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Sample records for labelling synthesis radioanalysis

  1. Synthesis Of Labeled Metabolites

    DOEpatents

    Martinez, Rodolfo A.; Silks, III, Louis A.; Unkefer, Clifford J.; Atcher, Robert

    2004-03-23

    The present invention is directed to labeled compounds, for example, isotopically enriched mustard gas metabolites including: [1,1',2,2'-.sup.13 C.sub.4 ]ethane, 1,1'-sulfonylbis[2-(methylthio); [1,1',2,2'-.sup.13 C.sub.4 ]ethane, 1-[[2-(methylsulfinyl)ethyl]sulfonyl]-2-(methylthio); [1,1',2,2'-.sup.13 C.sub.4 ]ethane, 1,1'-sulfonylbis[2-(methylsulfinyl)]; and, 2,2'-sulfinylbis([1,2-.sup.13 C.sub.2 ]ethanol of the general formula ##STR1## where Q.sup.1 is selected from the group consisting of sulfide (--S--), sulfone (--S(O)--), sulfoxide (--S(O.sub.2)--) and oxide (--O--), at least one C* is .sup.13 C, X is selected from the group consisting of hydrogen and deuterium, and Z is selected from the group consisting of hydroxide (--OH), and --Q.sup.2 --R where Q.sup.2 is selected from the group consisting of sulfide (--S--), sulfone(--S(O)--), sulfoxide (--S(O.sub.2)--) and oxide (--O--), and R is selected from the group consisting of hydrogen, a C.sub.1 to C.sub.4 lower alkyl, and amino acid moieties, with the proviso that when Z is a hydroxide and Q.sup.1 is a sulfide, then at least one X is deuterium.

  2. Synthesis of exemestane labelled with (13)C.

    PubMed

    Fontana, Erminia; Pignatti, Alberto; Giribone, Danilo; Di Salle, Enrico

    2008-08-01

    The synthesis of exemestane Aromasin, an irreversible steroidal aromatase inhibitor, specifically labelled with (13)C is reported. The preparation of [(13)C(3)]exemestane was achieved according to an eight-step procedure starting from the commercially available testosterone.

  3. Enzymic synthesis of labelled chiral substances.

    PubMed

    Battersby, A R

    1985-01-01

    The enzymic synthesis of chiral substances in which one hydrogen atom of a methylene group has been replaced by deuterium or tritium is illustrated. Such labelled products can be used to determine the stereochemistry of other enzyme-catalysed reactions.

  4. Affinity labeling of protein synthesis factors

    SciTech Connect

    Anthony, D.D.; Dever, T.E.; Abramson, R.D.; Lobur, M.; Merrick, W.C.

    1986-05-01

    The authors laboratory is interested in determining those eukaryotic protein synthesis factors which interact with nucleotides and mRNA. To study the binding the authors have used the nucleotides, their analogs, and mRNA analogs as listed below: (1) UV cross-linking with normal (/sup 32/P)XTP; (2) Oxidized GTP; (3) 3'p-azido benzoyl GDP (GTP); (4) 5'p-fluoro sulfonyl benzoyl guanosine; (5) 5'p-fluoro sulfonyl benzoyl adenosine; (6) oxidized mRNA. Currently, they are continuing their efforts to specifically label the proteins, and they are also trying to isolate a single labeled tryptic peptide from the proteins.

  5. Enzymatic synthesis of isotopically labeled isoprenoid diphosphates.

    PubMed

    Christensen, D J; Poulter, C D

    1994-07-01

    Recombinant yeast isopentenyl diphosphate (IPP) isomerase and avian farnesyl diphosphate (FPP) synthase from overproducing strains of Escherichia coli were used to synthesize FPP from IPP and dimethylallyl diphosphate (DMAPP). [2,4,5-13C3]IPP and [2,4,5-13C3]DMAPP were synthesized from ethyl [2-13C]bromoacetate and [1,3-13C2]acetone. Thes compounds were used as substrates for enzymatic synthesis of FPP selectivity labeled at the first or third isoprene residue or at all three.

  6. Synthesis of γ-Phosphate-Labeled and Doubly Labeled Adenosine Triphosphate Analogs.

    PubMed

    Hacker, Stephan M; Welter, Moritz; Marx, Andreas

    2015-03-09

    This unit describes the synthesis of γ-phosphate-labeled and doubly labeled adenosine triphosphate (ATP) analogs and their characterization using the phosphodiesterase I from Crotalus adamanteus (snake venom phosphodiesterase; SVPD). In the key step of the synthesis, ATP or an ATP analog, bearing a linker containing a trifluoroacetamide group attached to the nucleoside, are modified with an azide-containing linker at the terminal phosphate using an alkylation reaction. Subsequently, different labels are introduced to the linkers by transformation of one functional group to an amine and coupling to an N-hydroxysuccinimide ester. Specifically, the Staudinger reaction of the azide is employed as a straightforward means to obtain an amine in the presence of various labels. Furthermore, the fluorescence characteristics of a fluorogenic, doubly labeled ATP analog are investigated following enzymatic cleavage by SVPD.

  7. Direct Synthesis of ESBO Derivatives-18O Labelled with Dioxirane

    PubMed Central

    Tommasi, Immacolata; Fusco, Caterina

    2013-01-01

    This work addresses a new approach developed in our laboratory, consisting in the application of isolated dimethyldioxirane (DDO, 1a) labelled with 18O for synthesis of epoxidized glyceryl linoleate (Gly-LLL, 2). We expect that this work could contribute in improving analytical methods for the determination of epoxidized soybean oil (ESBO) in complex food matrices by adopting an 18O-labelled-epoxidized triacylglycerol as an internal standard. PMID:24163617

  8. Stereoselective synthesis of stable-isotope-labeled amino acids

    SciTech Connect

    Unkefer, C.J.; Martinez, R.A.; Silks, L.A. III; Lodwig, S.N.

    1994-12-01

    For magnetic resonance and vibrational spectroscopies to reach their full potential, they must be used in combination with sophisticated site-specific stable isotope labeling of biological macromolecules. Labeled amino acids are required for the study of the structure and function of enzymes and proteins. Because there are 20 common amino acids, each with its own distinguishing chemistry, they remain a synthetic challenge. The Oppolzer chiral auxiliary provides a general tool with which to approach the synthesis of labeled amino acids. By using the Oppolzer auxiliary, amino acids can be constructed from several small molecules, which is ideal for stable isotope labeling. In addition to directing the stereochemistry at the {alpha}-carbon, the camphorsultam can be used for stereo-specific isotope labeling at prochiral centers in amino acids. By using the camphorsultam auxiliary we have the potential to synthesize virtually any isotopomer of all of the common amino acids.

  9. General approach to the synthesis of specifically deuterium-labeled nucleosides

    SciTech Connect

    DeVoss, J.J.; Hangeland, J.J.; Townsend, C.A.

    1994-05-20

    Synthesis routes for labelled nucleosides starting with ribose have been studied. The method is viable for producing sufficient quantities of D/T labelled nucleosides for oligonucleotide synthesis. 1 tab.

  10. Synthesis of deuterium labeled 17-methyl-testosterone

    SciTech Connect

    Shinohara, Y.; Baba, S.; Kasuya, Y.

    1984-09-01

    The synthesis of two forms of selectively deuterated 17-methyl-testosterone is described. 17-Methyl-d3-testosterone was prepared by the Grignard reaction of dehydroepiandrosterone with deuterium labeled methyl magnesium iodide followed by an Oppenauer oxidation. 17-Methyl-d3-testosterone-19,19,19-d3 was prepared by treating 3,3-ethylenedioxy-5,10-epoxy-5 alpha, 10 alpha-estran-17-one with deuterium labeled methyl magnesium bromide followed by hydrolysis and dehydration of the 5 alpha-hydroxyandrostane derivative.

  11. Synthesis of nucleoside 5'-triphosphates labelled with radioactive phosphorus isotopes

    NASA Astrophysics Data System (ADS)

    Skoblov, Yurii S.; Korolev, A. E.; Maslova, R. N.

    1995-08-01

    The review presents an analysis of the chemical chemicoenzymic, and enzymic methods of synthesis of nucleoside 5'-triphosphates labelled with phosphorus isotopes ( 32P and 33P) in the α- and γ-positions. The major part of the review is devoted to enzymic methods of synthesis, because they have undoubted advantages. The enzymatic reactions are described in detail, the specificity and availability of the enzymes are considered, and data on the radiation stability of certain enzymes are presented. The bibliography includes 29 references.

  12. Synthesis of labeled oxalic acid derivatives

    DOEpatents

    Martinez, Rodolfo A.; Unkefer, Clifford J.; Alvarez, Marc A.

    2004-06-22

    The present invention is directed to labeled compounds, specifically ##STR1## where each C* is selected from the group consisting of a carbon-12, i.e., .sup.12 C, or a carbon-13, i.e., .sup.13 C and at least one C* is .sup.13 C, R.sup.1 is selected from the group of C.sub.1 -C.sub.4 lower alkyl and aryl, and X is selected from the group of --NR.sup.2 R.sup.3 where R.sup.2 and R.sup.3 are each independently selected from the group of C.sub.1 -C.sub.4 lower alkyl, alkoxy and aryl, --SR.sup.4 where R.sup.4 is selected from the group of C.sub.1 -C.sub.4 lower alkyl, alkoxy and aryl, and --OR.sup.5 where R.sup.5 is selected from the group of C.sub.1 -C.sub.4 lower alkyl, alkoxy and aryl with the proviso that when R.sup.1 is methyl then R.sup.5 is other than methyl, when R.sup.1 is ethyl then R.sup.5 is other than ethyl, and when R.sup.1 is benzyl then R.sup.5 is other than benzyl.

  13. Synthesis and properties of differently charged chemiluminescent acridinium ester labels.

    PubMed

    Natrajan, Anand; Sharpe, David

    2013-02-14

    Chemiluminescent acridinium dimethylphenyl esters containing N-sulfopropyl groups in the acridinium ring are highly sensitive, hydrophilic labels that are used in automated immunoassays for clinical diagnostics. Light emission from these labels is triggered with alkaline peroxide in the presence of a cationic surfactant. At physiological pH, N-sulfopropyl acridinium esters exist as water adducts that are commonly referred to as pseudobases. Pseudobase formation, which results from addition of water to the zwitterionic N-sulfopropyl acridinium ring, neutralizes the positive charge on the acridinium nitrogen and imparts a net negative charge to the label due to the sulfonate moiety. As a consequence, N-sulfopropyl acridinium ester conjugates of small molecule haptens as well as large molecules such as proteins gain negative charges at neutral pH. In the current study, we describe the synthesis and properties of two new hydrophilic acridinium dimethylphenyl ester labels where the net charge in the labels was altered. In one label, the structure of the hydrophilic N-alkyl group attached to the acridinium ring was changed so that the pseudobase of the label contains no net charge. In the second acridinium ester, two additional negative charges in the form of sulfopropyl groups were added to the acridinium ring to make this label's pseudobase strongly anionic. Chemiluminescence measurements of these labels, as well as their conjugates of an antibody with a neutral pI, indicate that acridinium ester charge while having a modest effect on emission kinetics has little influence on light output. However, our results demonstrate that acridinium ester charge can affect protein pI, apparent chemiluminescence stability and non-specific binding of protein conjugates to microparticles. These results emphasize the need for careful consideration of acridinium ester charge in order to optimize reagent stability and performance in immunoassays. In the current study, we observed that

  14. Efficient synthesis of deuterium labeled hydroxyzine and aripiprazole.

    PubMed

    Vohra, Mohit; Sandbhor, Mahendra; Wozniak, Andrew

    2015-06-15

    Hydroxyzine and aripiprazole are active pharmaceutical ingredients that have been largely acknowledged for their antipsychotic properties. Deuterium labeled isotopes of hydroxyzine and aripiprazole are internal standards that can aid in the further research of non-isotopic forms via quantification analysis using HPLC-MS/MS. The synthesis of hydroxyzine-d8 was accomplished by coupling piperazine-d8 with 4-chlorobenzhydryl chloride followed by the reaction of the first intermediate with 2-(2-chloroethoxy) ethanol to afford 11.7% of hydroxyzine-d8 with 99.5% purity. The synthesis of aripiprazole-d8 was also achieved in two steps. 1,4-Dibromobutane-d8 reacted with 7-hydroxy-3,4-dihydro-2(1H)-quinolinone. The first intermediate was then coupled with 1-(2, 3-dichlorophenyl)piperazine hydrochloride to produce 33.4% of aripiprazole-d8 with 99.93% purity.

  15. Synthesis of F-18 labeled resazurin by direct electrophilic fluorination.

    PubMed

    Kachur, Alexander V; Arroyo, Alejandro D; Popov, Anatoliy V; Saylor, Sarah J; Delikatny, E James

    2015-10-01

    We present the synthesis and characterization of F18-labeled fluorinated derivatives of resazurin, a probe for cell viability. The compounds were prepared by direct fluorination of resazurin with diluted [F18]-F2 gas under acidic conditions. The fluorination occurs into the ortho-positions to the hydroxyl group producing various mono-, di-, and trifluorinated derivatives. The properties of the fluorinated resazurins are similar to the parent compound with the addition of fluorine leading to decreased pKa values and a bathochromic shift of the absorption maxima. The fluorinated resazurin derivatives can be used as probes for observation of cell viability in various cells, tissues and organs using a combination of positron emission tomography and direct optical imaging of Cerenkov luminescence.

  16. Synthesis of F-18 labeled resazurin by direct electrophilic fluorination

    PubMed Central

    Kachur, Alexander V.; Arroyo, Alejandro D.; Popov, Anatoliy V.; Saylor, Sarah J.; Delikatny, E. James

    2015-01-01

    We present the synthesis and characterization of F18-labeled fluorinated derivatives of resazurin, a probe for cell viability. The compounds were prepared by direct fluorination of resazurin with diluted [F18]-F2 gas under acidic conditions. The fluorination occurs into the ortho-positions to the hydroxyl group producing various mono-, di-, and trifluorinated derivatives. The properties of the fluorinated resazurins are similar to the parent compound with the addition of fluorine leading to decreased pKa values and a bathochromic shift of the absorption maxima. The fluorinated resazurin derivatives can be used as probes for observation of cell viability in various cells, tissues and organs using a combination of positron emission tomography and direct optical imaging of Cerenkov luminescence. PMID:26504251

  17. Synthesis and detection of oxygen-18 labeled phosphate.

    PubMed

    Melby, Eric S; Soldat, Douglas J; Barak, Phillip

    2011-04-04

    Phosphorus (P) has only one stable isotope and therefore tracking P dynamics in ecosystems and inferring sources of P loading to water bodies have been difficult. Researchers have recently employed the natural abundance of the ratio of (18)O/(16)O of phosphate to elucidate P dynamics. In addition, phosphate highly enriched in oxygen-18 also has potential to be an effective tool for tracking specific sources of P in the environment, but has so far been used sparingly, possibly due to unavailability of oxygen-18 labeled phosphate (OLP) and uncertainty in synthesis and detection. One objective of this research was to develop a simple procedure to synthesize highly enriched OLP. Synthesized OLP is made up of a collection of species that contain between zero and four oxygen-18 atoms and, as a result, the second objective of this research was to develop a method to detect and quantify each OLP species. OLP was synthesized by reacting either PCl(5) or POCl(3) with water enriched with 97 atom % oxygen-18 in ambient atmosphere under a fume hood. Unlike previous reports, we observed no loss of oxygen-18 enrichment during synthesis. Electrospray ionization mass spectrometry (ESI-MS) was used to detect and quantify each species present in OLP. OLP synthesized from POCl(3) contained 1.2% P(18)O(16)O(3), 18.2% P(18)O(2) (16)O(2), 67.7% P(18)O(3) (16)O, and 12.9% P(18)O(4), and OLP synthesized from PCl(5) contained 0.7% P(16)O(4), 9.3% P(18)O(3) (16)O, and 90.0% P(18)O(4). We found that OLP can be synthesized using a simple procedure in ambient atmosphere without the loss of oxygen-18 enrichment and ESI-MS is an effective tool to detect and quantify OLP that sheds light on the dynamics of synthesis in ways that standard detection methods cannot.

  18. Synthesis and Detection of Oxygen-18 Labeled Phosphate

    PubMed Central

    Melby, Eric S.; Soldat, Douglas J.; Barak, Phillip

    2011-01-01

    Phosphorus (P) has only one stable isotope and therefore tracking P dynamics in ecosystems and inferring sources of P loading to water bodies have been difficult. Researchers have recently employed the natural abundance of the ratio of 18O/16O of phosphate to elucidate P dynamics. In addition, phosphate highly enriched in oxygen-18 also has potential to be an effective tool for tracking specific sources of P in the environment, but has so far been used sparingly, possibly due to unavailability of oxygen-18 labeled phosphate (OLP) and uncertainty in synthesis and detection. One objective of this research was to develop a simple procedure to synthesize highly enriched OLP. Synthesized OLP is made up of a collection of species that contain between zero and four oxygen-18 atoms and, as a result, the second objective of this research was to develop a method to detect and quantify each OLP species. OLP was synthesized by reacting either PCl5 or POCl3 with water enriched with 97 atom % oxygen-18 in ambient atmosphere under a fume hood. Unlike previous reports, we observed no loss of oxygen-18 enrichment during synthesis. Electrospray ionization mass spectrometertry (ESI-MS) was used to detect and quantify each species present in OLP. OLP synthesized from POCl3 contained 1.2% P18O16O3, 18.2% P18O216O2, 67.7% P18O316O, and 12.9% P18O4, and OLP synthesized from PCl5 contained 0.7% P16O4, 9.3% P18O316O, and 90.0% P18O4. We found that OLP can be synthesized using a simple procedure in ambient atmosphere without the loss of oxygen-18 enrichment and ESI-MS is an effective tool to detect and quantify OLP that sheds light on the dynamics of synthesis in ways that standard detection methods cannot. PMID:21483747

  19. Methods for the synthesis of tritium-labelled fatty acids and their derivatives, oxylipins and steroids

    NASA Astrophysics Data System (ADS)

    Shevchenko, Valerii P.; Nagaev, Igor Yu; Myasoedov, Nikolai F.

    1999-10-01

    The achievements in the field of synthesis and application of tritium-labelled oxylipins, steroids, fatty acids, phospho-, sphingo- and other lipids are reviewed. The importance of these studies for the solution of current problems of biochemistry, biology and pharmacology is exemplified in the application of labelled compounds. The bibliography includes 148 references.

  20. Synthesis of Site-Specifically (13)C Labeled Linoleic Acids.

    PubMed

    Offenbacher, Adam R; Zhu, Hui; Klinman, Judith P

    2016-10-12

    Soybean lipoxygenase-1 (SLO-1) catalyzes the C-H abstraction from the reactive carbon (C-11) in linoleic acid as the first and rate-determining step in the formation of alkylhydroperoxides. While previous labeling strategies have focused on deuterium labeling to ascertain the primary and secondary kinetic isotope effects for this reaction, there is an emerging interest and need for selectively enriched (13)C isotopologues. In this report, we present synthetic strategies for site-specific (13)C labeled linoleic acid substrates. We take advantage of a Corey-Fuchs formyl to terminal (13)C-labeled alkyne conversion, using (13)CBr4 as the labeling source, to reduce the number of steps from a previous fatty acid (13)C synthetic labeling approach. The labeled linoleic acid substrates are useful as nuclear tunneling markers and for extracting active site geometries of the enzyme-substrate complex in lipoxygenase.

  1. Synthesis of isotopically labeled versions of L-MTP-PE (mifamurtide) and MDP.

    PubMed

    Li, Yuexian; Plesescu, Mihaela; Prakash, Shimoga R

    2013-01-01

    L-MTP-PE (1), an immunomodulator and its metabolite MDP (4) were synthesized from labeled l-alanine and its protected derivative, respectively. The key intermediate product for the labeled L-MTP-PE synthesis, [(13) C3 ,D4 ]-alanyl-cephalin (2A), was synthesized from [(13) C3 ,D4 ]-l-alanine (3A) in three steps. The key intermediate product for labeled MDP synthesis, amine 11, was prepared from [(13) C3 ,(15) N]-Boc-l-alanine (5A) in two steps.

  2. Synthesis and NMR of {sup 15}N-labeled DNA fragments

    SciTech Connect

    Jones, R.A.

    1994-12-01

    DNA fragments labeled with {sup 15}N at the ring nitrogens and at the exocyclic amino groups can be used to obtain novel insight into interactions such as base pairing, hydration, drug binding, and protein binding. A number of synthetic routes to {sup 15}N-labeled pyrimidine nucleosides, purines, and purine nucleosides have been reported. Moreover, many of these labeled bases or monomers have been incorporated into nucleic acids, either by chemical synthesis or by biosynthetic procedures. The focus of this chapter will be on the preparation of {sup 15}N-labeled purine 2{prime}-deoxynucleosides, their incorporation into DNA fragments by chemical synthesis, and the results of NMR studies using these labeled DNA fragments.

  3. Synthesis and characterization of oligonucleotide conjugates bearing electroactive labels.

    PubMed

    Moreau, Julie; Dendane, Nabil; Schöllhorn, Bernd; Spinelli, Nicolas; Fave, Claire; Defrancq, Eric

    2013-02-15

    Oxime bond formation has been applied to the preparation of oligonucleotides labeled with electrochemical ferrocene and viologen labels. Aminooxy functionalized ferrocene and viologen derivatives were prepared by a straightforward route and efficiently conjugated with aldehyde containing oligonucleotides either at 3' or 5' end. Both labels were found to not disturb the recognition properties of the oligonucleotide. The versatility of the method was further demonstrated by preparing bi-functionalized conjugates with a disulfide at 3' end and an electrochemical label at 5' end. Copyright © 2012 Elsevier Ltd. All rights reserved.

  4. Synthesis of labeled compounds using recovered tritium from expired beta light sources

    SciTech Connect

    Matei, L.; Postolache, C.; Bubueanu, G.; Podina, C.

    2008-07-15

    In this paper, the technological procedures for extracting tritium from beta light source are highlighted. The recovered tritium was used in the synthesis of organically labeled compounds and in the preparation of tritiated water (HTO) with high specific activity. Technological procedures for treatment of beta light sources consist of: envelope breaking into evacuated enclosure, the radioactive gaseous mixture pumping and its storage on metallic sodium. The mixtures of T{sub 2} and {sup 3}He were used in the synthesis of tritium labeled steroid hormones, nucleosides analogues and for the preparation of HTO with high radioactivity concentrations. (authors)

  5. Synthesis of tritium-labeled vitamin A and its analogs

    SciTech Connect

    Rhee, S.W.; Bubb, J.E.

    1985-11-01

    Metabolic and pharmacologic studies of Vitamin A and its analogs related to the prevention of lung cancer and other epithelial cancers required tritium-labeled Vitamin A analogs and ..beta..-carotene at high specific activity. Syntheses of some of the isomers were therefore developed in the laboratory, as described in the paper. The advantages of the scheme shown are that : 1. Tritiums are introduced into the molecule by catalytic hydrogenation, thus affording high specific activity. 2. It uses an allylic rearrangement of tritiated vinyl-..beta..-ionol to C/sub 15/-phosphonium salt, which is condensed with C/sub 5/-nitrile to give C/sub 20/-skeleton of retinonitrile. 3. It permits the development of milder methods to convert tritium-labeled retinaldehyde, as a common intermediate, to the other retinoids (i.e., retinoic acid, retinol, and retinyl acetate). Furthermore, tritium-labeled all-trans-..beta..-carotene, an important carotenoid, has been obtained from the retinaldehyde.

  6. Synthesis, DTPA coupling and radio labeling of cationic aminodextran

    SciTech Connect

    Subramanian, G.; McAfee, J.G.; Schneider, R.F.; Zapf-Longo, C.; Palladino, E.; Lyons, B.J.; Roskopf, M.

    1984-01-01

    In glomerular diseases, the normal anionic charge of the basement membrane is lost at an early stage. Glomerular damage in rats has been detected more readily with cationic dextrans than with inulin. Hence, the authors attempted to demonstrate this phenomenon in vivo in rats with labeled cationic dextran. Aminated Dextran (AMDEX) was prepared by treating Dextran(mol. wt approx. = 15k) with sodium methoxide followed by a bromethylamine HBr in DMSO resulting in 10-25 aminogroups per mole. DTPA cyclic dianhydride was coupled to AMDEX using a weight ratio of 1:10 in 0.2 - 1.0 ml 0.42 M Hepes buffer at pH 7.4. Free DTPA was removed by gel filtration (Sephadex P6DG) or by using Centricon-10 (AMICON) centrifugal microconcentrators. AMDEX coupled with DTPA was labeled with Indium-111 in 0.25 M acetate buffer. Labeling yields were >90% by gel chromatography and electrophoresis (pH8.2 Barbitol buffer). AMEXDTPA was labeled also by ligand exchange with Tc-99m-Sn-citrate at neutral pH with a labeling yield of 30%. On electrophoresis, all the labeled samples retained their cationic character. The distribution of purified In-111 AMDEX, was compared with simultaneously IV injected Tc-99m DTPA in rats. The 2 hour urinary excretion, and renal clearance (calculated from the biexponential plasma clearance) were slower (70 to 80%) than those of DTPA, due to the larger molecular size of AMDEX. By 1 hr., 5% of the administered activity was retained in each kidney, probably due to adherence to anionic binding sites.

  7. Synthesis and preliminary biological evaluations of fluorescent or 149Promethium labeled Trastuzumab-polyethylenimine

    DOE PAGES

    Fitzsimmons, Jonathan; Nayak, Tapan; Cutler, Cathy; ...

    2015-12-30

    Radioimmunotherapy utilize a targeting antibody coupled to a therapeutic isotope to target and treat a tumor or disease. In this study we examine the synthesis and cell binding of a polymer scaffold containing a radiotherapeutic isotope and a targeting antibody. Methods: The multistep synthesis of a fluorescent or 149Promethium-labeled Trastuzumab-polyethyleneimine (PEI), Trastuzumab, or PEI is described. In vitro uptake, internalization and/or the binding affinity to the Her2/neu expressing human breast adenocarcinoma SKBr3 cells was investigated with the labeled compounds. Fluorescent-labeled Trastuzumab-PEI was internalized more into cells at 2 and 18 h than fluorescent-labeled Trastuzumab or PEI. The fluorescent-labeled Trastuzumab wasmore » concentrated on the cell surface at 2 and 18 h and the labeled PEI had minimal uptake. DOTA-PEI was prepared and contained an average of 16 chelates per PEI; the compound was radio-labeled with 149Promethium and conjugated to Trastuzumab. The purified 149Pm-DOTA-PEI-Trastuzumab had a radiochemical purity of 96.7% and a specific activity of 0.118 TBq/g. The compound demonstrated a dissociation constant for the Her2/neu receptor of 20.30 ± 6.91 nM. In conclusion, the results indicate the DOTA-PEI-Trastuzumab compound has potential as a targeted therapeutic carrier, and future in vivo studies should be performed.« less

  8. Synthesis of deuterium-labelled isotopomer of deferasirox.

    PubMed

    Havaldar, Freddy H; Dabholkar, Bhushan Vasant; Mule, Ganesh Baban; Kulkarni, Suhas

    2015-04-01

    A d4 -labeled isotopomer of deferasirox was synthesized as internal standard for use in a LC/mass spectroscopy (MS)/MS method developed for the simultaneous quantitative determination of deferasirox in human serum. d4 -deferasirox was synthesized from d8 -toluene. Copyright © 2015 John Wiley & Sons, Ltd.

  9. Routine synthesis of carbon-11-carboxyl-labeled L-dopa

    SciTech Connect

    Adam, M.J.; Grierson, J.R.; Ruth, T.J.; Pedersen, K.; Pate, B.D.

    1987-10-01

    Carbon-11-carboxyl-labeled L-dopa has been synthesized by the modified Bucherer-Strecker method. The reaction mixture was first purified by chiral HPLC followed by deprotection using hydriodic acid. The entire procedure was performed in a remotely operated system which gave the product in 28% radiochemical yield (decay corrected) in an overall synthesis time of 55-60 min.

  10. Synthesis and applications of RNAs with position-selective labeling and mosaic composition

    PubMed Central

    Liu, Yu; Holmstrom, Erik; Zhang, Jinwei; Yu, Ping; Wang, Jinbu; Dyba, Marzena A.; Chen, De; Ying, Jinfa; Lockett, Stephen; Nesbitt, David J.; Ferré-D'Amaré, Adrian R.; Sousa, Rui; Stagno, Jason R.; Wang, Yun-Xing

    2015-01-01

    Knowledge of the structure and dynamics of RNA molecules is critical to understand their many biological functions. Furthermore, synthetic RNAs have applications as therapeutics and molecular sensors. Both research and technological applications of RNA would be significantly enhanced by methods that enable incorporation of modified or labeled nucleotides into specifically designated positions or regions of RNA. However, the synthesis of tens of milligrams of such RNAs using existing methods has been impossible. We have developed a hybrid solid-liquid phase transcription method and automated robotic platform for the synthesis of RNAs with position-selective labeling. We demonstrate its utility by successfully preparing various isotope- or fluorescently-labeled versions of the 71-nucleotide aptamer domain of an adenine riboswitch1 for nuclear magnetic resonance (NMR) spectroscopy or single molecule Förster resonance-energy transfer (smFRET), respectively. Those RNAs include molecules that were selectively isotope-labeled in specific loops, linkers, a helix, several discrete positions, or a single internal position, as well as RNA molecules that were fluorescently-labeled in and near kissing loops. These selectively labeled RNAs have the same fold as those transcribed using conventional methods, but greatly simplified the interpretation of NMR spectra. The single-position isotope-labeled and fluorescently-labeled RNA samples revealed multiple conformational states of the adenine riboswitch. Lastly, we describe a robotic platform and the operation that automates this technology. Our selective labeling method may be useful for studying RNA structure and dynamics and for making RNA sensors for a variety of applications including cell-biological studies, substance detection2 and disease diagnostics3,4. PMID:25938715

  11. The “Speedy” Synthesis of Atom-Specific 15N Imino/Amido-Labeled RNA

    PubMed Central

    Kreutz, Christoph; Micura, Ronald

    2016-01-01

    Although numerous reports on the synthesis of atom-specific 15N-labeled nucleosides exist, fast and facile access to the corresponding phosphoramidites for RNA solid-phase synthesis is still lacking. This situation represents a severe bottleneck for NMR spectroscopic investigations on functional RNAs. Here, we present optimized procedures to speed up the synthesis of 15N(1) adenosine and 15N(1) guanosine amidites, which are the much needed counterparts of the more straightforward-to-achieve 15N(3) uridine and 15N(3) cytidine amidites in order to tap full potential of 1H/15N/15N-COSY experiments for directly monitoring individual Watson–Crick base pairs in RNA. Demonstrated for two preQ1 riboswitch systems, we exemplify a versatile concept for individual base-pair labeling in the analysis of conformationally flexible RNAs when competing structures and conformational dynamics are encountered. PMID:26237536

  12. Synthesis of an Isotopically Labeled Naphthalene Derivative That Supports a Long-Lived Nuclear Singlet State

    PubMed Central

    2015-01-01

    The synthesis of an octa-alkoxy substituted isotopically labeled naphthalene derivative, shown to have excellent properties in singlet NMR experiments, is described. This highly substituted naphthalene system, which incorporates an adjacent 13C spin pair, is readily accessed from a commercially available 13C2-labeled building block via sequential thermal alkynyl- and arylcyclobutenone rearrangements. The synthetic route incorporates a simple desymmetrization approach leading to a small difference in the chemical shifts of the 13C spin pair, a design constraint crucial for accessing nuclear singlet order. PMID:25898076

  13. Synthesis of 14C-labeled perfluorooctanoic and perfluorodecanoic acids; Purification of perfluorodecanoic acid

    SciTech Connect

    Reich, I.L.; Reich, H.J.; Menahan, L.A.; Peterson, R.E.

    1987-01-01

    Perfluorooctanoic and -decanoic acids are representative of a series of perfluorinated acids that have been used for a variety of industrial purposes primarily due to their surfactant properties. The toxicity of these compounds is being investigated in a number of laboratories. 14C-labeled materials would be useful in these studies but are not commercially available. Johncock prepared unlabeled PFOA in low yield by carbonation of the unstable perfluoroheptyllithium at -90 degrees Centigrade. We anticipated several problems in applying this procedure to the synthesis of the 14C-labeled material. Johncock's procedure was run on a fairly large scale (10 mmol) with excess CO2.

  14. A novel synthesis of polyacrolein microspheres and their application for cell labeling and cell separation.

    PubMed

    Margel, S; Beitler, U; Ofarim, M

    1981-01-01

    A novel method for the synthesis of polyacrolein microspheres with fluorescent or magnetic properties is described. These microspheres carry reactive aldehyde groups on their surface, which are used for covalent binding of various proteins at physiological pH. Polyacrolein microspheres may be used as a simple tool for cell labeling and cell separation. The feasibility of specific labeling of fresh human red blood cells and of the separation of human red blood cells from turkey red blood cells by means of a magnetic field is discussed.

  15. Escherichia coli cell-free protein synthesis and isotope labeling of mammalian proteins.

    PubMed

    Terada, Takaho; Yokoyama, Shigeyuki

    2015-01-01

    This chapter describes the cell-free protein synthesis method, using an Escherichia coli cell extract. This is a cost-effective method for milligram-scale protein production and is particularly useful for the production of mammalian proteins, protein complexes, and membrane proteins that are difficult to synthesize by recombinant expression methods, using E. coli and eukaryotic cells. By adjusting the conditions of the cell-free method, zinc-binding proteins, disulfide-bonded proteins, ligand-bound proteins, etc., may also be produced. Stable isotope labeling of proteins can be accomplished by the cell-free method, simply by using stable isotope-labeled amino acid(s) in the cell-free reaction. Moreover, the cell-free protein synthesis method facilitates the avoidance of stable isotope scrambling and dilution over the recombinant expression methods and is therefore advantageous for amino acid-selective stable isotope labeling. Site-specific stable isotope labeling is also possible with a tRNA molecule specific to the UAG codon. By the cell-free protein synthesis method, coupled transcription-translation is performed from a plasmid vector or a PCR-amplified DNA fragment encoding the protein. A milligram quantity of protein can be produced with a milliliter-scale reaction solution in the dialysis mode. More than a thousand solution structures have been determined by NMR spectroscopy for uniformly labeled samples of human and mouse functional domain proteins, produced by the cell-free method. Here, we describe the practical aspects of mammalian protein production by the cell-free method for NMR spectroscopy. © 2015 Elsevier Inc. All rights reserved.

  16. A fully automated synthesis for the preparation of 68Ga-labelled peptides.

    PubMed

    Decristoforo, Clemens; Knopp, Roger; von Guggenberg, Elisabeth; Rupprich, Marco; Dreger, Thorsten; Hess, Andre; Virgolini, Irene; Haubner, Roland

    2007-11-01

    Generator-produced Ga has attracted increasing interest for radiolabelling peptides used in PET applications. So far, the synthesis of Ga-peptide radiopharmaceuticals is mainly based on semi-automated systems. Here we describe a fully automated approach for the synthesis of Ga-labelled peptides. A commercially available Ga generator was eluted with 0.1 mol . l HCl. Reaction parameters such as buffer conditions, pH range, reaction temperature and time, volume of reaction solution and generator fraction were optimized for labelling DOTA-Tyr-octreotide (DOTATOC). Reaction yields, pH, radiochemical purity, sterility, endotoxins, breakthrough of Ge and final Ge content were determined. A fully automated radiopharmaceutical synthesis device based on a modular concept for remote-controlled processing was developed and evaluated for a number of DOTA-derivatized peptides. DOTATOC could be labelled in almost quantitative yields by heating 10-50 nmol peptide at pH 3.5-4.0 for 5 min at 95 degrees C in 1.5 ml. Purification using a reversed-phase cartridge was required to avoid any potential Ge breakthrough: final activities of Ge were below 100 Bq . ml. Automated synthesis resulted in overall decay-corrected reaction yields of about 60% within 10 min. Even after 1 year using a 1110 MBq generator more than 130 MBq Ga-DOTATOC could be obtained. Moreover, it was demonstrated that a variety of DOTA-derivatized peptides can be labelled using identical reaction conditions with high yields. The system described allows the fully automated, efficient and rapid preparation of Ga-DOTA-derivatized peptides. It has been used successfully and reliably for routine preparations in clinical studies.

  17. Synthesis of carbon-11, fluorine-18, and nitrogen-13 labeled radiotracers for biomedical applications

    SciTech Connect

    Fowler, J.S.; Wolf, A.P.

    1981-01-01

    A number of reviews, many of them recent, have appeared on various aspects of /sup 11/C, /sup 18/F and /sup 13/N-labeled radiotracers. This monograph treats the topic principally from the standpoint of synthetic organic chemistry while keeping in perspective the necessity of integrating the organic chemistry with the design and ultimate application of the radiotracer. Where possible, recent examples from the literature of organic synthesis are introduced to suggest potentially new routes which may be applied to problems in labeling organic molecules with the short-lived positron emitters, carbon-11, fluorine-18, and nitrogen-13. The literature survey of carbon-11, fluorine-18 and nitrogen-13 labeled compounds presented are of particular value to scientists working in this field. Two appendices are also included to provide supplementary general references. A subject index concludes this volume.

  18. The synthesis and characterization of biotin-silver-dendrimer nanocomposites as novel bioselective labels.

    PubMed

    Malý, J; Lampová, H; Semerádtová, A; Stofik, M; Kovácik, L

    2009-09-23

    This paper presents a synthesis of a novel nanoparticle label with selective biorecognition properties based on a biotinylated silver-dendrimer nanocomposite (AgDNC). Two types of labels, a biotin-AgDNC (bio-AgDNC) and a biotinylated AgDNC with a poly(ethylene)glycol spacer (bio-PEG-AgDNC), were synthesized from a generation 7 (G7) hydroxyl-terminated ethylenediamine-core-type (2-carbon core) PAMAM dendrimer (DDM) by an N,N'-dicyclohexylcarbodiimide (DDC) biotin coupling and a NaBH(4) silver reduction method. Synthesized conjugates were characterized by several analytical methods, such as UV-vis, FTIR, AFM, TEM, ELISA, HABA assay and SPR. The results show that stable biotinylated nanocomposites can be formed either with internalized silver nanoparticles (AgNPs) in a DMM polymer backbone ('type I') or as externally protected ('type E'), depending on the molar ratio of the silver/DMM conjugate and type of conjugate. Furthermore, the selective biorecognition function of the biotin is not affected by the AgNPs' synthesis step, which allows a potential application of silver nanocomposite conjugates as biospecific labels in various bioanalytical assays, or potentially as fluorescence cell biomarkers. An exploitation of the presented label in the development of electrochemical immunosensors is anticipated.

  19. The synthesis and characterization of biotin-silver-dendrimer nanocomposites as novel bioselective labels

    NASA Astrophysics Data System (ADS)

    Malý, J.; Lampová, H.; Semerádtová, A.; Štofik, M.; Kováčik, L.

    2009-09-01

    This paper presents a synthesis of a novel nanoparticle label with selective biorecognition properties based on a biotinylated silver-dendrimer nanocomposite (AgDNC). Two types of labels, a biotin-AgDNC (bio-AgDNC) and a biotinylated AgDNC with a poly(ethylene)glycol spacer (bio-PEG-AgDNC), were synthesized from a generation 7 (G7) hydroxyl-terminated ethylenediamine-core-type (2-carbon core) PAMAM dendrimer (DDM) by an N,N'-dicyclohexylcarbodiimide (DDC) biotin coupling and a NaBH4 silver reduction method. Synthesized conjugates were characterized by several analytical methods, such as UV-vis, FTIR, AFM, TEM, ELISA, HABA assay and SPR. The results show that stable biotinylated nanocomposites can be formed either with internalized silver nanoparticles (AgNPs) in a DMM polymer backbone ('type I') or as externally protected ('type E'), depending on the molar ratio of the silver/DMM conjugate and type of conjugate. Furthermore, the selective biorecognition function of the biotin is not affected by the AgNPs' synthesis step, which allows a potential application of silver nanocomposite conjugates as biospecific labels in various bioanalytical assays, or potentially as fluorescence cell biomarkers. An exploitation of the presented label in the development of electrochemical immunosensors is anticipated.

  20. Radiochemical synthesis of 105gAg-labelled silver nanoparticles

    NASA Astrophysics Data System (ADS)

    Ichedef, C.; Simonelli, F.; Holzwarth, U.; Bagaria, J. Piella; Puntes, V. F.; Cotogno, G.; Gilliland, D.; Gibson, N.

    2013-11-01

    A method for synthesis of radiolabelled silver nanoparticles is reported. The method is based on proton activation of silver metal powder, enriched in 107Ag, with a 30.7 MeV proton beam. At this proton energy 105gAg is efficiently created, mainly via the 107Ag(p,3n)105Cd → 105gAg reaction. 105gAg has a half-life of 41.29 days and emits easily detectable gamma radiation on decay to 105Pd. This makes it very useful as a tracing radionuclide for experiments over several weeks or months. Following activation and a period to allow short-lived radionuclides to decay, the powder was dissolved in concentrated nitric acid in order to form silver nitrate (AgNO3), which was used to synthesise radiolabelled silver nanoparticles via the process of sodium borohydride reduction. For comparison, non-radioactive silver nanoparticles were synthesised using commercially supplied AgNO3 in order to check if the use of irradiated Ag powder as a starting material would alter in any way the final nanoparticle characteristics. Both nanoparticle types were characterised using dynamic light scattering, zeta-potential and X-ray diffraction measurements, while additionally the non-radioactive samples were analysed by transmission electron microscopy and UV-Vis spectrometry. A hydrodynamic diameter of about 16 nm was determined for both radiolabelled and non-radioactive nanoparticles, while the electron microscopy on the non-radioactive samples indicated that the physical size of the metal NPs was (7.3 ± 1.4) nm.

  1. Stable-isotope-labeled carbohydrates and nucleosides: Synthesis and applications in chemistry and biology

    SciTech Connect

    Serianni, A.S.

    1994-12-01

    Carbohydrates play important roles in many key biochemical processes in living cells. For example, they are metabolized to produce energy, mediate cell-cell recognition, and play an indirect role (as constituents of DNA and RNA) in DNA replication, RNA transcription, and protein synthesis. These roles, and others of comparable biochemical significance, have been studied to varying extends with the use of stable isotopically labeled molecules, usually in conjunction with NMR spectroscopy and/or mass spectrometry. For example, carbohydrate metabolism has been monitored in vitro and in vivo with the use of isotopically labeled compounds. Molecular aspects of cell-cell recognition, mediated by cell-surface glycoproteins and glycolipids, have been probed through NMR studies of isotopically labeled oligosaccharides. More recently, the solution behavior of DNA and RNA has been examined through the use of labeled oligonucleotides. In all of these pursuits, the effort and expense to prepare labeled molecules, both of which can be substantial, are more than offset by the wealth of information derived from these studies. This information often cannot be accessed, or can be accessed only with great difficulty, using natural (unlabeled) compounds.

  2. High-throughput synthesis of stable isotope-labeled transmembrane proteins for targeted transmembrane proteomics using a wheat germ cell-free protein synthesis system.

    PubMed

    Takemori, Nobuaki; Takemori, Ayako; Matsuoka, Kazuhiro; Morishita, Ryo; Matsushita, Natsuki; Aoshima, Masato; Takeda, Hiroyuki; Sawasaki, Tatsuya; Endo, Yaeta; Higashiyama, Shigeki

    2015-02-01

    Using a wheat germ cell-free protein synthesis system, we developed a high-throughput method for the synthesis of stable isotope-labeled full-length transmembrane proteins as proteoliposomes to mimic the in vivo environment, and we successfully constructed an internal standard library for targeted transmembrane proteomics by using mass spectrometry.

  3. Design, synthesis, and functional activity of labeled CD1d glycolipid agonists.

    PubMed

    Jervis, Peter J; Polzella, Paolo; Wojno, Justyna; Jukes, John-Paul; Ghadbane, Hemza; Garcia Diaz, Yoel R; Besra, Gurdyal S; Cerundolo, Vincenzo; Cox, Liam R

    2013-04-17

    Invariant natural killer T cells (iNKT cells) are restricted by CD1d molecules and activated upon CD1d-mediated presentation of glycolipids to T cell receptors (TCRs) located on the surface of the cell. Because the cytokine response profile is governed by the structure of the glycolipid, we sought a method for labeling various glycolipids to study their in vivo behavior. The prototypical CD1d agonist, α-galactosyl ceramide (α-GalCer) 1, instigates a powerful immune response and the generation of a wide range of cytokines when it is presented to iNKT cell TCRs by CD1d molecules. Analysis of crystal structures of the TCR-α-GalCer-CD1d ternary complex identified the α-methylene unit in the fatty acid side chain, and more specifically the pro-S hydrogen at this position, as a site for incorporating a label. We postulated that modifying the glycolipid in this way would exert a minimal impact on the TCR-glycolipid-CD1d ternary complex, allowing the labeled molecule to function as a good mimic for the CD1d agonist under investigation. To test this hypothesis, the synthesis of a biotinylated version of the CD1d agonist threitol ceramide (ThrCer) was targeted. Both diastereoisomers, epimeric at the label tethering site, were prepared, and functional experiments confirmed the importance of substituting the pro-S, and not the pro-R, hydrogen with the label for optimal activity. Significantly, functional experiments revealed that biotinylated ThrCer (S)-10 displayed behavior comparable to that of ThrCer 5 itself and also confirmed that the biotin residue is available for streptavidin and antibiotin antibody recognition. A second CD1d agonist, namely α-GalCer C20:2 4, was modified in a similar way, this time with a fluorescent label. The labeled α-GalCer C20:2 analogue (11) again displayed functional behavior comparable to that of its unlabeled substrate, supporting the notion that the α-methylene unit in the fatty acid amide chain should be a suitable site for attaching

  4. Design, Synthesis, and Functional Activity of Labeled CD1d Glycolipid Agonists

    PubMed Central

    2013-01-01

    Invariant natural killer T cells (iNKT cells) are restricted by CD1d molecules and activated upon CD1d-mediated presentation of glycolipids to T cell receptors (TCRs) located on the surface of the cell. Because the cytokine response profile is governed by the structure of the glycolipid, we sought a method for labeling various glycolipids to study their in vivo behavior. The prototypical CD1d agonist, α-galactosyl ceramide (α-GalCer) 1, instigates a powerful immune response and the generation of a wide range of cytokines when it is presented to iNKT cell TCRs by CD1d molecules. Analysis of crystal structures of the TCR−α-GalCer–CD1d ternary complex identified the α-methylene unit in the fatty acid side chain, and more specifically the pro-S hydrogen at this position, as a site for incorporating a label. We postulated that modifying the glycolipid in this way would exert a minimal impact on the TCR–glycolipid–CD1d ternary complex, allowing the labeled molecule to function as a good mimic for the CD1d agonist under investigation. To test this hypothesis, the synthesis of a biotinylated version of the CD1d agonist threitol ceramide (ThrCer) was targeted. Both diastereoisomers, epimeric at the label tethering site, were prepared, and functional experiments confirmed the importance of substituting the pro-S, and not the pro-R, hydrogen with the label for optimal activity. Significantly, functional experiments revealed that biotinylated ThrCer (S)-10 displayed behavior comparable to that of ThrCer 5 itself and also confirmed that the biotin residue is available for streptavidin and antibiotin antibody recognition. A second CD1d agonist, namely α-GalCer C20:2 4, was modified in a similar way, this time with a fluorescent label. The labeled α-GalCer C20:2 analogue (11) again displayed functional behavior comparable to that of its unlabeled substrate, supporting the notion that the α-methylene unit in the fatty acid amide chain should be a suitable site for

  5. Base-Mediated One-Pot Synthesis of Aliphatic Diazirines for Photoaffinity Labeling.

    PubMed

    Wang, Lei; Tachrim, Zetryana Puteri; Kurokawa, Natsumi; Ohashi, Fumina; Sakihama, Yasuko; Hashidoko, Yasuyuki; Hashimoto, Makoto

    2017-08-22

    Aliphatic diazirines have been widely used as prominent photophores for photoaffinity labeling owing to their relatively small size which can reduce the steric effect on the natural interaction between ligands and proteins. Based on our continuous efforts to develop efficient methods for the synthesis of aliphatic diazirines, we present here a comprehensive study about base-mediated one-pot synthesis of aliphatic diazirines. It was found that potassium hydroxide (KOH) can also promote the construction of aliphatic diazirine with good efficiency. Importantly, KOH is cheaper, highly available, and easily handled and stored compared with the previously used base, potassium tert-butoxide (t-BuOK). Gram-scale study showed that it owned great advantages in being used for the large-scale production of aliphatic diazirines. This protocol is highly neat and the desired products can be easily isolated and purified. As the first comprehensive study of the base-mediated one-pot synthesis of aliphatic diazirines, this work provided good insight into the preparation and utilization of diazirine-based photoaffinity labeling probes.

  6. Synthesis of (6-(13)C)pyrimidine nucleotides as spin-labels for RNA dynamics.

    PubMed

    Wunderlich, Christoph H; Spitzer, Romana; Santner, Tobias; Fauster, Katja; Tollinger, Martin; Kreutz, Christoph

    2012-05-02

    We present a (13)C-based isotope labeling protocol for RNA. Using (6-(13)C)pyrimidine phosphoramidite building blocks, site-specific labels can be incorporated into a target RNA via chemical oligonucleotide solid-phase synthesis. This labeling scheme is particularly useful for studying milli- to microsecond dynamics via NMR spectroscopy, as an isolated spin system is a crucial prerequisite to apply Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion type experiments. We demonstrate the applicability for the characterization and detection of functional dynamics on various time scales by incorporating the (6-(13)C)uridine and -cytidine labels into biologically relevant RNAs. The refolding kinetics of a bistable terminator antiterminator segment involved in the gene regulation process controlled by the preQ(1) riboswitch class I was investigated. Using (13)C CPMG relaxation dispersion NMR spectroscopy, the milli- to microsecond dynamics of the HIV-1 transactivation response element RNA and the Varkud satellite stem loop V motif was addressed. © 2012 American Chemical Society

  7. Synthesis of ¹⁸F-labelled β-lactams by using the Kinugasa reaction.

    PubMed

    Zlatopolskiy, Boris D; Krapf, Philipp; Richarz, Raphael; Frauendorf, Holm; Mottaghy, Felix M; Neumaier, Bernd

    2014-04-14

    Owing to their broad spectrum of biological activities and low toxicity, β-lactams are attractive lead structures for the design of novel molecular probes. However, the synthesis of positron emission tomography (PET)-isotope-labelled β-lactams has not yet been reported. Herein, we describe the simple preparation of radiofluorinated β-lactams by using the fast Kinugasa reaction between (18)F-labelled nitrone [(18)F]-1 and alkynes of different reactivity. Additionally, (18)F-labelled fused β-lactams were obtained through the reaction of a cyclic nitrone 7 with radiofluorinated alkynes [(18)F]-6 a,b. Radiochemical yields of the Kinugasa reaction products could be significantly increased by the use of different Cu(I) ligands, which additionally allowed a reduction in the amount of precursor and/or reaction time. Model radiofluorinated β-lactam-peptide and protein conjugates ([(18)F]-10 and (18)F-labelled BSA conjugate) were efficiently obtained in high yield under mild conditions (aq. MeCN, ambient temperature) within a short reaction time, demonstrating the suitability of the developed method for radiolabelling of sensitive molecules such as biopolymers. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Synthesis and properties of chemiluminescent acridinium ester labels with fluorous tags.

    PubMed

    Natrajan, Anand; Wen, David; Sharpe, David

    2014-06-21

    Acridinium dimethylphenyl esters are highly sensitive chemiluminescent labels that are used in clinical diagnostics. Light emission from these labels is triggered with alkaline peroxide in the presence of the cationic surfactant cetyltrimethylammonium chloride (CTAC). CTAC compresses emission times of these labels to <5 seconds and also increases overall light yield 3-4 fold. The observed enhancement in acridinium ester chemiluminescence (light yield) is quite sensitive to the polarity of the micellar interface. In the current study, we report the synthesis of new acridinium ester labels with fluorous tags of varying fluorine content and their chemiluminescence in the presence of cationic micelles of CTAC, anionic micelles of sodium perfluorooctanoate (SPFO) as well as mixed micelles of CTAC and SPFO. These studies indicate that in the presence of the mixed micelle system of CTAC and SPFO and at low mole fractions of SPFO, polarity of the mixed micelle interface is lower than that of CTAC leading to a greater enhancement of chemiluminescence for both fluorinated acridinium esters as well as a structurally analogous but non-fluorinated acridinium ester. Chemiluminescence stability of the fluorinated acridinium esters was either comparable to or better than the stability of the non-fluorinated acridinium ester. Non-specific binding to paramagnetic microparticles was higher for fluorinated acridinium esters requiring a surfactant wash to reduce their non-specific binding to the same extent as that observed for the non-fluorinated acridinium ester.

  9. Synthesis of fluorine-18 labeled rhodamine B: A potential PET myocardial perfusion imaging agent

    PubMed Central

    Heinrich, Tobias K.; Gottumukkala, Vijay; Snay, Erin; Dunning, Patricia; Fahey, Frederic H; Treves, S. Ted; Packard, Alan B.

    2009-01-01

    There is considerable interest in developing an 18F-labeled PET myocardial perfusion agent. Rhodamine dyes share several properties with 99mTc-MIBI, the most commonly used single-photon myocardial perfusion agent, suggesting that an 18F-labeled rhodamine dye might prove useful for this application. In addition to being lipophilic cations, like 99mTc-MIBI, rhodamine dyes are known to accumulate in the myocardium and are substrates for Pgp, the protein implicated in MDR1 multidrug resistance. As the first step in determining whether 18F-labeled rhodamines might be useful as myocardial perfusion agents for PET, our objective was to develop synthetic methods for preparing the 18F-labeled compounds so that they could be evaluated in vivo. Rhodamine B was chosen as the prototype compound for development of the synthesis because the ethyl substituents on the amine moieties of rhodamine B protect them from side reactions, thus eliminating the need to include (and subsequently remove) protecting groups. The 2′-[18F]fluoroethyl ester of rhodamine B was synthesized by heating rhodamine B lactone with [18F]fluoroethyltosylate in acetonitrile at 165°C for 30 min.using [18F]fluoroethyl tosylate, which was prepared by the reaction of ethyleneglycol ditosylate with Kryptofix 2.2.2, K2CO3, and [18F]NaF in acetonitrile for 10 min. at 90°C. The product was purified by semi-preparative HPLC to produce the 2′-[18F]-fluoroethylester in >97% radiochemical purity with a specific activity of 1.3 GBq/μmol, an isolated decay corrected yield of 35%, and a total synthesis time of 90 min. PMID:19783150

  10. Synthesis of carrier-free tritium-labeled queen bee pheromone

    SciTech Connect

    Webster, F.X.; Prestwich, G.D.

    1988-03-01

    A short synthesis of (4,5-/sup 3/H/sub 2/) (E)-9-oxo-2-decenoic acid (ODA), a high-specific-activity tritium-containing isotopomer of the queen bee pheromone, is described. Catalytic tritiation of the ketal of ethyl 9-oxo-4-decenoate introduces tritium into two positions, one of which is completely unactivated. Subsequent transformation by selenation, oxidation, and hydrolysis affords the labeled 9-ODA at >60 Ci/mmol. The material is suitable for biochemical studies of binding and catabolism in ovarian, antennal, and other target tissues.

  11. Convergent Synthesis of a Deuterium Labeled Serine Dipeptide Lipid for Analysis of Biological Samples.

    PubMed

    Dietz, Christopher; Clark, Robert B; Nichols, Frank C; Smith, Michael B

    2017-03-08

    Bacterial serine dipeptide lipids are known to promote inflammatory processes and are detected in human tissues associated with periodontal disease or atherosclerosis. Accurate quantification of bacterial serine lipid, specifically lipid 654 [((S)-15-methyl-3-((13-methyltetradecanoyl)oxy)hexadecanoyl)glycyl-L-serine, (3S)-L-serine] isolated from Porphyromonas gingivalis,(1) in biological samples requires the preparation of a stable isotope internal standard for sample supplementation and subsequent mass spectrometric analysis. This report describes the convergent synthesis of a deuterium-substituted serine dipeptide lipid, which is an isotopically labeled homologue that represents a dominant form of serine dipeptide lipid recovered in bacteria.

  12. Development of a modular system for the synthesis of PET [(11)C]labelled radiopharmaceuticals.

    PubMed

    Boschi, Stefano; Lodi, Filippo; Cicoria, Gianfranco; Raul Ledesma, Jorge; Knopp, Roger; Rizzello, Anna; Di Pierro, Donato; Trespidi, Silvia; Marengo, Mario

    2009-10-01

    [((11))C]labelled radiopharmaceuticals as N-[(11)C]methyl-choline ([(11)C]choline), l-(S-methyl-[(11)C])methionine ([(11)C]methionine) and [(11)C]acetate have gained increasing importance in clinical PET and for the routine production of these radiopharmaceuticals, simple and reliable modules are needed to produce clinically relevant radioactivity. On the other hand, flexible devices are needed not only for the routine synthesis but also for more complex applications as the development of new tracers. The aim of this work was the adaptation of an Eckert Ziegler modular system for easy routine synthesis of [(11)C]choline, [(11)C]methionine and [(11)C]acetate using components that account for straightforward scaling up and upgrades.

  13. Measurement of protein synthesis using heavy water labeling and peptide mass spectrometry: Discrimination between major histocompatibility complex allotypes

    PubMed Central

    De Riva, Alessandra; Deery, Michael J.; McDonald, Sarah; Lund, Torben; Busch, Robert

    2010-01-01

    Methodological limitations have hampered the use of heavy water (2H2O), a convenient, universal biosynthetic label, for measuring protein synthesis. Analyses of 2H-labeled amino acids are sensitive to contamination; labeling of peptides has been measured for a few serum proteins, but this approach awaits full validation. Here we describe a method for quantifying protein synthesis by peptide mass spectrometry (MS) after 2H2O labeling, as applied to various proteins of the major histocompatibility complex (MHC). Human and murine antigen-presenting cells were cultured in medium containing 5% 2H2O; class I and class II MHC proteins were immunoprecipitated, bands were excised, and Ala-/Gly-rich, allele-specific tryptic peptides were identified by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Mass isotopomer distributions were quantified precisely by LC–MS and shifted markedly on 2H2O labeling. Experimental data agreed closely with models obtained by mass isotopomer distribution analysis (MIDA) and were consistent with contributions from Ala, Gly, and other amino acids to labeling. Estimates of fractional protein synthesis from peptides of the same protein were precise and internally consistent. The method was capable of discriminating between MHC isotypes and alleles, applicable to primary cells, and readily extendable to other proteins. It simplifies measurements of protein synthesis, enabling novel applications in physiology, in genotype/phenotype interactions, and potentially in kinetic proteomics. PMID:20406617

  14. Design, construction and testing of a low-cost automated (68)Gallium-labeling synthesis unit for clinical use.

    PubMed

    Heidari, Pedram; Szretter, Alicia; Rushford, Laura E; Stevens, Maria; Collier, Lee; Sore, Judit; Hooker, Jacob; Mahmood, Umar

    2016-01-01

    The interest in (68)Gallium labeled PET probes continues to increase around the world. Widespread use in Europe and Asia has led to great interest for use at numerous sites in the US. One barrier to entry is the cost of the automated synthesis units for relatively simple labeling procedures. We describe the construction and testing of a relatively low-cost automated (68)Ga-labeling unit for human-use. We provide a guide for construction, including part lists and synthesis timelists to facilitate local implementation. Such inexpensive systems could help increase use around the globe and in the US in particular by removing one of the barriers to greater widespread availability. The developed automated synthesis unit reproducibly synthesized (68)Ga-DOTATOC with average yield of 71 ± 8% and a radiochemical purity ≥ 95% in a synthesis time of 25 ± 1 minutes. Automated product yields are comparable to that of manual synthesis. We demonstrate in-house construction and use of a low-cost automated synthesis unit for labeling of DOTATOC and similar peptides with (68)Gallium.

  15. Design, construction and testing of a low-cost automated 68Gallium-labeling synthesis unit for clinical use

    PubMed Central

    Heidari, Pedram; Szretter, Alicia; Rushford, Laura E; Stevens, Maria; Collier, Lee; Sore, Judit; Hooker, Jacob; Mahmood, Umar

    2016-01-01

    The interest in 68Gallium labeled PET probes continues to increase around the world. Widespread use in Europe and Asia has led to great interest for use at numerous sites in the US. One barrier to entry is the cost of the automated synthesis units for relatively simple labeling procedures. We describe the construction and testing of a relatively low-cost automated 68Ga-labeling unit for human-use. We provide a guide for construction, including part lists and synthesis timelists to facilitate local implementation. Such inexpensive systems could help increase use around the globe and in the US in particular by removing one of the barriers to greater widespread availability. The developed automated synthesis unit reproducibly synthesized 68Ga-DOTATOC with average yield of 71 ± 8% and a radiochemical purity ≥ 95% in a synthesis time of 25 ± 1 minutes. Automated product yields are comparable to that of manual synthesis. We demonstrate in-house construction and use of a low-cost automated synthesis unit for labeling of DOTATOC and similar peptides with 68Gallium. PMID:27508104

  16. Fatty acid and cholesterol synthesis from specifically labeled leucine by isolated rat hepatocytes.

    PubMed

    Mathias, M M; Sullivan, A C; Hamilton, J G

    1981-10-01

    Hepatocytes isolated from female rats meal-fed a high-glucose diet were incubated in Krebs-Henseleit bicarbonate medium containing 16.5 mM glucose, 3H2O, and 14C-labeled amino acids (-)-Hydroxycitrate depressed the incorporation of 3H2O and [14C] alanine into fatty acids and cholesterol. Incorporation of [U-14C]leucine into lipids was not affected but incorporation of 3H2O into lipids was decreased significantly by (-)-hydroxycitrate. (-)-Hydroxycitrate depressed the incorporation of radioactivity from [2-14C]leucine into fatty acids and cholesterol by 61 and 38%, respectively, and stimulated the incorporation of radioactivity from [4,5-3H]leucine 35 and 28%. As [2-14C]leucine labels the acetyl-CoA pool and [4,5-3H]leucine labels the acetoacetate pool, it was concluded that mitochondrial 3-hydroxy-3-methylglutaryl-CoA is not incorporated intact into cholesterol, and that acetoacetate can be activated effectively in the liver cytosol for support of cholesterol and fatty acid synthesis.

  17. Convergent synthesis and evaluation of (18)F-labeled azulenic COX2 probes for cancer imaging.

    PubMed

    Nolting, Donald D; Nickels, Michael; Tantawy, Mohammed N; Yu, James Y H; Xie, Jingping; Peterson, Todd E; Crews, Brenda C; Marnett, Larry; Gore, John C; Pham, Wellington

    2012-01-01

    The overall objectives of this research are to (i) develop azulene-based positron emission tomography (PET) probes and (ii) image COX2 as a potential biomarker of breast cancer. Several lines of research have demonstrated that COX2 is overexpressed in breast cancer and that its presence correlates with poor prognoses. While other studies have reported that COX2 inhibition can be modulated and used beneficially as a chemopreventive strategy in cancer, no viable mechanism for achieving that approach has yet been developed. This shortfall could be circumvented through in vivo imaging of COX2 activity, particularly using sensitive imaging techniques such as PET. Toward that goal, our laboratory focuses on the development of novel (18)F-labled COX2 probes. We began the synthesis of the probes by transforming tropolone into a lactone, which was subjected to an [8 + 2] cycloaddition reaction to yield 2-methylazulene as the core ring of the probe. After exploring numerous synthetic routes, the final target molecule and precursor PET compounds were prepared successfully using convergent synthesis. Conventional (18)F labeling methods caused precursor decomposition, which prompted us to hypothesize that the acidic protons of the methylene moiety between the azulene and thiazole rings were readily abstracted by a strong base such as potassium carbonate. Ultimately, this caused the precursors to disintegrate. This observation was supported after successfully using an (18)F labeling strategy that employed a much milder phosphate buffer. The (18)F-labeled COX2 probe was tested in a breast cancer xenograft mouse model. The data obtained via successive whole-body PET/CT scans indicated probe accumulation and retention in the tumor. Overall, the probe was stable in vivo and no defluorination was observed. A biodistribution study and Western blot analysis corroborate with the imaging data. In conclusion, this novel COX2 PET probe was shown to be a promising agent for cancer imaging

  18. Convergent synthesis and evaluation of 18F-labeled azulenic COX2 probes for cancer imaging

    PubMed Central

    Nolting, Donald D.; Nickels, Michael; Tantawy, Mohammed N.; Yu, James Y. H.; Xie, Jingping; Peterson, Todd E.; Crews, Brenda C.; Marnett, Larry; Gore, John C.; Pham, Wellington

    2013-01-01

    The overall objectives of this research are to (i) develop azulene-based positron emission tomography (PET) probes and (ii) image COX2 as a potential biomarker of breast cancer. Several lines of research have demonstrated that COX2 is overexpressed in breast cancer and that its presence correlates with poor prognoses. While other studies have reported that COX2 inhibition can be modulated and used beneficially as a chemopreventive strategy in cancer, no viable mechanism for achieving that approach has yet been developed. This shortfall could be circumvented through in vivo imaging of COX2 activity, particularly using sensitive imaging techniques such as PET. Toward that goal, our laboratory focuses on the development of novel 18F-labled COX2 probes. We began the synthesis of the probes by transforming tropolone into a lactone, which was subjected to an [8 + 2] cycloaddition reaction to yield 2-methylazulene as the core ring of the probe. After exploring numerous synthetic routes, the final target molecule and precursor PET compounds were prepared successfully using convergent synthesis. Conventional 18F labeling methods caused precursor decomposition, which prompted us to hypothesize that the acidic protons of the methylene moiety between the azulene and thiazole rings were readily abstracted by a strong base such as potassium carbonate. Ultimately, this caused the precursors to disintegrate. This observation was supported after successfully using an 18F labeling strategy that employed a much milder phosphate buffer. The 18F-labeled COX2 probe was tested in a breast cancer xenograft mouse model. The data obtained via successive whole-body PET/CT scans indicated probe accumulation and retention in the tumor. Overall, the probe was stable in vivo and no defluorination was observed. A biodistribution study and Western blot analysis corroborate with the imaging data. In conclusion, this novel COX2 PET probe was shown to be a promising agent for cancer imaging and

  19. Microwave accelerated labeling methods in the synthesis of radioligands for positron emission tomography imaging.

    PubMed

    Kallmerten, Amy E; Alexander, Abigail; Wager, Krista M; Jones, Graham B

    2011-10-01

    Nuclear imaging using positron emission tomography [PET] is a powerful technique with clinical applications which include oncology, cardiovascular disease and CNS disorders. Conventional chemical syntheses of the short half-life radionuclides used in the process however imposes numerous limitations on scope of available ligands. By utilizing microwave assisted synthesis methods many of these limitations can be overcome, paving the way for the design of diverse families of agents with defined cellular targets. This review will survey recent developments in the field with emphasis on the period 2006-2011. Positron emission tomography [PET] has become one of the most powerful in vivo imaging modalities, capable of delivering mm3 resolution of radiotracer distribution and metabolism [1]. When combined with anatomic imaging methods (MRI, CT) co-registered multimode images offer the potential to track metabolic and physiologic events in diseased states and guide and accelerate clinical trials of investigational new drugs. Also, this same methodology can be used to evaluate first pass pharmacokinetics/pharmacodynamics in early stage drug discovery. Though powerful as a technique only a limited number of drugs have seen clinical use and to date only one drug 2-fluoro-deoxy-D-glucose (FDG) has received FDA approval [2]. One of the drawbacks of PET imaging is the need for tracers labeled with an appropriate nuclide and the half-lives of these agents places special constraints on the chemical synthesis. Among the most popular are 11C (t½ =20.4 min) and 18F (t ½ =109.8 min) labeled compounds and this has resulted in a resurgence of interest in practical application of their chemistries [3,4]. This review will focus on microwave mediated methods of acceleration of organic reactions used for the production of labeled PET image contrast agents, with emphasis on the five year period 2006 to 2011.

  20. Mild and regiospecific synthesis of 18F-labelled vinyl fluoride using [18F]fluorine reacted with silane.

    PubMed

    Di Raddo, P; Diksic, M

    1985-12-01

    A labelled vinyl fluoride of biological interest was prepared in good radiochemical yield by direct fluorination of the corresponding silane. The synthesis of 18F-labelled 4-fluoroantipyrine, a cerebral blood flow tracer, involved the reaction of 4-(trimethylsilyl) antipyrine with [18F]F2 (0.5% fluorine in neon) or 5% F2 in nitrogen in freon-11 as solvent. A radiochemical (chemical) yield of about 18% (42%) was obtained in a 25-min synthesis. The radiochemical and chemical purity of the final products was 99% after purification by HPLC.

  1. A class of novel nitronyl nitroxide labeling basic and acidic amino acids: synthesis, application for preparing ESR optionally labeling peptides, and bioactivity investigations.

    PubMed

    Zhang, Jianwei; Zhao, Ming; Cui, Guohui; Peng, Shiqi

    2008-04-01

    Aimed at optional ESR label 2-(4'-hydroxyl)phenyl-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl was introduced into the guanido of L-Arg-OH, the omega-amino group of L-Lys-OH with methylcarboxyl as a linker, and into the beta-carboxyl of L-Asp-OH and the gamma-carboxyl of L-Glu-OH with ethylamino as a linker. It was explored that the synthetic 30 novel ESR labeling amino acid derivatives were stable enough to the reaction conditions of peptide synthesis. Their incorporation led to 12 novel ESR optionally labeling PAK, RGDS, RGDV, and ECG. A series of NO related chemical tests, the in vitro and in vivo assays of these peptides confirmed that this strategy was practical.

  2. Synthesis and preliminary biological evaluations of fluorescent or 149Promethium labeled Trastuzumab-polyethylenimine

    SciTech Connect

    Fitzsimmons, Jonathan; Nayak, Tapan; Cutler, Cathy; Atcher, Robert

    2015-12-30

    Radioimmunotherapy utilize a targeting antibody coupled to a therapeutic isotope to target and treat a tumor or disease. In this study we examine the synthesis and cell binding of a polymer scaffold containing a radiotherapeutic isotope and a targeting antibody. Methods: The multistep synthesis of a fluorescent or 149Promethium-labeled Trastuzumab-polyethyleneimine (PEI), Trastuzumab, or PEI is described. In vitro uptake, internalization and/or the binding affinity to the Her2/neu expressing human breast adenocarcinoma SKBr3 cells was investigated with the labeled compounds. Fluorescent-labeled Trastuzumab-PEI was internalized more into cells at 2 and 18 h than fluorescent-labeled Trastuzumab or PEI. The fluorescent-labeled Trastuzumab was concentrated on the cell surface at 2 and 18 h and the labeled PEI had minimal uptake. DOTA-PEI was prepared and contained an average of 16 chelates per PEI; the compound was radio-labeled with 149Promethium and conjugated to Trastuzumab. The purified 149Pm-DOTA-PEI-Trastuzumab had a radiochemical purity of 96.7% and a specific activity of 0.118 TBq/g. The compound demonstrated a dissociation constant for the Her2/neu receptor of 20.30 ± 6.91 nM. In conclusion, the results indicate the DOTA-PEI-Trastuzumab compound has potential as a targeted therapeutic carrier, and future in vivo studies should be performed.

  3. Synthesis of isotopically labeled daclatasvir for use in human clinical studies.

    PubMed

    Easter, John A; Burrell, Richard C; Bonacorsi, Samuel J

    2016-04-01

    Daclatasvir is a novel hepatitis C virus NS5A inhibitor developed by Bristol-Myers Squibb and marketed as Daklinza®. The need to support the development of daclatasvir required the synthesis of carbon-14 labeled material for use in human absorption, distribution, metabolism, and excretion studies. A total of 7.53 mCi of [(14) C]-daclatasvir was synthesized in eight steps from commercially available [(14) C]-copper cyanide. The radiochemical purity was 99.6%, and specific activity was 3.86 μCi/mg. To support a human absolute bioavailability study, 5.56 g of [(13) C2 , (15) N4 ]-daclatasvir was synthesized in four steps. Copyright © 2016 John Wiley & Sons, Ltd.

  4. Synthesis and applications of selectively {sup 13}C-labeled RNA

    SciTech Connect

    SantaLucia, J. Jr.; Shen, L.X.; Lewis, H.; Cai, Z.; Tinoci, I. Jr.

    1994-12-01

    Spectral overlap is a substantial problem in NMR studies of RNA molecules >30 nucleotides. To overcome this difficulty, we synthesized selectively {sup 13}C-labeled RNAs and adapted several isotope-edited two- and three-dimensional NMR experiments originally developed for protein studies. We optimized protocols for synthesis of multi-gram quantities of CTP, UTp, ATP, and GTP using a combination of synthetic organic and enzymatic methods. Uracil is prepared in 40 to 50% yield from {sup 13}C-cyanide in two steps. Using acetyl- tribenzoyl-ribose and standard chemistry uracil is then attached to the sugar (90% yield). The tribenzoyl-uridine intermediate is converted into uridine or cytidine quantitatively, depending on the deblocking protocol. Labeled purines are synthesized using simple pyrimidine precursors and reacting with {sup 13}C-formic acid (80% yield). Purine nucleosides are then synthesized using uridine phosphorylase and purine nucleoside phosphorylase. The nucleosides were converted to NMPs by treatment with POC1{sub 3} in triethylphosphate. We converted NMPs to NTPs by standard enzymatic methods. Selectively labeled RNAs were synthesized by run-off transcription using {sup 13}C-labeled NTPs. Several different strategies help solve over-lap problems in larger RNAs. Isotope-edited two-dimensional NMR experiments such as {omega}1-1/2 X-filtered NOESY simplify NMR spectra by dividing the normal NOESY spectrum into two subspectra-one involving NOEs from protons bound to {sup 12}C and one from protons bound to {sup 13}C. For example, we labeled A and U residues of a 34-nucleotide pseudoknot, and the {sup 12}C subspectrum of the 1/2 X-filtered NOESY contained NOEs only from G and C residues (along with adenine 2H); the {sup 13}C subspectrum contained NOEs only from A and U residues. Each subspectrum has less overlap than the NOESY of an unlabeled sample; the editing strategy allows each resonance to be identified by residue type (A, C, G, or U).

  5. Synthesis of fluorescein-labelled O-mannosylated peptides as components for synthetic vaccines: comparison of two synthetic strategies.

    PubMed

    Brimble, Margaret A; Kowalczyk, Renata; Harris, Paul W R; Dunbar, P Rod; Muir, Victoria J

    2008-01-07

    Mannose-binding proteins on the surface of antigen-presenting cells (APCs) are capable of recognizing and internalizing foreign agents in the early stages of immune response. These receptors offer a potential target for synthetic vaccines, especially vaccines designed to stimulate T cells. We set out to synthesize a series of fluorescein-labelled O-mannosylated peptides using manual solid phase peptide synthesis (SPPS) on pre-loaded Wang resin, in order to test their ability to bind mannose receptors on human APCs in vitro. A flexible and reliable method for the synthesis of fluorescein-labelled O-mannosylated glycopeptides was desired in order to study their lectin-binding properties using flow cell cytometry. Two synthetic strategies were investigated: incorporation of a fluorescein label into the peptide chain via a lysine side chain epsilon-amino group at the final stage of standard Fmoc solid phase peptide synthesis or attachment of the fluorescein label to the N(alpha)-amino group of a lysine with further incorporation of a mannosylated peptide unit through the side chain N(epsilon)-amino group. The latter strategy proved more effective in that it facilitated SPPS by positioning the growing mannosylated peptide chain further removed from the fluorescein label.

  6. Synthesis and application of water-soluble, photoswitchable cyanine dyes for bioorthogonal labeling of cell-surface carbohydrates.

    PubMed

    Mertsch, Alexander; Letschert, Sebastian; Memmel, Elisabeth; Sauer, Markus; Seibel, Jürgen

    2016-09-01

    The synthesis of cyanine dyes addressing absorption wavelengths at 550 and 648 nm is reported. Alkyne functionalized dyes were used for bioorthogonal click reactions by labeling of metabolically incorporated sugar-azides on the surface of living neuroblastoma cells, which were applied to direct stochastic optical reconstruction microscopy (dSTORM) for the visualization of cell-surface glycans in the nm-range.

  7. Synthesis and evaluation of fluorescent cap analogues for mRNA labelling

    PubMed Central

    Ziemniak, Marcin; Szabelski, Mariusz; Lukaszewicz, Maciej; Nowicka, Anna; Darzynkiewicz, Edward; Rhoads, Robert E.; Wieczorek, Zbigniew; Jemielity, Jacek

    2013-01-01

    We describe the synthesis and properties of five dinucleotide fluorescent cap analogues labelled at the ribose of the 7-methylguanosine moiety with either anthraniloyl (Ant) or N-methylanthraniloyl (Mant), which have been designed for the preparation of fluorescent mRNAs via transcription in vitro. Two of the analogues bear a methylene modification in the triphosphate bridge, providing resistance against either the Dcp2 or DcpS decapping enzymes. All these compounds were prepared by ZnCl2-mediated coupling of a nucleotide P-imidazolide with a fluorescently labelled mononucleotide. To evaluate the utility of these compounds for studying interactions with cap-binding proteins and cap-related cellular processes, both biological and spectroscopic features of those compounds were determined. The results indicate acceptable quantum yields of fluorescence, pH independence, environmental sensitivity, and photostability. The cap analogues are incorporated by RNA polymerase into mRNA transcripts that are efficiently translated in vitro. Transcripts containing fluorescent caps but unmodified in the triphosphate chain are hydrolysed by Dcp2 whereas those containing a α-β methylene modification are resistant. Model studies exploiting sensitivity of Mant to changes of local environment demonstrated utility of the synthesized compounds for studying cap-related proteins. PMID:24273643

  8. Native SILAC: Metabolic Labeling of Proteins in Prototroph Microorganisms Based on Lysine Synthesis Regulation*

    PubMed Central

    Fröhlich, Florian; Christiano, Romain; Walther, Tobias C.

    2013-01-01

    Mass spectrometry (MS)-based quantitative proteomics has matured into a methodology able to detect and quantitate essentially all proteins of model microorganisms, allowing for unprecedented depth in systematic protein analyses. The most accurate quantitation approaches currently require lysine auxotrophic strains, which precludes analysis of most existing mutants, strain collections, or commercially important strains (e.g. those used for brewing or for the biotechnological production of metabolites). Here, we used MS-based proteomics to determine the global response of prototrophic yeast and bacteria to exogenous lysine. Unexpectedly, down-regulation of lysine synthesis in the presence of exogenous lysine is achieved via different mechanisms in different yeast strains. In each case, however, lysine in the medium down-regulates its biosynthesis, allowing for metabolic proteome labeling with heavy-isotope-containing lysine. This strategy of native stable isotope labeling by amino acids in cell culture (nSILAC) overcomes the limitations of previous approaches and can be used for the efficient production of protein standards for absolute SILAC quantitation in model microorganisms. As proof of principle, we have used nSILAC to globally analyze yeast proteome changes during salt stress. PMID:23592334

  9. Versatile Synthesis and Fluorescent Labeling of ZIF-90 Nanoparticles for Biomedical Applications.

    PubMed

    Jones, Christopher G; Stavila, Vitalie; Conroy, Marissa A; Feng, Patrick; Slaughter, Brandon V; Ashley, Carlee E; Allendorf, Mark D

    2016-03-01

    We describe a versatile method for the synthesis and fluorescent labeling of ZIF-90 nanoparticles (NPs). Gram-scale quantities of NPs can be produced under mild conditions, circumventing the need for high temperatures and extended reaction periods required by existing procedures. Monitoring the reaction in situ using UV-vis spectroscopy reveals that ZIF-90 NP nucleation in solution starts within seconds. In addition to reporting a method to reproducibly form sub-100 nm ZIF-90 particles, we show that particles of various sizes can be produced, ranging from 30 to 1000 nm, by altering amine chemistry or reaction temperature. The presence of linker aldehyde groups on the NP surface allows for postsynthetic labeling with amine-functionalized fluorescent dyes, providing utility for imaging within biological systems. In vitro cell studies show that ZIF-90 NPs have a high rate of cellular internalization, provide finite degradation periods of the order of several weeks, and are biocompatible with six different cell lines (>90% viable when incubated with NPs for up to 7 days). These features highlight the potential for use of ZIF-90 nanostructures in bioimaging and targeted drug delivery applications.

  10. Synthesis of Biotin-Labeled RNA for Gene Expression Measurements Using Oligonucleotide Arrays

    PubMed Central

    Vázquez, Ana E.; Nie, Liping; Yamoah, Ebenezer N.

    2010-01-01

    Using gene arrays, it is currently possible to simultaneously measure mRNA levels of many genes in any tissue of interest. Undoubtedly, comprehensive measurements of gene expression as part of carefully designed experiments will continue to further our understanding of audition and have the potential to open up new avenues of research. This chapter describes a reliable protocol to prepare high-quality biotin-labeled RNA target, specifically for oligonucleotide array experiments. The procedure includes isolation of high-quality total RNA, synthesis of double-stranded cDNA engineered for in vitro transcription with T7 RNA polymerase, subsequent in vitro transcription in the presence of biotin-labeled ribonucleotides, and fractionation of the RNA to ~ 500 bp fragments, suitable for oligonucleotide array experiments. Because the membranous labyrinth is composed of functionally interdependent cellular structures, which themselves contain numerous, highly differentiated cell types, comprehensive analysis of gene expression in the cochlea is best complemented by immunohistotochemical studies or, if no suitable antibodies are available, by in situ hybridization studies. Either one of these techniques will identify the specific cell types that express the genes of interests. PMID:18839339

  11. Determining synthesis rates of individual proteins in zebrafish (Danio rerio) with low levels of a stable isotope labelled amino acid.

    PubMed

    Geary, Bethany; Magee, Kieran; Cash, Phillip; Young, Iain S; Whitfield, Phillip D; Doherty, Mary K

    2016-05-01

    The zebrafish is a powerful model organism for the analysis of human cardiovascular development and disease. Understanding these processes at the protein level not only requires changes in protein concentration to be determined but also the rate at which these changes occur on a protein-by-protein basis. The ability to measure protein synthesis and degradation rates on a proteome-wide scale, using stable isotope labelling in conjunction with mass spectrometry is now a well-established experimental approach. With the advent of more selective and sensitive mass spectrometers, it is possible to accurately measure lower levels of stable isotope incorporation, even when sample is limited. In order to challenge the sensitivity of this approach, we successfully determined the synthesis rates of over 600 proteins from the cardiac muscle of the zebrafish using a diet where either 30% or 50% of the L-leucine was replaced with a stable isotope labelled analogue ([(2) H7 ]L-leucine]. It was possible to extract sufficient protein from individual zebrafish hearts to determine the incorporation rate of the label into hundreds of proteins simultaneously, with the two labelling regimens showing a good correlation of synthesis rates.

  12. Synthesis of the E and Z isomers of the antiestrogen tamoxifen and its metabolite, hydroxytamoxifen, in tritium-labeled form

    SciTech Connect

    Robertson, D.W.; Katzenellenbogen, J.A.

    1982-06-04

    Both isomers of the potent antiestrogen tamoxifen (1,2-diphenyl-1-(4-(2-(dimethylamino)ethoxy)phenyl)-1-butene: E isomer = ICI-47699; Z isomer = ICI-46474, Nolvadex) and its metabolite, hydroxytamoxifen (1-(4-(2-(dimethylamino)ethoxy)phenyl)-1-(4-hydroxyphenyl)-2-phenyl-1-butene), have been synthesized in a high specific activity, tritium-labeled form by catalytic tritium-halogen exchange performed on brominated precursors. The synthesis of another precursor to labeled tamoxifen which would enable the incorporation of three tritium atoms into the molecule by tritium-halogen exchange is reported.

  13. Synthesis of stable isotopically labelled 3-methylfuran-2(5H)-one and the corresponding strigolactones.

    PubMed

    Cheng, Yun; Ding, Wen-hui; Long, Qin; Zhao, Min; Yang, Jun; Li, Xiao-qiang

    2015-07-01

    Conventional synthetic procedures of strigolactones (SLs) involve the independent synthesis of ring ABC and ring D, followed by a coupling of the two fragments. Here we prepared three kinds of stable, isotopically labelled D-ring analogues productively using a facile protocol. Then, a coupling of the D-rings to ring ABC produced three isotope-labelled SL derivatives. Moreover, (+)-D3-2'-epi-1A and (-)-ent-D3-2'-epi-1A with high enantiomeric purity were obtained via chiral resolution.

  14. Convenient synthesis of stable deuterium-labeled alkylpyrazines for use in stable isotope dilution assays.

    PubMed

    Fang, Mingchih; Cadwallader, Keith R

    2013-04-17

    Stable isotope dilution assays (SIDA) provide for accurate and precise quantitation of aroma components, such as alkylpyrazines, which are often present in low concentrations in complex food matrices. The unavailability of labeled standards is the main limitation to the widespread use of SIDA. This study describes the chlorination of several alkylpyrazines to form the corresponding chloroalkylpyrazine compounds, which are efficient starting materials for the synthesis of deuterium-labeled alkylpyrazines, namely [²H₃]-2-methylpyrazine (d-1), [²H₅]-2-ethylpyrazine (d-2), [²H₃]-2,3(or 6)-dimethylpyrazine (d-3A, d-3B), [²H₃]-2,[²H₃]-6-dimethylpyrazine (d-3C), [²H₅]-2,[²H₅]-6-diethylpyrazine (d-4), [²H₅]-2-ethyl-3(or 6)-methylpyrazine (d-5A, d-5B), 2,[²H₃]-3,5-trimethylpyrazine (d-6), [²H₅]-2-ethyl-3,6-dimethylpyrazine (d-7), [²H₅]-2-ethyl-3,5-dimethylpyrazine (d-8), and 2,3-diethyl-[²H₃]-5-methylpyrazine (d-9), which were obtained in good yields (57-100%) and high purities (86-98%). These stable isotopes were used as internal standards in SIDA to accurately and precisely determine selected alkylpyrazines in commercial peanut butter, cocoa powder, and instant coffee. 2,3-Diethyl-5-methylpyrazine (p-9) and 2-ethyl-3,5-dimethylpyrazine (p-8), despite their low abundance, had the highest odor-active values among the 13 pyrazines quantified in all products due to their very low odor thresholds.

  15. Stable isotope-labeled vitamin D, metabolites and chemical analogs: Synthesis and use in mass spectrometric studies

    SciTech Connect

    Coldwell, R.D.; Trafford, D.J.; Varley, M.J.; Kirk, D.N.; Makin, H.L. )

    1990-10-01

    Methods for the measurement of vitamin D and its metabolites using stable isotope-labeled internal standards and mass spectrometry are reviewed. The synthesis of both labeled and unlabeled standards is illustrated, and details of the synthesis of (26,26,27,27,27(-2)H5)-25,26-dihydroxyvitamin D3 and (28,28,28(-2)H3)-24,25-dihydroxyvitamin D2 are given. The use of in vitro biologic systems for the production of further metabolites of deuterated 25-hydroxyvitamin D3 is discussed. Use of deuterated 25-hydroxydihydrotachysterol3 as a substrate in the isolated perfused rat kidney has provided valuable data for the assignment of structure to a number of metabolites of 25-hydroxydihydrotachysterol3 formed in this system. 51 refs.

  16. Shape-Controlled Synthesis of Isotopic Yttrium-90-Labeled Rare Earth Fluoride Nanocrystals for Multimodal Imaging.

    PubMed

    Paik, Taejong; Chacko, Ann-Marie; Mikitsh, John L; Friedberg, Joseph S; Pryma, Daniel A; Murray, Christopher B

    2015-09-22

    Isotopically labeled nanomaterials have recently attracted much attention in biomedical research, environmental health studies, and clinical medicine because radioactive probes allow the elucidation of in vitro and in vivo cellular transport mechanisms, as well as the unambiguous distribution and localization of nanomaterials in vivo. In addition, nanocrystal-based inorganic materials have a unique capability of customizing size, shape, and composition; with the potential to be designed as multimodal imaging probes. Size and shape of nanocrystals can directly influence interactions with biological systems, hence it is important to develop synthetic methods to design radiolabeled nanocrystals with precise control of size and shape. Here, we report size- and shape-controlled synthesis of rare earth fluoride nanocrystals doped with the β-emitting radioisotope yttrium-90 ((90)Y). Size and shape of nanocrystals are tailored via tight control of reaction parameters and the type of rare earth hosts (e.g., Gd or Y) employed. Radiolabeled nanocrystals are synthesized in high radiochemical yield and purity as well as excellent radiolabel stability in the face of surface modification with different polymeric ligands. We demonstrate the Cerenkov radioluminescence imaging and magnetic resonance imaging capabilities of (90)Y-doped GdF3 nanoplates, which offer unique opportunities as a promising platform for multimodal imaging and targeted therapy.

  17. Fluorescent dye-labelled polymer synthesis by nitroxide mediated radical polymerization

    NASA Astrophysics Data System (ADS)

    Kollár, Jozef; Chmela, Štefan; Hrčková, Ľudmila; Hrdlovič, Pavol

    2012-07-01

    New applications of polymers at advanced technologies demand increased requirements on their properties. These properties are influenced by molecular as well as supramolecular structure. Controlled radical polymerization mediated by stable nitroxides (NMP) or substituted alkoxyamines offers simple method for preparation of polymers with programmable structure of macromolecules which possess remarkable better physical as well as chemical properties. They can be used as a macro initiators for the synthesis of block copolymers. At the present time it has been generally accepted that the extent of "livingness" is high for all conversions [1-4]. To verify this statement a series of fluorescent dye-labelled regulators has been synthesized, spectrally characterized and used as the mediators of styrene and n-butyl acrylate polymerization. Direct quantification of dormant species concentration (extent of livingness) and calculation of molar mass of marked polymers was performed by absorption and/or emission spectroscopy. Controlled radical polymerization mediated by stable nitroxides bearing fluorescence mark represents unconventional approach for monitoring and evaluation of mechanism and kinetics of polymerization process. Results indicate that the extent of livingness is strongly influenced by conversion as well as mediator concentration. There is a clear tendency toward to decreasing amount of dormant species with increasing monomer conversion. Moreover, lower mediator concentration decreases livingness of polymerization process.

  18. Selected scientific topics of the 11th International Isotope Symposium on the Synthesis and Applications of Isotopes and Isotopically Labeled Compounds.

    PubMed

    Atzrodt, Jens; Derdau, Volker

    2013-01-01

    This micro-review describes hot topics and new trends in isotope science discussed at the 11th International Isotope Symposium on the Synthesis and Applications of Isotopes and Isotopically Labeled Compounds from a personal perspective.

  19. Synthesis of carbon-14, carbon-13 and deuterium labeled forms of thioacetamide and thioacetamide S-oxide

    PubMed Central

    Sarma, Diganta; Hanzlik, Robert P.

    2013-01-01

    Thioacetamide (TA) is a model hepatotoxin that undergoes metabolic activation via two successive S-oxidations. The ultimate toxic metabolite thioacetamide S,S-dioxide, or its tautomer acetimidoyl sulfinic acid CH3C(NH)SO2H, then acylates lysine side chains on cellular proteins leading to cellular dysfunction or death. To identify individual target proteins, quantitate the extent of their modification and elucidate the structural details of their modification we required both radio-labeled and stable-labeled forms of TA and its intermediate metabolite thioacetamide S-oxide (TASO). The latter is stable when purified but can be difficult to isolate. Considering currently available isotopic precursors we devised and report here methods for the synthesis and isolation of TA and TASO labeled with C-14, C-13 and/or deuterium. The methods are straightforward, utilize readily available precursors and are amenable to small scale. PMID:26069392

  20. Economical synthesis of 13C-labeled opiates, cocaine derivatives and selected urinary metabolites by derivatization of the natural products.

    PubMed

    Karlsen, Morten; Liu, Huiling; Johansen, Jon Eigill; Hoff, Bård Helge

    2015-03-25

    The illegal use of opiates and cocaine is a challenge world-wide, but some derivatives are also valuable pharmaceuticals. Reference samples of the active ingredients and their metabolites are needed both for controlling administration in the clinic and to detect drugs of abuse. Especially, (13)C-labeled compounds are useful for identification and quantification purposes by mass spectroscopic techniques, potentially increasing accuracy by minimizing ion alteration/suppression effects. Thus, the synthesis of [acetyl-(13)C4]heroin, [acetyl-(13)C4-methyl-(13)C]heroin, [acetyl-(13)C2-methyl-(13)C]6-acetylmorphine, [N-methyl-(13)C-O-metyl-(13)C]codeine and phenyl-(13)C6-labeled derivatives of cocaine, benzoylecgonine, norcocaine and cocaethylene was undertaken to provide such reference materials. The synthetic work has focused on identifying (13)C atom-efficient routes towards these derivatives. Therefore, the (13)C-labeled opiates and cocaine derivatives were made from the corresponding natural products.

  1. Synthesis and characterization of colloidal gold particles as labels for antibodies as used in lateral flow devices.

    PubMed

    Cvak, Barbara; Pum, Dietmar; Molinelli, Alexandra; Krska, Rudolf

    2012-04-21

    Based on well established citrate reduction protocols for the synthesis of colloidal gold particles, this work focuses on the characterization of these colloids for further use as color labels in lateral flow devices. A reproducible production method has been developed for the synthesis of well characterized colloidal gold particles to be employed in Lateral Flow Devices (LFDs). It has been demonstrated that when undertaking chemical reduction of gold salts with sodium citrate, the amount of reducing agent employed could be used to directly control the size of the resultant particles. A protocol was thereby developed for the synthesis of colloidal gold particles of pre-defined diameters in the range of 15 to 60 nm and of consistent size distribution. The absorption maxima (λ(max)) of the reaction solutions were analyzed by UV/VIS measurements to determine approximate particle sizes, which were confirmed with transmission electron microscopy (TEM) measurements. Colloidal gold particles of about 40 nm in diameter were synthesized and used for labeling monoclonal anti-mycotoxin antibodies (e.g. zearalenone). To deduce the extent of antibody coupling to these particles, smaller colloids with 15 nm diameter were labeled with anti-species specific antibodies. Both solutions were mixed and then scanned by TEM to obtain information about the success of coupling.

  2. New synthesis of fluorine-18-labeled 6-fluoro-L-dopa by cleaving the carbon-silicon bond with fluorine

    SciTech Connect

    Diksic, M.; Farrokhzad, S.

    1985-11-01

    A new synthesis of 3,4-dihydroxy-6-( YF)fluoro-L-phenylalanine using 6-trimethylsilyl-3,4-dimethoxy-L-dopa-ethylester as a fluorination substrate is described. The silane is prepared from the corresponding bromo compound by reacting the latter with magnesium and trimethylsilyl chloride. Reaction of the silane with ( YF)F2 in a mixture of freon-11/CCl4 (1:1) kept in a dry ice bath, subsequent hydrolysis with concentrated HBr in a bath at 140 degrees C, and simple chromatographic purification yielded YF-labeled 6-fluoro-L-dopa. A radiochemical yield of about 8% was achieved at the end of the 1-hr synthesis. The specific activity at the end of the synthesis was about 680 mCi/mmol after a 30-min irradiation.

  3. Synthesis and evaluation of 18F labeled alanine derivatives as potential tumor imaging agents

    PubMed Central

    Wang, Limin; Zha, Zhihao; Qu, Wenchao; Qiao, Hongwen; Lieberman, Brian P.; Plössl, Karl; Kung, Hank F.

    2012-01-01

    Introduction This paper reports the synthesis and labeling of 18F alanine derivatives. We also investigate their biological characteristics as potential tumor imaging agents mediated by alanine-serine-cysteine preferring (ASC) transporter system. Methods Three new 18F alanine derivatives were prepared from corresponding tosylate-precursors through a two-step labelling reaction. In vitro uptake studies to evaluate and to compare these three analogs were carried out in 9L glioma and PC-3 prostate cancer cell lines. Potential transport mechanisms, protein incorporation and stability of 3-(1-[18F]fluoromethyl)-L-alanine (L[18F]FMA) were investigated in 9L glioma cells. Its biodistribution was determined in a rat-bearing 9L tumor model. PET imaging studies were performed on rat bearing 9L glioma tumors and transgenic mouse carrying spontaneous generated M/tomND tumor (mammary gland adenocarcinoma). Results New 18F alanine derivatives were prepared with 7–34% uncorrected radiochemical yields, excellent enantiomeric purity (>99%) and good radiochemical purity (>99%). In vitro uptake of the L-[18F]FMA in 9L glioma and PC-3 prostate cancer cells was higher than those observed for other two alanine derivatives and [18F]FDG in first 1 h. Inhibition of cell uptake studies suggested that L-[18F]FMA uptake in 9L glioma was predominantly via transport system ASC. After entering into cells, L-[18F]FMA remained stable and was not incorporated into protein within 2 h. In vivo biodistribution studies demonstrated that L-[18F]FMA had relatively high uptake in liver and kidney. Tumor uptake was fast, reaching a maximum within 30 min. The tumor-to-muscle, tumor-to-blood and tumor-to-brain ratios at 60 min post injection were 2.2, 1.9 and 3.0, respectively. In PET imaging studies, tumors were visualized with L-[18F]FMA in both 9L rat and transgenic mouse. Conclusion L-[18F]FMA showed promising properties as a PET imaging agent for up-regulated ASC transporter associated with tumor

  4. MEASURING OF PROTEIN SYNTHESIS USING METABOLIC 2H-LABELING, HIGH-RESOLUTION MASS SPECTROMETRY AND AN ALGORITHM

    PubMed Central

    Kasumov, Takhar; Ilchenko, Sergey; Li, Ling; Rachdaoui, Nadia; Sadigov, Rovshan; Willard, Belinda; McCullough, Arthur J.; Previs, Stephen

    2013-01-01

    We recently developed a method for estimating protin dynamics in vivo with 2H2O using MALDI-TOF MS (Rachdaoui N. et al., MCP, 8, 2653-2662, 2009) and we confirmed that 2H-labeling of many hepatic free amino acids rapidly equilibrated with body water. Although this is a reliable method, it required modest sample purification and necessitated the determination of tissue-specific amino acid labeling. Another approach for quantifying protein kinetics is to measure the 2H-enrichments of body water (precursor) and protein-bound amino acid or proteolytic peptide (product) and to estimate how many copies of deuterium are incorporated into a product. In this study we have used nanospray LTQ-FTICR mass spectrometry to simultaneously measure the isotopic enrichment of peptides and protein-bound amino acids. A mathematical algorithm was developed to aid the data processing. The most notable improvement centers on the fact that the precursor:product labeling ratio can be obtained by measuring the labeling of water and a protein(s) (or peptides) of interest, therein minimizing the need to measure the amino acid labeling. As a proof of principle, we demonstrate that this approach can detect the effect of nutritional status on albumin synthesis in rats given 2H2O. PMID:21256107

  5. Measuring protein synthesis using metabolic ²H labeling, high-resolution mass spectrometry, and an algorithm.

    PubMed

    Kasumov, Takhar; Ilchenko, Serguey; Li, Ling; Rachdaoui, Nadia; Sadygov, Rovshan G; Willard, Belinda; McCullough, Arthur J; Previs, Stephen

    2011-05-01

    We recently developed a method for estimating protein dynamics in vivo with heavy water ((2)H(2)O) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) [16], and we confirmed that (2)H labeling of many hepatic free amino acids rapidly equilibrated with body water. Although this is a reliable method, it required modest sample purification and necessitated the determination of tissue-specific amino acid labeling. Another approach for quantifying protein kinetics is to measure the (2)H enrichments of body water (precursor) and protein-bound amino acid or proteolytic peptide (product) and to estimate how many copies of deuterium are incorporated into a product. In the current study, we used nanospray linear trap Fourier transform ion cyclotron resonance mass spectrometry (LTQ FT-ICR MS) to simultaneously measure the isotopic enrichment of peptides and protein-bound amino acids. A mathematical algorithm was developed to aid the data processing. The most notable improvement centers on the fact that the precursor/product labeling ratio can be obtained by measuring the labeling of water and a protein (or peptide) of interest, thereby minimizing the need to measure the amino acid labeling. As a proof of principle, we demonstrate that this approach can detect the effect of nutritional status on albumin synthesis in rats given (2)H(2)O. Copyright © 2011 Elsevier Inc. All rights reserved.

  6. Synthesis of [18F]-labelled Maltose Derivatives as PET Tracers for Imaging Bacterial Infection

    PubMed Central

    Namavari, Mohammad; Gowrishankar, Gayatri; Hoehne, Aileen; Jouannot, Erwan; Gambhir, Sanjiv S

    2015-01-01

    Purpose To develop novel positron emission tomography (PET) agents for visualization and therapy monitoring of bacterial infections. Procedures It is known that maltose and maltodextrins are energy sources for bacteria. Hence, 18F-labelled maltose derivatives could be a valuable tool for imaging bacterial infections. We have developed methods to synthesize 4-O-(α-D-glucopyranosyl)-6-deoxy-6-[18F]fluoro-D-glucopyranoside (6-[18F]fluoromaltose) and 4-O-(α-D-glucopyranosyl)-1-deoxy-1-[18F]fluoro-D-glucopyranoside (1-[18F]fluoromaltose) as bacterial infection PET imaging agents. 6-[18F]fluoromaltose was prepared from precursor 1,2,3-tri-O-acetyl-4-O-(2′,3′,-di-O-acetyl-4′,6′-benzylidene-α-D-glucopyranosyl)-6-deoxy-6-nosyl-D-glucopranoside (5). The synthesis involved the radio-fluorination of 5 followed by acidic and basic hydrolysis to give 6-[18F]fluoromaltose. In an analogous procedure, 1-[18F]fluoromaltose was synthesized from 2,3, 6-tri-O-acetyl-4-O-(2′,3′,4′,6-tetra-O-acetyl-α-D-glucopyranosyl)-1-deoxy-1-O-triflyl-D-glucopranoside (9). Stability of 6-[18F]fluoromaltose in phosphate-buffered saline (PBS) and human and mouse serum at 37 °C was determined. Escherichia coli uptake of 6-[18F]fluoromaltose was examined. Results A reliable synthesis of 1- and 6-[18F]fluoromaltose has been accomplished with 4–6 and 5–8 % radiochemical yields, respectively (decay-corrected with 95 % radiochemical purity). 6-[18F]fluoromaltose was sufficiently stable over the time span needed for PET studies (~96 % intact compound after 1-h and ~65 % after 2-h incubation in serum). Bacterial uptake experiments indicated that E. coli transports 6-[18F]fluoromaltose. Competition assays showed that the uptake of 6-[18F]fluoromaltose was completely blocked by co-incubation with 1 mM of the natural substrate maltose. Conclusion We have successfully synthesized 1- and 6-[18F]fluoromaltose via direct fluorination of appropriate protected maltose precursors. Bacterial uptake

  7. Synthesis of fluorine-18 radio-labeled serum albumins for PET blood pool imaging.

    PubMed

    Basuli, Falguni; Li, Changhui; Xu, Biying; Williams, Mark; Wong, Karen; Coble, Vincent L; Vasalatiy, Olga; Seidel, Jurgen; Green, Michael V; Griffiths, Gary L; Choyke, Peter L; Jagoda, Elaine M

    2015-03-01

    We sought to develop a practical, reproducible and clinically translatable method of radiolabeling serum albumins with fluorine-18 for use as a PET blood pool imaging agent in animals and man. Fluorine-18 radiolabeled fluoronicotinic acid-2,3,5,6-tetrafluorophenyl ester, [(18)F]F-Py-TFP was prepared first by the reaction of its quaternary ammonium triflate precursor with [(18)F]tetrabutylammonium fluoride ([(18)F]TBAF) according to a previously published method for peptides, with minor modifications. The incubation of [(18)F]F-Py-TFP with rat serum albumin (RSA) in phosphate buffer (pH9) for 15 min at 37-40 °C produced fluorine-18-radiolabeled RSA and the product was purified using a mini-PD MiniTrap G-25 column. The overall radiochemical yield of the reaction was 18-35% (n=30, uncorrected) in a 90-min synthesis. This procedure, repeated with human serum albumin (HSA), yielded similar results. Fluorine-18-radiolabeled RSA demonstrated prolonged blood retention (biological half-life of 4.8 hours) in healthy awake rats. The distribution of major organ radioactivity remained relatively unchanged during the 4 hour observation periods either by direct tissue counting or by dynamic PET whole-body imaging except for a gradual accumulation of labeled metabolic products in the bladder. This manual method for synthesizing radiolabeled serum albumins uses fluorine-18, a widely available PET radionuclide, and natural protein available in both pure and recombinant forms which could be scaled up for widespread clinical applications. These preclinical biodistribution and PET imaging results indicate that [(18)F]RSA is an effective blood pool imaging agent in rats and might, as [(18)F]HSA, prove similarly useful as a clinical imaging agent.

  8. Synthesis of radioactively labelled CdSe/CdS/ZnS quantum dots for in vivo experiments.

    PubMed

    Stachowski, Gordon M; Bauer, Christoph; Waurisch, Christian; Bargheer, Denise; Nielsen, Peter; Heeren, Jörg; Hickey, Stephen G; Eychmüller, Alexander

    2014-01-01

    During the last decades of nanoparticles research, many nanomaterials have been developed for applications in the field of bio-labelling. For the visualization of transport processes in the body, organs and cells, luminescent quantum dots (QDs) make for highly useful diagnostic tools. However, intercellular routes, bio-distribution, metabolism during degradation or quantification of the excretion of nanoparticles, and the study of the biological response to the QDs themselves are areas which to date have not been fully investigated. In order to aid in addressing those issues, CdSe/CdS/ZnS QDs were radioactively labelled, which allows quantification of the QD concentration in the whole body or in ex vivo samples by γ-counting. However, the synthesis of radioactively labelled QDs is not trivial since the coating process must be completely adapted, and material availability, security and avoidance of radioactive waste must be considered. In this contribution, the coating of CdSe/CdS QDs with a radioactive (65)ZnS shell using a modified, operator-safe, SILAR procedure is presented. Under UV illumination, no difference in the photoluminescence of the radioactive and non-radioactive CdSe/CdS/ZnS colloidal solutions was observed. Furthermore, a down-scaled synthesis for the production of very small batches of 5 nmol QDs without loss in the fluorescence quality was developed. Subsequently, the radio-labelled QDs were phase transferred by encapsulation into an amphiphilic polymer. γ-counting of the radioactivity provided confirmation of the successful labelling and phase transfer of the QDs.

  9. Synthesis of radioactively labelled CdSe/CdS/ZnS quantum dots for in vivo experiments

    PubMed Central

    Stachowski, Gordon M; Bauer, Christoph; Waurisch, Christian; Bargheer, Denise; Nielsen, Peter; Heeren, Jörg; Hickey, Stephen G

    2014-01-01

    Summary During the last decades of nanoparticles research, many nanomaterials have been developed for applications in the field of bio-labelling. For the visualization of transport processes in the body, organs and cells, luminescent quantum dots (QDs) make for highly useful diagnostic tools. However, intercellular routes, bio-distribution, metabolism during degradation or quantification of the excretion of nanoparticles, and the study of the biological response to the QDs themselves are areas which to date have not been fully investigated. In order to aid in addressing those issues, CdSe/CdS/ZnS QDs were radioactively labelled, which allows quantification of the QD concentration in the whole body or in ex vivo samples by γ-counting. However, the synthesis of radioactively labelled QDs is not trivial since the coating process must be completely adapted, and material availability, security and avoidance of radioactive waste must be considered. In this contribution, the coating of CdSe/CdS QDs with a radioactive 65ZnS shell using a modified, operator-safe, SILAR procedure is presented. Under UV illumination, no difference in the photoluminescence of the radioactive and non-radioactive CdSe/CdS/ZnS colloidal solutions was observed. Furthermore, a down-scaled synthesis for the production of very small batches of 5 nmol QDs without loss in the fluorescence quality was developed. Subsequently, the radio-labelled QDs were phase transferred by encapsulation into an amphiphilic polymer. γ-counting of the radioactivity provided confirmation of the successful labelling and phase transfer of the QDs. PMID:25551066

  10. Apparent Catalase Synthesis in Sunflower Cotyledons during the Change in Microbody Function: A Mathematical Approach for the Quantitative Evaluation of Density-labeling Data.

    PubMed

    Betsche, T; Gerhardt, B

    1978-10-01

    Density-labeling with 10 mm K(15)NO(3)/70% (2)H(2)O has been used to investigate catalase synthesis in different developmental stages of sunflower (Helianthus annuus L.) cotyledons. A mathematical approach is introduced for the quantitative evaluation of the density-labeling data. The method allows, in the presence of preexisting enzyme activity, calculation of this synthesized activity (apparent enzyme synthesis) which results from the balance between actual enzyme synthesis and the degradation of newly synthesized enzyme at a given time. During greening of the cotyledons, when the catalase activity declines and the population of leaf peroxisomes is formed, the apparent catalase synthesis is lower than, or at best equal to, that occurring during a developmental stage when the leaf peroxisome population is established and catalase synthesis and degradation of total catalase are in equilibrium. This result suggests a formation, in fatty cotyledons, of the leaf peroxisomes by transformation of the glyoxysomes rather than by de novo synthesis.

  11. Automated synthesis of radiopharmaceuticals for positron emission tomography: an apparatus for labelling with [11C] methyl iodide (MIASA)

    PubMed Central

    Cork, D. G.; Yamato, H.; Yajima, K.; Hayashi, N.; Sugawara, T.; Kato, S.

    1994-01-01

    A fully automated apparatus for the routine synthesis and formulation of short-lived 11C (t1/2 = 20 min) labelled radiopharmaceuticals for positron emission tomography (PET) has been developed. [11C]Carbon dioxide is converted to [11C]methyl iodide, which can be used to label a wide variety of substrates by methylation at C, N, O, or S electron rich centres. The apparatus, MIASA (methyl iodide automated synthesis apparatus), was designed to operate as part of an automated labelling system in a shielded ‘hot’ laboratory. The apparatus was designed without the size constraints of typical instrumentation used in hot cells, although it is compact where necessary. Ample use of indicators and sensors, together with compact design of the reaction flasks for small dead space and efficient evaporation, led to good reliability and performance. The design of the hardware and software is described in this paper, together with a preparation of 3-N-[11C]methylspiperone as a sterile injectable solution in physiological saline. PMID:18924994

  12. Labeling of hepatic glycogen after short- and long-term stimulation of glycogen synthesis in rats injected with 3H-galactose

    SciTech Connect

    Michaels, J.E.; Garfield, S.A.; Hung, J.T.; Cardell, R.R. Jr. )

    1990-08-01

    The effects of short- and long-term stimulation of glycogen synthesis elicited by dexamethasone were studied by light (LM) and electron (EM) microscopic radioautography (RAG) and biochemical analysis. Adrenalectomized rats were fasted overnight and pretreated for short- (3 hr) or long-term (14 hr) periods with dexamethasone prior to intravenous injection of tracer doses of 3H-galactose. Analysis of LM-RAGs from short-term rats revealed that about equal percentages (44%) of hepatocytes became heavily or lightly labeled 1 hr after labeling. The percentage of heavily labeled cells increased slightly 6 hr after labeling, and unlabeled glycogen became apparent in some hepatocytes. The percentage of heavily labeled cells had decreased somewhat 12 hr after labeling, and more unlabeled glycogen was evident. In the long-term rats 1 hr after labeling, a higher percentage of heavily labeled cells (76%) was observed compared to short-term rats, and most glycogen was labeled. In spite of the high amount of labeling seen initially, the percentage of heavily labeled hepatocytes had decreased considerably to 55% by 12 hr after injection; and sparsely labeled and unlabeled glycogen was prevalent. The EM-RAGs of both short- and long-term rats were similar. Silver grains were associated with glycogen patches 1 hr after labeling; 12 hr after labeling, the glycogen patches had enlarged; and label, where present, was dispersed over the enlarged glycogen clumps. Analysis of DPM/mg tissue corroborated the observed decrease in label 12 hr after administration in the long-term animals. The loss of label observed 12 hr after injection in the long-term pretreated rats suggests that turnover of glycogen occurred during this interval despite the net accumulation of glycogen that was visible morphologically and evident from biochemical measurement.

  13. A facile synthesis of deuterium labeled 2,2-dimethyl-[2H6]-succinic acid and its anhydride.

    PubMed

    Srinivas, G; Unny, V K P; Mukkanti, K; Choudary, B M

    2013-05-30

    Deuterium labeled 2,2-dimethyl-[(2)H(6)]-succinic anhydride by a sequence of reactions involving Knoevenagel condensation of [(2)H(6)]-acetone with ethyl cyanoacetate in the presence of piperidine, Michael addition of cyanide, HCl hydrolysis, simultaneous decarboxylation, and subsequent dehydration using acetic anhydride in an overall yield of 34.23% based on [(2)H(6)]-acetone utilized in the reaction is reported. The title compounds were characterized and confirmed spectroscopically by Fourier transform infrared, (1) H-NMR, and Mass. The chemical purity as determined by HPLC was 99%. To the best of our knowledge, the synthesis of these specifically deuterium labeled compounds has not been reported so far. Copyright © 2013 John Wiley & Sons, Ltd.

  14. Synthesis and luminescence properties of new red-shifted absorption lanthanide(III) chelates suitable for peptide and protein labelling.

    PubMed

    Maindron, Nicolas; Poupart, Séverine; Hamon, Maxime; Langlois, Jean-Baptiste; Plé, Nelly; Jean, Ludovic; Romieu, Anthony; Renard, Pierre-Yves

    2011-04-07

    The synthesis and photo-physical properties of an original bis-pyridinylpyrazine chromophore efficiently sensitising europium(III) and samarium(III) are described. The corresponding lanthanide(III) complexes display in aqueous solutions a maximum excitation wavelength which is significantly red-shifted compared to the usual terpyridine-based chelates, and a valuable luminescence brightness above 2,000 dm(3) mol(-1) cm(-1) at 345 nm was obtained with a europium(III) derivative. Further functionalisation with three different bioconjugatable handles was also investigated and their ability to efficiently label a model hexapeptide was evaluated and compared. Finally, the best bioconjugatable europium(III) chelate was used in representative labelling experiments involving monoclonal antibodies and the luminescence features of the corresponding bioconjugates remained satisfactory.

  15. Estimation of Whole Body Radiation Exposure to Nuclear Medicine Personnel During Synthesis of (177)Lutetium-labeled Radiopharmaceuticals.

    PubMed

    Arora, Geetanjali; Mishra, Rajesh; Kumar, Praveen; Yadav, Madhav; Ballal, Sanjana; Bal, Chandrasekhar; Damle, Nishikant Avinash

    2017-01-01

    With rapid development in the field of nuclear medicine therapy, radiation safety of the personnel involved in synthesis of radiopharmaceuticals has become imperative. Few studies have been done on estimating the radiation exposure of personnel involved in the radio labeling of (177)Lu-compounds in western countries. However, data from the Indian subcontinent are limited. We have estimated whole body radiation exposure to the radiopharmacist involved in the labeling of: (177)Lu-DOTATATE, (177)Lu-PSMA-617, and (177)Lu-EDTMP. Background radiation was measured by keeping a pocket dosimeter around the workbench when no radioactive work was conducted. The same pocket dosimeter was given to the radiopharmacist performing the labeling of (177)Lu-compounds. All radiopharmaceuticals were synthesized by the same radiopharmacist with 3, 1 and 3 year experience, respectively, in radiolabeling the above compounds. One Curie (1 Ci) of (177)Lu was received fortnightly by our department. Data were collected for 12 syntheses of (177)Lu-DOTATATE, 8 syntheses of (177)Lu-PSMA-617, and 3 syntheses of (177)Lu-EDTMP. Mean time required to complete the synthesis was 0.81, 0.65, and 0.58 h, respectively. Mean whole body radiation exposure was 0.023 ± 0.01 mSv, 0.01 ± 0.002 mSv, and 0.002 ± 0.0006 mSv, respectively. Overall mean radiation dose for all the three (177)Lu-compounds was 0.014 mSv. Highest exposure was obtained during the synthesis of (177)Lu-DOTATATE. Our data suggest that the manual radiolabeling of (177)Lu compounds is safe, and the whole body radiation exposure to the involved personnel is well within prescribed limits.

  16. Synthesis and properties of acridone-labeled base-discriminating fluorescent (BDF) nucleosides.

    PubMed

    Saito, Yoshio; Hanawa, Kazuo; Bag, Subhendu Sekhar; Motegi, Kaori; Saito, Isao

    2006-01-01

    We have developed novel acridone-labelled BDF probe which showed its potential in recognizing opposite matched base from its target sequence via enhancement of fiuorescence intensity. This probe emit at a longer wavelength than previously reported pyrene-labelled BDF probe and thus can be used in DNA chip.

  17. Synthesis and evaluation of radioactive and fluorescent residualizing labels for identifying sites of plasma protein catabolism

    SciTech Connect

    Maxwell, J.L.; Baynes, J.W.; Thorpe, S.R.

    1986-05-01

    Inulin and lactose were each coupled to tyramine by reductive amination with NaBH/sub 3/CN and the tyramine then labeled with /sup 125/I. Dilactitol-/sup 125/I-tyramine (DLT) and inulin-/sup 125/I-tyramine (InTn) were coupled by reductive amination and cyanuric chloride, respectively, to asialofetuin (ASF), fetuin and rat serum albumin (RSA). Attachment of either label had no effect on the circulating half-lives of the proteins. Radioactivity from labeled ASF was recovered in rat liver (> 90%) by 1 h post-injection and remained in liver with half-lives of 2 and 6 days, respectively, for the DLT and InTn labels. Whole body recoveries of radioactivity from DLT- and InTn labels. Whole body recoveries of radioactivity from DLT- and InTn-labeled RSA were 5 and 6.5 days, respectively, again indicating that the larger glycoconjugate label residualized more efficiently in cells following protein degradation. (Lactitol)/sub 2/-N-CH/sub 2/-CH/sub 2/-NH-fluroescein (DLF) was also coupled to ASF by reductive amination and recovered quantitatively in liver at 1 h post-injection. Native ASF was an effective competitor for clearance of DLF-ASF from the circulation. Fluorescent degradation products were retained in liver with a half-life of 1.2 days. Residualizing fluorescent labels should be useful for identification and sorting of cells active in the degradation of plasma proteins.

  18. Synthesis and properties of peptide nucleic acid labeled at the N-terminus with HiLyte Fluor 488 fluorescent dye.

    PubMed

    Hnedzko, Dziyana; McGee, Dennis W; Rozners, Eriks

    2016-09-15

    Fluorescently labeled peptide nucleic acids (PNAs) are important tools in fundamental research and biomedical applications. However, synthesis of labeled PNAs, especially using modern and expensive dyes, is less explored than similar preparations of oligonucleotide dye conjugates. Herein, we present a simple procedure for labeling of the PNA N-terminus with HiLyte Fluor 488 as the last step of solid phase PNA synthesis. A minimum excess of 1.25equiv of activated carboxylic acid achieved labeling yields close to 90% providing a good compromise between the price of dye and the yield of product and significant improvement over previous literature procedures. The HiLyte Fluor 488-labeled PNAs retained the RNA binding ability and in live cell fluorescence microscopy experiments were brighter and significantly more photostable than PNA labeled with carboxyfluorescein. In contrast to fluorescein-labeled PNA, the fluorescence of PNAs labeled with HiLyte Fluor 488 was independent of pH in the biologically relevant range of 5-8. The potential of HiLyte Fluor 488-labeling for studies of PNA cellular uptake and distribution was demonstrated in several cell lines.

  19. Synthesis and optical properties of pyrrolidinyl peptide nucleic acid carrying a clicked Nile red label.

    PubMed

    Yotapan, Nattawut; Charoenpakdee, Chayan; Wathanathavorn, Pawinee; Ditmangklo, Boonsong; Wagenknecht, Hans-Achim; Vilaivan, Tirayut

    2014-01-01

    DNA or its analogues with an environment-sensitive fluorescent label are potentially useful as a probe for studying the structure and dynamics of nucleic acids. In this work, pyrrolidinyl peptide nucleic acid (acpcPNA) was labeled at its backbone with Nile red, a solvatochromic benzophenoxazine dye, by means of click chemistry. The optical properties of the Nile red-labeled acpcPNA were investigated by UV-vis and fluorescence spectroscopy in the absence and in the presence of DNA. In contrast to the usual quenching observed in Nile red-labeled DNA, the hybridization with DNA resulted in blue shifting and an enhanced fluorescence regardless of the neighboring bases. More pronounced blue shifts and fluorescence enhancements were observed when the DNA target carried a base insertion in close proximity to the Nile red label. The results indicate that the Nile red label is located in a more hydrophobic environment in acpcPNA-DNA duplexes than in the single-stranded acpcPNA. The different fluorescence properties of the acpcPNA hybrids of complementary DNA and DNA carrying a base insertion are suggestive of different interactions between the Nile red label and the duplexes.

  20. Synthesis and optical properties of pyrrolidinyl peptide nucleic acid carrying a clicked Nile red label

    PubMed Central

    Yotapan, Nattawut; Charoenpakdee, Chayan; Wathanathavorn, Pawinee; Ditmangklo, Boonsong

    2014-01-01

    Summary DNA or its analogues with an environment-sensitive fluorescent label are potentially useful as a probe for studying the structure and dynamics of nucleic acids. In this work, pyrrolidinyl peptide nucleic acid (acpcPNA) was labeled at its backbone with Nile red, a solvatochromic benzophenoxazine dye, by means of click chemistry. The optical properties of the Nile red-labeled acpcPNA were investigated by UV–vis and fluorescence spectroscopy in the absence and in the presence of DNA. In contrast to the usual quenching observed in Nile red-labeled DNA, the hybridization with DNA resulted in blue shifting and an enhanced fluorescence regardless of the neighboring bases. More pronounced blue shifts and fluorescence enhancements were observed when the DNA target carried a base insertion in close proximity to the Nile red label. The results indicate that the Nile red label is located in a more hydrophobic environment in acpcPNA–DNA duplexes than in the single-stranded acpcPNA. The different fluorescence properties of the acpcPNA hybrids of complementary DNA and DNA carrying a base insertion are suggestive of different interactions between the Nile red label and the duplexes. PMID:25246975

  1. Polycationic nanoparticles: (1) synthesis of a polylysine-MION conjugate and its application in labeling fibroblasts.

    PubMed

    Groman, Ernest V; Yang, Meiheng; Reinhardt, Christopher P; Weinberg, James S; Vaccaro, Dennis E

    2009-03-01

    Nanoparticles are increasingly used to label cells to track them by imaging or to quantify them in vivo. However, normal cellular uptake mechanisms are inadequate to load cells with tracking label. We propose a simple method to coat nanoparticles, such as monocrystalline iron oxide nanoparticle (MION), with the transfection agent polylysine in order to facilitate rapid, uniform, and heavy labeling of fibroblasts. The method is based on commercially available reagents, requires no more than 1 h of laboratory contact time, and can be accomplished safely without a chemical hood. A suspension of MION was treated by addition of solid sodium periodate to oxidize glucose residues of dextran and introduced aldehyde groups to the dextran coat surrounding MION's crystalline magnetite core. After a 30-min incubation to effect oxidation, unreacted periodate was quenched with glycerol. The preparation was dialyzed to remove reactants and diluted to a final concentration of 2 mg Fe/ml. Poly-L-lysine was added to the oxidized MION (MION-A) to form reversible covalent Schiff base linkages. The resulting conjugate, a polylysine iron oxide nano-particle is abbreviated PLION. NIH3T3 fibroblasts labeled with either MION, MION-A, or MION plus polylysine showed minimal uptake of iron while cells labeled with PLION acquired a brown hue demonstrating strong labeling with iron. Microscopic assessment of iron labeling was confirmed using Prussian blue staining. In some cells, the concentration of iron was sufficiently high and localized to suggest association with cytoplasmic vacuoles. The nucleus of the cell was not labeled. Cell labeling increased when the ratio of polylysine to MION increased and with increasing amount of PLION.

  2. Biotin-labeled synthetic oligodeoxyribonucleotides: chemical synthesis and uses as hybridization probes.

    PubMed Central

    Chollet, A; Kawashima, E H

    1985-01-01

    Oligodeoxynucleotides have been selectively labeled with biotin at their 5'-termini through an aminoalkylphosphoramide linker arm by an efficient chemical method. The reactions were performed in aqueous solution on unprotected oligonucleotides and were insensitive of the sequence and length of the oligonucleotide. 5'-biotin-labeled oligonucleotides were hybridized to dot, Southern and genomic blots of target plasmid DNA immobilized on nitrocellulose filters. Detection level is about 2 fmole. There is no noticeable disturbance of the strength and selectivity of hybridization of the 5'-biotin-labeled probes in comparison with non-modified DNA. Images PMID:4000941

  3. Synthesis and solid-state NMR structural characterization of 13C-labeled graphite oxide.

    PubMed

    Cai, Weiwei; Piner, Richard D; Stadermann, Frank J; Park, Sungjin; Shaibat, Medhat A; Ishii, Yoshitaka; Yang, Dongxing; Velamakanni, Aruna; An, Sung Jin; Stoller, Meryl; An, Jinho; Chen, Dongmin; Ruoff, Rodney S

    2008-09-26

    The detailed chemical structure of graphite oxide (GO), a layered material prepared from graphite almost 150 years ago and a precursor to chemically modified graphenes, has not been previously resolved because of the pseudo-random chemical functionalization of each layer, as well as variations in exact composition. Carbon-13 (13C) solid-state nuclear magnetic resonance (SSNMR) spectra of GO for natural abundance 13C have poor signal-to-noise ratios. Approximately 100% 13C-labeled graphite was made and converted to 13C-labeled GO, and 13C SSNMR was used to reveal details of the chemical bonding network, including the chemical groups and their connections. Carbon-13-labeled graphite can be used to prepare chemically modified graphenes for 13C SSNMR analysis with enhanced sensitivity and for fundamental studies of 13C-labeled graphite and graphene.

  4. Isotopic labeling of mouse interferon by incorporation of radioactive amino acids during synthesis

    SciTech Connect

    DeMaeyer-Guignard, J.; Cachard, A.; DeMaeyer, E.

    1982-07-30

    Mouse interferon produced by C-243 cells induced with Newcastle disease virus was isotopically labeled by adding either (/sup 35/S)methionine or a /sup 14/C-labeled amino acid mixture to the culture medium. A method combining butyric acid and theophylline treatment and resulting in high interferon yields was used. Following purification by two-step affinity chromatography on poly(U) and antibody columns, the resulting material was analyzed on SDS-PAGE. The migration pattern of radioactivity and interferon coincided well and autoradiography revealed three major bands at migration distances corresponding, respectively, to 35, 28, and 22 K. Interferon represented 3.8% of all (/sup 35/S)methionine-labeled proteins and 2.6% of all /sup 14/C-amino acid-labeled proteins released into the medium.

  5. Synthesis and NMR studies of (13)C-labeled vitamin D metabolites.

    PubMed

    Okamura, William H; Zhu, Gui-Dong; Hill, David K; Thomas, Richard J; Ringe, Kerstin; Borchardt, Daniel B; Norman, Anthony W; Mueller, Leonard J

    2002-03-08

    Isotope-labeled drug molecules may be useful for probing by NMR spectroscopy the conformation of ligand associated with biological hosts such as membranes and proteins. Triple-labeled [7,9,19-(13)C(3)]-vitamin D(3) (56), its 25-hydroxylated and 1 alpha,25-dihydroxylated metabolites (58 and 68, respectively), and other labeled materials have been synthesized via coupling of [9-(13)C]-Grundmann's ketone 39 or its protected 25-hydroxy derivative 43 with labeled A ring enyne fragments 25 or 26. The labeled CD-ring fragment 39 was prepared by a sequence involving Grignard addition of [(13)C]-methylmagnesium iodide to Grundmann's enone 28, oxidative cleavage, functional group modifications leading to seco-iodide 38, and finally a kinetic enolate S(N)2 cycloalkylation. The C-7,19 double labeling of the A-ring enyne was achieved by the Corey-Fuchs/Wittig processes on keto aldehyde 11. By employing these labeled fragments in the Wilson-Mazur route, the C-7,9,19 triple-(13)C-labeled metabolites 56, 58, and 68 as well as other (13)C-labeled metabolites have been prepared. In an initial NMR investigation of one of the labeled metabolites prepared in this study, namely [7,9,19-(13)C(3)]-25-hydroxyvitamin D(3) (58), the three (13)C-labeled carbons of the otherwise water insoluble steroid could be clearly detected by (13)C NMR analysis at 0.1 mM in a mixture of CD(3)OD/D(2)O (60/40) or in aqueous dimethylcyclodextrin solution and at 2 mM in 20 mM sodium dodecyl sulfate (SDS) aqueous micellar solution. In the SDS micellar solution, a double half-filter NOESY experiment revealed that the distance between the H(19Z) and H(7) protons is significantly shorter than that of the corresponding distance calculated from the solid state (X-ray) structure of the free ligand. The NMR data in micelles reveals that 58 exists essentially completely in the alpha-conformer with the 3 beta-hydroxyl equatorially oriented, just as in the solid state. The shortened distance (H(19Z))-H(7)) in micellar

  6. Synthesis and Reactivity of (18)F-Labeled α,α-Difluoro-α-(aryloxy)acetic Acids.

    PubMed

    Khotavivattana, Tanatorn; Calderwood, Samuel; Verhoog, Stefan; Pfeifer, Lukas; Preshlock, Sean; Vasdev, Neil; Collier, Thomas L; Gouverneur, Véronique

    2017-02-03

    In this work, we describe the (18)F-labeling of α,α-difluoro-α-(aryloxy)acetic acid derivatives and demonstrate that these building blocks are amenable to post-(18)F-fluorination functionalization. Protodecarboxylation offers a new entry to (18)F-difluoromethoxyarene, and the value of this approach is further demonstrated with coupling processes leading to representative (18)F-labeled TRPV1 inhibitors and TRPV1 antagonists.

  7. Synthesis of isotopically labeled R- or S-[.sup.13C, .sup.2H] glycerols

    DOEpatents

    Martinez, Rodolfo A.; Unkefer, Clifford J.; Alvarez, Marc A.

    2008-01-22

    The present invention is directed to asymmetric chiral labeled glycerols including at least one chiral atom, from one to two .sup.13C atoms and from zero to four deuterium atoms bonded directly to a carbon atom, e.g., (2S) [1,2-.sup.13C.sub.2]glycerol and (2R) [1,2-.sup.13C.sub.2]glycerol, and to the use of such chiral glycerols in the preparation of labeled amino acids.

  8. Synthesis and characterization of fluorescence-labelled silica core-shell and noble metal-decorated ceria nanoparticles

    PubMed Central

    Rennhak, Markus; Reller, Armin

    2014-01-01

    Summary The present review article covers work done in the cluster NPBIOMEM in the DFG priority programme SPP 1313 and focuses on synthesis and characterization of fluorescent silica and ceria nanoparticles. Synthetic methods for labelling of silica and polyorganosiloxane/silica core–shell nanoparticles with perylenediimide derivatives are described, as well as the modification of the shell with thiol groups. Photometric methods for the determination of the number of thiol groups and an estimate for the number of fluorescent molecules per nanoparticles, including a scattering correction, have been developed. Ceria nanoparticles decorated with noble metals (Pt, Pd, Rh) are models for the decomposition products of automobile catalytic converters which appear in the exhaust gases and finally interact with biological systems including humans. The control of the degree of agglomeration of small ceria nanoparticles is the basis for their synthesis. Almost monodisperse agglomerates (40 ± 4–260 ± 40 nm diameter) can be prepared and decorated with noble metal nanoparticles (2–5 nm diameter). Fluorescence labelling with ATTO 647N gave the model particles which are now under biophysical investigation. PMID:25671137

  9. Synthesis and characterization of fluorescence-labelled silica core-shell and noble metal-decorated ceria nanoparticles.

    PubMed

    Herrmann, Rudolf; Rennhak, Markus; Reller, Armin

    2014-01-01

    The present review article covers work done in the cluster NPBIOMEM in the DFG priority programme SPP 1313 and focuses on synthesis and characterization of fluorescent silica and ceria nanoparticles. Synthetic methods for labelling of silica and polyorganosiloxane/silica core-shell nanoparticles with perylenediimide derivatives are described, as well as the modification of the shell with thiol groups. Photometric methods for the determination of the number of thiol groups and an estimate for the number of fluorescent molecules per nanoparticles, including a scattering correction, have been developed. Ceria nanoparticles decorated with noble metals (Pt, Pd, Rh) are models for the decomposition products of automobile catalytic converters which appear in the exhaust gases and finally interact with biological systems including humans. The control of the degree of agglomeration of small ceria nanoparticles is the basis for their synthesis. Almost monodisperse agglomerates (40 ± 4-260 ± 40 nm diameter) can be prepared and decorated with noble metal nanoparticles (2-5 nm diameter). Fluorescence labelling with ATTO 647N gave the model particles which are now under biophysical investigation.

  10. Synthesis of enantiomerically pure [(14) C]-labelled morpholine derivatives for a class of trace amine-associate receptor 1 agonists.

    PubMed

    Edelmann, Martin R; Hartung, Thomas; Trussardi, René; Iding, Hans; Galley, Guido; Pflieger, Philippe; Norcross, Roger D

    2016-12-01

    Various agonists of the trace amine-associate receptor 1, under consideration as potential clinical development candidates, were labelled with carbon-14 for use in preclinical in vitro and in vivo drug metabolism studies. Herein, the [(14) C]-radiosynthesis of 2-phenyl-substituted morpholines 1 is described. After evaluating and optimizing different synthetic routes, 4-iodonitrobenzene 3 was selected as starting material for the 14-step synthesis. Incorporation of carbon-14 into the acetyl moiety allowed a safe and efficient synthesis of [(14) C]-labelled 4-nitroacetophenone 2 in five steps and 38% yield. Further transformation of 2 to the target compounds 1 was achieved in a 9-step synthesis. In a representative example, [(14) C]-labelled 1 was obtained in an overall yield of 11% and was isolated in >99% radiochemical purity and a specific activity of 47 mCi/mmol.

  11. Evaluation of DNA synthesis with carbon-11-labeled 4′-thiothymidine

    PubMed Central

    Toyohara, Jun

    2016-01-01

    In the cancer research field, the preferred method for evaluating the proliferative activity of cancer cells in vivo is to measure DNA synthesis rates. The cellular proliferation rate is one of the most important cancer characteristics, and represents the gold standard of pathological diagnosis. Positron emission tomography (PET) has been used to evaluate in vivo DNA synthetic activity through visualization of enhanced nucleoside metabolism. However, methods for the quantitative measurement of DNA synthesis rates have not been fully clarified. Several groups have been engaged in research on 4′-[methyl-11C]-thiothymidine (11C-4DST) in an effort to develop a PET tracer that allows quantitative measurement of in vivo DNA synthesis rates. This mini-review summarizes the results of recent studies of the in vivo measurement of cancer DNA synthesis rates using 11C-4DST. PMID:27721942

  12. A Solution to the Common Problem of the Synthesis and Applications of Hexachlorofluorescein Labeled Oligonucleotides

    PubMed Central

    Chuvilin, Andrey N.; Smirnov, Igor P.; Mosina, Alena G.; Varizhuk, Anna M.; Pozmogova, Galina E.

    2016-01-01

    A common problem of the preparation of hexachlorofluorescein labeled oligonucleotides is the transformation of the fluorophore to an arylacridine derivative under standard ammonolysis conditions. We show here that the arylacridine byproduct with distinct optical characteristics cannot be efficiently separated from the major product by HPLC or electrophoretic methods, which hampers precise physicochemical experiments with the labeled oligonucleotides. Studies of the transformation mechanism allowed us to select optimal conditions for avoiding the side reaction. The novel method for the post-synthetic deblocking of hexachlorofluorescein-labeled oligodeoxyribonucleotides described in this paper prevents the formation of the arylacridine derivative, enhances the yield of target oligomers, and allows them to be proper real-time PCR probes. PMID:27861573

  13. Synthesis and evaluation of new thiodigalactoside-based chemical probes to label galectin-3.

    PubMed

    van Scherpenzeel, Monique; Moret, Ed E; Ballell, Lluis; Liskamp, Rob M J; Nilsson, Ulf J; Leffler, Hakon; Pieters, Roland J

    2009-07-06

    New chemical probes were synthesized to label galectin-3. They are based on the high affinity thiodigalactoside ligand. The probes were synthesized with benzophenone or acetophenone moieties as the photolabel for covalent attachment to the protein. Besides labeling the protein, these aromatic photolabels also greatly enhance the affinity of the probes towards galectin-3, due to the interaction of the photolabel with two arginine guanidinium groups of the protein. The linkage between the sugar and the photolabel was varied as an ester, an amide, and a triazole. For the amide and triazole derivatives, a versatile synthetic route towards a symmetrical 3-azido-3-deoxy-thiodigalactoside was developed. The new probes were evaluated for their binding affinity of human galectin-3. They were subsequently tested for their labeling efficiency, as well as specificity in the presence of a protein mixture and a human cancer cell lysate.

  14. 13C-labeled D-ribose: chemi-enzymic synthesis of various isotopomers.

    PubMed

    Serianni, A S; Bondo, P B

    1994-04-01

    Current interest in the use of heteronuclear multidimensional NMR methods to assess the structures, conformations and/or dynamics of oligonucleotides in solution has created an immediate need for nucleosides and their derivatives labeled in various ways with stable isotopes (13C, 2H, 15N and/or 17,18O). This short review focuses exclusively on chemienzymic methods to introduce one or more 13C labels into D-ribose, a precursor to ribo- and 2'-deoxyribonucleosides. It will be demonstrated that five convenient reactions, applied in specific sequences, provide access to 26 of the 32 13C-labeled isotopomers of D-ribose in acceptable yields. While not explicitly discussed herein, these same reactions, appropriately modified, can also be used to insert one or more 2H and/or 17,18O isotopes into this aldopentose.

  15. Straightforward preparation of labeled potassium cyanate by ozonation and application to the synthesis of [13C] or [14C]ureidocarboxylic acids.

    PubMed

    Loreau, Olivier; Marlière, Philippe

    2013-06-15

    The development of new efficient syntheses of labeled reagents is a great challenge. Avoidance of overcomplicated procedures, availability and cost of starting materials are important considerations in choosing the synthetic route. In this report, we describe a facile and rapid preparation of labeled cyanate by ozonation of cyanide, a basic precursor. The crude cyanate was used without purification for the synthesis of various [(13)C] or [(14)C]ureidocarboxylic acids (20-68% yield from potassium cyanide). According to these results, cyanide ozonation may prove to be a promising alternative to traditional preparations of labeled cyanate.

  16. Expedited Synthesis of Fluorine-18 Labeled Phenols. A Missing Link in PET Radiochemistry

    SciTech Connect

    Katzenellenbogen, John A.; Zhou, Dong

    2015-03-26

    Fluorine-18 (F-18) is arguably the most valuable radionuclide for positron emission tomographic (PET) imaging. However, while there are many methods for labeling small molecules with F-18 at aliphatic positions and on electron-deficient aromatic rings, there are essentially no reliable and practical methods to label electron-rich aromatic rings such as phenols, with F-18 at high specific activity. This is disappointing because fluorine-labeled phenols are found in many drugs; there are also many interesting plant metabolites and hormones that contain either phenols or other electron-rich aromatic systems such as indoles whose metabolism, transport, and distribution would be interesting to study if they could readily be labeled with F-18. Most approaches to label phenols with F-18 involve the labeling of electron-poor precursor arenes by nucleophilic aromatic substitution, followed by subsequent conversion to phenols by oxidation or other multi-step sequences that are often inefficient and time consuming. Thus, the lack of good methods for labeling phenols and other electron-rich aromatics with F-18 at high specific activity represents a significant methodological gap in F-18 radiochemistry that can be considered a “Missing Link in PET Radiochemistry”. The objective of this research project was to develop and optimize a series of unusual synthetic transformations that will enable phenols (and other electron-rich aromatic systems) to be labeled with F-18 at high specific activity, rapidly, reliably, and conveniently, thereby bridging this gap. Through the studies conducted with support of this project, we have substantially advanced synthetic methodology for the preparation of fluorophenols. Our progress is presented in detail in the sections below, and much has been published or presented publication; other components are being prepared for publication. In essence, we have developed a completely new method to prepare o-fluorophenols from non-aromatic precursors

  17. Synthesis of amino-group functionalized superparamagnetic iron oxide nanoparticles and applications as biomedical labeling probes

    NASA Astrophysics Data System (ADS)

    Ma, Ming; Zhan, Yanqiang; Shen, Yaqi; Xia, Xing; Zhang, Suming; Liu, Zuli

    2011-08-01

    Superparamagnetic iron oxide (SPIO) nanoparticles were synthesized by coprecipitation technique and further functionalized with amino-group to obtain amino-group functionalized (amino-SPIO) nanoparticles. The X-ray diffraction results reveal the structure of amino-SPIO nanoparticles, from which the average iron core diameter is approximately 10 nm by calculation; while Zetasizer reveals their hydrodynamic diameter are mainly distributed in the range of 40-60 nm. These nanoparticles can be taken up by liver tissue, resulting in dramatically darkening of liver tissue under T2-magnetic resonance imaging (MRI). The spin-spin relaxivity coefficient of these nanoparticles is 179.20 mM-1 s-1 in a 1.5 T magnetic resonance system. In addition, amino-SPIO nanoparticles were conjugated to Tat (FITC) peptide and incubated with neural stem cells in vitro, the authors can detect the positive-labeling (labeled) neural stem cells showing green fluorescence, which indicates Tat (FITC) peptide-derivated amino-SPIO nanoparticles are able to enter cells. Furthermore, it was also find significant negative T2 contrast enhancement when compared with the non-nanoparticles-labeled neural stem cells in T2-weighted MRI. The amino-SPIO nanoparticles show promising potential as a new type of labeling probes, which can be used in magnetic resonance-enhanced imaging and fluorescence diagnosis.

  18. Synthesis of deuterium-labelled halogen derivatives of L-tryptophan catalysed by tryptophanase.

    PubMed

    Winnicka, Elżbieta; Szymańska, Jolanta; Kańska, Marianna

    2016-06-01

    The isotopomers of halogen derivatives of l-tryptophan (l-Trp) (4'-F-, 7'-F-, 5'-Cl- and 7'-Br-l-Trp), specifically labelled with deuterium in α-position of the side chain, were obtained by enzymatic coupling of the corresponding halogenated derivatives of indole with S-methyl-l-cysteine in (2)H2O, catalysed by enzyme tryptophanase (EC 4.1.99.1). The positional deuterium enrichment of the resulting tryptophan derivatives was controlled using (1)H NMR. In accordance with the mechanism of the lyase reaction, a 100% deuterium labelling was observed in the α-position; the chemical yields were between 23 and 51%. Furthermore, β-F-l-alanine, synthesized from β-F-pyruvic acid by the l-alanine dehydrogenase reaction, has been tested as a coupling agent to obtain the halogenated deuterium-labelled derivatives of l-Trp. The chemical yield (∼30%) corresponded to that as observed with S-methyl-l-cysteine but the deuterium label was only 63%, probably due to the use of a not completely deuterated incubation medium.

  19. Synthesis and biodistribution of radioarsenic labeled dimethylarsinothiols: derivatives of penicillamine and mercaptoethanol.

    PubMed

    Emran, A; Hosain, F; Spencer, R P; Kolstad, K S

    1984-01-01

    Arsenic analogs of sulfhydryl containing biomolecules can be derived from dimethylchloroarsine as a precursor. Arsenic-76 labeled dimethylarsinothiols (dimethylarsinopenicillamine and dimethylarsinomercaptoethanol) were synthesized, purified by chromatography, and their biodistributions obtained in mice. The present study demonstrates the possibility of developing a group of radioarsenicals from SH-containing biomolecules.

  20. Synthesis of isotopically labelled 2-isopropylthioxanthone from 2,2'-dithiosalicylic acid and deuterium cumene.

    PubMed

    Fang, Chao; Yang, Weicheng; Yang, Chao; Wang, Haoran; Sun, Kai; Luo, Yong

    2016-06-30

    Two efficient synthetic routes of stable deuterium labelled 2-isopropylthioxanthone were presented with 98.1% and 98.8% isotopic abundance in acceptable yields and excellent chemical purities. Their structures and the isotope-abundance were confirmed according to proton nuclear magnetic resonance and liquid chromatography-mass spectrometry.

  1. Synthesis of a Fluorescently Labeled (68)Ga-DOTA-TOC Analog for Somatostatin Receptor Targeting.

    PubMed

    Ghosh, Sukhen C; Hernandez Vargas, Servando; Rodriguez, Melissa; Kossatz, Susanne; Voss, Julie; Carmon, Kendra S; Reiner, Thomas; Schonbrunn, Agnes; Azhdarinia, Ali

    2017-07-13

    Fluorescently labeled imaging agents can identify surgical margins in real-time to help achieve complete resections and minimize the likelihood of local recurrence. However, photon attenuation limits fluorescence-based imaging to superficial lesions or lesions that are a few millimeters beneath the tissue surface. Contrast agents that are dual-labeled with a radionuclide and fluorescent dye can overcome this limitation and combine quantitative, whole-body nuclear imaging with intraoperative fluorescence imaging. Using a multimodality chelation (MMC) scaffold, IRDye 800CW was conjugated to the clinically used somatostatin analog, (68)Ga-DOTA-TOC, to produce the dual-labeled analog, (68)Ga-MMC(IRDye 800CW)-TOC, with high yield and specific activity. In vitro pharmacological assays demonstrated retention of receptor-targeting properties for the dual-labeled compound with robust internalization that was somatostatin receptor (SSTR) 2-mediated. Biodistribution studies in mice identified the kidneys as the primary excretion route for (68)Ga-MMC(IRDye 800CW)-TOC, along with clearance via the reticuloendothelial system. Higher uptake was observed in most tissues compared to (68)Ga-DOTA-TOC but decreased as a function of time. The combination of excellent specificity for SSTR2-expressing cells and suitable biodistribution indicate potential application of (68)Ga-MMC(IRDye 800CW)-TOC for intraoperative detection of SSTR2-expressing tumors.

  2. Synthesis of azidotubulin: a photoaffinity label for tubulin-binding proteins.

    PubMed

    Balczon, R D; Brinkley, B R

    1989-10-17

    A photoaffinity label for the identification of tubulin-binding proteins was synthesized from phosphocellulose-purified bovine brain tubulin and (N-hydroxysuccinimidyl)-4-azidosalicylic acid. The azidotubulin derivative retained the ability to undergo temperature-dependent microtubule assembly and disassembly. When incubated with purified tau protein, the azidotubulin and tau formed cross-linked complexes upon photoactivation. When 125I-labeled azidotubulin was used to photoaffinity label tubulin-binding proteins within the kinetochore of isolated mammalian chromosomes, a 130-kDa band was identified on autoradiographs of SDS-polyacrylamide gels of the 125I-labeled azidotubulin/chromosome preparations. The 130-kDa complex was isolated by antitubulin affinity chromatography and analyzed by immunoblotting using both antitubulin and kinetochore-specific sera obtained from human patients with the autoimmune disease scleroderma CREST. The immunoblots demonstrated that the 130-kDa band that was observed on autoradiographs was a complex of a subunit of the tubulin dimer and an 80-kDa CREST-specific kinetochore protein. The binding of azidotubulin to the 80-kDa kinetochore protein was significantly decreased when chromosomes were treated with a mixture of 9 parts underivatized tubulin to 1 part azidotubulin prior to photolysis. The formation of the 130-kDa azidotubulin/kinetochore protein complex was not inhibited by pretreating the chromosomes with CREST serum prior to incubation with azidotubulin. Azidotubulin should be a useful probe for the identification and characterization of tubulin-binding proteins.

  3. Controlled synthesis of conjugated microporous polymer films: versatile platforms for highly sensitive and label-free chemo- and biosensing.

    PubMed

    Gu, Cheng; Huang, Ning; Gao, Jia; Xu, Fei; Xu, Yanhong; Jiang, Donglin

    2014-05-05

    Conjugated microporous polymers (CMPs), in which rigid building blocks form robust networks, are usually synthesized as insoluble and unprocessable powders. We developed a methodology using electropolymerization for the synthesis of thin CMP films. The thickness of these films is synthetically controllable, ranging from nanometers to micrometers, and they are obtained on substrates or as freestanding films. The CMP films combine a number of striking physical properties, including high porosity, extended π conjugation, facilitated exciton delocalization, and high-rate electron transfer. We explored the CMP films as versatile platforms for highly sensitive and label-free chemo- and biosensing of electron-rich and electron-poor arenes, metal ions, dopamine, and hypochloroic acid, featuring rapid response, excellent selectivity, and robust reusability.

  4. Vanadyl complexes with dansyl-labelled di-picolinic acid ligands: synthesis, phosphatase inhibition activity and cellular uptake studies.

    PubMed

    Collins, Juliet; Cilibrizzi, Agostino; Fedorova, Marina; Whyte, Gillian; Mak, Lok Hang; Guterman, Inna; Leatherbarrow, Robin; Woscholski, Rudiger; Vilar, Ramon

    2016-04-28

    Vanadium complexes have been previously utilised as potent inhibitors of cysteine based phosphatases (CBPs). Herein, we present the synthesis and characterisation of two new fluorescently labelled vanadyl complexes (14 and 15) with bridged di-picolinic acid ligands. These compounds differ significantly from previous vanadyl complexes with phosphatase inhibition properties in that the metal-chelating part is a single tetradentate unit, which should afford greater stability and scope for synthetic elaboration than the earlier complexes. These new complexes inhibit a selection of cysteine based phosphatases (CBPs) in the nM range with some selectivity. Fluorescence spectroscopic studies (including fluorescence anisotropy) were carried out to demonstrate that the complexes are not simply acting as vanadyl delivery vehicles but they interact with the proteins. Finally, we present preliminary fluorescence microscopy studies to demonstrate that the complexes are cell permeable and localise throughout the cytoplasm of NIH3T3 cells.

  5. Metabolism of Nonessential N15-Labeled Amino Acids and the Measurement of Human Whole-Body Protein Synthesis Rates

    NASA Technical Reports Server (NTRS)

    Stein, T. P.; Settle, R. G.; Albina, J. A.; Dempsey, D. T.; Melnick, G.

    1991-01-01

    Eight N-15 labeled nonessential amino acids plus (15)NH4Cl were administered over a 10 h period to four healthy adult males using a primed-constant dosage regimen. The amount of N-15 excreted in the urine and the urinary ammonia, hippuric acid, and plasma alanine N-15 enrichments were measured. There was a high degree of consistency across subjects in the ordering of the nine compounds based on the fraction of N-15 excreted (Kendall coefficient of concordance W = 0.83, P is less than 0.01). Protein synthesis rates were calculated from the urinary ammonia plateau enrichment and the cumulative excretion of N-15. Glycine was one of the few amino acids that gave similar values by both methods.

  6. Synthesis and incorporation of 13C-labeled DNA building blocks to probe structural dynamics of DNA by NMR

    PubMed Central

    Nußbaumer, Felix; Juen, Michael Andreas; Gasser, Catherina; Kremser, Johannes; Müller, Thomas; Tollinger, Martin

    2017-01-01

    Abstract We report the synthesis of atom-specifically 13C-modified building blocks that can be incorporated into DNA via solid phase synthesis to facilitate investigations on structural and dynamic features via NMR spectroscopy. In detail, 6-13C-modified pyrimidine and 8-13C purine DNA phosphoramidites were synthesized and incorporated into a polypurine tract DNA/RNA hybrid duplex to showcase the facile resonance assignment using site-specific labeling. We also addressed micro- to millisecond dynamics in the mini-cTAR DNA. This DNA is involved in the HIV replication cycle and our data points toward an exchange process in the lower stem of the hairpin that is up-regulated in the presence of the HIV-1 nucleocapsid protein 7. As another example, we picked a G-quadruplex that was earlier shown to exist in two folds. Using site-specific 8-13C-2′deoxyguanosine labeling we were able to verify the slow exchange between the two forms on the chemical shift time scale. In a real-time NMR experiment the re-equilibration of the fold distribution after a T-jump could be monitored yielding a rate of 0.012 min−1. Finally, we used 13C-ZZ-exchange spectroscopy to characterize the kinetics between two stacked X-conformers of a Holliday junction mimic. At 25°C, the refolding process was found to occur at a forward rate constant of 3.1 s−1 and with a backward rate constant of 10.6 s−1.

  7. Design and synthesis of novel spin-labeled camptothecin derivatives as potent cytotoxic agents.

    PubMed

    Zhao, Xiao-Bo; Wu, Dan; Wang, Mei-Juan; Goto, Masuo; Morris-Natschke, Susan L; Liu, Ying-Qian; Wu, Xiao-Bing; Song, Zi-Long; Zhu, Gao-Xiang; Lee, Kuo-Hsiung

    2014-11-15

    In our continuing search for natural product-based spin-labeled antitumor drugs, 20 novel spin-labeled camptothecin derivatives were synthesized via a Cu-catalyzed one pot reaction and evaluated for cytotoxicity against four human tumor cell lines (A-549, MDA-MB-231, KB, and KBvin). Eighteen of the target compounds (9a, 9b, 9d-9k, 9m-9t) exhibited significant in vitro antiproliferative activity against these four tested tumor cell lines. Compounds 9e and 9j (IC50 0.057 and 0.072μM, respectively) displayed the greatest cytotoxicity against the multidrug-resistant (MDR) KBvin cell line and merit further development into preclinical and clinical drug candidates for treating cancer including MDR phenotype.

  8. Bovine serum albumin-directed synthesis of biocompatible CdSe quantum dots and bacteria labeling.

    PubMed

    Wang, Qisui; Ye, Fangyun; Fang, Tingting; Niu, Wenhan; Liu, Peng; Min, Xinmin; Li, Xi

    2011-03-01

    A simple method was developed for preparing CdSe quantum dots (QDs) using a common protein (bovine serum albumin (BSA)) to sequester QD precursors (Cd(2+)) in situ. Fluorescence (FL) and absorption spectra showed that the chelating time between BSA and Cd(2+), the molar ratio of BSA/Cd(2+), temperature, and pH are the crucial factors for the quality of QDs. The average QD particle size was estimated to be about 5 nm, determined by high-resolution transmission electron microscopy. With FL spectra, Fourier transform infrared spectra, and thermogravimetric analysis, an interesting mechanism was discussed for the formation of the BSA-CdSe QDs. The results indicate that there might be conjugated bonds between CdSe QDs and -OH, -NH, and -SH groups in BSA. In addition, fluorescence imaging suggests that the QDs we designed can successfully label Escherichia coli cells, which gives us a great opportunity to develop biocompatible tools to label bacteria cells.

  9. Pre-malbrancheamide: Synthesis, Isotopic Labeling, Biosynthetic Incorporation, and Detection in Cultures of Malbranchea aurantiaca

    PubMed Central

    Ding, Yousong; Greshock, Thomas J.; Miller, Kenneth A.

    2009-01-01

    An advanced metabolite, named pre-malbrancheamide, involved in the biosynthesis of malbrancheamide (1) and malbrancheamide B (2) has been synthesized in double 13C-labeled form and was incorporated into the indole alkaloid 2 by Malbranchea aurantiaca. In addition, pre-malbrancheamide has been detected as a natural metabolite in cultures of M. aurantiaca. The biosynthetic implications of these experiments are discussed. PMID:18844365

  10. Cryptophycin affinity labels: synthesis and biological activity of a benzophenone analogue of cryptophycin-24.

    PubMed

    Vidya, Ramdas; Eggen, MariJean; Georg, Gunda I; Himes, Richard H

    2003-02-24

    An efficient synthesis of a C16 side chain benzophenone analogue of cryptophycin-24 using a crotylboration reaction and Heck coupling as key steps is described. In an in vitro tubulin assembly assay, the benzophenone analogue of the beta isomer (IC(50)=7.4 microM) is twice as active as cryptophycin-24 (IC(50)=15 microM).

  11. Facile synthesis of (55)Fe-labeled well-dispersible hematite nanoparticles for bioaccumulation studies in nanotoxicology.

    PubMed

    Huang, Bin; Xiao, Lin; Yang, Liu-Yan; Ji, Rong; Miao, Ai-Jun

    2016-06-01

    Although water-dispersible engineered nanoparticles (ENPs) have a wide range of applications, the ENPs used in many nanotoxicological studies tend to form micron-sized aggregates in the exposure media and thus cannot reflect the toxicity of real nanoparticles. Here we described the synthesis of bare hematite nanoparticles (HNPs-0) and two poly(acrylic acid) (PAA)-coated forms (HNPs-1 and HNPs-2). All three HNPs were well dispersed in deionized water, but HNPs-0 quickly aggregated in the three culture media tested. By contrast, the suspensions of HNPs-1 and HNPs-2 remained stable, with negligible amounts of PAA and Fe(3+) liberated from either one under the investigated conditions. To better quantify the accumulation of the coated HNPs, a relatively innocuous (55)Fe-labeled form of HNPs-2 was synthesized as an example and its accumulation in three phytoplankton species was tested. Consistent with the uptake kinetics model for conventional pollutants, the cellular accumulation of HNPs-2 increased linearly with exposure time for two of the three phytoplankton species. These results demonstrate the utility of (55)Fe-labeled well-dispersible HNPs as a model material for nanoparticle bioaccumulation studies in nanotoxicology. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. One-Pot Synthesis of Biocompatible CdSe/CdS Quantum Dots and Their Applications as Fluorescent Biological Labels.

    PubMed

    Zhai, Chuanxin; Zhang, Hui; Du, Ning; Chen, Bingdi; Huang, Hai; Wu, Yulian; Yang, Deren

    2011-12-01

    We developed a novel one-pot polyol approach for the synthesis of biocompatible CdSe quantum dots (QDs) using poly(acrylic acid) (PAA) as a capping ligand at 240°C. The morphological and structural characterization confirmed the formation of biocompatible and monodisperse CdSe QDs with several nanometers in size. The encapsulation of CdS thin layers on the surface of CdSe QDs (CdSe/CdS core-shell QDs) was used for passivating the defect emission (650 nm) and enhancing the fluorescent quantum yields up to 30% of band-to-band emission (530-600 nm). Moreover, the PL emission peak of CdSe/CdS core-shell QDs could be tuned from 530 to 600 nm by the size of CdSe core. The as-prepared CdSe/CdS core-shell QDs with small size, well water solubility, good monodispersity, and bright PL emission showed high performance as fluorescent cell labels in vitro. The viability of QDs-labeled 293T cells was evaluated using a 3-(4,5-dimethylthiazol)-2-diphenyltertrazolium bromide (MTT) assay. The results showed the satisfactory (>80%) biocompatibility of as-synthesized PAA-capped QDs at the Cd concentration of 15 μg/ml.

  13. A chemical synthesis of a multiply (13) C-labeled hexasaccharide: a high-mannose N-glycan fragment.

    PubMed

    Zhang, Wenhui; Pan, Qingfeng; Serianni, Anthony S

    2016-12-01

    As covalent modifiers of proteins, high-mannose N-glycans are important in maintaining protein structure and function in vivo. The conformations of these glycans can be studied by nuclear magnetic resonance spectroscopy using spin-spin couplings (J-couplings; scalar couplings) and other nuclear magnetic resonance parameters that are sensitive to the geometries of their constituent glycosidic linkages and other mobile elements in their structures. These analyses often require (13) C-labeling at specific carbon atoms, especially when measurements of (13) C-(13) C J-couplings are of interest. The selection of particular (13) C isotopomers of a glycan depends on the type of question under scrutiny. A chemical synthesis of a mannose-containing hexasaccharide, α[1-(13) C]Man(1→2)α[1,2-(13) C2 ]Man(1→6)[α[1-(13) C]Man(1→2)α[1,2-(13) C2 ]Man(1→3)]α[1,2-(13) C2 ]Man(1→6)βManOCH3 , which is a nested fragment of the high-mannose N-glycans of human glycoproteins and contains eight (13) C-enriched carbon sites, is described in this report. The selected (13) C isotopomer was chosen to maximize the measurement of J-couplings sensitive to linkage conformations. This work demonstrates that chemical syntheses of multiply (13) C-labeled oligosaccharides are technically feasible and practical using present synthetic methods. The availability of this and other multiply (13) C-labeled mannose-containing oligosaccharides will promote future studies of their conformations in solution and in the bound state. Copyright © 2016 John Wiley & Sons, Ltd.

  14. Synthesis of BODIPY-Labeled Cholesterylated Glycopeptides by Tandem Click Chemistry for Glycocalyxification of Giant Unilamellar Vesicles (GUVs).

    PubMed

    Stuhr-Hansen, Nicolai; Vagianou, Charikleia-Despoina; Blixt, Ola

    2017-07-18

    The glycocalyx cover membrane surfaces of all living cells. These complex architectures render their interaction mechanisms on the membrane surface difficult to study. Artificial cell-sized membranes with selected and defined glycosylation patterns may serve as a minimalistic approach to systematically study cell surface glycan interactions. The development of a facile general synthetic procedure for the synthesis of BODIPY-labeled cholesterylated glycopeptides, which can coat cell-size giant unilamellar vesicles (GUVs), is described. These peptide constructs were synthesized by: 1) solid-phase peptide synthesis (SPPS) using cholesterylated Fmoc-amino acids (Fmoc=9-fluorenylmethoxycarbonyl) followed by tandem click reactions, 2) attachment of a BODIPY-bicyclononyne (BCN) (prepared by Mitsunobu chemistry via novel aryl BCN-ethers) in the absence of a catalyst, and 3) glycosylation by means of copper(I)-catalyzed click reaction of an azidoglycan. Seven different GUV-glycoforms were prepared and four of these were evaluated with their corresponding four specific anti-glycan binding lectins. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Enzyme-Activated G-Quadruplex Synthesis for in Situ Label-Free Detection and Bioimaging of Cell Apoptosis.

    PubMed

    Liu, Zhuoliang; Luo, Xingyu; Li, Zhu; Huang, Yan; Nie, Zhou; Wang, Hong-Hui; Yao, Shouzhuo

    2017-02-07

    Fluorogenic probes targeting G-quadruplex structures have emerged as the promising toolkit for functional research of G-quadruplex and biosensor development. However, their biosensing applications are still largely limited in in-tube detection. Herein, we proposed a fluorescent bioimaging method based on enzyme-generated G-quadruplexes for detecting apoptotic cells at the cell and tissue level, namely, terminal deoxynucleotidyl transferase (TdT)-activated de novo G-quadruplex synthesis (TAGS) assay. The detection target is genomic DNA fragmentation, a biochemical hallmark of apoptosis. The TAGS assay can efficiently "tag" DNA fragments via using their DNA double-strand breaks (DSBs) to initiate the de novo synthesis of G-quadruplexes by TdT with an unmodified G-rich dNTP pool, followed by a rapid fluorescent readout upon the binding of thioflavin T (ThT), a fluorogenic dye highly specific for G-quadruplex. The feasibility of the TAGS assay was proved by in situ sensitive detection of individual apoptotic cells in both cultured cells and tissue sections. The TAGS assay has notable advantages, including being label-free and having quick detection, high sensitivity and contrast, mix-and-read operation without tedious washing, and low cost. This method not only shows the feasibility of G-quadruplex in tissue bioanalysis but also provides a promising tool for basic research of apoptosis and drug evaluation for antitumor therapy.

  16. Abstracts of the 24th international isotope society (UK group) symposium: synthesis and applications of labelled compounds 2015.

    PubMed

    Aigbirhio, F I; Allwein, S; Anwar, A; Atzrodt, J; Audisio, D; Badman, G; Bakale, R; Berthon, F; Bragg, R; Brindle, K M; Bushby, N; Campos, S; Cant, A A; Chan, M Y T; Colbon, P; Cornelissen, B; Czarny, B; Derdau, V; Dive, V; Dunscombe, M; Eggleston, I; Ellis-Sawyer, K; Elmore, C S; Engstrom, P; Ericsson, C; Fairlamb, I J S; Georgin, D; Godfrey, S P; He, L; Hickey, M J; Huscroft, I T; Kerr, W J; Lashford, A; Lenz, E; Lewinton, S; L'Hermite, M M; Lindelöf, Å; Little, G; Lockley, W J S; Loreau, O; Maddocks, S; Marguerit, M; Mirabello, V; Mudd, R J; Nilsson, G N; Owens, P K; Pascu, S I; Patriarche, G; Pimlott, S L; Pinault, M; Plastow, G; Racys, D T; Reif, J; Rossi, J; Ruan, J; Sarpaki, S; Sephton, S M; Simonsson, R; Speed, D J; Sumal, K; Sutherland, A; Taran, F; Thuleau, A; Wang, Y; Waring, M; Watters, W H; Wu, J; Xiao, J

    2016-04-01

    The 24th annual symposium of the International Isotope Society's United Kingdom Group took place at the Møller Centre, Churchill College, Cambridge, UK on Friday 6th November 2015. The meeting was attended by 77 delegates from academia and industry, the life sciences, chemical, radiochemical and scientific instrument suppliers. Delegates were welcomed by Dr Ken Lawrie (GlaxoSmithKline, UK, chair of the IIS UK group). The subsequent scientific programme consisted of oral presentations, short 'flash' presentations in association with particular posters and poster presentations. The scientific areas covered included isotopic synthesis, regulatory issues, applications of labelled compounds in imaging, isotopic separation and novel chemistry with potential implications for isotopic synthesis. Both short-lived and long-lived isotopes were represented, as were stable isotopes. The symposium was divided into a morning session chaired by Dr Rebekka Hueting (University of Oxford, UK) and afternoon sessions chaired by Dr Sofia Pascu (University of Bath, UK) and by Dr Alan Dowling (Syngenta, UK). The UK meeting concluded with remarks from Dr Ken Lawrie (GlaxoSmithKline, UK). Copyright © 2016 John Wiley & Sons, Ltd.

  17. Synthesis of 6-(F-18)L-fluoro-dopa using F-18 labelled acetyl hypofluorite

    SciTech Connect

    Adam, M.J.; Abeysekera, B.; Ruth, T.J.; Grierson, J.R.; Pate, B.D.

    1985-05-01

    The synthesis of (F-18)6-fluoro-dopa via acetyl hypofluorite has recently been reported. The authors have modified this procedure by adding an acetate protecting group on the dopa ring and have treated this new starting material with either solution or gas phase F-18 acetyl hypofluorite. Using this starting material the yield has been significantly increased over the published method. The authors routinely prepare 4-5 mCi of pure (F-18)6-fluoro-dopa (3-4% radiochemical yield, at EOS) in an overall synthesis time of 2 hours. Both 2 and 6 fluoro-dopa are produced in nearly equivalent amounts by this method as determined by /sup 19/F nmr. These are easily separated by HPLC after deblocking with HI. The final isolated product is >99% in the L-isomer form and fluorinated in >97% in the 6 position.

  18. Fluoride-Cleavable, Fluorescently Labelled Reversible Terminators: Synthesis and Use in Primer Extension

    PubMed Central

    Knapp, Diana C; Serva, Saulius; D'Onofrio, Jennifer; Keller, Angelika; Lubys, Arvydas; Kurg, Ants; Remm, Maido; Engels, Joachim W

    2011-01-01

    Fluorescent 2′-deoxynucleotides containing a protecting group at the 3′-O-position are reversible terminators that enable array-based DNA sequencing-by-synthesis (SBS) approaches. Herein, we describe the synthesis and full characterisation of four reversible terminators bearing a 3′-blocking moiety and a linker-dye system that is removable under the same fluoride-based treatment. Each nucleotide analogue has a different fluorophore attached to the base through a fluoride-cleavable linker and a 2-cyanoethyl moiety as the 3′-blocking group, which can be removed by using a fluoride treatment as well. Furthermore, we identified a DNA polymerase, namely, RevertAid M-MuLV reverse transcriptase, which can incorporate the four modified reversible terminators. The synthesised nucleotides and the optimised DNA polymerase were used on CodeLink slides spotted with hairpin oligonucleotides to demonstrate their potential in a cyclic reversible terminating approach. PMID:21294195

  19. Synthesis and characterization of photoaffinity labelling reagents towards the Hsp90 C-terminal domain.

    PubMed

    Simon, Binto; Huang, Xuexia; Ju, Huangxian; Sun, Guoxuan; Yang, Min

    2017-02-21

    Glucosyl-novobiocin-based diazirine photoaffinity labelling reagents (PALs) were designed and synthesized to probe the Hsp90 C-terminal domain unknown binding pocket and the structure-activity relationship. Five PALs were successfully synthesized from novobiocin in six consecutive steps employing phase transfer catalytic glycosylation. Reactions were monitored and guided by analytical LC/MS which led to different strategies of adding either a PAL precursor or a sugar moiety first. The structures and bonding linkages of these compounds were characterised by various 2D-NMR spectroscopy and MS techniques. Synthetic techniques provide powerful probes for unknown protein binding pockets.

  20. Design, synthesis and 64Cu labeling of fatty acid analogs containing dithiosemicarbazone chelate.

    PubMed

    Arano, Y; Magata, Y; Horiuchi, K; Matsumoto, K; Fujibayashi, Y; Ohmomo, Y; Tanaka, C; Saji, H; Yokoyama, A

    1989-01-01

    For the development of 62Cu labeled fatty acid analogs, two fatty acid analogs, containing dithiosemicarbazone (DTS) molecule as the 62Cu coordinating site, were designed and synthesized: a fatty acid analog containing DTS molecule at the omega-position, (a) the 12,13-dioxotetradecanoic acid di(N-methyl-thiosemicarbazone) (FA-DTS), and an omega-phenyl fatty acid analog containing DTS molecule at the para-position, (b) the p-carboxyundecylphenylglyoxal-di (N-methylthiosemicarbazone] (PFA-DTS). FA-DTS was synthesized by the reaction of ethyl diethoxyacetate with ethyl 11-bromonundecanate by successive decarboxylation and hydrolysis and final condensation with N-methylthiosemicarbazide. PFA-DTS was synthesized by the Friedel-Craft acylation of ethyl 11-phenylundecanate, selenium oxidation of the acetophenone derivative, followed by the condensation with N-methylthiosemicarbazide. Radiolabeling of FA-DTS and PFA-DTS with [64Cu]copper acetate was simple, rapid and quantitative. When injected into mice, both compounds were distributed and retained in the myocardium. These results offer a good basis for further development of 62Cu labeled fatty acid analogs.

  1. Synthesis, characterization, and protein labeling of difunctional magnetic nanoparticles modified with thiazole orange dye

    NASA Astrophysics Data System (ADS)

    Fei, Xuening; Zhu, Huifang; Zhou, Jianguo; Yu, Lu

    2014-03-01

    A dual functional nanoparticle was designed and synthesized by encapsulating magnetic core inside silica particles and subsequently a thiazole orange (TO) dye derivative was modified on the surface of the nanoparticles. The obtained particles were characterized by Fourier transform infrared spectroscope, Uv-Vis spectrophotometer, fluorescence spectrophotometer, transmission electron microscope, dynamic light scattering, etc. The size of preliminary magnetic particles is ca. 7 nm, but after coating a silica layer and dye, the size of particles is increased to ca. 60 nm. The hydrodynamic diameter, water dispersibility, and zeta potential were also determined. The hydrodynamic diameter of particles with silica and dye is 65.2 and 70.5 nm, respectively, with positive zeta potential (25.1, 38.5 mV). Furthermore magnetic properties of the particles were measured and the experimental results suggested that it could meet the requirement of application as magnetic resonance imaging agent. Finally to verify the availability of the particles as fluorescent labeling, protein labeling experiment was performed using bovine serum albumin (BSA) protein and the results showed that the dual functional particle has higher affinity with BSA than TO molecule itself.

  2. Synthesis of selectively labeled histidine and its methylderivatives with deuterium, tritium, and carbon-14.

    PubMed

    Šamonina-Kosicka, J; Kańska, M

    2013-05-30

    Isotopologues of l-histidine and its N-methylderivatives labeled with deuterium and tritium at the 5-position in the imidazole ring were obtained using the isotope exchange method. The deuterium-labeled isotopologues [5-(2)H]-l-histidine, [5-(2)H]-N(τ) -methyl-l-histidine, [5-(2)H]-N(π) -methyl-l-histidine, and [2,5-(2)H(2)]-l-histidine were synthesized by isotope exchange method carried out in a fully deuterated medium with. The same reaction conditions were applied to synthesize [5-(3)H]-N(τ) -methyl-l-histidine, [5-(3)H]-N(π) -methyl-l-histidine, and [5-(3)H]-l-histidine with specific activity of 2.0, 5.0, and 2.6 MBq/mmol, respectively. The N(π) -[methyl-(14)C]-histamine was obtained with specific activity of 0.23 MBq/mmol in a one-step reaction by the direct methylation of histamine by [(14)C]iodomethane. Copyright © 2013 John Wiley & Sons, Ltd.

  3. Synthesis, characterization and theranostic evaluation of Indium-111 labeled multifunctional superparamagnetic iron oxide nanoparticles.

    PubMed

    Zolata, Hamidreza; Abbasi Davani, Fereydoun; Afarideh, Hossein

    2015-02-01

    Indium-111 labeled, Trastuzumab-Doxorubicin Conjugated, and APTES-PEG coated magnetic nanoparticles were designed for tumor targeting, drug delivery, controlled drug release, and dual-modal tumor imaging. Superparamagnetic iron oxide nanoparticles (SPIONs) were synthesized by thermal decomposition method to obtain narrow size particles. To increase SPIONs circulation time in blood and decrease its cytotoxicity in healthy tissues, SPIONs surface was modified with 3-Aminopropyltriethoxy Silane (APTES) and then were functionalized with N-Hydroxysuccinimide (NHS) ester of Polyethylene Glycol Maleimide (NHS-PEG-Mal) to conjugate with thiolated 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3,6,9,-triacetic acid (PCTA) bifunctional chelator (BFC) and Trastuzumab antibody. In order to tumor SPECT/MR imaging, SPIONs were labeled with Indium-111 (T1/2=2.80d). NHS ester of monoethyl malonate (MEM-NHS) was used for conjugation of Doxorubicin (DOX) chemotherapeutic agent onto SPIONs surface. Mono-Ethyl Malonate allows DOX molecules to be attached to SPIONs via pH-sensitive hydrazone bonds which lead to controlled drug release in tumor region. Active and passive tumor targeting were achieved through incorporated anti-HER2 (Trastuzumab) antibody and EPR effect of solid tumors for nanoparticles respectively. In addition to in vitro assessments of modified SPIONs in SKBR3 cell lines, their theranostic effects were evaluated in HER2 + breast tumor bearing BALB/c mice via biodistribution study, dual-modal molecular imaging and tumor diameter measurements.

  4. Quantifying protein synthesis and degradation in Arabidopsis by dynamic 13CO2 labeling and analysis of enrichment in individual amino acids in their free pools and in protein.

    PubMed

    Ishihara, Hirofumi; Obata, Toshihiro; Sulpice, Ronan; Fernie, Alisdair R; Stitt, Mark

    2015-05-01

    Protein synthesis and degradation represent substantial costs during plant growth. To obtain a quantitative measure of the rate of protein synthesis and degradation, we supplied (13)CO2 to intact Arabidopsis (Arabidopsis thaliana) Columbia-0 plants and analyzed enrichment in free amino acids and in amino acid residues in protein during a 24-h pulse and 4-d chase. While many free amino acids labeled slowly and incompletely, alanine showed a rapid rise in enrichment in the pulse and a decrease in the chase. Enrichment in free alanine was used to correct enrichment in alanine residues in protein and calculate the rate of protein synthesis. The latter was compared with the relative growth rate to estimate the rate of protein degradation. The relative growth rate was estimated from sequential determination of fresh weight, sequential images of rosette area, and labeling of glucose in the cell wall. In an 8-h photoperiod, protein synthesis and cell wall synthesis were 3-fold faster in the day than at night, protein degradation was slow (3%-4% d(-1)), and flux to growth and degradation resulted in a protein half-life of 3.5 d. In the starchless phosphoglucomutase mutant at night, protein synthesis was further decreased and protein degradation increased, while cell wall synthesis was totally inhibited, quantitatively accounting for the inhibition of growth in this mutant. We also investigated the rates of protein synthesis and degradation during leaf development, during growth at high temperature, and compared synthesis rates of Rubisco large and small subunits of in the light and dark. © 2015 American Society of Plant Biologists. All Rights Reserved.

  5. Microwave-assisted synthesis of deuterium labeled estrogen fatty acid esters.

    PubMed

    Kiuru, Paula S; Wähälä, Kristiina

    2006-01-01

    Deuterated analogs of estrogen fatty acid esters are needed as internal standards for isotope dilution GC/MS analyses. We have developed a rapid and efficient synthesis for 2,4,16,16-D4-estrone palmitate, stearate, oleate, linoleate, and linolenate and the corresponding 2,4,16,16,17alpha-D5-estradiol fatty acid 17-mono and 3,17-diesters using analogous fatty acid chlorides or fatty acid anhydrides and 4-(dimethylamino)pyridine under microwave irradiation. Chemoselective hydrolysis of fatty acid diesters was carried out by KOH in t-BuOH.

  6. Design, synthesis and cytotoxic activity of novel spin-labeled rotenone derivatives.

    PubMed

    Liu, Ying-Qian; Ohkoshi, Emika; Li, Lin-Hai; Yang, Liu; Lee, Kuo-Hsiung

    2012-01-15

    Three series of novel spin-labeled rotenone derivatives were synthesized and evaluated for cytotoxicity against four tumor cell lines, A-549, DU-145, KB and KBvin. All of the derivatives showed promising in vitro cytotoxic activity against the tumor cell lines tested, with IC(50) values ranging from 0.075 to 0.738μg/mL. Remarkably, all of the compounds were more potent than paclitaxel against KBvin in vitro, and compounds 3a and 3d displayed the highest cytotoxicity against this cell line (IC(50) 0.075 and 0.092μg/mL, respectively). Based on the observed cytotoxicity, structure-activity relationships have been described.

  7. Hydroxylated Fluorescent Dyes for Live-Cell Labeling: Synthesis, Spectra and Super-Resolution STED.

    PubMed

    Butkevich, Alexey N; Belov, Vladimir N; Kolmakov, Kirill; Sokolov, Viktor V; Shojaei, Heydar; Sidenstein, Sven C; Kamin, Dirk; Matthias, Jessica; Vlijm, Rifka; Engelhardt, Johann; Hell, Stefan W

    2017-09-07

    Hydroxylated rhodamines, carbopyronines, silico- and germanorhodamines with absorption maxima in the range of 530-640 nm were prepared and applied in specific labeling of living cells. The direct and high-yielding entry to germa- and silaxanthones tolerates the presence of protected heteroatoms and may be considered for the syntheses of various sila- and germafluoresceins, as well as -rhodols. Application in stimulated emission depletion (STED) fluorescence microscopy revealed a resolution of 50-75 nm in one- and two-color imaging of vimentin-HaloTag fused protein and native tubulin. The established structure-property relationships allow for prediction of the spectral properties and the positions of spirolactone/zwitterion equilibria for the new analogues of rhodamines, carbo-, silico-, and germanorhodamines using simple additive schemes. © 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

  8. Synthesis, characterization, and biodistribution of multiple 89Zr-labeled pore-expanded mesoporous silica nanoparticles for PET

    NASA Astrophysics Data System (ADS)

    Miller, Larissa; Winter, Gordon; Baur, Benjamin; Witulla, Barbara; Solbach, Christoph; Reske, Sven; Lindén, Mika

    2014-04-01

    Functional nanoparticles are highly interesting imaging agents for positron emission tomography (PET) due to the possibility of multiple incorporation of positron emitting radionuclides thus increasing the signal strength. Furthermore, long-term nanoparticle biodistribution tests with increased signal-to-noise ratio can be achieved with nanoparticles carrying long-lived isotopes. Mesoporous silica nanoparticles, MSNs, have recently attracted a lot of interest as both imaging agents and carriers for drugs in vitro and in vivo. Here we present results related to the synthesis of PET imageable MSNs carrying the long-lived 89Zr isotope (half-life of 78.4 hours). Here, 89Zr4+ was immobilized through covalent attachment of the complexing agent p-isothiocyanatobenzyldesferrioxamine (DFO-NCS) to large-pore MSNs. Due to the presence of the high DFO content on the MSNs, quantitative 89Zr4+ labeling was achieved within just a few minutes, and no subsequent purification step was needed in order to remove non-complexed 89Zr4+. The stability of the 89Zr-labeled MSNs against leaching of 89Zr4+ was verified for 24 hours. The high signal strength of the 89Zr-DFO-MSNs was evidenced by successful PET imaging using a mouse model at particle loadings one order of magnitude lower than those previously applied in PET-MSN studies. The biodistribution followed the same trends as previously observed for MSNs of different sizes and surface functionalities. Taken together, our results suggest that 89Zr-DFO-MSNs are promising PET imaging agents for long-term in vivo imaging.Functional nanoparticles are highly interesting imaging agents for positron emission tomography (PET) due to the possibility of multiple incorporation of positron emitting radionuclides thus increasing the signal strength. Furthermore, long-term nanoparticle biodistribution tests with increased signal-to-noise ratio can be achieved with nanoparticles carrying long-lived isotopes. Mesoporous silica nanoparticles, MSNs, have

  9. Synthesis and in vivo evaluation of gallium-68-labeled glycine and hippurate conjugates for positron emission tomography renography.

    PubMed

    Pathuri, Gopal; Hedrick, Andria F; January, Spenser E; Galbraith, Wendy K; Awasthi, Vibhudutta; Arnold, Charles D; Cowley, Benjamin D; Gali, Hariprasad

    2015-01-01

    The objective of this study was to evaluate four new (68) Ga-labeled 1,4,7,10-cyclododeca-1,4,7,10-tetraacetic acid (DOTA)/1,4,7-triazacyclononane-1,4,7-triacetic acid derived (NODAGA)-glycine/hippurate conjugates and select a lead candidate for potential application in positron emission tomography (PET) renography. The non-metallated conjugates were synthesized by a solid phase peptide synthesis method. The (68) Ga labeling was achieved by reacting an excess of the non-metallated conjugate with (68) GaCl4 (-) at pH -4.5 and 10-min incubation either at room temperature for NODAGA or 90 °C for DOTA. Radiochemical purity of all (68) Ga conjugates was found to be >98%. (68) Ga-NODAGA-glycine displayed the lowest serum protein binding (0.4%) in vitro among the four (68) Ga conjugates. Biodistribution of (68) Ga conjugates in healthy Sprague Dawley rats at 1-h post-injection revealed an efficient clearance from circulation primarily through the renal-urinary pathway with <0.2% of injected dose per gram remaining in the blood. The kidney/blood and kidney/muscle ratios of (68) Ga-NODAGA-glycine were significantly higher than other (68) Ga conjugates. On the basis of these results, (68) Ga-NODAGA-glycine was selected as the lead candidate. (68) Ga-NODAGA-glycine PET renograms obtained in healthy rats suggest (68) Ga-NODAGA-glycine as a PET alternate of (99m) Tc-Diethylenetriaminepentaacetic acid (DTPA).

  10. Synthesis of acetylene-substituted probes with benzene-phosphate backbones for RNA labeling.

    PubMed

    Kitamura, Yoshiaki; Ueno, Yoshihito; Kitade, Yukio

    2014-06-24

    Conversion of dimethyl 5-aminoisophthalate into the iodoarene via the corresponding diazonium intermediate, followed by Sonogashira coupling with trimethylsilylacetylene afford the alkynylarene, which is reduced with LiAlH4 to give 5-ethynyl-1,3-benzenedimethanol (B(E)). One hydroxyl group is protected with a 4,4'-dimethoxytrityl (DMTr) group and subsequently another hydroxyl group is phosphitylated to produce the phosphoramidite. The mono-DMTr compound is also modified to afford the corresponding succinate, which is then reacted with controlled pore glass (CPG) to provide the solid support. Either the phosphoramidite or the solid support is employed in solid-phase synthesis of RNA containing B(E). RNA oligomers bearing B(E) rapidly react with 4-fluorobenzylazide to produce the cycloaddition products in good to excellent yield.

  11. Elucidating the Stereochemistry of Enzymatic Benzylsuccinate Synthesis with Chirally Labeled Toluene.

    PubMed

    Seyhan, Deniz; Friedrich, Peter; Szaleniec, Maciej; Hilberg, Markus; Buckel, Wolfgang; Golding, Bernard T; Heider, Johann

    2016-09-12

    Benzylsuccinate synthase is a glycyl radical enzyme that initiates anaerobic toluene metabolism by adding fumarate to the methyl group of toluene to yield (R)-benzylsuccinate. To investigate whether the reaction occurs with retention or inversion of configuration at the methyl group of toluene, we synthesized both enantiomers of chiral toluene with all three H isotopes in their methyl groups. The chiral toluenes were converted into benzylsuccinates preferentially containing (2) H and (3) H at their benzylic C atoms, owing to a kinetic isotope effect favoring hydrogen abstraction from the methyl groups. The configuration of the products was analyzed by enzymatic CoA-thioester synthesis and stereospecific oxidation using enzymes involved in benzylsuccinate degradation. Assessment of the configurations of the benzylsuccinate isomers based on loss or retention of tritium showed that inversion of configuration at the methyl group occurs when the chiral toluenes react with fumarate.

  12. Bacteria-instructed synthesis of polymers for self-selective microbial binding and labelling.

    PubMed

    Magennis, E Peter; Fernandez-Trillo, Francisco; Sui, Cheng; Spain, Sebastian G; Bradshaw, David J; Churchley, David; Mantovani, Giuseppe; Winzer, Klaus; Alexander, Cameron

    2014-07-01

    The detection and inactivation of pathogenic strains of bacteria continues to be an important therapeutic goal. Hence, there is a need for materials that can bind selectively to specific microorganisms for diagnostic or anti-infective applications, but that can be formed from simple and inexpensive building blocks. Here, we exploit bacterial redox systems to induce a copper-mediated radical polymerization of synthetic monomers at cell surfaces, generating polymers in situ that bind strongly to the microorganisms that produced them. This 'bacteria-instructed synthesis' can be carried out with a variety of microbial strains, and we show that the polymers produced are self-selective binding agents for the 'instructing' cell types. We further expand on the bacterial redox chemistries to 'click' fluorescent reporters onto polymers directly at the surfaces of a range of clinical isolate strains, allowing rapid, facile and simultaneous binding and visualization of pathogens.

  13. Synthesis and preliminary evaluation of [18F]-labeled 2-oxoquinoline derivatives for PET imaging of cannabinoid CB2 receptor

    PubMed Central

    Turkman, Nashaat; Paolillo, Vincenzo; Shavrin, Aleksander; Yeh, Hsin Hsien; Flores, Leo; Soghomonian, Suren; Ravinovich, Brian; Volgin, Andrei; Gelovani, Juri; Alauddin, Mian

    2011-01-01

    Introduction The cannabinoid receptor type 2 (CB2) is an important target for development of drugs and imaging agents for diseases, such as neuroinflammation, neurodegeneration, and cancer. Recently we reported synthesis and results of in vitro receptor binding of a focused library of fluorinated 2-oxoquinoline derivatives as CB2 receptor ligands. Some of the compounds demonstrated as good CB2-specific ligands with Ki values in the nanomolar to sub-nanomolar concentrations; therefore, we pursued the development of their 18F-labeled analogues that should be useful for PET imaging of CB2 receptor expression. Here, we report the radiosynthesis of two 18F-labeled 2-oxoquinoline derivatives, and preliminary in vitro and ex-vivo evaluation of one compound as a CB2-specific radioligand. Methods 4-[18F]Fluorobenzyl amine [18F]-3 was prepared by radiofluorination of 4-cyano-N,N,N-trimethylanilinium triflate salt followed by reduction with LiAlH4 and then coupled with acid chlorides 11 and 12 to afford [18F]-13 and [18F]-14. In vitro CB2 receptor binding assay was performed using U87 cells transduced with CB2- and CB1-receptor. Ex-vivo autoradiography was performed with [18F]-14 on spleen, CB2- and CB1-expressing and wild type U87 subcutaneous tumors grown in mice. Results The radiochemical yields of [18F]-13 and [18F]-14 were 10%-15.0% with an average of 12% (n=10); radiochemical purity was > 99% with specific activity 1200 mCi/μmole. The dissociation constant Kd for [18F]-14 was 3.4 nM. Ex-vivo autoradiography showed accumulation of [18F]-14 in the CB2-expressing tumor. Conclusion Two new [18F]-labeled CB2 ligands have been synthesized. Compound [18F]-14 appears to be a potential PET imaging agent for the assessment of CB2 receptor expression in vivo. PMID:22226022

  14. Synthesis and positron emission tomography studies of carbon-11-labeled imatinib (Gleevec)

    PubMed Central

    Kil, Kun-Eek; Ding, Yu-Shin; Lin, Kuo-Shyan; Alexoff, David; Kim, Sung Won; Shea, Colleen; Xu, Youwen; Muench, Lisa; Fowler, Joanna S.

    2010-01-01

    Introduction Imatinib mesylate (Gleevec) is a well known drug for treating chronic myeloid leukemia and gastrointestinal stromal tumors. Its active ingredient, imatinib ([4-[(4-methyl-1-piperazinyl)methyl]-N-[4-methyl-3-[[4-(3-pyridyl)-2-pyrimidinyl]amino]phenyl]benzamide), blocks the activity of several tyrosine kinases. Here we labeled imatinib with carbon-11 as a tool for determining the drug distribution and pharmacokinetics of imatinib, and we carried out positron emission tomography (PET) studies in baboons. Methods [N-11C-methyl]imatinib was synthesized from [11C]methyl iodide and norimatinib was synthesized by the demethylation of imatinib (isolated from Gleevec tablets) according to a patent procedure [Collins JM, Klecker RW Jr, Anderson LW. Imaging of drug accumulation as a guide to antitumor therapy. US Patent 20030198594A1, 2003]. Norimatinib was also synthesized from the corresponding amine and acid. PET studies were carried out in three baboons to measure pharmacokinetics in the brain and peripheral organs and to determine the effect of a therapeutic dose of imatinib. Log D and plasma protein binding were also measured. Results [N-11C-methyl]imatinib uptake in the brain is negligible (consistent with P-glycoprotein-mediated efflux); it peaks and clears rapidly from the heart, lungs and spleen. Peak uptake and clearance occur more slowly in the liver and kidneys, followed by accumulation in the gallbladder and urinary bladder. Pretreatment with imatinib did not change uptake in the heart, lungs, kidneys and spleen, and increased uptake in the liver and gallbladder. Conclusions [N-11C-methyl]imatinib has potential for assessing the regional distribution and kinetics of imatinib in the human body to determine whether the drug targets tumors and to identify other organs to which the drug or its labeled metabolites distribute. Paired with tracers such as 2-deoxy-2-[18F]fluoro-D-glucose (18FDG) and 3′-deoxy-3′-[18F]fluorothymidine (18FLT), [N-11C

  15. Synthesis of fluorine-18-labeled biotin derivatives: Biodistribution and infection localization

    SciTech Connect

    Shoup, T.M.; Fischman, A.J.; Jaywook, S.

    1994-10-01

    Recently there has been much interest in the exploitation of the high binding affinity of avidin/biotin as a means of targeting drugs and radionuclides for in vivo applications. We are interested in broadening the application of the avidin/biotin complex to PET. To this end we set out to prepare {sup 18}F-labeled biotin analogs. Two {sup 18}F biotin derivatives, [3aS-(3a{alpha},4{beta},6a{alpha})]-hexa-hydro-2-oxo-1H-thieno[3,4-d]imidazole-4-(N-3-(1-[{sup 18F}]fluoropropyl))pentanamide (1) and [3aS-(3a{alpha},4{beta},6a{alpha})]-tetrahydro - 4 - 5-(1-[{sup 18}F]fluoropentyl)-1H-thieno[3,4-d]imidazol-2(3H)-F(2) were prepared with high specific activity (NCA) and evaluated for their potential in infection localization. Compound 1 binds to avidin and the biodistribution of these derivatives were studied in Escherichia coli infected rats. Half of the infected rats were treated with avidin 24 hr prior to intravenous injection of the {sup 18}F-labeled biotin analogs. Biotin 1, without avidin pretreatment, showed a selectivity of 6.08 {plus_minus} 1.12 for infection compared to normal muscle. With avidin pretreatment, selectivity increased slightly, giving an infection to normal muscle ratio of 6.39 {plus_minus} 0.96. In contrast, the biodistribution of biotin 2 indicated more binding to normal muscle with an infection to normal muscle ratio of 0.58 {plus_minus} 0.07. This lack of selectivity illustrates the importance of the side-chain amide group in infection localization. There was some defluorination of 1 and 2, as evidenced by increased {sup 18}F bone uptake after 60 min: 2.94 {plus_minus} 0.37 and 1.17 {plus_minus} 0.21%IG/g {plus_minus}s.d., respectively. Biotin derivatives could be radiofluorinated with high specific activity. Biotin 1, is a potential positron tomography tracer for infection imaging. 16 refs., 2 figs., 6 tabs.

  16. The synthesis of 2 14C-labelled 2-(3-alkoxyphenyl)-5,6-dihydro-5-trazolo[5, 1-a] isoquinoline compounds, novel antifertility agents.

    PubMed

    Sartori, G; Consonni, P; Omodei-sale, A

    1981-04-01

    2 new compounds, L-10503, 2-(3-methoxyphenyl)-5,6-dihydro-s-triazolo [5,1-a] isoquinoline and DL-204 IT, 2-(3-ethoxyphenyl)-5,6-dihydro-s-triazolo [5,1-a] isoquinoline have been developed in the Lepetit Research Laboratories in Milan, Italy. These compounds have been tested in monkeys and rats and have been shown to terminate pregnancy after a single intramuscular injection. Pharmocokinetical, metabolic, and placental absorption studies of these compounds required synthesis of 14 C labelled forms for both. This article describes in details the laboratory procedures to obtain synthesis of these compounds.

  17. Diselenide-Labeled Cyclic Polystyrene with Multiple Responses: Facile Synthesis, Tunable Size, and Topology.

    PubMed

    Cai, Zhaoxiong; Lu, Weihong; Gao, Feng; Pan, Xiangqiang; Zhu, Jian; Zhang, Zhengbiao; Zhu, Xiulin

    2016-05-01

    Diselenide-containing polymers have attracted more and more attention due to their redox sensitivity and bioapplication. In this work, a bifunctional diselenocarbonate is prepared and used to mediate the reversible addition-fragmentation chain transfer (RAFT) polymerization, producing α,ω-selenocarbonate-labeled telechelic polystyrene. Based on effective aminolysis of the terminal selenocarbonates and the followed spontaneous oxidation coupling reaction of diselenols, monoblock cyclic polystyrene linked by one diselenide bond and multiblock cyclic copolymer linked by several diselenide bonds are prepared by manipulating the concentration of α,ω-telechelic polystyrene in solution. The progress of aminolysis and the subsequent spontaneous oxidation of selenols to diselenides are monitored by UV-vis, gel permeation chromatography (GPC), and NMR characterizations, confirming the cyclic topologies of the resultant polymers (monocyclic or multiblock cyclic polymer). The monoblock cyclic or multiblock polymers show redox sensitivity, which can be converted to linear polymer by reducing or oxidizing agent. Moreover, the obtained monoblock cyclic polymer or multiblock cyclic copolymer can be transformed to each other under UV irradiation by adjusting the concentration of the cyclic polystyrene. For the first time, this work provides an alternative and promising approach to realize the topological transformation of polymers by installing multiresponsive diselenide moities into the backbone of cyclic polymer. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Synthesis, spectroscopic properties, and biological applications of eight novel chlorinated fluorescent proteins-labeling probes.

    PubMed

    Wu, Xianglong; Tian, Min; Fan, Wutu; Pan, Yalei; Zhai, Yuankun; Niu, Yinbo; Li, Chenrui; Lu, Tingli; Mei, Qibing

    2014-05-01

    Eight novel chlorinated fluorescent proteins-labeling probes with a linker and reactive group were prepared in 7 steps by the reaction of chlorinated resorcinols with 3, 6-dichloro-4-carboxyphthalic anhydride in the presence of methanesulfonic acid. Structures of target compounds and intermediates were determined via IR, MS, (1)H NMR and element analysis. The spectral properties of the chlorinated fluoresceins were studied. These fluorescent probes showed absorbance peaks at 508-536 nm and fluorescence peaks at 524-550 nm. It was found that they have absorption and emission maxima at long wavelengths and high fluorescence quantum yields. Emission spectra of chlorinated fluoresceins shifted towards long wavelength with increase in chlorine. The probes were used for fluorescence imaging of cells in order to investigate whether they can conjugate to cells. The fluorescence imaging of living cells showed that they were localized in cell nucleus. However, they were localized in cytosol of chemically fixed cells. These probes will be useful reagents for the preparation of stable fluorescent conjugates.

  19. One-step synthesis of potassium ferricyanide-doped polyaniline nanoparticles for label-free immunosensor.

    PubMed

    He, Sijing; Wang, Qiyou; Yu, Yanyan; Shi, Qiujia; Zhang, Lin; Chen, Zuanguang

    2015-06-15

    A novel, label-free and inherent electroactive redox immunosensor for ultrasensitive detection of carcinoembryonic antigen (CEA) was proposed based on gold nanoparticles (AuNPs) and potassium ferricyanide-doped polyaniline (FC-PANI) nanoparticles. FC-PANI composite was synthesized via oxidative polymerization of aniline, using potassium ferricyanide (K3[Fe(CN)6]) as both oxidant and dopant. FC-PANI acting as the signal indicator was first fixed on a gold electrode (GE) to be the signal layer. Subsequently, the negatively charged AuNPs could be adsorbed on the positively charged FC-PANI modified GE surface by electrostatic adsorption, and then to immobilize CEA antibody (anti-CEA) for the assay of CEA. The CEA concentration was measured through the decrease of amperometric signals in the corresponding specific binding of antigen and antibody. The wide linear range of the immunosensor was from 1.0 pg mL(-1) to 500.0 ng mL(-1) with a low detection limit of 0.1 pg mL(-1) (S/N=3). The proposed method would have a potential application in clinical immunoassays with the properties of facile procedure, stability, high sensitivity and selectivity.

  20. Selective synthesis and labeling of the polysialic acid capsule in Escherichia coli K1 strains with mutations in nanA and neuB.

    PubMed Central

    Vimr, E R

    1992-01-01

    The enzymes required for polysialic acid capsule synthesis in Escherichia coli K1 are encoded by region 2 neu genes of the multigenic kps cluster. To facilitate analysis of capsule synthesis and translocation, an E. coli K1 strain with mutations in nanA and neuB, affecting sialic acid degradation and synthesis, respectively, was constructed by transduction. The acapsular phenotype of the mutant was corrected in vivo by exogenous addition of sialic acid. By blocking sialic acid degradation, the nanA mutation allows intracellular metabolite accumulation, while the neuB mutation prevents dilution by the endogenous sialic acid pool and allows capsule synthesis to be controlled experimentally by the exogenous addition of sialic acid to the growth medium. Complementation was detected by bacteriophage K1F adsorption or infectivity assays. Polysialic acid translocation was observed within 2 min after addition of sialic acid to the growth medium, demonstrating the rapidity in vivo of sialic acid transport, activation, and polymerization and translocation of polysaccharide to the cell surface. Phage adsorption was not inhibited by chloramphenicol, demonstrating that de novo protein synthesis was not required for polysialic acid synthesis or translocation at 37 degrees C. Exogenous radiolabeled sialic acid was incorporated exclusively into capsular polysaccharide. The polymeric nature of the labeled capsular material was confirmed by gel permeation chromatography and susceptibility of sialyl polymers to K1F endo-N-acylneuraminidase. The ability to experimentally manipulate capsule expression provides new approaches for investigating polysialic acid synthesis and membrane translocation mechanisms. PMID:1400168

  1. Testing Models of Fatty Acid Transfer and Lipid Synthesis in Spinach Leaf Using in Vivo Oxygen-18 Labeling1

    PubMed Central

    Pollard, Mike; Ohlrogge, John

    1999-01-01

    Oxygen-18 labeling has been applied to the study of plant lipid biosynthesis for the first time. [13C218O2]Acetate was incubated with spinach (Spinacia oleracea) leaves and the 18O content in fatty acid methyl esters isolated from different lipid classes measured by gas chromatography-mass spectometry. Fatty acids isolated from lipids synthesized within the plastid, such as monogalactosyldiacylglycerol, show an 18O content consistent with the exogenous acetate undergoing a single activation step and with the direct utilization of acyl-acyl carrier protein by the acyl transferases of the chloroplast. In contrast, fatty acids isolated from lipids assembled in the cytosol, such as phosphatidylcholine, show a 50% reduction in the 18O content. This is indicative of export of the fatty acyl groups from the plastid via a free carboxylate anion, and is consistent with the acyl-acyl carrier protein thioesterase:acyl-coenzyme A (CoA) synthetase mediated export mechanism. If this were not the case and the acyl group was transferred directly from acyl-acyl carrier protein to an acyl acceptor on the cytosolic side, there would be either complete retention of 18O or, less likely, complete loss of 18O, but not a 50% loss of 18O. Thus, existing models for fatty acid transfer from the plastid and for spatially separate synthesis of “prokaryotic” and “eukaryotic” lipids have both been confirmed. PMID:10594108

  2. Design considerations for the synthesis of polymer coated iron oxide nanoparticles for stem cell labelling and tracking using MRI.

    PubMed

    Barrow, Michael; Taylor, Arthur; Murray, Patricia; Rosseinsky, Matthew J; Adams, Dave J

    2015-10-07

    Iron oxide nanoparticles (IONPs, sometimes called superparamagnetic iron oxide nanoparticles or SPIONs) have already shown promising results for in vivo cell tracking using magnetic resonance imaging (MRI). To fully exploit the potential of these materials as contrast agents, there is still a need for a greater understanding of how they react to physiological conditions. A key aspect is the specific nature of the surface coating, which can affect important properties of the IONPs such as colloidal stability, toxicity, magnetism and labelling efficiency. Polymers are widely used as coatings for IONPs as they can increase colloidal stability in hydrophilic conditions, as well as protect the iron oxide core from degradation. In this tutorial review, we will examine the design and synthesis approaches currently being employed to produce polymer coated IONPs as cell tracking agents, and what considerations must be made. We will also give some perspective on the challenges and limitations that remain for polymer coated IONPs as MRI contrast agents for stem cell tracking.

  3. Testing models of fatty acid transfer and lipid synthesis in spinach leaf using in vivo oxygen-18 labeling

    SciTech Connect

    Pollard, M.; Ohlrogge, J.

    1999-12-01

    Oxygen-18 labeling has been applied to the study of plant lipid biosynthesis for the first time. [{sup 13}C{sub 2}{sup 18}O{sub 2}]Acetate was incubated with spinach (Spinacia oleracea) leaves and the {sup 18}O content in fatty acid methyl esters isolated from different lipid classes measured by gas chromatography-mass spectrometry. Fatty acids isolated from lipids synthesized within the plastid, such as monogalactosyldiacylglycerol, show an {sup 18}O content consistent with the exogenous acetate undergoing a single activation step and with the direct utilization of acyl-acyl carrier protein by the acyl transferases of the chloroplast. In contrast, fatty acids isolated from lipids assembled in the cytosol, such as phosphatidylcholine, show a 50% reduction in the {sup 18}O content. This is indicative of export of the fatty acyl groups from the plastid via a free carboxylate anion, and is consistent with the acyl-acyl carrier protein thioesterase:acyl-coenzyme A (CoA) synthetase mediated export mechanism. If this were not the case and the acyl group was transferred directly from acyl-acyl carrier protein to an acyl acceptor on the cytosolic side, there would be either complete retention of {sup 18}O or, less likely, complete loss of {sup 18}O, but not a 50% loss of {sup 18}O. Thus, existing models for fatty acid transfer from the plastid and for spatially separate synthesis of prokaryotic and eukaryotic lipids have both been confirmed.

  4. Synthesis of ferrocene-labeled steroids via copper-catalyzed azide-alkyne cycloaddition. Reactivity difference between 2β-, 6β- and 16β-azido-androstanes.

    PubMed

    Fehér, Klaudia; Balogh, János; Csók, Zsolt; Kégl, Tamás; Kollár, László; Skoda-Földes, Rita

    2012-06-01

    Copper-catalyzed cycloaddition of steroidal azides and ferrocenyl-alkynes were found to be an efficient methodology for the synthesis of ferrocene-labeled steroids. At the same time, a great difference between the reactivity of 2β- or 16β-azido-androstanes and a sterically hindered 6β-azido steroid toward both ferrocenyl-alkynes and simple alkynes, such as phenylacetylene, 1-octyne, propargyl acetate and methyl propiolate, was observed. Copyright © 2012 Elsevier Inc. All rights reserved.

  5. Synthesis of biotin-labelled core glycans of GPI anchors and their application in the study of GPI interaction with pore-forming bacterial toxins.

    PubMed

    Gao, Jian; Zhou, Zhifang; Guo, Jiatong; Guo, Zhongwu

    2017-06-06

    A convergent strategy was developed for the first-time synthesis of biotin-labeled GPI core glycans. These GPI conjugates are useful for various biological studies showcased by their application in the scrutiny of pore-forming bacterial toxin-GPI interaction, revealing that the phosphate group at the GPI inositol 1-O-position had a significant impact on GPI-toxin binding.

  6. PEG-Labeled Nucleotides and Nanopore Detection for Single Molecule DNA Sequencing by Synthesis

    PubMed Central

    Kumar, Shiv; Tao, Chuanjuan; Chien, Minchen; Hellner, Brittney; Balijepalli, Arvind; Robertson, Joseph W. F.; Li, Zengmin; Russo, James J.; Reiner, Joseph E.; Kasianowicz, John J.; Ju, Jingyue

    2012-01-01

    We describe a novel single molecule nanopore-based sequencing by synthesis (Nano-SBS) strategy that can accurately distinguish four bases by detecting 4 different sized tags released from 5′-phosphate-modified nucleotides. The basic principle is as follows. As each nucleotide is incorporated into the growing DNA strand during the polymerase reaction, its tag is released and enters a nanopore in release order. This produces a unique ionic current blockade signature due to the tag's distinct chemical structure, thereby determining DNA sequence electronically at single molecule level with single base resolution. As proof of principle, we attached four different length PEG-coumarin tags to the terminal phosphate of 2′-deoxyguanosine-5′-tetraphosphate. We demonstrate efficient, accurate incorporation of the nucleotide analogs during the polymerase reaction, and excellent discrimination among the four tags based on nanopore ionic currents. This approach coupled with polymerase attached to the nanopores in an array format should yield a single-molecule electronic Nano-SBS platform. PMID:23002425

  7. PEG-labeled nucleotides and nanopore detection for single molecule DNA sequencing by synthesis.

    PubMed

    Kumar, Shiv; Tao, Chuanjuan; Chien, Minchen; Hellner, Brittney; Balijepalli, Arvind; Robertson, Joseph W F; Li, Zengmin; Russo, James J; Reiner, Joseph E; Kasianowicz, John J; Ju, Jingyue

    2012-01-01

    We describe a novel single molecule nanopore-based sequencing by synthesis (Nano-SBS) strategy that can accurately distinguish four bases by detecting 4 different sized tags released from 5'-phosphate-modified nucleotides. The basic principle is as follows. As each nucleotide is incorporated into the growing DNA strand during the polymerase reaction, its tag is released and enters a nanopore in release order. This produces a unique ionic current blockade signature due to the tag's distinct chemical structure, thereby determining DNA sequence electronically at single molecule level with single base resolution. As proof of principle, we attached four different length PEG-coumarin tags to the terminal phosphate of 2'-deoxyguanosine-5'-tetraphosphate. We demonstrate efficient, accurate incorporation of the nucleotide analogs during the polymerase reaction, and excellent discrimination among the four tags based on nanopore ionic currents. This approach coupled with polymerase attached to the nanopores in an array format should yield a single-molecule electronic Nano-SBS platform.

  8. Chemo-enzymatic synthesis of selectively ¹³C/¹⁵N-labeled RNA for NMR structural and dynamics studies.

    PubMed

    Alvarado, Luigi J; Longhini, Andrew P; LeBlanc, Regan M; Chen, Bin; Kreutz, Christoph; Dayie, T Kwaku

    2014-01-01

    RNAs are an important class of cellular regulatory elements, and they are well characterized by X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy in their folded or bound states. However, the apo or unfolded states are more difficult to characterize by either method. Particularly, effective NMR spectroscopy studies of RNAs in the past were hampered by chemical shift overlap of resonances and associated rapid signal loss due to line broadening for RNAs larger than the median size found in the PDB (~25 nt); most functional riboswitches are bigger than this median size. Incorporation of selective site-specific (13)C/(15)N-labeled nucleotides into RNAs promises to overcome this NMR size limitation. Unlike previous isotopic enrichment methods such as phosphoramidite, de novo, uniform-labeling, and selective-biomass approaches, this newer chemical-enzymatic selective method presents a number of advantages for producing labeled nucleotides over these other methods. For example, total chemical synthesis of nucleotides, followed by solid-phase synthesis of RNA using phosphoramidite chemistry, while versatile in incorporating isotope labels into RNA at any desired position, faces problems of low yields (<10%) that drop precipitously for oligonucleotides larger than 50 nt. The alternative method of de novo pyrimidine biosynthesis of NTPs is also a robust technique, with modest yields of up to 45%, but it comes at the cost of using 16 enzymes, expensive substrates, and difficulty in making some needed labeling patterns such as selective labeling of the ribose C1' and C5' and the pyrimidine nucleobase C2, C4, C5, or C6. Biomass-produced, uniformly or selectively labeled NTPs offer a third method, but suffer from low overall yield per labeled input metabolite and isotopic scrambling with only modest suppression of (13)C-(13)C couplings. In contrast to these four methods, our current chemo-enzymatic approach overcomes most of these shortcomings and allows

  9. Microscale synthesis of isotopically labeled R-[6-xH]N5,N10-methylene-5,6,7,8-tetrahydrofolate as a cofactor for thymidylate synthase.

    PubMed

    Agrawal, Nitish; Mihai, Cornelia; Kohen, Amnon

    2004-05-01

    A one-pot synthesis of isotopically labeled R-[6-xH]N5,N10-methylene-5,6,7,8-tetrahydrofolate (CH2H4F) is presented, where x=1, 2, or 3 represents hydrogen, deuterium, or tritium, respectively. The current procedure offers high-yield, high-purity, and microscale-quantity synthesis. In this procedure, two enzymes were used simultaneously in the reaction mixture. The first was Thermoanaerobium brockii alcohol dehydrogenase, which stereospecifically catalyzed a hydride transfer from C-2-labeled isopropanol to the re face of oxidized nicotinamide adenine dinucleotide phosphate to form R-[4-xH]-labeled reduced nicotinamide adenine dinucleotide phosphate. The second enzyme, Escherichia coli dihydrofolate reductase, used the xH to reduce 7,8-dihydrofolate (H2F) to form S-[6-xH]5,6,7,8-tetrahydrofolate (S-[6-xH]H4F). The enzymatic reactions were followed by chemical trapping of S-[6-xH]H4F with formaldehyde to form the final product. Product purification was carried out in a single step by reverse phase high-pressure liquid chromatography separation followed by lyophilization. Two analytical methods were developed to follow the reaction progress. Finally, the utility of the labeled cofactor in mechanistic studies of thymidylate synthase is demonstrated by measuring the tritium kinetic isotope effect on the enzyme's second order rate constant.

  10. An improved strategy for the synthesis of [18F]-labeled arabinofuranosyl nuclosides

    PubMed Central

    Zhang, Hanwen; Cantorias, Melchor V.; Pillarsetty, NagaVaraKishore; Burnazi, Eva M.; Cai, Shangde; Lewis, Jason S.

    2012-01-01

    The expression of the herpes simplex virus type-1 thymidine kinase (HSV1-tk) gene can be imaged efficaciously using a variety of 2′-[18F]fluoro-2′-deoxy-1-b-D-arabinofuranosyl-uracil derivatives [[18F]-FXAU, X= I(iodo), E(ethyl), and M(methyl)]. However, the application of these derivatives in clinical and translational studies has been impeded by their complicated and long syntheses (3–5 h). To remedy these issues, in the study at hand we have investigated whether microwave or combined catalysts could facilitate the coupling reaction between sugar and nucleobase and, further, have probed the feasibility of establishing a novel approach for [18F]-FXAU synthesis. We have demonstrated that the rate of the trimethylsilyl trifluoromethanesulfonate (TMSOTf)-catalyzed coupling reaction between the 2-deoxy-sugar and uracil derivatives at 90°C can be significantly accelerated by microwave-driven heating or by the addition of Lewis acid catalyst (SnCl4). Further, we have observed that the stability of the α- and β-anomers of [18F]-FXAU derivatives differs during the hydrolysis step. Using the microwave-driven heating approach, overall decay-corrected radiochemical yields of 19–27% were achieved for [18F]-FXAU in 120 min at a specific activity of >22 MBq/nmol (595 Ci/mmol). Ultimately, we believe that these high yielding syntheses of [18F]-FIAU, [18F]-FMAU and [18F]-FEAU will facilitate routine production for clinical applications. PMID:22819195

  11. /sup 18/O isotope shift in /sup 15/N NMR spectroscopy. 2. Synthesis of /sup 15/N, /sup 18/O-labeled hydroxylamine hydrochloride

    SciTech Connect

    Rajendran, G.; Van Etten, R.L.

    1986-03-12

    Since hydroxylamine can serve as a key intermediate in the synthesis of a variety of compounds, the synthesis of (/sup 15/N, /sup 18/O)-labelled hydroxylamine hydrochloride was undertaken. Published procedures for the synthesis of hydroxylamine resulted in poor yields in some cases and in lower percentage of /sup 18/O in the product than expected in other cases. The compound was synthesized in dry tetrahydrofuran (THF) by treating NaNO/sub 2/ with borane-methyl sulfide. The course of the reaction was examined using /sup 11/B NMR spectroscopy, and the product yield was 74%. The /sup 18/O enrichment was demonstrated by both mass spectrometry and /sup 15/N NMR of the isolated acetoxime. 23 references, 1 figure.

  12. Synthesis of deuterium-labeled 17-hydroxyprogesterone suitable as an internal standard for isotope dilution mass spectrometry

    SciTech Connect

    Shimizu, K.; Yamaga, N.; Kohara, H.

    1988-03-01

    A synthesis is reported of 17-hydroxyprogesterone, labeled with four atoms of deuterium at ring C and suitable for use as an internal standard for isotope dilution mass spectrometry. Base-catalyzed equilibration of methyl 3 alpha-acetoxy-12-oxo-cholanate (III) with /sup 2/H/sub 2/O, followed by reduction of the 12-oxo group by the modified Wolff-Kisher method using (/sup 2/H)diethylene glycol and (/sup 2/H)hydrazine hydrate afforded (11,11,12,12,23,23(-2)H)lithocholic acid (V). The Meystre-Miescher degradation of the side chain of V yielded 3 alpha-hydroxy-5 beta-(11,11,12,12(-2)H)pregnan-20-one (X). Oxidation of the 3,20-enol-diacetate of X with perbenzoic acid followed by saponification afforded 3 alpha,17-dihydroxy-5 beta-(11,11,12,12(-2)H)pregnan-20-one (XI). Oxidation of XI with N-bromoacetamide yielded 17-hydroxy-5 beta-(11,11,12,12(-2)H)pregnane-3,20-dione (XII). Bromination of XII followed by dehydrobromination yielded 17-hydroxy-(11,11,12,12(-2)H) progesterone (XIV), consisting of 0.3% /sup 2/H0-, 1.1% /sup 2/H/sub 1/-, 8.6% /sup 2/H/sub 2/-, 37.1% /sup 2/H/sub 3/-, 52.1% /sup 2/H/sub 4/-, and 0.8% /sup 2/H/sub 5/-species.

  13. Synthesis and evaluation of radioactive and fluorescent residualizing labels for monitoring protein degradation in vivo and in vitro

    SciTech Connect

    Maxwell, J.L.

    1988-01-01

    Residualizing labels for proteins, such as dilactitol-{sup 125}I-tyramine, are tracers which have been used to identify the tissue and cellular sites of catabolism of long-lived plasma proteins, such as albumin. The radioactive degradation products formed from labeled proteins are relatively large and hydrophilic. These tracers accumulate in lysosomes following uptake and catabolism of the carrier protein. However, the gradual loss of the catabolites from cells has limited the usefulness of these radioactive labels in studies on longer-lived proteins. The objective of this dissertation was to design a radioactive residualizing label, Inulin-{sup 125}I-tyramine ({sup 125}I-InTn), that would be retained more efficiently in cells than existing labels and to develop and evaluate the first fluorescent residualizing label, N,N-dilactitol-N{prime}-fluoresceinyl-ethylenediamine (DLF).

  14. Sci—Fri AM: Mountain — 04: Label-free Raman spectroscopy of single tumour cells detects early radiation-induced glycogen synthesis associated with increased radiation resistance

    SciTech Connect

    Matthews, Q; Lum, JJ; Isabelle, M; Harder, S; Jirasek, A; Brolo, AG

    2014-08-15

    Purpose: To use label-free Raman spectroscopy (RS) for early treatment monitoring of tumour cell radioresistance. Methods: Three human tumour cell lines, two radioresistant (H460, SF{sub 2} = 0.57 and MCF7, SF{sub 2} = 0.70) and one radiosensitive (LNCaP, SF{sub 2} = 0.36), were irradiated with single fractions of 2, 4, 6, 8 or 10 Gy. In additional experiments, H460 and MCF7 cells were irradiated under co-treatment with the anti-diabetic drug metformin, a known radiosensitizing agent. Treated and control cultures were analyzed with RS daily for 3 days post-treatment. Single-cell Raman spectra were acquired from 20 live cells per sample, and experiments were repeated in triplicate. The combined data sets were analyzed with principal component analysis using standard algorithms. Cells from each culture were also subjected to standard assays for viability, proliferation, cell cycle, and radiation clonogenic survival. Results: The radioresistant cells (H460, MCF7) exhibited a RS molecular radiation response signature, detectable as early as 1 day post-treatment, of which radiation-induced glycogen synthesis is a significant contributor. The radiosensitive cells (LNCaP) exhibited negligible glycogen synthesis. Co-treatment with metformin in MCF7 cells blocked glycogen synthesis, reduced viability and proliferation, and increased radiosensitivity. Conversely, metformin co-treatment in H460 cells did not produce these same effects; importantly, both radiation-induced synthesis of glycogen and radiosensitivity were unaffected. Conclusions: Label-free RS can detect early glycogen synthesis post-irradiation, a previously undocumented metabolic mechanism associated with tumour cell radioresistance that can be targeted to increase radiosensitivity. RS monitoring of intratumoral glycogen may provide new opportunities for personalized combined modality radiotherapy treatments.

  15. Synthesis of no carrier added F-18 16-fluorohexadecanoic acid (FHDA) and investigation of its labeled metabolites and its kinetics in the heart

    SciTech Connect

    DeGrado, T.R.; Bernstein, D.R.; Gatley, S.J.; Ng, C.K.; Holden, J.E.

    1984-01-01

    No carrier added FHDA was prepared via saponification of the product of silver oxide assisted reaction of near-anhydrous tetraethylammonium fluoride with methyl 16-iodohexadecanoate. The labeled fatty acid was injected into isolated perfused rat hearts. Coronary perfusate was collected for 4-9 minutes, when hearts were chilled and homogenized. F-18 in perfusate was analysed by HPLC (NH column; 50mM amm. acetate in 50% acetonitrile). Material with the same retention time as F-18 fluoroacetate (prepared by F-for-I exchange with ethyl iodoacetate) was found. Some F-18 stuck permanently to the column and was assigned as fluoride since the same fraction of label in perfusate was retained on alumina columns eluted with water. Anion exchange HPLC (SAX column; 20mM pot. phosphate, pH 7) of homogenates gave peaks corresponding to fluoroacetate plus fluoride and minor peaks which could be fluoroacetylCoA and fluorocitrate. The authors interpret their data as follows. Beta-oxidation of FHDA results in fluoroacetylCoA which either undergoes ''lethal synthesis'' to fluorocitrate or is hydrolysed to fluoroacetate which diffuses out of the heart. The source of the fluoride is not yet clear, but could complicate interpretation of FHDA kinetics measured in vivo with positron tomography. Clearance of label from FHDA in isolated perfused hearts was faster than for labeled 16-iodohexadecanoic acid, indicating that the F-18 tracer may be a more sensitive probe of myocardial fatty acid metabolism.

  16. Efficient enzymatic synthesis and dual-colour fluorescent labelling of DNA probes using long chain azido-dUTP and BCN dyes.

    PubMed

    Ren, Xiaomei; El-Sagheer, Afaf H; Brown, Tom

    2016-05-05

    A sterically undemanding azide analogue of dTTP (AHP dUTP) with an alkyl chain and ethynyl attachment to the nucleobase was designed and incorporated into DNA by primer extension, reverse transcription and polymerase chain reaction (PCR). An azide-modified 523 bp PCR amplicon with all 335 thymidines replaced by AHP dU was shown to be a perfect copy of the template from which it was amplified. Replacement of thymidine with AHP dU increases duplex stability, accounting in part for the high incorporation efficiency of the azide-modified triphosphate. Single-stranded azide-labelled DNA was conveniently prepared from PCR products by λ-exonuclease digestion and streptavidin magnetic bead isolation. Efficient fluorescent labelling of single and double-stranded DNA was carried out using dyes functionalized with bicyclo[6.1.0]non-4-yne (BCN) via the strain-promoted alkyne-azide cycloaddition (SPAAC) reaction. This revealed that the degree of labelling must be carefully controlled to achieve optimum fluorescence and avoid fluorescence quenching. Dual-coloured probes were obtained in a single tube fluorescent labelling reaction; and varying the ratios of the two dyes provides a simple method to prepare DNA probes with unique fluorescent signatures. AHP dUTP is a versatile clickable nucleotide with potentially wide applications in biology and nanotechnology including single molecule studies and synthesis of modified aptamer libraries via SELEX.

  17. Efficient enzymatic synthesis and dual-colour fluorescent labelling of DNA probes using long chain azido-dUTP and BCN dyes

    PubMed Central

    Ren, Xiaomei; El-Sagheer, Afaf H.; Brown, Tom

    2016-01-01

    A sterically undemanding azide analogue of dTTP (AHP dUTP) with an alkyl chain and ethynyl attachment to the nucleobase was designed and incorporated into DNA by primer extension, reverse transcription and polymerase chain reaction (PCR). An azide-modified 523 bp PCR amplicon with all 335 thymidines replaced by AHP dU was shown to be a perfect copy of the template from which it was amplified. Replacement of thymidine with AHP dU increases duplex stability, accounting in part for the high incorporation efficiency of the azide-modified triphosphate. Single-stranded azide-labelled DNA was conveniently prepared from PCR products by λ-exonuclease digestion and streptavidin magnetic bead isolation. Efficient fluorescent labelling of single and double-stranded DNA was carried out using dyes functionalized with bicyclo[6.1.0]non-4-yne (BCN) via the strain-promoted alkyne-azide cycloaddition (SPAAC) reaction. This revealed that the degree of labelling must be carefully controlled to achieve optimum fluorescence and avoid fluorescence quenching. Dual-coloured probes were obtained in a single tube fluorescent labelling reaction; and varying the ratios of the two dyes provides a simple method to prepare DNA probes with unique fluorescent signatures. AHP dUTP is a versatile clickable nucleotide with potentially wide applications in biology and nanotechnology including single molecule studies and synthesis of modified aptamer libraries via SELEX. PMID:26819406

  18. Design, Synthesis, and Evaluation of a Low-Molecular-Weight (11)C-Labeled Tetrazine for Pretargeted PET Imaging Applying Bioorthogonal in Vivo Click Chemistry.

    PubMed

    Denk, Christoph; Svatunek, Dennis; Mairinger, Severin; Stanek, Johann; Filip, Thomas; Matscheko, Dominik; Kuntner, Claudia; Wanek, Thomas; Mikula, Hannes

    2016-07-20

    A low-molecular-weight tetrazine labeled with the short-lived positron emitter carbon-11 was developed as a bioorthogonal PET probe for pretargeted imaging. A method for efficient and fast synthesis of this imaging agent is presented using radiolabeling of a readily available precursor. High reactivity with trans-cyclooctenes was observed and in vivo investigations including PET/MR scanning showed homogeneous biodistribution, good metabolic stability, and rapid excretion in naive mice. These properties are key to the success of bioorthogonal (11)C-PET imaging, which has been shown in a simple pretargeting experiment using TCO-modified mesoporous silica nanoparticles. Overall, this (11)C-labeled tetrazine represents a highly versatile and advantageous chemical tool for bioorthogonal PET imaging and enables pretargeting approaches using carbon-11 for the first time.

  19. Labeled oxazaphosphorines for applications in mass spectrometry studies. 2. Synthesis of deuterium-labeled 2-dechloroethylcyclophosphamides and 2- and 3-dechloroethylifosfamides.

    PubMed

    Springer, James B; Colvin, O Michael; Ludeman, Susan M

    2014-02-01

    The prodrugs cyclophosphamide (CP) and ifosfamide (IF) each metabolize to an active alkylating agent through a cytochrome P450-mediated oxidation at the C-4 position. Competing with this activation pathway are enzymatic oxidations at the exocyclic α and α' carbons, which result in dechloroethylation of CP and IF. The incidence of oxidation at one position relative to another is believed to be at least one factor underlying the high degree of interpatient variability in both CP and IF pharmacokinetics. As standards for the mass spectrometry quantification of dechloroethylation, the following were synthesized: (1) [4,4,5,5-(2) H4 ]-2-dechloroethylcyclophosphamide (equivalent to [4,4,5,5-(2) H4 ]-3-dechloroethylifosfamide); (2) [α,α,4,4,5,5-(2) H6 ]-2-dechloroethylcyclophosphamide (equivalent to [α,α,4,4,5,5-(2) H6 ]-3-dechloroethylifosfamide); and (3) [α,α,4,4,5,5-(2) H6 ]-2-dechloroethylifosfamide. The common precursor to all of the target compounds was [2,2,3,3-(2) H4 ]-3-aminopropanol. A one-pot reaction of this compound with POCl3 and unlabeled or labeled 2-chloroethylamine hydrochloride gave the d4 and d6 labeled 2-dechloroethylcyclophosphamides. The construction of the 2-dechloroethylifosfamide from the aminopropanol required five discreet steps. Optimization of the synthetic pathways and stability studies are discussed.

  20. Synthesis of ¹⁸O-labeled photosynthetically active chlorophylls at the 3- or 7-carbonyl group with high regioselectivity.

    PubMed

    Morishita, Hidetada; Mizoguchi, Tadashi; Tamiaki, Hitoshi

    2010-09-01

    The 3- and 7-formyl groups of chlorophyll-d (Chl-d) and bacteriochlorophyll-e (BChl-e), respectively, were regioselectively labeled with an isotopically stable oxygen-18 (¹⁸O) atom to give 3¹-¹⁸O-labeled Chl-d and 7¹-¹⁸O-labeled BChl-e (ca. 90% ¹⁸O) by exchanging the carbonyl oxygen atoms in the presence of acidic H₂ ¹⁸O (ca. 95% ¹⁸O). Another photosynthetically active chlorophyll, BChl-a possessing the 3-acetyl group was treated under similar acidic conditions to afford a trace amount of 3¹-¹⁸O-labeled BChl-a and further demetallated compound, the corresponding 3¹-¹⁸O-labeled bacteriopheophytin-a as the major product with 55% ¹⁸O-degree. The FT-IR spectra of ¹⁸O-(un)labeled chlorophylls in the solution and the solid states showed that the 3- and 7-carbonyl stretching vibration modes moved to about a 30-cm⁻¹ lower wavenumber by ¹⁸O-labeling at the 3¹- and 7¹-oxo moieties. In artificial chlorosome-like self-aggregates of BChl-e, the ¹⁸O-labeled 7-carbonyl stretching mode was completely resolved from the specially hydrogen-bonded 13-C=O stretching mode, evidently indicating no interaction of the 7-CHO with other functional groups in the supramolecules.

  1. Amino Acid Synthesis in Photosynthesizing Spinach Cells: Effects of Ammonia on Pool Sizes and Rates of Labeling from 14CO2

    SciTech Connect

    Larsen, Peder Olesen; Cornwell, Karen L.; Gee, Sherry L.; Bassham, James A.

    1981-08-01

    In this paper, isolated cells from leaves of Spinacia oleracea have been maintained in a state capable of high rates of photosynthetic CO2 fixation for more than 60 hours. The incorporation of 14CO2 under saturating CO2 conditions into carbohydrates, carboxylic acids, and amino acids, and the effect of ammonia on this incorporation have been studied. Total incorporation, specific radioactivity, and pool size have been determined as a function of time for most of the protein amino acids and for γ-aminobutyric acid. The measurements of specific radio-activities and of the approaches to 14C “saturation” of some amino acids indicate the presence and relative sizes of metabolically active and passive pools of these amino acids. Added ammonia decreased carbon fixation into carbohydrates and increased fixation into carboxylic acids and amino acids. Different amino acids were, however, affected in different and highly specific ways. Ammonia caused large stimulatory effects in incorporation of 14C into glutamine (a factor of 21), aspartate, asparagine, valine, alanine, arginine, and histidine. No effect or slight decreases were seen in glycine, serine, phenylalanine, and tyrosine labeling. In the case of glutamate, 14C labeling decreased, but specific radioactivity increased. The production of labeled γ-aminobutyric acid was virtually stopped by ammonia. The results indicate that added ammonia stimulates the reactions mediated by pyruvate kinase and phosphoenolpyruvate carboxylase, as seen with other plant systems. Finally, the data on the effects of added ammonia on total labeling, pool sizes, and specific radioactivities of several amino acids provides a number of indications about the intracellular sites of principal synthesis from carbon skeletons of these amino acids and the selective nature of effects of increased intracellular ammonia concentration on such synthesis.

  2. Hepatic albumin and urea synthesis: The mathematical modelling of the dynamics of [14C]carbonate-derived guanidine-labelled arginine in the isolated perfused rat liver.

    PubMed Central

    Tavill, A S; Nadkarni, D; Metcalfe, J; Black, E; Hoffenberg, R; Carson, E R

    1975-01-01

    A mathematical model was constructed to define the dynamics of incorporation of radioactivity into urea carbon and the guanidine carbon of arginine in plasma albumin after the rapid intraportal-venous administration of Na214CO3 in the isolated perfused rat liver. 2. The model was formulated in terms of compartmental analysis and additional experiments were designed to provide further information on subsystem dynamics and to discriminate between alternative model structures. 3. Evidence for the rapid-time-constant of labelling of intracellular arginine was provided by precursor-product analysis of precursor [14C]carboante and product [14C]urea in the perfusate. 4. Compartmental analysis of the dynamics of newly synthesized urea was based on the fate of exogenous [13C]urea, endogenous [14C]urea and the accumulation of [12C]urea in perfusate water, confirming the early completion of urea carbon labelling, the absence of continuing synthesis of labelled urea, and the presence of a small intrahepatic urea-delay pool. 5. Analysis of the perfusate dynamics of endogenously synthesized and exogenously administered [6-14C]arginine indicated that although the capacity for extrahepatic formation of [14C]-urea exists, little or no arginine formed within the intrahepatic urea cycle was transported out of the liver. However, the presence of a rapidly turning-over intrahepatic arginine pool was confirmed. 6. On the basis of these subsystem analyses it was possible to offer feasible estimations for the parameters of the mathematical model. However, it was not possible to stimulate the form and magnitude of the dynamics of newly synthesized labelled urea and albumin which were simultaneously observed after administration of [14C]carbonate on the basis of a preliminary model which postulated that both products were derived from a single hepatic pool of [16-14C]arginine. On the other hand these observed dynamics could be satisfied to a two-compartment arginine model, which also

  3. Analysis of 18F-labelled synthesis products on TLC plates: comparison of radioactivity scanning, film autoradiography, and a phosphoimaging technique.

    PubMed

    Kämäräinen, Eeva-Liisa; Haaparanta, Merja; Siitari-Kauppi, Marja; Koivula, Teija; Lipponen, Tiina; Solin, Olof

    2006-09-01

    We compared radioactivity scanning, film autoradiography, and digital photostimulated luminescence (PSL) autoradiography (phosphoimaging technique) in detection of radioactivity on thin-layer chromatography (TLC) plates. TLC combined with radioactivity detection is rapid, simple, and relatively flexible. Here, (18)F-labelled synthesis products were analyzed by TLC and the radioactivity distribution on the plates determined using the three techniques. Radioactivity scanning is appropriate only with good chromatographic resolution and previously validated scanning parameters. Film autoradiography exhibits poor linearity if radioactivity varies greatly. PSL provides high sensitivity and resolution and superior linearity compared with the other methods.

  4. Synthesis and pharmacological characterization of a europium-labelled single-chain antagonist for binding studies of the relaxin-3 receptor RXFP3.

    PubMed

    Haugaard-Kedström, Linda M; Wong, Lilian L L; Bathgate, Ross A D; Rosengren, K Johan

    2015-06-01

    Relaxin-3 and its endogenous receptor RXFP3 are involved in fundamental neurological signalling pathways, such as learning and memory, stress, feeding and addictive behaviour. Consequently, this signalling system has emerged as an attractive drug target. Development of leads targeting RXFP3 relies on assays for screening and ligand optimization. Here, we present the synthesis and in vitro characterization of a fluorescent europium-labelled antagonist of RXFP3. This ligand represents a cheap and safe but powerful tool for future mechanistic and cell-based receptor-ligand interaction studies of the RXFP3 receptor.

  5. Isotope-Labeled Amyloids via Synthesis, Expression, and Chemical Ligation for Use in FTIR, 2D IR, and NMR Studies.

    PubMed

    Zhang, Tianqi O; Grechko, Maksim; Moran, Sean D; Zanni, Martin T

    2016-01-01

    This chapter provides protocols for isotope-labeling the human islet amyloid polypeptide (hIAPP or amylin) involved in type II diabetes and γD-crystallin involved in cataract formation. Because isotope labeling improves the structural resolution, these protocols are useful for experiments using Fourier transform infrared (FTIR), two-dimensional infrared (2D IR), and NMR spectroscopies. Our research group specializes in using 2D IR spectroscopy and isotope labeling. 2D IR spectroscopy provides structural information by measuring solvation from 2D diagonal lineshapes and vibrational couplings from cross peaks. Infrared spectroscopy can be used to study kinetics, membrane proteins, and aggregated proteins. Isotope labeling provides greater certainty in the spectral assignment, which enables new structural insights that are difficult to obtain with other methods. For amylin, we provide a protocol for (13)C/(18)O labeling backbone carbonyls at one or more desired amino acids in order to obtain residue-specific structural resolution. We also provide a protocol for expressing and purifying amylin from E. coli, which enables uniform (13)C or (13)C/(15)N labeling. Uniform labeling is useful for measuring the monomer infrared spectrum in an amyloid oligomer or fiber as well as amyloid protein bound to another polypeptide or protein, such as a chaperone or an inhibitor. In addition, our expression protocol results in 2-2.5 mg of amylin peptide per 1 L cell culture, which is a high enough yield to straightforwardly obtain the 2-10 mg needed for high resolution and solid-state NMR experiments. Finally, we provide a protocol to isotope-label either of the two domains of γD-crystallin using expressed protein ligation. Domain labeling makes it possible to resolve the structures of the two halves of the protein in FTIR and 2D IR spectra. With modifications, these strategies and protocols for isotope labeling can be applied to other amyloid polypeptides and proteins.

  6. First synthesis of mitomycins completely labeled at the C-6-methyl by [sup 13]CH[sub 3] and CD[sub 3

    SciTech Connect

    Arai, Hitoshi; Kasai, Masaji ); Kanda, Yutaka )

    1993-02-12

    The C-6-methyl group of mitomycins was completely labeled with carbon-13 or deuterium for the first time. The synthesis was accomplished by the C-6-methylation of 6-demethyl-7,7-(ethylenedioxy)-6-(phenylseleno)mitosane 8 that was formed by a novel replacement of the methylene moiety of 6-demethyl-7,7-(ethylenedioxy)-6-methylenemitosane 10 by a phenylseleno group, and followed by conversion to the mitomycins. For the synthesis of 8, the authors found that selenamide 11 is an excellent reagent for the replacement. They have also determined that the replacement proceeded via the addition of 11 to the methylene of 10 with a subsequent retro-Mannich reaction. 15 refs.

  7. Regioselective synthesis of isotopically labeled Δ9-tetrahydrocannabinolic acid A (THCA-A-D3) by reaction of Δ9-tetrahydrocannabinol-D3 with magnesium methyl carbonate.

    PubMed

    Roth, Nadine; Wohlfarth, Ariane; Müller, Michael; Auwärter, Volker

    2012-10-10

    For the reliable quantification of Δ9-tetrahydrocannabinolic acid A (THCA-A), the biogenetic precursor of Δ9-tetrahydrocannabinol (THC), in biological matrices by LC-MS/MS and GC-MS(/MS), an isotopically labeled internal standard was synthesized starting from Δ9-tetrahydrocannabinol-D(3) (THC-D(3)). Synthesis strategy was based on a method reported by Mechoulam et al. in 1969 using magnesium methyl carbonate (MMC) as carboxylation reagent for the synthesis of cannabinoid acids. Preliminary experiments with THC to optimize yield of the product (THCA-A) resulted in the synthesis of the positional isomer tetrahydrocannabinolic acid B (THCA-B) as a byproduct. Using the optimized conditions for the desired isomer, THCA-A-D(3) was prepared and isolated with a yield of approx. 10% after two synthesis cycles. Isotope purity was estimated to be >99% by relative abundance of the molecular ions. The synthesized compound proved to be suitable as an internal standard for quantification of THCA-A in serum and hair samples of cannabis consumers.

  8. Synthesis and characterization of a 'fluorous' (fluorinated alkyl) affinity reagent that labels primary amine groups in proteins/peptides.

    PubMed

    Qian, Jiang; Cole, Richard B; Cai, Yang

    2011-01-01

    Strong non-covalent interactions such as biotin-avidin affinity play critical roles in protein/peptide purification. A new type of 'fluorous' (fluorinated alkyl) affinity approach has gained popularity due especially to its low level of non-specific binding to proteins/peptides. We have developed a novel water-soluble fluorous labeling reagent that is reactive (via an active sulfo-N-hydroxylsuccinimidyl ester group) to primary amine groups in proteins/peptides. After fluorous affinity purification, the bulky fluorous tag moiety and the long oligoethylene glycol (OEG) spacer of this labeling reagent can be trimmed via the cleavage of an acid labile linker. Upon collision-induced dissociation, the labeled peptide ion yields a characteristic fragment that can be retrieved from the residual portion of the fluorous affinity tag, and this fragment ion can serve as a marker to indicate that the relevant peptide has been successfully labeled. As a proof of principle, the newly synthesized fluorous labeling reagent was evaluated for peptide/protein labeling ability in phosphate-buffered saline (PBS). Results show that both the aqueous environment protein/peptide labeling and the affinity enrichment/separation process were highly efficient.

  9. Sodium-proton exchanger isoform-1: synthesis of a potent inhibitor labeled with deuterium and carbon-14.

    PubMed

    Latli, Bachir; Haddad, Nizar; Hrapchak, Matt; Wei, Xudong; Tang, Wenjun; Song, Jinhua J; Senanayake, Chris H

    2013-03-01

    Sodium-proton exchangers, NHEs are plasma membrane proteins that are essential in the regulation of intracellular pH of the myocardium. There are nine known variously expressed isoforms of NHEs with NHE-1 being the predominant isoform in the heart. N-[4-(1-acetyl-piperidin-4-yl)-3-trifluoromethyl-benzoyl]-guanidine (1) is a potent NHE 1-inhibitor with good pharmacokinetics. It was prepared labeled with deuterium and carbon-14 to aid in drug metabolism, pharmacokinetics, and other studies. The combination of Comins' reaction and reduction under deuterium gas was used to access deuterium labeled (1) starting from deuterium labeled pyridine. Carbon-14 labeled zinc cyanide was used to prepare [(14)C]-(1) in three steps, with a specific activity of 55.6 mCi/mmol.

  10. Perylene-labeled silica nanoparticles: synthesis and characterization of three novel silica nanoparticle species for live-cell imaging.

    PubMed

    Blechinger, Julia; Herrmann, Rudolf; Kiener, Daniel; García-García, F Javier; Scheu, Christina; Reller, Armin; Bräuchle, Christoph

    2010-11-05

    The increasing exposure of humans to nanoscaled particles requires well-defined systems that enable the investigation of the toxicity of nanoparticles on the cellular level. To facilitate this, surface-labeled silica nanoparticles, nanoparticles with a labeled core and a silica shell, and a labeled nanoparticle network-all designed for live-cell imaging-are synthesized. The nanoparticles are functionalized with perylene derivatives. For this purpose, two different perylene species containing one or two reactive silica functionalities are prepared. The nanoparticles are studied by transmission electron microscopy, widefield and confocal fluorescence microscopy, as well as by fluorescence spectroscopy in combination with fluorescence anisotropy, in order to characterize the size and morphology of the nanoparticles and to prove the success and homogeneity of the labeling. Using spinning-disc confocal measurements, silica nanoparticles are demonstrated to be taken up by HeLa cells, and they are clearly detectable inside the cytoplasm of the cells.

  11. Determining Degradation and Synthesis Rates of Arabidopsis Proteins Using the Kinetics of Progressive 15N Labeling of Two-dimensional Gel-separated Protein Spots*

    PubMed Central

    Li, Lei; Nelson, Clark J.; Solheim, Cory; Whelan, James; Millar, A. Harvey

    2012-01-01

    The growth and development of plant tissues is associated with an ordered succession of cellular processes that are reflected in the appearance and disappearance of proteins. The control of the kinetics of protein turnover is central to how plants can rapidly and specifically alter protein abundance and thus molecular function in response to environmental or developmental cues. However, the processes of turnover are largely hidden during periods of apparent steady-state protein abundance, and even when proteins accumulate it is unclear whether enhanced synthesis or decreased degradation is responsible. We have used a 15N labeling strategy with inorganic nitrogen sources coupled to a two-dimensional fluorescence difference gel electrophoresis and mass spectrometry analysis of two-dimensional IEF/SDS-PAGE gel spots to define the rate of protein synthesis (KS) and degradation (KD) of Arabidopsis cell culture proteins. Through analysis of MALDI-TOF/TOF mass spectra from 120 protein spots, we were able to quantify KS and KD for 84 proteins across six functional groups and observe over 65-fold variation in protein degradation rates. KS and KD correlate with functional roles of the proteins in the cell and the time in the cell culture cycle. This approach is based on progressive 15N labeling that is innocuous for the plant cells and, because it can be used to target analysis of proteins through the use of specific gel spots, it has broad applicability. PMID:22215636

  12. Heavy-Atom Labeled Transmembrane β-Peptides: Synthesis, CD-Spectroscopy, and X-ray Diffraction Studies in Model Lipid Multilayer.

    PubMed

    Rost, Ulrike; Xu, Yihui; Salditt, Tim; Diederichsen, Ulf

    2016-08-18

    Transmembrane β-peptides are promising candidates for the design of well-controlled membrane anchors in lipid membranes. Here, we present the synthesis of transmembrane β-peptides with and without tryptophan anchors, as well as a novel iodine-labeled d-β(3) -amino acid. By using one or more of the heavy-atom labeled amino acids as markers, the orientation of the helical peptide was inferred based on the electron-density profile determined by X-ray reflectivity. The β-peptides were synthesized through manual Fmoc-based solid-phase peptide synthesis (SPPS) and reconstituted in unilamellar vesicles forming a right-handed 314 -helix secondary structure, as shown by circular dichroism spectroscopy. We then integrated the β-peptide into solid-supported membrane stacks and carried out X-ray reflectivity and grazing incidence small-angle X-ray scattering to determine the β-peptide orientation and its effect on the membrane bilayers. These β-peptides adopt a well-ordered transmembrane motif in the solid-supported model membrane, maintaining the basic structure of the original bilayer with some distinct alterations. Notably, the helical tilt angle, which accommodates the positive hydrophobic mismatch, induces a tilt of the acyl chains. The tilted chains, in turn, lead to a membrane thinning effect.

  13. Synthesis of RNAs with up to 100 Nucleotides Containing Site-Specific 2-methylseleno Labels for use in X-ray Crystallography

    SciTech Connect

    Hobartner,C.; Rieder, R.; Kreutz, C.; Puffer, B.; Lang, K.; Polonskaia, A.; Serganov, A.; Micura, R.

    2005-01-01

    The derivatization of nucleic acids with selenium is a new and highly promising approach to facilitate their three-dimensional structure determination by X-ray crystallography. Here, we report a comprehensive study on the chemical and enzymatic syntheses of RNAs containing 2'-methylseleno (2'-Se-methyl) nucleoside labels. Our approach includes the first synthesis of an appropriate purine nucleoside phosphoramidite building block. Most importantly, a substantially changed RNA solid-phase synthesis cycle, comprising treatment with threo-1, 4-dimercapto-2, 3-butanediol (DTT) after the oxidation step, is required for a reliable strand elongation. This novel operation allows for the chemical syntheses of multiple Se-labeled RNAs in sizes that can typically be achieved only for nonmodified RNAs. In combination with enzymatic ligation, biologically important RNA targets become accessible for crystallography. Exemplarily, this has been demonstrated for the Diels-Alder ribozyme and the add adenine riboswitch sequences. We point out that the approach documented here has been the chemical basis for the very recent structure determination of the Diels-Alder ribozyme which represents the first novel RNA fold that has been solved via its Se-derivatives.

  14. Determining degradation and synthesis rates of arabidopsis proteins using the kinetics of progressive 15N labeling of two-dimensional gel-separated protein spots.

    PubMed

    Li, Lei; Nelson, Clark J; Solheim, Cory; Whelan, James; Millar, A Harvey

    2012-06-01

    The growth and development of plant tissues is associated with an ordered succession of cellular processes that are reflected in the appearance and disappearance of proteins. The control of the kinetics of protein turnover is central to how plants can rapidly and specifically alter protein abundance and thus molecular function in response to environmental or developmental cues. However, the processes of turnover are largely hidden during periods of apparent steady-state protein abundance, and even when proteins accumulate it is unclear whether enhanced synthesis or decreased degradation is responsible. We have used a (15)N labeling strategy with inorganic nitrogen sources coupled to a two-dimensional fluorescence difference gel electrophoresis and mass spectrometry analysis of two-dimensional IEF/SDS-PAGE gel spots to define the rate of protein synthesis (K(S)) and degradation (K(D)) of Arabidopsis cell culture proteins. Through analysis of MALDI-TOF/TOF mass spectra from 120 protein spots, we were able to quantify K(S) and K(D) for 84 proteins across six functional groups and observe over 65-fold variation in protein degradation rates. K(S) and K(D) correlate with functional roles of the proteins in the cell and the time in the cell culture cycle. This approach is based on progressive (15)N labeling that is innocuous for the plant cells and, because it can be used to target analysis of proteins through the use of specific gel spots, it has broad applicability.

  15. Synthesis of water-dispersible zinc oxide quantum dots with antibacterial activity and low cytotoxicity for cell labeling

    NASA Astrophysics Data System (ADS)

    Hsu, Shan-hui; Lin, Ying Yi; Huang, Sherry; Lem, Kwok Wai; Huong Nguyen, Dinh; Lee, Dai Soo

    2013-11-01

    Typical photoluminescent semiconductor nanoparticles, called quantum dots (QDs), have potential applications in biological labeling. When used to label stem cells, QDs may impair the differentiation capacity of the stem cells. In this study, we synthesized zinc oxide (ZnO) QDs in methanol with an average size of ∼2 nm. We then employed two different types of polyethylene glycol (PEG) molecules (SH-PEG-NH2 and NH2-PEG-NH2) to conjugate ZnO QDs and made them water-dispersible. Fourier transform infrared spectroscopy spectra indicated the attachment of PEG molecules on ZnO QDs. No obvious size alteration was observed for ZnO QDs after PEG conjugation. The water-dispersible ZnO QDs still retained the antibacterial activity and fluorescence intensity. The cytotoxicity evaluation revealed that ZnO QDs at higher concentrations decreased cell viability but were generally safe at 30 ppm or below. Cell lines of hepatocytes (HepG2), osteoblasts (MC3T3-E1) and mesenchymal stem cells (MSCs) were successfully labeled by the water-dispersible ZnO QDs at 30 ppm. The ZnO QD-labeled MSCs maintained their stemness and differentiation capacity. Therefore, we conclude that the water-dispersible ZnO QDs developed in this study have antibacterial activity, low cytotoxicity, and proper labeling efficiency, and can be used to label a variety of cells including stem cells.

  16. Synthesis of GDP-mannose and mannosylglycerate from labeled mannose by genetically engineered Escherichia coli without loss of specific isotopic enrichment.

    PubMed

    Sampaio, Maria-Manuel; Santos, Helena; Boos, Winfried

    2003-01-01

    We report the construction of an Escherichia coli mutant that harbors two compatible plasmids and that is able to synthesize labeled 2-O-alpha-D-mannosyl-D-glycerate from externally added labeled mannose without the loss of specific isotopic enrichment. The strain carries a deletion in the manA gene, encoding phosphomannose isomerase. This deletion prevents the formation of fructose-6-phosphate from mannose-6-phosphate after the uptake of mannose from the medium by mannose-specific enzyme II of the phosphotransferase system (PtsM). The strain also has a deletion of the cps gene cluster that prevents the synthesis of colanic acid, a mannose-containing polymer. Plasmid-encoded phosphomannomutase (cpsG) and mannose-1-phosphate guanylyltransferase (cpsB) ensure the formation of GDP-mannose. A second plasmid harbors msg, a gene from Rhodothermus marinus that encodes mannosylglycerate synthase, which catalyzes the formation of 2-O-alpha-D-mannosyl-D-glycerate from GDP-mannose and endogenous glycerate. The rate-limiting step in 2-O-alpha-D-mannosyl-D-glycerate formation is the transfer of GDP-mannose to glycerate. 2-O-alpha-D-mannosyl-D-glycerate can be released from cells by treatment with cold-water shock. The final product is formed in a yield exceeding 50% the initial quantity of labeled mannose, including loss during preparation and paper chromatography.

  17. Synthesis of GDP-Mannose and Mannosylglycerate from Labeled Mannose by Genetically Engineered Escherichia coli without Loss of Specific Isotopic Enrichment

    PubMed Central

    Sampaio, Maria-Manuel; Santos, Helena; Boos, Winfried

    2003-01-01

    We report the construction of an Escherichia coli mutant that harbors two compatible plasmids and that is able to synthesize labeled 2-O-α-d-mannosyl-d-glycerate from externally added labeled mannose without the loss of specific isotopic enrichment. The strain carries a deletion in the manA gene, encoding phosphomannose isomerase. This deletion prevents the formation of fructose-6-phosphate from mannose-6-phosphate after the uptake of mannose from the medium by mannose-specific enzyme II of the phosphotransferase system (PtsM). The strain also has a deletion of the cps gene cluster that prevents the synthesis of colanic acid, a mannose-containing polymer. Plasmid-encoded phosphomannomutase (cpsG) and mannose-1-phosphate guanylyltransferase (cpsB) ensure the formation of GDP-mannose. A second plasmid harbors msg, a gene from Rhodothermus marinus that encodes mannosylglycerate synthase, which catalyzes the formation of 2-O-α-d-mannosyl-d-glycerate from GDP-mannose and endogenous glycerate. The rate-limiting step in 2-O-α-d-mannosyl-d-glycerate formation is the transfer of GDP-mannose to glycerate. 2-O-α-d-mannosyl-d-glycerate can be released from cells by treatment with cold-water shock. The final product is formed in a yield exceeding 50% the initial quantity of labeled mannose, including loss during preparation and paper chromatography. PMID:12514000

  18. [Microbial synthesis of deuterium labelled L-phenylalanine with different levels of isotopic enrichment by facultative methylotrophic bacterium Brevibacterium methylicum with RMP assimilation of carbon].

    PubMed

    Mosin, O V; Shvets, V I; Skladnev, D A; Ignatov, I

    2014-01-01

    The preparative microbial synthesis of amino acids labelled with stable isotopes, including deuterium ( 2 H), suitable for biomedical applications by methylotrophic bacteria was studied using L-phenylalanine as example. This amino acid is secreted by Gram-negative aerobic facultative methylotrophic bacteria Brevibacterium methylicum, assimilating methanol via ribulose-5-monophosphate (RMP) cycle of assimilation of carbon, The data on adaptation of L-phenylalanine secreted by methylotrophic bacterium В. methylicum to the maximal concentration of deuterium in the growth medium with 98% 2 Н 2 O and 2% [ 2 Н]methanol, and biosynthesis of deuterium labelled L-phenylalanine With different levels of enrichment are presented. The strain was adapted by means of plating initial cells on firm (2% agarose) minimal growth media with an increasing gradient of 2 Н 2 O concentration from 0; 24.5; 49.0; 73.5 up to 98% 2 Н 2 O followed by subsequent selection of separate colonies stable to the action of 2 Н 2 O. These colonies were capable to produce L-phenylalanine. L-phenylalanine was extracted from growth medium by extraction with isopropanol with the subsequent crystallization in ethanol (output 0.65 g/l). The developed method of microbial synthesis allows to obtain deuterium labelled L-phenylalanine with different levels of isotopic enrichment, depending on concentration of 2 Н 2 O in growth media, from 17% (on growth medium with 24,5% 2 Н 2 O) up to 75% (on growth medium with 98% 2 Н 2 O) of deuterium in the molecule that is confirmed with the data of the electron impact (EI) mass- spectrometry analysis of methyl ethers of N-dimethylamino(naphthalene)-5-sulfochloride (dansyl) phenylalanine in these experimental conditions.

  19. Synthesis and evaluation of an (18)F-labeled pyrimidine-pyridine amine for targeting CXCR4 receptors in gliomas.

    PubMed

    Demoin, Dustin Wayne; Shindo, Masahiro; Zhang, Hanwen; Edwards, Kimberly J; Serganova, Inna; Pillarsetty, Naga Vara Kishore; Lewis, Jason S; Blasberg, Ronald G

    2016-10-01

    Chemokine receptor-4 (CXCR4, fusin, CD184) is expressed on several tissues involved in immune regulation and is upregulated in many diseases including malignant gliomas. A radiolabeled small molecule that readily crosses the blood-brain barrier can aid in identifying CXCR4-expressing gliomas and monitoring CXCR4-targeted therapy. In the current work, we have synthesized and evaluated an [(18)F]-labeled small molecule based on a pyrimidine-pyridine amine for its ability to target CXCR4. The nonradioactive standards and the nitro precursor used in this study were prepared using established methods. An HPLC method was developed to separate the nitro-precursor from the nonradioactive standard and radioactive product. The nitro-precursor was radiolabeled with (18)F under inert, anhydrous conditions using the [(18)F]-kryptofix 2.2.2 complex to form the desired N-(4-(((6-[(18)F]fluoropyridin-2-yl)amino)methyl)benzyl)pyrimidin-2-amine ([(18)F]-3). The purified radiolabeled compound was used in serum stability, partition coefficient, cellular uptake, and in vivo cancer targeting studies. [(18)F]-3 was synthesized in 4-10% decay-corrected yield (to start of synthesis). [(18)F]-3 (tR ≈ 27 min) was separated from the precursor (tR ≈ 30 min) using a pentafluorophenyl column with an isocratic solvent system. [(18)F]-3 displayed acceptable serum stability over 2 h. The amount of [(18)F]-3 bound to the plasma proteins was determined to be > 97%. The partition coefficient (LogD7.4) is 1.4 ± 0.5. Competitive in vitro inhibition indicated 3 does not inhibit uptake of (67)Ga-pentixafor. Cell culture media incubation and ex vivo urine analysis indicate rapid metabolism of [(18)F]-3 into hydrophilic metabolites. Thus, in vitro uptake of [(18)F]-3 in CXCR4 overexpressing U87 cells (U87 CXCR4) and U87 WT indicated no specific binding. In vivo studies in mice bearing U87 CXCR4 and U87 WT tumors on the left and right shoulders were carried out using [(18)F]-3 and (68)Ga-pentixafor on

  20. Synthesis and evaluation of glycosylated octreotate analogues labeled with radioiodine and 211At via a tin precursor.

    PubMed

    Vaidyanathan, G; Affleck, D J; Schottelius, M; Wester, H; Friedman, H S; Zalutsky, M R

    2006-01-01

    Carbohydration of N-terminus and substitution of a threonine for the threoninol residue at the C-terminus of Tyr3-octreotide (TOC) has resulted in improved pharmacokinetics and tumor targeting of its radioiodinated derivatives. Yet, these peptides are very susceptible to in vivo deiodination due to the similarity of monoiodotyrosine (MIT) to thyroid hormone. The goal of this work was to develop octreotate analogues containing both a sugar moiety and a nontyrosine prosthetic group on which a radioiodine or 211At can be introduced. Solid-phase synthesis and subsequent modifications delivered an iodo standard of the target peptide N(alpha)-(1-deoxy-D-fructosyl)-N(epsilon)-(3-iodobenzoyl)-Lys0-octreotate (GIBLO) and the corresponding tin precursor N(alpha)-(1-deoxy-D-fructosyl)-N(epsilon)-[(3-tri-n-butylstannyl)benzoyl]-Lys0-octreotate (GTBLO). GIBLO displaced [125I]TOC from somatostatin receptor subtype 2 (SSTR2)-positive AR42J rat pancreatic tumor cell membranes with an IC50 of 0.46 +/- 0.05 nM suggesting that GIBLO retained affinity to SSTR2. GTBLO was radiohalogenated to [131I]GIBLO and N(alpha)-(1-deoxy-D-fructosyl)-N(epsilon)-(3-[211At]astatobenzoyl)-Lys0-octreotate ([211At]GABLO) in 21.2 +/- 4.9% and 46.8 +/- 9.5% radiochemical yields, respectively. From a paired-label internalization assay using D341 Med medulloblastoma cells, the maximum specific internalized radioactivity from [131I]GIBLO was 1.78 +/- 0.8% of input dose compared to 9.67 +/- 0.43% for N(alpha)-(1-deoxy-D-fructosyl)-[125I]iodo-Tyr3-octreotate ([125I]I-Gluc-TOCA). Over a 4 h period, the extent of internalization of [131I]GIBLO and [211At]GABLO was similar in this cell line. In D341 Med murine subcutaneous xenografts, the uptake of [125I]I-Gluc-TOCA at 0.5, 1 and 4 h was 21.5 +/- 4.0% ID/g, 18.8 +/- 7.7% ID/g, and 0.9 +/- 0.4% ID/g, respectively. In comparison, these values for [131I]GIBLO were 6.9 +/- 1.2% ID/g, 4.7 +/- 1.4% ID/g, and 0.8 +/- 0.5% ID/g. Both in vitro and in vivo catabolism

  1. Novel indium-111 labeled gastrin peptide analogues (MG-CL1-4): synthesis and quality control.

    PubMed

    Naqvi, Syed Ali-Raza; Khan, Zulfiqar Ali; Nagra, Saeed Ahmad; Yar, Muhammad; Sherazi, Tauqir A; Shahzad, Sohail Shahzad; Shah, Syed Qaiser; Mahmood, Nasir; Ishfaq, Malik Muhammad; Mather, Stephen John

    2013-03-01

    Radiolabeled neuropeptides are widely investigated to diagnose and therapy of tumors. These peptides get internalization after binding with particular receptors at the surface of cells and finally move to lysosome. Internalization into tumor cells helps in mapping the infected site. Minigastrin peptide analogues (MG-CL1-4) were synthesised and labeled with 111-In radioisotope under different sets of conditions for imaging CCk-2 receptor bearing tumors. Different parameters such as temperature (80-100°C), pH (4-12), incubation time (5-30 minutes) and dilution effect were investigated to get the maximum labeling yield and stability. The results indicated that MG-CL1-4 is successfully labeled with indium-111 at pH 4.5 with heating at 98°C for 15 minute. At these conditions i.e. heating, pH and incubation minimum oxidized and maximum labeling yield, more than 94 %, was obtained. The labeling stability was studied by incubating the radiolabeled complex for predefined time points in PBSA and blood serum. Results show that more than 90% radiolabeled MG-CL1-4 remained intact.

  2. Simplified NaCl based (68)Ga concentration and labeling procedure for rapid synthesis of (68)Ga radiopharmaceuticals in high radiochemical purity.

    PubMed

    Mueller, Dirk; Klette, Ingo; Baum, Richard P; Gottschaldt, M; Schultz, Michael K; Breeman, Wouter A P

    2012-08-15

    A simple sodium chloride (NaCl) based (68)Ga eluate concentration and labeling method that enables rapid, high-efficiency labeling of DOTA conjugated peptides in high radiochemical purity is described. The method utilizes relatively few reagents and comprises minimal procedural steps. It is particularly well-suited for routine automated synthesis of clinical radiopharmaceuticals. For the (68)Ga generator eluate concentration step, commercially available cation-exchange cartridges and (68)Ga generators were used. The (68)Ga generator eluate was collected by use of a strong cation exchange cartridge. 98% of the total activity of (68)Ga was then eluted from the cation exchange cartridge with 0.5 mL of 5 M NaCl solution containing a small amount of 5.5 M HCl. After buffering with ammonium acetate, the eluate was used directly for radiolabeling of DOTATOC and DOTATATE. The (68)Ga-labeled peptides were obtained in higher radiochemical purity compared to other commonly used procedures, with radiochemical yields greater than 80%. The presence of (68)Ge could not be detected in the final product. The new method obviates the need for organic solvents, which eliminates the required quality control of the final product by gas chromatography, thereby reducing postsynthesis analytical effort significantly. The (68)Ga-labeled products were used directly, with no subsequent purification steps, such as solid-phase extraction. The NaCl method was further evaluated using an automated fluid handling system and it routinely facilitates radiochemical yields in excess of 65% in less than 15 min, with radiochemical purity consistently greater than 99% for the preparation of (68)Ga-DOTATOC.

  3. Synthesis of a fluorescently labeled compound for the detection of arsenic-induced apoptotic HL60 cells.

    PubMed

    Femia, A Lis; Temprana, C Facundo; Amor, M Silvia; Grasselli, Mariano; Alonso, Silvia Del V

    2012-03-01

    Arsenic compounds have shown medical usefulness since they proved to be effective in causing complete remission of acute promyelocytic leukemia. In this work we obtained a fluorescently labeled arsenic compound that can be used with current fluorescence techniques for basic and applied research, focused on arsenic-induced apoptosis studies. This compound is an arsanilic acid bearing a covalently linked FITC that was chemically synthesized and characterized by fluorescence, UV-Vis, mass and FTIR spectrometry. In addition, we assessed its apoptotic activity as well as its fluorescent labeling properties in HL60 cell line as a leukemia cell model through flow cytometry. We obtained a compound with a 1:1 FITC:arsenic ratio and a 595 m/z, confirming its structure by FTIR. This compound proved to be useful at inducing apoptosis in the leukemia cell model and labeling this apoptotic cell population, in such a way that the highest FITC fluorescence correlated with the highest arsenic amount.

  4. Efficient, Traceless Semi-Synthesis of α-Synuclein Labeled with a Fluorophore/Thioamide FRET Pair

    PubMed Central

    Wissner, Rebecca F.; Wagner, Anne M.; Warner, John B.; Petersson, E. James

    2015-01-01

    We have shown that thioamides can be incorporated into proteins through semi-synthesis and used as probes to monitor structural changes. To date, our methods have required the presence of a cysteine at the peptide ligation site, which may not be present in the native peptide sequence. Here, we present a strategy for the semi-synthesis of thioproteins using homocysteine as a ligation point with subsequent masking as methionine, making the ligation “traceless.” PMID:26893537

  5. Gram-scale synthesis and efficient purification of 13C-labeled levoglucosan from 13C glucose.

    PubMed

    Alexander, Lisa; Hoyt, Caroline; Michalczyk, Ryszard; Wu, Ruilian; Thorn, Dave L; Silks, L A Pete

    2013-01-01

    (13)C-Labeled levoglucosan has been synthesized and purified in good yield, and on the gram scale in one step from commercially available (13)C glucose. This one-step protocol uses 2-chloro-1,3-dimethylimidazolinium chloride that serves to selectively activate the anomeric carbon toward substitution reactions. The labeled glucose is then smoothly converted to the anhydroglucose. Purification is efficiently achieved on large scale by chromatography on silica gel. Published 2012. This article is a US Government work and is in the public domain in the USA.

  6. Synthesis of carbon-14 and deuterium labeled N-nitroso-2 (3',7'-dimethyl-2',6'-octadienyl) aminoethanols

    USGS Publications Warehouse

    Abidi, S.L.; Idelson, A.L.

    1981-01-01

    Methods of preparation of carbon-14 and deuterium labeled N-nitroso-2(3,7-dimethyl-2,6-octadienyl) aminoethanols are described. The primary synthetic method involved alkylation of ethanolamine or ethylglycine with suitable chlorides and subsequent mild nitrosation. Isomeric 14C-nitrosamines were also prepared by selective -cleavage of the di-substituted ethanolamine with nitrous acid.

  7. Synthesis and biological evaluation of technetium-labeled D-glucose-MAG3 derivative as agent for tumor diagnosis.

    PubMed

    de Barros, André Luís Branco; Cardoso, Valbert Nascimento; Mota, Luciene das Graças; Leite, Elaine Amaral; Oliveira, Mônica Cristina de; Alves, Ricardo José

    2009-05-01

    A d-glucose-MAG(3) derivative was successfully synthesized and radiolabeled in high labeling yield. Biodistribution studies in Ehrlich tumor-bearing mice were performed. This compound showed high accumulation in tumor tissue with high tumor-to-muscle ratio and moderate tumor-to-blood ratio. Thus, d-glucose-MAG(3) is a potential agent for tumor diagnosis.

  8. Biochemical synthesis of uniformly (13)C-labeled diterpene hydrocarbons and their bioconversion to diterpenoid phytoalexins in planta.

    PubMed

    Ye, Zhongfeng; Nakagawa, Kazuya; Natsume, Masahiro; Nojiri, Hideaki; Kawaide, Hiroshi; Okada, Kazunori

    2017-06-01

    Phytocassanes and momilactones are the major diterpenoid phytoalexins inductively produced in rice as bioactive substances. Regardless of extensive studies on the biosynthetic pathways of these phytoalexins, bioconversion of diterpene hydrocarbons is not shown in planta. To elucidate the entire biosynthetic pathways of these phytoalexins, uniformly (13)C-labeled ent-cassadiene and syn-pimaradiene were enzymatically synthesized with structural verification by GC-MS and (13)C-NMR. Application of the (13)C-labeled substrates on rice leaves led to the detection of (13)C-labeled metabolites using LC-MS/MS. Further application of this method in the moss Hypnum plumaeforme and the nearest out-group of Oryza species Leersia perrieri, respectively, resulted in successful bioconversion of these labeled substrates into phytoalexins in these plants. These results demonstrate that genuine biosynthetic pathways from these diterpene hydrocarbons to the end product phytoalexins occur in these plants and that enzymatically synthesized [U-(13)C20] diterpene substrates are a powerful tool for chasing endogenous metabolites without dilution with naturally abundant unlabeled compounds.

  9. Universal chitosan-assisted synthesis of Ag-including heterostructured nanocrystals for label-free in situ SERS monitoring.

    PubMed

    Cai, Kai; Xiao, Xiaoyan; Zhang, Huan; Lu, Zhicheng; Liu, Jiawei; Li, Qin; Liu, Chen; Foda, Mohamed F; Han, Heyou

    2015-12-07

    A universal chitosan-assisted method was developed to synthesize various Ag-including heterostructured nanocrystals, in which chelation probably plays a vital role. The as-prepared Ag/Pd heterostructured nanocrystals show outstanding properties when used as bifunctional nanocomposites in label-free in situ SERS monitoring of Pd-catalyzed reaction.

  10. New Synthesis and Tritium Labeling of a Selective Ligand for Studying High-affinity γ-Hydroxybutyrate (GHB) Binding Sites

    PubMed Central

    Vogensen, Stine B.; Marek, Aleš; Bay, Tina; Wellendorph, Petrine; Kehler, Jan; Bundgaard, Christoffer; Frølund, Bente; Pedersen, Martin H.F.; Clausen, Rasmus P.

    2013-01-01

    3-Hydroxycyclopent-1-enecarboxylic acid (HOCPCA, 1) is a potent ligand for the high-affinity GHB binding sites in the CNS. An improved synthesis of 1 together with a very efficient synthesis of [3H]-1 is described. The radiosynthesis employs in situ generated lithium trimethoxyborotritide. Screening of 1 against different CNS targets establishes a high selectivity and we demonstrate in vivo brain penetration. In vitro characterization of [3H]-1 binding shows high specificity to the high-affinity GHB binding sites. PMID:24053696

  11. Synthesis and characterization of C@CdS dots in aqueous solution and their application in labeling human gastric carcinoma cells

    NASA Astrophysics Data System (ADS)

    Dong, Wei; Zhou, Siqi; Dong, Yan; Wang, Jingwen; Liu, Shuang; Zhu, Pengxia

    2015-03-01

    Colloidal carbon spheres coated with cadmium sulfide nanoparticle quantum dots (C@CdS dots) with the particle size smaller than 50 nm were synthesized by an aqueous approach. The effects of different reaction times, temperatures, and pH values were carefully investigated to optimize the synthesis conditions. The as-prepared C@CdS dots were linked with mouse anti-human carcinoembryonic antigen antibody and goat anti-mouse immunoglobulin (IgG) to directly and indirectly label fixed human gastric carcinoma cells, respectively. The cytotoxicity of the C@CdS dots was also tested using the human gastric carcinoma cells. No apparent cytotoxicity was observed, which suggested the potential application of the as-prepared C@CdS dots in bioimaging.

  12. Cell-based assay of MGAT2-driven diacylglycerol synthesis for profiling inhibitors: use of a stable isotope-labeled substrate and high-resolution LC/MS.

    PubMed

    Onorato, Joelle M; Chu, Ching-Hsuen; Ma, Zhengping; Kopcho, Lisa M; Chao, Hannguang J; Lawrence, R Michael; Cheng, Dong

    2015-03-01

    To demonstrate monoacylglycerol acyltransferase 2 (MGAT2)-mediated enzyme activity in a cellular context, cells of the murine secretin tumor cell-1 line of enteroendocrine origin were used to construct human MGAT2-expressing recombinant cell lines. Low throughput and utilization of radiolabeled substrate in a traditional TLC technique were circumvented by development of a high-resolution LC/MS platform. Monitoring incorporation of stable isotope-labeled D31-palmitate into diacylglycerol (DAG) allowed selective tracing of the cellular DAG synthesis activity. This assay format dramatically reduced background interference and increased the sensitivity and the signal window compared with the TLC method. Using this assay, several MGAT2 inhibitors from different chemotypes were characterized. The described cell-based assay adds a new methodology for the development and evaluation of MGAT2 inhibitors for the treatment of obesity and type 2 diabetes. Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.

  13. A semi-preparative enzymic synthesis of malonyl-CoA from [14C]acetate and 14CO2: labelling in the 1, 2 or 3 position.

    PubMed

    Roughan, G

    1994-06-01

    A semi-preparative enzymic synthesis of [1-14C]malonyl-CoA from [1-14C]acetate and bicarbonate, and of [3-14C]malonyl-CoA from Na2(14)CO3 and acetate, was achieved by using chloroplasts rapidly isolated from 7-8-day-old pea shoots. Around 70% of the [1-14C]acetate was converted into malonyl-CoA in 2-3 h, and the specific radioactivity of [3-14C]malonyl-CoA synthesized in the system was 25-30 Ci/mol. Reactions were monitored and labelled products were purified by h.p.l.c.

  14. Fluorescent vancomycin and terephthalate comodified europium-doped layered double hydroxides nanoparticles: synthesis and application for bacteria labelling

    NASA Astrophysics Data System (ADS)

    Sun, Jianchao; Fan, Hai; Wang, Nan; Ai, Shiyun

    2014-09-01

    Vancomycin (Van)- and terephthalate (TA)-comodified europium-doped layered double hydroxides (Van-TA-Eu-LDHs) nanoparticles were successfully prepared by a two-step method, in which, TA acted as a sensitizer to enhance the fluorescent property and Van was modified on the surface of LDH to act as an affinity reagent to bacteria. The obtained products were characterized by X-ray diffraction, transmission electron microscope and fluorescent spectroscopy. The results demonstrated that the prepared Van- and TA-comodified europium-doped layered double hydroxides (Van-TA-Eu-LDHs) nanoparticles with diameter of 50 nm in size showed highly efficient fluorescent property. Furthermore, due to the high affinity of Van to bacteria, the prepared Van-TA-Eu-LDHs nanoparticles showed efficient bacteria labelling by fluorescent property. The prepared nanoparticles may have wide applications in the biological fields, such as biomolecular labelling and cell imaging.

  15. Metabolism of nonessential N-15-labeled amino acids and the measurement of human whole-body protein synthesis rates

    NASA Technical Reports Server (NTRS)

    Stein, T. P.; Settle, R. G.; Albina, J. A.; Melnick, G.; Dempsey, D. T.

    1991-01-01

    Eight N-15-labeled nonessential amino acids plus (N-15)H4Cl were administered over a 10-h period to four healthy adult males using a primed-constant dosage regimen. The amount of N-15 excreted in the urine and the urinary ammonia, hippuric acid, and plasma alanine N-15 enrichments were measured. There was a high degree of consistency across subjects in the ordering of the nine compounds based on the fraction of N-15 excreted.

  16. Metabolism of nonessential N-15-labeled amino acids and the measurement of human whole-body protein synthesis rates

    NASA Technical Reports Server (NTRS)

    Stein, T. P.; Settle, R. G.; Albina, J. A.; Melnick, G.; Dempsey, D. T.

    1991-01-01

    Eight N-15-labeled nonessential amino acids plus (N-15)H4Cl were administered over a 10-h period to four healthy adult males using a primed-constant dosage regimen. The amount of N-15 excreted in the urine and the urinary ammonia, hippuric acid, and plasma alanine N-15 enrichments were measured. There was a high degree of consistency across subjects in the ordering of the nine compounds based on the fraction of N-15 excreted.

  17. Synthesis, Radiolabelling and In Vitro Characterization of the Gallium-68-, Yttrium-90- and Lutetium-177-Labelled PSMA Ligand, CHX-A''-DTPA-DUPA-Pep.

    PubMed

    Baur, Benjamin; Solbach, Christoph; Andreolli, Elena; Winter, Gordon; Machulla, Hans-Jürgen; Reske, Sven N

    2014-04-29

    Since prostate-specific membrane antigen (PSMA) has been identified as a diagnostic target for prostate cancer, many urea-based small PSMA-targeting molecules were developed. First, the clinical application of these Ga-68 labelled compounds in positron emission tomography (PET) showed their diagnostic potential. Besides, the therapy of prostate cancer is a demanding field, and the use of radiometals with PSMA bearing ligands is a valid approach. In this work, we describe the synthesis of a new PSMA ligand, CHX-A''-DTPA-DUPA-Pep, the subsequent labelling with Ga-68, Lu-177 and Y-90 and the first in vitro characterization. In cell investigations with PSMA-positive LNCaP C4-2 cells, KD values of ≤14.67 ± 1.95 nM were determined, indicating high biological activities towards PSMA. Radiosyntheses with Ga-68, Lu-177 and Y-90 were developed under mild reaction conditions (room temperature, moderate pH of 5.5 and 7.4, respectively) and resulted in nearly quantitative radiochemical yields within 5 min.

  18. Chemo-Enzymatic Synthesis of (13)C Labeled Complex N-Glycans As Internal Standards for the Absolute Glycan Quantification by Mass Spectrometry.

    PubMed

    Echeverria, Begoña; Etxebarria, Juan; Ruiz, Nerea; Hernandez, Álvaro; Calvo, Javier; Haberger, Markus; Reusch, Dietmar; Reichardt, Niels-Christian

    2015-11-17

    Methods for the absolute quantification of glycans are needed in glycoproteomics, during development and production of biopharmaceuticals and for the clinical analysis of glycan disease markers. Here we present a strategy for the chemo-enzymatic synthesis of (13)C labeled N-glycan libraries and provide an example for their use as internal standards in the profiling and absolute quantification of mAb glycans by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. A synthetic biantennary glycan precursor was (13)C-labeled on all four amino sugar residues and enzymatically derivatized to produce a library of 15 glycan isotopologues with a mass increment of 8 Da over the natural products. Asymmetrically elongated glycans were accessible by performing enzymatic reactions on partially protected UV-absorbing intermediates, subsequent fractionation by preparative HPLC, and final hydrogenation. Using a preformulated mixture of eight internal standards, we quantified the glycans in a monoclonal therapeutic antibody with excellent precision and speed.

  19. One-pot synthesis of quantum dot-labeled hydrophilic molecularly imprinted polymer nanoparticles for direct optosensing of folic acid in real, undiluted biological samples.

    PubMed

    Yang, Yaqiong; Wang, Zhengzheng; Niu, Hui; Zhang, Huiqi

    2016-12-15

    A facile and efficient one-pot approach for the synthesis of quantum dot (QD)-labeled hydrophilic molecularly imprinted polymer (MIP) nanoparticles for direct optosensing of folic acid (FA) in the undiluted bovine and porcine serums is described. Hydrophilic macromolecular chain transfer agent-mediated reversible addition-fragmentation chain transfer (RAFT) precipitation polymerization was used to implement the molecular imprinting of FA in the presence of CdTe quantum dots (QDs). The resulting FA-imprinted polymer nanoparticles with surface-grafted hydrophilic poly(glyceryl monomethacrylate) brushes and QDs labeling not only showed outstanding specific molecular recognition toward FA in biological samples, but also exhibited good photostability, rapid binding kinetics, and obvious template binding-induced fluorescence quenching. These characteristics make them a useful fluorescent chemosensor for directly and selectively optosensing FA in the undiluted bovine and porcine serums, with its limit of detection being 0.025μM and average recoveries ranging from 98% to 102%, even in the presence of several interfering compounds. This advanced fluorescent MIP chemosensor is highly promising for rapid quantification of FA in such applications as clinical diagnostics and food analysis.

  20. Synthesis, Radiolabelling and In Vitro Characterization of the Gallium-68-, Yttrium-90- and Lutetium-177-Labelled PSMA Ligand, CHX-A''-DTPA-DUPA-Pep

    PubMed Central

    Baur, Benjamin; Solbach, Christoph; Andreolli, Elena; Winter, Gordon; Machulla, Hans-Jürgen; Reske, Sven N.

    2014-01-01

    Since prostate-specific membrane antigen (PSMA) has been identified as a diagnostic target for prostate cancer, many urea-based small PSMA-targeting molecules were developed. First, the clinical application of these Ga-68 labelled compounds in positron emission tomography (PET) showed their diagnostic potential. Besides, the therapy of prostate cancer is a demanding field, and the use of radiometals with PSMA bearing ligands is a valid approach. In this work, we describe the synthesis of a new PSMA ligand, CHX-A''-DTPA-DUPA-Pep, the subsequent labelling with Ga-68, Lu-177 and Y-90 and the first in vitro characterization. In cell investigations with PSMA-positive LNCaP C4-2 cells, KD values of ≤14.67 ± 1.95 nM were determined, indicating high biological activities towards PSMA. Radiosyntheses with Ga-68, Lu-177 and Y-90 were developed under mild reaction conditions (room temperature, moderate pH of 5.5 and 7.4, respectively) and resulted in nearly quantitative radiochemical yields within 5 min. PMID:24787458

  1. Methanol synthesis via CO₂ hydrogenation over a Au/ZnO catalyst: an isotope labelling study on the role of CO in the reaction process.

    PubMed

    Hartadi, Yeusy; Widmann, Daniel; Behm, R Jürgen

    2016-04-28

    Methanol synthesis for chemical energy storage, via hydrogenation of CO2 with H2 produced by renewable energies, is usually accompanied by the undesired formation of CO via the reverse water-gas shift reaction. Aiming at a better mechanistic understanding of methanol formation from CO2/H2 on highly selective supported Au/ZnO catalysts we have investigated the role of CO in the reaction process using isotope labelling experiments. Using (13)C-labelled CO2, we found for reaction at 5 bar and 240 °C that (i) the methanol formation rate is significantly higher in CO2-containing gas mixtures than in a CO2-free mixture and (ii) in mixtures containing both CO2 and CO methanol formation from CO increases with the CO content up to 1% CO, and then remains at 20% of the total methanol formation up to a CO2/CO ratio of 1/1, making CO2 the preferred carbon source in these mixtures. A shift in the preferred carbon source for MeOH from CO2 towards CO is observed with increasing reaction temperatures between 240 °C and 300 °C. At even higher temperatures CO is expected to become the dominant carbon source. The consequences of these findings for the application of Au/ZnO catalysts for chemical storage of renewable energies are discussed.

  2. Synthesis of four carbon-13-labeled type a trichothecene mycotoxins and their application as internal standards in stable isotope dilution assays.

    PubMed

    Asam, Stefan; Rychlik, Michael

    2006-09-06

    The first stable isotope dilution assay (SIDA) for the simultaneous quantitation of the most abundant type A trichothecenes in foods and feeds was developed. Synthesis of carbon-13-labeled T2-toxin, HT2-toxin, diacetoxyscirpenol, and monoacetoxyscirpenol was accomplished by [13C2]-acetylation of T2-triol and scirpentriol, respectively. Scirpentriol was prepared from diacetoxyscirpenol by complete alkaline hydrolysis and subsequently was converted to [13C6]-triacetoxyscirpentriol by peracetylation with [13C4]-acetic anhydride. The latter compound was selectively hydrolyzed using ammonium hydroxide to give [13C4]-diacetoxyscirpenol and [13C2]-monoacetoxyscirpenol in reasonable yields. Analogously, [13C6]-T2-triacetate was prepared from T2-triol and subjected to controlled hydrolysis to yield [13C4]-T2-toxin and [13C2]-HT2-toxin. All synthesized products were characterized by NMR and MS experiments. Using the prepared isotopically labeled standards, SIDAs were developed for the quantitation of type A trichothecenes in food and feeds. The mycotoxins were quantified by LC-single and tandem MS after cleanup on multifunctional columns. The method revealed good sensitivity with low detection and quantification limits along with excellent recovery and good precision in interassay studies. Food samples were analyzed using the developed SIDA and showed substantial contamination of oat products with T2-toxin and HT2-toxin. Diacetoxyscirpenol was detected on potatoes, whereas monoacetoxyscirpenol was not present in the analyzed samples.

  3. Synthesis of 81Br-labeled polybrominated diphenyl ethers and their characterization using GC(EI)MS and GC(ICP)MS.

    PubMed

    González-Gago, Adriana; Marchante-Gayón, Juan Manuel; Ferrero, Miguel; Garcia Alonso, J Ignacio

    2010-04-01

    A mixture of different (81)Br-labeled polybrominated diphenyl ethers (PBDEs) was prepared and characterized for its future use as spike for the isotope dilution analysis of PBDEs. The synthesis was carried out by direct bromination of diphenyl ether using (81)Br enriched Br(2) obtained after aqueous oxidation of bromide with potassium peroxymonosulfate and extraction into dichloromethane. The number of bromine atoms introduced in the diphenyl ether molecule depended on the molar ratio between bromine and diphenyl ether. The final mixture prepared contained a mixture of tri-, tetra-, penta-, and hexabrominated PBDEs with a larger concentration of the tetrabrominated congener BDE-47. The isotopic composition of bromine in the resulting PBDEs mixture was determined by GC(ICP)MS and resulted in a 99.53% enrichment of the isotope 81 of bromine. The concentration of three of the PBDE congeners (28, 47, and 99) in the mixture was determined by reverse isotope dilution analysis using a certified, natural abundance, PBDEs mixture and both GC(ICP)MS and GC(EI)MS. For this purpose, the fragmentation and isotope distribution patterns of the different PBDE cogeners in the positive electron ionization source were studied in detail both for natural abundance and labeled compounds. A procedure based on isotope pattern deconvolution was developed which allowed the direct determination of the concentration of the labeled PBDEs in the spike mixture by GC(EI)MS. Finally, the GC(EI)MS isotope pattern deconvolution procedure was applied for the determination of natural abundance congeners 28, 47, and 99 in spiked waters at ng L(-1) levels. Detection limits below 0.5 ng L(-1) could be obtained for all compounds using only 100 mL of sample and liquid-liquid extraction with isooctane.

  4. In situ DNA-templated synthesis of silver nanoclusters for ultrasensitive and label-free electrochemical detection of microRNA.

    PubMed

    Yang, Cuiyun; Shi, Kai; Dou, Baoting; Xiang, Yun; Chai, Yaqin; Yuan, Ruo

    2015-01-21

    On the basis of the use of silver nanoclusters (AgNCs) in situ synthesized by cytosine (C)-rich loop DNA templates as signal amplification labels, the development of a label-free and highly sensitive method for electrochemical detection of microRNA (miRNA-199a) is described. The target miRNA-199a hybridizes with the partial dsDNA probes to initiate the target-assisted polymerization nicking reaction (TAPNR) amplification to produce massive intermediate sequences, which can be captured on the sensing electrode by the self-assembled DNA secondary probes. These surface-captured intermediate sequences further trigger the hybridization chain reaction (HCR) amplification to form dsDNA polymers with numerous C-rich loop DNA templates on the electrode surface. DNA-templated synthesis of AgNCs can be realized by subsequent incubation of the dsDNA polymer-modified electrode with AgNO3 and sodium borohydride. With this integrated TAPNR and HCR dual amplification strategy, the amount of in situ synthesized AgNCs is dramatically enhanced, leading to substantially amplified current response for highly sensitive detection of miRNA-199a down to 0.64 fM. In addition, the developed method also shows high selectivity toward the target miRNA-199a. Featured with high sensitivity and label-free capability, the proposed sensing scheme can thus offer new opportunities for achieving sensitive, selective, and simple detection of different types of microRNA targets.

  5. Food Labels

    MedlinePlus

    ... Loss Surgery? A Week of Healthy Breakfasts Shyness Food Labels KidsHealth > For Teens > Food Labels Print A ... have at least 95% organic ingredients. continue Making Food Labels Work for You The first step in ...

  6. Synthesis of a potent and selective (18)F-labeled delta-opioid receptor antagonist derived from the Dmt-Tic pharmacophore for positron emission tomography imaging.

    PubMed

    Ryu, Eun Kyoung; Wu, Zhanhong; Chen, Kai; Lazarus, Lawrence H; Marczak, Ewa D; Sasaki, Yusuke; Ambo, Akihiro; Salvadori, Severo; Ren, Chuancheng; Zhao, Heng; Balboni, Gianfranco; Chen, Xiaoyuan

    2008-03-27

    Identification and pharmacological characterization of two new selective delta-opioid receptor antagonists, derived from the Dmt-Tic pharmacophore, of potential utility in positron emission tomography (PET) imaging are described. On the basis of its high delta selectivity, H-Dmt-Tic--Lys(Z)-OH (reference compound 1) is a useful starting point for the synthesis of (18)F-labeled compounds prepared by the coupling of N-succinimidyl 4-[ (18)F]fluorobenzoate ([(18)F]SFB) with Boc-Dmt-Tic--Lys(Z)-OH under slightly basic conditions at 37 degrees C for 15 min, deprotection with TFA, and HPLC purification. The total synthesis time was 120 min, and the decay-corrected radiochemical yield of [(18)F]- 1 was about 25-30% ( n = 5) starting from [(18)F]SFB ( n = 5) with an effective specific activity about 46 GBq/micromol. In vitro autoradiography studies showed prominent uptake of [ (18)F]- 1 in the striatum and cortex with significant blocking by 1 and UFP-501 (selective delta-opioid receptor antagonist), suggesting high specific binding of [(18)F]- 1 to delta-opioid receptors. Noninvasive microPET imaging studies revealed the absence of [(18)F]- 1 in rat brain, since it fails to cross the blood-brain barrier. This study demonstrates the suitability of [ (18)F]- 1 for imaging peripheral delta-opioid receptors.

  7. Synthesis and in vitro and in vivo evaluation of an (18)F-labeled neuropeptide Y analogue for imaging of breast cancer by PET.

    PubMed

    Hofmann, Sven; Maschauer, Simone; Kuwert, Torsten; Beck-Sickinger, Annette G; Prante, Olaf

    2015-04-06

    Imaging of Y1R expression in breast cancer is still a challenging task. Herein, we report a suitable (18)F-labeled high-molecular-weight glycopeptide for imaging of peripheral neuropeptide Y (NPY) Y1 receptor (Y1R)-positive tumors by preclinical small-animal positron emission tomography (PET). The Y1R-preferring NPY [F(7),P(34)]NPY analogue was functionalized with an alkyne-bearing propargylglycine (Pra) in position 4. The corresponding fluoroglycosylated (FGlc) peptide analogue [Pra(4)(FGlc),F(7),P(34)]NPY and its (18)F-labeled analogue were synthesized by click chemistry-based fluoroglycosylation. The radiosynthesis was performed by (18)F-fluoroglycosylation starting from the 2-triflate of the β-mannosylazide and the alkyne peptide [Pra(4),F(7),P(34)]NPY. The radiosynthesis of the(18)F-labeled analogue was optimized using a minimum amount of peptide precursor (40 nmol), proceeding with an overall radiochemical yield of 20-25% (nondecay corrected) in a total synthesis time of 75 min with specific activities of 40-70 GBq/μmol. In comparison to NPY and [F(7),P(34)]NPY, in vitro Y1R and Y2R activation studies with the cold [Pra(4)(FGlc),F(7),P(34)]NPY on stably transfected COS-7 cells displayed a high potency for the induction of Y1R-specific inositol accumulation (pEC50 = 8.5 ± 0.1), whereas the potency at Y2R was significantly decreased. Internalization studies on stably transfected HEK293 cells confirmed a strong glycopeptide-mediated Y1R internalization and a substantial Y1R subtype selectivity over Y2R. In vitro autoradiography with Y1R-positive MCF-7 tumor tissue slices indicated high specific binding of the (18)F-labeled glycopeptide, when binding was reduced by 95% ([Pra(4),F(7),P(34)]NPY) and by 86% (BIBP3226 Y1R antagonist) in competition studies. Biodistribution and small-animal PET studies on MCF-7 breast tumor-bearing nude mice revealed radiotracer uptake in the MCF-7 tumor of 1.8%ID/g at 20 min p.i. and 0.7%ID/g at 120 min p.i. (n = 3-4), increasing

  8. Synthesis of /sup 13/C-labeled standards for use in coal liquefaction studies. II. Dissolving metal reactions applied to naphthalenes and indoles: effect of sonication

    SciTech Connect

    Pickering, R.E.

    1986-01-01

    High yield syntheses of /sup 13/C-labeled standards for later use in coal liquefaction studies are described. An alternate route for the synthesis of 1-cyclohexanone-1-/sup 13/C is discussed. Naphthalene and 2,3-dimethylnaphthalene were reduced with metal-amine solutions. The effect of sonication on product distribution was investigated. The effect of different amine solvent and different metals also was studied. A series of indoles were reduced with lithium and ethylenediamine to study the effect of sonication. To aid identification of some reduction products, independent syntheses were conducted. Naphthalenol-1-/sup 13/C, phenol-1-/sup 13/C, 1-indanone-1-/sup 13/C, 3,4-dihydro-2(1H)-quinolinone-2-/sup 13/C, 1,2,3,4-tetrahydroquinoline-2-/sup 13/C and carbazole-9a-/sup 13/C were synthesized in good yield. The carbonation of 1,5-pentanedimagnesium bromide was shown to be a viable alternative route for the synthesis of 1-cyclohexanone-1-/sup 13/C. Sonication was found to be an efficient agitation method for the reductive dimerization or the reductive amination of naphthalene and 2,3-dimethylnaphthalene. The product distribution was found to be dependent on the selection of the amine solvent. Sodium was shown to be a better reagent for reductive dimerization than potassium, lithium, calcium, or magnesium. The synthesis of 6,6',7,7'-tetramethyl-,1'-binaphthyl, 6,6',7,7'-tetramethyl-1,2'-binaphthyl, 6,6',7,7'-tetramethyl-2,2'-binaphthyl and some of their hydro-derivatives are described. The reduction of indoles with lithium-ethylenediamine was found to be influenced by sonication which significant increased the yield of the 4,5,6,7-tetrahydro-derivative. N-Substituted indoles were shown to undergo reductive cleavage with lithium-ethylenediamine while being treated with ultrasound.

  9. Chemoenzymatic Strategy for the Synthesis of Site-Specifically Labeled Immunoconjugates for Multimodal PET and Optical Imaging

    PubMed Central

    2015-01-01

    The complementary nature of positron emission tomography (PET) and optical imaging (OI) has fueled increasing interest in the development of multimodal PET/OI probes that can be employed during the diagnosis, staging, and surgical treatment of cancer. Due to their high selectivity and affinity, antibodies have emerged as promising platforms for the development of hybrid PET/OI agents. However, the lack of specificity of many bioconjugation reactions can threaten immunoreactivity and lead to poorly defined constructs. To circumvent this issue, we have developed a chemoenzymatic strategy for the construction of multimodal PET/OI immunoconjugates that have been site-specifically labeled on the heavy chain glycans. The methodology consists of four steps: (1) the enzymatic removal of the terminal galactose residues on the heavy chain glycans; (2) the enzymatic incorporation of azide-bearing galactose (GalNAz) residues into the heavy chain glycans; (3) the strain-promoted click conjugation of chelator- and fluorophore-modified dibenzocyclooctynes to the azide-modified sugars; and (4) the radiolabeling of the immunoconjugate. For proof-of-concept, a model system was created using the colorectal cancer-targeting antibody huA33, the chelator desferrioxamine (DFO), the positron-emitting radiometal 89Zr, and the near-infrared fluorescent dye Alexa Fluor 680. The bioconjugation strategy is robust and reproducible, reliably producing well-defined and immunoreactive conjugates labeled with 89Zr, Alexa Fluor 680, or an easily and precisely tuned mixture of the two reporters. In in vivo PET and fluorescence imaging experiments, a hybrid 89Zr- and Alexa Fluor 680-labeled huA33 conjugate displayed high levels of specific uptake (>45% ID/g) in athymic nude mice bearing A33 antigen-expressing SW1222 colorectal cancer xenografts. PMID:25418333

  10. Chemoenzymatic strategy for the synthesis of site-specifically labeled immunoconjugates for multimodal PET and optical imaging.

    PubMed

    Zeglis, Brian M; Davis, Charles B; Abdel-Atti, Dalya; Carlin, Sean D; Chen, Aimei; Aggeler, Robert; Agnew, Brian J; Lewis, Jason S

    2014-12-17

    The complementary nature of positron emission tomography (PET) and optical imaging (OI) has fueled increasing interest in the development of multimodal PET/OI probes that can be employed during the diagnosis, staging, and surgical treatment of cancer. Due to their high selectivity and affinity, antibodies have emerged as promising platforms for the development of hybrid PET/OI agents. However, the lack of specificity of many bioconjugation reactions can threaten immunoreactivity and lead to poorly defined constructs. To circumvent this issue, we have developed a chemoenzymatic strategy for the construction of multimodal PET/OI immunoconjugates that have been site-specifically labeled on the heavy chain glycans. The methodology consists of four steps: (1) the enzymatic removal of the terminal galactose residues on the heavy chain glycans; (2) the enzymatic incorporation of azide-bearing galactose (GalNAz) residues into the heavy chain glycans; (3) the strain-promoted click conjugation of chelator- and fluorophore-modified dibenzocyclooctynes to the azide-modified sugars; and (4) the radiolabeling of the immunoconjugate. For proof-of-concept, a model system was created using the colorectal cancer-targeting antibody huA33, the chelator desferrioxamine (DFO), the positron-emitting radiometal (89)Zr, and the near-infrared fluorescent dye Alexa Fluor 680. The bioconjugation strategy is robust and reproducible, reliably producing well-defined and immunoreactive conjugates labeled with (89)Zr, Alexa Fluor 680, or an easily and precisely tuned mixture of the two reporters. In in vivo PET and fluorescence imaging experiments, a hybrid (89)Zr- and Alexa Fluor 680-labeled huA33 conjugate displayed high levels of specific uptake (>45% ID/g) in athymic nude mice bearing A33 antigen-expressing SW1222 colorectal cancer xenografts.

  11. Ibogaine labeling with 99mTc-tricarbonyl: synthesis and transport at the mouse blood-brain barrier.

    PubMed

    Tournier, Nicolas; André, Pascal; Blondeel, Sandy; Rizzo-Padoin, Nathalie; du Moulinet d'Hardemarre, Amaury; Declèves, Xavier; Scherrmann, Jean-Michel; Cisternino, Salvatore

    2009-12-01

    The (99m)Tc-tricarbonyl core may be used as an ideal tool for gamma-labeling ligands in noninvasive SPECT imaging. However, most (99m)Tc-tricarbonyl-labeled agents have difficulty crossing the blood-brain barrier (BBB). We radiolabeled the neuroactive indole ibogaine with (99m)Tc-tricarbonyl and measured its transport into the mouse brain by in situ brain perfusion. We measured the interactions of [(99m)Tc(CO)(3)-ibogaine](+) and (99m)Tc-tricarbonyl with the main BBB efflux transporters P-gp and BCRP in vitro and in vivo. Ibogaine was radiolabeled (yield: over 95%). [(99m)Tc(CO)(3)-ibogaine](+) entered the brain (K(in)) poorly (0.18 microL/g/s), at about the same rate as (99m)Tc-tricarbonyl (0.16 microL/g/s) and [(99m)Tc-sestamibi](+) (0.10 microL/g/s). The CNS tracer [(99m)Tc-HMPAO](0) entered the brain approximately 70-times higher than [(99m)Tc(CO)(3)-ibogaine](+). In vitro studies revealed that neither [(99m)Tc(CO)(3)-ibogaine](+) nor (99m)Tc-tricarbonyl ion were substrates for P-gp or BCRP. But lowering the membrane dipole potential barrier with phloretin enhanced the brain transport of [(99m)Tc(OH(2))(3)(CO)(3)](+) approximately 3-fold. Thus, ibogaine directly labeled with (99m)Tc-tricarbonyl is not suitable for CNS imaging because of its poor uptake. Brain transport is not restricted by efflux transporters but is reduced by its lipophilicity and interaction with the membrane-positive dipole potential.

  12. Continuous-Flow Synthesis of Deuterium-Labeled Antidiabetic Chalcones: Studies towards the Selective Deuteration of the Alkynone Core.

    PubMed

    Ötvös, Sándor B; Hsieh, Chi-Ting; Wu, Yang-Chang; Li, Jih-Heng; Chang, Fang-Rong; Fülöp, Ferenc

    2016-03-07

    Flow chemistry-based syntheses of deuterium-labeled analogs of important antidiabetic chalcones were achieved via highly controlled partial C≡C bond deuteration of the corresponding 1,3-diphenylalkynones. The benefits of a scalable continuous process in combination with on-demand electrolytic D2 gas generation were exploited to suppress undesired over-reactions and to maximize reaction rates simultaneously. The novel deuterium-containing chalcone derivatives may have interesting biological effects and improved metabolic properties as compared with the parent compounds.

  13. Intrinsically germanium-69-labeled iron oxide nanoparticles: synthesis and in-vivo dual-modality PET/MR imaging.

    PubMed

    Chakravarty, Rubel; Valdovinos, Hector F; Chen, Feng; Lewis, Christina M; Ellison, Paul A; Luo, Haiming; Meyerand, M Elizabeth; Nickles, Robert J; Cai, Weibo

    2014-08-13

    Intrinsically germanium-69-labeled super-paramagnetic iron oxide nanoparticles are synthesized via a newly developed, fast and highly specific chelator-free approach. The biodistribution pattern and the feasibility of (69) Ge-SPION@PEG for in vivo dual-modality positron emission tomography/magnetic resonance (PET/MR) imaging and lymph-node mapping are investigated, which represents the first example of the successful utilization of a (69) Ge-based agent for PET/MR imaging. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Synthesis and site-directed fluorescence labeling of azido proteins using eukaryotic cell-free orthogonal translation systems.

    PubMed

    Quast, Robert B; Claussnitzer, Iris; Merk, Helmut; Kubick, Stefan; Gerrits, Michael

    2014-04-15

    Eukaryotic cell-free systems based on wheat germ and Spodoptera frugiperda insect cells were equipped with an orthogonal amber suppressor tRNA-synthetase pair to synthesize proteins with a site-specifically incorporated p-azido-l-phenylalanine residue in order to provide their chemoselective fluorescence labeling with azide-reactive dyes by Staudinger ligation. The specificity of incorporation and bioorthogonality of labeling within complex reaction mixtures was shown by means of translation and fluorescence detection of two model proteins: β-glucuronidase and erythropoietin. The latter contained the azido amino acid in proximity to a signal peptide for membrane translocation into endogenous microsomal vesicles of the insect cell-based system. The results indicate a stoichiometric incorporation of the azido amino acid at the desired position within the proteins. Moreover, the compatibility of cotranslational protein translocation, including glycosylation and amber suppression-based incorporation of p-azido-l-phenylalanine within a cell-free system, is demonstrated. The presented approach should be particularly useful for providing eukaryotic and membrane-associated proteins for investigation by fluorescence-based techniques.

  15. PCR synthesis of double stranded DNA labeled with 5-bromouridine. A step towards finding a bromonucleoside for clinical trials.

    PubMed

    Michalska, Barbara; Sobolewski, Ireneusz; Polska, Katarzyna; Zielonka, Justyna; Zylicz-Stachula, Agnieszka; Skowron, Piotr; Rak, Janusz

    2011-12-05

    Incorporation of 5-bromouridine (5BrdU) into DNA makes it sensitive to UV and ionizing radiation, which opens up a prospective route for the clinical usage of 5-bromouridine and other halonucleosides. In the present work the polymerase chain reaction (PCR) protocol, which enables a long DNA fragment (resembling DNA synthesized in the cell in the presence of halonucleosides) to be completely substituted with 5BrdU, was optimized. Using HPLC coupled to enzymatic digestion, it was demonstrated that the actual amounts of native nucleosides and 5BrdU correspond very well to those calculated from the sequence of PCR products. The synthesized DNA is photosensitive to photons of 300nm. HPLC analysis demonstrated that the photolysis of labeled PCR products leads to a significant decrease in the 5BrdU signal and the simultaneous occurrence of a uridine peak. Agarose and polyacrylamide gel electrophoresis suggest that single strand breaks and cross-links are formed as a result of UV irradiation. The PCR protocol described in the current paper may be employed for labeling DNA not only with BrdU but also with other halonucleosides.

  16. Synthesis and biological evaluation of (68) Ga-labeled Pteroyl-Lys conjugates for folate receptor-targeted tumor imaging.

    PubMed

    Zhang, Xuran; Yu, Qian; He, Yingfang; Zhang, Chun; Zhu, Hua; Yang, Zhi; Lu, Jie

    2016-07-01

    In order to develop novel (68) Ga-labeled PET tracers for folate receptor imaging, two DOTA-conjugated Pteroyl-Lys derivatives, Pteroyl-Lys-DOTA and Pteroyl-Lys-DAV-DOTA, were designed, synthesized and radiolabeled with (68) Ga. Biological evaluations of the two radiotracers were performed with FR-positive KB cell line and athymic nude mice bearing KB tumors. Both (68) Ga-DOTA-Lys-Pteroyl and (68) Ga-DOTA-DAV-Lys-Pteroyl exhibited receptor specific binding in KB cells in vitro. The tumor uptake values of (68) Ga-DOTA-Lys-Pteroyl and (68) Ga-DOTA-DAV-Lys-Pteroy were 10.06 ± 0.59%ID/g and 11.05 ± 0.60%ID/g at 2 h post-injection, respectively. Flank KB tumor was clearly visualized with (68) Ga-DOTA-DAV-Lys-Pteroyl by Micro-PET imaging at 2 h post-injection, suggesting the feasibility of using (68) Ga-labeled Pteroyl-Lys conjugates as a novel class of FR targeted probes.

  17. Synthesis and biosynthesis of {sup 13}C-, {sup 15}N-labeled deoxynucleosides useful for biomolecular structural determinations

    SciTech Connect

    Ashburn, D.A.; Garcia, K.; Hanners, J.L.; Silks, L.A. III; Unkefer, C.J.

    1994-12-01

    Currently, there is a great emphasis on elucidating the structure, function, and dynamics of DNA. Much of the research involved in this study uses nuclear magnetic resonance (NMR) spectroscopy. Effective use of NMR spectroscopy for DNA molecules with mw > 10,000 requires stable isotope enrichment. We present strategies for site-specific isotopic labeling of the purine bases adenosine and guanosine and the biosynthesis of (U-{sup 13}C, {sup 15}N) DNA from methylotropic bacteria. With commercially available 6-chloropurine, an effective two-step route leads to 2{prime}-deoxy-(amino-{sup 15}N)adenosine (dA). The resulting d(amino-{sup 15}N)A is used in a series of reactions to synthesize 2{prime}-deoxy-(2-{sup 13}C,1,amino-{sup 15}N{sub 2})guanosine or any combination thereof. An improved biosynthesis of labeled DNA has been accomplished using Methylobacterium extorquens AS1. Each liter of growth medium contains 4 g of methanol to yield 1 g of lyophilized cells. As much as 200 mg of RNA per liter of culture has been obtained. We are currently developing large-scale isolation protocols. General synthetic pathways to oligomeric DNA will be presented.

  18. Quantifying Protein Synthesis and Degradation in Arabidopsis by Dynamic 13CO2 Labeling and Analysis of Enrichment in Individual Amino Acids in Their Free Pools and in Protein1[OPEN

    PubMed Central

    Fernie, Alisdair R.; Stitt, Mark

    2015-01-01

    Protein synthesis and degradation represent substantial costs during plant growth. To obtain a quantitative measure of the rate of protein synthesis and degradation, we supplied 13CO2 to intact Arabidopsis (Arabidopsis thaliana) Columbia-0 plants and analyzed enrichment in free amino acids and in amino acid residues in protein during a 24-h pulse and 4-d chase. While many free amino acids labeled slowly and incompletely, alanine showed a rapid rise in enrichment in the pulse and a decrease in the chase. Enrichment in free alanine was used to correct enrichment in alanine residues in protein and calculate the rate of protein synthesis. The latter was compared with the relative growth rate to estimate the rate of protein degradation. The relative growth rate was estimated from sequential determination of fresh weight, sequential images of rosette area, and labeling of glucose in the cell wall. In an 8-h photoperiod, protein synthesis and cell wall synthesis were 3-fold faster in the day than at night, protein degradation was slow (3%–4% d−1), and flux to growth and degradation resulted in a protein half-life of 3.5 d. In the starchless phosphoglucomutase mutant at night, protein synthesis was further decreased and protein degradation increased, while cell wall synthesis was totally inhibited, quantitatively accounting for the inhibition of growth in this mutant. We also investigated the rates of protein synthesis and degradation during leaf development, during growth at high temperature, and compared synthesis rates of Rubisco large and small subunits of in the light and dark. PMID:25810096

  19. Part 1: Progress Towards the Synthesis of a Lemonose Derivative Part 2: Synthesis and Characterization of Antibiotic-Labeled Graphite Nanofibers

    NASA Astrophysics Data System (ADS)

    Briegel, Alicia Christine

    Lemonose is a carbohydrate that is part of the natural product lemonomycin, which has shown activity against strains of bacteria such as methicillin-resistant Staphylococcus aureus (MRSA). Lemonose is 2,4,6-trideoxy-4-(dimethylamino)-3- C-methyl-L-lyxohexopyranose. Previous studies on carbohydrate-containing antibiotics showed that structural modifications on the sugar unit changed the activity and/or toxicity of the parent compound. The goal of this work is to synthesize 2,4,6-trideoxy-4-amino-3-C-methyl-L-lyxohexopyranose, a derivative of lemonose (shown below). The key synthetic challenge is the formation of the cis amino alcohol. Two strategies were investigated in this research: epoxidation-reduction and electrophilic cyclization. Graphite nanofibers (GNFs) are novel nanoscale materials that can be prepared inexpensively, in gram quantities, via the catalytic decomposition of carbon monoxide or hydrocarbons over mono- or bi-metallic catalysts. GNFs have potential for applications across a diverse spectrum of research areas in chemistry, biology, medicine, and energy storage. Surface functionalization and characterization are both critical to the further development of GNFs as biomaterials. The covalent functionalization of the GNF surface with antibiotics was carried out in this study. Fibers labeled with antibiotics including amikacin and ciprofloxacin were prepared and studied for their potential biological activity against the common bacterium Pseudomonas aeruginosa. Serial dilution assays and optical density measurements revealed that antibiotic-labeled GNFs possess antibacterial activity.

  20. An improved method for the synthesis of mercurated dUTP. Enzymic synthesis of Hg-labelled DNA of high molecular weight suitable for use in an image based DNA sequencing strategy.

    PubMed

    Bridgman, A J; Petersen, G B

    1996-01-01

    The development of high-resolution scanning-probe microscopes has reawakened interest in the possibility of sequencing large nucleic acid molecules by direct imaging. Such an approach would be facilitated by the availability of effective methods for increasing contrast by labelling specific nucleotides, and the utility of introducing mercury atoms into complete DNA molecules through the enzymic polymerisation of mercurated pyrimidine deoxynucleoside triphosphates has been re-investigated. A simplified and improved method for the synthesis of a heat- and thiol-stable, mercurated derivative of deoxyuridine triphosphate in high yield and the incorporation of this precursor into full-length copies of a single-stranded phage M13 template are described. The DNA product has been fully characterised and the quantitative and specific replacement of thymidylic acid residues by the mercurated analogue demonstrated.

  1. Design, synthesis, modeling, biological evaluation and photoaffinity labeling studies of novel series of photoreactive benzamide probes for histone deacetylase 2

    PubMed Central

    Vaidya, Aditya Sudheer; Karumudi, Bhargava; Mendonca, Emma; Madriaga, Antonett; Abdelkarim, Hazem; van Breemen, Richard B.; Petukhov, Pavel A.

    2012-01-01

    The design, modeling, synthesis, biological evaluation of a novel series of photoreactive benzamide probes for class I HDAC isoforms is reported. The probes are potent and selective for HDAC1 and 2 and are efficient in crosslinking to HDAC2 as demonstrated by photolabeling experiments. The probes exhibit a time-dependent inhibition of class I HDACs. The inhibitory activities of the probes were influenced by the positioning of the aryl and alkyl azido groups necessary for photocrosslinking and attachment of the biotin tag. The probes inhibited the deacetylation of H4 in MDA-MB-231 cell line, indicating that they are cell permeable and target the nuclear HDACs. PMID:22771007

  2. Synthesis and evaluation of novel F-18 labeled quinazoline derivatives with low lipophilicity for tumor PET imaging.

    PubMed

    Chong, Yan; Chang, Jin; Zhao, Wenwen; He, Yong; Li, Yuqiao; Zhang, Huabei; Qi, Chuanmin

    2017-08-18

    Four novel F-18 labeled quinazoline derivatives with low lipophilicity, [(18) F]4-(2-fluoroethoxy)-6,7-dimethoxyquinazoline ([(18) F]I), [(18) F]4-(3-((4-(2-fluoroethoxy)-7-methoxyquinazolin-6-yl)oxy)propyl)morpholine ([(18) F]II), [(18) F]4-(2-fluoroethoxy)-7-methoxy-6-(2-methoxyethoxy)quinazoline ([(18) F]III) and [(18) F]4-(2-fluoroethoxy)-6,7-bis(2-methoxyethoxy)quinazoline ([(18) F]IV), were synthesized via a two-step radiosynthesis procedure with an overall radiochemical yield of 10-38% (without decay correction) and radiochemical purities of > 98%. The lipophilicity and stability of labeled compounds were tested in vitro. The log P values of the four radiotracers ranged from 0.52 to 1.07. We then performed ELISA to measure their affinities to EGFR-TK. ELISA assay results indicated that each inhibitor was specifically bound to EGFR-TK in a dose-dependent manner. The EGFR-TK autophosphorylation IC50 values of [(18) F]I, [(18) F]II, [(18) F]III, and [(18) F]IV were 7.732 μM, 0.4698 μM, 0.1174 μM, and 0.1176 μM, respectively. All labeled compounds were evaluated via cellular uptake and blocking studies in HepG2 cell lines in vitro. Cellular uptake and blocking experiment results indicated that [(18) F]I and [(18) F]III had excellent cellular uptake at 120 min post-injection in HepG2 carcinoma cells (51.80±3.42 %ID/mg protein and 27.31±1.94 %ID/mg protein, respectively). Additionally, biodistribution experiments in S180 tumor-bearing mice in vivo indicated that [(18) F]I had a very fast clearance in blood and a relatively high uptake ratio of tumor to blood (4.76) and tumor to muscle (1.82) at 60 min post-injection. [(18) F]III had a quick clearance in plasma, and its highest uptake ratio of tumor to muscle was 2.55 at 15 min post-injection. These experimental results and experiences were valuable for the further exploration of novel radiotracers of quinazoline derivatives. This article is protected by copyright. All rights reserved.

  3. Synthesis and preliminary in vivo evaluation of well-dispersed biomimetic nanocrystalline apatites labeled with positron emission tomographic imaging agents.

    PubMed

    Sandhöfer, Benedikt; Meckel, Marian; Delgado-López, José Manuel; Patrício, Tatiana; Tampieri, Anna; Rösch, Frank; Iafisco, Michele

    2015-05-20

    In recent years, biomimetic synthetic apatite nanoparticles (AP-NPs), having chemical similarity with the mineral phase of bone, have attracted a great interest in nanomedicine as potential drug carriers. To evaluate the therapeutic perspectives of AP-NPs through the mechanisms of action and organs they interact with, the noninvasive monitoring of their in vivo behavior is of paramount importance. To this aim, here the feasibility to radiolabel AP-NPs ("naked" and surface-modified with citrate to reduce their aggregation) with two positron emission tomographic (PET) imaging agents ([(18)F]NaF and (68)Ga-NO2AP(BP)) was investigated. [(18)F]NaF was used for the direct incorporation of the radioisotope into the crystal lattice, while the labeling by surface functionalization was accomplished by using (68)Ga-NO2AP(BP) (a new radio-metal chelating agent). The labeling results with both tracers were fast, straightforward, and reproducible. AP-NPs demonstrated excellent ability to bind relevant quantities of both radiotracers and good in vitro stability in clinically relevant media after the labeling. In vivo PET studies in healthy Wistar rats established that the radiolabeled AP-NPs gave significant PET signals and they were stable over the investigated time (90 min) since any tracer desorption was detected. These preliminary in vivo studies furthermore showed a clear ability of citrated versus naked AP-NPs to accumulate in different organs. Interestingly, contrary to naked AP-NPs, citrated ones, which unveiled higher colloidal stability in aqueous suspensions, were able to escape the first physiological filter, i.e., the lungs, being then accumulated in the liver and, to a lesser extent, in the spleen. The results of this work, along with the fact that AP-NPs can be also functionalized with targeting ligands, with therapeutic agents, and also with metals for a combination of different imaging modalities, make AP-NPs very encouraging materials for further investigations

  4. Synthesis of stereoarray isotope labeled (SAIL) lysine via the "head-to-tail" conversion of SAIL glutamic acid.

    PubMed

    Terauchi, Tsutomu; Kamikawai, Tomoe; Vinogradov, Maxim G; Starodubtseva, Eugenia V; Takeda, Mitsuhiro; Kainosho, Masatsune

    2011-01-07

    A stereoarray isotope labeled (SAIL) lysine, (2S,3R,4R,5S,6R)-[3,4,5,6-(2)H(4);1,2,3,4,5,6-(13)C(6);2,6-(15)N(2)]lysine, was synthesized by the "head-to-tail" conversion of SAIL-Glu, (2S,3S,4R)-[3,4-(2)H(2);1,2,3,4,5-(13)C(5);2-(15)N]glutamic acid, with high stereospecificities for all five chiral centers. With the SAIL-Lys in hand, the unambiguous simultaneous stereospecific assignments were able to be established for each of the prochiral protons within the four methylene groups of the Lys side chains in proteins.

  5. Synthesis and steady-state photophysical properties of dye-labeled dendrimers having novel oligothiophene cores: A comparative study

    SciTech Connect

    Adronov, A.; Malenfant, P.R.L.; Frechet, J.M.J.

    2000-05-01

    Novel chromophore-labeled dendrimers with penta- and heptathiophene cores and coumarin-2 chromophores at their periphery have been shown to be very efficient light-harvesting systems. Excitation of the peripheral coumarin-2 chromophores results in energy transfer to the oligothiophene cores as a result of the large overlap between the donor emission spectrum and the acceptor absorption spectrum, as well as the large transition dipole moments of the oligothiophenes. Although these core dyes have low fluorescence quantum yields, their emission intensity is significantly enhanced by the ability of the large light-harvesting dendron to funnel absorbed energy to the core. Because of the large Stokes shift of the oligothiophenes, the emission spectrum of the dendrimers was red-shifted by 200 nm from the excitation wavelength. Additionally, it was found that oligothiophene orientation--end functionalization vs central functionalization--did not have a significant effect on energy-transfer efficiency.

  6. Dansyl-labeled anionic amphiphile with a hexadecanoic carbon chain: synthesis and detection for shape transitions in organized molecular assemblies.

    PubMed

    Gao, Lining; Xia, Huiyun; Wang, Xiaoman; Li, Li; Chen, Huaxin

    2015-03-15

    The probing properties of a new fluorophore-labeled anionic surfactant, sodium 16-(N-dansyl)aminocetylate (16-DAN-ACA) were investigated systematically in molecular assemblies, especially in the transitions between micelles and vesicles. 16-DAN-ACA can efficiently differentiate the two different aggregate types in mixed cationic and anionic surfactant systems. The fluorescence anisotropy of 16-DAN-ACA was found to be sensitive for directly detecting the micellar growth in micelles containing oppositely charged surfactants; both cationic cetyltrimethylammonium bromide (CTAB) systems and anionic sodium dodecyl sulfate (SDS) systems were studied. The results indicated that the 16-DAN-ACA is a good fluorescent probe for differentiating the different aggregates, and even more can be used to detect the micellar growth.

  7. Synthesis of deuterium-labelled substrates for the study of oleuropein biosynthesis in Olea europaea callus cultures.

    PubMed

    Serrilli, Anna Maria; Maggi, Agnese; Casagrande, Valentina; Bianco, Armandodoriano

    2016-01-01

    We propose the cell culture approach to investigate oleuropein (1) biogenesis in Olea europaea L. We suggest employing olive callus cultures to identify the iridoidic precursor of oleuropein. In fact, we confirmed that callus cells from olive shoot explants are able to produce key secoiridoid as 1. To enable this approach, we synthesised and characterised deuterium-labelled iridoidic precursors belonging both to the loganin and the 8-epiloganin series. These iridoids are [7,8-(2)H2]-7-deoxy-8-epi-loganin (2(D)), [8,10-(2)H2]-8-epi-loganin (4(D)) and [7,8-(2)H2]-7-deoxy-loganin (3(D)).

  8. Simultaneous Synthesis and Biotinylation of Proteins Using Puromycin-Based Labeling Technology for Fabrication of Protein Array Chip

    NASA Astrophysics Data System (ADS)

    Kumal, Subhashini Raj; Biyani, Manish; Ueno, Shingo; Akagi, Takanori; Ichiki, Takanori

    2013-06-01

    Protein arrays represent a class of devices that are of growing importance in the field of proteomics. These arrays enable screening of a large amount of proteins in a short time and at a lower cost. Here we present a method to fabricate protein array using biotin-conjugated puromycin to simultaneously synthesize and label proteins followed by immobilization onto streptavidin-functionalized surface based on the noncovalent biotin-streptavidin interaction. This method demonstrates the fabrication of protein array based on cell-free transcription/translation system using unmodified DNA as a starting genetic material. As a consequence, the procedure of protein arraying has been greatly simplified over the conventional approaches that require tedious and multi-step reactions. Further, an integrated approach of micro reactor array technology makes this method very simple and robust for achieving high-density protein arrays.

  9. Synthesis of a (68)ga-labeled peptoid-Peptide hybrid for imaging of neurotensin receptor expression in vivo.

    PubMed

    Maschauer, Simone; Einsiedel, Jürgen; Hocke, Carsten; Hübner, Harald; Kuwert, Torsten; Gmeiner, Peter; Prante, Olaf

    2010-08-12

    The neurotensin receptor subtype 1 (NTS1) represents an attractive molecular target for imaging various tumors. Positron emission tomography (PET) gained widespread importance due to its sensitivity. We combined the design of a metabolically stable neurotensin analogue with a (68)Ga-radiolabeling approach. The (68)Ga-labeled peptoid-peptide hybrid [(68)Ga]3 revealed high stability, specific tumor uptake (0.7%ID/g, 65 min p.i.), and advantageous biokinetics in vivo using HT29 tumor-bearing nude mice. Because of the ability to internalize into NTS1-expressing tumor cells, [(68)Ga]3 proved to be highly suitable for a reliable and practical visualization of NTS1-expressing tumors in vivo by small animal PET.

  10. Synthesis of a 68Ga-Labeled Peptoid−Peptide Hybrid for Imaging of Neurotensin Receptor Expression in Vivo

    PubMed Central

    2010-01-01

    The neurotensin receptor subtype 1 (NTS1) represents an attractive molecular target for imaging various tumors. Positron emission tomography (PET) gained widespread importance due to its sensitivity. We combined the design of a metabolically stable neurotensin analogue with a 68Ga-radiolabeling approach. The 68Ga-labeled peptoid−peptide hybrid [68Ga]3 revealed high stability, specific tumor uptake (0.7%ID/g, 65 min p.i.), and advantageous biokinetics in vivo using HT29 tumor-bearing nude mice. Because of the ability to internalize into NTS1-expressing tumor cells, [68Ga]3 proved to be highly suitable for a reliable and practical visualization of NTS1-expressing tumors in vivo by small animal PET. PMID:24900199

  11. Synthesis of iodine-125 labeled (+/-)-15-(4-azidobenzyl)carazolol: a potent beta-adrenergic photoaffinity probe

    SciTech Connect

    Heald, S.L.; Jeffs, P.W.; Lavin, T.N.; Nambi, P.; Lefkowitz, R.J.; Caron, M.G.

    1983-06-01

    (+/-)-15-(4-Azidobenzyl)carazolol (2), a potent beta-adrenergic photoaffinity ligand has been radioiodinated to theoretical specific activity (2175 Ci/mmol) and shown to label covalently beta-adrenergic receptor peptides in avian and amphibian erythrocyte membrane preparations. The radioiodinated analogues of the desired compound (2) were optimally prepared by two synthetic steps from (+/-)-15-(4-aminobenzyl)carazolol (8). The latter was iodinated with carrier-free Na/sup 125/I and chloramine T to yield two major isotopomers (the monoiodinated derivatives 9 and 10), which were separated by thin-layer chromatography and converted via diazonium salt formation to their respective 4-azides, 12 and 6. These azides can be used interchangeably in ligand binding or photoaffinity labeling experiments. Compound 8 was obtained by catalytic reduction of the nitro derivative (7), which was arrived at by direct reaction of 1,1-dimethyl-2-(4-nitrophenyl)ethylamine (3) with 4-(2,3-epoxypropoxy)carbazole (5). Of the desired isomers, (+/-)-15-(4-azido-3-iodobenzyl)carazolol (6) could be synthesized from 1,1-dimethyl-2-(4-azido-3-iodophenyl)ethylamine (4) by direct reaction with 5. This and the preceding sequence of reactions were carried out by using nonradioactive materials, and separation and purification of products were accomplished by high-performance liquid chromatography. The compounds described have been shown to be potent beta-adrenergic antagonistsec The photoactive azide derivatives of these compounds (6 and 12) have been shown to covalently incorporate into the beta-adrenergic receptor binding subunit of frog and turkey erythrocyte membrane preparations. Incorporation of the ligands into these polypeptides can be blocked specifically by both beta-adrenergic agonists and antagonists.

  12. Co-precipitation of DEAE-dextran coated SPIONs: how synthesis conditions affect particle properties, stem cell labelling and MR contrast.

    PubMed

    Barrow, Michael; Taylor, Arthur; García Carrión, Jaime; Mandal, Pranab; Park, B Kevin; Poptani, Harish; Murray, Patricia; Rosseinsky, Matthew J; Adams, Dave J

    2016-09-01

    Superparamagnetic iron oxide nanoparticles (SPIONs) are widely used as contrast agents for stem cell tracking using magnetic resonance imaging (MRI). The total mass of iron oxide that can be internalised into cells without altering their viability or phenotype is an important criterion for the generation of contrast, with SPIONs designed for efficient labelling of stem cells allowing for an increased sensitivity of detection. Although changes in the ratio of polymer and iron salts in co-precipitation reactions are known to affect the physicochemical properties of SPIONs, particularly core size, the effects of these synthesis conditions on stem cell labelling and magnetic resonance (MR) contrast have not been established. Here, we synthesised a series of cationic SPIONs with very similar hydrodynamic diameters and surface charges, but different polymer content. We have investigated how the amount of polymer in the co-precipitation reaction affects core size and modulates not only the magnetic properties of the SPIONs but also their uptake into stem cells. SPIONs with the largest core size and lowest polymer content presented the highest magnetisation and relaxivity. These particles also had the greatest uptake efficiency without any deleterious effect on either the viability or function of the stem cells. However, for all particles internalised in cells, the T2 and T2(*) relaxivity was independent of the SPION's core size. Our results indicate that the relative mass of iron taken up by cells is the major determinant of MR contrast generation and suggest that the extent of SPION uptake can be regulated by the amount of polymer used in co-precipitation reactions. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  13. Synthesis and evaluation of the racemate and individual enantiomers of C-11 labeled methylphenidate as radioligands for the presynaptic dopaminergic neuron

    SciTech Connect

    Ding, Y.S.; Fowler, J.S.; Volkow, N.D.

    1994-05-01

    Methylphenidate (MP, ritalin) is a psychostimulant drug widely used to treat attention deficit hyperactivity disorder and narcolepsy. Its therapeutic properties are attributed to inhibition of the dopamine (DA) transporter enhancing synaptic DA. MP has two chiral centers and is marketed as the dl-threo racemic form. However, its pharmacological activity is believed due solely to the d-enantiomer. We have synthesized [{sup 11}C]d,l-threo-methylphenidate ([{sup 11}C]MP) in order to examine its pharmacokinetics in vivo and to examine its suitability as a radioligand for PET studies of the presynaptic DA neuron. [{sup 11}C]MP was prepared by O-{sup 11}C-alkylation of a protected derivative of ritalinic acid with labeled methyl iodide. Serial studies at baseline and after treatment with methylphenidate (0.5 mg/kg, 20 min prior); GBR 12909 (1.5 mg/kg; 30 min prior); tomoxetine (1.5 mg/kg, 20 min prior) and citalopram (2.0 mg/kg, 30 min prior) were performed to assess non-specific binding and binding to the DA, norepinephrine and serotonin transporters respectively. Only MP and GBR 12909 changed the SR/CB distribution volume ratio (decrease of 38 and 37% respectively) demonstrating selectivity for DA transporters over other monoamine transporters. We then pursued the synthesis of enantiomerically pure C-{sup 11} labeled d- and l-MP by using enantiomerically pure protected d- and l-ritalinic acids as precursors. A striking difference in SR/CB ratio (3.3 and 1.1 for d- and l-respectively at 1 hr. after i.v. injections) strongly suggests that the pharmacological specificity of MP resides entirely in the d-isomer and the binding of l-isomer was mostly non-specific. Further evaluations are underway. Radioligand reversibility, selectivity and the fact that MP is an approved drug are advantages of using [{sup 11}C]MP.

  14. Synthesis, characterization, and biological evaluation of (99m) Tc(CO)3 -labeled peptides for potential use as tumor targeted radiopharmaceuticals.

    PubMed

    Baishya, Rinku; Nayak, Dipak K; Chatterjee, Nabanita; Halder, Kamal K; Karmakar, Sanmoy; Debnath, Mita C

    2014-01-01

    During the past decade, several peptides containing Arg-Gly-Asp sequence have been conjugated with different chelating agents for labeling with various radionuclides for the diagnosis of tumor development. In this study, we report the synthesis of two tetrapeptides (Asp-Gly-Arg-His and Asp-Gly-Arg-Cys) and one hexapeptide [Asp-Gly-Arg-D-Tyr-Lys-His] by changing the amino acid sequence of the Arg-Gly-Asp motif. Peptide synthesis was initiated from aspartic acid. Aspartic acid placed at C-terminal end of the peptide chain can be conjugated with different drug molecules facilitating their transport to the site of action. The peptides were synthesized in excellent yield and labeled using freshly prepared [(99m) Tc(CO)3 (H2 O)3 ](+) intermediate. A complexation yield of over 97% was achieved under mild conditions even at low ligand concentrations of 10(-2)  m. Radiolabeled peptides were characterized by HPLC and were found to be substantially stable in saline, in His solution as well as in rat serum and tissue (kidney, liver) homogenates. Internalization studies using Ehrlich ascites carcinoma cell line showed rapid and significant internalization (30-35% at 30 min of incubation attaining maximum value of about 40-60% after 2-4 h incubation). A good percentage of quick internalization was also observed in αv β3 -receptor-positive B16F10 mouse melanoma cell line (14-16% after 30 min of incubation and 25-30% after 2-4 h incubation). Imaging and biodistribution studies were performed in Swiss albino mice bearing Ehrlich ascites tumor in right thigh. Radiolabeled peptides exhibited fast blood clearance and rapid elimination through the urinary systems. (99m) Tc(CO)3 -tetra-Pep2 exhibited remarkable localization at tumor site (1.15%, 1.17%, and 1.37% ID/g at 2, 4, and 6 h p.i., respectively) which could be due to slow clearance of the radiolabeled peptide from blood in comparison with the other two radiolabeled peptides. However, (99m) Tc(CO)3 -hexa-Pep exhibited the

  15. One-pot green synthesis of N-doped carbon quantum dots for cell nucleus labelling and copper (Ⅱ) detection.

    PubMed

    Ci, Jiliang; Tian, Ye; Kuga, Shigenori; Niu, Zhongwei; Wu, Min; Huang, Yong

    2017-09-21

    The doping of nitrogen into carbon quantum dots was vitally important for improvement of fluorescence performance. However, the synthesis of nitrogen-doped carbon quantum dots (N-CQDs) was usually conducted under strong acid and high temperature, which would result in the environmental pollution and energy consumption. Herein, the N-CQDs were prepared by a mild one-pot hydrothermal process. The hydrothermal reaction temperature was adjusted to control the particle size, N/C atomic ratio and quantum yield. The products were water-soluble with narrow particle size distribution and good dispersion stability in wide pH range. The N-CQDs could penetrate into HeLa cell nucleus without any further functionalization. Moreover, the fluorescence of N-CQDs could be selectively quenched by Cu2+, suggesting application of detecting Cu2+ in human plasma. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Synthesis and Preclinical Evaluation of Three Novel Fluorine-18 Labeled Radiopharmaceuticals for P-Glycoprotein PET Imaging at the Blood-Brain Barrier.

    PubMed

    Savolainen, Heli; Cantore, Mariangela; Colabufo, Nicola A; Elsinga, Philip H; Windhorst, Albert D; Luurtsema, Gert

    2015-07-06

    P-Glycoprotein (P-gp), along with other transporter proteins at the blood-brain barrier (BBB), limits the entry of many pharmaceuticals into the brain. Altered P-gp function has been found in several neurological diseases. To study the P-gp function, many positron emission tomography (PET) radiopharmaceuticals have been developed. Most P-gp radiopharmaceuticals are labeled with carbon-11, while labeling with fluorine-18 would increase their applicability due to longer half-life. Here we present the synthesis and in vivo evaluation of three novel fluorine-18 labeled radiopharmaceuticals: 4-((6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)methyl)-2-(4-fluorophenyl)oxazole (1a), 2-biphenyl-4-yl-2-fluoroethoxy-6,7-dimethoxy-1,2,3,4-tetrahydro-isoquinoline (2), and 5-(1-(2-fluoroethoxy))-[3-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-propyl]-5,6,7,8-tetrahydronaphthalen (3). Compounds were characterized as P-gp substrates in vitro, and Mdr1a/b((-/-))Bcrp1((-/-)) and wild-type mice were used to assess the substrate potential in vivo. Comparison was made to (R)-[(11)C]verapamil, which is currently the most frequently used P-gp substrate. Compound [(18)F]3 was performing the best out of the new radiopharmaceuticals; it had 2-fold higher brain uptake in the Mdr1a/b((-/-))Bcrp1((-/-)) mice compared to wild-type and was metabolically quite stable. In the plasma, 69% of the parent compound was intact after 45 min and 96% in the brain. Selectivity of [(18)F]3 to P-gp was tested by comparing the uptake in Mdr1a/b((-/-)) mice to uptake in Mdr1a/b((-/-))Bcrp1((-/-)) mice, which was statistically not significantly different. Hence, [(18)F]3 was found to be selective for P-gp and is a promising new radiopharmaceutical for P-gp PET imaging at the BBB.

  17. Fluorescent magnetic nanoparticles for cell labeling: flux synthesis of manganite particles and novel functionalization of silica shell.

    PubMed

    Kačenka, Michal; Kaman, Ondřej; Kikerlová, Soňa; Pavlů, Barbora; Jirák, Zdeněk; Jirák, Daniel; Herynek, Vít; Černý, Jan; Chaput, Frédéric; Laurent, Sophie; Lukeš, Ivan

    2015-06-01

    Novel synthetic approaches for the development of multimodal imaging agents with high chemical stability are demonstrated. The magnetic cores are based on La0.63Sr0.37MnO3 manganite prepared as individual grains using a flux method followed by additional thermal treatment in a protective silica shell allowing to enhance their magnetic properties. The cores are then isolated and covered de novo with a hybrid silica layer formed through the hydrolysis and polycondensation of tetraethoxysilane and a fluorescent silane synthesized from rhodamine, piperazine spacer, and 3-iodopropyltrimethoxysilane. The aminoalkyltrialkoxysilanes are strictly avoided and the resulting particles are hydrolytically stable and do not release dye. The high colloidal stability of the material and the long durability of the fluorescence are reinforced by an additional silica layer on the surface of the particles. Structural and magnetic studies of the products using XRD, TEM, and SQUID magnetometry confirm the importance of the thermal treatment and demonstrate that no mechanical treatment is required for the flux-synthesized manganite. Detailed cell viability tests show negligible or very low toxicity at concentrations at which excellent labeling is achieved. Predominant localization of nanoparticles in lysosomes is confirmed by immunofluorescence staining. Relaxometric and biological studies suggest that the functionalized nanoparticles are suitable for imaging applications.

  18. Facile synthesis of cuprous oxide nanowires decorated graphene oxide nanosheets nanocomposites and its application in label-free electrochemical immunosensor.

    PubMed

    Wang, Huan; Zhang, Yong; Wang, Yulan; Ma, Hongmin; Du, Bin; Wei, Qin

    2017-01-15

    In this work, the assembly between one-dimensional (1D) nanomaterials and two-dimensional (2D) nanomaterials was achieved by a simple method. Cuprous oxide nanowires decorated graphene oxide nanosheets (Cu2O@GO) nanocomposites were synthesized for the first time by a simple electrostatic self-assembly process. The nanostructure was well confirmed by scanning electron microscope (SEM) and transmission electron microscope (TEM) images. Taking advantages of good electrocatalytic activity and high specific surface area of Cu2O@GO nanocomposites, a label-free electrochemical immunosensor was developed by employing Cu2O@GO as signal amplification platform for the quantitative detection of alpha fetoprotein (AFP). In addition, toluidine blue (TB) was used as the electron transfer mediator to provide the electrochemical signal, which was adsorbed on graphene oxide nanosheets (GO NSs) by electrostatic attraction. The detection mechanism was based on the monitoring of the electrochemical current response change of TB by the square wave voltammetry (SWV) when immunoreaction occurred on the surface of electrode. Under optimal conditions, the proposed immunosensor displayed a high sensitivity and a low detection limit. This designed method may provide an effective method in the clinical diagnosis of AFP and other tumor markers.

  19. Synthesis and Evaluation of Technetium-99m- and Rhenium-Labeled Inhibitors of the Prostate-Specific Membrane Antigen (PSMA)

    PubMed Central

    Banerjee, Sangeeta R.; Foss, Catherine A.; Castanares, Mark; Mease, Ronnie C.; Byun, Youngjoo; Fox, James J.; Hilton, John; Lupold, Shawn E.; Kozikowski, Alan P.; Pomper, Martin G.

    2012-01-01

    The prostate-specific membrane antigen (PSMA) is increasingly recognized as a viable target for imaging and therapy of cancer. We prepared seven 99mTc/Re-labeled compounds by attaching known Tc/Re chelating agents to an amino-functionalized PSMA inhibitor (lys-NHCONH-glu) with or without a variable length linker moiety. Ki values ranged from 0.17 to 199 nM. Ex vivo biodistribution and in vivo imaging demonstrated the degree of specific binding to engineered PSMA+ PC3 PIP tumors. PC3-PIP cells are derived from PC3 that have been transduced with the gene for PSMA. Despite demonstrating nearly the lowest PSMA inhibitory potency of this series, [99mTc(CO)3(L1)]+ (L1 = (2-pyridylmethyl)2N(CH2)4CH(CO2H)-NHCO-(CH2)6CO-NH-lys-NHCONH-glu) showed the highest, most selective PIP tumor uptake, at 7.9 ± 4.0% injected dose per gram of tissue at 30 min postinjection. Radioactivity cleared from nontarget tissues to produce a PIP to flu (PSMA-PC3) ratio of 44:1 at 120 min postinjection. PSMA can accommodate the steric requirements of 99mTc/Re complexes within PSMA inhibitors, the best results achieved with a linker moiety between the ε amine of the urea lysine and the chelator. PMID:18637669

  20. Design, synthesis and evaluation of (18)F-labeled bradykinin B1 receptor-targeting small molecules for PET imaging.

    PubMed

    Zhang, Zhengxing; Kuo, Hsiou-Ting; Lau, Joseph; Jenni, Silvia; Zhang, Chengcheng; Zeisler, Jutta; Bénard, François; Lin, Kuo-Shyan

    2016-08-15

    Two fluorine-18 ((18)F) labeled bradykinin B1 receptor (B1R)-targeting small molecules, (18)F-Z02035 and (18)F-Z02165, were synthesized and evaluated for imaging with positron emission tomography (PET). Z02035 and Z02165 were derived from potent antagonists, and showed high binding affinity (0.93±0.44 and 2.80±0.50nM, respectively) to B1R. (18)F-Z02035 and (18)F-Z02165 were prepared by coupling 2-[(18)F]fluoroethyl tosylate with their respective precursors, and were obtained in 10±5 (n=4) and 22±14% (n=3), respectively, decay-corrected radiochemical yield with >99% radiochemical purity. (18)F-Z02035 and (18)F-Z02165 exhibited moderate lipophilicity (LogD7.4=1.10 and 0.59, respectively), and were stable in mouse plasma. PET imaging and biodistribution studies in mice showed that both tracers enabled visualization of the B1R-positive HEK293T::hB1R tumor xenografts with better contrast than control B1R-negative HEK293T tumors. Our data indicate that small molecule antagonists can be used as pharmacophores for the design of B1R-targeting PET tracers.

  1. Synthesis and Evaluation of Novel Tc-99m Labeled Probestin Conjugates for Imaging APN/CD13 Expression In Vivo

    PubMed Central

    Pathuri, Gopal; Hedrick, Andria F.; Disch, Bryan; Doan, John; Ihnat, Michael A.; Awasthi, Vibhudutta; Gali, Hariprasad

    2011-01-01

    The enzyme aminopeptidase N (APN, also known as CD13) is known to play an important role in tumor proliferation, attachment, angiogenesis, and tumor invasion. In this study, we hypothesized that a radiolabeled high affinity APN inhibitor could be potentially useful for imaging APN expression in vivo. Here we report synthesis, radiolabeling, and biological evaluation of new probestin conjugates containing a tripeptide, N,N-dimethylglycyl-L-lysinyl-L-cysteinylamide (N3S), chelator. New probestin conjugates were synthesized by solid-phase peptide synthesis method, purified by reversed-phase HPLC, and characterized by electrospray mass spectrometry. The conjugates were complexed with Re(V) and 99mTc(V) by transmetallation using corresponding Re(V) or 99mTc(V) gluconate synthon. The mass spectral analyses of ReO-N3S-Probestin conjugates were consistent with the formation of neutral Re(V)O-N3S complexes. Initial biological activity of ReO-N3S-Probestin conjugates determined by performing an in vitro APN enzyme assay using intact HT-1080 cells demonstrated higher inhibition of APN enzyme activity than bestatin. In vivo biodistribution and whole body planar imaging studies of 99mTcO-N3S-PEG2-Probestin performed in nude mice xenografted with human fibrosarcoma tumors derived from HT-1080 cells demonstrated a tumor uptake value of 2.88 ± 0.64 %ID/g with tumor-to-blood and tumor-to-muscle ratios of 4.8 and 5.3 respectively at 1 hr post-injection (p.i.). Tumors were clearly visible in whole-body planar image obtained at 1 hr p.i., but not when the APN was competitively blocked with a co-injection of excess non-radioactive ReO-N3S-PEG2-Probestin conjugate. These results demonstrate the feasibility of using high affinity APN inhibitor conjugates as targeting vectors for in vivo targeting of APN. PMID:22148582

  2. Hydrothermal synthesis of GSH-TGA co-capped CdTe quantum dots and their application in labeling colorectal cancer cells.

    PubMed

    Yu, Yongli; Xu, Linru; Chen, Jing; Gao, Huanyu; Wang, Shuo; Fang, Jin; Xu, Shukun

    2012-06-15

    We have successfully synthesized GSH and TGA co-capped CdTe quantum dots (QDs) with good biological compatibility and high fluorescence intensity. The effects of different reaction time, temperature, pH value, ligand concentration and the molar ratio of GSH/TGA were carefully investigated to optimize the synthesis condition. The optical properties of as-prepared CdTe QDs were studied by UV-visible absorption spectrum and fluorescence spectrum, meanwhile their structure and morphology were characterized using transmission electron microscope (TEM), Fourier transform infrared spectra (FT-IR) and X-ray powder diffraction (XRD). Compared with the CdTe QDs that are single-capped with either GSH or TGA, the GSH-TGA co-capped CdTe QDs demonstrated significantly improved fluorescence intensity and optical stability. In addition, GSH-TGA co-capped CdTe QDs were conjugated to amonoclonal antibody ND-1. The GSH-TGA co-capped CdTe QDs-antibody probe was successfully used to label colorectal cancer cells, CCL187, in vitro. Copyright © 2012 Elsevier B.V. All rights reserved.

  3. Synthesis and characterization of a new photocrosslinking CTP analog and its use in photoaffinity labeling E. coli and T7 RNA polymerases.

    PubMed Central

    Hanna, M M; Zhang, Y; Reidling, J C; Thomas, M J; Jou, J

    1993-01-01

    A new photocrosslinking CTP analog that functioned as a substrate during transcription was synthesized and used to photoaffinity label E. coli and bacteriophage T7 RNA polymerases. This analog, 5-((4-azidophenacyl)thio) cytidine-5'-triphosphate (5-APAS-CTP) contains an aryl azide group approximately 10 A from the nucleotide base and specifically replaced CTP during synthesis of RNA by both polymerases. Analog was placed at the 3' end or internally within RNA. Both polymerases inefficiently incorporated two 5-APAS-CMP molecules sequentially, as was found for the related 5-APAS-UMP. Analog was placed at the 3' end of RNA in transcription complexes paused at the site of Q-modification of E. coli RNA polymerase, downstream of the lambda PR' promoter (+16), a pause that requires specific DNA sequences but no apparent RNA hairpin. Crosslinking was examined in the presence and absence of the NusA protein, which enhances the transcriptional pause at this site and is required for Q modification of the polymerase. Crosslinking of the 3' end of the RNA to NusA was not observed, consistent with our earlier results involving a NusA-enhanced pause site downstream from an RNA hairpin. Images PMID:7684833

  4. Synthesis, dynamic NMR characterization and XRD studies of novel N,N’-substituted piperazines for bioorthogonal labeling

    PubMed Central

    Pretze, Marc; Gott, Matthew; Köckerling, Martin

    2016-01-01

    Novel, functionalized piperazine derivatives were successfully synthesized and fully characterized by 1H/13C/19F NMR, MS, elemental analysis and lipophilicity. All piperazine compounds occur as conformers resulting from the partial amide double bond. Furthermore, a second conformational shape was observed for all nitro derivatives due to the limited change of the piperazine chair conformation. Therefore, two coalescence points were determined and their resulting activation energy barriers were calculated using 1H NMR. To support this result, single crystals of 1-(4-nitrobenzoyl)piperazine (3a, monoclinic, space group C2/c, a = 24.587(2), b = 7.0726(6), c = 14.171(1) Å, β = 119.257(8)°, V = 2149.9(4) Å3, Z = 4, D obs = 1.454 g/cm3) and the alkyne derivative 4-(but-3-yn-1-yl)-1-(4-fluorobenzoyl)piperazine (4b, monoclinic, space group P21/n, a = 10.5982(2), b = 8.4705(1), c = 14.8929(3) Å, β = 97.430(1)°, V = 1325.74(4) Å3, Z = 4, D obs = 1.304 g/cm3) were obtained from a saturated ethyl acetate solution. The rotational conformation of these compounds was also verified by XRD. As proof of concept for future labeling purposes, both nitropiperazines were reacted with [18F]F–. To test the applicability of these compounds as possible 18F-building blocks, two biomolecules were modified and chosen for conjugation either using the Huisgen-click reaction or the traceless Staudinger ligation. PMID:28144316

  5. Synthesis and preliminary evaluation of a (99m) Tc-labeled folate-PAMAM dendrimer for FR imaging.

    PubMed

    Song, Manli; Guo, Zhide; Gao, Mengna; Shi, Changrong; Xu, Duo; You, Linyi; Wu, Xiaowei; Su, Xinhui; Zhuang, Rongqiang; Pan, Weimin; Liu, Ting; Zhang, Xianzhong

    2017-05-01

    Folate receptor is an ideal target for tumor-specific diagnostic and therapeutic. The aim of this study was to synthesize (99m) Tc-labeled folate-polyamidoamine dendrimer modified with 2-hydrazinonicotinic acid ((99m) Tc-HP3 FA) for FR imaging. The (99m) Tc-HP3 FA conjugate was prepared using N-tris-(hydroxymethyl)-methylglycine and trisodium triphenylphosphine-3,3',3″-trisulfonate as coligands. Physicochemical properties, in vitro cell uptake study, and in vivo micro-single-photon emission computed tomography/CT imaging were performed. The radiolabeled (99m) Tc-HP3 FA conjugate was prepared with high radiolabeling yield, good stability, and water solubility (logP = -1.70 ± 0.21). In cell uptake study, the radiolabeled conjugate showed high uptakes in the FR-abundant KB cells and could be blocked significantly by excess folic acid. The 7721 cells which served as control group substantially had no uptakes. The results of micro-single-photon emission computed tomography/CT imaging exhibited that high accumulation of activity was found in FR-overexpressed KB tumor, and the tumor-to-muscle ratio was approximately 25.78, while, using free FA as inhibitor, the uptakes of (99m) Tc-HP3 FA in KB tumor and kidney were obviously inhibited. In summary, a new radiocompound was synthesized successfully with specific FR targeting ability. The feasibility of (99m) Tc-HP3 FA for early diagnosis of FR-positive tumors with non-invasive single-photon emission computed tomography imaging was demonstrated and the possibility of imaging-guided drug delivery based on multifunctional polyamidoamine will be studied in the future. © 2016 John Wiley & Sons A/S.

  6. Synthesis and characterization of a stable, label-free optical biosensor from TiO2-coated porous silicon.

    PubMed

    Li, Jianlin; Sailor, Michael J

    2014-05-15

    A nanoscale layer of TiO2 is coated on the inner pore walls of a porous silicon (PSi) film by room-temperature infiltration of a TiO2 sol-gel precursor and firing at 500 °C. The PSi:TiO2 composite films are characterized by Fourier transform infrared (FTIR), X-ray diffraction (XRD), energy dispersive X-ray spectral analysis (EDS), scanning electron microscopy (SEM) and reflective interferometric Fourier transform spectroscopy (RIFTS). The analysis indicates that TiO2 conformally coats the inner pore surfaces of the PSi film. The film displays greater aqueous stability in the pH range 2-12 relative to a PSi:SiO2 surface. A label-free optical interference immunosensor based on the TiO2-coated PSi film is demonstrated by real-time monitoring of the physical adsorption of protein A, followed by the specific binding of rabbit anti-sheep immunoglobulin (IgG) and then specific capture of sheep IgG. The time to achieve equilibrium for the physical adsorption of protein A on the surface of TiO2-coated PSi film is significantly greater than that of PSi film. The specificity of the protein A and rabbit anti-sheep IgG construct on the sensor is confirmed by tests with non-binding chicken IgG. The sensitivity of the immunosensor is shown to be 8210 ± 170 nm/refractive index unit (RIU). Copyright © 2013 Elsevier B.V. All rights reserved.

  7. Synthesis of fluorescent label, DBD-beta-proline, and the resolution efficiency for chiral amines by reversed-phase chromatography.

    PubMed

    Min, Jun Zhe; Toyo'oka, Toshimasa; Kato, Masaru; Fukushima, Takeshi

    2005-01-01

    DBD-d(and l)-beta-proline, new fluorescent chiral derivatization reagents, were synthesized from the reaction of 4-(N,N-dimethylaminosulfonyl)-7- fl uoro-2,1,3-benzoxadiazole (DBD-F) with beta-proline. The racemic mixture synthesized was separated by a chiral stationary phase (CSP) column, Chiralpak AD-H, with n-hexane-EtOH-TFA-diethylamine (70:30:0.1:0.1) as the mobile phase. The dl-forms were decided according to the results obtained from a circular dichroism (CD) detector after separation by the CSP column. The fractionated enantiomers reacted with chiral amine to produce a couple of diastereomers. The labeling proceeded in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and pyridine as the activation reagents. The reaction conditions were mild and no racemization occurred during the diastereomer formation. The resulting diastereomers fluoresced at around 570 nm (excitation at around 460 nm). Good linearity of the calibration curves was obtained in the range 1-75 pmol and the detection limits on chromatogram were less than 1 pmol. The separability of the diastereomers was compared with the diastereomers derived from DBD-d(or l)-proline. The resolution values (Rs) obtained from the diastereomers of three chiral amines with DBD-d(or l)-beta-proline were higher than those derived from DBD-d(or l)-proline, e.g. dl-phenylalanine methylester (dl-PAME), 2.23 vs 1.37; (R)(S)-1-phenylethylamine [(R)(S)-PEA], 2.09 vs 1.13; and (R)(S)-1-(1-naphthyl)ethylamines [(R)(S)-NEA], 5.19 vs 1.23. The results suggest that the position of COOH group on pyrrolidine moiety in the structures is one of the important factors for the efficient separation of a couple of the diastereomers.

  8. Suppression of isotope scrambling in cell-free protein synthesis by broadband inhibition of PLP enymes for selective 15N-labelling and production of perdeuterated proteins in H2O.

    PubMed

    Su, Xun-Cheng; Loh, Choy-Theng; Qi, Ruhu; Otting, Gottfried

    2011-05-01

    Selectively isotope labelled protein samples can be prepared in vivo or in vitro from selectively labelled amino acids but, in many cases, metabolic conversions between different amino acids result in isotope scrambling. The best results are obtained by cell-free protein synthesis, where metabolic enzymes are generally less active, but isotope scrambling can never be suppressed completely. We show that reduction of E. coli S30 extracts with NaBH(4) presents a simple and inexpensive way to achieve cleaner selective isotope labelling in cell-free protein synthesis reactions. The purpose of the NaBH(4) is to inactivate all pyridoxal-phosphate (PLP) dependent enzymes by irreversible reduction of the Schiff bases formed between PLP and lysine side chains of the enzymes or amino groups of free amino acids. The reduced S30 extracts retain their activity of protein synthesis, can be stored as well as conventional S30 extracts and effectively suppress conversions between different amino acids. In addition, inactivation of PLP-dependent enzymes greatly stabilizes hydrogens bound to α-carbons against exchange with water, minimizing the loss of α-deuterons during cell-free production of proteins from perdeuterated amino acids in H(2)O solution. This allows the production of highly perdeuterated proteins that contain protons at all exchangeable positions, without having to back-exchange labile deuterons for protons as required for proteins that have been synthesized in D(2)O.

  9. Fluorescently labelled glycans and their applications.

    PubMed

    Yan, Hongbin; Yalagala, Ravi Shekar; Yan, Fengyang

    2015-11-01

    This review summarises the literature on the synthesis and applications of fluorescently labelled carbohydrates. Due to the sensitivity of fluorescent detection, this approach provides a useful tool to study processes involving glycans. A few general categories of labelling are presented, in situ labelling of carbohydrates with fluorophores, fluorescently labelled glycolipids, fluorogenic glycans, pre-formed fluorescent glycans for intracellular applications, glycan-decorated fluorescent polymers, fluorescent glyconanoparticles, and other functional fluorescent glycans.

  10. Synthesis of (68)Ga-labeled NOTA-RGD-GE11 heterodimeric peptide for dual integrin and epidermal growth factor receptor-targeted tumor imaging.

    PubMed

    Yu, Hung-Man; Chen, Jyun-Hong; Lin, Kun-Liang; Lin, Wuu-Jyh

    2015-06-15

    Radiolabeled Arg-Gly-Asp (RGD) peptide analogs have been extensively studied for αvβ3 integrin-targeted angiogenesis imaging. According to recently presented evidence, the dodecapeptide GE11 has high affinity to the epidermal growth factor receptor (EGFR), which is overexpressed in many types of cancer. Dual-receptor molecular imaging probes with two different heterodimeric peptides exhibit improved cancer targeting efficacy. In the present study, the design and synthesis of a new RGD-GE11 peptide heterodimer for dual αvβ3 integrin/EGFR-targeted cancer imaging are described. The RGD-GE11 heterodimer was linked with 6-aminohexanoic acid (6-Ahx) and cysteine and conjugated with 1,4,7-triazacyclononane-N,N',N″-triacetic acid (NOTA) to form NOTA-RGD-cys-6-Ahx-GE11. The monomeric peptides, NOTA-cys-6-Ahx-GE11 and c(RGDyK), were formed by a peptide synthesizer. The peptide heterodimer NOTA-RGD-GE11 was obtained by NOTA-cys-6-Ahx-GE11 and maleimidopropyl-c(RGDyK) conjugation with a thioether linkage. The NOTA peptide conjugate was labeled with freshly eluted (68)Ga and purified using reversed-phase high-performance liquid chromatography. The (68)Ga-NOTA-RGD-cys-6-Ahx-GE11 was successfully prepared, in this study, with a radiochemical yield of 85% and a radiochemical purity of >98%. These results warrant further investigation of this heterodimeric peptide's binding affinity to the receptors. Copyright © 2015 John Wiley & Sons, Ltd.

  11. Nutrition Labeling

    NASA Astrophysics Data System (ADS)

    Metzger, Lloyd E.

    Nutrition labeling regulations differ in countries around the world. The focus of this chapter is on nutrition labeling regulations in the USA, as specified by the Food and Drug Administration (FDA) and the Food Safety and Inspection Service (FSIS) of the United States Department of Agriculture (USDA). A major reason for analyzing the chemical components of foods in the USA is nutrition labeling regulations. Nutrition label information is not only legally required in many countries, but also is of increasing importance to consumers as they focus more on health and wellness.

  12. Design and operations at the National Tritium Labelling Facility

    SciTech Connect

    Morimoto, H.; Williams, P.G.

    1991-09-01

    The National Tritium Labelling Facility (NTLF) is a multipurpose facility engaged in tritium labeling research. It offers to the biomedical research community a fully equipped laboratory for the synthesis and analysis of tritium labeled compounds. The design of the tritiation system, its operations and some labeling techniques are presented.

  13. Synthesis and characterization of boron fenbufen and its F-18 labeled homolog for boron neutron capture therapy of COX-2 overexpressed cholangiocarcinoma.

    PubMed

    Yeh, Chun-Nan; Chang, Chi-Wei; Chung, Yi-Hsiu; Tien, Shi-Wei; Chen, Yong-Ren; Chen, Tsung-Wen; Huang, Ying-Cheng; Wang, Hsin-Ell; Chou, You-Cheng; Chen, Ming-Huang; Chiang, Kun-Chun; Huang, Wen-Sheng; Yu, Chung-Shan

    2017-09-30

    Boron neutron capture therapy (BNCT) is a binary therapy that employs neutron irradiation on the boron agents to release high-energy helium and alpha particles to kill cancer cells. An optimal response to BNCT depends critically on the time point of maximal (10)B accumulation and highest tumor to normal ratio (T/N) for performing the neutron irradiation. The aggressive cholangiocarcinoma (CCA) representing a liver cancer that overexpresses COX-2 enzyme is aimed to be targeted by COX-2 selective boron carrier, fenbufen boronopinacol (FBPin). Two main works were performed including: 1) chemical synthesis of FBPin as the boron carrier and 2) radiochemical labeling with F-18 to provide the radiofluoro congener, m-[(18)F]fluorofenbufen ester boronopinacol (m-[(18)F]FFBPin), to assess the binding affinity, cellular accumulation level and distribution profile in CCA rats. FBPin was prepared from bromofenbufen via 3 steps with 82% yield. The binding assay employed [(18)F]FFBPin to compete FBPin for binding to COX-1 (IC50=0.91±0.68μM) and COX-2 (IC50=0.33±0.24μM). [(18)F]FFBPin-derived 60-min dynamic PET scans predict the (10)B-accumulation of 0.8-1.2ppm in liver and 1.2-1.8ppm in tumor and tumor to normal ratio=1.38±0.12. BNCT was performed 40-55min post intravenous administration of FBPin (20-30mg) in the CCA rats. CCA rats treated with BNCT display more tumor reduction than that by NCT with respect of 2-[(18)F]fluoro-2-deoxy glucose uptake in the tumor region of interest, 20.83±3.00% (n=12) vs. 12.83±3.79% (n=10), P=0.05. The visualizing agent [(18)F]FFBPin resembles FBPin to generate the time-dependent boron concentration profile. Optimal neutron irradiation period is thus determinable for BNCT. A boron-substituted agent based on COX-2-binding features has been prepared. The moderate COX-2/COX-1 selectivity index of 2.78 allows a fair tumor selectivity index of 1.38 with a mild cardiovascular effect. The therapeutic effect from FBPin with BNCT warrants a proper

  14. Synthesis of [13C4]-labeled ∆9-tetrahydrocannabinol and 11-nor-9-carboxy-∆9-tetrahydrocannabinol as internal standards for reducing ion suppressing/alteration effects in LC/MS-MS quantification.

    PubMed

    Karlsen, Morten; Liu, Huiling; Johansen, Jon Eigill; Hoff, Bård Helge

    2014-09-01

    (-)-∆9-Tetrahydrocannabinol is the principal psychoactive component of the cannabis plant and also the active ingredient in some prescribed drugs. To detect and control misuse and monitor administration in clinical settings, reference samples of the native drugs and their metabolites are needed. The accuracy of liquid chromatography/mass spectrometric quantification of drugs in biological samples depends among others on ion suppressing/alteration effects. Especially, 13C-labeled drug analogues are useful for minimzing such interferences. Thus, to provide internal standards for more accurate quantification and for identification purpose, synthesis of [13C4]-∆9-tetrahydro-cannabinol and [13C4]-11-nor-9-carboxy-∆9-tetrahydrocannabinol was developed via [13C4]-olivetol. Starting from [13C4]-olivetol the synthesis of [13C4]-11-nor-9-carboxy-∆9-tetrahydrocannabinol was shortened from three to two steps by employing nitromethane as a co-solvent in condensation with (+)-apoverbenone.

  15. Highly efficient click labeling using 2-[18F]fluoroethyl azide and synthesis of an 18F N-hydroxysuccinimide ester as conjugation agent

    PubMed Central

    Zhou, Dong; Chu, Wenhua; Dence, Carmen S.; Mach, Robert H.; Welch, Michael J.

    2012-01-01

    Introduction Click labeling using 2-[18F]fluoroethyl azide has been proven to be promising methods of radiolabeling small molecules and peptides, some of which are undergoing clinical evaluations. However, the previously reported method afforded low yield, poor purities and under desirable reproducibility. Methods A vacuum distillation method was used to isolate 2-[18F]fluoroethyl azide, and the solvent effect of acetonitrile (ACN) and dimethylformamide (DMF) on the click labeling using Cu(I) from copper sulfate/sodium ascorbate was studied. The labeling conditions were optimized to radiosynthesize a hydroxysuccinimide ester (NHS). Results 2-[18F]fluoroethyl azide was isolated by the vacuum distillation method with > 80% yield within 10 min in a “pure” and click-ready form. It was found that the amount of DMF was critical for maintaining high levels of Cu(I) from copper sulfate/sodium ascorbate in order to rapidly complete the click labeling reaction. The addition of bathophenanthrolinedisulfonic acid disodium salt (BPDS) to the mixture of copper sulfate/sodium ascorbate also greatly improved the click labeling efficiency. Through exploiting these optimizations, a base-labile N-hydroxysuccinimide (NHS) ester was rapidly radiosynthesized in 90% isolated yield with good chemical and radiochemical purities. Conclusions We have developed a general method to click-label small molecules efficiently using [18F]2 for research and clinical use. This NHS ester can be used for conjugation chemistry to label antibodies, peptides and small molecules as PET tracers. PMID:22770647

  16. Solid-phase synthesis of peptide radiopharmaceuticals using Fmoc-N-epsilon-(hynic-Boc)-lysine, a technetium-binding amino acid: application to Tc-99m-labeled salmon calcitonin.

    PubMed

    Greenland, William E P; Howland, Kevin; Hardy, Judith; Fogelman, Ignac; Blower, Philip J

    2003-04-24

    Labeling of proteins with metallic radionuclides for use in radiopharmaceuticals involves covalently attaching a bifunctional chelator. In principle, use of smaller peptides allows this chelator to be incorporated during solid-phase peptide synthesis (SPPS) with total site specificity. To realize the advantages of this approach, a lysine-hynic conjugate Fmoc-N-epsilon-(Hynic-Boc)-Lys was synthesized for incorporating the well-known technetium-99m-binding hydrazinonicotinamide ligand into peptides during SPPS. It was used to synthesize a technetium-99m-labeled salmon calcitonin with the hynic-linked amino acid in place of lysine-18. A trifluoroacetate group protected the hynic during alkaline oxidation to the cyclic disulfide and was readily removed by mild acid treatment. The peptide was efficiently labeled (91-98% radiochemical yield) with Tc-99m in the presence of tricine and SnCl(2) with high specific activity (>100 MBq/microg). The product showed good serum stability and specific affinity for human calcitonin receptors. Fmoc-N-epsilon-(Hynic-Boc)-Lys is a highly versatile technetium-binding amino acid for incorporation into peptides during SPPS. This allows total flexibility and control in the site of attachment and is suitable for a combinatorial approach to peptide radiopharmaceuticals.

  17. Synthesis and migration of /sup 3/H-fucose-labeled glycoproteins in the retinal pigment epithelium of albino rats, as visualized by radioautography

    SciTech Connect

    Haddad, A.; Bennett, G.

    1987-03-01

    /sup 3/H-fucose was injected into the vitreous body of the eye(s) of 250-gm rats, which were then killed by means of an intracardiac perfusion with glutaraldehyde after intervals of 10 min, 1 and 4 hr, and 1 and 7 days. The eyes were removed and further fixed, and pieces of retina were processed for light and electron microscope radioautography. Light microscope radioautography showed that the pigment epithelial cells actively incorporated /sup 3/H-fucose label. The intensity of reaction peaked at 4 hr after injection of the label and then slowly declined. Quantitative electron microscope radioautography revealed that, at 10 min after /sup 3/H-fucose injection, over 70% of the label was localized to the Golgi apparatus, indicating that fucose residues are added to newly synthesized glycoproteins principally at this site. With time the proportion of label associated with the Golgi apparatus decreased, but that assigned to the infolded basal plasma membrane, the apical microvilli, and various apical lysosomes increased. These results indicate that in retinal pigment epithelial cells newly synthesized glycoproteins continuously migrate from the Golgi apparatus to lysosomes and to various regions of the plasma membrane. In this case, the membrane glycoproteins may play specific roles in receptor functions of the basal plasma membrane or phagocytic activities at the apical surface. Very little label migrated to Bruch's membrane, indicating either a very slow turnover or a paucity of fucose-containing glycoproteins at this site.

  18. Synthesis and application of a N-1' fluorescent biotinyl derivative inducing the specific carboxy-terminal dual labeling of a novel RhoB-selective scFv.

    PubMed

    Chaisemartin, L; Chinestra, P; Favre, G; Blonski, C; Faye, J C

    2009-05-20

    The fluorescent site-specific labeling of protein would provide a new, easy-to-use alternative to biochemical and immunochemical methods. We used an intein-mediated strategy for covalent labeling of the carboxy-terminal amino acid of a RhoB-selective scFv previously isolated from a phage display library (a human synthetic V(H) + V(L) scFv phage library). The scFv fused to the Mxe intein was produced in E. coli and purified and was then labeled with a newly synthesized fluorescent biotinyl cysteine derivative capable of inducing scFv-Mxe intein splicing. In this study, we investigated the splicing and labeling properties of various amino acids in the hinge domain between scFv and Mxe under thiol activation. In this dual labeling system, the fluorescein is used for antibody detection and biotin is used for purification, resulting in a high specific activity for fluorescence. We then checked that the purified biotinylated fluorescent scFv retained its selectivity for RhoB without modification of its affinity.

  19. Toward a biorelevant structure of protein kinase C bound modulators: design, synthesis, and evaluation of labeled bryostatin analogues for analysis with rotational echo double resonance NMR spectroscopy.

    PubMed

    Loy, Brian A; Lesser, Adam B; Staveness, Daryl; Billingsley, Kelvin L; Cegelski, Lynette; Wender, Paul A

    2015-03-18

    Protein kinase C (PKC) modulators are currently of great importance in preclinical and clinical studies directed at cancer, immunotherapy, HIV eradication, and Alzheimer's disease. However, the bound conformation of PKC modulators in a membrane environment is not known. Rotational echo double resonance (REDOR) NMR spectroscopy could uniquely address this challenge. However, REDOR NMR requires strategically labeled, high affinity ligands to determine interlabel distances from which the conformation of the bound ligand in the PKC-ligand complex could be identified. Here we report the first computer-guided design and syntheses of three bryostatin analogues strategically labeled for REDOR NMR analysis. Extensive computer analyses of energetically accessible analogue conformations suggested preferred labeling sites for the identification of the PKC-bound conformers. Significantly, three labeled analogues were synthesized, and, as required for REDOR analysis, all proved highly potent with PKC affinities (∼1 nM) on par with bryostatin. These potent and strategically labeled bryostatin analogues are new structural leads and provide the necessary starting point for projected efforts to determine the PKC-bound conformation of such analogues in a membrane environment, as needed to design new PKC modulators and understand PKC-ligand-membrane structure and dynamics.

  20. Design and synthesis of fluorescence-labeled closo-dodecaborate lipid: its liposome formation and in vivo imaging targeting of tumors for boron neutron capture therapy.

    PubMed

    Nakamura, Hiroyuki; Ueda, Noriko; Ban, Hyun Seung; Ueno, Manabu; Tachikawa, Shoji

    2012-02-21

    The fluorescence-labeled closo-dodecaborane lipid (FL-SBL) was synthesized from (S)-(+)-1,2-isopropylideneglycerol as a chiral starting material. FL-SBL was readily accumulated into the PEGylated DSPC liposomes prepared from DSPC, CH, and DSPE-PEG-OMe by the post insertion protocol. The boron concentrations and the fluorescent intensities of the FL-SBL-labeled DSPC liposomes increased with the increase of the additive FL-SBL, and the maximum emission wavelength of the liposomes appeared at 531 nm. A preliminary in vivo imaging study of tumor-bearing mice revealed that the FL-SBL-labeled DSPC liposomes were delivered to the tumor tissue but not distributed to hypoxic regions.

  1. Food labeling

    MedlinePlus

    ... States Food and Drug Administration (FDA) has proposed making changes to the food labels that may correct these problems. AMOUNTS PER SERVING The total calories and the calories from fat are listed. These numbers help consumers make decisions about fat intake. The list of nutrients includes ...

  2. Sequence- and structure-dependent DNA base dynamics: Synthesis, structure, and dynamics of site and sequence specifically spin-labeled DNA

    SciTech Connect

    Spaltenstein, A.; Robinson, B.H.; Hopkins, P.B. )

    1989-11-28

    A nitroxide spin-labeled analogue of thymidine (1a), in which the methyl group is replaced by an acetylene-tethered nitroxide, was evaluated as a probe for structural and dynamics studies of sequence specifically spin-labeled DNA. Residue 1a was incorporated into synthetic deoxyoligonucleotides by using automated phosphite triester methods. {sup 1}H NMR, CD, and thermal denaturation studies indicate that 1a (T) does not significantly alter the structure of 5{prime}-d(CGCGAATT*CGCG) from that of the native dodecamer. EPR studies on monomer, single-stranded, and duplexed DNA show that 1a readily distinguishes environments of different rigidity. Comparison of the general line-shape features of the observed EPR spectra of several small duplexes (12-mer, 24-mer) with simulated EPR spectra assuming isotropic motion suggests that probe 1a monitors global tumbling of small duplexes. Increasing the length of the DNA oligomers results in significant deviation from isotropic motion, with line-shape features similar to those of calculated spectra of objects with isotropic rotational correlation times of 20-100 ns. EPR spectra of a spin-labeled GT mismatch and a T bulge in long DNAs are distinct from those of spin-labeled Watson-Crick paired DNAs, further demonstrating the value of EPR as a tool in the evaluation of local dynamic and structural features in macromolecules.

  3. Synthesis and positron emission tomography studies of C-11 labeled isotopomers and metabolites of GTS-21, a partial α7 nicotinic cholinergic agonist drug

    PubMed Central

    Kim, Sung Won; Ding, Yu-Shin; Alexoff, David; Patel, Vinal; Logan, Jean; Lin, Kuo-Shyan; Shea, Colleen; Muench, Lisa; Xu, Youwen; Carter, Pauline; King, Payton; Constanzo, Jasmine R.; Ciaccio, James A.; Fowler, Joanna S.

    2009-01-01

    Introduction GTS-21 ((3E)-3-[(2,4-dimethoxyphenyl)methylene]-3,4,5,6-tetrahydro-2,3′-bipyridine), a partial α7 nicotinic acetylcholine receptor agonist drug, has recently been shown to improve cognition in schizophrenia and Alzheimer’s disease. One of its two major demethylated metabolites, 4-OH-GTS-21, has been suggested to contribute to its therapeutic effects. Methods We labeled GTS-21 in two different positions with carbon-11 ([2-methoxy-11C]GTS-21 and [4-methoxy-11C]GTS-21) along with two corresponding demethylated metabolites ([2-methoxy-11C]4-OH-GTS-21 and [4-methoxy-11C]2-OH-GTS-21) for pharmacokinetic studies in baboons and mice with PET. Results Both [2-methoxy-11C]GTS-21 and [4-methoxy-11C]GTS-21 showed similar initial high rapid uptake in baboon brain, peaking from 1–3.5 min (0.027–0.038 %ID/cc) followed by rapid clearance (t1/2 <15 min), resulting in low brain retention by 30 min. However, after 30 min, [2-methoxy-11C]GTS-21 continued to clear while [4-methoxy-11C]GTS-21 plateaued, suggesting the entry of a labeled metabolite into the brain. Comparison of the pharmacokinetics of the two labeled metabolites confirmed expected higher brain uptake and retention of [4-methoxy-11C]2-OH-GTS-21 (the labeled metabolite of [4-methoxy-11C]GTS-21) relative to [2-methoxy-11C]4-OH-GTS-21 (the labeled metabolite of [2-methoxy-11C]GTS-21) which had negligible brain uptake. Ex vivo studies in mice showed that GTS-21 is the major chemical form in the mouse brain. Whole body dynamic PET imaging in baboon and mouse showed that the major route of excretion of C-11 is through the gallbladder. Conclusions The major findings are (1) extremely rapid uptake and clearance of [2-methoxy-11C]GTS-21 from the brain which may need to be considered in developing optimal dosing of GTS-21 for patients, and (2) significant brain uptake of 2-OH-GTS-21, suggesting that it might contribute to the therapeutic effects of GTS-21. This study illustrates the value of comparing

  4. Synthesis and positron emission tomography studies of C-11-labeled isotopomers and metabolites of GTS-21, a partial alpha7 nicotinic cholinergic agonist drug.

    PubMed

    Kim, Sung Won; Ding, Yu-Shin; Alexoff, David; Patel, Vinal; Logan, Jean; Lin, Kuo-Shyan; Shea, Colleen; Muench, Lisa; Xu, Youwen; Carter, Pauline; King, Payton; Constanzo, Jasmine R; Ciaccio, James A; Fowler, Joanna S

    2007-07-01

    (3E)-3-[(2,4-dimethoxyphenyl)methylene]-3,4,5,6-tetrahydro-2,3'-bipyridine (GTS-21), a partial alpha7 nicotinic acetylcholine receptor agonist drug, has recently been shown to improve cognition in schizophrenia and Alzheimer's disease. One of its two major demethylated metabolites, 4-OH-GTS-21, has been suggested to contribute to its therapeutic effects. We labeled GTS-21 in two different positions with carbon-11 ([2-methoxy-(11)C]GTS-21 and [4-(11)C]GTS-21) along with two corresponding demethylated metabolites ([2-methoxy-(11)C]4-OH-GTS-21 and [4-methoxy-(11)C]2-OH-GTS-21) for pharmacokinetic studies in baboons and mice with positron emission tomography (PET). Both [2-(11)C]GTS-21 and [4-methoxy-(11)C]GTS-21 showed similar initial high rapid uptake in baboon brain, peaking from 1 to 3.5 min (0.027-0.038%ID/cc) followed by rapid clearance (t(1/2)<15 min), resulting in low brain retention by 30 min. However, after 30 min, [2-methoxy-(11)C]GTS-21 continued to clear while [4-methoxy-(11)C]GTS-21 plateaued, suggesting the entry of a labeled metabolite into the brain. Comparison of the pharmacokinetics of the two labeled metabolites confirmed expected higher brain uptake and retention of [4-methoxy-(11)C]2-OH-GTS-21 (the labeled metabolite of [4-methoxy-(11)C]GTS-21) relative to [2-methoxy-(11)C]4-OH-GTS-21 (the labeled metabolite of [2-methoxy-(11)C]GTS-21), which had negligible brain uptake. Ex vivo studies in mice showed that GTS-21 is the major chemical form in the mouse brain. Whole-body dynamic PET imaging in baboon and mouse showed that the major route of excretion of C-11 is through the gallbladder. The major findings are as follows: (a) extremely rapid uptake and clearance of [2-methoxy-(11)C]GTS-21 from the brain, which may need to be considered in developing optimal dosing of GTS-21 for patients, and (b) significant brain uptake of 2-OH-GTS-21, suggesting that it might contribute to the therapeutic effects of GTS-21. This study illustrates the value of

  5. Short-lived positron emitter labeled radiotracers - present status

    SciTech Connect

    Fowler, J.S.; Wolf, A.P.

    1982-01-01

    The preparation of labelled compounds is important for the application of positron emission transaxial tomography (PETT) in biomedical sciences. This paper describes problems and progress in the synthesis of short-lived positron emitter (/sup 11/C, /sup 18/F, /sup 13/N) labelled tracers for PETT. Synthesis of labelled sugars, amino acids, and neurotransmitter receptors (pimozide and spiroperidol tagged with /sup 11/C) is discussed in particular. (DLC)

  6. Design and synthesis of a new class of fluorescent photoaffinity label with specific reference to 4-benzoyl-1-benzamidofluorescein: a new photolabel for adenine nucleotide binding domains on enzymes

    NASA Astrophysics Data System (ADS)

    Rosen, Jane E.

    1993-05-01

    Benzophenone was used as the photoreactive moiety in the synthesis of several water soluble, fluorescent photoaffinity labels. The following compounds were synthesized: 5-(2-(p- benzoylbenzamido)ethylamino-1-napthalenesulfonate (BzEDANS); 5-(2-(p- benzoylbenzamido)hexylamino-1-napthalenesulfonate (BzHDANS); and 4-benzoyl-1- benzamidofluorescein (BzAF). BzEDANS and BzHDANS were found to be unsuitable for use as photochemical probes. They were incapable of photoinduced covalent binding to methylene carbon due to intramolecular triplet-triplet energy transfer. BzAF was synthesized because its fluorescent moiety, fluorescein, is an inefficient acceptor for intramolecular quenching of the benzophenone triplet state diradical intermediate. BzAF was found to be a suitable and efficient photolabel and is presently a prototype for a new class of fluorescent photolabel.

  7. [18F]- and [11C]-Labeled N-benzyl-isatin sulfonamide analogues as PET tracers for apoptosis: synthesis, radiolabeling mechanism, and in vivo imaging of apoptosis in Fas-treated mice

    PubMed Central

    Zhou, Dong; Chu, Wenhua; Chen, Delphine L.; Wang, Qi; Reichert, David E.; Rothfuss, Justin; D'Avignon, Andre; Welch, Michael J.; Mach, Robert H.

    2011-01-01

    Summary The radiolabeled isatin sulfonamide caspase-3 inhibitor, [18F]2 (WC-II-89), is a potential PET radiotracer for noninvasive imaging of apoptosis. The radiolabeling mechanism was studied by 13C NMR, ESI/MS, and computational calculations. It was found that the high electrophilicity of the C3 carbonyl group in the isatin ring, which served as a trap for [18F]fluoride, was responsible for the failure of the radiolabeling via nucleophilic substitution of the mesylate group in 7a by [18F]fluoride. Once treated with a strong base, 7a opened the isatin ring completely to form an isatinate intermediate 16, which lost the ability to trap [18F]fluoride, thereby allowing the displacement of the mesylate group to afford the 18F-labeled isatinate 17. [18F]17 can be converted to isatin [18F]2 efficiently under acidic conditions. The ring-opening and re-closure of the isatin ring under basic and acidic conditions were confirmed by reversed phase HPLC analysis, ESI/MS and 13C NMR studies. Computational studies of model compounds also support the above proposed mechanism. Similarly, the ring-opening and re-closure method was used successfully in the synthesis of the 11C labeled isatin sulfonamide analogue [11C]4 (WC-98). A microPET imaging study using [11C]4 in the Fas liver apoptosis model demonstrated retained activity in the target organ (liver) of the treated mice. Increased caspase-3 activation in the liver was verified by the fluorometric caspase-3 enzyme assay. Therefore, this study provides a useful method for radio-synthesis of isatin derivative radiotracers for PET and SPECT studies, and [11C]4 is a potential PET radiotracer for noninvasive imaging of apoptosis. PMID:19300818

  8. Synthesis of 11C‐labeled Sulfonyl Carbamates through a Multicomponent Reaction Employing Sulfonyl Azides, Alcohols, and [11C]CO

    PubMed Central

    Stevens, Marc Y.; Chow, Shiao Y.; Estrada, Sergio; Eriksson, Jonas; Asplund, Veronika; Orlova, Anna; Mitran, Bogdan; Antoni, Gunnar; Larhed, Mats; Åberg, Ola

    2016-01-01

    Abstract We describe the development of a new methodology focusing on 11C‐labeling of sulfonyl carbamates in a multicomponent reaction comprised of a sulfonyl azide, an alkyl alcohol, and [11C]CO. A number of 11C‐labeled sulfonyl carbamates were synthesized and isolated, and the developed methodology was then applied in the preparation of a biologically active molecule. The target compound was obtained in 24±10 % isolated radiochemical yield and was evaluated for binding properties in a tumor cell assay; in vivo biodistribution and imaging studies were also performed. This represents the first successful radiolabeling of a non‐peptide angiotensin II receptor subtype 2 agonist, C21, currently in clinical trials for the treatment of idiopathic pulmonary fibrosis. PMID:28032026

  9. Chemical synthesis of glycoproteins with the specific installation of gradient enriched 15N-labeled amino acids for getting insight into glycoprotein behavior.

    PubMed

    Kajihara, Yasuhiro; Nguyen, Minh Hien; Izumi, Masayuki; Sato, Hajime; Okamoto, Ryo

    2017-03-09

    We propose a novel partially 15N-labelling method for the amide backbone of a synthetic glycoprotein. By use of a chemical approach utilizing SPPS and NCL, we inserted thirteen 15N-labeled amino acids at specific positions of the protein backbone, while intentionally varying the enrichment of 15N atoms. This idea enables us to discriminate even the same type of amino acid based on the intensities of 1H-15N HSQC signals, thus allowing us to understand the dynamics of the local conformation of a synthetic homogeneous glycoprotein. Results suggested that the attachment of an oligosaccharide of either a bi-antennary complex-type or a high-mannose-type did not disturb protein conformation. However, T1 values suggested that the oligosaccharide influenced dynamics at the local conformation. Temperature-varied CD spectra and T1 values clearly indicated that oligosaccharides appeared to inhibit protein fluctuation or, in other words, stabilize protein structure.

  10. Synthesis of deuterium-labelled 5'-O-[N-(Salicyl)sulfamoyl]adenosine (Sal-AMS-d(4)) as an internal standard for quantitation of Sal-AMS.

    PubMed

    Gupte, Amol; Subramanian, Murali; Remmel, Rory P; Aldrich, Courtney C

    2008-02-01

    5'-O-[N-(Salicyl)sulfamoyl]adenosine (Sal-AMS, 1) is a potent inhibitor of the bifunctional enzyme salicyl-AMP ligase in Mycobacterium tuberculosis. This inhibitor acts by disrupting the biosynthesis of the mycobactin siderophores that are essential for the process of iron acquisition. To aid with in vitro metabolism and in vivo pharmacokinetic studies of Sal-AMS, a stable deuterium-labelled Sal-AMS analog (Sal-AMS-d(4)) was synthesized. This deuterium-labelled analog was used as an internal standard to conduct in vitro plasma and microsomal stability studies. Sal-AMS was found to be stable for 24 h in human plasma and 1 h in human liver microsomes at 37 degrees C.

  11. Mass spectrometric measurements of norepinephrine synthesis in man from infusion of stable isotope-labelled L-threo-3,4-dihydroxyphenylserine

    SciTech Connect

    Suzuki, T.; Sakoda, S.; Ueji, M.; Kishimoto, S.

    1985-02-04

    The kinetics of stable isotope-labelled L-threo-3,4-dihydroxyphenylserine (L-threo-DOPS), an immediate precursor of (-)-norepinephrine, was studied to investigate the pharmacologic mechanism of its therapeutic effect on orthostatic hypotension in familial amyloid polyneuropathy (FAP) and on akinesia and freezing in parkinsonism. (/sup 13/C,D)-L-threo-DOPS was synthesized, and 100 mg of the compound was infused for 2 h into two normal subjects, two FAP patients and two patients with the degenerative diseases of the central nervous system. Labelled and endogenous norepinephrine in urine and plasma was assayed simultaneously by gas chromatography/mass spectrometry. The results indicate that the increase in norepinephrine in biological fluids after administration of L-threo-DOPS is attributable mostly to norepinephrine derived from L-threo-DOPS, not to pre-formed endogenous norepinephrine released by L-threo-DOPS.

  12. N-(2-chloroethyl)-N-nitrosoureas covalently bound to nonionic and monocationic lexitropsin dipeptides. Synthesis, DNA affinity binding characteristics, and reactions with sup 32 P-end-labeled DNA

    SciTech Connect

    Church, K.M.; Wurdeman, R.L.; Zhang, Yi; Chen, Faxian; Gold, B. )

    1990-07-24

    The synthesis and characterization of a series of compounds that contain an N-alkyl-N-nitrosourea functionality linked to DNA minor groove binding bi- and tripeptides (lexitropsins or information-reading peptides) based on methylpyrrole-2-carboxamide subunits are described. The lexitropsins (lex) synthesized have either a 3-(dimethylamino)propyl or propyl substituent on the carboxyl terminus. The preferred DNA affinity binding sequences of these compounds were footprinted in {sup 32}P-end-labeled restriction fragments with methidiumpropyl-EDTA{center dot}Fe(II), and in common with other structural analogues, e.g., distamycin and netropsin, these nitrosoureas recognize A-T-rich runs. The affinity binding of the compound with the dimethylamino terminus, which is ionized at near-neutral pH, appeared stronger than that observed for the neutral dipeptide. The sequence specificity for DNA alkylation by (2-chloroethyl)nitrosourea-lex dipeptides (Cl-ENU-lex), with neutral and charged carboxyl termini, using {sup 32}P-end-labeled restriction fragments, was determined by the conversion of the adducted sites into single-strand breaks by sequential heating at neutral pH and exposure to base. The DNA cleavage sites were visualized by polyacrylamide gel electrophoresis and autoradiography. Linking the Cl-ENU moiety to minor groove binders is a viable strategy to qualitatively and quantitatively control the delivery and release of the ultimate DNA alkylating agent in a sequence-dependent fashion.

  13. Synthesis, characterization and bioevaluation of technetium-99m labeled N-(2-Hydroxybenzyl)-2-amino-2-deoxy-D-glucose as a tumor imaging agent.

    PubMed

    Nadeem, Qaisar; Khan, Irfanullah; Javed, Muhammad; Mahmood, Zaid; Dar, Ume-Kalsoom; Ali, Muhammad; Hyder, Syed Waqar; Murad, Sohail

    2013-03-01

    N-(2-Hydroxybenzyl)-2-amino-2-deoxy-D-glucose (NHADG) was synthesized by conjugation of salicylaldehyde to glucosamine. The obtained compound was well characterized via different analytical techniques. Labeling of the synthesized compound with technetium-99m ((99m)Tc) in pertechnetate form ((99m)Tc O4-) was carried out via chelation reaction in the presence of stannous chloride dihydrate. Maximum radiochemical yield of (99m)Tc-NHADG complex (99%) was obtained by using 1 mg NHADG, 200 μg SnCl2.2H2O, at pH 9.5 and reaction time of 15 min. The radiochemical purity of the (99m)Tc-NHADG complex was measured by instant thin layer chromatography (ITLC) and paper chromatography (PC), without any notable decomposition at room temperature over a period of 4h. The biological evaluation results show that the (99m)Tc labeled NHADG conjugate is able to specifically target mammary carcinoma in mice models, thus highlighting its potential as an effective (99m)Tc labeled glucose-derived agent for tumor imaging.

  14. CdTe/CdS-MPA quantum dots as fluorescent probes to label yeast cells: synthesis, characterization and conjugation with Concanavalin A

    NASA Astrophysics Data System (ADS)

    Kato, Ilka T.; Santos, Camila C.; Benetti, Endi; Tenório, Denise P. L. A.; Cabral Filho, Paulo E.; Sabino, Caetano P.; Fontes, Adriana; Santos, Beate S.; Prates, Renato A.; Ribeiro, Martha S.

    2012-03-01

    Candida albicans is the most frequent human opportunistic pathogenic fungus and one of the most important causes of nosocomial infections. In fact, diagnosis of invasive candidiasis presents unique problems. The aim of this work was to evaluate, by fluorescence image analysis, cellular labeling of C. albicans with CdTe/CdS quantum dots conjugated or not to concanavalin A (ConA). Yeast cells were incubated with CdTe/CdS quantum dots (QD) stabilized with mercaptopropionic acid (MPA) (emission peak at 530 nm) for 1 hour. In the overall study we observed no morphological alterations. The fluorescence microscopic analysis of the yeast cells showed that the non-functionalized QDs do not label C. albicans cells, while for the QD conjugated to ConA the cells showed a fluorescence profile indicating that the membrane was preferentially marked. This profile was expected since Concanavalin A is a protein that binds specifically to terminal carbohydrate residues at the membrane cell surface. The results suggest that the QD-labeled Candida cells represent a promising tool to open new possibilities for a precise evaluation of fungal infections in pathological conditions.

  15. Introduction to Pesticide Labels

    EPA Pesticide Factsheets

    Pesticide product labels provide critical information about how to safely and legally handle and use pesticide products. Unlike most other types of product labels, pesticide labels are legally enforceable. Learn about pesticide product labels.

  16. Cellular uptake of PLA nanoparticles studied by light and electron microscopy: synthesis, characterization and biocompatibility studies using an iridium(III) complex as correlative label.

    PubMed

    Reifarth, Martin; Pretzel, David; Schubert, Stephanie; Weber, Christine; Heintzmann, Rainer; Hoeppener, Stephanie; Schubert, Ulrich S

    2016-03-21

    We present the synthesis of polylactide by ring-opening polymerization using a luminescent iridium(III) complex acting as initiator. The polymer was formulated into nanoparticles, which were taken up by HEK-293 cells. We could show that the particles provided an appropriate contrast in both superresolution fluorescence and electron microscopy, and, moreover, are non-toxic, in contrast to the free iridium complex.

  17. A 99mTc-Labelled Tetrazine for Bioorthogonal Chemistry. Synthesis and Biodistribution Studies with Small Molecule trans-Cyclooctene Derivatives

    PubMed Central

    Vito, Alyssa; Alarabi, Hussain; Czorny, Shannon; Beiraghi, Omid; Kent, Jeff; Janzen, Nancy; Genady, Afaf R.; Alkarmi, Salma A.; Rathmann, Stephanie; Naperstkow, Zoya; Blacker, Megan; Llano, Lisset; Berti, Paul J.

    2016-01-01

    A convenient strategy to radiolabel a hydrazinonicotonic acid (HYNIC)-derived tetrazine with 99mTc was developed, and its utility for creating probes to image bone metabolism and bacterial infection using both active and pretargeting strategies was demonstrated. The 99mTc-labelled HYNIC-tetrazine was synthesized in 75% yield and exhibited high stability in vitro and in vivo. A trans-cyclooctene (TCO)-labelled bisphosphonate (TCO-BP) that binds to regions of active calcium metabolism was used to evaluate the utility of the labelled tetrazine for bioorthogonal chemistry. The pretargeting approach, with 99mTc-HYNIC-tetrazine administered to mice one hour after TCO-BP, showed significant uptake of radioactivity in regions of active bone metabolism (knees and shoulders) at 6 hours post-injection. For comparison, TCO-BP was reacted with 99mTc-HYNIC-tetrazine before injection and this active targeting also showed high specific uptake in the knees and shoulders, whereas control 99mTc-HYNIC-tetrazine alone did not. A TCO-vancomycin derivative was similarly employed for targeting Staphylococcus aureus infection in vitro and in vivo. Pretargeting and active targeting strategies showed 2.5- and 3-fold uptake, respectively, at the sites of a calf-muscle infection in a murine model, compared to the contralateral control muscle. These results demonstrate the utility of the 99mTc-HYNIC-tetrazine for preparing new technetium radiopharmaceuticals, including those based on small molecule targeting constructs containing TCO, using either active or pretargeting strategies. PMID:27936007

  18. Synthesis and characterization of (68)Ga-labeled curcumin and curcuminoid complexes as potential radiotracers for imaging of cancer and Alzheimer's disease.

    PubMed

    Asti, Mattia; Ferrari, Erika; Croci, Stefania; Atti, Giulia; Rubagotti, Sara; Iori, Michele; Capponi, Pier C; Zerbini, Alessandro; Saladini, Monica; Versari, Annibale

    2014-05-19

    Curcumin (CUR) and curcuminoids complexes labeled with fluorine-18 or technetium-99m have recently shown their potential as diagnostic tools for Alzheimer's disease. Gallium-68 is a positron-emitting, generator-produced radionuclide, and its properties can be exploited in situ in medical facilities without a cyclotron. Moreover, CUR showed a higher uptake in tumor cells compared to normal cells, suggesting potential diagnostic applications in this field. In spite of this, no studies using labeled CUR have been performed in this direction, so far. Herein, (68)Ga-labeled complexes with CUR and two curcuminoids, namely diacetyl-curcumin (DAC) and bis(dehydroxy)curcumin (bDHC), were synthesized and characterized by means of experimental and theoretical approaches. Moreover, a first evaluation of their affinity to synthetic β-amyloid fibrils and uptake by A549 lung cancer cells was performed to show the potential application of these new labeled curcuminoids in these diagnostic fields. The radiotracers were prepared by reacting (68)Ga(3+) obtained from a (68)Ge/(68)Ga generator with 1 mg/mL curcuminoids solutions. Reaction parameters (precursor amount, reaction temperature, and pH) were optimized to obtain high and reproducible radiochemical yield and purity. Stoichiometry and formation of the curcuminoid complexes were investigated by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, NMR, ultraviolet-visible, and fluorescence spectroscopy on the equivalent (nat)Ga-curcuminoids (nat = natural) complexes, and their structure was computed by theoretical density functional theory calculations. The analyses evidenced that CUR, DAC, and bDHC were predominantly in the keto-enol form and attested to Ga(L)2(+) species formation. Identity of the (68)Ga(L)2(+) complexes was confirmed by coelution with the equivalent (nat)Ga(L)2(+) complexes in ultrahigh-performance liquid chromatography analyses.(68)Ga(CUR)2(+), (68)Ga(DAC)2(+), and (68)Ga(bDHC)2

  19. Synthesis and opioid receptor binding of indium (III) and [(111)In]-labeled macrocyclic conjugates of diprenorphine: novel ligands designed for imaging studies of peripheral opioid receptors.

    PubMed

    Srivastava, Shefali; Fergason-Cantrell, Emily A; Nahas, Roger I; Lever, John R

    2016-10-06

    Radiolabeled diprenorphine (DPN) and analogs are widely used ligands for non-invasive brain imaging of opioid receptors. To develop complementary radioligands optimized for studies of the peripheral opioid receptors, we prepared a pair of hydrophilic DPN derivatives, conjugated to the macrocyclic chelator DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), for complexation with trivalent metals. The non-radioactive indium (III) complexes, tethered to the C6-oxygen position of the DPN scaffold by 6- to 9-atom spacers, displayed high affinities for binding to μ, δ and κ opioid receptors in vitro. Use of the 9-atom linker conferred picomolar affinities equipotent to those of the parent ligand DPN. The [(111)In]-labeled complexes were prepared in good yield (>70%), with high radiochemical purity (~99%) and high specific radioactivity (>4000 mCi/μmol). Their log D7.4 values were -2.21 to -1.66. In comparison, DPN is lipophilic, with a log D7.4 of +2.25. Further study in vivo is warranted to assess the suitability of these [(111)In]-labeled DPN-DOTA conjugates for imaging trials.

  20. Synthesis of radioiodine-labeled 2-phenylethyl 1-thio-beta-D-galactopyranoside for imaging of LacZ gene expression.

    PubMed

    Choi, Joon Hun; Choe, Yearn Seong; Lee, Kyung-Han; Choi, Yong; Kim, Sang Eun; Kim, Byung-Tae

    2003-01-02

    A potent inhibitor of beta-galactosidase (EC 3.2.1.23), 2-phenylethyl 1-thio-beta-D-galactopyranoside (PETG), was radioiodinated for noninvasive imaging of LacZ gene expression. In order to introduce radioiodine to the phenyl ring of PETG, 2-(4-bromophenyl)ethanethiol was prepared and attached to the C-1 position of beta-D-galactose pentaacetate under conditions that resulted in the exclusive formation of the beta anomer. The bromo group of PETG was converted to the tributylstannyl group where radioiododemetallation was carried out. Radioiodine-labeled PETG tetraacetate was purified by HPLC, which can be used as a prodrug for biological evaluation or hydrolyzed to 2-(4-[123I/125I]iodophenyl)ethyl 1-thio-beta-D-galactopyranoside ([123I/125I]7) under basic conditions. The resulting radioiodine-labeled PETG was obtained in overall 62% radiochemical yield (decay-corrected) and with specific activity of 46-74 GBq/micromol.

  1. Synthesis and opioid receptor binding of indium (III) and [111In]-labeled macrocyclic conjugates of diprenorphine: novel ligands designed for imaging studies of peripheral opioid receptors

    PubMed Central

    Srivastava, Shefali; Fergason-Cantrell, Emily A.; Nahas, Roger I.; Lever, John R.

    2016-01-01

    Radiolabeled diprenorphine (DPN) and analogs are widely used ligands for non-invasive brain imaging of opioid receptors. To develop complementary radioligands optimized for studies of the peripheral opioid receptors, we prepared a pair of hydrophilic DPN derivatives, conjugated to the macrocyclic chelator DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), for complexation with trivalent metals. The non-radioactive indium (III) complexes, tethered to the C6-oxygen position of the DPN scaffold by 6- to 9-atom spacers, displayed high affinities for binding to μ, δ and κ opioid receptors in vitro. Use of the 9-atom linker conferred picomolar affinities equipotent to those of the parent ligand DPN. The [111In]-labeled complexes were prepared in good yield (>70%), with high radiochemical purity (~99%) and high specific radioactivity (>4000 mCi/μmol). Their log D7.4 values were −2.21 to −1.66. In comparison, DPN is lipophilic, with a log D7.4 of +2.25. Further study in vivo is warranted to assess the suitability of these [111In]-labeled DPN-DOTA conjugates for imaging trials. PMID:28190898

  2. Synthesis and pre-clinical evaluation of a new class of high-affinity (18)F-labeled PSMA ligands for detection of prostate cancer by PET imaging.

    PubMed

    Kelly, James; Amor-Coarasa, Alejandro; Nikolopoulou, Anastasia; Kim, Dohyun; Williams, Clarence; Ponnala, Shashikanth; Babich, John W

    2017-04-01

    Current clinical imaging of PSMA-positive prostate cancer by positron emission tomography (PET) mainly features (68)Ga-labeled tracers, notably [(68)Ga]Ga-PSMA-HBED-CC. The longer half-life of fluorine-18 offers significant advantages over Ga-68, clinically and logistically. We aimed to develop high-affinity PSMA inhibitors labeled with fluorine-18 as alternative tracers for prostate cancer. Six triazolylphenyl ureas and their alkyne precursors were synthesized from the Glu-urea-Lys PSMA binding moiety. PSMA affinity was determined in a competitive binding assay using LNCaP cells. The [(18)F]triazoles were isolated following a Cu(I)-catalyzed click reaction between the alkynes and [(18)F]fluoroethylazide. The (18)F-labeled compounds were evaluated in nude mice bearing LNCaP tumors and compared to [(68)Ga]Ga-PSMA-HBED-CC and [(18)F]DCFPyL. Biodistribution studies of the two tracers with the highest imaged-derived tumor uptake and highest PSMA affinity were undertaken at 1 h, 2 h and 4 h post-injection (p.i.), and co-administration of PMPA was used to determine whether uptake was PSMA-specific. F-18-labeled triazolylphenyl ureas were prepared with a decay-corrected RCY of 20-40 %, >98 % radiochemical and chemical purity, and specific activity of up to 391 GBq/μmol. PSMA binding (IC50) ranged from 3-36 nM. The position of the triazole influenced tumor uptake (3 > 4 > 2), and direct conjugation of the triazole with the phenylurea moiety was preferred to insertion of a spacer group. Image-derived tumor uptake ranged from 6-14 %ID/g at 2 h p.i., the time of maximum tumor uptake; uptake of [(68)Ga]Ga-PSMA-HBED-CC and [(18)F]DCFPyL was 5-6 %ID/g at 1-3 h p.i., the time of maximum tumor uptake. Biodistribution studies of the two most promising compounds gave maximum tumor uptakes of 10.9 ± 1.0 % and 14.3 ± 2.5 %ID/g, respectively, as compared to 6.27 ± 1.44 %ID/g for [(68)Ga]Ga-PSMA-HBED-CC. Six [(18)F]triazolylphenyl ureas were prepared in

  3. Synthesis of a phenolic precursor and its efficient O-[18F]fluoroethylation with purified no-carrier-added [18F]2-fluoroethyl brosylate as the labeling agent.

    PubMed

    Jarkas, Nashwa; Voll, Ronald J; Goodman, Mark M

    2013-09-01

    [(18)F]2-Fluoroethyl-p-toluenesulfonate also called [(18)F]2-fluoroethyl tosylate has been widely used for labeling radioligands for positron emission tomography (PET). [(18)F]2-Fluoroethyl-4-bromobenzenesulfonate, also called [(18)F]2-fluoroethyl brosylate ([(18)F]F(CH2)2OBs), was used as an alternative radiolabeling agent to prepare [(18)F]FEOHOMADAM, a fluoroethoxy derivative of HOMADAM, by O-fluoroethylating the phenolic precursor. Purified by reverse-phase HPLC, the no-carrier-added [(18)F]F(CH2)2OBs was obtained in an average radiochemical yield (RCY) of 35%. The reaction of the purified and dried [(18)F]F(CH2)2OBs with the phenolic precursor was performed by heating in DMF and successfully produced [(18)F]FEOHOMADAM, after HPLC purification, in RCY of 21%.

  4. Synthesis and in vitro cellular uptake of 11C-labeled 5-aminolevulinic acid derivative to estimate the induced cellular accumulation of protoporphyrin IX.

    PubMed

    Suzuki, Chie; Kato, Koichi; Tsuji, Atsushi B; Kikuchi, Tatsuya; Zhang, Ming-Rong; Arano, Yasushi; Saga, Tsuneo

    2013-08-15

    Protoporphyrin IX (PpIX) accumulation induced by exogenous 5-aminolevulinic acid (ALA) in tumors affects the therapeutic efficacy of ALA-based photodynamic and sonodynamic therapies. To develop a new imaging probe to estimate the ALA-induced PpIX accumulation, (11)C-labeled ALA analog (4), an ALA-dehydratase inhibitor, was radiosynthesized via (11)C-methylation of a Schiff-base-activated precursor in the presence of tetrabutylammonium fluoride, followed by the hydrolysis of ester and imine groups. The cellular uptake of 4 linearly increased with time and was inhibited by ALA and other transporter competitors. Monitoring analog 4 with positron emission tomography might be useful to estimate the ALA-induced PpIX accumulation in tumors. Copyright © 2013 Elsevier Ltd. All rights reserved.

  5. Azido-iodo-N-benzyl derivatives of threo-methylphenidate (Ritalin, Concerta): Rational design, synthesis, pharmacological evaluation, and dopamine transporter photoaffinity labeling.

    PubMed

    Lapinsky, David J; Velagaleti, Ranganadh; Yarravarapu, Nageswari; Liu, Yi; Huang, Yurong; Surratt, Christopher K; Lever, John R; Foster, James D; Acharya, Rejwi; Vaughan, Roxanne A; Deutsch, Howard M

    2011-01-01

    In contrast to tropane-based compounds such as benztropine and cocaine, non-tropane-based photoaffinity ligands for the dopamine transporter (DAT) are relatively unexplored. Towards addressing this knowledge gap, ligands were synthesized in which the piperidine nitrogen of 3- and 4-iodomethylphenidate was substituted with a benzyl group bearing a photoreactive azide. Analog (±)-3a demonstrated modest DAT affinity and a radioiodinated version was shown to bind covalently to rat striatal DAT and hDAT expressed in cultured cells. Co-incubation of (±)-3a with nonradioactive d-(+)-methylphenidate or (-)-2-β-carbomethoxy-3-β-(4-fluorophenyl)tropane (β-CFT, WIN-35,428, a cocaine analog) blocked DAT labeling. Compound (±)-3a represents the first successful example of a DAT photoaffinity ligand based on the methylphenidate scaffold. Such ligands are expected to assist in mapping non-tropane ligand-binding pockets within plasma membrane monoamine transporters.

  6. Azido-Iodo-N-Benzyl Derivatives of threo-Methylphenidate (Ritalin, Concerta): Rational Design, Synthesis, Pharmacological Evaluation, and Dopamine Transporter Photoaffinity Labeling

    PubMed Central

    Lapinsky, David J.; Velagaleti, Ranganadh; Yarravarapu, Nageswari; Liu, Yi; Huang, Yurong; Surratt, Christopher K.; Lever, John R.; Foster, James D.; Acharya, Rejwi; Vaughan, Roxanne A.; Deutsch, Howard M.

    2010-01-01

    In contrast to tropane-based compounds such as benztropine and cocaine, non-tropane-based photoaffinity ligands for the dopamine transporter (DAT) are relatively unexplored. Towards addressing this knowledge gap, ligands were synthesized in which the piperidine nitrogen of 3- and 4-iodomethylphenidate was substituted with a benzyl group bearing a photoreactive azide. Analog (±)-3a demonstrated modest DAT affinity and a radioiodinated version was shown to bind covalently to rat striatal DAT and hDAT expressed in cultured cells. Co-incubation of (±)-3a with nonradioactive D-(+)-methylphenidate or (−)-2-β-carbomethoxy-3-β-(4-fluorophenyl)tropane (β-CFT, WIN-35,428, a cocaine analog) blocked DAT labeling. Compound (±)-3a represents the first successful example of a DAT photoaffinity ligand based on the methylphenidate scaffold. Such ligands are expected to assist in mapping non-tropane ligand-binding pockets within plasma membrane monoamine transporters. PMID:21129986

  7. Bioinspired synthesis and characterization of gadolinium-labeled magnetite nanoparticles for dual contrast t1- and T2-weighted magnetic resonance imaging.

    PubMed

    Bae, Ki Hyun; Kim, Young Beom; Lee, Yuhan; Hwang, Jinyoung; Park, Hyunwook; Park, Tae Gwan

    2010-03-17

    Gadolinium-labeled magnetite nanoparticles (GMNPs) were synthesized via a bioinspired manner to use as dual contrast agents for T1- and T2-weighted magnetic resonance imaging. A mussel-derived adhesive moiety, 3,4-dihydroxy-l-phenylalanine (DOPA), was utilized as a robust anchor to form a mixed layer of poly(ethylene glycol) (PEG) chains and dopamine molecules on the surface of iron oxide nanoparticles. Gadolinium ions were subsequently complexed at the distal end of the dopamine molecules that were prefunctionalized with a chelating ligand for gadolinium. The resultant GMNPs exhibited high dispersion stability in aqueous solution. Crystal structure and superparamagnetic properties of magnetite nanocrystals were also maintained after the complexation of gadolinium. The potential of GMNPs as dual contrast agents for T1 and T2-weighted magnetic resonance imaging was demonstrated by conducting in vitro and in vivo imaging and relaxivity measurements.

  8. Low molecular weight poly (2-dimethylamino ethylmethacrylate) polymers with controlled positioned fluorescent labeling: Synthesis, characterization and in vitro interaction with human endothelial cells.

    PubMed

    Flebus, Luca; Lombart, François; Sevrin, Chantal; Defraigne, Jean-Olivier; Peters, Pierre; Parhamifar, Ladan; Molin, Daniel G M; Grandfils, Christian

    2015-01-15

    Poly (2-dimethylamino ethylmethacrylate) (PDMAEMA) is an attractive non-degradable polymer studied as nonviral vector for gene delivery but it can be also adopted for delivery of other biopharmaceutical drugs. As a parenteral carrier, the PDMAEMA free form (FF) might interact with tissues and cells. Few data are available on its selective internalization and efflux from cells, while the majority of studies published have followed the distribution of DNA complexed with PDMAEMA. In order to address polycation safety, the first aim was to synthesize by atom transfer radical polymerisation (ATRP) fluorescent labeled PDMAEMA of low molecular weight (Mw) (below 15 kDa), controlling the position and density of fluorescein. The second goal was to analyze the possible difference in uptake and subcellular distribution of this labeled FF polycation between human umbilical vein endothelial cells (HUVEC) and hCMEC/D3 cells. These two cell lines have been chosen in order to detect selectivity towards the blood-brain barrier (BBB). In both cases, polycation was detected along the plasma membrane followed by progressive migration to the peri-nuclear region, where it overlapped with lysosomal structures. The analysis by fluorescence-activated cell sorting (FACS) of the PDMAEMA uptake by hCMEC/D3 cells showed a significant (p<0.05) inhibition (40%) in presence of 2-dexoxy-D-glucose inhibitor, a result supporting an energy-dependence mechanism(s). Cytotoxicity study showed that low Mw PDMAEMA (10 kDa) lead to a minor cytotoxicity compared to the higher ones. As main conclusion this study highlights the similitude in cell trafficking of FF PDMAEMA and data previously reported for PDMAEMA/DNA complexes.

  9. Synthesis and pre-clinical evaluation of an 18F-labeled single-chain antibody fragment for PET imaging of epithelial ovarian cancer

    PubMed Central

    Sharma, Sai Kiran; Wuest, Melinda; Way, Jenilee D; Bouvet, Vincent R; Wang, Monica; Wuest, Frank R

    2016-01-01

    Anti-CA125 antibodies have been used in immunoassays to quantify levels of shed antigen in the serum of patients who are under surveillance for epithelial ovarian cancer (EOC). However, there is currently no molecular imaging probe in the clinic for the assessment of CA125 expression in vivo. The present study describes the development of an 18F-labeled single-chain variable fragment (scFv) for PET imaging of CA125 in preclinical EOC models. Anti-CA125 scFv was derived from MAb-B43.13 by recombinant expression of the fragment in E.coli. Fragment scFv-B43.13 was purified via immobilized metal affinity chromatography and characterized for antigen binding via immuno-staining and flow cytometry. Prosthetic group N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB) was used for radiolabeling of scFv-B43.13. Preclinical ovarian cancer models were developed based on ovarian cancer cell lines OVCAR3 (CA125-positive) and SKOV3 (CA125-negative) in NIH-III mice. The radiopharmacological profile of 18F-labeled scFv-B43.13 ([18F]FBz-scFv-B43.13) was studied with PET. [18F]FBz-scFv-B43.13 was prepared in radiochemical yields of 3.7 ± 1.8% (n = 5) at an effective specific activity of 3.88 ± 0.76 GBq/µmol (n = 5). The radiotracer demonstrated selective uptake in CA125-positive OVCAR3 cells and virtually no uptake in CA125-negative SKOV3 cells. Standardized uptake values (SUV) of radioactivity uptake in OVCAR3 tumors was 0.5 (n = 3) and 0.3 (n = 2) in SKOV3 tumors after 60 min post injection (p.i.). PMID:27508105

  10. Synthesis of 14N and 15N-labeled trityl-nitroxide biradicals with strong spin-spin interaction and improved sensitivity to redox status and oxygen

    PubMed Central

    Liu, Yangping; Villamena, Frederick A.; Song, Yuguang; Sun, Jian; Rockenbauer, Antal

    2014-01-01

    Simultaneous evaluation redox status and oxygenation in biological systems is of great importance for the understanding of biological functions. Electron paramagnetic resonance spectroscopy coupled with the use of the nitroxide radicals have been an indispensable technique for this application but are still limited by its low oxygen sensitivity, and low EPR resolution in part due to the moderately broad EPR triplet and spin quenching through bioreduction. In this study, we showed that these drawbacks can be overcome through the use of trityl-nitroxide biradicals allowing for the simultaneous measurement of redox status and oxygenation. A new trityl-nitroxide biradical TNN14 composed of a pyrrolidinyl-nitroxide and a trityl, and its isotopically labeled 15N analogue TNN15 were synthesized and characterized. Both biradicals exhibited much stronger spin-spin interaction with J > 400 G than the previous synthesized trityl-nitroxide biradicals TN1 (~160 G) and TN2 (~52 G) with longer linker chain length. The enhanced stability of TNN14 was evaluated using ascorbate as reductant and the effect of different types of cyclodextrins on its stability in the presence of ascorbate was also investigated. Both biradicals are sensitive to redox status, and their corresponding trityl-hydroxylamines resulting from the reduction of the biradicals by ascorbate share the same oxygen sensitivity. Of note is that the 15N-labeled TNN15-H with an EPR doublet exhibits improved EPR signal amplitude as compared to TNN14-H with an EPR triplet. In addition, cyclic voltammetric studies verify the characteristic electrochemical behaviors of the trityl-nitroxide biradicals. PMID:21028905

  11. Synthesis and sensing integration: A novel enzymatic reaction modulated Nanoclusters Beacon (NCB) "Illumination" strategy for label-free biosensing and logic gate operation.

    PubMed

    Hong, Lu; Zhou, Fu; Wang, Guangfeng; Zhang, Xiaojun

    2016-12-15

    A novel fluorescent label-free "turn-on" NAD(+) and adenosine triphosphate (ATP) biosensing strategy is proposed by fully exploiting ligation triggered Nanocluster Beacon (NCB). In the presence of the target, the split NCB was brought to intact, which brought the C-rich sequence and enhancer sequence in close proximity resulting in the lightening of dark DNA/AgNCs ("On" mode). Further application was presented for logic gate operation and aptasensor construction. The feasibility was investigated by Ultraviolet-visible spectroscopy (UV-vis), Fluorescence, lifetime and High Resolution Transmission Electron Microscopy (HRTEM) etc. The strategy displayed good performance in the detection of NAD(+) and ATP, with the detection limit of 0.002nM and 0.001mM, the linear range of 10-1000nM and 0.003-0.01mM, respectively. Due to the DNA/AgNCs as fluorescence reporter, the completely label-free fluorescent strategy boasts the features of simplicity and low cost, and showing little reliance on the sensing environment. Meanwhile, the regulation by overhang G-rich sequence not relying on Förster energy transfer quenching manifests the high signal-to-background ratios (S/B ratios). This method not only provided a simple, economical and reliable fluorescent NAD(+) assay but also explored a flexible G-rich sequence regulated NCB probe for the fluorescent biosensors. Furthermore, this sensing mode was expanded to the application of a logic gate design, which exhibited a high performance for not only versatile biosensors construction but also for molecular computing application. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. A Label-Free Microelectrode Array Based on One-Step Synthesis of Chitosan–Multi-Walled Carbon Nanotube–Thionine for Ultrasensitive Detection of Carcinoembryonic Antigen

    PubMed Central

    Xu, Huiren; Wang, Yang; Wang, Li; Song, Yilin; Luo, Jinping; Cai, Xinxia

    2016-01-01

    Carcinoembryonic antigen (CEA) has been an extensively used tumor marker responsible for clinical early diagnosis of cervical carcinomas, and pancreatic, colorectal, gastric and lung cancer. Combined with micro-electro mechanical system (MEMS) technology, it is important to develop a novel immune microelectrode array (MEA) not only for rapid analysis of serum samples, but also for cell detection in vitro and in vivo. In this work, we depict a simple approach to modify chitosan–multi-walled carbon nanotubes–thionine (CS–MWCNTs–THI) hybrid film through one-step electrochemical deposition and the CS-MWCNTs-THI hybrid films are successfully employed to immobilize anti-CEA for fabricating simple, label-free, and highly sensitive electro-chemical immune MEAs. The detection principle of immune MEA was based on the fact that the increasing formation of the antigen-antibody immunocomplex resulted in the decreased response currents and the relationship between the current reductions with the corresponding CEA concentrations was directly proportional. Experimental results indicated that the label-free MEA had good selectivity and the limit of detection for CEA is 0.5 pg/mL signal to noise ratio (SNR) = 3. A linear calibration plot for the detection of CEA was obtained in a wide concentration range from 1 pg/mL to 100 ng/mL (r = 0.996). This novel MEA has potential applications for detecting CEA for the research on cancer cells and cancer tissue slices as well as for effective early diagnosis. PMID:28335260

  13. Reusable electrochemical cell for rapid separation of [18F]fluoride from [18O]water for flow-through synthesis of 18F-labeled tracers

    PubMed Central

    Sadeghi, Saman; Liang, Vincent; Cheung, Shilin; Woo, Suh; Wu, Curtis; Ly, Jimmy; Deng, Yuliang; Eddings, Mark; van Dam, R. Michael

    2015-01-01

    A brass-platinum electrochemical micro flow cell was developed to extract [18F]fluoride from an aqueous solution and release it into an organic based solution, suitable for subsequent radio-synthesis, in a fast and reliable manner. This cell does not suffer electrode erosion and is thus reusable while operating faster by enabling increased voltages. By optimizing temperature, trapping and release potentials, flow rates, and electrode materials, an overall [18F]fluoride trapping and release efficiency of 84±5% (n=7) was achieved. X-ray photoelectron spectroscopy (XPS) was used to analyze electrode surfaces of various metal-metal systems and the findings were correlated with the performance of the electrochemical cell. To demonstrate the reactivity of the released [18F]fluoride, the cell was coupled to a flow-through reactor and automated synthesis of [18F]FDG with a repeatable decay-corrected yield of 56±4% (n=4) was completed in <15 min. A multi-human dose of 5.92 GBq [18F]FDG was also demonstrated. PMID:23474380

  14. Stable isotope labeling methods for DNA.

    PubMed

    Nelissen, Frank H T; Tessari, Marco; Wijmenga, Sybren S; Heus, Hans A

    2016-08-01

    NMR is a powerful method for studying proteins and nucleic acids in solution. The study of nucleic acids by NMR is far more challenging than for proteins, which is mainly due to the limited number of building blocks and unfavorable spectral properties. For NMR studies of DNA molecules, (site specific) isotope enrichment is required to facilitate specific NMR experiments and applications. Here, we provide a comprehensive review of isotope-labeling strategies for obtaining stable isotope labeled DNA as well as specifically stable isotope labeled building blocks required for enzymatic DNA synthesis. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Studies toward labeling cytisine with [11C]phosgene: rapid synthesis of a delta-lactam involving a new chemoselective lithiation-annulation method.

    PubMed

    Rouden, Jacques; Seitz, Thomas; Lemoucheux, Laurent; Lasne, Marie-Claire

    2004-05-28

    With the aim of the radiolabeling of cytisine, a potent agonist of nicotinic receptors, with [(11)C]phosgene, the rapid synthesis of a lactam model of our target has been studied. The key step of the delta-lactam formation is a new chemoselective lithiation-annulation method, under high dilution, of a suitable piperidinylcarbamoyl chloride. This precursor was obtained from (2-hydroxyethyl)piperidine in a linear synthetic sequence involving a Corey-Fuchs olefination of the corresponding aldehyde, followed by a selective reduction, using a diimide equivalent, of an iodoalkyne into a (Z)-iodopropene piperidine. This alkene served as main precursor to study the cyclization according to several procedures using phosgene as the required carbonylating reagent.

  16. An efficient solid-state synthesis of fluorescent surface carboxylated carbon dots derived from C60 as a label-free probe for iron ions in living cells.

    PubMed

    Lan, Jing; Liu, Chunfang; Gao, Mingxuan; Huang, Chengzhi

    2015-11-01

    In order to achieve the simple, easily repeated, and large scale preparation of fluorescent CDs, a new solid-state synthesis (SSS) approach was developed by calcining the mixture of fullerenes (C60) and solid sodium hydroxide. The cage of fullerenes could be opened and the hydroxyl and carboxyl were successfully introduced in the presence of sodium hydroxide under high temperature. The as-prepared surface carboxylated CDs possess many good properties, such as high water solubility, good photostability, salt tolerance, and nontoxicity. Especially, the fluorescence of CDs could be highly quenched by Fe(3+) because of the strong interaction of hydroxyl or carboxyl on the as-obtained CDs with Fe(3+), which realized a sensitive detection of Fe(3+) in the linear range of 0.02-0.6 μmol/L. What is more, we further applied the obtained CDs into the intracellular imaging of Fe(3+).

  17. Synthesis of 3H, 13C,2H3,15N and 14C-labelled SCH 466036, a histamine 3 receptor antagonist.

    PubMed

    Hesk, D; Borges, S; Dumpit, R; Hendershot, S; Koharski, D; Lavey, C; McNamara, P; Voronin, K

    2015-02-01

    The synthesis of [(3)H]SCH 466036, [Me-(3)H3]SCH 466036, [(13)C,(2)H3,(15)N]SCH 466036 and [(14)C]SCH 466036 is described. [(3)H]SCH 466036 was prepared in two steps via Raney Ni-catalysed exchange with tritiated water. [Me-(3)H3]SCH 466036 was prepared in a single step from [(3)H]methyl iodide in 45% yield. [(13)C,(2)H3,(15)N]SCH 466036 was prepared in two steps from [(15)N]hydroxylamine and [(13)C,(2)H3]methyl iodide with an overall yield of 16%. [(14)C]SCH 466036 was prepared in seven steps from [(14)C]potassium cyanide in an overall yield of 13%.

  18. Synthesis and in Vivo Biological Evaluation of (68)Ga-Labeled Carbonic Anhydrase IX Targeting Small Molecules for Positron Emission Tomography.

    PubMed

    Sneddon, Deborah; Niemans, Raymon; Bauwens, Matthias; Yaromina, Ala; van Kuijk, Simon J A; Lieuwes, Natasja G; Biemans, Rianne; Pooters, Ivo; Pellegrini, Paul A; Lengkeek, Nigel A; Greguric, Ivan; Tonissen, Kathryn F; Supuran, Claudiu T; Lambin, Philippe; Dubois, Ludwig; Poulsen, Sally-Ann

    2016-07-14

    Tumor hypoxia contributes resistance to chemo- and radiotherapy, while oxygenated tumors are sensitive to these treatments. The indirect detection of hypoxic tumors is possible by targeting carbonic anhydrase IX (CA IX), an enzyme overexpressed in hypoxic tumors, with sulfonamide-based imaging agents. In this study, we present the design and synthesis of novel gallium-radiolabeled small-molecule sulfonamides targeting CA IX. The compounds display favorable in vivo pharmacokinetics and stability. We demonstrate that our lead compound, [(68)Ga]-2, discriminates CA IX-expressing tumors in vivo in a mouse xenograft model using positron emission tomography (PET). This compound shows specific tumor accumulation and low uptake in blood and clears intact to the urine. These findings were reproduced in a second study using PET/computed tomography. Small molecules investigated to date utilizing (68)Ga for preclinical CA IX imaging are scarce, and this is one of the first effective (68)Ga compounds reported for PET imaging of CA IX.

  19. Aqueous synthesis of type-II CdTe/CdSe core-shell quantum dots for fluorescent probe labeling tumor cells.

    PubMed

    Zeng, Ruosheng; Zhang, Tingting; Liu, Jincheng; Hu, Song; Wan, Qiang; Liu, Xuanming; Peng, Zhiwei; Zou, Bingsuo

    2009-03-04

    In this paper, we report a two-step aqueous synthesis of highly luminescent CdTe/CdSe core/shell quantum dots (QDs) via a simple method. The emission range of the CdTe/CdSe QDs can be tuned from 510 to 640 nm by controlling the thickness of the CdSe shell. Accordingly, the photoluminescence quantum yield (PL QY) of CdTe/CdSe QDs with an optimized thickness of the CdSe shell can reach up to 40%. The structures and compositions of the core/shell QDs were characterized by transmission electron microscopy, x-ray diffraction, and x-ray photoelectron spectroscopy experiments, and their formation mechanism is discussed. Furthermore, folate conjugated CdTe/CdSe QDs in Hela cells were assessed with a fluorescence microscope. The results show that folate conjugated CdTe/CdSe QDs could enter tumor cells efficiently.

  20. Fluroine-18 labeled 28-carbomethoxy-3{beta}-(4-chlorophenyl)-8-[-3-fluoropropyl] nortropane(FPT): Synthesis and tissue distribution of a potential, radioligand for mapping cocaine receptor sites by PET

    SciTech Connect

    Keil, R.; Goodman, M.M.; Shoup, T.

    1995-05-01

    Highly potent and selective radioligands for the dopamine transporter labeled with fluorine-18 (t {1/2}=110 min) are attractive probes for longitudinal in vivo mapping of cocaine receptor sites in the caudate by PET. Recently, we reported an iodine-123 labeled 3{beta}-aryl analog of cocaine, 2{beta}-carbomethoxy-3{beta}-(4-chlorophenyl)-8-((E)-3-iodopropen-1-yl)nortropane, which was 125 times more potent than cocaine in inhibiting [I-125] RTI-55 binding to rat striatal homogenates and which showed high striatal (S) uptake (0.61% dose/g) and high S to cerebellum (C) ratio S/C=16.5 at 120 min in rats. These results demonstrated bulk tolerance at the 8-position of this I-123 analog. These findings prompted us to synthesize a new radioligand fluorine-18 labeled 2{beta}-carbomethoxy-3{beta}-(4-chlorophenyl)-8-3-fluoropropylnortropane (FPT) as a potential cocaine receptor PET imaging agent. Treatment of 2{beta}-carbomethoxy-3{beta}-(chlorophenyl) nortropane (1) with 1-bromo-3-fluoropropane (2) in CH3CN at 80{degrees}C afforded FPT (3). In Vitro binding studies in rat striatal homogenates using [I-125] RTI-55 resulted in a Ki (nM) of 8.2 for FPT. [F-18]FPT (3) was prepared by treating 1,3-diiodopropane (4) with NCA K[F-18]/K222 for 5 min in CH3CN at 85{degrees}C to give [F-18] 1-fluoro-3-iodopropane (5) in 50% E.O.B. yield. Coupling of [F-18] 5 with 1 in CH3CN at 60{degrees}C afforded [F-18]FPT in 5% yield (not optimized) E.O.B. following HPLC purification in a total synthesis time of 100 min.. [F-18]5 was >99% radiochemically pure with a specific activity of 8 Ci/{mu}mole. Following intravenous administration to rats [F-18]FPT showed high uptake in the striatum (S) with rapid washout from the cerebellum to afford a high S/C ratios=6.2 at 120 min. Primate imaging will also be presented. These results suggest that FPT is an excellent candidate for mapping cocaine receptor sites by PET.

  1. Synthesis and in vitro/in vivo evaluation of novel mono- and trivalent technetium-99m labeled ghrelin peptide complexes as potential diagnostic radiopharmaceuticals.

    PubMed

    Koźmiński, Przemysław; Gniazdowska, Ewa

    2015-01-01

    Ghrelin is an endogenous hormone present in blood. It is released from the oxyntic cells (X/A-like cells) of the stomach and fundus and can exist in two forms: as an acylated and des-acylated ghrelin. Ghrelin is an endogenous ligand of the growth hormone receptor (growth hormone secretagogue receptor, GHS-R). Overexpression of GHS-R1a receptor was identified in cells of different types of tumors (e.g. pituitary adenoma, neuroendocrine tumors of the thyroid, lung, breast, gonads, prostate, stomach, colorectal, endocrine and non-endocrine pancreatic tumors). This fact suggests that gamma radionuclide labeled ghrelin peptide may be considered as a potential diagnostic radiopharmaceutical. Ghrelin peptide labeled with mono- and trivalent technetium-99m complexes, (99m)Tc-Lys-GHR, has been prepared on the n.c.a. scale. The physicochemical (stability, charge, shape, lipophilicity) and biological (receptor affinity, biodistribution) properties of the conjugates have been studied relevant to use the conjugates as receptor-based diagnostic radiopharmaceuticals. The obtained conjugates [(99m)Tc(CO)3LN,O(CN-Lys-GHR)](+), (99m)Tc(CO)3LS,O(CN-Lys-GHR) and (99m)Tc(NS3)(CN-Lys-GHR) show different shape, charge, lipophilicity and two of them, (99m)Tc(CO)3LS,O(CN-Lys-GHR) and (99m)Tc(NS3)(CN-Lys-GHR), high stability in neutral aqueous solutions, even in the presence of excess concentration of histidine/cysteine competitive standard ligands or human serum. The in vitro binding affinity of (99m)Tc-Lys-GHR conjugates with respect to growth hormone secretagogue receptor (GHS-R1a) present on DU-145 cells was in the range of IC50 from 45 to 54 nM. The conjugate (99m)Tc(CO)3LS,O(CN-Lys-GHR) exhibited excretion route by the liver and kidney in comparable degree, while the more lipophilic conjugate (99m)Tc(NS3)(CN-Lys-GHR)-mainly by the liver. Basing on the results concerning physicochemical and biochemical properties, the conjugates (99m)Tc(CO)3LS,O(CN-Lys-GHR) and (99m)Tc(NS3)(CN

  2. Pesticide Label Review Training

    EPA Pesticide Factsheets

    This training will help ensure that reviewers evaluate labels according to four core principles. It also will help pesticide registrants developing labels understand what EPA expects of pesticide labels, and what the Agency generally finds acceptable.

  3. Synthesis and Evaluation of 18F-labeled Pyridaben Analogues for Myocardial Perfusion Imaging in Mice, Rats and Chinese mini-swine

    PubMed Central

    Mou, Tiantian; Zhao, Zuoquan; You, Linyi; Li, Yesen; Wang, Qian; Fang, Wei; Lu, Jie; Peng, Cheng; Zhang, Xianzhong

    2016-01-01

    This study reports three novel 18F-labeled pyridaben analogues for potential myocardial perfusion imaging (MPI). Three precursors and the corresponding nonradioactive compounds were synthesized and characterized. The radiolabeled tracers were obtained by substituting tosyl with 18F. The total radiosynthesis time of these tracers was 70–90 min. Typical decay-corrected radiochemical yields were 47–58%, with high radiochemical purities (>98%). Tracers were evaluated as MPI agents in vitro, ex vivo and in vivo. In the mouse biodistribution study, all three radiotracers showed high initial heart uptake (34–54% ID/g at 2 min after injection) and fast liver clearance. In the microPET imaging study, [18F]Fmpp2 produced heart images with good quality in both mice and rats. In the whole-body PET/CT images of mini-swine, [18F]Fmpp2 showed excellent initial heart standardized uptake value (SUV) (7.12 at 5 min p.i.) and good retention (5.75 at 120 min p.i.). The heart/liver SUV ratios were 4.12, 5.42 and 5.99 at 30, 60 and 120 min after injection, respectively. The favorable biological properties of [18F]Fmpp2 suggest that it is worth further investigation as a potential MPI agent. PMID:27646847

  4. Facile synthesis of boron- and nitride-doped MoS2 nanosheets as fluorescent probes for the ultrafast, sensitive, and label-free detection of Hg(2+).

    PubMed

    Liu, Xiaojia; Li, Liping; Wei, Yuanjie; Zheng, Yizhi; Xiao, Qian; Feng, Bo

    2015-07-07

    Bulk MoS2, a prototypical transition metal chalcogenide material, is an indirect band gap semiconductor with negligible photoluminescence. In this study, we have developed, for the first time, a simple and low-cost synthetic strategy to prepare boron- and nitrogen-doped MoS2 (B,N-MoS2) nanosheets. Through boron and nitrogen doping, the band gap of MoS2 increases from 1.20 eV to 1.61 eV, and the obtained B,N-MoS2 nanosheets exhibit an enhanced fluorescence. The B,N-MoS2 nanosheets can be used as a green and facile sensing platform for label-free detection of Hg(2+) because of their high sensitivity and selectivity toward Hg(2+). In addition, detection can be easily accomplished through one-step rapid (within 2 min) operation, with a limit as low as 1 nM. This study demonstrates that the introduction of boron and nitrogen elements into ultrathin MoS2 nanosheets for enhanced fluorescence properties is feasible through a facile and general preparation strategy and may also offer a unique idea as a potential way to design more efficient MoS2-based sensors and fluorescent materials.

  5. Synthesis and evaluation in monkey of two sensitive 11C-labeled aryloxyanilide ligands for imaging brain peripheral benzodiazepine receptors in vivo.

    PubMed

    Briard, Emmanuelle; Zoghbi, Sami S; Imaizumi, Masao; Gourley, Jonathan P; Shetty, H Umesha; Hong, Jinsoo; Cropley, Vanessa; Fujita, Masahiro; Innis, Robert B; Pike, Victor W

    2008-01-10

    We sought to develop (11)C-labeled ligands for sensitive imaging of brain peripheral benzodiazepine receptors (PBR) in vivo. Two aryloxyanilides with high affinity for PBR were identified and synthesized, namely, N-acetyl- N-(2-methoxycarbonylbenzyl)-2-phenoxyaniline ( 3, PBR01) and N-(2-methoxybenzyl)- N-(4-phenoxypyridin-3-yl)acetamide ( 10, PBR28). 3 was hydrolyzed to 4, which was esterified with [ (11)C]iodomethane to provide [ (11)C] 3. The O-desmethyl analogue of 10 was converted into [ (11)C] 10 with [ (11)C]iodomethane. [ (11)C] 3 and [ (11)C] 10 were each injected into monkey to assess their brain kinetics with positron emission tomography (PET). After administration of either radioligand there was moderately high brain uptake of radioactivity. Receptor blocking and displacement experiments showed that a high proportion of this radioactivity was bound specifically to PBR. In monkey and rat, 3 and 10 were rapidly metabolized by ester hydrolysis and N-debenzylation, respectively, each to a single polar radiometabolite. [ (11)C] 3 and [ (11)C] 10 are effective for imaging PBR in monkey brain. [ (11)C] 10 especially warrants further evaluation in human subjects.

  6. Enzymic synthesis of steroid sulphates. XI. Study of the oestrogen binding site of oestrogen sulphotransferase by affinity labelling with 4-mercuri-17beta-oestradiol.

    PubMed

    Dodsworth, A I; Jackson, D E

    1975-04-19

    Oistrogen sulphotransferase (3"-phosphoadenylylsulphate: oestrone sulphotransferase, EC 2.8.2.4) contains asingle sulphydryl group thought to be at, or near, the oestrogen-binding site. 4-mercuri-17beta-oestradiol, the activity of the enzyme decreased with increasing concentration of the oestrogen derivative. However, some 40% of the activity remained when all the sulphydryl had reacted to form mercaptide. Formation of mercaptide was only marginally decreased in the presence of the substrate 17beta-oestradiol. Other steroids, such as 11-deoxycorticosterone and testosterone, which are non-substrates for the enzyme, were more effective than 17beta-oestradiol in inhibiting mercaptide formation. Bovine serum albumin also reacted with 4-mercure-17beta-oestradiol and the effects of various steroids on mercaptide formation by the affinity label closely paralleled those found for the enzyme. 2t is concluded that the single sulphydryl group in the enzyme is not directly involved in the binding of oestrogen at the active site but is perhaps in closer proximity to a second site capable of binding certain non-substrate steroids.

  7. Design, synthesis, radiolabeling and in vivo evaluation of carbon-11 labeled N-[2-[4-(3-cyanopyridin-2-yl)piperazin-1-yl]ethyl]-3-methoxybenzamide, a potential Positron Emission Tomography tracer for the dopamine D4 receptors

    PubMed Central

    Lacivita, Enza; De Giorgio, Paola; Lee, Irene T.; Rodeheaver, Sean I.; Weiss, Bryan A.; Fracasso, Claudia; Caccia, Silvio; Berardi, Francesco; Perrone, Roberto; Zhang, Ming-Rong; Maeda, Jun; Higuchi, Makoto; Suhara, Tetsuya; Schetz, John A.; Leopoldo, Marcello

    2010-01-01

    Here we describe the design, synthesis, physicochemical, and pharmacological evaluation of D4 dopamine receptor ligands related to N-[2-[4-(4-chlorophenyl)piperazin-1-yl]ethyl]-3-methoxybenzamide (2). Structural features were incorporated to increase affinity for the target receptor, to improve selectivity over D2 and sigma1 receptors, to enable labeling with carbon-11 or fluorine-18, and to adjust lipophilicity within the range considered optimal for brain penetration and low nonspecific binding. Compounds 7 and 13 showed the overall best characteristics: nanomolar affinity for the D4 receptor, > 100-fold selectivity over D2 and D3 dopamine receptor 5-HT1A, 5-HT2A and 5-HT2C serotonin receptors and sigma1 receptors, and logP = 2.37–2.55. Following intraperitoneal administration, both compounds rapidly entered the central nervous system. The methoxy of N-[2-[4-(3-cyanopyridin-2-yl)piperazin-1-yl]ethyl]-3-methoxybenzamide (7) was radiolabelled with carbon-11 and subjected to PET analysis in non-human primate. [11C]7 time-dependently accumulated to saturation in the posterior eye in the region of the retina, a tissue containing a high density of D4 receptors. PMID:20873719

  8. Synthesis and uptake of fluorescence-labeled Combi-molecules by P-glycoprotein-proficient and -deficient uterine sarcoma cells MES-SA and MES-SA/DX5.

    PubMed

    Larroque-Lombard, Anne-Laure; Todorova, Margarita; Golabi, Nahid; Williams, Christopher; Jean-Claude, Bertrand J

    2010-03-11

    Here, we report on the first synthesis of fluorescent-labeled epidermal growth factor receptor-DNA targeting combi-molecules, and we studied the influence of P-glycoprotein status of human sarcoma MES-SA cells on their growth inhibitory effect and cellular uptake. The results showed that 6, bearing a longer spacer between the quinazoline ring and the dansyl group, was more stable and more cytotoxic than 4. In contrast to the latter, it induced significant levels of DNA damage in human tumor cells. Moreover, in contrast to doxorubicin, a drug known to be actively effluxed by P-gp, the more stable combi-molecule 6 induced almost identical levels of drug uptake and DNA damage in P-gp-proficient and -deficient cells. Likewise, in contrast to doxorubicin, 4 and 6 exerted equal levels of antiproliferative activity against the two cell types. The results in toto suggest that despite their size, the antiproliferative effects of 4 and 6 were independent of P-gp status of the cells.

  9. (18)F-Labeled 1,4-Dioxa-8-azaspiro[4.5]decane Derivative: Synthesis and Biological Evaluation of a σ1 Receptor Radioligand with Low Lipophilicity as Potent Tumor Imaging Agent.

    PubMed

    Xie, Fang; Bergmann, Ralf; Kniess, Torsten; Deuther-Conrad, Winnie; Mamat, Constantin; Neuber, Christin; Liu, Boli; Steinbach, Jörg; Brust, Peter; Pietzsch, Jens; Jia, Hongmei

    2015-07-23

    We report the syntheses and evaluation of series of novel piperidine compounds with low lipophilicity as σ1 receptor ligands. 8-(4-(2-Fluoroethoxy)benzyl)-1,4-dioxa-8-azaspiro[4.5]decane (5a) possessed high affinity (K(i) = 5.4 ± 0.4 nM) for σ1 receptors and selectivity for σ2 receptors (30-fold) and the vesicular acetylcholine transporter (1404-fold). [(18)F]5a was prepared using a one-pot, two-step labeling procedure in an automated synthesis module, with a radiochemical purity of >95%, and a specific activity of 25-45 GBq/μmol. Cellular association, biodistribution, and autoradiography with blocking experiments indicated specific binding of [(18)F]5a to σ1 receptors in vitro and in vivo. Small animal positron emission tomography (PET) imaging using mouse tumor xenograft models demonstrated a high accumulation in human carcinoma and melanoma. Treatment with haloperidol significantly reduced the accumulation of the radiotracer in tumors. These findings suggest that radiotracer with suitable lipophilicity and appropriate affinity for σ1 receptors could be used for tumor imaging.

  10. Synthesis, biological evaluation, and molecular dynamics (MD) simulation studies of three novel F-18 labeled and focal adhesion kinase (FAK) targeted 5-bromo pyrimidines as radiotracers for tumor.

    PubMed

    Fang, Yu; Wang, Dawei; Xu, Xingyu; Liu, Jianping; Wu, Aiqin; Li, Xiang; Xue, Qianqian; Wang, Huan; Wang, Hang; Zhang, Huabei

    2017-02-15

    Focal adhesion kinase (FAK) is considered as an attractive target for oncology. A series of F-18 labeled 5-bromo-N(2)-(4-(2-fluoro-pegylated (FPEG))-3,5-dimethoxyphenyl)-N(4)-(4-methoxyphenyl)pyrimidine-2,4-diamine derivatives were prepared and evaluated as the FAK targeted radiotracers for the early diagnoses of tumor. For the study of the FAK targeted drug molecules, this was the first attempt to develop the tumor diagnostic imaging agents on the radiopharmaceutical level. They inhibited the activity of FAK with IC50 in the range of 91.4-425.7 nM, and among which the result of the [(19)F]2 was relatively good and had a modest IC50 of 91.4 nM. The [(19)F]2 was also profiled in vitro against some other kinds of cancer-related kinases (including two kinds of non-receptor tyrosine kinase: PYK2 and JAK2, and three kinds of receptor tyrosine kinase: IGF-1R, EGFR and PDGFRβ). It displayed 25.2 folds selectivity against PYK2, 35.1 folds selectivity against EGFR, and more than 100 folds selectivity against IGF-1R, JAK2 and PDGFRβ. For the biodistribution in S180 bearing mice, the corresponding [(18)F]2 were also relatively good, with modest tumor uptake of 5.47 ± 0.19 and 5.80 ± 0.06 %ID/g at 15 and 30 min post-injection, respectively. Furthermore, its tumor/muscle, tumor/bone and tumor/blood ratio at 15 min post-injection were 3.16, 2.53 and 4.52, respectively. And its tumor/muscle, tumor/bone and tumor/blood ratio at 30 min post-injection were 3.14, 2.76 and 4.43, respectively. In addition, coronal micro-PET/CT images of a mouse bearing S180 tumor clearly confirmed that [(18)F]2 could be accumulated in tumor, especially at 30 min post-injection. Besides, for the [(18)F]2, both the biodistribution data and the micro-PET/CT imaging study showed significantly reduced uptake of the radiotracer in the tumor tissue at 30 min post-injection in mice that received PF-562,271 (one of the reported best selective FAK inhibitor which was developed by Pfitzer Inc. and

  11. "Click"-cyclized (68)Ga-labeled peptides for molecular imaging and therapy: synthesis and preliminary in vitro and in vivo evaluation in a melanoma model system.

    PubMed

    Martin, Molly E; Sue O'Dorisio, M; Leverich, Whitney M; Kloepping, Kyle C; Walsh, Susan A; Schultz, Michael K

    2013-01-01

    Cyclization techniques are used often to impart higher in vivo stability and binding affinity to peptide targeting vectors for molecular imaging and therapy. The two most often used techniques to impart these qualities are lactam bridge construction and disulfide bond formation. While these techniques have been demonstrated to be effective, orthogonal protection/deprotection steps can limit achievable product yields. In the work described in this chapter, new α-melanocyte stimulating hormone (α-MSH) peptide analogs were synthesized and cyclized by copper-catalyzed terminal azide-alkyne cycloaddition "click" chemistry techniques. The α-MSH peptide and its cognate receptor (melanocortin receptor subtype 1, MC1R) represent a well-characterized model system to examine the effect of the triazole linkage for peptide cyclization on receptor binding in vitro and in vivo. Four new DOTA-conjugated α-MSH analogs were cyclized and evaluated by in vitro competitive binding assays, serum stability testing, and in vivo imaging by positron emission tomography (PET) of tumor-bearing mice. These new DOTA-conjugated click-cyclized analogs exhibited selective high binding affinity (<2 nM) for MC1R on melanoma cells in vitro, high stability in human serum, and produced high-contrast PET/CT images of tumor xenografts. (68)Ga-labeled DOTA bioconjugates displayed rapid pharmacokinetics with receptor-mediated tumor accumulation of up to 16 ± 5% ID/g. The results indicate that the triazole ring is an effective bioisosteric replacement for the standard lactam bridge assemblage for peptide cyclization. Radiolabeling results confirm that Cu catalyst is sufficiently removed prior to DOTA chelator addition to enable insertion of radio metals or stable metals for molecular imaging and therapy. Thus, these click-chemistry-cyclized variants show promise as agents for melanocortin receptor-targeted imaging and radionuclide therapy.

  12. Synthesis and evaluation of an (18) F-labeled derivative of F3 for targeting surface-expressed nucleolin in cancer and tumor endothelial cells.

    PubMed

    Lam, Phoebe Y H; Hillyar, Christopher R T; Able, Sarah; Vallis, Katherine A

    2016-10-01

    The surface overexpression of nucleolin provides an anchor for the specific attachment of biomolecules to cancer and angiogenic endothelial cells. The peptide F3 is a high-affinity ligand of the nucleolin receptor (NR) that has been investigated as a carrier to deliver biologically active molecules to tumors for both therapeutic and imaging applications. A site-specific PEGylated F3 derivative was radiolabeled with [(18) F]Al-F. The binding affinity and cellular distribution of the compound was assessed in tumor (H2N) and tumor endothelial (2H-11) cells. Specific uptake via the NR was demonstrated by the siRNA knockdown of nucleolin in both cell lines. The partition and the plasma stability of the compound were assessed at 37°C. The enzyme-mediated site-specific modification of F3 to give NODA-PEG-F3 (NP-F3) was achieved. Radiolabeling with [(18) F]Al-F gave (18) F-NP-F3. (18) F-NP-F3 demonstrated high affinity for cancer and tumor endothelial cells. The siRNA knockdown of nucleolin resulted in a binding affinity reduction of 50% to 60%, confirming cell surface binding via the NR. NP-F3 was stable in serum for 2 h. (18) F-NP-F3 is reported as the first (18) F-labeled F3 derivative. It was obtained in a site-specific, high-yield, and efficient manner and binds to surface NR in the low nanomolar range, suggesting it has potential as a tumor and angiogenesis tracer.

  13. Synthesis and electrocatalytic effect of Ag@Pt core-shell nanoparticles supported on reduced graphene oxide for sensitive and simple label-free electrochemical aptasensor.

    PubMed

    Mazloum-Ardakani, Mohammad; Hosseinzadeh, Laleh; Taleat, Zahra

    2015-12-15

    Bimetallic Ag@Pt core-shell nanoparticles supported on reduced graphene oxide nanosheets (Ag@Pt-GRs) was synthesized and used as novel desirable sensor platform and electrocatalyst for catechol as probe in aptasensor. Gold screen-printed electrodes modified with Ag@Pt-GRs and applied to advance enzyme-free and label-free electrochemical aptasensor for detection of protein biomarker tumor necrosis factor-alpha (TNF-α). The morphology of the Ag@Pt-GRs could be characterized by transmission electron microscopy, X-ray diffraction and UV-vis spectra. The results showed that these nanocomposite exhibited attractive electrocatalytic activity and also yielded large surface area, which improve the amount of immobilized TNF-α aptamer. Due to the excellent electrocatalytic activity of Ag@Pt-GRs towards the oxidation of catechol, determination of TNF-α antigen was based on its obstruction to the electrocatalytic oxidation of catechol by Ag@Pt-GRs after binding to the surface of electrode through interaction with the aptamer. The calibration curve was obtained by differential pulse voltammetry and square wave voltammetry. Under optimum conditions, the results demonstrated that this electrochemical aptasensor possessed a dynamic range from 0.0 pg/mL to 60 pg/mL with a low detection limit of 2.07 pg/mL for TNF-α. The analytical usefulness of the aptasensor was finally demonstrated analyzing serum samples. The simple fabrication method, high sensitivity, specificity, good reproducibility and stability as well as acceptable accuracy for TNF-α detection in human serum samples are the main advantages of this aptasensor, which might have broad applications in protein diagnostics and bioassay.

  14. “Click” cyclized gallium-68 labeled peptides for molecular imaging and therapy: Synthesis and preliminary in vitro and in vivo evaluation in a melanoma model system

    PubMed Central

    Martin, Molly E.; O'Dorisio, M. Sue; Leverich, Whitney M.; Kloepping, Kyle C.; Schultz, Michael K.

    2013-01-01

    Cyclization techniques are used often to impart higher in vivo stability and binding affinity to peptide targeting vectors for molecular imaging and therapy. The two most often used techniques to impart these qualities are lactam bridge construction and disufide bond formation. While these techniques have been demonstrated to be effective, orthogonal protection/deprotection steps can limit achievable product yields. In this chapter, new α-melanocyte stimulating hormone (α-MSH) peptide analogs were synthesized and cyclized by copper-catalyzed terminal azide-alkyne cycloaddition “click” chemistry techniques. The α-MSH peptide and its cognate receptor (melanocortin receptor subtype 1, MC1R) represents a well-characterized model system to examine the effect of the triazole linkage for peptide cyclization on receptor binding in vitro and in vivo. Four new DOTA-conjugated α-MSH analogs were cyclized and evaluated by in vitro competitive binding assays, serum stability testing, and in vivo imaging by positron emission tomography (PET) of tumor bearing mice. These new DOTA-conjugated click-cyclized analogs exhibited selective high binding affinity (<2 nM) MC1R to melanoma cells in vitro, high stability in human serum, and produced high contrast PET/CT images of tumor xenografts. Gallium-68 labeled DOTA bioconjugates displayed rapid pharmacokinetics with receptor mediated tumor accumulation of up to 16±5 %ID/g. The results indicate that the triazole ring is an effective bioisosteric replacement for the standard lactam bridge assemblage for peptide cyclization. Radiolabeling results confirm that Cu catalyst is sufficiently removed prior to DOTA chelator addition to enable insertion of radiometals or stable metals for molecular imaging and therapy. Thus, these click-chemistry-cyclized variants show promise as agents for melanocortin receptor-targeted imaging and radionuclide therapy. PMID:22918759

  15. Synthesis and evaluation of 18F-labeled ATP competitive inhibitors of topoisomerase II as probes for imaging topoisomerase II expression

    PubMed Central

    Daumar, Pierre; Zeglis, Brian M.; Ramos, Nicholas; Divilov, Vadim; Sevak, Kuntal Kumar; Pillarsetty, NagaVaraKishore; Lewis, Jason S.

    2015-01-01

    Type II topoisomerase (Topo-II) is an ATP-dependent enzyme that is essential in the transcription, replication, and chromosome segregation processes and, as such, represents an attractive target for cancer therapy. Numerous studies indicate that the response to treatment with Topo-II inhibitors is highly dependent on both the levels and the activity of the enzyme. Consequently, a non-invasive assay to measure tumoral Topo-II levels has the potential to differentiate responders from non-responders. With the ultimate goal of developing a radiofluorinated tracer for positron emission tomography (PET) imaging, we have designed, synthesized, and evaluated a set of fluorinated compounds based on the structure of the ATP-competitive Topo-II inhibitor QAP1. Compounds 18 and 19b showed inhibition of Topo-II in in vitro assays and exhibited moderate, Topo-II level dependent cytotoxicity in SK-BR-3 and MCF-7 cell lines. Based on these results, 18F-labeled analogs of these two compounds were synthesized and evaluated as PET probes for imaging Topo-II overexpression in mice bearing SK-BR-3 xenografts. [18F]-18 and [18F]-19b were synthesized from their corresponding protected tosylated derivatives by fluorination and subsequent deprotection. Small animal PET imaging studies indicated that both compounds do not accumulate in tumors and exhibit poor pharmacokinetics, clearing from the blood pool very rapidly and getting metabolized over. The insights gained from the current study will surely aid in the design and construction of future generations of PET agents for the non-invasive delineation of Topo-II expression. PMID:25240701

  16. Synthesis and evaluation of an 18F‐labeled derivative of F3 for targeting surface‐expressed nucleolin in cancer and tumor endothelial cells

    PubMed Central

    Lam, Phoebe Y.H.; Hillyar, Christopher R.T.; Able, Sarah

    2016-01-01

    The surface overexpression of nucleolin provides an anchor for the specific attachment of biomolecules to cancer and angiogenic endothelial cells. The peptide F3 is a high‐affinity ligand of the nucleolin receptor (NR) that has been investigated as a carrier to deliver biologically active molecules to tumors for both therapeutic and imaging applications. A site‐specific PEGylated F3 derivative was radiolabeled with [18F]Al‐F. The binding affinity and cellular distribution of the compound was assessed in tumor (H2N) and tumor endothelial (2H‐11) cells. Specific uptake via the NR was demonstrated by the siRNA knockdown of nucleolin in both cell lines. The partition and the plasma stability of the compound were assessed at 37°C. The enzyme‐mediated site‐specific modification of F3 to give NODA‐PEG‐F3 (NP‐F3) was achieved. Radiolabeling with [18F]Al‐F gave 18F‐NP‐F3. 18F‐NP‐F3 demonstrated high affinity for cancer and tumor endothelial cells. The siRNA knockdown of nucleolin resulted in a binding affinity reduction of 50% to 60%, confirming cell surface binding via the NR. NP‐F3 was stable in serum for 2 h. 18F‐NP‐F3 is reported as the first 18F‐labeled F3 derivative. It was obtained in a site‐specific, high‐yield, and efficient manner and binds to surface NR in the low nanomolar range, suggesting it has potential as a tumor and angiogenesis tracer. PMID:27594091

  17. 2′-Azido RNA, a Versatile Tool for Chemical Biology: Synthesis, X-ray Structure, siRNA Applications, Click Labeling

    PubMed Central

    2012-01-01

    Chemical modification can significantly enrich the structural and functional repertoire of ribonucleic acids and endow them with new outstanding properties. Here, we report the syntheses of novel 2′-azido cytidine and 2′-azido guanosine building blocks and demonstrate their efficient site-specific incorporation into RNA by mastering the synthetic challenge of using phosphoramidite chemistry in the presence of azido groups. Our study includes the detailed characterization of 2′-azido nucleoside containing RNA using UV-melting profile analysis and CD and NMR spectroscopy. Importantly, the X-ray crystallographic analysis of 2′-azido uridine and 2′-azido adenosine modified RNAs reveals crucial structural details of this modification within an A-form double helical environment. The 2′-azido group supports the C3′-endo ribose conformation and shows distinct water-bridged hydrogen bonding patterns in the minor groove. Additionally, siRNA induced silencing of the brain acid soluble protein (BASP1) encoding gene in chicken fibroblasts demonstrated that 2′-azido modifications are well tolerated in the guide strand, even directly at the cleavage site. Furthermore, the 2′-azido modifications are compatible with 2′-fluoro and/or 2′-O-methyl modifications to achieve siRNAs of rich modification patterns and tunable properties, such as increased nuclease resistance or additional chemical reactivity. The latter was demonstrated by the utilization of the 2′-azido groups for bioorthogonal Click reactions that allows efficient fluorescent labeling of the RNA. In summary, the present comprehensive investigation on site-specifically modified 2′-azido RNA including all four nucleosides provides a basic rationale behind the physico- and biochemical properties of this flexible and thus far neglected type of RNA modification. PMID:22273279

  18. On-resin synthesis of an acylated and fluorescence-labeled cyclic integrin ligand for modification of poly(lactic-co-glycolic acid).

    PubMed

    Hassert, Rayk; Hoffmeister, Peter-Georg; Pagel, Mareen; Hacker, Michael; Schulz-Siegmund, Michaela; Beck-Sickinger, Annette G

    2012-11-01

    Cyclic Arg-Gly-Asp (RGD) peptides show remarkable affinity and specificity to integrin receptors and mediate important physiological effects in tumor angiogenesis. Additionally, they are one of the keyplayers in improving the biocompatibility of biomaterials. The fully biodegradable polymer poly(lactic-co-glycolic acid) (PLGA) is frequently used for biomedical implants and can be applied as nanoparticles for drug delivery. The aim of this work was the generation of a lipidated c[RGDfK] peptide including a second functionality for coating of hydrophobic PLGA. Therefore, we established a general and straightforward strategy for the introduction of two different modifications into the same c[RGDfK] peptide. This allowed the generation of a palmitoylated integrin-binding lipopeptide that shows high affinity to PLGA. Additionally, we coupled 5(6)-carboxyfluorescein to the second site for modification to enable sensitive quantification of the immobilized lipopeptide on PLGA. In conclusion, we present a synthesis protocol that enables the preparation of c[RGDfK] lipopeptides with a strong affinity to PLGA and an additional site for modifications. This will provide the opportunity to introduce a variety of effector molecules site-specifically to the c[RGDfK] lipopeptide, which will enable the introduction of multifunctionality into c[RGDfK]-coated PLGA devices or nanoparticles.

  19. Deep Label Distribution Learning With Label Ambiguity

    NASA Astrophysics Data System (ADS)

    Gao, Bin-Bin; Xing, Chao; Xie, Chen-Wei; Wu, Jianxin; Geng, Xin

    2017-06-01

    Convolutional Neural Networks (ConvNets) have achieved excellent recognition performance in various visual recognition tasks. A large labeled training set is one of the most important factors for its success. However, it is difficult to collect sufficient training images with precise labels in some domains such as apparent age estimation, head pose estimation, multi-label classification and semantic segmentation. Fortunately, there is ambiguous information among labels, which makes these tasks different from traditional classification. Based on this observation, we convert the label of each image into a discrete label distribution, and learn the label distribution by minimizing a Kullback-Leibler divergence between the predicted and ground-truth label distributions using deep ConvNets. The proposed DLDL (Deep Label Distribution Learning) method effectively utilizes the label ambiguity in both feature learning and classifier learning, which help prevent the network from over-fitting even when the training set is small. Experimental results show that the proposed approach produces significantly better results than state-of-the-art methods for age estimation and head pose estimation. At the same time, it also improves recognition performance for multi-label classification and semantic segmentation tasks.

  20. Stable isotope labeling of oligosaccharide cell surface antigens

    SciTech Connect

    Unkefer, C.J.; Silks, L.A. III; Martinez, R.A.

    1998-12-31

    The overall goal of this Laboratory Directed Research and Development (LDRD) project was to develop new methods for synthesis of {sup 13}C-labeled oligosaccharides that are required for nuclear magnetic resonance (NMR) studies of their solution conformation. Oligosaccharides are components of the cell`s outer surface and are involved in important processes such as cell-cell recognition and adhesion. Recently, Danishefsky and coworkers at Slone-Kettering Cancer Center developed a method for the solid-phase chemical synthesis of oligosaccharides. The specific goal of this LDRD project was to prepare uniform {sup 13}C-labeled aldohexose precursors required for the solid-phase synthesis of the Lewis blood-group antigenic determinants. We report the synthesis of {sup 13}C-labeled D-glucal, D-galactal and Fucosyl precursors. We have been collaborating with the Danishefsky group on the synthesis of the Lewis oligosaccharides and the NMR analysis of their solution conformation.

  1. Design, synthesis and evaluation in an LPS rodent model of neuroinflammation of a novel (18)F-labelled PET tracer targeting P2X7.

    PubMed

    Fantoni, Enrico Raffaele; Dal Ben, Diego; Falzoni, Simonetta; Di Virgilio, Francesco; Lovestone, Simon; Gee, Antony

    2017-12-01

    The P2X7 receptor has been shown to play a fundamental role in the initiation and sustenance of the inflammatory cascade. The development of a novel fluorine-18 PET tracer superior and with a longer half-life to those currently available is a promising step towards harnessing the therapeutic and diagnostic potential offered by this target. Inspired by the known antagonist A-804598, the present study outlines the design via molecular docking, synthesis and biological evaluation of the novel P2X7 tracer [(18)F]EFB. The tracer was radiolabelled via a three-step procedure, in vitro binding assessed in P2X7-transfected HEK293 and in B16 cells by calcium influx assays and an initial preclinical evaluation was performed in a lipopolysaccharide (LPS)-injected rat model of neuroinflammation. The novel tracer [(18)F]EFB was synthesised in 210 min in 3-5% decay-corrected radiochemical yield (DC RCY), >99% radiochemical purity (RCP) and >300 GBq/μmol and fully characterised. Functional assays showed that the compound binds with nM K i to human, rat and mouse P2X7 receptors. In vivo, [(18)F]EFB displayed a desirable distribution profile, and while it showed low blood-brain barrier penetration, brain uptake was quantifiable and displayed significantly higher mean longitudinal uptake in inflamed versus control rat CNS regions. [(18)F]EFB demonstrates strong in vitro affinity to human and rodent P2X7 and limited yet quantifiable BBB penetration. Considering the initial promising in vivo data in an LPS rat model with elevated P2X7 expression, this work constitutes an important step in the development of a radiotracer useful for the diagnosis and monitoring of clinical disorders with associated neuroinflammatory processes.

  2. Synthesis of AS1411-aptamer-conjugated CdTe quantum dots with high fluorescence strength for probe labeling tumor cells.

    PubMed

    Alibolandi, Mona; Abnous, Khalil; Ramezani, Mohammad; Hosseinkhani, Hossein; Hadizadeh, Farzin

    2014-09-01

    In this paper, we report microwave-assisted, one-stage synthesis of high-quality functionalized water-soluble cadmium telluride (CdTe) quantum dots (QDs). By selecting sodium tellurite as the Te source, cadmium chloride as the Cd source, mercaptosuccinic acid (MSA) as the capping agent, and a borate-acetic acid buffer solution with a pH range of 5-8, CdTe nanocrystals with four colors (blue to orange) were conveniently prepared at 100 °C under microwave irradiation in less than one hour (reaction time: 10-60 min). The influence of parameters such as the pH, Cd:Te molar ratio, and reaction time on the emission range and quantum yield percentage (QY%) was investigated. The structures and compositions of the prepared CdTe QDs were characterized by transmission electron microscopy, energy-dispersive X-ray spectroscopy, selective area electron diffraction, and X-ray powder diffraction experiments. The formation mechanism of the QDs is discussed in this paper. Furthermore, AS1141-aptamer-conjugated CdTe QDs in the U87MG glioblastoma cell line were assessed with a fluorescence microscope. The obtained results showed that the best conditions for obtaining a high QY of approximately 87% are a pH of 6, a Cd:Te molar ratio of 5:1, and a 30-min reaction time at 100 °C under microwave irradiation. The results showed that AS1141-aptamer-conjugated CdTe QDs could enter tumor cells efficiently. It could be concluded that a facile high-fluorescence-strength QD conjugated with a DNA aptamer, AS1411, which can recognize the extracellular matrix protein nucleolin, can specifically target U87MG human glioblastoma cells. The qualified AS1411-aptamer-conjugated QDs prepared in this study showed excellent capabilities as nanoprobes for cancer targeting and molecular imaging.

  3. 177Lu-labeled HPMA Copolymers Utilizing Cathepsin B and S Cleavable Linkers: Synthesis, Characterization and Preliminary In Vivo Investigation in a Pancreatic Cancer Model

    PubMed Central

    Ogbomo, Sunny M.; Shi, Wen; Wagh, Nilesh K; Zhou, Zhengyuan; Brusnahan, Susan K.; Garrison, Jered C.

    2013-01-01

    Introduction A major barrier to the advancement of therapeutic nanomedicines has been the non-target toxicity caused by the accumulation of the drug delivery systems in organs associated with the reticuloendothelial system, particularly the liver and spleen. Herein, we report the development of peptide based metabolically active linkers (MALs) that are enzymatically cleaved by cysteine cathepsin B and S, two proteases highly expressed in the liver and spleen. The overall goal of this approach is to utilize the MALs to lower the non-target retention and toxicity of radiolabeled drug delivery systems, thus resulting in higher diagnostic and radiotherapeutic efficacy. Methods In this study three MALs (MAL0, MAL1 and MAL2) were investigated. MAL1 and MAL2 are composed of known substrates of cathepsin B and S, respectively, while MAL0 is a non-cleavable control. Both MAL1 and MAL2 were shown to undergo enzymatic cleavage with the appropriate cathepsin protease. Subsequent to conjugation to the HPMA copolymer and radiolabeling with 177Lu, the peptide-polymer conjugates were renamed 177Lu- metabolically active copolymers (177Lu-MACs) with the corresponding designation 177Lu-MAC0, 177Lu-MAC1 and 177Lu-MAC2. Results In vivo evaluation of the 177Lu-MACs was performed in a HPAC human pancreatic cancer xenograft mouse model. 177Lu-MAC1 and 177Lu-MAC2 demonstrated 3.1 and 2.1 fold lower liver retention, respectively, compared to control (177Lu-MAC0) at 72 h post-injection. With regard to spleen retention, 177Lu-MAC1 and 177Lu-MAC2 each exhibited a nearly fourfold lower retention, relative to control, at the 72 h time point. However, the tumor accumulation of the 177Lu-MAC0 was two to three times greater than 177Lu-MAC1 and 177Lu-MAC2 at the same time point. The MAL approach demonstrated the capability of substantially reducing the non-target retention of the 177Lu-labeled HPMA copolymers. Conclusions While further studies are needed to optimize the pharmacokinetics of the 177Lu

  4. Poly-arginine conjugated triarylmethyl radical as intracellular spin label.

    PubMed

    Driesschaert, Benoit; Bobko, Andrey A; Eubank, Timothy D; Samouilov, Alexandre; Khramtsov, Valery V; Zweier, Jay L

    2016-04-01

    Stable triarylmethyl radicals are ideal spin labels used for biomedical electron paramagnetic resonance applications. Previously reported structures exhibit polar charged functions for water solubilization preventing them from crossing the cell membrane. We report the synthesis of a triarylmethyl radical conjugated to poly-arginine peptide allowing intracellular delivery of the paramagnetic label.

  5. Synthesis of Cryptophycin Affinity Labels and Tubulin Labeling

    DTIC Science & Technology

    2006-05-01

    Nostoc sp.), are a new and potent tumor-selective class of tubulin-binding antimitotic agents1 that show excellent activity against MDR cancer cell...lines and were exceptionally active against mammary derived tumors.2,3 Cryptophycin-1 (1, Fig. 1) is the major cytotoxin in Nostoc sp.4,5 and...arenastatin A), isolated from the Okinawan marine sponge Dysidea arenaria6 and later from Nostoc sp. strain GSV 224,7 is also a potent inhibitor of tubulin

  6. Synthesis of Cryptophycin Affinity Labels and Tubulin Labeling

    DTIC Science & Technology

    2004-05-01

    isolated from blue-green algae ( Nostoc sp.), are a new and potent tumor-selective class of tubulin-binding antimitotic agents that show excellent activity... Nostoc sp.3𔃾 and displays IC 50 values in the pM range. Of special importance is the reduced susceptibility of the cryptophycins to P-glycoprotein...also named arenastatin A), isolated from the Okinawan marine sponge Dysidea arenaria5 and later from Nostoc sp. strain GSV 224,6 is also a potent

  7. Synthesis of Cryptophycin Affinity Labels and Tubulin Labeling

    DTIC Science & Technology

    2005-05-01

    in one step from coumalic acid,26 however, the equipment needed for the required decarboxylation reaction was not readily. available to us and...purification for the Heck coupling reaction to furnish 41% of the desired octadienoate ester. However, the moderate yield of these reactions led us to...a traditional Yamaguchi reaction where the acid 33 is activated as the Yamaguchi chloride and reacted with alcohol 20. The rest of the synthetic

  8. Hemoglobin Labeled by Radioactive Lysine

    DOE R&D Accomplishments Database

    Bale, W. F.; Yuile, C. L.; DeLaVergne, L.; Miller, L. L.; Whipple, G. H.

    1949-12-08

    This paper reports on the utilization of tagged epsilon carbon of DL-lysine by a dog both anemic and hypoproteinemic due to repeated bleeding plus a diet low in protein. The experiment extended over period of 234 days, a time sufficient to indicate an erythrocyte life span of at least 115 days based upon the rate of replacement of labeled red cell proteins. The proteins of broken down red cells seem not to be used with any great preference for the synthesis of new hemoglobin.

  9. Adapter Reagents for Protein Site Specific Dye Labeling

    PubMed Central

    Thompson, Darren A.; Evans, Eric G. B.; Kasza, Tomas; Millhauser, Glenn L.; Dawson, Philip E.

    2016-01-01

    Chemoselective protein labeling remains a significant challenge in chemical biology. Although many selective labeling chemistries have been reported, the practicalities of matching the reaction with appropriately functionalized proteins and labeling reagents is often a challenge. For example, we encountered the challenge of site specifically labeling the cellular form of the murine Prion protein with a fluorescent dye. To facilitate this labeling, a protein was expressed with site specific p-acetylphenylalanine. However, the utility of this aceto-phenone reactive group is hampered by the severe lack of commercially available aminooxy fluorophores. Here we outline a general strategy for the efficient solid phase synthesis of adapter reagents capable of converting maleimido-labels into aminooxy or azide functional groups that can be further tuned for desired length or solubility properties. The utility of the adapter strategy is demonstrated in the context of fluorescent labeling of the murine Prion protein through an adapted aminooxy-Alexa dye. PMID:24599728

  10. 99mTc: Labeling Chemistry and Labeled Compounds

    NASA Astrophysics Data System (ADS)

    Alberto, R.; Abram, U.

    This chapter reviews the radiopharmaceutical chemistry of technetium related to the synthesis of perfusion agents and to the labeling of receptor-binding biomolecules. To understand the limitations of technetium chemistry imposed by future application of the complexes in nuclear medicine, an introductory section analyzes the compulsory requirements to be considered when facing the incentive of introducing a novel radiopharmaceutical into the market. Requirements from chemistry, routine application, and market are discussed. In a subsequent section, commercially available 99mTc-based radiopharmaceuticals are treated. It covers the complexes in use for imaging the most important target organs such as heart, brain, or kidney. The commercially available radiopharmaceuticals fulfill the requirements outlined earlier and are discussed with this background. In a following section, the properties and perspectives of the different generations of radiopharmaceuticals are described in a general way, covering characteristics for perfusion agents and for receptor-specific molecules. Technetium chemistry for the synthesis of perfusion agents and the different labeling approaches for target-specific biomolecules are summarized. The review comprises a general introduction to the common approaches currently in use, employing the N x S4-x , [3+1] and 2-hydrazino-nicotinicacid (HYNIC) method as well as more recent strategies such as the carbonyl and the TcN approach. Direct labeling without the need of a bifunctional chelator is briefly reviewed as well. More particularly, recent developments in the labeling of concrete targeting molecules, the second generation of radiopharmaceuticals, is then discussed and prominent examples with antibodies/peptides, neuroreceptor targeting small molecules, myocardial imaging agents, vitamins, thymidine, and complexes relevant to multidrug resistance are given. In addition, a new approach toward peptide drug development is described. The section

  11. Syntheses of isotope-labeled SGLT2 inhibitor canagliflozin (JNJ-28431754).

    PubMed

    Lin, Ronghui; Hoerr, David C; Weaner, Larry E; Salter, Rhys

    2017-08-18

    Canagliflozin (Invokana, JNJ-28431754) is an orally bioavailable and selective SGLT2 (subtype 2 sodium-glucose transport protein) inhibitor approved for the treatment of type 2 diabetes. Herein, we report the synthesis of (13) C and (14) C-labeled canagliflozin. Stable isotope-labeled [(13) C6 ]canagliflozin was synthesized in 4 steps starting from [(13) C6 ]-labeled glucose. The [(14) C]-Labeled canagliflozin was synthesized by incorporation of [(14) C] into the benzylic position between the thiophene and benzene rings of the compound. Detailed synthesis of the isotope-labeled compounds is reported. Copyright © 2017 John Wiley & Sons, Ltd.

  12. Mental Labels and Tattoos

    ERIC Educational Resources Information Center

    Hyatt, I. Ralph

    1977-01-01

    Discusses the ease with which mental labels become imprinted in our system, six basic axioms for maintaining negative mental tattoos, and psychological processes for eliminating mental tattoos and labels. (RK)

  13. Pesticide Labeling Questions & Answers

    EPA Pesticide Factsheets

    Pesticide manufacturers, applicators, state regulatory agencies, and other stakeholders raise questions or issues about pesticide labels. The questions on this page are those that apply to multiple products or address inconsistencies among product labels.

  14. Soil Fumigant Labels - Chloropicrin

    EPA Pesticide Factsheets

    Search by EPA registration number, product name, or company name, and follow the link to the Pesticide Product Label System (PPLS) for details on each fumigant. Updated labels include new safety requirements for buffer zones and related measures.

  15. Soil Fumigant Labels - Dazomet

    EPA Pesticide Factsheets

    Updated labels include new safety requirements for buffer zones and related measures. Find information from the Pesticide Product Labeling System (PPLS) for products such as Basamid G, manufactured by Amvac.

  16. Mental Labels and Tattoos

    ERIC Educational Resources Information Center

    Hyatt, I. Ralph

    1977-01-01

    Discusses the ease with which mental labels become imprinted in our system, six basic axioms for maintaining negative mental tattoos, and psychological processes for eliminating mental tattoos and labels. (RK)

  17. Soil Fumigant Labels

    EPA Pesticide Factsheets

    The 2012 updated pesticide labels include new safety requirements for buffer zones and related measures. Find labels for each different type of fumigant: chloropicrin, dazomet, dimethyl disulfide, metam sodium/potassium, and methyl bromide.

  18. Electronic Submission of Labels

    EPA Pesticide Factsheets

    Pesticide registrants can provide draft and final labels to EPA electronically for our review as part of the pesticide registration process. The electronic submission of labels by registrants is voluntary but strongly encouraged.

  19. The Labelling of Chemicals.

    ERIC Educational Resources Information Center

    Education in Science, 1979

    1979-01-01

    Describes the impact on chemistry laboratories and teachers in the United Kingdom of the Packaging and Labelling of Dangerous Substances Regulations 1978. These regulations require suppliers to label containers in particular ways. (HM)

  20. Semiotic labelled deductive systems

    SciTech Connect

    Nossum, R.T.

    1996-12-31

    We review the class of Semiotic Models put forward by Pospelov, as well as the Labelled Deductive Systems developed by Gabbay, and construct an embedding of Semiotic Models into Labelled Deductive Systems.

  1. Label Review Training: Module 1: Label Basics, Page 16

    EPA Pesticide Factsheets

    This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. Learn about the importance of labels and the role in enforcement.

  2. Label Review Training: Module 1: Label Basics, Page 14

    EPA Pesticide Factsheets

    This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. Learn about positive effects from proper labeling.

  3. Label Review Training: Module 1: Label Basics, Page 15

    EPA Pesticide Factsheets

    This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. Learn about the consequences of improper labeling.

  4. Label Review Training: Module 1: Label Basics, Page 21

    EPA Pesticide Factsheets

    This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. Learn about types of labels.

  5. Label Review Training: Module 1: Label Basics, Page 19

    EPA Pesticide Factsheets

    This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. This section covers supplemental distributor labeling.

  6. Label Review Training: Module 1: Label Basics, Page 17

    EPA Pesticide Factsheets

    This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. See an overview of the importance of labels.

  7. Label Review Training: Module 1: Label Basics, Page 22

    EPA Pesticide Factsheets

    This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. Learn about what labels require review.

  8. Label Review Training: Module 1: Label Basics, Page 27

    EPA Pesticide Factsheets

    This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. See examples of mandatory and advisory label statements.

  9. Label Review Training: Module 1: Label Basics, Page 26

    EPA Pesticide Factsheets

    This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. Learn about mandatory and advisory label statements.

  10. Label Review Training: Module 1: Label Basics, Page 24

    EPA Pesticide Factsheets

    This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. This page is about which labels require review.

  11. Label Review Training: Module 1: Label Basics, Page 18

    EPA Pesticide Factsheets

    This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. This section discusses the types of labels.

  12. Label Review Training: Module 1: Label Basics, Page 23

    EPA Pesticide Factsheets

    This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. Lists types of labels that do not require review.

  13. Sample Pesticide Label for Label Review Training

    EPA Pesticide Factsheets

    Pesticide labels translate results of our extensive evaluations of pesticide products into conditions, directions and precautions that define parameters for use of a pesticide with the goal of ensuring protection of human health and the environment.

  14. Pesticide Product Label System

    EPA Pesticide Factsheets

    The Pesticide Product Label System (PPLS) provides a collection of pesticide product labels (Adobe PDF format) that have been approved by EPA under Section 3 of the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA). New labels were added to PPLS on November 21, 2014. Pesticide product labels provide critical information about how to safely handle and use registered pesticide products. An approved pesticide product label represents the full content of EPAs registration decision regarding that product. Pesticide labels contain detailed information on the use, storage, and handling of a product. This information will be found on EPA stamped-approved labels and, in some cases, in subsequent related correspondence, which is also included in PPLS. You may need to review several PDF files for a single product to determine the complete current terms of registration.

  15. Synthesis of tritium labeled Ac-(Nle/sup 4/, D-Phe/sup 7/)-. cap alpha. -MSH/sub 4-11/-NH/sub 2/: a superpotent melanotropin with prolonged biological activity

    SciTech Connect

    Wilkes, B.D.; Hruby, V.J.; Yamamura, H.I.; Akiyama, K.; Castrucci, A.M. de; Hadley, M.E.; Andrews, J.R.; Wan, Y.P.

    1984-03-05

    Ac-(Nle/sup 4/, D-Phe/sup 7/)-..cap alpha..-MSH/sub 4-11/-NH/sub 2/ an octapeptide, is a melanotropin analogue (Ac-Nle-Glu-His-D-Phe-Arg-Trp-Gly-Lys-NH/sub 2/), which is a superpotent agonist of frog and lizard skin melanocytes and mouse S 91 (Cloudman) melanoma cells. This melanotropin possesses ultraprolonged activity on melanocytes, both in vitro and in vivo, and the peptide is resistant to inactivation by serum enzymes. The tritium-labeled congener was prepared by direct incorporation of (/sup 3/H)-labeled norleucine into the peptide. The melanotropic activity of the labeled peptide is identical to the unlabeled analogue. This labeled peptide should be useful for studies on the localization and characterization of melanotropin receptors.

  16. Synthesis and evaluation of an (125)I-labeled azide prosthetic group for efficient and bioorthogonal radiolabeling of cyclooctyne-group containing molecules using copper-free click reaction.

    PubMed

    Choi, Mi Hee; Shim, Ha Eun; Nam, You Ree; Kim, Hye Rim; Kang, Jung Ae; Lee, Dong-Eun; Park, Sang Hyun; Choi, Dae Seong; Jang, Beom-Su; Jeon, Jongho

    2016-02-01

    Herein we report the radiosynthesis of a pyridine derived azide prosthetic group for iodine radioisotope labeling of dibenzocyclooctyne (DBCO) conjugated molecules. The radiolabeling of the stannylated precursor 2 was conducted using [(125)I]NaI and chloramine-T to give (125)I-labeled azide ([(125)I]1) with high radiochemical yield (72±8%, n=4) and radiochemical purity (>99%). Using (125)I-labeled azide ([(125)I]1), cyclic RGD peptide and near infrared fluorescent molecule were efficiently labeled with modest to good radiochemical yields. The biodistribution study and SPECT/CT images showed that [(125)I]1 underwent rapid renal clearance. These results clearly demonstrated that [(125)I]1 could be used as an useful radiotracer for in vivo pre-targeted imaging as well as efficient in vitro radiolabeling of DBCO containing molecules.

  17. Synthesis and characterization of tritium labeled N-((R)-1-((S)-4-(4-chlorophenyl)-4-hydroxy-3,3-dimethylpiperidin-1-yl)-3-methyl-1-oxobutan-2-yl)-3-sulfamoylbenzamide.

    PubMed

    Hong, Yang; Hynes, John; Tian, Yuan; Balasubramanian, Balu; Bonacorsi, Samuel

    2015-08-01

    N-((R)-1-((S)-4-(4-chlorophenyl)-4-hydroxy-3,3-dimethylpiperidin-1-yl)-3-methyl-1-oxobutan-2-yl)-3-sulfamoylbenzamide is a potent C-C chemokine receptor 1 (CCR1) antagonist. The compound, possessing benzamide functionality, successfully underwent tritium/hydrogen (T/H) exchange with an organoiridium catalyst (Crabtree's catalyst). The labeling pattern in the product was studied with liquid chromatography-mass spectrometry, time-of-flight mass spectrometry, and (3) H-NMR. Overall, multiple labeled species were identified. In addition to the anticipated incorporation of tritium in the benzamide moiety, tritium labeling was observed in the valine portion of the molecule including substitution at its chiral carbon. Using authentic standards, liquid chromatography analysis of the labeled compound showed complete retention of stereochemical configuration.

  18. Labeling of Patient Specimens

    DTIC Science & Technology

    2011-01-26

    printers in each clinic to print labels .JDI Capt Cutter Research compatible printer, Cost, Time Frame Develop standard training for all clinics...Standardize label content, automate with inkless printers once process is proven c . Place visual reminders for providers and support staff 2. Event

  19. Labeling and Delinquency.

    ERIC Educational Resources Information Center

    Adams, Mike S.; Robertson, Craig T.; Gray-Ray, Phyllis; Ray, Melvin C.

    2003-01-01

    Index comprised of six contrasting descriptive adjectives was used to measure incarcerated youths' perceived negative labeling from the perspective of parents, teachers, and peers. Results provided partial support for hypothesis that juveniles who choose a greater number of negative labels will report more frequent delinquent involvement. Labeling…

  20. Labeling and Delinquency.

    ERIC Educational Resources Information Center

    Adams, Mike S.; Robertson, Craig T.; Gray-Ray, Phyllis; Ray, Melvin C.

    2003-01-01

    Index comprised of six contrasting descriptive adjectives was used to measure incarcerated youths' perceived negative labeling from the perspective of parents, teachers, and peers. Results provided partial support for hypothesis that juveniles who choose a greater number of negative labels will report more frequent delinquent involvement. Labeling…

  1. Government perspective: food labeling.

    PubMed

    Philipson, Tomas

    2005-07-01

    The Food and Drug Administration acknowledges the severity of the obesity epidemic. The Food and Drug Administration recognizes the importance of food labeling as a vehicle for dietary messages and, thus, enforces stringent guidelines to maintain the integrity of the food label. As food labels await another upgrade to make them more effective and easier to understand, the Food and Drug Administration considers what information will be most useful for consumers to make healthy choices. The causal relationship between food labels and subsequent diet choice is not well understood; more research in this area is needed. The Commissioner of the Food and Drug Administration has recently appointed an Obesity Working Group to develop proposals on pertinent topics of obesity, including the role of food labeling as a dietary guide.

  2. Mining Multi-label Data

    NASA Astrophysics Data System (ADS)

    Tsoumakas, Grigorios; Katakis, Ioannis; Vlahavas, Ioannis

    A large body of research in supervised learning deals with the analysis of single-label data, where training examples are associated with a single label λ from a set of disjoint labels L. However, training examples in several application domains are often associated with a set of labels Y ⊆ L. Such data are called multi-label.

  3. Label Review Training: Module 1: Label Basics, Page 29

    EPA Pesticide Factsheets

    This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review. This page is a quiz on Module 1.

  4. Label Review Training: Module 1: Label Basics, Page 25

    EPA Pesticide Factsheets

    This module of the pesticide label review training provides basic information about pesticides, their labeling and regulation, and the core principles of pesticide label review: clarity, accuracy, consistency with EPA policy, and enforceability.

  5. Evaluation of the efficiency of Pd/H2 -catalyzed benzylic H/D exchange of dehydroabietinal with D(2) O and synthesis of a tritium-labeled analogue.

    PubMed

    Petros, Robby A; Shah, Jyoti

    2014-01-01

    Dehydroabietinal (DA) has been identified as an important signaling molecule in systemic acquired resistance in plants. Deuterium and tritium-labeled DA were synthesized to confirm its role in signaling and to further elucidate the mechanism by which DA induces systemic acquired resistance. Pd/H2 -catalyzed exchange of benzylic hydrogen atoms of DA with (2) H-H2 O or (3) H-H2 O was conducted with >97% label incorporation for (2) H-DA and a specific activity of 12.6 mCi/mmol for (3) H-DA synthesized from 90 mCi/mmol (3) H-H2 O. The extent of deuterium labeling at each benzylic position was determined via an inverse-gated (13) C NMR experiment. C7 and C15 were 87% and 81% labeled, respectively. Isotope-induced chemical shift changes at C6 were used to approximate the amount of singly (66%) and doubly (17%) labeled (2) H-DA at C7. Results also indicated that two of the three benzylic protons in DA underwent facile exchange. Exchange at the remaining position was likely hampered by steric interactions of nearby methyl groups at the surface of the Pd catalyst.

  6. Soil Fumigant Labels - Methyl Bromide

    EPA Pesticide Factsheets

    Search soil fumigant pesticide labels by EPA registration number, product name, or company, and follow the link to The Pesticide Product Label System (PPLS) for details. Updated labels include new safety requirements for buffer zones and related measures.

  7. Off-Label Drug Use

    MedlinePlus

    ... their drugs for off-label uses. Off-label marketing is very different from off-label use. Why ... Employment Become a Supplier Report Fraud or Abuse Global Health ACS CAN Sign Up for Email Policies ...

  8. Capacitive label reader

    DOEpatents

    Arlowe, H.D.

    1983-07-15

    A capacitive label reader includes an outer ring transmitting portion, an inner ring transmitting portion, and a plurality of insulated receiving portions. A label is the mirror-image of the reader except that identifying portions corresponding to the receiving portions are insulated from only one of two coupling elements. Positive and negative pulses applied, respectively, to the two transmitting rings biased a CMOS shift register positively to either a 1 or 0 condition. The output of the CMOS may be read as an indication of the label.

  9. Capacitive label reader

    DOEpatents

    Arlowe, H.D.

    1985-11-12

    A capacitive label reader includes an outer ring transmitting portion, an inner ring transmitting portion, and a plurality of insulated receiving portions. A label is the mirror-image of the reader except that identifying portions corresponding to the receiving portions are insulated from only one of two coupling elements. Positive and negative pulses applied, respectively, to the two transmitting rings biased a CMOS shift register positively to either a 1 or 0 condition. The output of the CMOS may be read as an indication of the label. 5 figs.

  10. Capacitive label reader

    DOEpatents

    Arlowe, H. Duane

    1985-01-01

    A capacitive label reader includes an outer ring transmitting portion, an inner ring transmitting portion, and a plurality of insulated receiving portions. A label is the mirror-image of the reader except that identifying portions corresponding to the receiving portions are insulated from only one of two coupling elements. Positive and negative pulses applied, respectively, to the two transmitting rings biased a CMOS shift register positively to either a 1 or 0 condition. The output of the CMOS may be read as an indication of the label.

  11. Like your labels?

    PubMed

    Field, Michele

    2010-01-01

    The descriptive “conventions” used on food labels are always evolving. Today, however, the changes are so complicated (partly driven by legislation requiring disclosures about environmental impacts, health issues, and geographical provenance) that these labels more often baffle buyers than enlighten them. In a light-handed manner, the article points to how sometimes reading label language can be like deciphering runes—and how if we are familiar with the technical terms, we can find a literal meaning, but still not see the implications. The article could be ten times longer because food labels vary according to cultures—but all food-exporting cultures now take advantage of our short attention-span when faced with these texts. The question is whether less is more—and if so, in this contest for our attention, what “contestant” is voted off.

  12. Label Review Training - Resources

    EPA Pesticide Factsheets

    Pesticide labels translate results of our extensive evaluations of pesticide products into conditions, directions and precautions that define parameters for use of a pesticide with the goal of ensuring protection of human health and the environment.

  13. Oligonucleotide labeling methods. 3. Direct labeling of oligonucleotides employing a novel, non-nucleosidic, 2-aminobutyl-1,3-propanediol backbone.

    PubMed Central

    Nelson, P S; Kent, M; Muthini, S

    1992-01-01

    Novel CE-phosphoramidite (7a-e) and CPG (8a, c, d, e) reagents have been prepared from a unique 2-aminobutyl-1,3-propanediol backbone. The reagents have been used to directly label oligonucleotides with fluorescein, acridine, and biotin via automated DNA synthesis. The versatile 2-aminobutyl-1,3-propanediol backbone allows for labeling at any position (5', internal, and 3') during solid phase oligonucleotide synthesis. Multiple labels can be achieved by repetitive coupling cycles. Furthermore, the 3-carbon atom internucleotide phosphate distance is retained when inserted internally. Using this method, individual oligonucleotides possessing two and three different reporter molecules have been prepared. PMID:1475185

  14. Routing and Label Space Reduction in Label Switching Networks

    NASA Astrophysics Data System (ADS)

    Solano, Fernando; Caro, Luis Fernando; Stidsen, Thomas; Papadimitriou, Dimitri

    This chapter is devoted to the analysis and modeling of some problems related to the optimal usage of the label space in label switching networks. Label space problems concerning three different technologies and architectures - namely Multi-protocol Label Switching (MPLS), Ethernet VLAN-Label Switching (ELS) and All-Optical Label Switching (AOLS) - are discussed in this chapter. Each of these cases yields to different constraints of the general label space reduction problem. We propose a generic optimization model and, then, we describe some adaptations aiming at modeling each particular case. Simulation results are briefly discussed at the end of this chapter.

  15. Fluorine-18 labeling of small molecules: the use of 18F-labeled aryl fluorides derived from no-carrier-added [18F]fluoride as labeling precursors.

    PubMed

    Wuest, F

    2007-01-01

    The favourable long-half life, the ease of production and the low energy of the emitted positron make 18F an ideal radionuclide for PET imaging. Radiochemistry of 18F basically relies on two distinctive types of reactions: nucleophilic and electrophilic reactions. All syntheses of 18F-labeled radiotracers are based on either [18F]fluoride ion or [18F]fluorine gas as simple primary labeling precursors which are obtained directly from the cyclotron. They can be applied either directly to the radiosynthesis or they can be transformed into more complex labeling precursors enabling the multi-step build-up of organic tracer molecules. The topic of this review is a survey on the application of several 18F-labeled aryl fluorides as building blocks derived from no-carrier-added (n.c.a.) [18F] fluoride to build up small monomeric PET radiotracers at high specific radioactivity by multi-step synthesis procedures.

  16. Everyone Feels Empowered: Understanding Feminist Self-Labeling

    ERIC Educational Resources Information Center

    Liss, Miriam; Erchull, Mindy J.

    2010-01-01

    Research findings raise questions about whether the feminist identity development model provides information about women's social identification as a feminist. Specifically, the penultimate stage, Synthesis, has been theorized to capture when feminist identity formation coalesces and women take on the feminist label. However, available data have…

  17. F-18 labeled 3-fluorodiazepam

    SciTech Connect

    Luxen, A.; Barrio, J.R.; Bida, G.T.; Satyamurthy, N.; Phelps, M.E.

    1985-05-01

    3-Fluorodiazepam is a new and potent antianxiety agent with prolonged action. The authors found that molecular fluorine (0.5% in Ne) reacts cleanly with diazepam in freon or chloroform at room temperature to produce 3-fluorodiazepam in good yields. Successful syntheses have employed 2:1 to 5:1 molar ratios diazepam: fluorine to minimize the formation of byproducts. (/sup 18/F) 3-Fluorodiazepam, a potential candidate for PET studies, (specific activity 3-5 Ci/mmol) has been synthesized from /sup 18/F-F/sub 2/ using the same procedure, followed by column chromatographic purification (Silicagel, dichloromethane: ethyl acetate, 5:1) with a radiochemical yield of 12-20% (50% maximum) and a chemical and radiochemical purity >99% as judged by reversed-phase high pressure liquid chromatography analysis (Ultrasyl octyl column, 10 ..mu.. m, 4.6 x 250 mm i.d., 60% MeOH 40% water; flow rate, 1.0 ml/min; retention time for (/sup 18/F) fluorodiazepam, 11.4 min; for diazepam, 13.5 min; radioactivity and ultraviolet detectors). Lower radiochemical yields (5-7%), and significant formation of by-products were observed when (/sup 18/F)acetylhypofluorite, prepared in the gasphase, was used as the reagent. Readily accessible routes to /sup 18/F-labeled benzodiazepines of higher specific activity were also investigated. Approaches to the synthesis of high specific activity (>200 Ci/mmol) (/sup 18/F)3-fluorodiazepam involve nucleophilic displacement at carbon-3 (e.g. from 3-chlorodiazepam) with (/sup 18/F)fluoride ion. The results presented here demonstrate the synthetic accessibility of /sup 18/F-labeled benzodiazepines for application in neurotransmitter ligand studies with PET.

  18. Nanostructured luminescently labeled nucleic acids.

    PubMed

    Kricka, Larry J; Fortina, Paolo; Park, Jason Y

    2017-03-01

    Important and emerging trends at the interface of luminescence, nucleic acids and nanotechnology are: (i) the conventional luminescence labeling of nucleic acid nanostructures (e.g. DNA tetrahedron); (ii) the labeling of bulk nucleic acids (e.g. single-stranded DNA, double-stranded DNA) with nanostructured luminescent labels (e.g. copper nanoclusters); and (iii) the labeling of nucleic acid nanostructures (e.g. origami DNA) with nanostructured luminescent labels (e.g. silver nanoclusters). This review surveys recent advances in these three different approaches to the generation of nanostructured luminescently labeled nucleic acids, and includes both direct and indirect labeling methods. Copyright © 2016 John Wiley & Sons, Ltd.

  19. Label Distribution in Tissues of Wheat Seedlings Cultivated with Tritium-Labeled Leonardite Humic Acid

    PubMed Central

    Kulikova, Natalia A.; Abroskin, Dmitry P.; Badun, Gennady A.; Chernysheva, Maria G.; Korobkov, Viktor I.; Beer, Anton S.; Tsvetkova, Eugenia A.; Senik, Svetlana V.; Klein, Olga I.; Perminova, Irina V.

    2016-01-01

    Humic substances (HS) play important roles in the biotic-abiotic interactions of the root plant and soil contributing to plant adaptation to external environments. However, their mode of action on plants remains largely unknown. In this study the HS distribution in tissues of wheat seedlings was examined using tritium-labeled humic acid (HA) derived from leonardite (a variety of lignites) and microautoradiography (MAR). Preferential accumulation of labeled products from tritiated HA was found in the roots as compared to the shoots, and endodermis was shown to be the major control point for radial transport of label into vascular system of plant. Tritium was also found in the stele and xylem tissues indicating that labeled products from tritiated HA could be transported to shoot tissues via the transpiration stream. Treatment with HA lead to an increase in the content of polar lipids of photosynthetic membranes. The observed accumulation of labeled HA products in root endodermis and positive impact on lipid synthesis are consistent with prior reported observations on physiological effects of HS on plants such as enhanced growth and development of lateral roots and improvement/repairs of the photosynthetic status of plants under stress conditions. PMID:27350412

  20. Label Distribution in Tissues of Wheat Seedlings Cultivated with Tritium-Labeled Leonardite Humic Acid

    NASA Astrophysics Data System (ADS)

    Kulikova, Natalia A.; Abroskin, Dmitry P.; Badun, Gennady A.; Chernysheva, Maria G.; Korobkov, Viktor I.; Beer, Anton S.; Tsvetkova, Eugenia A.; Senik, Svetlana V.; Klein, Olga I.; Perminova, Irina V.

    2016-06-01

    Humic substances (HS) play important roles in the biotic-abiotic interactions of the root plant and soil contributing to plant adaptation to external environments. However, their mode of action on plants remains largely unknown. In this study the HS distribution in tissues of wheat seedlings was examined using tritium-labeled humic acid (HA) derived from leonardite (a variety of lignites) and microautoradiography (MAR). Preferential accumulation of labeled products from tritiated HA was found in the roots as compared to the shoots, and endodermis was shown to be the major control point for radial transport of label into vascular system of plant. Tritium was also found in the stele and xylem tissues indicating that labeled products from tritiated HA could be transported to shoot tissues via the transpiration stream. Treatment with HA lead to an increase in the content of polar lipids of photosynthetic membranes. The observed accumulation of labeled HA products in root endodermis and positive impact on lipid synthesis are consistent with prior reported observations on physiological effects of HS on plants such as enhanced growth and development of lateral roots and improvement/repairs of the photosynthetic status of plants under stress conditions.

  1. A facile synthesis of 5,5-dideutero-4-dimethyl(phenyl)silyl-6-undecyl-tetrahydropyran-2-one as a deuterium labeled synthon for (-)-tetrahydrolipstatin and (+)-δ-hexadecanolide.

    PubMed

    Wagh, Sandip J; Chowdhury, Raghunath; Mukhopadhyay, Sulekha; Ghosh, Sunil K

    2013-11-01

    Deuterium-labeled biologically active compounds are gaining importance because they can be utilized as tracers or surrogate compounds to understand the mechanism of action, absorption, distribution, metabolism, and excretion. Deuterated drug molecules (heavy drugs) become novel as well as popular because of better stability and bioavailability compared with their hydrogen analogs. Labeling of organic molecules with deuterium at specific positions is thus gaining popularity. In this work, we have exploited a highly regioselective and enantioselective direct Michael addition of methyl-d3 alkyl ketones to dimethyl(phenyl)silylmethylene malonate that was catalyzed by (S)-N-(2-pyrrolidinylmethyl)pyrrolidine/trifluoroacetic acid/ D2 O combination with high yield and isotopic purity. The 5,5-dideutero-4-dimethyl(phenyl)silyl-6-undecyl-tetrahydropyran-2-one was obtained from the adduct of methyl-d3 undecanyl ketone and dimethyl(phenyl)silylmethylene malonate by a silicon controlled diastereoselective ketone reduction, lactonization, and deethoxycarbonylation. The dideuterated silylated tetrahydropyran-2-one is the precursor for geminal (2) H2 -labeled (+)-4-hydroxy-6-undecyl-tetrahydropyran-2-one, an advanced intermediate for gem-dideutero (-)-tetrahydrolipstatin and (+)-δ-hexadecanolide syntheses. Copyright © 2013 John Wiley & Sons, Ltd.

  2. 21 CFR 820.120 - Device labeling.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... QUALITY SYSTEM REGULATION Labeling and Packaging Control § 820.120 Device labeling. Each manufacturer shall establish and maintain procedures to control labeling activities. (a) Label integrity. Labels... accuracy including, where applicable, the correct expiration date, control number, storage instructions...

  3. 2'-Alkynylnucleotides: A Sequence- and Spin Label-Flexible Strategy for EPR Spectroscopy in DNA.

    PubMed

    Haugland, Marius M; El-Sagheer, Afaf H; Porter, Rachel J; Peña, Javier; Brown, Tom; Anderson, Edward A; Lovett, Janet E

    2016-07-27

    Electron paramagnetic resonance (EPR) spectroscopy is a powerful method to elucidate molecular structure through the measurement of distances between conformationally well-defined spin labels. Here we report a sequence-flexible approach to the synthesis of double spin-labeled DNA duplexes, where 2'-alkynylnucleosides are incorporated at terminal and internal positions on complementary strands. Post-DNA synthesis copper-catalyzed azide-alkyne cycloaddition (CuAAC) reactions with a variety of spin labels enable the use of double electron-electron resonance experiments to measure a number of distances on the duplex, affording a high level of detailed structural information.

  4. Full automation of (68)Ga labelling of DOTA-peptides including cation exchange prepurification.

    PubMed

    Ocak, M; Antretter, M; Knopp, R; Kunkel, F; Petrik, M; Bergisadi, N; Decristoforo, C

    2010-02-01

    Here we describe a fully automated approach for the synthesis of (68)Ga-labelled DOTA-peptides based on pre-concentration and purification of the generator eluate by using a cation exchange-cartridge and its comparison with fully automated direct labelling applying fractionated elution. Pre-concentration of the eluate on a cation exchange cartridge both using a resin-based and a disposable cation-exchange cartridge efficiently removed (68)Ge as well as major metal contaminations with Fe and Zn. This resulted in a high labelling efficiency of DOTA-peptides at high specific activity (SA) with short synthesis times. Copyright (c) 2009 Elsevier Ltd. All rights reserved.

  5. Clickable fluorophores for biological labeling--with or without copper.

    PubMed

    Kele, Péter; Li, Xiaohua; Link, Martin; Nagy, Krisztina; Herner, András; Lorincz, Krisztián; Béni, Szabolcs; Wolfbeis, Otto S

    2009-09-07

    The synthesis of a set of new clickable fluorophores that virtually cover the whole visible spectrum reaching the near infra-red regime is presented herein. Besides dyes that are capable of participating in classical copper catalyzed 1,3-dipolar cycloaddition reactions with the counterparting function we have also prepared dyes containing a cyclooctyne moiety, an alkyne derivative that enables copper free clicking to azides. The suitability of these dyes for fluorescent labeling of biomolecules is presented by examples on model frameworks representing major biopolymer building blocks. The versatility of these dyes is presented in cell labeling experiments as well as by labeling the azide modified surface glycans of CHO-cells either by copper catalyzed or copper-free click reaction. These dyes are expected to have a large variety of applications in (bio)orthogonal labeling schemes both in vivo and in vitro.

  6. A Deceiving Label?

    ERIC Educational Resources Information Center

    Lum, Lydia

    2009-01-01

    The author reports on the growing debate among educators on whether the umbrella Asian Pacific Islander label conceals disparities among Asian American students or provides political power in numbers. Nationally, experts say that support services aimed at not only Southeast Asians, but all Asian Pacific Islander students, remain scarce in higher…

  7. A Deceiving Label?

    ERIC Educational Resources Information Center

    Lum, Lydia

    2009-01-01

    The author reports on the growing debate among educators on whether the umbrella Asian Pacific Islander label conceals disparities among Asian American students or provides political power in numbers. Nationally, experts say that support services aimed at not only Southeast Asians, but all Asian Pacific Islander students, remain scarce in higher…

  8. From Labels to Opportunities

    ERIC Educational Resources Information Center

    Wolter, Deborah

    2017-01-01

    The author argues that to truly help young students who struggle with reading and writing--including those with identified disabilities or conditions that effect building literacy--teachers should avoid the approach of focusing on a student's deficits and creating labels for him or her (dyslexic, English language learner, and so on). A rush to…

  9. Photoaffinity-labeled Cytokinins

    PubMed Central

    Theiler, Jane B.; Leonard, Nelson J.; Schmitz, Ruth Y.; Skoog, Folke

    1976-01-01

    Two new azidopurine derivatives, 2-azido-N6-(Δ2-isopentenyl)adenine and 2-azido-N6-benzyladenine, have been synthesized as potential photoaffinity labels for probing cytokinin-binding sites. The preparation and the biological activity of these compounds are described. PMID:16659772

  10. Synthesis of a water-soluble analog of 6-methyl-3-N-alkyl catechol labeled with carbon 13: NMR approach to the reactivity of poison ivy/oak sensitizers toward proteins.

    PubMed

    Goetz, G; Meschkat, E; Lepoittevin, J P

    1999-04-19

    A 13-C labeled water soluble derivative of alkylcatechol was synthesized and reacted with human serum albumin in phosphate buffer at pH 7.4 in air to allow a slow oxidation of the catechol into orthoquinone. The formation of several adducts was evidenced by a combination of 13C and 1H-13C correlation NMR. Although some adducts could result from a classical o-quinone formation - Michael type addition, our results suggest that a second pathway, involving a direct reaction of a carbon centered radical with proteins could be an important mechanism in the formation of modified proteins.

  11. Label Review Training: Module 1: Label Basics, Page 7

    EPA Pesticide Factsheets

    Page 7, Label Training, Pesticide labels translate results of our extensive evaluations of pesticide products into conditions, directions and precautions that define parameters for use of a pesticide with the goal of ensuring protection of human he

  12. Dual mode fluorescent (18)F-PET tracers: efficient modular synthesis of rhodamine-[cRGD]2-[(18)F]-organotrifluoroborate, rapid, and high yielding one-step (18)F-labeling at high specific activity, and correlated in vivo PET imaging and ex vivo fluorescence.

    PubMed

    Liu, Zhibo; Radtke, Mark Alex; Wong, May Q; Lin, Kuo-Shyan; Yapp, Donald T; Perrin, David M

    2014-11-19

    The design of dual mode fluorescent-PET peptidic tracers that can be labeled with [(18)F]fluoride at high specific activity and high yield has been challenged by the short half-life of (18)F and its aqueous indolence toward nucleophilic displacement, that often necessitates multistep reactions that start with punctiliously dry conditions. Here we present a modular approach to constructing a fluorescent dimeric peptide with a pendant radioprosthesis that is labeled in water with [(18)F]fluoride ion in a single, user-friendly step. The modular approach starts with grafting a new zwitterionic organotrifluoroborate radioprosthesis onto a pentaerythritol core with three pendent alkynes that enable successive grafting of a bright fluorophore (rhodamine) followed by two peptides (cylcoRGD). The construct is labeled with [(18)F]fluoride via isotope exchange within 20 min in a single step at high specific activity (>3 Ci/μmol) and in good yield to provide 275 mCi and high radiochemical purity. Neither drying of the [(18)F]fluoride ion solution nor HPLC purification of the labeled tracer is required. Facile chemical synthesis of this dual mode tracer along with a user-friendly one-step radiolabeling method affords very high specific activity. In vivo PET images of the dual mode tracer are acquired at both high and low specific activities. At very high specific activity, i.e., 3.5 Ci/μmol, tumor uptake is relatively high (5.5%ID/g), yet the associated mass is below the limits of fluorescent detection. At low specific activity, i.e., 0.01 Ci/μmol, tumor uptake in the PET image is reduced by approximately 50% (2.9%ID/g), but the greater associated mass enables fluorescence detection in the tumor. These data highlight a facile production of a dual mode fluorescent-PET tracer which is validated with in vivo and ex vivo images. These data also define critical limitations for the use of dual mode tracers in small animals.

  13. Synthesis of Fluorine-Containing Phosphodiesterase 10A (PDE10A) Inhibitors and the In Vivo Evaluation of F-18 Labeled PDE10A PET Tracers in Rodent and Nonhuman Primate

    PubMed Central

    Li, Junfeng; Zhang, Xiang; Jin, Hongjun; Fan, Jinda; Flores, Hubert; Perlmutter, Joel S.; Tu, Zhude

    2015-01-01

    A series of fluorine-containing PDE10A inhibitors were designed and synthesized to improve the metabolic stability of [11C]MP-10. Twenty of the 22 new analogues had high potency and selectivity for PDE10A: 18a–j, 19d–j, 20a–b, and 21b had IC50 values <5 nM for PDE10A. Seven F-18 labeled compounds [18F]18a–e, [18F]18g, and [18F]20a were radiosynthesized by 18F-introduction onto the quinoline rather than the pyrazole moiety of the MP-10 pharmacophore and performed in vivo evaluation. Biodistribution studies in rats showed ~2-fold higher activity in the PDE10A-enriched striatum than nontarget brain regions; this ratio increased from 5 to 30 min postinjection, particularly for [18F]18a–d and [18F]20a. Micro-PET studies of [18F]18d and [18F]20a in nonhuman primates provided clear visualization of striatum with suitable equilibrium kinetics and favorable metabolic stability. These results suggest this strategy may identify a 18F-labeled PET tracer for quantifying the levels of PDE10A in patients with CNS disorders including Huntington’s disease and schizophrenia. PMID:26430878

  14. Use the Nutrition Facts Label

    MedlinePlus

    ... Features Spokespeople News Archive eNewsletters Calendar Use the Nutrition Facts Label You can help your family eat ... to some of their favorite foods. Use the Nutrition Facts label found on food packages to make ...

  15. Decode the Sodium Label Lingo

    MedlinePlus

    ... For Preschooler For Gradeschooler For Teen Decode the Sodium Label Lingo Published January 24, 2013 Print Email Reading food labels can help you slash sodium. Here's how to decipher them. "Sodium free" or " ...

  16. Labeling lake water with tritium

    USGS Publications Warehouse

    Frederick, B.J.

    1963-01-01

    A method of packaging tritiated water in a manner that facilitates safe handling in environmental labeling operations, and procedures followed in labeling a large body of water with a small volume of tritiated water are described. ?? 1963.

  17. Collective Multi-Label Classification

    DTIC Science & Technology

    2005-01-01

    there is one output random variable . We begin by de- scribing this traditional classifier, then we describe its common ex- tension to the multi- label ...dependencies among the output variables . In addition to having feature for each label -term pair, CML main- tains features accounting for label co...over all possible multi- labelings — that is, over all subsets of Y . This method is intuitively appealing: it is easy to explain, and it is informative

  18. Microgravity Science Glovebox - Labels

    NASA Technical Reports Server (NTRS)

    1997-01-01

    Labels are overlaid on a photo (0003837) of the Microgravity Science Glovebox (MSG). The MSG is being developed by the European Space Agency (ESA) and NASA are developing the MSG for use aboard the International Space Station (ISS). Scientists will use the MSG to carry out multidisciplinary studies in combustion science, fluid physics and materials science. The MSG is managed by NASA's Marshall Space Flight Center (MSFC). Photo Credit: NASA/MSFC

  19. Food Labels Tell the Story!

    MedlinePlus

    ... My World From the Label to the Table! Food Labels Tell the Story! What is in food? Food provides your body with all of the ... your food choices. Nutrition Facts—the Labels on Food Products Beginning in 1994, the US government began ...

  20. Synthesis and biological studies of positron-emitting radiopharmaceuticals

    SciTech Connect

    Dischino, D.D.

    1983-01-01

    The development and clinical evaluation of two-positron emitting radiopharmaceuticals designed to image myelin in humans is reported. Carbon-11-labeled benzyl methyl ether was synthesized by the reaction of carbon-11-labeled methanol and benzyl chloride in dimethyl sulfoxide containing powdered potassium hydroxide in a radiochemical yield of 43% and a synthesis and purification time of 40 minutes. Carbon-11-labeled diphenylmethanol was synthesized by the reaction of carbon-11-labeled carbon dioxide and phenyllithium followed by the reduction of the carbon-11-labeled intermediate to diphenylmethanol via lithium aluminum hydride in a radiochemical yield of 71% and a synthesis and purification time of 38 minutes. Carbon-11-labeled benzyl methyl ether and diphenylmethanol were each evaluated as myelin imaging agents in three patients with multiple sclerosis via positron-emission tomography. In two out of three patients studied with carbon-11-labeled benzyl methyl ether, the distribution of activity in the brain was not consistent with local lipid content. A new synthesis of carbon-11-labeled-DL-phenylalanine labeled in the benzylic position and the synthesis of fluorine-18-labeled 1-(2-nitro-1-imidazolyl)-3-fluoro-2-propanol, a potential in vivo marker of hypoxic tissue, are reported.

  1. Synthesis and biochemical evaluation of tritium-labeled 1-methyl-N-(8-methyl-8-azabicyclo(3. 2. 1)oct-3-yl)-1H-indazole-3-carboxa mide, a useful radioligand for 5HT3 receptors

    SciTech Connect

    Robertson, D.W.; Bloomquist, W.; Cohen, M.L.; Reid, L.R.; Schenck, K.; Wong, D.T. )

    1990-12-01

    The advent of potent, highly selective 5HT3 receptor antagonists has stimulated considerable interest in 5HT3 receptor mediated physiology and pharmacology. To permit detailed biochemical studies regarding interaction of the indazole class of serotonin (5HT) antagonists with 5HT3 receptors in multiple tissues, we synthesized 1-methyl-N-(8-methyl-8-azabicyclo(3.2.1)oct-3-yl)-1H-indazole- 3-carboxamide (LY278584, compound 9) in high specific activity, tritium-labeled form. This radioligand was selected as a synthetic target because of its potency as a 5HT3-receptor antagonist, its selectivity for this receptor viz a viz other 5HT-receptor subtypes, and the ability to readily incorporate three tritia via the indazole N-CH3 substituent. Alkylation of N-(8-methyl-8-azabicyclo(3.2.1)oct-3-yl)-1H-indazole-3-carboxamide (8) with sodium hydride and tritium-labeled iodomethane, followed by HPLC purification, resulted in (3H)-9 with a radiochemical purity of 99% and a specific activity of 80.5 Ci/mmol. This radioligand bound with high affinity to a single class of saturable recognition sites in membranes isolated from cerebral cortex of rat brain. The Kd was 0.69 nM and the Bmax was 16.9 fmol/mg of protein. The specific binding was excellent, and accounted for 83-93% of total binding at concentrations of 2 nM or less. The potencies of known 5HT3-receptor antagonists as inhibitors of (3H)-9 binding correlated well with their pharmacological receptor affinities as antagonists of 5HT-induced decreases in heart rate and contraction of guinea pig ileum, suggesting the central recognition site for this radioligand may be extremely similar to or identical with peripheral 5HT3 receptors.

  2. Learning with imperfectly labeled patterns

    NASA Technical Reports Server (NTRS)

    Chittineni, C. B.

    1979-01-01

    The problem of learning in pattern recognition using imperfectly labeled patterns is considered. The performance of the Bayes and nearest neighbor classifiers with imperfect labels is discussed using a probabilistic model for the mislabeling of the training patterns. Schemes for training the classifier using both parametric and non parametric techniques are presented. Methods for the correction of imperfect labels were developed. To gain an understanding of the learning process, expressions are derived for success probability as a function of training time for a one dimensional increment error correction classifier with imperfect labels. Feature selection with imperfectly labeled patterns is described.

  3. Review of nutrition labeling formats.

    PubMed

    Geiger, C J; Wyse, B W; Parent, C R; Hansen, R G

    1991-07-01

    This article examines nutrition labeling history as well as the findings of nine research studies of nutrition labeling formats. Nutrition labeling regulations were announced in 1973 and have been periodically amended since then. In response to requests from consumers and health care professionals for revision of the labeling system, the Food and Drug Administration initiated a three-phase plan for reform of nutrition labeling in 1990. President Bush signed the Nutrition Labeling and Education Act in November 1990. Literature analysis revealed that only nine studies with an experimental design have focused on nutrition labeling since 1971. Four were conducted before 1975, which was the year that nutrition labeling was officially implemented, two were conducted in 1980, and three were conducted after 1986. Only two of the nine studies supported the traditional label format mandated by the Code of Federal Regulations, and one study partially supported it. Four of the nine studies that evaluated graphic presentations of nutrition information found that consumer comprehension of nutrition information was improved with a graphic format for nutrition labeling: three studies supported the use of bar graphs and one study supported the use of a pie chart. Full disclosure (ie, complete nutrient and ingredient labeling) was preferred by consumers in two of the three studies that examined this variable. The third study supported three types of information disclosure dependent upon socioeconomic class. In those studies that tested graphics, a bar graph format was significantly preferred and showed better consumer comprehension than the traditional format.

  4. Map labeling and its generalizations

    SciTech Connect

    Doddi, S. |; Marathe, M.V.; Mirzaian, A.; Moret, B.M.E.; Zhu, B. |

    1997-01-01

    Map labeling is of fundamental importance in cartography and geographical information systems and is one of the areas targeted for research by the ACM Computational Geometry Impact Task Force. Previous work on map labeling has focused on the problem of placing maximal uniform, axis-aligned, disjoint rectangles on the plane so that each point feature to be labeled lies at the corner of one rectangle. Here, we consider a number of variants of the map labeling problem. We obtain three general types of results. First, we devise constant-factor polynomial-time-approximation algorithms for labeling point features by rectangular labels, where the feature may lie anywhere on the boundary of its label region and where labeling rectangles may be placed in any orientation. These results generalize to the case of elliptical labels. Secondly, we consider the problem of labeling a map consisting of disjoint rectilinear fine segments. We obtain constant-factor polynomial-time approximation algorithms for the general problem and an optimal algorithm for the special case where all segments are horizontal. Finally, we formulate a bicriteria version of the map-labeling problem and provide bicriteria polynomial- time approximation schemes for a number of such problems.

  5. Labeling and Functionalizing Amphipols for Biological Applications

    PubMed Central

    Bon, Christel Le; Popot, Jean-Luc; Giusti, Fabrice

    2014-01-01

    Amphipols (APols) are short amphipathic polymers developed as an alternative to detergents for handling membrane proteins (MPs) in aqueous solution. MPs are, as a rule, much more stable following trapping with APols than they are in detergent solutions. The best-characterized APol to date, called A8-35, is a mixture of short-chain sodium polyacrylates randomly derivatized with octylamine and isopropylamine. Its solution properties have been studied in detail, and it has been used extensively for biochemical and biophysical studies of MPs. One of the attractive characteristics of APols is that it is relatively easy to label them, isotopically or otherwise, without affecting their physical-chemical properties. Furthermore, several variously modified APols can be mixed, achieving multiple functionalization of MP/APol complexes in the easiest possible manner. Labeled or tagged APols are being used to study the solution properties of APols, their miscibility, their biodistribution upon injection into living organisms, their association with MPs and the composition, structure and dynamics of MP/APol complexes, examining the exchange of surfactants at the surface of MPs, labeling MPs to follow their distribution in fractionation experiments or to immobilize them, increasing the contrast between APols and solvent or MPs in biophysical experiments, improving NMR spectra, etc. Labeling or functionalization of APols can take various courses, each of which has its specific constraints and advantages regarding both synthesis and purification. The present review offers an overview of the various derivatives of A8-35 and its congeners that have been developed in our laboratory and discusses the pros and cons of various synthetic routes. PMID:24696186

  6. Labeling and functionalizing amphipols for biological applications.

    PubMed

    Le Bon, Christel; Popot, Jean-Luc; Giusti, Fabrice

    2014-10-01

    Amphipols (APols) are short amphipathic polymers developed as an alternative to detergents for handling membrane proteins (MPs) in aqueous solution. MPs are, as a rule, much more stable following trapping with APols than they are in detergent solutions. The best-characterized APol to date, called A8-35, is a mixture of short-chain sodium polyacrylates randomly derivatized with octylamine and isopropylamine. Its solution properties have been studied in detail, and it has been used extensively for biochemical and biophysical studies of MPs. One of the attractive characteristics of APols is that it is relatively easy to label them, isotopically or otherwise, without affecting their physical-chemical properties. Furthermore, several variously modified APols can be mixed, achieving multiple functionalization of MP/APol complexes in the easiest possible manner. Labeled or tagged APols are being used to study the solution properties of APols, their miscibility, their biodistribution upon injection into living organisms, their association with MPs and the composition, structure and dynamics of MP/APol complexes, examining the exchange of surfactants at the surface of MPs, labeling MPs to follow their distribution in fractionation experiments or to immobilize them, increasing the contrast between APols and solvent or MPs in biophysical experiments, improving NMR spectra, etc. Labeling or functionalization of APols can take various courses, each of which has its specific constraints and advantages regarding both synthesis and purification. The present review offers an overview of the various derivatives of A8-35 and its congeners that have been developed in our laboratory and discusses the pros and cons of various synthetic routes.

  7. 18F: Labeling Chemistry and Labeled Compounds

    NASA Astrophysics Data System (ADS)

    Ross, T. L.; Wester, H. J.

    Positron emission tomography (PET) is a unique tool for the investigation, localization, and quantification of physiological activities in vivo by tracing the involved or accompanying biochemical processes. Because of its nuclear and chemical properties, fluorine-18, which is commonly produced by a cyclotron using the 18O(p,n)18F or the 20Ne(d,α)18F nuclear process, is a nearly ideal positron emitting radionuclide. Its half-life of 109.7 min permits tracer syntheses and imaging protocols extending over hours and allows distribution of 18F-radiopharmaceuticals to hospitals and facilities lacking a cyclotron. The low maximum positron energy of 635 keV results in low radiation doses, short ranges in tissue, and therefore in excellent imaging resolution. Introduction of 18F-fluorine, either via nucleophilic strategies using [18F]F- or electrophilic routes using molecular [18F]F2, permits the synthesis of a broad spectrum of compounds within a time compatible with the half-life. Although fluorine is only slightly larger than a hydrogen atom, changes in the physiological behavior of bioactive compounds as a result of alteration in metabolic stability, lipophilicity, affinity to the target, or other structures, etc., are often observed even after F-for-H or F-for-OH substitutions. In this chapter, an overview of the scope and limitations of the 18F-chemistry is given. Fluorination strategies, routes, and synthetic aspects are exemplified, as far as possible, by established and selected 18F-radiopharmaceuticals with clinical relevance or with potential for further clinical application.

  8. Synthesis and Biological Evaluation of Novel Carbon-11 Labeled Pyridyl Ethers: Candidate Ligands for In Vivo Imaging of α4β2 Nicotinic Acetylcholine Receptors (α4β2-nAChRs) in the brain with Positron Emission Tomography

    PubMed Central

    Gao, Yongjun; Ravert, Hayden T.; Kuwabara, Hiroto; Xiao, Yingxian; Endres, Christopher J.; Hilton, John; Holt, Daniel P.; Kumar, Anil; Alexander, Mohab; Wong, Dean F.; Dannals, Robert F.; Horti, Andrew G.

    2009-01-01

    The most abundant subtype of cerebral nicotinic acetylcholine receptors (nAChR), α4β2, plays a critical role in various brain functions and pathological states. Imaging agents suitable for visualization and quantification of α4β2 nAChRs by positron emission tomography (PET) would present unique opportunities to define the function and pharmacology of the nAChRs in the living human brain. In this study, we report the synthesis, nAChR binding affinity, and pharmacological properties of several novel 3-pyridyl ether compounds. Most of these derivatives displayed a high affinity to the nAChR and a high subtype selectivity for α4β2-nAChR. Three of these novel nAChR ligands were radiolabeled with the positron-emitting isotope 11C and evaluated in animal studies as potential PET radiotracers for imaging of cerebral nAChRs with improved brain kinetics. PMID:19481945

  9. Supplementing national menu labeling.

    PubMed

    Hodge, James G; White, Lexi C

    2012-12-01

    The US Food and Drug Administration's forthcoming national menu labeling regulations are designed to help curb the national obesity epidemic by requiring calorie counts on restaurants' menus. However, posted calories can be easily ignored or misunderstood by consumers and fail to accurately describe the healthiness of foods. We propose supplemental models that include nutritional information (e.g., fat, salt, sugar) or specific guidance (e.g., "heart-healthy" graphics). The goal is to empower restaurant patrons with better data to make healthier choices, and ultimately to reduce obesity prevalence.

  10. 2'-modified nucleosides for site-specific labeling of oligonucleotides

    NASA Technical Reports Server (NTRS)

    Krider, Elizabeth S.; Miller, Jeremiah E.; Meade, Thomas J.

    2002-01-01

    We report the synthesis of 2'-modified nucleosides designed specifically for incorporating labels into oligonucleotides. Conversion of these nucleosides to phosphoramidite and solid support-bound derivatives proceeds in good yield. Large-scale synthesis of 11-mer oligonucleotides possessing the 2'-modified nucleosides is achieved using these derivatives. Thermal denaturation studies indicate that the presence of 2'-modified nucleosides in 11-mer duplexes has minimal destabilizing effects on the duplex structure when the nucleosides are placed at the duplex termini. The powerful combination of phosphoramidite and support-bound derivatives of 2'-modified nucleosides affords the large-scale preparation of an entirely new class of oligonucleotides. The ability to synthesize oligonucleotides containing label attachment sites at 3', intervening, and 5' locations of a duplex is a significant advance in the development of oligonucleotide conjugates.

  11. 2'-modified nucleosides for site-specific labeling of oligonucleotides

    NASA Technical Reports Server (NTRS)

    Krider, Elizabeth S.; Miller, Jeremiah E.; Meade, Thomas J.

    2002-01-01

    We report the synthesis of 2'-modified nucleosides designed specifically for incorporating labels into oligonucleotides. Conversion of these nucleosides to phosphoramidite and solid support-bound derivatives proceeds in good yield. Large-scale synthesis of 11-mer oligonucleotides possessing the 2'-modified nucleosides is achieved using these derivatives. Thermal denaturation studies indicate that the presence of 2'-modified nucleosides in 11-mer duplexes has minimal destabilizing effects on the duplex structure when the nucleosides are placed at the duplex termini. The powerful combination of phosphoramidite and support-bound derivatives of 2'-modified nucleosides affords the large-scale preparation of an entirely new class of oligonucleotides. The ability to synthesize oligonucleotides containing label attachment sites at 3', intervening, and 5' locations of a duplex is a significant advance in the development of oligonucleotide conjugates.

  12. 49 CFR 583.5 - Label requirements.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... of the fuel economy label required by 15 U.S.C. 2006, or a separate label. A separate label may... case of a label that is included as part of the Monroney price information label or fuel economy label... motor vehicle equipment and that, to the best of the requester's knowledge, the outside supplier is...

  13. Synthesis of carbon-13 labelled carbonaceous deposits and their evaluation for potential use as surrogates to better understand the behaviour of the carbon-14-containing deposit present in irradiated PGA graphite

    NASA Astrophysics Data System (ADS)

    Payne, L.; Walker, S.; Bond, G.; Eccles, H.; Heard, P. J.; Scott, T. B.; Williams, S. J.

    2016-03-01

    The present work has used microwave plasma chemical vapour deposition to generate suitable isotopically labelled carbonaceous deposits on the surface of Pile Grade A graphite for use as surrogates for studying the behaviour of the deposits observed on irradiated graphite extracted from UK Magnox reactors. These deposits have been shown elsewhere to contain an enhanced concentration of 14C compared to the bulk graphite. A combination of Raman spectroscopy, ion beam milling with scanning electron microscopy and secondary ion mass spectrometry were used to determine topography and internal morphology in the formed deposits. Direct comparison was made against deposits found on irradiated graphite samples trepanned from a Magnox reactor core and showed a good similarity in appearance. This work suggests that the microwave plasma chemical vapour deposition technique is of value in producing simulant carbon deposits, being of sufficiently representative morphology for use in non-radioactive surrogate studies of post-disposal behaviour of 14C-containing deposits on some irradiated Magnox reactor graphite.

  14. Synthesis and Characterization of High-Affinity 4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene-Labeled Fluorescent Ligands for Human β-Adrenoceptors

    PubMed Central

    2011-01-01

    The growing practice of exploiting noninvasive fluorescence-based techniques to study G protein-coupled receptor pharmacology at the single cell and single molecule level demands the availability of high-quality fluorescent ligands. To this end, this study evaluated a new series of red-emitting ligands for the human β-adrenoceptor family. Upon the basis of the orthosteric ligands propranolol, alprenolol, and pindolol, the synthesized linker-modified congeners were coupled to the commercially available fluorophore BODIPY 630/650-X. This yielded high-affinity β-adrenoceptor fluorescent ligands for both the propranolol and alprenolol derivatives; however, the pindolol-based products displayed lower affinity. A fluorescent diethylene glycol linked propranolol derivative (18a) had the highest affinity (log KD of −9.53 and −8.46 as an antagonist of functional β2- and β1-mediated responses, respectively). Imaging studies with this compound further confirmed that it can be employed to selectively label the human β2-adrenoceptor in single living cells, with receptor-associated binding prevented by preincubation with the nonfluorescent β2-selective antagonist 3-(isopropylamino)-1-[(7-methyl-4-indanyl)oxy]butan-2-ol (ICI 118551) (J. Cardiovasc. Pharmacol.1983, 5, 430–437.) PMID:21870877

  15. Synthesis and application of (13)C-labeled 2-acetyl-4-((1R,2S,3R)-1,2,3,4-tetrahydroxybutyl)imidazole (THI), an immunosuppressant observed in caramel food colorings.

    PubMed

    Elsinghorst, Paul W; Raters, Marion; Dingel, Anna; Fischer, Jochen; Matissek, Reinhard

    2013-08-07

    2-Acetyl-4-((1R,2S,3R)-1,2,3,4-tetrahydroxybutyl)imidazole (THI) is a minor toxic contaminant observed in caramel food colorings and was shown to exert immunosuppressant activity when fed to rodents. Because of this toxicity, maximum levels of THI in caramel food colorings have been defined by international and European authorities. Several reports of THI analysis using external standardization have been published for liquid foods such as beers and soft drinks. However, no suitable internal standard has yet been described allowing THI analysis in more complex samples. In this paper we describe the preparation of a labeled [(13)C6]THI analogue and its application for the successful validation of the first stable isotope dilution assay (SIDA) of THI in caramel food colorings. A brief survey of THI levels in commercially available caramel class III (E 150c) and IV (E 150d) food colorings is also included, corroborating that THI occurs only in caramel class III food colorings.

  16. Food labels: a critical assessment.

    PubMed

    Temple, Norman J; Fraser, Joy

    2014-03-01

    Foods sold in packages have both front-of-package (FOP) labels and back-of-package (BOP) labels. The aim of this review is to determine the role they play in informing consumers as to the composition of foods in order to help select a healthy diet. Recent literature was evaluated and findings combined with assessments made by the authors of food labels used in the United States and Canada. Research shows that most consumers have difficulty understanding the information provided by both FOP and BOP food labels used in the United States and Canada. Research has evaluated the merits of alternative designs. FOP labels should be based on a clear and simple design. They should present information on key nutrients (total fat, saturated fat, sugar, and sodium or salt) and also energy value. They should have color and words that indicate "high," "medium," and "low" levels. Labels can also state quantity per serving. The traffic light system is the best example of this design. An extra traffic light indicating the overall health value of the food should be added. A clearer BOP label also is needed. Implementation of a new food labeling system will probably be opposed by the food industry. More research is needed into which food label designs are most effective, especially for persuading consumers to select healthier food. Both FOP and BOP food labels used in the United States and Canada need to be redesigned using a traffic light system. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. Posttranscriptional chemical labeling of RNA by using bioorthogonal chemistry.

    PubMed

    George, Jerrin Thomas; Srivatsan, Seergazhi G

    2017-02-17

    Recent developments in RNA labeling technology have provided viable tools to analyze RNA synthesis, processing and function in cell-free and cellular environments. Notably, emerging methodologies based on posttranscriptional chemical labeling by using bioorthogonal chemistry have enabled the visualization and profiling of exogenous and endogenous RNA transcripts. In this review, we first give an overview of different RNA labeling strategies based on chemical as well as genetically encoded systems. Subsequently, we provided a detailed discussion on methodologies that have been developed to introduce various bioorthogonal reactive groups into RNA transcripts, which are compatible for posttranscriptional functionalization. Finally, the utility of these techniques in imaging and studying the dynamics of RNA production, distribution and decay in complex cellular environment is discussed.

  18. Semiconductor Quantum Rods as Single Molecule FluorescentBiological Labels

    SciTech Connect

    Fu, Aihua; Gu, Weiwei; Boussert, Benjamine; Koski, Kristie; Gerion, Daniele; Manna, Liberato; Le Gros, Mark; Larabell, Carolyn; Alivisatos, A. Paul

    2006-05-29

    In recent years, semiconductor quantum dots have beenapplied with great advantage in a wide range of biological imagingapplications. The continuing developments in the synthesis of nanoscalematerials and specifically in the area of colloidal semiconductornanocrystals have created an opportunity to generate a next generation ofbiological labels with complementary or in some cases enhanced propertiescompared to colloidal quantum dots. In this paper, we report thedevelopment of rod shaped semiconductor nanocrystals (quantum rods) asnew fluorescent biological labels. We have engineered biocompatiblequantum rods by surface silanization and have applied them fornon-specific cell tracking as well as specific cellular targeting. Theproperties of quantum rods as demonstrated here are enhanced sensitivityand greater resistance for degradation as compared to quantum dots.Quantum rods have many potential applications as biological labels insituations where their properties offer advantages over quantumdots.

  19. Calorimetric evidence for phase transitions in spin-label lipid bilayers.

    PubMed

    Chen, S C; Sturtevant, J M; Conklin, K; Gaffney, B J

    1982-09-28

    Dispersions of pure, spin-label phosphatidylcholines in aqueous buffer have been investigated with the Privalov high-sensitivity differential scanning calorimeter. The lipids studied are mixed-chain ones in which C-2 of glycerol bears a spin-label derivative of stearic acid and the fatty acid group at C-1 is palmitate. A well-defined phase transition is observed at 30.3-30.7 degrees C for the phosphatidylcholine labeled near the polar end of the stearate chain (label at C-5). A sharp transition (32-34 degrees C) is also observed for the lipid spin-labeled near the terminal methyl of stearate (label at C-16), but the thermodynamic parameters for this lipid depend strongly on the history of the sample. Calorimetric evidence for hysteresis in the phase transition of the C-16-labeled lipid is presented. In contrast to the above spin-label lipids, the lipid labeled at C-12 does not show a sharp transition in the region 5-35 degrees C. In general, therefore, the thermal behavior of the spin-label phosphatidylcholines resembles that of phosphatidylcholines bearing double bonds or branched methyl groups at similar locations on acyl chains. During synthesis of mixed-chain lipids, migration of acyl chains occurs. Methyl esterification procedures which are compatible with the acid-labile spin-label group are described. Gas chromatographic analysis of methyl esters shows that chain migration during synthesis gives 15-20% of the spin-label fatty acid at the glycerol C-1 position.

  20. Optimizing connected component labeling algorithms

    SciTech Connect

    Wu, Kesheng; Otoo, Ekow; Shoshani, Arie

    2005-01-16

    This paper presents two new strategies that can be used to greatly improve the speed of connected component labeling algorithms. To assign a label to a new object, most connected component labeling algorithms use a scanning step that examines some of its neighbors. The first strategy exploits the dependencies among them to reduce the number of neighbors examined. When considering 8-connected components in a 2D image, this can reduce the number of neighbors examined from four to one in many cases. The second strategy uses an array to store the equivalence information among the labels. This replaces the pointer based rooted trees used to store the same equivalence information. It reduces the memory required and also produces consecutive final labels. Using an array instead of the pointer based rooted trees speeds up the connected component labeling algorithms by a factor of 5 {approx} 100 in our tests on random binary images.

  1. Optimizing connected component labeling algorithms

    NASA Astrophysics Data System (ADS)

    Wu, Kesheng; Otoo, Ekow; Shoshani, Arie

    2005-04-01

    This paper presents two new strategies that can be used to greatly improve the speed of connected component labeling algorithms. To assign a label to a new object, most connected component labeling algorithms use a scanning step that examines some of its neighbors. The first strategy exploits the dependencies among them to reduce the number of neighbors examined. When considering 8-connected components in a 2D image, this can reduce the number of neighbors examined from four to one in many cases. The second strategy uses an array to store the equivalence information among the labels. This replaces the pointer based rooted trees used to store the same equivalence information. It reduces the memory required and also produces consecutive final labels. Using an array instead of the pointer based rooted trees speeds up the connected component labeling algorithms by a factor of 5 ~ 100 in our tests on random binary images.

  2. Principles of protein labeling techniques.

    PubMed

    Obermaier, Christian; Griebel, Anja; Westermeier, Reiner

    2015-01-01

    Protein labeling methods prior to separation and analysis have become indispensable approaches for proteomic profiling. Basically, three different types of tags are employed: stable isotopes, mass tags, and fluorophores. While proteins labeled with stable isotopes and mass tags are measured and differentiated by mass spectrometry, fluorescent labels are detected with fluorescence imagers. The major purposes for protein labeling are monitoring of biological processes, reliable quantification of compounds and specific detection of protein modifications and isoforms in multiplexed samples, enhancement of detection sensitivity, and simplification of detection workflows. Proteins can be labeled during cell growth by incorporation of amino acids containing different isotopes, or in biological fluids, cells or tissue samples by attaching specific groups to the ε-amino group of lysine, the N-terminus, or the cysteine residues. The principles and the modifications of the different labeling approaches on the protein level are described; benefits and shortcomings of the methods are discussed.

  3. Synthesis of internal labeled standards of melatonin and its metabolite N1-acetyl-N2-formyl-5-methoxykynuramine for their quantification using an on-line liquid chromatography-electrospray tandem mass spectrometry system.

    PubMed

    Almeida, Eduardo A; Klitzke, Clécio F; Martinez, Glaucia R; Medeiros, Marisa H G; Di Mascio, Paolo

    2004-01-01

    Melatonin (N-acetyl-5-methoxytryptamine) is implicated in physiologic changes related to light-dark cycles and has been recently found to display antioxidant properties. It is known that the reaction of melatonin with certain reactive oxygen and nitrogen species, such as hydrogen peroxide and singlet oxygen, produces N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK). We report herein on the development of a new liquid chromatography/tandem mass spectrometry (LC/ESI/MS-MS) assay to quantitatively determine melatonin and AFMK. The stable isotopic internal standard of melatonin-D3 was synthesized by the reaction of 5-methoxytryptamine with deuterated acetyl chloride (CD3COCl). Labeled AFMK (AFMK-D3) was obtained after photooxidation of melatonin-D3. The predominant ion [M + H]+ in the full scan mass spectra of melatonin, melatonin-D3, AFMK and AFMK-D3 were located, respectively, at m/z = 233, 236, 265 and 268. The collision-induced dissociation of the molecules revealed a predominant fragment at m/z = 174 for melatonin and melatonin-D3 (loss of the N-acetyl group), and at m/z = 178 for AFMK and AFMK-D3 (loss of both the N-acetyl and the N-formyl groups). The m/z transitions from 233 to 174 (melatonin), from 236 to 174 (melatonin-D3), from 265 to 178 (AFMK), and from 268 to 178 (AFMK-D3) were therefore chosen for the multiple reaction monitoring detection experiments, ensuring a high specificity and an accurate quantification of melatonin and AFMK in human plasma.

  4. Synthesis of 2'-deoxy-2'-[18F]fluoro-beta-D-arabinofuranosyl nucleosides, [18F]FAU, [18F]FMAU, [18F]FBAU and [18F]FIAU, as potential PET agents for imaging cellular proliferation. Synthesis of [18F]labelled FAU, FMAU, FBAU, FIAU.

    PubMed

    Mangner, Thomas J; Klecker, Raymond W; Anderson, Lawrence; Shields, Anthony F

    2003-04-01

    An efficient and reliable synthesis of 2'-deoxy-2'-[(18)F]fluoro-beta-D-arabinofuranosyl nucleosides is presented. Overall decay-corrected radiochemical yields of 35-45% of 4 analogs, FAU, FMAU, FBAU and FIAU are routinely obtained in >98% radiochemical purity and with specific activities of greater than 3 Ci/micromol (110 MBq/micromol) in a synthesis time of approximately 3 hours. When approximately 220 mCi (8.15 GBq) of starting [(18)F]fluoride is used, 25 -30 mCi (0.93 -1.11 GBq) of product (enough to image two patients sequentially) is typically obtained.

  5. Label Structured Cell Proliferation Models

    DTIC Science & Technology

    2010-06-16

    variable as a mass-like quantity. The specific model for the dynamics of life and death processes of a population of cells labeled with CFSE is proposed in... variables = + where < 0 is label degradation velocity. Because we really don’t understand completely the degradation process (there appears to be...little agreement as to what variables on which this velocity might depend) and to allow for generality (other labels that might be used may well

  6. Label Ranking Algorithms: A Survey

    NASA Astrophysics Data System (ADS)

    Vembu, Shankar; Gärtner, Thomas

    Label ranking is a complex prediction task where the goal is to map instances to a total order over a finite set of predefined labels. An interesting aspect of this problem is that it subsumes several supervised learning problems, such as multiclass prediction, multilabel classification, and hierarchical classification. Unsurprisingly, there exists a plethora of label ranking algorithms in the literature due, in part, to this versatile nature of the problem. In this paper, we survey these algorithms.

  7. GEO label: The General Framework for Labeling and Certification

    NASA Astrophysics Data System (ADS)

    Bye, B. L.; McCallum, I.; Maso, J.

    2012-04-01

    The Group on Earth Observations (GEO) is coordinating efforts to build a Global Earth Observation System of Systems, or GEOSS. As part of a strategy to increase the involvement of the science and technology community in GEOSS, both as users and developers of GEOSS itself, GEO decided to develop a GEO label concept related to the scientific relevance, quality, acceptance and societal needs for services and data sets of GEOSS. The development of a GEO label is included in the GEO work plan and several projects address the challenges of developing a GEO label concept. Within the different projects developing the GEO label, various perspectives and approaches are being applied. In order to arrive at a generally accepted GEO label concept, a common understanding and basic knowledge of labeling is necessary. Assessment of quality of internationally standardized Earth observation data products implies possible certification. A general understanding of the framework for international standards and certification will also contribute to a more coherent discussion and more efficient development of a GEO label. We will describe the general labeling and certification framework emphasizing the relation to the three elements of the GEO label: quality, user acceptance and relevance. Based on a survey of international labels done by the EGIDA project, we have analyzed the legal framework and organization of labels and certification. We will discuss the frameworks for certification, user ratings, registration and analysis of user requirements. Quality assessment is a particular focus of the analysis and is based on the work done by the GeoViQua project. A GEO label will function both as a data distribution strategy and as a general management system for data. Through a label users can compare different data sets and get access to more information about the relevant data, including quality. A label will provide traceability of data both in the interest of users as well as data

  8. Labeling conventions in isoelectronic sequences

    SciTech Connect

    Maniak, S.T.; Curtis, L.J. )

    1990-08-01

    The isoelectronic exposition of atomic structure properties involves labeling ambiguities when more than one level of the same total angular momentum and parity is present, and an energy ordered labeling of these levels can lead to apparent isoelectronic discontinuities. For example, in the recent oscillator strength calculations for S-like ions by Saloman and Kim (Phys. Rev. A 38, 577 (1988)), abrupt changes in the rates were sometimes observed between one isoelectronic element and the next. We suggest an alternative labeling scheme that removes these discontinuities and produces a smooth isoelectronic variation. This alternative labeling offers advantages for data exposition and for semiempirical interpolation and extrapolation.

  9. Labeled Cocaine Analogs

    DOEpatents

    Goodman, Mark M.; Shi, Bing Zhi; Keil, Robert N.

    1999-03-30

    Novel methods for positron emission tomography or single photon emission spectroscopy using tracer compounds having the structure: ##STR1## where X in .beta. configuration is phenyl, naphthyl; 2,3 or 4-iodophenyl; 2,3 or 4-(trimethylsilyl)phenyl; 3,4,5 or 6-iodonaphthyl; 3,4,5 or 6-(trimethylsilyl)naphthyl; 2,3 or 4-(trialkylstannyl)phenyl; or 3,4,5 or 6-(trialkylstannyl)napthyl Y in .beta. configuration is 2-fluoroethoxy, 3-fluoropropoxy, 4-fluorobutoxy, 2-fluorocyclopropoxy, 2 or 3-fluorocyclobutoxy, R,S 1'-fluoroisopropoxy, R 1'-fluoroisopropoxy, S 1'-fluoroisopropoxy, 1',3'-difluoroisopropoxy, R,S 1'-fluoroisobutoxy, R 1'-fluoroisobutoxy, S 1'-fluoroisobutoxy, R,S 4'-fluoroisobutoxy, R 4'-fluoroisobutoxy, S 4'-fluoroisobutoxy, or 1',1'-di(fluoromethyl)isobutoxy, The compounds bind dopamine transporter protein and can be labeled with .sup.18 F or .sup.123 I for imaging.

  10. Modeling the contribution of individual proteins to mixed skeletal muscle protein synthetic rates over increasing periods of label incorporation.

    PubMed

    Miller, Benjamin F; Wolff, Christopher A; Peelor, Fredrick F; Shipman, Patrick D; Hamilton, Karyn L

    2015-03-15

    Advances in stable isotope approaches, primarily the use of deuterium oxide ((2)H2O), allow for long-term measurements of protein synthesis, as well as the contribution of individual proteins to tissue measured protein synthesis rates. Here, we determined the influence of individual protein synthetic rates, individual protein content, and time of isotopic labeling on the measured synthesis rate of skeletal muscle proteins. To this end, we developed a mathematical model, applied the model to an established data set collected in vivo, and, to experimentally test the impact of different isotopic labeling periods, used (2)H2O to measure protein synthesis in cultured myotubes over periods of 2, 4, and 7 days. We first demonstrated the influence of both relative protein content and individual protein synthesis rates on measured synthesis rates over time. When expanded to include 286 individual proteins, the model closely approximated protein synthetic rates measured in vivo. The model revealed a 29% difference in measured synthesis rates from the slowest period of measurement (20 min) to the longest period of measurement (6 wk). In support of these findings, culturing of C2C12 myotubes with isotopic labeling periods of 2, 4, or 7 days revealed up to a doubling of the measured synthesis rate in the shorter labeling period compared with the longer period of labeling. From our model, we conclude that a 4-wk period of labeling is ideal for considering all proteins in a mixed-tissue fraction, while minimizing the slowing effect of fully turned-over proteins. In addition, we advocate that careful consideration must be paid to the period of isotopic labeling when comparing mixed protein synthetic rates between studies.

  11. Laser labeling, a safe technology to label produce

    USDA-ARS?s Scientific Manuscript database

    Laser labeling of fruits and vegetables is an alternative means to label produce. Low energy CO2 laser beams etch the surface showing the contrasting underlying layer. These etched surfaces can promote water loss and potentially allow for entry of decay organisms. The long-term effects of laser labe...

  12. Laser labeling, a safe technology to label produce

    USDA-ARS?s Scientific Manuscript database

    Labeling of the produce has gained marked attention in recent years. Laser labeling technology involves the etching of required information on the surface using a low energy CO2 laser beam. The etching forms alphanumerical characters by pinhole dot matrix depressions. These openings can lead to wat...

  13. Directly labeled DNA probes using fluorescent nucleotides with different length linkers.

    PubMed Central

    Zhu, Z; Chao, J; Yu, H; Waggoner, A S

    1994-01-01

    Directly labeled fluorescent DNA probes have been made by nick translation and PCR using dUTP attached to the fluorescent label, Cy3, with different length linkers. With preparation of probes by PCR we find that linker length affects the efficiency of incorporation of Cy3-dUTP, the yield of labeled probe, and the signal intensity of labeled probes hybridized to chromosome target sequences. For nick translation and PCR, both the level of incorporation and the hybridization fluorescence signal increased in parallel when the length of the linker arm is increased. Under optimal conditions, PCR yielded more densely labeled probes, however, the yield of PCR labeled probe decreased with greater linear density of labeling. By using a Cy3-modified dUTP with the longest linker under optimal conditions it was possible to label up to 28% of the possible substitution sites on the target DNA with reasonable yield by PCR and 18% by nick translation. A mechanism involving steric interactions between the polymerase, cyanine-labeled sites on template and extending chains and the modified dUTP substrate is proposed to explain the inverse correlation between the labeling efficiency and the yield of DNA probe synthesis by PCR. Images PMID:8078779

  14. The elusive structure of Pd2(dba)3. Examination by isotopic labeling, NMR spectroscopy, and X-ray diffraction analysis: synthesis and characterization of Pd2(dba-Z)3 complexes.

    PubMed

    Kapdi, Anant R; Whitwood, Adrian C; Williamson, David C; Lynam, Jason M; Burns, Michael J; Williams, Thomas J; Reay, Alan J; Holmes, Jordan; Fairlamb, Ian J S

    2013-06-05

    Pd(0)2(dba)3 (dba = E,E-dibenzylidene acetone) is the most widely used Pd(0) source in Pd-mediated transformations. Pd(0)2(dba-Z)3 (Z = dba aryl substituents) complexes exhibit remarkable and differential catalytic performance in an eclectic array of cross-coupling reactions. The precise structure of these types of complexes has been confounding, since early studies in 1970s to the present day. In this study the solution and solid-state structures of Pd(0)2(dba)3 and Pd(0)2(dba-Z)3 have been determined. Isotopic labeling ((2)H and (13)C) has allowed the solution structures of the freely exchanging major and minor isomers of Pd(0)2(dba)3 to be determined at high field (700 MHz). DFT calculations support the experimentally determined major and minor isomeric structures, which show that the major isomer of Pd(0)2(dba)3 possesses bridging dba ligands found exclusively in a s-cis,s-trans conformation. For the minor isomer one of the dba ligands is found exclusively in a s-trans,s-trans conformation. Single crystal X-ray diffraction analysis of Pd(0)2(dba)3·CHCl3 (high-quality data) shows that all three dba ligands are found over two positions. NMR spectroscopic analysis of Pd(0)2(dba-Z)3 reveals that the aryl substituent has a profound effect on the rate of Pd-olefin exchange and the global stability of the complexes in solution. Complexes containing the aryl substituents, 4-CF3, 4-F, 4-t-Bu, 4-hexoxy, 4-OMe, exhibit well-resolved (1)H NMR spectra at 298 K, whereas those containing 3,5-OMe and 3,4,5-OMe exhibit broad spectra. The solid-state structures of three Pd(0)2(dba-Z)3 complexes (4-F, 4-OMe, 3,5-OMe) have been determined by single crystal X-ray diffraction methods, which have been compared with Goodson's X-ray structure of Pd(0)2(dba-4-OH)3.

  15. Synthesis and evaluation of bombesin derivatives on the basis of pan-bombesin peptides labeled with indium-111, lutetium-177, and yttrium-90 for targeting bombesin receptor-expressing tumors.

    PubMed

    Zhang, Hanwen; Chen, Jianhua; Waldherr, Christian; Hinni, Karin; Waser, Beatrice; Reubi, Jean Claude; Maecke, Helmut R

    2004-09-15

    Bombesin receptors are overexpressed on a variety of human tumors like prostate, breast, and lung cancer. The aim of this study was to develop radiolabeled (Indium-111, Lutetium-177, and Yttrium-90) bombesin analogues with affinity to the three bombesin receptor subtypes for targeted radiotherapy. The following structures were synthesized: diethylenetriaminepentaacetic acid-gamma-aminobutyric acid-[D-Tyr6, beta-Ala11, Thi13, Nle14] bombesin (6-14) (BZH1) and 1,4,7,10-tetraazacyclododecane-N,N',N",N"' -tetraacetic acid-gamma-aminobutyric acid-[D-Tyr6, beta-Ala11, Thi13, Nle14] bombesin (6-14) (BZH2). [111In]-BZH1 and in particular [90Y]-BZH2 were shown to have high affinity to all three human bombesin receptor subtypes with binding affinities in the nanomolar range. In human serum metabolic cleavage was found between beta-Ala11 and His12 with an approximate half-life of 2 hours. The metabolic breakdown was inhibited by EDTA and beta-Ala11-His12 (carnosine) indicating that carnosinase is the active enzyme. Both 111In-labeled peptides were shown to internalize into gastrin-releasing peptide-receptor-positive AR4-2J and PC-3 cells with similar high rates, which were independent of the radiometal. The biodistribution studies of [111In]-BZH1 and [111In]-BZH2 ([177Lu]-BZH2) in AR4-2J tumor-bearing rats showed specific and high uptake in gastrin-releasing peptide-receptor-positive organs and in the AR4-2J tumor. A fast clearance from blood and all of the nontarget organs except the kidneys was found. These radiopeptides were composed of the first pan-bombesin radioligands, which show great promise for the early diagnosis of tumors bearing not only gastrin-releasing peptide-receptors but also the other two bombesin receptor subtypes and may be of use in targeted radiotherapy of these tumors.

  16. Nutrition Marketing on Food Labels

    ERIC Educational Resources Information Center

    Colby, Sarah E.; Johnson, LuAnn; Scheett, Angela; Hoverson, Bonita

    2010-01-01

    Objective: This research sought to determine how often nutrition marketing is used on labels of foods that are high in saturated fat, sodium, and/or sugar. Design and Setting: All items packaged with food labels (N = 56,900) in all 6 grocery stores in Grand Forks, ND were surveyed. Main Outcome Measure(s): Marketing strategy, nutrient label…

  17. Nutrition Marketing on Food Labels

    ERIC Educational Resources Information Center

    Colby, Sarah E.; Johnson, LuAnn; Scheett, Angela; Hoverson, Bonita

    2010-01-01

    Objective: This research sought to determine how often nutrition marketing is used on labels of foods that are high in saturated fat, sodium, and/or sugar. Design and Setting: All items packaged with food labels (N = 56,900) in all 6 grocery stores in Grand Forks, ND were surveyed. Main Outcome Measure(s): Marketing strategy, nutrient label…

  18. Health claims on food labels.

    PubMed

    Tollefson, L

    1994-03-01

    Food and drug law requires that the ingredients in most foods be disclosed on their labels, but until recently there was no requirement that nutrition information be provided. The Nutrition Labeling and Education Act of 1990 (NLEA), passed on November 8, 1990, mandated the Food and Drug Administration to establish regulations requiring most foods to have a uniform nutrition label showing the amount of calories, calories from fat, total fat, saturated fatty acids, cholesterol, total carbohydrates, complex carbohydrates, sugars, fiber, protein, and sodium. The Act also establishes the circumstances under which content claims and disease claims may be made about nutrients in food. This paper briefly discusses recent changes in the food label brought about by the NLEA and focuses on health claims on food labels.

  19. Synthesis and Evaluation of Two 18F-Labeled 6-Iodo-2-(4′-N,N-dimethylamino)phenylimidazo[1,2-a]pyridine Derivatives as Prospective Radioligands for β-Amyloid in Alzheimer’s Disease

    PubMed Central

    Cai, Lisheng; Chin, Frederick T.; Pike, Victor W.; Toyama, Hiroshi; Liow, Jeih-San; Zoghbi, Sami S.; Modell, Kendra; Briard, Emmanuelle; Shetty, H. Umesha; Sinclair, Kathryn; Donohue, Sean; Tipre, Dnyanesh; Kung, Mei-Ping; Dagostin, Claudio; Widdowson, David A.; Green, Michael; Gao, Weiyi; Herman, Mary M.; Ichise, Masanori; Innis, Robert B.

    2014-01-01

    This study evaluated 18F-labeled IMPY [6-iodo-2-(4′-N,N-dimethylamino)phenylimidazo[1,2-a]pyridine] derivatives as agents for imaging β-amyloid plaque with positron emission tomography (PET). The precursor for radiolabeling and reference compounds was synthesized in up to five steps from commercially accessible starting materials. One of the two N-methyl groups of IMPY was substituted with either a 3-fluoropropyl (FPM-IMPY) or a 2-fluoroethyl (FEM-IMPY) group. FPM-IMPY and FEM-IMPY were found to have moderate affinity for Aβ- aggregates with Ki = 27 ± 8 and 40 ± 5 nM, respectively. A “one-pot” method for 18F-2-fluoroethylation and 18F-3-fluoropropylation of the precursor was developed. The overall decay-corrected radiochemical yields were 26–51%. In PET experiments with normal mouse, high uptake of activity was obtained in the brain after iv injection of each probe: 6.4% ID/g for [18F]FEM-IMPY at 1.2 min, and 5.7% ID/g for [18F]FPM-IMPY at 0.8 min. These values were similar to those of [123I/125I]IMPY (7.2% ID/g at 2 min). Polar and nonpolar radioactive metabolites were observed in both plasma and brain homogenates after injection of [18F]FEM or [18F]FPM-IMPY. In contrast to the single-exponential washout of [123I/125I]IMPY, the washouts of brain activity for the two fluorinated analogues were biphasic, with an initial rapid phase over 20 min and a subsequent much slower phase. Residual brain activity at 2 h, which may represent polar metabolites trapped in the brain, was 4.5% ID/g for [18F]FEM-IMPY and 2.1% ID/g for [18F]FPM-IMPY. Substantial skull uptake of [18F]fluoride was also clearly observed. With a view to slow the metabolism of [18F]FEM-IMPY, an analogue was prepared with deuteriums substituted for the four ethyl hydrogens. However, D4-[18F]FEM-IMPY showed the same brain uptake and clearance as the protio analogue. Metabolism of the [18F]FEM-IMPY was appreciably slower in rhesus monkey than in mouse. Autoradiography of postmortem brain sections

  20. 21 CFR 201.72 - Potassium labeling.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 4 2012-04-01 2012-04-01 false Potassium labeling. 201.72 Section 201.72 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.72 Potassium labeling. (a) The labeling of over-the-counter (OTC) drug products intended for oral ingestion shall contain the potassium content...

  1. 21 CFR 201.72 - Potassium labeling.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 4 2014-04-01 2014-04-01 false Potassium labeling. 201.72 Section 201.72 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.72 Potassium labeling. (a) The labeling of over-the-counter (OTC) drug products intended for oral ingestion shall contain the potassium content...

  2. 21 CFR 201.72 - Potassium labeling.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 4 2013-04-01 2013-04-01 false Potassium labeling. 201.72 Section 201.72 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.72 Potassium labeling. (a) The labeling of over-the-counter (OTC) drug products intended for oral ingestion shall contain the potassium content...

  3. 16 CFR 1633.12 - Labeling.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... the Standard shall bear a permanent, conspicuous, and legible label(s) containing the following... with black text. The label text shall comply with the following format requirements: (1) All... as needed for varying information. The label must be white with black text. The label shall contain...

  4. 21 CFR 201.64 - Sodium labeling.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... contains sodium bicarbonate, sodium phosphate, or sodium biphosphate as an active ingredient for oral... 21 Food and Drugs 4 2014-04-01 2014-04-01 false Sodium labeling. 201.64 Section 201.64 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.64 Sodium labeling. (a) The labeling of...

  5. 21 CFR 201.64 - Sodium labeling.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... contains sodium bicarbonate, sodium phosphate, or sodium biphosphate as an active ingredient for oral... 21 Food and Drugs 4 2012-04-01 2012-04-01 false Sodium labeling. 201.64 Section 201.64 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.64 Sodium labeling. (a) The labeling of...

  6. 21 CFR 201.64 - Sodium labeling.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... contains sodium bicarbonate, sodium phosphate, or sodium biphosphate as an active ingredient for oral... 21 Food and Drugs 4 2013-04-01 2013-04-01 false Sodium labeling. 201.64 Section 201.64 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.64 Sodium labeling. (a) The labeling of...

  7. 21 CFR 201.72 - Potassium labeling.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 4 2011-04-01 2011-04-01 false Potassium labeling. 201.72 Section 201.72 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.72 Potassium labeling. (a) The labeling of over-the-counter (OTC) drug products intended for oral ingestion shall contain the potassium...

  8. 21 CFR 201.71 - Magnesium labeling.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 4 2014-04-01 2014-04-01 false Magnesium labeling. 201.71 Section 201.71 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.71 Magnesium labeling. (a) The labeling of over-the-counter (OTC) drug products intended for oral ingestion shall contain the magnesium content...

  9. 21 CFR 201.71 - Magnesium labeling.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 4 2012-04-01 2012-04-01 false Magnesium labeling. 201.71 Section 201.71 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.71 Magnesium labeling. (a) The labeling of over-the-counter (OTC) drug products intended for oral ingestion shall contain the magnesium content...

  10. 21 CFR 201.71 - Magnesium labeling.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 4 2013-04-01 2013-04-01 false Magnesium labeling. 201.71 Section 201.71 Food and... LABELING Labeling Requirements for Over-the-Counter Drugs § 201.71 Magnesium labeling. (a) The labeling of over-the-counter (OTC) drug products intended for oral ingestion shall contain the magnesium content...

  11. Fluorine-18 labeled tracers for PET studies in the neurosciences

    SciTech Connect

    Ding, Yu-Shin; Fowler, J.S.

    1995-12-31

    This chapter focuses on fluorine-18, the positron emitter with the longest half-life, the lowest positron energy and probably, the most challenging chemistry. The incorporation of F-18 into organic compounds presents many challenges, including: the need to synthesize and purify the compound within a 2--3 hour time frame; the limited number of labeled precursor molecules; the need to work on a microscale; and the need to produce radiotracers which are chemically and radiochemically pure, sterile and pyrogen-free, and suitable for intravenous injection. The PET method and F-18 labeling of organic molecules are described followed by highlights of the applications of F-18 labeled compounds in the neurosciences and neuropharmacology. It is important to emphasize the essential and pivotal role that organic synthesis has played in the progression of the PET field over the past twenty years from one in which only a handful of institutions possessed the instrumentation and staff to carry out research to the present-day situation where there are more than 200 PET centers worldwide. During this period PET has become an important scientific tool in the neurosciences, cardiology and oncology. It is important to point out that PET is by no means a mature field. The fact that a hundreds of different F-18 labeled compounds have been developed but only a few possess the necessary selectivity and sensitivity in vivo to track a specific biochemical process illustrates this and underscores a major difficulty in radiotracer development, namely the selection of priority structures for synthesis and the complexities of the interactions between chemical compounds and living systems. New developments in rapid organic synthesis are needed in order to investigate new molecular targets and to improve the quantitative nature of PET experiments.

  12. Labeled Cocaine Analogs

    DOEpatents

    Goodman, Mark M.; Shi, Bing Zhi; Keil, Robert N.

    1999-01-26

    Novel compounds having the structure: ##STR1## where X in .beta. configuration is phenyl, naphthyl; 2,3 or 4-iodophenyl; 2,3 or 4-(trimethylsilyl)phenyl; 3,4,5 or 6-iodonaphthyl; 3,4,5 or 6-(trimethylsilyl)naphthyl; 2,3 or 4-(trialkylstannyl)phenyl; or 3,4,5 or 6-(trialkylstannyl)naphthyl Y in .beta. configuration is Y.sub.1 or Y.sub.2, where Y.sub.1 is 2-fluoroethoxy, 3-fluoropropoxy, 4-fluorobutoxy, 2-fluorocyclopropoxy, 2 or 3-fluorocyclobutoxy, R,S 1'-fluoroisopropoxy, R 1'-fluoroisopropoxy, S 1'-fluoroisopropoxy, 1',3'-difluoroisopropoxy, R,S 1'-fluoroisobutoxy, R 1'-fluoroisobutoxy, S 1'-fluoroisobutoxy, R,S 4'-fluoroisobutoxy, R 4'-fluoroisobutoxy, S 4'-fluoroisobutoxy, or 1',1'-di(fluoromethyl)isobutoxy, and Y.sub.2 is 2-methanesulfonyloxy ethoxy, 3-methanesulfonyloxy propoxy, 4-methanesulfonyloxy butoxy, 2-methanesulfonyloxy cyclopropoxy, 2 or 3-methanesulfonyloxy cyclobutoxy, 1'methanesulfonyloxy isopropoxy, 1'-fluoro, 3'-methanesulfonyloxy isopropoxy, 1'-methanesulfonyloxy, 3'-fluoro isopropoxy, 1'-methanesulfonyloxy isobutoxy, or 4'-methanesulfonyloxy isobutoxy bind dopamine transporter protein and can be labeled with .sup.18 F or .sup.123 I for imaging.

  13. Astatine-211-labeled radiotherapeutics: an emerging approach to targeted alpha-particle radiotherapy.

    PubMed

    Zalutsky, M R; Vaidyanathan, G

    2000-09-01

    Targeted radiotherapy or endoradiotherapy is an appealing approach to cancer treatment because of the potential for delivering curative doses of radiation to tumor while sparing normal tissues. Radionuclides that decay by the emission of alpha-particles such as the heavy halogen astatine-211 (211At) offer the exciting prospect of combining cell-specific molecular targets with radiation having a range in tissue of only a few cell diameters. Herein, the radiobiological advantages of alpha-particle targeted radiotherapy will be reviewed, and the rationale for using 211At for this purpose will be described. The chemistry of astatine is similar to that of iodine; however, there are important differences which make the synthesis and evaluation of 211At-labeled compounds more challenging. Perhaps the most successful approach that has been developed involves the astatodemetallation of tin, silicon or mercury precursors. Astatine-211 labeled agents that have been investigated for targeted radiotherapy include [211At]astatide, 211At- labeled particulates, 211At-labeled naphthoquinone derivatives, 211At-labeled methylene blue, 211At-labeled DNA precursors, meta-[211At]astatobenzylguanidine, 211At-labeled biotin conjugates, 211At-labeled bisphosphonates, and 211At-labeled antibodies and antibody fragments. The status of these 211At-labeled compounds will be discussed in terms of their labeling chemistry, cytotoxicity in cell culture, as well as their tissue distribution and therapeutic efficacy in animal models of human cancers. Finally, an update on the status of the first clinical trial with an 211At-labeled targeted therapeutic, 211At-labeled chimeric anti-tenascin antibody 81C6, will be provided.

  14. Aryldiazomethanes for universal labeling of nucleic acids and analysis on DNA chips.

    PubMed

    Laayoun, Ali; Kotera, Mitsuharu; Sothier, Isabelle; Trévisiol, Emmanuelle; Bernal-Méndez, Eloy; Bourget, Cécile; Menou, Lionel; Lhomme, Jean; Troesch, Alain

    2003-01-01

    DNA and RNA labeling and detection are key steps in nucleic acid-based technologies, used in medical research and molecular diagnostics. We report here the synthesis, reactivity, and potential of a new type of labeling molecule, m-(N-Biotinoylamino)phenylmethyldiazomethane (m-BioPMDAM), that reacts selectively and efficiently with phosphates in nucleotide monomers, oligonucleotides, DNA, and RNA. This molecule contains a biotin as detectable unit and a diazomethyl function as reactive moiety. We demonstrate that this label fulfills the requirements of stability, solubility, reactivity, and selectivity for hybridization-based analysis and especially for detection on high-density DNA chips.

  15. Applications of Stereospecifically-labeled Fatty Acids in Oxygenase and Desaturase Biochemistry

    PubMed Central

    Brash, Alan R.; Schneider, Claus; Hamberg, Mats

    2012-01-01

    Oxygenation and desaturation reactions are inherently associated with the abstraction of a hydrogen from the fatty acid substrate. Since the first published application in 1965, stereospecific placement of a labeled hydrogen isotope (deuterium or tritium) at the reacting carbons has proven a highly effective strategy for investigating the chemical mechanisms catalyzed by lipoxygenases, hemoprotein fatty acid dioxygenases including cyclooxygenases, cytochromes P450, and also the desaturases and isomerases. This review presents a synopsis of all published studies through 2010 on the synthesis and use of stereospecifically labeled fatty acids (70 references), and highlights some of the mechanistic insights gained by application of stereospecifically labeled fatty acids. PMID:21971646

  16. Studying the Conformation of a Receptor Tyrosine Kinase in Solution by Inhibitor‐Based Spin Labeling

    PubMed Central

    Yin, Dongsheng M.; Hannam, Jeffrey S.; Schmitz, Anton; Schiemann, Olav

    2017-01-01

    Abstract The synthesis of a spin label based on PD168393, a covalent inhibitor of a major anticancer drug target, the epidermal growth factor receptor (EGFR), is reported. The label facilitates the analysis of the EGFR structure in solution by pulsed electron paramagnetic resonance (EPR) spectroscopy. For various EGFR constructs, including near‐full‐length EGFR, we determined defined distance distributions between the two spin labels bound to the ATP binding sites of the EGFR dimer. The distances are in excellent agreement with an asymmetric dimer of the EGFR. Based on crystal structures, this dimer had previously been proposed to reflect the active conformation of the receptor but structural data demonstrating its existence in solution have been lacking. More generally, our study provides proof‐of‐concept that inhibitor‐based spin labeling enables the convenient introduction of site‐specific spin labels into kinases for which covalent or tight‐binding small‐molecule modulators are available. PMID:28628261

  17. Indium-111 labeled gold nanoparticles for in-vivo molecular targeting.

    PubMed

    Ng, Quinn K T; Olariu, Cristina I; Yaffee, Marcus; Taelman, Vincent F; Marincek, Nicolas; Krause, Thomas; Meier, Lorenz; Walter, Martin A

    2014-08-01

    The present report describes the synthesis and biological evaluation of a molecular imaging platform based on gold nanoparticles directly labeled with indium-111. The direct labeling approach facilitated radiolabeling with high activities while maintaining excellent stability within the biological environment. The resulting imaging platform exhibited low interference of the radiolabel with targeting molecules, which is highly desirable for in-vivo probe tracking and molecular targeted tumor imaging. The indium-111 labeled gold nanoparticles were synthesized using a simple procedure that allowed stable labeling of the nanoparticle core with various indium-111 activities. Subsequent surface modification of the particle cores with RGD-based ligands at various densities allowed for molecular targeting of the αvß3 integrin in-vitro and for molecular targeted imaging in human melanoma and glioblastoma models in-vivo. The results demonstrate the vast potential of direct labeling with radioisotopes for tracking gold nanoparticles within biological systems. Copyright © 2014 Elsevier Ltd. All rights reserved.

  18. Algorithms for Labeling Focus Regions.

    PubMed

    Fink, M; Haunert, Jan-Henrik; Schulz, A; Spoerhase, J; Wolff, A

    2012-12-01

    In this paper, we investigate the problem of labeling point sites in focus regions of maps or diagrams. This problem occurs, for example, when the user of a mapping service wants to see the names of restaurants or other POIs in a crowded downtown area but keep the overview over a larger area. Our approach is to place the labels at the boundary of the focus region and connect each site with its label by a linear connection, which is called a leader. In this way, we move labels from the focus region to the less valuable context region surrounding it. In order to make the leader layout well readable, we present algorithms that rule out crossings between leaders and optimize other characteristics such as total leader length and distance between labels. This yields a new variant of the boundary labeling problem, which has been studied in the literature. Other than in traditional boundary labeling, where leaders are usually schematized polylines, we focus on leaders that are either straight-line segments or Bezier curves. Further, we present algorithms that, given the sites, find a position of the focus region that optimizes the above characteristics. We also consider a variant of the problem where we have more sites than space for labels. In this situation, we assume that the sites are prioritized by the user. Alternatively, we take a new facility-location perspective which yields a clustering of the sites. We label one representative of each cluster. If the user wishes, we apply our approach to the sites within a cluster, giving details on demand.

  19. Label Review Training: Module 1: Label Basics, Page 5

    EPA Pesticide Factsheets

    Pesticide labels translate results of our extensive evaluations of pesticide products into conditions, directions and precautions that define parameters for use of a pesticide with the goal of ensuring protection of human health and the environment.

  20. 76 FR 75809 - Prior Label Approval System: Generic Label Approval

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-12-05

    ... Protection Reference Center was launched as a Web page in February 1999. The Web page includes a PowerPoint presentation titled ``Labeling 101,'' which is used by the Agency as a teaching tool at workshops on meat and...

  1. Label Review Training: Module 1: Label Basics, Page 2

    EPA Pesticide Factsheets

    Pesticide labels translate results of our extensive evaluations of pesticide products into conditions, directions and precautions that define parameters for use of a pesticide with the goal of ensuring protection of human health and the environment.

  2. Label Review Training: Module 1: Label Basics, Page 9

    EPA Pesticide Factsheets

    Pesticide labels translate results of our extensive evaluations of pesticide products into conditions, directions and precautions that define parameters for use of a pesticide with the goal of ensuring protection of human health and the environment.

  3. Label Review Training: Module 1: Label Basics, Page 8

    EPA Pesticide Factsheets

    Pesticide labels translate results of our extensive evaluations of pesticide products into conditions, directions and precautions that define parameters for use of a pesticide with the goal of ensuring protection of human he

  4. Label Review Training: Module 1: Label Basics, Page 6

    EPA Pesticide Factsheets

    Page 6, Pesticide labels translate results of our extensive evaluations of pesticide products into conditions, directions and precautions that define parameters for use of a pesticide with the goal of ensuring protection of human health and the environment

  5. Label Review Training: Module 1: Label Basics, Page 4

    EPA Pesticide Factsheets

    Pesticide labels translate results of our extensive evaluations of pesticide products into conditions, directions and precautions that define parameters for use of a pesticide with the goal of ensuring protection of human health and the environment.

  6. Label Review Training: Module 1: Label Basics, Page 3

    EPA Pesticide Factsheets

    Pesticide labels translate results of our extensive evaluations of pesticide products into conditions, directions and precautions that define parameters for use of a pesticide with the goal of ensuring protection of human health and the environment.

  7. 21 CFR 331.80 - Professional labeling.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... HUMAN USE ANTACID PRODUCTS FOR OVER-THE-COUNTER (OTC) HUMAN USE Labeling § 331.80 Professional labeling..., muscle weakness, and osteomalacia. (b) Professional labeling for an antacid-antiflatulent combination...

  8. 21 CFR 331.80 - Professional labeling.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... HUMAN USE ANTACID PRODUCTS FOR OVER-THE-COUNTER (OTC) HUMAN USE Labeling § 331.80 Professional labeling..., muscle weakness, and osteomalacia. (b) Professional labeling for an antacid-antiflatulent combination...

  9. 21 CFR 331.80 - Professional labeling.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... HUMAN USE ANTACID PRODUCTS FOR OVER-THE-COUNTER (OTC) HUMAN USE Labeling § 331.80 Professional labeling..., muscle weakness, and osteomalacia. (b) Professional labeling for an antacid-antiflatulent combination...

  10. Mobile Application for Pesticide Label Matching

    EPA Pesticide Factsheets

    The label matching application will give inspectors the ability to instantly compare pesticide product labels against state and federal label databases via their cell phone, tablet or other mobile device.

  11. 40 CFR 94.212 - Labeling.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... shall be of a color that contrasts with the background of the label: (1) The label heading: Marine...) to be designated as Blue Sky Series engines must contain the statement on the label: “Blue Sky...

  12. 40 CFR 94.212 - Labeling.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... shall be of a color that contrasts with the background of the label: (1) The label heading: Marine...) to be designated as Blue Sky Series engines must contain the statement on the label: “Blue Sky...

  13. 40 CFR 94.212 - Labeling.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... shall be of a color that contrasts with the background of the label: (1) The label heading: Marine...) to be designated as Blue Sky Series engines must contain the statement on the label: “Blue Sky...

  14. 49 CFR 172.442 - CORROSIVE label.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... ADMINISTRATION, DEPARTMENT OF TRANSPORTATION HAZARDOUS MATERIALS REGULATIONS HAZARDOUS MATERIALS TABLE, SPECIAL... SECURITY PLANS Labeling § 172.442 CORROSIVE label. (a) Except for size and color, the CORROSIVE label...

  15. 49 CFR 172.442 - CORROSIVE label.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... ADMINISTRATION, DEPARTMENT OF TRANSPORTATION HAZARDOUS MATERIALS REGULATIONS HAZARDOUS MATERIALS TABLE, SPECIAL... SECURITY PLANS Labeling § 172.442 CORROSIVE label. (a) Except for size and color, the CORROSIVE label...

  16. 49 CFR 172.442 - CORROSIVE label.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... ADMINISTRATION, DEPARTMENT OF TRANSPORTATION HAZARDOUS MATERIALS REGULATIONS HAZARDOUS MATERIALS TABLE, SPECIAL... SECURITY PLANS Labeling § 172.442 CORROSIVE label. (a) Except for size and color, the CORROSIVE label...

  17. Soil Fumigant Labels - Dimethyl Disulfide (DMDS)

    EPA Pesticide Factsheets

    Search by EPA registration number, product name, or company and follow the link to the Pesticide Product Labeling System (PPLS) for label details. Updated labels include new safety requirements for buffer zones and related measures.

  18. Chronological protein synthesis in regenerating rat liver.

    PubMed

    He, Jinjun; Hao, Shuai; Zhang, Hao; Guo, Fuzheng; Huang, Lingyun; Xiao, Xueyuan; He, Dacheng

    2015-07-01

    Liver regeneration has been studied for decades; however, its regulation remains unclear. In this study, we report a dynamic tracing of protein synthesis in rat regenerating liver with a new proteomic technique, (35) S in vivo labeling analysis for dynamic proteomics (SiLAD). Conventional proteomic techniques typically measure protein alteration in accumulated amounts. The SiLAD technique specifically detects protein synthesis velocity instead of accumulated amounts of protein through (35) S pulse labeling of newly synthesized proteins, providing a direct way for analyzing protein synthesis variations. Consequently, protein synthesis within short as 30 min was visualized and protein regulations in the first 8 h of regenerating liver were dynamically traced. Further, the 3.5-5 h post partial hepatectomy (PHx) was shown to be an important regulatory turning point by acute regulation of many proteins in the initiation of liver regeneration. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. 21 CFR 1302.04 - Location and size of symbol on label and labeling.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 9 2011-04-01 2011-04-01 false Location and size of symbol on label and labeling... AND PACKAGING REQUIREMENTS FOR CONTROLLED SUBSTANCES § 1302.04 Location and size of symbol on label and labeling. The symbol shall be prominently located on the label or the labeling of the commercial...

  20. 78 FR 24211 - Draft Guidance for Industry on Safety Considerations for Container Labels and Carton Labeling...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-04-24

    ... Container Labels and Carton Labeling Design To Minimize Medication Errors; Availability AGENCY: Food and... Labels and Carton Labeling Design to Minimize Medication Errors.'' The draft guidance focuses on safety aspects of the container label and carton labeling design for prescription drug and biological products...