Trypsin as a catalyst for peptide synthesis

Trypsin-catalyzed syntheses of peptides were performed using various N-acylated amino acid or peptide esters as donors and amino acid derivatives, peptides, or their derivatives as acceptors. The synthesis was almost quantitative under optimal conditions. Considerably more enzyme and a more alkaline pH were necessary for synthesis than hydrolysis. Another very important condition was the concentration of the starting materials; higher concentrations resulted in much better product yields. The nucleophile specificity for synthesis was also important; hydrophobic or bulky amino acid residues were most efficient at the P1' position, and L-proline as well as D-amino acid residues were the worst choices. The synthesis was also dependent on the solubility of the products synthesized; the yield was higher with products of lower solubility. As donor esters, good substrates were all useful. Accordingly, fragment condensation was possible using N-acylated peptide esters and various peptides. The present study suggests that trypsin may become a useful tool for peptide synthesis.     

Peptide bond synthesis catalyzed by alpha-chymotrypsin.

alpha-Chymotrypsin [EC 3.4.21.1] catalyzed the syntheses of peptide bonds with various N-acylated amino acids or peptides having aromatic or hydrophobic amino acid residues at the C-terminal position as carboxyl components, and amino acid derivatives, peptides or their derivatives as amine components. A neutral pH was most efficient and quite high concentrations of alpha-chymotrypsin and starting materials were required for synthesis. Four amine components, hydrophobic or bulky amino acid residues were useful at the N-terminal position. Stereospecificity was also observed at the N-terminal position of amine components. Peptide synthesis was not usually seen when the products were soluble in the reaction mixture. This could be partly overcome by increasing the concentration of either the carboxyl or the amine component to more than ten times that of the other.     

Specificity of Proteinase K from Tritirachium album Limber for Synthetic Peptides

The specificity of proteinase K from Tritirachium album Limber was determined using various synthetic peptide substrates. The esterase activity against N-acylated amino acid esters indicated that the enzyme is primarily specific against aromatic or hydrophobic amino acid residues at the carboxyl side of the splitting point. Secondary interaction for hydrolysis was also studied using peptide esters or others, which showed that the enzyme activity is markedly promoted by elongating the peptide chain to the N-terminal from the splitting point. Thus, peptide chloromethyl ketone derivatives such as Cbz-Ala-Gly-PheCH₂Cl inactivated the enzyme activity markedly.     

Protease-catalysed peptide synthesis in solvent-free system

Summary A new method of enzymatic peptide synthesis without any liquid added has been proposed. The method is based on the admixing of N-acylamino acid (peptide) esters and nucleophiles (amides or tert.-butylesters of amino acids or peptides, peptides) with various proteolytic enzymes such as α-chymotrypsin, trypsin, proteinase K, subtilisin, elastase and papain in the presence of Na₂CO₃. 10H₂O. In most instances, acylating components were completely converted within a few hours giving satisfactory yields of desired peptide products.     

C-terminal amidation of acylamino acids and peptides using a transpeptidation method catalyzed by carboxypeptidase Y

The carboxypeptidase Y-catalyzed reaction of acyl transfer of acylamino acid and peptide residues from the corresponding esters to ammonia and to amides of amino acids has been studied, and conditions for obtaining amides of amino acids and peptides with the yields up to 90% found.     

Pepstatin-insenstive acid proteases from Scytalidium lignicolum. Kinetic study with synthetic peptides.

A kinetic study was conducted on the acid proteases A-1 and A-2 from Scytalidium lignicolum using synthetic peptides as substrates. Almost maximum activity was attained with N-acylated tetrapeptides as the molecular size of substrates was increased. Suitable amino acid residues were required at the P1-P2 and P1'-P2' positions [notation of Schechter and Berger (14)]. Hydrophobic or bulky residues such as leucine were specifically required at the P1 and P1' positions, with the specificity at the latter position being considerably lower than that at the former. For catalysis, the presence of certain amino acid residues at the P2 and P2' positions was essential, mainly in relation to kcat. An inhibition study supported this view. Stringent stereospecificity was observed at the P2 and P2' positions, but the side chain specificity was low. Study of the B enzyme from the same organism was very difficult owing to its low activity against the peptides used. The Scytalidium acid proteases A-1, A-2, and B showed considerably different behavior against peptide substrates in comparison with usual acid proteases, which are senstive to pepstatin.     

Enzymatic synthesis of chromogenic substrates for Glu,Asp-specific proteinases.

Glu,Asp-specific endopeptidases represent a new subfamily of chymotrypsin-like proteolytic enzymes. These enzymes prefer Glu or Asp residues in the P1 position of the substrates. p-Nitroanilides of N-acylated di-, tri- and tetrapeptides with C-terminal glutamic or aspartic acid residues have been obtained. Acyl peptide p-nitroanilides were synthesized via acylation of glutamic or aspartic acid p-nitroanilides using methyl esters of the respective N-acylated peptides, generally with good yields. The reactions were performed in organic solvents using subtilisin 72 sorbed on silica as a catalyst. The kinetic parameters for the hydrolysis of these p-nitroanilides with proteinases from Bacillus intermedius and Bacillus licheniformis were determined.     

Novel serine penicillocarboxypeptidase CPD-S3 from Penicillium janthinellum IBT 3991: Purification, characterization, and uses in peptide synthesis and modification

A novel carboxypeptidase (CPD-S3) from Penicillium janthinellum IBT 3991 has been isolated in a two-step purification procedure by cation exchange and affinity chromatography. The enzyme is a serine carboxypeptidase with a denatured molecular mass determined by SDS of 62 kDa of which 32% is carbohydrate. The isoelectric point is 5.1. CPD-S3 exhibits a high stability towards organic solvents and elevated temperatures. Besides the carboxypeptidase activity, CPD-S3 exhibits esterase, amidase, and carboxamidohydrolase activities. CPD-S3 favors substrates of -configuration with basic amino acid residues in either P₁ or P_1', and particularly dibasic substrates and medium-sized straight-chain alkyl esters for hydrolysis. In aminolysis of esters, amino acid amides and hydrazines coupled in good yield, but methyl esters poorly, and unlike other carboxypeptidases, free amino acids could not be coupled or transpeptidation effected to form amides. In ester semisynthesis, peptides with neutral, but not basic, residues in P₁ could be esterified. The scope of applicability for enzymatic peptide synthesis is limited.     

Determination of isokinetic ratios necessary for equimolar incorporation of carboxylic acids in the solid-phase synthesis of mixture-based combinatorial libraries

The methods used to study the relative reaction rates of 45 different aliphatic and aromatic carboxylic acids when coupled to resin-bound amino acid amides is described. Competition experiments involving the coupling of incoming carboxylic acids to resin-bound amino acid amides were performed. The relative composition of each N-acylated amino acid amide in the resulting mixtures was compared to controls prepared by physically mixing equal aliquots of individual compounds in order to study the relative reaction rates of the incoming carboxylic acids. The ratios of the incoming carboxylic acids were then iteratively adjusted to yield as close to equimolar products as possible. As expected, the steric and electronic nature of the incoming carboxylic acids was found to influence their relative reaction rates. The steric hindrance of the resin-bound amino acid appears to have a proportional effect on the reaction rates of the incoming carboxylic acids. N-acylated amino acid amides in the final mixtures, prepared using the final isokinetic ratios, were found to be approximately equimolar.     

Glycine flanked by hydrophobic bulky amino acid residues as minimal sequence for effective subtilisin catalysis.

The specificity of alkaline mesentericopeptidase (a proteinase closely related to subtilisin BPN') for the C-terminal moiety of the peptide substrate (Pi' specificity) has been studied in both hydrolysis and aminolysis reactions. N-Anthraniloylated peptide p-nitroanilides as fluorogenic substrates and amino acid or peptide derivatives as nucleophiles were used in the enzymic peptide hydrolysis and synthesis. Both hydrolysis and aminolysis kinetic data suggest a stringent specificity of mesentericopeptidase and related subtilisins to glycine as P1' residue and predilection for bulky hydrophobic P2' residues. A synergism in the action of S1' and S2'subsites has been observed. It appears that glycine flanked on both sides by hydrophobic bulky amino acid residues is the minimal amino acid sequence for an effective subtilisin catalysis.     

Pepsin as a catalyst for peptide synthesis: formation of peptide bonds not typical for pepsin substrate specificity.

Porcine pepsin in water solutions containing 15-28% of dimethylformamide at pH 5 and 20-37 degrees C catalysed the formation of peptide bonds between Z-Ala-Ala-Phe-OH and various amino acid or peptide derivatives. Substrate binding subsite S1' of pepsin demonstrated broad specificity in these reactions but revealed a certain preference for hydrophobic amino acid residues, including non-proteinous homophenylalanine, p-nitrophenylalanine, S-methylcysteine, as well as for those that contained, in addition to the hydrophobic elements, a group capable of donating a hydrogen bond, e.g. o-nitrotyrosine. This observation increases the range of peptides that might be prepared by pepsin-catalysed synthesis.     

Synthesis and solubility properties of peptide fragments of human hemoglobin alpha-chain (123-136)

The solubility prediction method for protected peptides was successfully applied to relatively small peptide fragments of human hemoglobin alpha-chain (123-136) which contained various polar amino acid residues such as Asp(OBzl), Glu(OBzl), Lys(Z), Ser(Bzl), and Thr(Bzl). As reported previously for hydrophobic peptides and human proinsulin C-peptide fragments, solubility data indicated that the insolubility of protected peptides having a mean value of Pc value below 0.90 appeared to begin at the octa- or nonapeptide sequence level and that beta-sheet structure played an important role in the insolubility of peptides. When a peptide has a beta-sheet structure in the solid state, we can clearly determine the critical chain length for peptide insolubility, the solubility dependence on solvent properties, and the solubility independence of amino acid compositions of peptides.     

Determination of enzyme specificity in a complex mixture of peptide substrates by N-terminal sequence analysis.

A method has been developed to determine preferred residue substitutions in the P' position of peptide substrates for proteolytic enzymes. The method has been validated with four different enzymes; the angiotensin I-converting enzyme, atrial dipeptidyl carboxyhydrolase, bacterial dipeptidyl carboxyhydrolase, and meprin A. A mixture of N-acylated potential peptide-substrates for each of the enzymes was prepared in a single synthesis procedure on the same solid-phase synthesis resin. The peptides were identical in all residue positions except the P' position to be studied, into which numerous amino acid residues were incorporated on a theoretical equimolar basis. After cleavage and extraction of the peptides from the resin, no attempt was made to purify them individually; the exact concentration of each peptide in the mixture was determined by quantitative amino acid analysis. Incubation of an enzyme with its peptide-substrate mixture at [S] much less than Km yielded peptide hydrolytic products with newly exposed N-termini. The identity and amount of each hydrolysis product was determined by automated N-terminal sequence analysis. One cycle of sequencing revealed preferred amino acid substitutions in the P'1 position, two cycles the P'2 position, and so forth. Comparison of the rates of production of the various products indicates the preferred substitution in that particular P' position. New information on the substrate specificities of each of the enzymes tested was obtained and it is clear that this approach can be applied to any protease with a defined (or suspected) point of cleavage in a peptide substrate.     

Polymer-supported biopolymer synthesis: 1. High load solid (gel) phase strategy for peptide synthesis with a phenolic poly(acryloylmorpholine)-based support

An efficient, low-cost, reaction strategy for the solid (gel) phase synthesis of peptides and protected peptide segments has been developed. The strategy involves the use of a new poly(acryloylmorpholine)-based phenolic support matrix, Koch-Light Peptide Resin A. Illustrative syntheses of N-terminal Boc- and Z-protected[Leu]- enkephalin derivatives, including C-terminal acid hydrazides and esters, are described. The strategy, which is effective for the synthesis of peptides at high matrix loadings, is adopted readily for large-scale application.     

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.     

Peptide synthesis catalyzed by papain at alkaline pH values

The synthesis of peptides in the presence of papain at pH 8-9.5 is described. Starting substances are acylamino acid alkyl esters (the carboxyl component) and amides or tert.-butylesters of amino acids, as well as peptide (the amino component). Under such conditions secondary hydrolysis is not essential, making the synthesis of peptides soluble in aqueous medium. The yield of peptides is 50-94%. The effect of different factors (temperature, solvents, reagent concentrations) on the result of the reaction has been studied. It has been found that an excess of the carboxyl component is expedient to increase the yield of peptides.     

Peptide synthesis in aqueous-organic solvent mixtures with alpha-chymotrypsin immobilized to tresyl chloride-activated agarose

alpha-Chymotrypsin was immobilized with a high coupling yield (up to 80%) to tresyl chloride activated Sepharose CL-4B.The immobilized enzyme was tested for its ability to synthesize soluble peptides from N-acetylated amino acid esters as acyl donors and amino acid amides as acceptor amines in water-water-miscible organic solvent mixtures. It was found that the yield of peptide increased with increasing concentration of organic cosolvent. Almost complete synthesis (97%) of Ac-Phe-Ala-NH(2) was obtained from Ac-Phe-OMe using a sixfold excess of Ala-NH(2). The rate of peptide formation in aqueous-organic solvent mixtures was good. Thus, 0.1M peptide was formed in less than 2 h in 50 vol% DMF with 0.1 mg immobilized chymotrypsin/mL reaction mixture. The immobilized enzyme distinguished between the L and D configurations of acceptor amino acid amides even in high concentration of nonaqueous component (90% 1,4-butanediol). The effect of temperature was studied. It was found that both the yield of peptide and the stability of immobilized enzyme increased when the temperature was lowered. Experiments could be performed at subzero temperatures in the aqueous-organic solvent mixtures resulting in very high yield of peptide. After three weeks continuous operation at 4 degrees C in 50% DMF, the immobilized enzyme retained 66%of its original synthetic activity. The activity of the immobilized enzyme was better conserved with a preparation made from agarose with a higher tresyl group content compared to a preparation made from a lower activated agarose, indicating that multiple point of attachment has a favorable effect on the stability of the enzyme in aqueous-organic solvent mixtures. The major advantage of using water-miscible instead of water-immiscible organic solvents to promote peptide syntheses appears to be the increased solubility of substrates and products, making continuous operation possible.     

Peptide enzymatic microsynthesis, using carboxypeptidase Y as the catalyst: application to stepwise synthesis of Leuenkephalin

In order to develop the use of carboxypeptidase Y (CPD-Y, EC 3.4.12.1) as a catalyst for radioactive hormonal synthesis, the stepwise synthesis of a pentapeptide, Leu-enkephalin, was studied on a microscale. Each peptide bond was formed by enzymatic catalysis, using microquantities of the precursors (amino acid or peptide esters as acyl-components and amino acid ester or amide as nucleophiles). The high condensation yields obtained suggests that CPD-Y can be a useful tool for preparation of radioactive hormonal peptides.     

Catalysis by human leukocyte elastase. Aminolysis of acyl-enzymes by amino acid amides and peptides

Acyl-enzymes of human leukocyte elastase (HLE) were generated in situ during the hydrolysis of peptide thiobenzyl esters and served as substrates for aminolysis by a variety of amino acid amides and short peptide nucleophiles. For amino acid amides, there is a positive correlation between nucleophilic reactivity toward N-methoxysuccinyl (MeOSuc)-Ala-Ala-Pro-Val-HLE and the hydrophobicity of the side chain. For peptides, nucleophilicity toward MeOSuc-Ala-Ala-Pro-Val-HLE decreases dramatically with increasing chain length. Combined, these results suggest that substrate specificity for the P1' residue may be more dependent on side chain hydrophobicity than on specific, structural features of the side chain and there may be no important binding interactions available past S1'. Kinetic parameters were also determined for the nucleophilic reactions of PheNH2 and TyrNH2 with MeOSuc-Pro-Val-HLE, MeOSuc-Ala-Pro-Val-HLE, MeOSuc-Ala-Ala-Pro-Val-HLE, and MeOSuc-Ala-Ala-Pro-Ala-HLE. Reactivity of these acyl-enzymes toward nucleophilic attack displays no dependence on peptide chain length but does increase significantly for the substrate with Ala at P1. This same correlation between reactivity and acyl-enzyme structure is also seen for nucleophilic attack by water.     

The applicability of subtilisin Carlsberg in peptide synthesis.

The synthesis of peptide bonds catalysed by subtilisin Carlsberg was studied in different hydrophilic organic solvents with variable H2O concentration. Z-Val-Trp-OMe and Z-Ala-Phe-OMe were used as acyl donors, and a series of amino acid derivatives, di- and tripeptides of the general structure Xaa-Gly, Gly-Xaa, Gly-Gly-Xaa (Xaa represents all natural L-amino acids except cysteine) and other peptides were used as nucleophiles. A comparative study of the enzymatic synthesis in aqueous DMF (50%, v/v) and acetonitrile containing 10% (v/v) of H2O demonstrated that the yields of peptide products were higher in most cases when acetonitrile with low H2O concentration was used. The acylation of weak nucleophiles was improved in organic solvents with very low H2O concentration (2%). The reactions in anhydrous Bu(t)-OH proceeded with substantially lower velocity. Generally, the restricted nucleophile specificity of the enzyme for glycine and hydrophilic amino acid residues in P1' position, as well as numerous side reactions, limit the utilization of subtilisin in peptide synthesis, especially in the case of the segment condensations. Contrary to the published data, we have proved that proline derivatives were not acylated in any media with the help of subtilisin Carlsberg. Effective ester hydrolysis of a protected nonapeptide corresponding to the N-terminal sequence of dicarba-eel-calcitonin catalysed by subtilisin was achieved.