α-Chymotrypsin-catalyzed synthesis of poly-l-cysteine in a frozen aqueous solution

Poly-l-cysteine (PLCys) is drawing attention as a potential sorbent of thiol (SH)-reactive toxic heavy metal ions in the wastewater and polluted soils. However, preparation of PLCys relies on chemically synthesized polymers, in which SH groups must be protected and deprotected prior to use. On the other hand, α-chymotrypsin polymerized l-cysteine ethyl ester in a frozen aqueous solution, provides PLCys with degree of polymerization from 6 to 11 without blocking of SH groups. Kinetic analyses suggested that the acylation of α-chymotrypsin with the initial substrate was a rate-limiting step in the enzymatic polymerization. The peptide yields reached 85% and 65% of SH groups in PLCys were assumed to be free forms. Although detail information on correlation between the state of SH groups and heavy metal adsorption properties of PLCys should be explored in further studies, the present study for the first time proposed an easy method for synthesis of PLCys requiring neither SH-protection nor -deprotection.     

Protease-catalyzed fragment condensation via substrate mimetic strategy: a useful combination of solid-phase peptide synthesis with enzymatic methods.

The concept of substrate mimetic strategy represents a new powerful method in the field of enzymatic peptide synthesis. This strategy takes advantage of the shift in the site-specific amino acid moiety from the acyl residue to the ester-leaving group of the carboxyl component enabling acylation of the enzyme by nonspecific acyl residues. As a result, peptide bond formation occurs independently of the primary specificity of proteases. Moreover, because of the coupling of nonspecific acyl residues, the newly formed peptide bond is not subject to secondary hydrolysis achieving irreversible peptide synthesis. Here, we report the combination of solid-phase peptide synthesis with substrate mimetic-mediated enzymatic peptide fragment condensations. First, the utility of the oxime resin strategy for the synthesis of peptide fragments in the form of substrate mimetics esterified as 4-guanidinophenyl-, phenyl- and mercaptopropionic acid esters was investigated. The study was completed by using the resulting N(alpha)-protected peptide esters as acyl donors in trypsin-, alpha-chymotrypsin- and V8 protease-catalyzed fragment condensations.     

Peptide antibiotic production through immobilized biocatalyst technology

The use of immobilized biocatalysts for producing known or new antibiotics is presented. An evaluation of the applicability of this concept in the fascinating field of peptide antibiotic bioconversions and fermentations is also given.The use of immobilized enzymes, organelles and cells to synthesize antibiotics as an alternative method to conventional fermentation is discussed. In vitro total enzymatic antibiotic synthesis is illustrated with the ‘multienzyme thiotemplate mechanism’ of Bacillus brevis, the producer of gramicidin S. Total synthesis of peptide antibiotics, based on immobilized living cells, has recently been demonstrated with penicillin, bacitracin, nisin and a few other antibiotics.As an industrial example of the use of enzymes or cells to convert peptide antibiotics into therapeutically useful derivatives, free and immobilized penicillin acylases, producing the penicillin nucleus 6-aminopenicillanic acid (6-APA), are reviewed as well as their potential to synthesize semisynthetic β-lactams (penicillins, cephalosporins).Acylases, acetylesterases and α-amino acid ester hydrolases acting on cephalosporin-compounds and yielding valuable intermediary or end products have also gained wide interest. Stereospecific enzymic side-chain preparations for semisynthetic penicillin and cephalosporin production have recently reached the industrial stage. Bioconversion possibilities with the novel β-lactam compounds are suggested.These examples of simple single-step, as well as complex multi-step, enzyme reactions point to the vast potential of immobilized biocatalyst technology in fermentation science, in organic synthesis and in biotechnological processes in general.     

Catalysis in peptide synthesis

Enzyme catalyzed formation of peptide bonds reached practical significance in partial syntheses such as the conversion of pork insulin to human insulin. Catalysis is important also in coupling with active esters. The earlier applied acid-base catalysis was replaced by the use of bifunctional catalysts, 1-hydroxybenzotriazole being an outstanding example: it brought about major improvements in peptide bond formation. Intramolecular catalysis can be discerned in the rapid acylation by certain active esters, e.g., esters of catechol, 4-nitroguaiacol or 8-hydroxyquinoline. The ready formation of symmetrical anhydrides in the reaction of acylamino acids with carbodiimides is best explained by intramolecular catalysis within the O-acylisourea intermediates. Catalysis plays important roles both in introduction and in removal of blocking groups. Preparation of alkyl esters through base-catalyzed transesterification of active aryl esters and the application of this reaction for the anchoring of peptides to polymeric supports are described. Enzyme catalyzed hydrolysis of alkyl esters and hydrolytic fission of the phenylacetyl group from lysine side chains with aid of penicillin amidohydrolase are characteristic examples of the application of catalysis for the removal of blocking groups. Acidolysis of the benzyl groups including the benzyloxcarbonyl group is catalyzed by thioanisole or by 4-methylthiophenol. The catalytic effect of solvents is demonstrated with the cleavage of triphenylmethyl and biphenylyl-isopropyloxycarbonyl groups by 1-hydroxybenzotriazole in trifluoroethanol. The increasing role of catalysis in peptide synthesis and its future application for the solution of fundamental problems, such as amine activation, are discussed.     

Progress in the preparation of peptide aldehydes via polymer supported IBX oxidation and scavenging by threonyl resin.

Peptide aldehydes are of interest due to their inhibitory properties toward numerous classes of proteolytic enzymes such as caspases or the proteasome. A novel access to peptide aldehydes is described using a combination of solid phase peptide synthesis with polymer-assisted solution phase synthesis based on the oxidation of peptide alcohols with a mild and selective polymer-bound IBX derivative. The oxidation is followed by selective purification via scavenging the peptide aldehyde in a capture-release procedure using threonine attached to an aminomethyl resin. Peptide aldehydes are obtained in excellent purity and satisfying yield. The optical integrity of the C-terminal residue is conserved in a high degree. The procedures are compatible with the use of common side-chain protecting groups. The potential for using the method in parallel approaches is very advantageous. A small collection of new and known peptide aldehydes has been tested for inhibitory activity against caspases 1 and 3.     

Nmr studies on linear homo-oligopeptides: A perspective view

The use of high-resolution nmr to determine the structure of linear homo-oligopeptides in solution is described. Complete assignments of NH and α-CH resonances were elucidated based upon a guest–host series of compounds and selective α-deuteration of residues in the chain. The conformations for oligomethionines and oligoglutamates in dimethylsulfoxide, chloroform, and trifluoroethanol are discussed, and molecular models are described. Recent studies of L -glutamate peptides attached to polyoxyethylene are also included. These peptide–polyoxyethylene conjugates are soluble in a broad range of solvents, such as dimethyl-sulfoxide, chloroform, trifluoroethanol, and water. Preliminary conclusions are drawn concerning the effects of amine terminal end groups and the nature of the solvent on the conformations of these peptide derivatives.     

Conformational analysis of polyoxyethylene-bound homo-oligo-L-glutamates in aqueous environment

A detailed conformational analysis of homo-oligo-L -glutamates was carried out in aqueous solution using ¹H-nmr spectroscopy. Three series of side-chain protected (α-OMe) glutamate oligopeptides, attached to polyoxyethylene (POE) to enhance their solubility, were synthesized. The effect of the N-terminal blocking groups—Boc, Ac, and pGlu—on the conformations of these peptides in water is discussed. Unequivocal assignments were obtained for all amide NH resonances through use of selectively α-deuterated oligo-glutamates. Analysis of vicinal coupling constants, temperature dependence of NH chemical shifts, transfer of saturation experiments, and titration studies with a denaturing solvent (DMSO) were used to investigate the peptide structure. These data suggest that the Glu¹ and Glu² NH protons of each heptamer are solvent exposed, while the NH protons of interior glutamate residues chain are solvent sequestered. The nmr data are consistent with the onset of helical structure at the heptamer of each series in aqueous solution. The POE-peptides with pGlu at the N-terminus showed considerably reduced stability of structure than those with the Boc or acetyl blocking groups. Peptide conformations and their stability in water are compared to those in other solvents.     

Synthesis of all-cis 2,5-imino-2,5-dideoxy-fucitol and its evaluation as a potent fucosidase and galactosidase inhibitor

We here describe a simple and efficient synthetic method for a non-hydrolysable precursor of a GDP-fucose analogue: The synthesis of the racemic aminofuranofucitol 3 from sorbic alcohol by nitroso-Diels-Alder reaction. This 'all-cis-pyrrolidine', with all substituents occupying a cis position, has been determined as a potent inhibitor of α-L-fucosidase and a moderate inhibitor of α- and β-D-galactosidase. The good recognition of this fucose moiety analogue by specific enzymes is thus confirmed. The C-anomeric bond in this particular structure is in the β-position and makes this compound an interesting candidate for further chemical modifications. Influence of the methyl and hydroxymethyl groups on the inhibition potency is discussed.     

The effect of an antiviral peptide on the ribosomal reactions of the peptide elongation enzymes, EF-I and EF-II

A basic peptide with antiviral properties isolated from pokeweed is shown to inhibit the synthesis of globin and phenylalanine peptides on ribosomes isolated from rabbit reticulocytes. The inhibition appears to involve a specific effect of the peptide inhibitor on the larger ribosomal subunit that can be produced at a ratio of inhibitor to ribosomes of less than one to one. Ribosomes treated with the inhibitor have a reduced capacity to support enzymatic binding of Phe-tRNA to ribosomes and GTP hydrolysis caused by the elongation enzyme, EF-I. Treated ribosomes exhibit a concomitant capacity for increased GTP hydrolysis by EF-II but do not efficiently support EF-II-dependent binding of [³H]GTP. Such binding appears to involve the formation of an EF-II·GDP·ribosome complex. Thus, the inhibitor has an effect on GTP-dependent reaction carried out by both of the peptide elongation enzymes. The relation between these effects in the reticulocyte system is discussed in relation to the effects of siomycin or thiostrepton in blocking GTP hydrolysis by EF-T and EF-G on prokaryotic ribosomes.     

Starch- and glycogen-debranching and branching enzymes: Prediction of structural features of the catalytic (β/α)8-barrel domain and evolutionary relationship to other amylolytic enzymes

Sequence alignment and structure prediction are used to locate catalytic α-amylase-type (β/α)₈-barrel domains and the positions of their β-strands and α-helices in isoamylase, pullulanase, neopullulanase, α-amylase-pullulanase, dextran glucosidase, branching enzyme, and glycogen branching enzymes—all enzymes involved in hydrolysis or synthesis of α-1,6-glucosidic linkages in starch and related polysaccharides. This has allowed identification of the transferase active site of the glycogen debranching enzyme and the locations of β ? α loops making up the active sites of all enzymes studied. Activity and specificity of the enzymes are discussed in terms of conserved amino acid residues and loop variations. An evolutionary distance tree of 47 amylolytic and related enzymes is built on 37 residues representing the four best conserved β-strands of the barrel. It exhibits clusters of enzymes close in specificity, with the branching and glycogen debranching enzymes being the most distantly related.     

Availability of tyrosine amide for α-chymotrypsin-catalyzed synthesis of oligo-tyrosine peptides

Oligo-tyrosine peptides such as Tyr-Tyr having angiotensin I-converting enzyme (ACE) inhibitory activity could be synthesized by α-chymotrypsin-catalyzed reaction with l-tyrosine ethyl ester in aqueous media. However, peptide yield in the reaction was below 10%. Since l-tyrosine amide showed highly nucleophilic activity for the deacylation of enzyme through which a new peptide bond was made, its application to the enzymatic peptide synthesis was evaluated in this study. Addition of tyrosine amide into the reaction produced Tyr-Tyr-NH₂, of which yield exceeded 130% on the basis of tyrosine ethyl ester. Although purified Tyr-Tyr-NH₂ did not inhibit ACE activity, α-chymotrypsin could act on the dipeptide amide and convert about 40% of it to Tyr-Tyr. The use of both ester and amide forms of tyrosine is expected to be a potent procedure for α-chymotrypsin-catalyzed synthesis of antihypertensive peptides.     

Utilization of Proteases from Aspergillus Species for Peptide Synthesis via the Kinetically Controlled Approach

We examined Aspergillus melleus protease (Amano protease P) and A. oryzae protease (Amano protease A) as catalysts for peptide bond formation via the kinetically controlled approach. As the coupling efficiency was only moderate, even with a good amino acid substrate as the carboxyl component, in acetonitrile as a solvent (with or without a small amount of added water) that we had mainly employed previously in α-chymotrypsin catalyzed couplings, other solvent systems were sought. In 1,1,1,3,3,3-hexafluoro-2-propanol-DMF (1:1) without added water, these Aspergillus proteases were found to remain active for a long period of time and to be utilizable for peptide synthesis when the carbamoylmethyl ester was employed as the acyl donor, though the coupling efficiencies were dependent rather largely on the combination of the amino acid residues at the coupling site. The superiority of the carbamoylmethyl ester to conventional esters, for example the methyl ester, was once again established. Furthermore, some segment condensations were also achieved by the same procedure.     

Mechanism and yields in enzyme catalysed equilibrium and kinetically controlled synthesis of β-lactam antibiotics,peptides and other condensation products

Hydrolases can be used to catalyse the synthesis of condensation products such as β-lactam antibiotics, peptides, oligosaccharides and glycerides. In biotechnological processes, synthesis to achieve maximum yields may be carried out either as an equilibrium controlled or kinetically controlled reaction. Only in the later case is the yield of condensation product influenced by the properties of the enzyme that act as a transferase in this reaction. With the same amount of enzyme the maximum yield is also obtained much more rapidly than in the equilibrium controlled process. Hydrolases with high ratios of transferase to hydrolase activity are favourable for use. Recent results on the mechanisms of enzyme catalysed condensations allow a rational analysis of how yield controlling factors (pH, temperature, ionic strength, enzyme and substrate properties) may be changed to obtain optimum yields. This can be used to evaluate whether these biotechnological processes can compete with the chemical methods currently used for the synthesis of these products. It can also be used to plan rational protein engineering of the enzymes that in kinetically controlled synthesis of the condensation products may give yields that can compete favourably with the existing chemical processes to produce these compounds.     

Preparation of protected peptide amides using the Fmoc chemical protocol. Comparison of resins for solid phase synthesis.

Different resins were examined for their potential use in the solid phase synthesis of protected peptide amides using the 9-fluorenylmethoxycarbonyl (Fmoc) chemical protocol. The model protected peptide amide BocTyr-Gly-Gly-Phe-Leu-Arg(Pmc)NH2 (1) was synthesized on both the acid-labile 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy resin (Rink amide resin) (2) and on resins containing the base-labile linker 4-hydroxymethylbenzoic acid. Of the resins examined only the methylbenzhydrylamine resin containing the 4-hydroxymethylbenzoic acid linkage, which was cleaved by ammonolysis in isopropanol, gave the model peptide 1 in good overall yield (53% including functionalization). Thus the synthesis of protected peptide amides by solid phase synthesis using Fmoc-protected amino acids with t-butyl-type side chain protecting groups is feasible. The choice of peptide-resin linkage and its cleavage conditions, however, are critical to the success of such syntheses. The potential application of this synthetic strategy to the preparation of novel peptide amides is discussed.     

Amino acid pyridoxyl esters in peptide synthesis

Pyridoxyl residue was suggested to be used as a multifunctional protective and modifying group in peptide synthesis. The modification was carried out by introducing the pyridoxyl residue in free or partially protected peptides or by the addition of amino acid pyridoxyl esters by the methods of conventional peptide synthesis without the removal of the pyridoxyl group at the terminal stages of the synthesis (the second approach is more convenient). Pyridoxyl residue was also used as a spacer in solid phase peptide synthesis. It was attached to the polymer by the alkylation of the hydroxyl groups or of the pyridine ring of the pyridoxyl derivatives with the chloromethylated styrene-divinylbenzene copolymer (the standard Merrifield resin). Potentials for the use of pyridoxyl derivatives in the synthesis of linear, multiplet, and cyclic peptides are discussed.     

Chemical modification of proteins: comments and perspectives

The use of chemical modification of proteins has increased exponentially during the past two decades. Today the many different uses of chemical modification include determination of relative reactivities of side chain groups, the quantitation of individual amino acids, development of affinity reagents, mechanism-based reagents for pharmaceutical uses, cross-linking reagents, special techniques for bioprostheses, blocking reagents for peptide synthesis, and reagents for specific cleavages of peptide bonds. Chemical modification should continue to be a primary tool in protein chemistry. It can supply information or products difficult or impossible to attain by the newer powerful technique of in vitro mutagenesis as well as serve as a supplementary procedure for the latter.     

Synthesis of peptide aldehydes.

The functionalization of peptides and proteins by aldehyde groups has become the subject of intensive research since the discovery of the inhibition properties of peptide aldehydes towards various enzymes. Furthermore, peptide aldehydes are of great interest for peptide backbone modification or ligation reactions. This review focuses upon their synthesis, which has been developed following two main strategies. The first strategy consists of prior synthesis of the peptide, followed by the introduction of the aldehyde function. The second possible strategy uses alpha-amino aldehydes as starting materials. After protection of the aldehyde, peptide elongation occurs. At the end of the synthesis, the aldehyde function can be unmasked.     

Alpha-amino acid ester hydrolases: Properties and applications

The review describes two major groups of α-amino acid ester hydrolases (AEHs)—enzymes with a similar active center structure, which determines their unique specificity to esters containing an amino group in the α position to the carbonyl. The first group comprises microbial AEHs of the β-lactam acylase type. Technical biocatalysts based on this group of enzymes are used for the production of semi-synthetic amino-β-lactam antibiotics. The second AEH group includes eukaryotic valacyclovirases, which activate in vivo a number of antiviral and anticancer prodrugs. The directed activity of these enzymes is used for the development of target pharmaceutical preparations for the therapy of viral and oncological diseases. The review summarizes and compares the available data on the structure and properties, substrate specificity, and the kinetic parameters of enzymes of these two groups. Experiments identifying the AEH active site and providing the molecular basis for the unique specificity of these enzymes are discussed. The data from the available scientific and patent publications concerning the aminopenicillin and aminocephalosporin synthesis catalyzed by β-lactam acylase AEHs are reviewed and systematized.     

Pyridoxyl amino acid esters in peptide synthesis

Pyridoxyl residue was suggested to be used as a multifunctional protective and modifying group in peptide synthesis. The modification was carried out by introducing the pyridoxyl residue in free or partially protected peptides or by the addition of amino acid pyridoxyl esters by the methods of conventional peptide synthesis without the removal of the pyridoxyl group at the terminal stages of the synthesis (the second approach is more convenient). Pyridoxyl residue was also used as a spacer in solid phase peptide synthesis. It was attached to the polymer by the alkylation of the hydroxyl groups or of the pyridine ring of the pyridoxyl derivatives with the chloromethylated styrene-divinylbenzene copolymer (the standard Merrifield resin). Potentials for the use of pyridoxyl derivatives in the synthesis of linear, multiplet, and cyclic peptides are discussed.