doped barium titanate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumar, Rohtash; School of Physical Sciences, Jawaharlal Nehru University, New Delhi; Asokan, K.

2016-05-23

We report on synthesis, microstructural and relaxor ferroelectric properties of Zirconium(Zr) doped Barium Titanate (BT) samples with general formula Ba(Ti{sub 1-x}Zr{sub x})O{sub 3} (x=0.20, 0.35). These lead-free ceramics were prepared by solid state reaction route. The phase transition behavior and temperature dependent dielectric properties and composition dependent ferroelectric properties were investigated. XRD analysis at room temperature confirms phase purity of the samples. SEM observations revealed retarded grain growth with increasing Zr mole fraction. Dielectric properties of BZT ceramics is influenced significantly by small addition of Zr mole fraction. With increasing Zr mole fraction, dielectric constant decreases while FWHM and frequencymore » dispersion increases. Polarization vs electric field hysteresis measurements reveal ferroelectric relaxor phase at room temperature. The advantages of such substitution maneuvering towards optimizing ferroelectric properties of BaTiO{sub 3} are discussed.« less

Photoaffinity labeling the substance P receptor using a derivative of substance P containing para-benzoylphenylalanine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Boyd, N.D.; White, C.F.; Leeman, S.E.

A novel photoreactive substance P (SP) analogue has been synthesized by solid-phase peptide synthesis methodology to incorporate the amino acid p-benzoyl-L-phenylalanine (L-Phe(pBz)) in place of the Phe{sup 8} residue of SP. (Phe{sup 8}(OpBz))SP was equipotent with SP in competing for SP binding sites on rat submaxillary gland membranes and had potent sialagogic activity in vivo. In the absence of light, the {sup 125}I-labeled Bolton-Hunter conjugate of (Phe{sup 8}(pBz))SP bound in a saturable and reversible manner to an apparently homogeneous class of binding sites with an affinity K{sub D} = 0.4 nM. The binding of {sup 125}I-(Phe{sup 8}(pBz))SP was inhibited competitivelymore » by various tachykinin peptides and analogues with the appropriate specificity for SP/NK-1 receptors. Upon photolysis, up to 70{percent} of the specifically bound {sup 125}I-(Phe{sup 8}(pBz))SP underwent covalent linkage to two polypeptides of M{sub r} = 53 000 and 46 000, identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Quantitative analysis of the inhibitory effects of SP and related peptides on {sup 125}I-(Phe{sup 8}(pBz))SP photoincorporation indicated that the binding sites of the two photolabeled polypeptides have the same peptide specificity, namely, that typical of NK-1-type SP receptors. Further information on the relationship between the two labeled SP binding sites was provided by enzymatic digestion studies. The highly specific and remarkably efficient photolabeling achieved with {sup 125}I-(Phe{sup 8}(pBz))SP suggests that this photoaffinity probe will be of considerable value for the characterization of the molecular structure of the SP receptor.« less

Chemical synthesis, structure-activity relationship, and properties of shepherin I: a fungicidal peptide enriched in glycine-glycine-histidine motifs.

PubMed

Remuzgo, César; Oewel, Thaís S; Daffre, Sirlei; Lopes, Thiago R S; Dyszy, Fabio H; Schreier, Shirley; Machado-Santelli, Gláucia M; Teresa Machini, M

2014-11-01

Although glycine-rich antimicrobial peptides (AMPs) are found in animals and plants, very little has been reported on their chemistry, structure activity-relationship, and properties. We investigated those topics for Shepherin I (Shep I), a glycine-rich AMP with the unique amino acid sequence G(1)YGGHGGHGGHGGHGGHGGHGHGGGGHG(28). Shep I and analogues were synthesized by the solid-phase method at 60 °C using conventional heating. Purification followed by chemical characterization confirmed the products' identities and high purity. Amino acid analysis provided their peptide contents. All peptides were active against the clinically important Candida species, but ineffective against bacteria and mycelia fungi. Truncation of the N- or C-terminal portion reduced Shep I antifungal activity, the latter being more pronounced. Carboxyamidation of Shep I did not affect the activity against C. albicans or C. tropicalis, but increased activity against S. cerevisiae. Carboxyamidated analogues Shep I (3-28)a and Shep I (6-28)a were equipotent to Shep I and Shep Ia against Candida species. As with most cationic AMPs, all peptides had their activity significantly reduced in high-salt concentrations, a disadvantage that is defeated if 10 µM ZnCl2 is present. At 100 µM, the peptides were practically not hemolytic. Shep Ia also killed C. albicans MDM8 and ATCC 90028 cells. Fluo-Shep Ia, an analogue labeled with 5(6)-carboxyfluorescein, was rapidly internalized by C. albicans MDM8 cells, a salt-sensitive process dependent on metabolic energy and temperature. Altogether, such results shed light on the chemistry, structural requirements for activity, and other properties of candidacidal glycine-rich peptides. Furthermore, they show that Shep Ia may have strong potential for use in topical application.

Synthesis and evaluation of novel Tc-99m labeled NGR-containing hexapeptides as tumor imaging agents.

PubMed

Kim, Dae-Weung; Kim, Woo Hyoung; Kim, Myoung Hyoun; Kim, Chang Guhn

2015-02-01

Asparagine-glycine-arginine (NGR)-containing peptides targeting aminopeptidase N (APN)/CD13 can be an excellent candidate for targeting ligands in molecular tumor imaging. In this study, we developed two NGR-containing hexapeptides, and evaluated the diagnostic performance of Tc-99m labeled hexapeptides as molecular imaging agents in an HT-1080 fibrosarcoma-bearing murine model. Peptides were synthesized using Fmoc solid-phase peptide synthesis. Radiochemical purity of Tc-99m was evaluated using instant thin-layer chromatography. The uptake of two NGR-containing hexapeptides within HT-1080 cells was evaluated in vitro. In HT-1080 fibrosarcoma tumor-bearing mice, gamma images were acquired. A biodistribution study was performed to calculate percentage of the injected dose per gram of tissue (%ID/g). Two hexapeptides, glutamic acid-cysteine-glycine (ECG)-NGR and NGR-ECG were successfully synthesized. After radiolabeling procedures with Tc-99m, the complexes Tc-99m hexapeptides were prepared in high yield. The uptake of Tc-99m ECG-NGR within the tumor cells had been assured by in vitro studies. The gamma camera imaging in the murine model showed that Tc-99m ECG-NGR was accumulated substantially in the subcutaneously engrafted tumor. However, Tc-99m NGR-ECG was accumulated minimally in the tumor. Two NGR-containing hexapeptides, ECG-NGR and NGR-ECG were developed as molecular imaging agents to target APN/CD13 in HT-1080 fibrosarcoma. Tc-99m ECG-NGR showed a significant uptake in the tumor, and it is a good candidate for tumor imaging. Copyright © 2015 John Wiley & Sons, Ltd.

High pressure synthesis of a hexagonal close-packed phase of the high-entropy alloy CrMnFeCoNi

NASA Astrophysics Data System (ADS)

Tracy, Cameron L.; Park, Sulgiye; Rittman, Dylan R.; Zinkle, Steven J.; Bei, Hongbin; Lang, Maik; Ewing, Rodney C.; Mao, Wendy L.

2017-05-01

High-entropy alloys, near-equiatomic solid solutions of five or more elements, represent a new strategy for the design of materials with properties superior to those of conventional alloys. However, their phase space remains constrained, with transition metal high-entropy alloys exhibiting only face- or body-centered cubic structures. Here, we report the high-pressure synthesis of a hexagonal close-packed phase of the prototypical high-entropy alloy CrMnFeCoNi. This martensitic transformation begins at 14 GPa and is attributed to suppression of the local magnetic moments, destabilizing the initial fcc structure. Similar to fcc-to-hcp transformations in Al and the noble gases, the transformation is sluggish, occurring over a range of >40 GPa. However, the behaviour of CrMnFeCoNi is unique in that the hcp phase is retained following decompression to ambient pressure, yielding metastable fcc-hcp mixtures. This demonstrates a means of tuning the structures and properties of high-entropy alloys in a manner not achievable by conventional processing techniques.

Synthesis of g-C3N4/Fe3O4 nanocomposites and application as a new sorbent for solid phase extraction of polycyclic aromatic hydrocarbons in water samples.

PubMed

Wang, Man; Cui, Shihai; Yang, Xiaodi; Bi, Wentao

2015-01-01

An easy preparation of g-C3N4/Fe3O4 nanocomposites by chemical co-precipitation has been demonstrated. The as-prepared materials were characterized by X-ray diffraction, transmission electron microscopy, Fourier transform infrared spectroscopy and thermogravimetric analysis. The high affinity of g-C3N4 toward polycyclic aromatic hydrocarbons and the magnetic behavior of Fe3O4 were combined to provide an efficient and simple magnetic solid phase extraction (MSPE). The adsorption and desorption of polycyclic aromatic hydrocarbons on g-C3N4/Fe3O4 were examined. Different factors affecting the magnetic solid phase extraction of polycyclic aromatic hydrocarbons were assessed in terms of adsorption, desorption, and recovery. Under the optimized conditions, the proposed method showed good limits of detection (LOD, S/N=3) in the range of 0.05-0.1 ng mL(-1) and precision in the range of 1.8-5.3% (RSDs, n=3). This method was also successfully applied to the analysis of real water samples; good spiked recoveries over the range of 80.0-99.8% were obtained. Copyright © 2014 Elsevier B.V. All rights reserved.

Ribosomal synthesis and folding of peptide-helical aromatic foldamer hybrids

NASA Astrophysics Data System (ADS)

Rogers, Joseph M.; Kwon, Sunbum; Dawson, Simon J.; Mandal, Pradeep K.; Suga, Hiroaki; Huc, Ivan

2018-03-01

Translation, the mRNA-templated synthesis of peptides by the ribosome, can be manipulated to incorporate variants of the 20 cognate amino acids. Such approaches for expanding the range of chemical entities that can be produced by the ribosome may accelerate the discovery of molecules that can perform functions for which poorly folded, short peptidic sequences are ill suited. Here, we show that the ribosome tolerates some artificial helical aromatic oligomers, so-called foldamers. Using a flexible tRNA-acylation ribozyme—flexizyme—foldamers were attached to tRNA, and the resulting acylated tRNAs were delivered to the ribosome to initiate the synthesis of non-cyclic and cyclic foldamer-peptide hybrid molecules. Passing through the ribosome exit tunnel requires the foldamers to unfold. Yet foldamers encode sufficient folding information to influence the peptide structure once translation is completed. We also show that in cyclic hybrids, the foldamer portion can fold into a helix and force the peptide segment to adopt a constrained and stretched conformation.

Aminosilane functionalizations of mesoporous oxidized silicon for oligonucleotide synthesis and detection

PubMed Central

De Stefano, Luca; Oliviero, Giorgia; Amato, Jussara; Borbone, Nicola; Piccialli, Gennaro; Mayol, Luciano; Rendina, Ivo; Terracciano, Monica; Rea, Ilaria

2013-01-01

Direct solid phase synthesis of peptides and oligonucleotides (ONs) requires high chemical stability of the support material. In this work, we have investigated the passivation ability of porous oxidized silicon multilayered structures by two aminosilane compounds, 3-aminopropyltriethoxysilane and 3-aminopropyldimethylethoxysilane (APDMES), for optical label-free ON biosensor fabrication. We have also studied by spectroscopic reflectometry the hybridization between a 13 bases ON, directly grown on the aminosilane modified porous oxidized silicon by in situ synthesis, and its complementary sequence. Even if the results show that both devices are stable to the chemicals (carbonate/methanol) used, the porous silica structure passivated by APDMES reveals higher functionalization degree due to less steric hindrance of pores. PMID:23536541

Preclinical evaluation of a bispecific low-molecular heterodimer targeting both PSMA and GRPR for improved PET imaging and therapy of prostate cancer.

PubMed

Eder, Matthias; Schäfer, Martin; Bauder-Wüst, Ulrike; Haberkorn, Uwe; Eisenhut, Michael; Kopka, Klaus

2014-05-01

It has recently been reported that metastases of prostate cancer usually show highly heterogeneous or partly lost prostate-specific membrane antigen (PSMA) expression. In order to image and treat both PSMA positive and negative tissues PSMA targeting probes need to be extended by a further specificity. Since prostate cancer cells usually express both PSMA and gastrin-releasing peptide receptor (GRPR) a bispecific low-molecular heterodimeric molecule, addressing both targets at the same time, may significantly improve prostate cancer imaging and therapy. The nonapeptide BZH3 representing the GRPR binding part was combined with the urea-based PSMA inhibitor Glu-urea-Lys(Ahx)-HBED-CC. The syntheses of the compounds were performed according to standard Fmoc-solid phase peptide synthesis. The binding properties were analyzed by competitive cell binding and internalization experiments. The in vivo targeting properties were investigated by means of biodistribution studies. Cell binding experiments revealed high binding affinities to both GRPR and PSMA expressing cell lines. The heterodimer bound with IC50 -values essentially matching the IC50 values of the respective monomers (25.0 ± 5.4 nM for PSMA and 9.0 ± 1.8 nM for GRPR, respectively). In vivo, the heterodimer showed dual targeting of PSMA (5.4%ID/g for PSMA-positive tumors) and GRPR receptors (3.3% ID/g for GRPR-positive tumors) while exhibiting fast pharmacokinetic properties. The clearance from background was comparable to the monomeric PSMA-targeting reference. The heterodimeric molecule is a promising agent for PET imaging of primary and recurrent prostate cancer covering two receptor entities which might lead to an improved diagnostic sensitivity and therapeutic efficiency. © 2014 Wiley Periodicals, Inc.

Pentavalent Bismuth-Mediated Glycosylation Methods to Activate Sialic and Uronic Acids and the Incorporation of Sialic Acids Into Insulin

NASA Astrophysics Data System (ADS)

Kabotso, Daniel Elorm Kwame

The negative charge at physiological pH of carboxylic acid-containing monosaccharides modulate the properties of many natural biomolecules such as oligosaccharides and glycoconjugates. Unfortunately, these altered electronic properties also make the incorporation of such acidic sugars more challenging as compared to the more commonly studied neutral sugars. Herein are reported the first demonstration of glycosylation reactions mediated by triphenylbis(1,1,1-trifluoromethanesulfonato)-bismuth with thioglycosides containing carboxylic acid substituents protected as esters. Unlike with many neutral sugar substrates, the addition of 1-propanethiol to the reactions proved critical to obtaining good yields of the desired glycosylation products using sialic acid, galacturonic acid, and glucuronic acid. The protocol was demonstrated to be amenable to automation using a liquid-handling platform. The consequences of artificially incorporating carboxylic-acid-containing sugars into proteins were tested by the design of a linker containing 1 to 4 sialic acids--a sugar found in many human proteins and brain tissues--that was attached via reductive amination of trityl thiopropylaldehyde at the phenyl alanine terminal end of the protein insulin produced through solid-phase peptide synthesis. Removal of the trityl group with neat trifluoroacetic acid furnished the thiol-free modified insulin that was ligated via a disulfide bond to the peptide scaffold bearing acetyl protected sialic acids. A 14-15% ammonium hydroxide solution was found to be effective in deprotecting the acetyl groups without degradation of the disulfide bond. In addition to maintaining the potency and bioactivity of insulin, the sialic acid-containing linker rendered insulin more resistant to aggregation due to heat and mechanical agitation compared to the unmodified protein.

Chimeric relaxin peptides highlight the role of the A-chain in the function of H2 relaxin.

PubMed

Hossain, Mohammed Akhter; Wade, John D; Bathgate, Ross A D

2012-05-01

Human gene-2 (H2) relaxin is a member of the insulin-relaxin peptide superfamily. Because of the potential clinical applications of H2 relaxin, there is a need for novel analogs that have improved biological activity and receptor specificity. In this respect, we have chemically assembled chimeric peptides consisting of the B-chain of H2 relaxin in combination with A-chains from other insulin/relaxin family members. The peptides were prepared using solid phase peptide synthesis together with regioselective disulfide bond formation and characterized by RP-HPLC, MALDI-TOF MS and amino acid analysis. Their in vitro activity was assessed in RXFP1 or RXFP2 expressing cells. Replacement of the H2 relaxin A-chain resulted in parallel losses of binding affinity and activity on RXFP1. Not surprisingly H1A-H2B demonstrated the highest activity as the H1 A-chain shares high homology with H2 relaxin whereas INSLA-H2B, which shows low homology, had very poor activity. Importantly A-chain replacements had a dramatic effect on RXFP2 activity similar to previous results demonstrating different modes of activation of A-chain variants on RXFP1 and RXFP2. H3A-H2B is particularly interesting as it displays moderate activity at RXFP1 but poor activity at RXFP2 indicating that it may be a template for specific RXFP1 agonist development. Our study confirms that the activity of H2 relaxin at both RXFP1 and RXFP2 relies on interactions with both the B- and A-chains, and also provide new biochemical insights into the mechanism of relaxin action that the A-chain needs to be in native or near-native form for strong RXFP1 or RXFP2 agonist activity. Copyright © 2012 Elsevier Inc. All rights reserved.

Synthesis of β-Peptide Standards for Use in Model Prebiotic Reactions

NASA Astrophysics Data System (ADS)

Forsythe, Jay G.; English, Sloane L.; Simoneaux, Rachel E.; Weber, Arthur L.

2018-05-01

A one-pot method was developed for the preparation of a series of β-alanine standards of moderate size (2 to ≥12 residues) for studies concerning the prebiotic origins of peptides. The one-pot synthesis involved two sequential reactions: (1) dry-down self-condensation of β-alanine methyl ester, yielding β-alanine peptide methyl ester oligomers, and (2) subsequent hydrolysis of β-alanine peptide methyl ester oligomers, producing a series of β-alanine peptide standards. These standards were then spiked into a model prebiotic product mixture to confirm by HPLC the formation of β-alanine peptides under plausible reaction conditions. The simplicity of this approach suggests it can be used to prepare a variety of β-peptide standards for investigating differences between α- and β-peptides in the context of prebiotic chemistry.

Aqueous-organic phase-transfer of highly stable gold, silver, and platinum nanoparticles and new route for fabrication of gold nanofilms at the oil/water interface and on solid supports.

PubMed

Feng, Xingli; Ma, Houyi; Huang, Shaoxin; Pan, Wei; Zhang, Xiaokai; Tian, Fang; Gao, Caixia; Cheng, Yingwen; Luo, Jingli

2006-06-29

A simple but effective aqueous-organic phase-transfer method for gold, silver, and platinum nanoparticles was developed on the basis of the decrease of the PVP's solubility in water with the temperature increase. The present method is superior in the transfer efficiency of highly stable nanoparticles to the common phase-transfer methods. The gold, silver, and platinum nanoparticles transferred to the 1-butanol phase dispersed well, especially silver and platinum particles almost kept the previous particle size. Electrochemical synthesis of gold nanoparticles in an oil-water system was achieved by controlling the reaction temperature at 80 degrees C, which provides great conveniences for collecting metal particles at the oil/water interface and especially for fabricating dense metal nanoparticle films. A technique to fabricate gold nanofilms on solid supports was also established. The shapes and sizes of gold nanoparticles as the building blocks may be controllable through changing reaction conditions.

Effect of synthesis methods on the Ca{sub 3}Co{sub 4}O{sub 9} thermoelectric ceramic performances

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sotelo, A.; Rasekh, Sh.; Torres, M.A.

2015-01-15

Three different synthesis methods producing nanometric grain sizes, coprecipitation with ammonium carbonate, oxalic acid, and by attrition milling have been studied to produce Ca{sub 3}Co{sub 4}O{sub 9} ceramics and compared with the classical solid state route. These three processes have produced high reactive precursors and all the organic material and CaCO{sub 3}·have been decomposed in a single thermal treatment. Coprecipitation leads to pure Ca{sub 3}Co{sub 4}O{sub 9} phase, while attrition milling and classical solid state produce small amounts of Ca{sub 3}Co{sub 2}O{sub 6} secondary phase. Power factor values are similar for all three samples, being slightly lower for the onesmore » produced by attrition milling. These values are much higher than the obtained in samples prepared by the classical solid state method, used as reference. The maximum power factor values determined at 800 °C (∼0.43 mW/K{sup 2} m) are slightly higher than the best reported values obtained in textured ones which also show much higher density values. - Graphical abstract: Impressive raise of PF in Ca{sub 3}Co{sub 4}O{sub 9} thermoelectric materials obtained from nanometric grains. - Highlights: • Ca{sub 3}Co{sub 4}O{sub 9} has been produced by four different methods. • Precursors particle sizes influences on the final performances. • Coprecipitation methods produce single Ca{sub 3}Co{sub 4}O{sub 9} phase. • Power factor reaches values comparable to high density textured materials.« less

The solubility and site preference of Fe3+ in Li7−3xFexLa3Zr2O12 garnets

PubMed Central

Rettenwander, D.; Geiger, C.A.; Tribus, M.; Tropper, P.; Wagner, R.; Tippelt, G.; Lottermoser, W.; Amthauer, G.

2015-01-01

A series of Fe3+-bearing Li7La3Zr2O12 (LLZO) garnets was synthesized using solid-state synthesis methods. The synthetic products were characterized compositionally using electron microprobe analysis and inductively coupled plasma optical emission spectroscopy (ICP-OES) and structurally using X-ray powder diffraction and 57Fe Mössbauer spectroscopy. A maximum of about 0.25 Fe3+ pfu could be incorporated in Li7−3xFexLa3Zr2O12 garnet solid solutions. At Fe3+ concentrations lower than about 0.16 pfu, both tetragonal and cubic garnets were obtained in the synthesis experiments. X-ray powder diffraction analysis showed only a garnet phase for syntheses with starting materials having intended Fe3+ contents lower than 0.52 Fe3+ pfu. Back-scattered electron images made with an electron microprobe also showed no phase other than garnet for these compositions. The lattice parameter, a0, for all solid-solution garnets is similar with a value of a0≈12.98 Å regardless of the amount of Fe3+. 57Fe Mössbauer spectroscopic measurements indicate the presence of poorly- or nano-crystalline FeLaO3 in syntheses with Fe3+ contents greater than 0.16 Fe3+ pfu. The composition of different phase pure Li7−3xFexLa3Zr2O12 garnets, as determined by electron microprobe (Fe, La, Zr) and ICP-OES (Li) measurements, give Li6.89Fe0.03La3.05Zr2.01O12, Li6.66Fe0.06La3.06Zr2.01O12, Li6.54Fe0.12La3.01Zr1.98O12, and Li6.19Fe0.19La3.02Zr2.04O12. The 57Fe Mössbauer spectrum of cubic Li6.54Fe0.12La3.01Zr1.98O12 garnet indicates that most Fe3+ occurs at the special crystallographic 24d position, which is the standard tetrahedrally coordinated site in garnet. Fe3+ in smaller amounts occurs at a general 96h site, which is only present for certain Li-oxide garnets, and in Li6.54Fe0.12La3.01Zr1.98O12 this Fe3+ has a distorted 4-fold coordination. PMID:26435549

Effect of arginine methylation on the RNA recognition and cellular uptake of Tat-derived peptides.

PubMed

Li, Jhe-Hao; Chiu, Wen-Chieh; Yao, Yun-Chiao; Cheng, Richard P

2015-05-01

Arginine (Arg) methylation is a common post-translational modification that regulates gene expression and viral infection. The HIV-1 Tat protein is an essential regulatory protein for HIV proliferation, and is methylated in the cell. The basic region (residues 47-57) of the Tat protein contains six Arg residues, and is responsible for two biological functions: RNA recognition and cellular uptake. In this study, we explore the effect of three different methylation states at each Arg residue in Tat-derived peptides on the two biological functions. The Tat-derived peptides were synthesized by solid phase peptide synthesis. TAR RNA binding of the peptides was assessed by electrophoresis mobility shift assays. The cellular uptake of the peptides into Jurkat cells was determined by flow cytometry. Our results showed that RNA recognition was affected by both methylation state and position. In particular, asymmetric dimethylation at position 53 decreased TAR RNA binding affinity significantly, but unexpectedly less so upon asymmetric dimethylation at position 52. The RNA binding affinity even slightly increased upon methylation at some of the flanking Arg residues. Upon Arg methylation, the cellular uptake of Tat-derived peptides mostly decreased. Interestingly, cellular uptake of Tat-derived peptides with a single asymmetrically dimethylated Arg residue was similar to the native all Arg peptide (at 120 μM). Based on our results, TAR RNA binding apparently required both guanidinium terminal NH groups on Arg53, whereas cellular uptake apparently required guanidinium terminal NH₂ groups instead. These results should provide insight into how nature uses arginine methylation to regulate different biological functions, and should be useful for the development of functional molecules with methylated arginines. Copyright © 2015. Published by Elsevier Ltd.

A new approach to phosphoserine and phosphothreonine analysis in peptides and proteins: chemical modification, enrichment via solid-phase reversible binding, and analysis by mass spectrometry.

PubMed

Thaler, Florian; Valsasina, Barbara; Baldi, Rosario; Xie, Jin; Stewart, Albert; Isacchi, Antonella; Kalisz, Henryk M; Rusconi, Luisa

2003-06-01

beta-Elimination of the phosphate group on phosphoserine and phosphothreonine residues and addition of an alkyldithiol is a useful tool for analysis of the phosphorylation states of proteins and peptides. We have explored the influence of several conditions on the efficiency of this PO(4)(3-) elimination reaction upon addition of propanedithiol. In addition to the described influence of different bases, the solvent composition was also found to have a major effect on the yield of the reaction. In particular, an increase in the percentage of DMSO enhances the conversion rate, whereas a higher amount of protic polar solvents, such as water or isopropanol, induces the opposite effect. We have also developed a protocol for enrichment of the modified peptides, which is based on solid-phase covalent capture/release with a dithiopyridino-resin. The procedure for beta-elimination and isolation of phosphorylated peptides by solid-phase capture/release was developed with commercially available alpha-casein. Enriched peptide fragments were characterized by MALDI-TOF mass spectrometric analysis before and after alkylation with iodoacetamide, which allowed rapid confirmation of the purposely introduced thiol moiety. Sensitivity studies, carried out in order to determine the detection limit, demonstrated that samples could be detected even in the low picomolar range by mass spectrometry. The developed solid-phase enrichment procedure based on reversible covalent binding of the modified peptides is more effective and significantly simpler than methods based on the interaction between biotin and avidin, which require additional steps such as tagging the modified peptides and work-up of the samples prior to the affinity capture step.

A study of the effects of synthesis conditions on Li5FeO4/carbon nanotube composites

PubMed Central

Lee, Suk-Woo; Kim, Hyun-Kyung; Kim, Myeong-Seong; Roh, Kwang Chul; Kim, Kwang-Bum

2017-01-01

Li5FeO4/carbon nanotube (LFO/CNT) composites composed of sub-micron sized LFO and a nanocarbon with high electrical conductivity were successfully synthesized for the use as lithium ion predoping source in lithium ion cells. The phase of LFO in the composite was found to be very sensitive to the synthesis conditions, such as the heat treatment temperature, type of lithium salt, and physical state of the precursors (powder or pellet), due to the carbothermic reduction of Fe3O4 by CNTs during high temperature solid state reaction. Under optimized synthesis conditions, LFO/CNT composites could be synthesized without the formation of impurities. To the best of our knowledge, this is the first report on the synthesis and characterization of a sub-micron sized LFO/CNT composites. PMID:28422146

Rapid determination of 54 pharmaceutical and personal care products in fish samples using microwave-assisted extraction-Hollow fiber-Liquid/solid phase microextraction.

PubMed

Zhang, Yi; Guo, Wen; Yue, Zhenfeng; Lin, Li; Zhao, Fengjuan; Chen, Peijin; Wu, Weidong; Zhu, Hong; Yang, Bo; Kuang, Yanyun; Wang, Jiong

2017-04-15

In this paper, a simple, rapid, solvent-less and environmental friendliness microextraction method, microwave-assisted extraction-hollow fiber-liquid/solid phase microextraction (MAE-HF-L/SME), was developed for simultaneous extraction and enrichment of 54 trace hydrophilic/lipophilic pharmaceutical and personal care products (PPCPs) from fish samples. A solid-phase extraction material, solid-phase microextraction (SPME) fiber, was synthesized. The SPME fiber had a homogeneous, loose structure and good mechanical properties, and they exhibited a good adsorption capacity for most PPCPs selected. The material formed the basis for the method of MAE-HF-L/SME. A method of liquid chromatography-high resolution mass spectroscopy (LC-HRMS) for analysis of 54 PPCPs. Under optimal synthesis and extraction conditions, the limits of detection (LODs, n=3) and the limits of quantitation (LOQs, n=10) for the 54 PPCPs were between 0.01-0.50μg·kg -1 and 0.052.00μg·kg -1 , respectively. Percent recoveries and the relative standard deviations (RSDs) in spiked fish samples (n=6) were between 56.3%-119.9% and 0.3%-17.1%, respectively. The microextraction process of 54 PPCPs in MAE-HF-L/SME took approximately 12min. The method has a low matrix interference and high enrichment factor and may be applicable for determination of 54 different PPCPs in fish samples. Copyright © 2017 Elsevier B.V. All rights reserved.

Sintering of Lead-Free Piezoelectric Sodium Potassium Niobate Ceramics

PubMed Central

Malič, Barbara; Koruza, Jurij; Hreščak, Jitka; Bernard, Janez; Wang, Ke; Fisher, John G.; Benčan, Andreja

2015-01-01

The potassium sodium niobate, K0.5Na0.5NbO3, solid solution (KNN) is considered as one of the most promising, environment-friendly, lead-free candidates to replace highly efficient, lead-based piezoelectrics. Since the first reports of KNN, it has been recognized that obtaining phase-pure materials with a high density and a uniform, fine-grained microstructure is a major challenge. For this reason the present paper reviews the different methods for consolidating KNN ceramics. The difficulties involved in the solid-state synthesis of KNN powder, i.e., obtaining phase purity, the stoichiometry of the perovskite phase, and the chemical homogeneity, are discussed. The solid-state sintering of stoichiometric KNN is characterized by poor densification and an extremely narrow sintering-temperature range, which is close to the solidus temperature. A study of the initial sintering stage revealed that coarsening of the microstructure without densification contributes to a reduction of the driving force for sintering. The influences of the (K + Na)/Nb molar ratio, the presence of a liquid phase, chemical modifications (doping, complex solid solutions) and different atmospheres (i.e., defect chemistry) on the sintering are discussed. Special sintering techniques, such as pressure-assisted sintering and spark-plasma sintering, can be effective methods for enhancing the density of KNN ceramics. The sintering behavior of KNN is compared to that of a representative piezoelectric lead zirconate titanate (PZT). PMID:28793702

Changes in Bone Alkaline Phosphatase and Procollagen Type-1 C-Peptide after Static and Dynamic Exercises

ERIC Educational Resources Information Center

Kubo, Keitaro; Yuki, Kazuhito; Ikebukuro, Toshihiro

2012-01-01

We investigated the effects of two types of nonweight-bearing exercise on changes in bone-specific alkaline phosphatase (BAP) and pro-collagen type 1 C-peptide (P1P). BAP is a specific marker of bone synthesis, whereas P1P reflects synthesis of type 1 collagen in other organs as well as bone. Eight participants performed static and dynamic…

Amino acid substrates impose polyamine, eIF5A, or hypusine requirement for peptide synthesis

PubMed Central

Shin, Byung-Sik; Katoh, Takayuki; Gutierrez, Erik; Kim, Joo-Ran; Suga, Hiroaki

2017-01-01

Abstract Whereas ribosomes efficiently catalyze peptide bond synthesis by most amino acids, the imino acid proline is a poor substrate for protein synthesis. Previous studies have shown that the translation factor eIF5A and its bacterial ortholog EF-P bind in the E site of the ribosome where they contact the peptidyl-tRNA in the P site and play a critical role in promoting the synthesis of polyproline peptides. Using misacylated Pro-tRNAPhe and Phe-tRNAPro, we show that the imino acid proline and not tRNAPro imposes the primary eIF5A requirement for polyproline synthesis. Though most proline analogs require eIF5A for efficient peptide synthesis, azetidine-2-caboxylic acid, a more flexible four-membered ring derivative of proline, shows relaxed eIF5A dependency, indicating that the structural rigidity of proline might contribute to the requirement for eIF5A. Finally, we examine the interplay between eIF5A and polyamines in promoting translation elongation. We show that eIF5A can obviate the polyamine requirement for general translation elongation, and that this activity is independent of the conserved hypusine modification on eIF5A. Thus, we propose that the body of eIF5A functionally substitutes for polyamines to promote general protein synthesis and that the hypusine modification on eIF5A is critically important for poor substrates like proline. PMID:28637321

Protease-catalyzed peptide bond formation: application to synthesis of the COOH-terminal octapeptide of cholecystokinin.

PubMed

Kullmann, W

1982-05-01

This study of protease-catalyzed peptide synthesis reports the preparation of the COOH-terminal octapeptide amide of cholecystokinin. The octapeptide was assembled by chemical condensation of two tetrapeptide segments that had been synthesized through the concerted catalytic reactions of several proteases of different specificities. The resulting octapeptide derivative was subjected to catalytic transfer hydrogenation, followed by sulfation of its tyrosine residue and removal of the N alpha-protecting group. The homogeneous target peptide was obtained after purification via partition chromatography, gel filtration, and ion-exchange chromatography. The synthetic octapeptide stimulated amylase release from pancreatic acinar cells.

A study of the effects of synthesis conditions on Li5FeO4/carbon nanotube composites.

PubMed

Lee, Suk-Woo; Kim, Hyun-Kyung; Kim, Myeong-Seong; Roh, Kwang Chul; Kim, Kwang-Bum

2017-04-19

Li 5 FeO 4 /carbon nanotube (LFO/CNT) composites composed of sub-micron sized LFO and a nanocarbon with high electrical conductivity were successfully synthesized for the use as lithium ion predoping source in lithium ion cells. The phase of LFO in the composite was found to be very sensitive to the synthesis conditions, such as the heat treatment temperature, type of lithium salt, and physical state of the precursors (powder or pellet), due to the carbothermic reduction of Fe 3 O 4 by CNTs during high temperature solid state reaction. Under optimized synthesis conditions, LFO/CNT composites could be synthesized without the formation of impurities. To the best of our knowledge, this is the first report on the synthesis and characterization of a sub-micron sized LFO/CNT composites.