- The role of methionine-192 of the chymotrypsin active site in the binding and catalysis of mono(amino acid) and peptide substrates.
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We find that specific oxidation for the Met-192 residue in delta-chymotrypsin to methionine sulfoxide results in a twofold increase in Km(app) and unchanged kcat in the hydrolysis of N-acetyl mono(amino acid) amide substrates. However, the catalyzed hydrolyses of N-acetyl dipeptide amide substrates by (methionine sulfoxide)-192-delta-chymotrypsin (MS-delta-Cht) shows a four- to fivefold decrease in kcat and unchanged Km(app) with respect to delta-chymotrypsin. Hydrolysis of alpha-casein by MS-delta-Cht shows a similar 4.2-fold decrease in kcat. These results imply that the Met-192 acts differently with substrates that bind only in the primary, S1, binding site (i.e., AcPheNH2) from those that bind to more extended regions of the enzyme active site. In the binding of c+AcPheNH2 and AcTrpNH2, the results support a mechanism in which the Met-192 acts to slow the rate of sustrate dissociation from the Michaelis complex to free substrate and enzyme. This is in agreement with the x-ray crystallographic structure of dioxane inhibited alpha-chymotrypsin (Steitz, T., et al. (1969), J. Mol. Biol. 46, 337). However, this mechanism is not apparent when peptide and protein substrates bind. The decrease in kcat on Met-192 modification of approximately fivefold in the hydrolysis of polypeptide substrates show a small, but significant, catalytic contribution of the Met-192 toward the lowering of the energy of activation polypeptide substrate hydrolysis by chymotrypsin. This may support the crystallographic model of Fersht et al. (Fersht, A., et al. (1973), Biochemistry 12, 2035) in which it is proposed that the Met-192 participates in the distortion of bound polypeptide substrates toward the reaction transition-state configuration and, thus, plays a role in catalysis. However, if this mechanism occurs, the effect is small, only contributing about 1 kcal/mol to the lowering of the reaction activation energy.
- Greadway Jr.,Schultz
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- Noncanonical amino acids to improve the pH response of pHLIP insertion at tumor acidity
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The pH low insertion peptide (pHLIP) offers the potential to deliver drugs selectively to the cytoplasm of cancer cells based on tumor acidosis. The WT pHLIP inserts into membranes with a pH50 of 6.1, while most solid tumors have extracellular pH (pHe) of 6.5-7.0. To close this gap, a SAR study was carried out to search for pHLIP variants with improved pH response. Replacing Asp25 with α-aminoadipic acid (Aad) adjusts the pH50 to 6.74, matching average tumor acidity, and replacing Asp14 with γ-carboxyglutamic acid (Gla) increases the sharpness of pH response (transition over 0.5 instead of 1 pH unit). These effects are additive: the Asp14Gla/Asp25Aad double variant shows a pH50 of 6.79, with sharper transition than Asp25Aad. Furthermore, the advantage of the double variant over WT pHLIP in terms of cargo delivery was demonstrated in turn-on fluorescence assays and anti-proliferation studies (using paclitaxel as cargo) in A549 lung cancer cells at pH 6.6.
- Onyango, Joab O.,Chung, Michael S.,Eng, Chee-Huat,Klees, Lukas M.,Langenbacher, Rachel,Yao, Lan,An, Ming
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- Development of n new class of rate-accelerating additives for nitroxide- mediated 'living' free radical polymerization
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Acylating agents have been identified as a new class of rate accelerating additives for nitroxide-mediated 'living' free radical polymerization. It is found that addition of 1 weight percent of acetic anhydride results in a significant decrease in the reaction time, 48 to 4 hours. This decrease in reaction time leads to greater control over the polymerization process with low polydispersity materials being obtained up to 150 000 a.m.u. A possible explanation for this effect is acylation of the alkoxyamine nitrogen leading to an increase in the lability of the C-ON bond.
- Malmstroem, Eva,Miller, Robert D.,Hawker, Craig J.
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- Synthesis of N-hydroxysuccinimide esters using polymer bound HOBT
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The preparation of the N-hydroxysuccinimide esters via reactions mediated by polymer bound 1-hydrozybenzotriazole (HOBT) is reported.
- Dendrinos, Kleanthis G.,Kalivretenos, Aristotle G.
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- A New Method for the Preparation of Active Esters Using Di-2-pyridyl Carbonate
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The use of di-2-pyridyl carbonate in the presence of a catalytic amount of 4-dimethylaminopyridine is found to be very effective in the preparation of various active esters.
- Kim, Sunggak,Ko, Young Kwan
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- Enhanced sample multiplexing for nitrotyrosine-modified proteins using combined precursor isotopic labeling and isobaric tagging
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Current strategies for identification and quantification of 3-nitrotyrosine (3NT) post-translationally modified proteins (PTM) generally rely on biotin/avidin enrichment. Quantitative approaches have been demonstrated which employ isotopic labeling or isobaric tagging in order to quantify differences in the relative abundances of 3NT-modified proteins in two or potentially eight samples, respectively. Here, we present a novel strategy which uses combined precursor isotopic labeling and isobaric tagging (cPILOT) to increase the multiplexing capability of quantifying 3NT-modified proteins to 12 or 16 samples using commercially available tandem mass tags (TMT) or isobaric tags for relative and absolute quantification (iTRAQ), respectively. This strategy employs "light" and "heavy" labeled acetyl groups to block both N-termini and lysine residues of tryptic peptides. Next, 3NT is reduced to 3-aminotyrosine (3AT) using sodium dithionite followed by derivatization of light and heavy labeled 3AT-peptides with either TMT or iTRAQ multiplex reagents. We demonstrate the proof-of-principle utility of cPILOT with in vitro nitrated bovine serum albumin (BSA) and mouse splenic proteins using TMT 0, TMT6, and iTRAQ8 reagents and discuss limitations of the strategy.
- Robinson, Ren? A. S.,Evans, Adam R.
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- NMR investigations of the conformation of new cyclodextrin-based amphiphilic transporters for hydrophobic drugs: molecular lollipops
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Amphiphilic compounds, obtained by grafting aliphatic acids onto a modified cyclodextrin, have been synthesized and studied by solution NMR.The large chain-length dependence of the NMR spectra in aqueous media is explained by the possible formation of auto-inclusion complexes.This process has been evidenced by extensive NMR experiments and by competition with potential guests.This new class of molecules ("lollipops") provides important information for the optimization of a design for amphiphilic transporters to be included in organized phases such as micelles or liposomes.
- Bellanger, Nathalie,Perly, Bruno
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- Formation of 1-hydroxymethylene-1,1-bisphosphinates through the addition of a silylated phosphonite on various trivalent derivatives
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An easily handled one-pot synthetic procedure was previously developed for the synthesis of bisphosphinates starting from acyl chlorides. Herein, other trivalent derivatives as acid anhydrides and activated esters were tested to form various bisphosphinates. This modulation of the reactivity can be controlled according to the nature of the acid derivative for the use of sensitive and functionalized substrates.
- Dussart-Gautheret, Jade,Deschamp, Julia,Monteil, Maelle,Gager, Olivier,Legigan, Thibaut,Migianu-Griffoni, Evelyne,Lecouvey, Marc
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p. 14559 - 14569
(2020/12/29)
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- Synthesis and In Vitro Neuroprotective Activity of Glycine Analogs of Gk-2 Dimeric Dipeptide Mimetic of Nerve Growth Factor 4th Loop
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A dimeric dipeptide mimetic of nerve growth factor (NGF), bis-(N-monosuccinyl-L-glutamyl-L-lysine) hexamethylenediamide (GK-2), was previously developed at V. V. Zakusov State Institute of Pharmacology, activated specific TrkA receptors, and exhibited neuroprotective activity in vitro (10–5 – 10–9 M) and in vivo (0.1 – 10 mg/kg i.p. and p.o.). GK-2 was designed based on the beta-turn (-Asp94-Glu95-Lys96-Gln97-) of the NGF 4th loop and preserved the central dipeptide fragment (-Glu95-Lys96-). The Asp94 residue was replaced by its monosuccinyl bioisostere. The dimeric structure of NGF was reproduced using a bivalent hexamethylenediamine spacer. The structure—activity (neuroprotective) relationship for GK-2 was studied in the present work using a glycine scan, i.e., successive replacement of the peptide side groups by H. The bis-(N-acetyl-L-glutamyl-L-lysine) (GK-2Ac), bis-(N-monosuccinylglycyl-L-lysine) (GK-2-Gly1), and bis-(N-monosuccinyl-L-glutamylglycine) hexamethylenediamides (GK-2-Gly2) were less active with neuroprotective activity in vitro under oxidative stress for HT22 cells at concentrations 10 – 100 times greater than GK-2. The conclusion was drawn that each side radical of GK-2 was important for manifestation of the full neuroprotective activity of dimeric dipeptide GK-2, a mimetic of the NGF 4th loop. However, removal of any of the side radicals would probably not change the active structure of the beta-turn so that the two remaining side radicals should retain the ability to bind to their TrkA subsites. This could explain the retention of neuroprotective activity in the GK-2 glycine analogs.
- Antipov, P. I.,Antipova, T. A.,Firsova, Yu. N.,Gudasheva, T. A.,Nikolaev, S. V.,Rebeko, A. G.,Sazonova, N. M.,Tarasyuk, A. V.,Zvyagintsev, A. A.
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- DIPEPTIDE MIMETICS OF NGF AND BDNF NEUROTROPHINS
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The invention relates to compounds having either agonist or antagonist activities for the neurotrophins NGF and BDNF and represented by monomeric or dimeric substituted dipeptides that are analogs of the exposed portions of loop 1 or loop 4 regions of these neurotrophins near or at a beta-turn of the respective loop. N-acylated substituents of these dipeptides are biostereoisomers of the amino acid residues preceding these dipeptide sequences in the neurotrophin primary structure. The dimeric structure is produced advantageously by using hexatnethylenediaanine to which dipeptides are attached via their carboxyl groups. The claimed compounds displayed neuroprotective and differentiation-inducing activities in cellular models and enhanced the amount of phosphorylated tyrosine kinase A and the heat shock proteins Hsp32 and Hsp70 in the concentration range of 10 -9 to 10 -5 M. They also displayed neuroprotective, anti-parkinsonian, anti-stroke, anti-ischemic, anti-depressant and anti-amnestic activities in animal models and were active in experimental models of Alzheimer's disease. These in vivo effects of the claimed compounds are displayed in the dose range of 0.01 to 10 mg/kg when administered intraperitoneally.
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Paragraph 0181; 0182
(2019/04/16)
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- Enzymatic synthesis of trideuterated sialosides
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Sialic acids are a family of acidic monosaccharides often found on the termini of cell surface proteins or lipid glycoconjugates of higher animals. Herein we describe the enzymatic synthesis of the two isotopically labeled sialic acid derivatives d3-X-Gal-a-2,3-Neu5Ac and d3-X-Gal-a-2,3-Neu5Gc. Using deuterium oxide as the reaction solvent, deuterium atoms could be successfully introduced during the enzymatic epimerization and aldol addition reactions when the sialosides were generated. NMR and mass spectrometric analyses confirmed that the resulting sialosides were indeed tri-deuterated. These compounds may be of interest as internal standards in liquid chromatography/mass spectrometric assays for biochemical or clinical studies of sialic acids. This was further exemplified by the use of this tri-deuterated sialosides as internal standards for the quantification of sialic acids in meat and egg samples.
- Cai, Zhi-P.,Conway, Louis P.,Huang, Ying Y.,Wang, Wen J.,Laborda, Pedro,Wang, Ting,Lu, Ai M.,Yao, Hong L.,Huang, Kun,Flitsch, Sabine L.,Liu, Li,Voglmeir, Josef
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- Design, synthesis and structure-activity relationship studies of a novel focused library of 2,3,4-substituted oxazolidines with antiproliferative activity against cancer cell lines
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In the present work we describe the synthesis and antiproliferative evaluation of a focused library of 30 novel oxazolidines designed by modification of N-substituent, by ring variation, by alkyl variation or by extension of the structure. It was noted that carbamate and N,O-aminal groups were essential for activity. In general, replacement of the phenyl ring with pyridinyl was not tolerated. However, the introduction of a second phenyl ring with an appropriate spacer at the 3- or 4-position of the first phenyl ring generally enhanced the cytotoxic profile. Among all the prepared compounds, 24 was the most potent compound found in this class, being active on four of five cancer cell lines and it was 5-fold and 10-fold more potent than the lead compounds against HL60 and JURKAT cells, respectively. In addition, it showed relevant activity against MCF-7 and HCT-116 cells, which were resistant to lead. Moreover, 24 showed little antiproliferative activity against VERO, indicating low toxicity to normal cells. Thus, this compound has the potential to be developed as an anticancer agent.
- Andrade, Saulo F.,Oliveira, Bárbara G.,Pereira, Larissa C.,Ramos, Jonas P.,Joaquim, Angélica R.,Steppe, Martin,Souza-Fagundes, Elaine M.,Alves, Ricardo J.
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- 5′-O-[(N-acyl)sulfamoyl]adenosines as antitubercular agents that inhibit MbtA: An adenylation enzyme required for siderophore biosynthesis of the mycobactins
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A study of the structure - activity relationships of 5′-O-[N- (salicyl)sulfamoyl]adenosine (6), a potent inhibitor of the bifunctional enzyme salicyl-AMP ligase (MbtA, encoded by the gene Rv2384) in Mycobacterium tuberculosis, is described, targeting the salicyl moiety. A systematic series of analogues was prepared exploring the importance of substitution at the C-2 position revealing that a hydroxy group is required for optimal activity. Examination of a series of substituted salicyl derivatives indicated that substitution at C-4 was tolerated. Consequently, a series of analogues at this position provided 4-fluoro derivative, which displayed an impressive MIC 99 of 0.098 μM against whole-cell M. tuberculosis under iron-limiting conditions. Examination of other heterocyclic, cycloalkyl, alkyl, and aminoacyl replacements of the salicyl moiety demonstrated that these nonconserative modifications were poorly tolerated, a result consistent with the fairly strict substrate specificities of related non-ribosomal peptide synthetase adenylation enzymes.
- Qiao, Chunhua,Gupte, Amol,Boshoff, Helena I.,Wilson, Daniel J.,Bennett, Eric M.,Somu, Ravindranadh V.,Barry III, Clifton E.,Aldrich, Courtney C.
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p. 6080 - 6094
(2008/09/18)
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- Synthesis and evaluation of peptidomimetics as selective inhibitors and active site probes of nitric oxide synthases
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Nitric oxide synthase (NOS) catalyzes the conversion of L-arginine to L- citrilline and nitric oxide (NO). Selective inhibition of the isoforms of NOS could have great therapeutic potential in the treatment of certain disease states arising from pathologically elevated synthesis of NO. Recently, we reported dipeptide amides containing a basic amine side chain as potent and selective inhibitors of neuronal NOS (Huang, H.; Martasek, P.; Roman, L. J.; Masters, B. S. S.; Silverman, R. B. J. Med. Chem. 1999, 42, 3147). The most potent nNOS inhibitor among these compounds is L-Arg(NO2)-L-Dbu-NH2 (1) (K(i) = 130 nM), which also exhibits the highest selectivity over eNOS (>1500-fold) with excellent selectivity over iNOS (190-fold). Here we describe the design and synthesis of a series of peptidomimetic analogues of this dipeptide as potential selective inhibitors of nNOS. The biochemical evaluation of these compounds also revealed the binding requirements of the dipeptide inhibitors with NOS. Incorporation of protecting groups at the N- terminus of the dipeptide amide 1 (compounds 4 and 5) resulted in dramatic decreases in the inhibitory potency of nNOS. Masking the NH group of the peptide bond (peptoids 6-8 and N-methylated compounds 9-11) also gave much poorer nNOS inhibitors than 1. Both of the results demonstrate the importance of the α-amine of the dipeptide and the NH moiety of the peptide bond for binding at the active site. Modifications at the C-terminus of the peptide included converting the amide to the methyl ester (12), tert-butyl ester (13), and carboxylic acid (14) and also descarboxamide analogues (15-17), which revealed less restricted binding requirements for the C-terminus of the dipeptide. Further optimization should be possible when we learn more about the binding requirements at the active sites of NOSs.
- Huang, Hui,Martásek, Pavel,Roman, Linda J.,Silverman, Richard B.
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p. 2938 - 2945
(2007/10/03)
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- Process for the preparation of carboxylic acid succinimidyl esters
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A process for the preparation of carboxylic acid succinimidyl esters by reaction of N-hydroxysuccinimide with a carboxylic acid and a halophosphoric acid ester of the formula STR1 is desired, in which R1 and R2 are identical or different and are a C2 - to C6 -alkyl radical or a phenyl radical, or R1 and R2 The process is carried out in the presence of a base in a diluent at a temperature of 0° C. up to 100° C. with isolation of the corresponding carboxylic acid succinimidyl ester.
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- One-pot formation of succinimidyl esters by the system chlorophosphate/hydroxysuccinimide/base
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Succinimidyl esters of various carboxylic acids are formed in high yield at ambient to slightly elevated temperature by the system chlorophosphate/hydroxysuccinimide/base.
- Poechlauer, Peter,Hendel, Wolfram
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p. 3489 - 3494
(2007/10/03)
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- REACTIVITY OF N-HYDROXYSUCCINIMIDE ESTERS
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The reactions of N-hydroxysuccinimide esters (OSu-esters) of benzyloxycarbonylamino acids and of simple carboxylic acid with p-anisidine in DMSO were studied kinetically at various temperatures.Activation parameters ΔH++ and ΔS++ for these data were determined.The rate constants for aminolysis of carboxylic acid esters fit the two-parameter Taft equation with reaction constants equal: ρ* = 1.08 and ρs = 1.1.The activation entropies are in the range -121.8 - -180.0 J/deg M.Transition state in these reactions is tetrahedral and zwitter ionic, in agreementwith the mechanism given by Cline and Hanna for aminolysis of succinimidyl benzoates.
- Stefanowicz, P.,Siemion, I. Z.
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p. 111 - 118
(2007/10/02)
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- Gram-Scale Synthesis of Uridine 5'-Diphospho-N-acetylglucosamine: Comparison of Enzymatic and Chemical Routes
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Practical chemoenzymatic and chemical routes to uridine 5'-diphospho-N-acetylglucosamine (UDP-GlcNAc) on a gram scale have been developed.The chemoenzymatic synthesis provided convenient access to glucosamine-6-phosphate and N-acetylglucosamine-6-phosphate (GlcNAc-6-P) in > 10 mmol quantities.The condensation between GlcNAc-6-P and UTP was catalyzed by readily available crude enzyme extract from dried cells of the yeast Candida utilis and afforded a 17 percent yield of UDP-GlcNAc from GlcNAc-6-P.The otherwise straightforward chemoenzymatic sequence was hampered by the need to purify the product from the final complex reaction mixture.The chemical synthesis of UDP-GlcNAc proceeded through five steps in an overall yield of 15 percent from pentaacetylglucosamine with the selective formation of tetraacetylglucosamine-α-1-phosphate as the key reaction.
- Heidlas, Juergen E.,Lees, Watson J.,Pale, Patrick,Whitesides, George M.
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p. 146 - 151
(2007/10/02)
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- Functionalized congeners of adenosine: Preparation of analogues with high affinity for A1-adenosine receptors
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A series of functionalized congeners of adenosine based on N6-phenyladenosine, a potent A1-adenosine receptor agonist, was synthesized. Derivatives of the various congeners should be useful as receptor and histochemical probes and for the preparation of radioligands and affinity columns or as targeted drugs. N6-[4-(Carboxymethyl)phenyl]adenosine served as the starting point for synthesis of the methyl ester, the methyl amide, the ethyl glycinate, and various substituted anilides. One of the latter, N6-[4-[[[4-(carbomethoxymethyl)anilino]carbonyl]methyl]phenyl]adenosine, served as the starting point for the synthesis of another series of congeners including the methyl amide, the hydrazide, and the aminoethyl amide. The terminal amino function of the last congener was acylated to provide further analogues. The various congeners were potent competitive antagonists of binding of N6-[3H]cyclohexyladenosine to A1-adenosine receptors in rat cerebral cortical membranes. The affinity of the congener for the A1 receptor was highly dependent on the nature of the spacer group and the terminal moiety with K(i) values ranging 1-100 nM. A biotinylated analogue had a K(i) value of 11 nM. A conjugate derived from the Bolton-Hunter reagent had a K(i) value of 4.5 nM. The most potent congener contained a terminal [(aminoethyl)amino]carbonyl function and had a K(i) value of less than 1 nM.
- Jacobson,Kirk,Padgett,Daly
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p. 1341 - 1346
(2007/10/02)
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- "Naked" Hydrogencarbonate Ion as a Bifunctional Catalyst toward Amide Substrates. Nucleophilic Ion Pairs. 9.
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The cleavage of amide substrates(p-nitro-N-methyltrifluoroacetanilide and p-nitro-N-methyldichloroacetanilide) with anionic nucleophiles was studied at 30 deg C in aprotic (acetonitrile and N,N-dimethylformamide) and protic (ethanol) media.Hydrogencarbona
- Shinkai, Seiji,Nakashima, Naotoshi,Kunitake, Toyoki
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p. 840 - 845
(2007/10/02)
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- Use of esters of N-hydroxysuccinimide in the synthesis of N-acylamino acids.
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Several crystalline N-hydroxysuccinimide esters of short- and long-chain fatty acids have been synthesized. These compounds react with free amino acids to form preferentially N-acylamino acids. The reaction of the N-hydroxysuccinimide esters with hydroxylamine and the behavior of the N-acylamino acids on thin-layer chromatography are described.
- Lapidot,Rappoport,Wolman
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p. 142 - 145
(2007/10/08)
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