Synthesis Of Dipeptides Research Articles

SYNTHESIS OF PHOSPHONO DIPEPTIDES, INHIBITORS OF CATHEPSIN C

Phosphono dipeptides containing glycine, glycylglycine or L-alanine at N-termini and racemic phosphonic acid analogues of aromatic amino acids, as well as racemic alicyclic aminophosphonates, exhibit moderate inhibitory activity towards cathepsin C. This activity is probably due to the binding of the phosphonate moiety by a positively charged part of the enzyme which is complementary to the carboxylate part of the synthetic dipeptide products of the enzymatic reaction.

Physical Characterization Of Porous Materials And Correlation With The Activity Of Immobilized Enzyme In Organic Medium

A series of commonly used porous supports was characterized by determination of particle size distribution, porosity, surface area (total and distributions with pore diameters) and skeletal density. The performance of immobilized α-chymotrypsin catalyzed dipeptide synthesis in an acetonitrile medium was correlated with these physical properties. At high enzyme loading, when internal mass transfer limitations are expected to occur, the activity can be correlated with the support characteristic parameter. This is a combination of physical properties such as particle size, porosity, and volumetric porosity, which influence the substrate diffusion rate. At low enzyme loading the important parameter is the accessible surface area, which will determine how the enzyme is distributed in the pores of the support. When assessing the effect of the support material on enzymatic activity, the geometric considerations studied here should always be contemplated before making any assumptions about direct effects of support material on enzymatic catalytic properties.

The Gene Encoding the Elongation Factor P Protein Is Essential for Viability and Is Required for Protein Synthesis

Elongation factor P (EFP) is a protein that stimulates the peptidyltransferase activity of fully assembled 70 S prokaryotic ribosomes and enhances the synthesis of certain dipeptides initiated by N-formylmethionine. This reaction appears conserved throughout species and is promoted in eukaryotic cells by a homologous protein, eIF5A. Here we ask whether the Escherichia coli gene encoding EFP is essential for cell viability. A kanamycin resistance (KanR) gene was inserted near the N-terminal end of the efp gene and was cloned into a plasmid, pMAK705, that has a temperature-sensitive origin of replication. After transformation into a recA+ E. coli strain, temperature-sensitive mutants were isolated, and their chromosomal DNA was sequenced. Mutants containing the efp-KanR gene in the chromosome grew at 33 degrees C only in the presence of the wild-type copy of the efp gene in the pMAK705 plasmid and were unable to grow at 44 degrees C. Incorporation of various isotopes in vivo suggests that translation is impaired in the efp mutant at 44 degrees C. At 44 degrees C, mutant cells are severely defective in peptide-bond formation. We conclude that the efp gene is essential for cell viability and is required for protein synthesis.

Biotransformation in organic media by enzymes and whole cells

Biotransformation of poorly water soluble compounds in organic media by immobilized enzyme and whole cells is illustrated in this paper taking the following examples from the author's laboratory: (1) controlled hydrolysis of triglycerides and synthesis reactions by a recombinant lipolytic enzyme (cutinase); (2) enzymatic synthesis of dipeptides; (3) continuous production of isovaleraldehyde by Gluconobacter oxydans in isooctane; and (4) sitosterol side chain cleavage by Mycobacterium sp. The role of water and organic solvent are evaluated, namely the increase in the volumetric productivity of the reaction system and the shift of the reaction equilibrium in favour of product synthesis. High product yields have been obtained due to the reduction of substrate/product inhibition. Biocatalyst stability in the presence of the organic phase was also performed.

Temperature effects on S 1- and S 1′-enantioselectivity of α-chymotrypsin

The temperature dependence of E (enantiomeric ratio or enantioselectivity, a quantitative measure for enzyme stereospecificity) has been studied for the α-chymotrypsin catalysed hydrolysis of the enantiomeric N-Boc- l/ d-TyrOMe, l/ d-TyrOMe, Ac- l/ d-PhgOMe, l/ d-PhgOMe and for the kinetically controlled synthesis of the diastereomeric dipeptides N-Ac- l-Tyr- l/ d-ArgNH 2 and N-Ac- l-Tyr- l/ d-ValNH 2. The results show that the S 1- and S 1′-enantioselectivity can be modulated by the temperature (3–15 fold for the studied substrates in the range 5–45°C). For l/ d-PhgOMe a reversal in stereospecificity was found in this temperature interval. For the studied substrates both an increase or decrease of the enantiomeric ratio with increasing temperature was observed. For these processes the following relation for the temperature dependence of E has been derived ln E= ln(k l /k d )=−( ΔΔH #− ΔΔH b )/RT+( ΔΔS #− ΔΔS b )/R where k l and k d are apparent second order rate constants for the reactions with the l- and d-enantiomers, respectively. ΔΔ denotes the differences between the thermodynamic parameters for transformation of the enantiomeric substrates. The subscript b applies for the binding of the substrate or the nucleophile and the superscript # for the formation of the transition state of the enzyme acylation or deacylation. For the studied processes either the enthalpy (ΔΔ H #−ΔΔ H b) or the entropy (ΔΔ S #−ΔΔ S b) term was found to control the discrimination. Thus, the enantioselectivity decreases or increases with temperature, respectively. The influence of ground-state interactions and transition-state stabilisation on enzyme enantioselectivity has been discussed.

Protease-catalyzed synthesis of new hydrophobic dipeptides containing non-proteinogenic amino acids

Several dipeptides containing nonproteinogenic amino acids (especially l-tertiary leucine or l-neopentyglycine) were synthesized enzymatically. Thermolysin was used in solid-to-solid conversions in an equilibrium-controlled reaction while α-chymotrypsin was applied in kinetically controlled synthesis. The dipeptide Z-Npg-Npg-NH 2 could be obtained using a protease mixture from Streptomyces. The active enzyme has not yet been purified.

Unambiguous Demonstration of Compression Promoted Dipeptide Synthesis in a Monolayer Film

Unambiguous Demonstration of Compression Promoted Dipeptide Synthesis in a Monolayer Film

Synthesis of dipeptides by suspension-to-suspension conversion via thermolysin catalysis: from analytical to preparative scale.

When using proteases in direct reversal of their normal hydrolytic function, the equilibrium position is very important in limiting the attainable yield in equilibrium-controlled enzymic peptide synthesis. Analysis of the equilibrium position reveals a favourable shift towards the peptide product if starting materials are largely undissolved in the reaction medium and the product precipitates. This approach enabled us to obtain high peptide yields in thermolysin-catalysed reactions in high-density aqueous media with an equimolar supply of substrates. The easy scale-up (up to mol-scale) of this approach is demonstrated by two examples. Z-His-Phe-NH2 and Z-Asp-Phe-OMe, precursors for cyclo-[-His-Phe-] and the low-calorie sweetener Aspartame, respectively, were synthesized in preparative yields of 84-88%.

Stereoselective synthesis of dipeptides—I

The alkylation of 7 occurs in moderate to good trans stereoselection (d.e.40–98%), while a poorer d.e. (10–90%) is observed for 8. On the contrary, in both substrates the alkylation with CH 3I produces a greater amount of the cis isomer (d.e48%). Cleavage of the lactims 9( a,e) gives enantiomerically pure dipeptides 14( a,e). The absolute configuration of the introduced stereogenic centres has been assigned on the basis of the 1H-NMR data in connection with the conformational analysis.

Dipeptide synthesis using acetylated trypsin derivative

A modified trypsin (AA-trypsin, acetylated with acetic acid N-hydroxysuccinimide ester) gave increased yields of Bzl-Arg-Leu-NH2 dipeptide (90% versus 59% for native trypsin) when used in 95% acetonitrile. AA-Trypsin had decreased Km and increased kcat values for amide and ester substrates. kcat/Km also increased for each substrate upon modification. AA-Trypsin showed enhanced esterase activity in hydrophilic solvents compared with native enzyme.

A new approach to peptide synthesis

A new approach to the synthesis of dipeptides is described based on the formation of the NHCHR1CONH–CHR2CO bond by carbenoid N–H insertion, rather than the formation of the peptide bond itself.

Corrigendum Dipeptide synthesis using acetylated trypsin derivative

Corrigendum Dipeptide synthesis using acetylated trypsin derivative

Effects of Acetonitrile-Water Mixtures on α-Chymotrypsin Catalyzed Dipeptide Synthesis

α-Chymotrpysin (EC 3.4 21.1) was immobilized by deposition on celite and subsequent cross-linking with glutaraldehyde. The effects of different mixtures of aqueous buffer and acetonitrile on the immobilized preparation were evaluated using a dipeptide synthesis as model reaction. The initial reaction rate at 6-95% of water increased with increasing water content. The maximum yield of peptide had two maxima; the first one at 6% of water (92%) and the second one at 80% of water (39%). The presence of two maxima was due to severe enzyme inactivation at intermediate water contents (50-60%). The immobilisation procedure slowed the inactivation of α-chymotrypsin. Cross-linked enzyme was inactivated to a lesser extent than both free enzyme and enzyme that had been deposited on celite. The increased resistance to inactivation was, however, not sufficient to make peptide synthesis attractive at intermediate water contents (50-60%). In order to obtain good peptide yields, low water contents (below 10%) should be used.

Kinetic behavior of activation of thermolysin by normal alcohols

This paper reports an insight into the kinetic mechanism of activation and then inhibition in a thermolysin - catalyzed peptide synthesis of dipeptide N -(benzyloxycarbonyl)-L-phenylalanyl-L-phenylalanine methyl ester in aqueous - organic one phase system containing n-alcohols as activator. The jump in catalytic efficiency - as an effect of alcoholic solvents and the kinetic mode of activation are discussed.

Genetics of glycopeptide resistance in enterococci.

Glycopeptide resistance in enterococci is phenotypically and genotypically heterogeneous. The genes responsible for inducible resistance to high levels of vancomycin and teicoplanin (VanA phenotype) are carried by the 10,851-bp Tn1546 transposon. Transposition of Tn1546 into self-transferable plasmids and subsequent transfer by conjugation appears to be responsible for the dissemination of this type of resistance. Nine polypeptides are encoded by Tn1546 that belong to five functional groups: transposition functions (ORF1 and ORF2), regulation of resistance gene expression (VanR and VanS), synthesis of depsipeptide D-Ala-D-lactate (VanH and VanA), hydrolysis of D-Ala-D-Ala-containing peptidoglycan precursors (VanX and VanY), and low-level teicoplanin resistance (VanZ). VanB-type resistance (various levels of resistance to vancomycin and susceptibility to teicoplanin) is also due to production of D-Ala-D-Lac. The VanB ligase of VanB-type strains is structurally and functionally similar to VanA. The vanB gene was found on composite transposon Tn1547, which, in turn, was part of larger conjugative chromosomally located elements (90 to 250 kb). In contrast to acquired VanA- and VanB-type resistance, VanC-type resistance (low level of resistance to vancomycin and susceptibility to teicoplanin) is an intrinsic property of motile enterococci. Resistance in these species is due to synthesis of dipeptide D-Ala-D-Ser by VanC ligases leading to production of cell wall precursors with reduced vancomycin affinity.

Enzymatic synthesis of peptides containing unnatural amino acids

Several proteases were studied as potential catalysts for the enzymatic synthesis of oligopeptides containing the unnatural amino acid allylglycine, the overall objective being the synthesis of a reactive tetrapeptide that could be chemically polymerized into a potentially biocompatible or biodegradable material. Commercially available enzymes were screened for esterase activity toward the methyl ester of the amino acid allylglycine ( dl-AgOMe) to identify potential catalysts for dipeptide synthesis. Proteases from Aspergillus oryzae and Aspergillus sojae, pronase E and protease Nagarse synthesized the protected dipeptide Cbz-allylglycine-phenylalaninamide(Cbz-L-Ag-L-PheNH 2) from Cbz- dl-AgOMe and L-PheNH 2. However, the same enzymes were not able to catalyze the synthesis of Cbz-phenylalanine-allylglycine ethyl ester (Cbz-L-Phe-L-AgOEt). Thus, although these enzymes could use allylglycine as the acyl donor they could not employ it as the acyl acceptor in peptide synthesis. In contrast, chymotrypsin was able to use allylglycine ethyl ester (DL-AgOEt) as the acyl acceptor in the synthesis of Cbz-L-Phe-L-AgOEt, but was not able to synthesize Cb-L-Ag-L-PheNH 2. The two dipeptides, Cbz-allylglycine-phenylalanine and phenylalanine-allylglycine ethyl ester, served as substrates for the thermolysin-catalyzed synthesis of the tetrapeptide Cbz-L-Ag-L-Phe-L-Phe-L-AgOEt.

ChemInform Abstract: A New Approach to the Synthesis of Dipeptides with Unnatural Amino Acids Using Organozinc Chemistry.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

δ- l-(α-Aminoadipoyl)- l-cysteinyl- d-valine synthetase: isolation of l-cysteinyl- d-valine, a ‘shunt’ product, and implications for the order of peptide bond formation

l-Cysteinyl- d-valine was isolated from incubations of l-glutamate, l-cysteine and l-valine with δ- l-(α-aminoadipoyl)- l-cysteinyl- d-valine synthetase and identified by 1H NMR and electrospray ionization MS. This is entirely consistent with our prior proposal (Shiau, C.-Y., Baldwin, J.E., Byford, M.F., Sobey, W.J. and Schofield, C.J. (1995) FEBS Lett. 358, 97–100) that the α-peptide bond between cysteine and valine is formed before the δ-peptide bond between α-aminoadipate and cysteine. The inclusion of l-glutamate, an analogue of l-α-aminoadipate, did not result in a detectable amount of tripeptide product, but did increase apparent yields of l-cysteinyl- d-valine. Conceivably, formation of the l-glutamyladenylate stimulates synthesis of the cysteinyl-valine dipeptide indirectly via a conformational change in the enzyme.

ChemInform Abstract: Synthesis of Dipeptides Containing α‐Substituted Amino Acids; Their Use as Chiral Ligands in Lewis Acid Catalyzed Reactions.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Synthesis of chromophoric dipeptides as substrates for papain

Chromophoric dipeptides (2) and (3) are found to be better substrates for papain in comparison to simpler substrates 1a or 1b. A synthetic strategy for obtaining α-NH(COOEt)-5-MTA-Et ( 1b), Ala-5-MTA-Et ( 2), Phe-5-MTA-Et ( 3) has been described.