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.     

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.     

Peptide synthesis catalyzed by polyethylene glycol-modified chymotrypsin in organic solvents

Chymotrypsin modified with polyethylene glycol was successfully used for peptide synthesis in organic solvents. The benzene-soluble modified enzyme readily catalyzed both aminolysis of N-benzoyl-L-tyrosine p-nitroanilide and synthesis of N-benzoyl-L-tyrosine butylamide in the presence of trace amounts of water. A quantitative reaction was obtained when either hydrophobic or bulky amides of L- as well as D-amino acids were used as acceptor nucleophiles, while almost no reaction occurred with free amino acids or ester derivatives. The acceptor nucleophile specificity of modified chymotrypsin as a catalyst in the formation of both amide and peptide bonds in organic solvents was quite comparable to that in aqueous solution as well as to that of the leaving group in hydrolysis reactions. By contrast, the substrate specificity of modified chymotrypsin in organic solvents was different from that in water since arginine and lysine esters were found to be as effective as aromatic amino acids to form the acyl-enzyme with subsequent synthesis of a peptide bond.     

Nucleophile specificity of subtilisin in an organic solvent with low water content: investigation via acyl transfer reactions

Nucleophile specificity of subtilisin (subtilopeptidase A) was studied via acyl transfer reactions in acetonitrile containing piperidine and 10 vol% of water. Ac-Tyr-OEt was used as acyl donor 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) were used as nucleophiles. The nucleophilic efficiencies of these peptides were characterized by the values of the apparent partition constants, p(app), determined from the HPLC analysis of the reactions. The order of preference for the P'(1) position was estimated to be: Gly > hydrophilic, positively charged > hydrophobic, aromatic > negatively charged > Leu > Pro side chain. For the P'(2) position the order of preference was: Gly > hydrophilic, charged > hydrophobic, aromatic > Pro side chain. The values of p(app) for Gly-Gly-Xaa tripeptides cover a range of only two orders of magnitude, with lower nucleophile efficiency for those with hydrophobic amino acid residues in the P'(3) position. The dipeptide with Pro in P'(1) did not react at all, but a tripeptide having Pro in P'(3) was a very good nucleophile. The negatively charged amino acid residues in the P'(1) position result in very weak nucleophilic behavior, whereas the peptides with Asp or Glu in P'(2) and P'(3) are well accepted. Generally, peptides of the Gly-Xaa or Gly-Gly-Xaa series were better nucleophiles than peptides of the Xaa-Gly series. The length of the peptide chain or amidation of alpha-carboxyl function had no influence on nucleophilic behavior. No significant difference in nucleophile specificity between subtilopeptidase A and nagarse was observed. (c) 1996 John Wiley & Sons, Inc.     

Peptide synthesis using proteases dissolved in organic solvents

Organic solvent-soluble -chymotrypsin (CT) and subtilisin Carlsberg (SC) are effective catalysts for peptide synthesis in homogeneous organic solutions. The soluble enzymes have values of k_cat/K_m for the reaction of N-Bz-L-Tyr-OEt with L-Leu-NH₂ to yield the dipeptide N-Bz-L-Tyr-L-Leu-NH₂ that are over 3 orders of magnitude higher than their suspended counterparts in isooctane (containing 30% (v/v) tetrahydrofuran (THF) to aid in substrate solubility). Both enzymes are substantially more active in hydrophobic organic solvents than hydrophilic solvents. Adding small concentrations of water (<0.2% and 1% (v/v) in isooctane-THF and ethyl acetate, respectively) results in up to a 150-fold activation of -chymotrypsin-catalyzed peptide synthesis. Importantly, added water does not promote hydrolysis in either isooctane-THF or ethyl acetate; thus, -chymotrypsin is highly selective toward peptide synthesis in the nearly anhydrous organic solutions. Unlike CT, the activation of subtilisin Carlsberg upon partial hydration of isooctane-THF or ethyl acetate was not significant and actually resulted in substantial hydrolysis. Using -chymotrypsin, a variety of tripeptides were produced from dipeptide amino acid esters. Reactivity of D-amino acid amides as acyl acceptors and partially unblocked amino acid acyl donors further expands the generality of the use of organic solvent-soluble enzymes as peptide synthesis catalysts.     

Increased nucleophile reactivity of amino acid beta-naphthylamides in alpha-chymotrypsin-catalyzed peptide synthesis

Alpha-chymotrypsin-catalyzed acyl transfer from Boc-L-MetONp, Ac-L-TyrOEt, Bz-L-TyrOMe, Mal-L-PheOMe to the C-protected amino acids (L-AlaNH2, L-LeuNH2, L-ArgOMe and beta-naphthylamides of L-Arg, L-Leu, L-Ala and L-Glu) has been studied. Modification of the carboxylic groups with beta-naphthylamide was shown to increase the reactivity of nucleophiles in these reactions by a factor of more than 100 in comparison with amides and esters of the same amino acids. This effect can be accounted for by the effective formation of the nucleophile-acylenzyme complex due to hydrophobic interactions of the beta-naphthylamide moiety with the corresponding subsite of alpha-chymotrypsin. The reaction kinetics follows the scheme involving hydrolysis of the nucleophile-acylenzyme intermediate. The contribution of this pathway depends on the structures of both the acyl-group donor and the added nucleophile. The competitive inhibition by amino acid beta-naphthylamides is also observed. The results obtained show that modification of the COOH-group of added nucleophiles by beta-naphthylamide strongly affects the reactivity of these compounds in the alpha-chymotrypsin-catalyzed peptide synthesis.     

Synthesis of N-protected peptide alcohols catalyzed by subtilisin or alpha-chymotrypsin in organic solvents

A series of N-protected peptide alcohols were synthesized using amino alcohols with unprotected hydroxy groups as amino components by the catalysis of subtilisin or alpha-chymotrypsin in organic solvents. N-protected aromatic amino acid esters were more suitable as acyl donors for subtilisin. The influences of different N-protecting groups, organic solvents, and content of water on synthesis of N-protected peptide alcohols were systematically studied.     

Unusual specificity of polyethylene glycol-modified thermolysin in peptide synthesis catalyzed in organic solvents

Summary Polyethylene glycol-modified thermolysin was found to efficiently catalyze peptide synthesis in organic solvents. As in aqueous media, the reaction occurred through a rapid equilibrium random bireactant mechanism. However, the substrate specificity of modified thermolysin was actually changed since hydrophilic as well as acidic amino acids were better carboxyl group donors than hydrophobic residues, contrary to what is observed in both the enzyme-catalyzed synthesis and hydrolysis of peptide bonds in water.     

Surfactant-protease complex as a novel biocatalyst for peptide synthesis in hydrophilic organic solvents*

The peptide synthesis from N-acetyl-L-phenylalanine ethyl ester with alaninamide catalyzed by a surfactant-protease complex has been performed in anhydrous hydrophilic organic solvents. Proteases derived from various sources were converted to surfactant-coated complexes with a nonionic surfactant. The surfactant-subtilisin Carlsberg (STC) complex had a higher enzymatic activity than the other protease complexes and the initial reaction rate in tert-amyl alcohol was 26-fold that of STC lyophilized from an optimum aqueous buffer solution. Native STC hardly catalyzed the same reaction. The addition of water to the reaction medium activated the lyophilized STC, however, the reaction rate was much lower than that of the STC complex, and a hydrolysis reaction preferentially proceeded. The STC complex exhibited a high catalytic activity in hydrophilic organic solvents (e.g. tertiary alcohol). The addition of dimethylformamide as a cosolvent improved the solubility of amino acid amides and further activated the STC complex due to the water mimicking effect. When hydrophilic amino acid amides were employed as an acyl acceptor, the peptide formation proceeded efficiently compared to that using hydrophobic substrates. The surfactant-STC complex is a powerful biocatalyst for peptide synthesis because the STC complexes display a high catalytic activity in anhydrous hydrophilic organic solvents and did not require the excess amount of water. Thus the side (hydrolysis) reaction is effectively suppressed and the yield in the dipeptide formation is considerably high.     

Enzymatic formation of Glu-Xaa and Asp-Xaa bonds using Glu/Asp-specific endopeptidase from Bacillus licheniformis in frozen aqueous systems.

The capability of Glu/Asp-specific endopeptidase from Bacillus licheniformis to form Glu/Asp-Xaa bonds in frozen aqueous systems was investigated. Under frozen state conditions, the enzyme was able to catalyse peptide bond formation more effectively than in liquid reaction mixtures. The acceptance of amino components which were completely inefficient nucleophiles at room temperature indicates a changed specificity of Glu/Asp-specific endopeptidase under frozen state conditions. Protease-catalysed coupling of two acidic amino acids was demonstrated for the first time. The utilization of Glu/Asp-specific endopeptidase from Bacillus licheniformis in frozen aqueous systems offers new possibilities in enzyme-catalysed peptide synthesis.     

Non-coded amino acids as acyl donor substrates for peptide bond formation catalyzed by thermoase in toluene

The peptide bond formation of N-protected non-coded amino acids having different structures as acyl donor substrates that is catalyzed by thermoase in organic media was investigated. In these reactions, N-protected l--non-coded amino acids, including l-Orn, l-Cit, -aminobutyric acid (l--Abu) and phenylalanine homologues, were used as the acyl donors and phenylalanine derivatives were used as the acyl acceptors. This kind of enzymatic reactions cannot be carried out in an aqueous buffer due to the rigid specificity of proteases to coded amino acids in water. The results demonstrated that the substrate specificity of proteases could be broadened in organic solvents. In addition, the factors that influenced these protease-catalyzed reactions, including structures of the substrates, water content and the bases used, were systematically studied. Our work provided important evidence for broadening the application of protease in organic synthesis.     

Peptide synthesis catalyzed by proteases. Analysis of a kinetic model for enzymes with acyl-enzyme mechanism of action

The kinetics of peptide synthesis via transfer of the acyl moiety from activated derivatives of amino acids or peptides (S) to nucleophiles (N) catalyzed by proteases forming an acyl-enzyme intermediate, was analysed. A kinetic model assumes enzymatic hydrolysis of the formed peptide (P), so the kinetic curve for P has a maximum (denoted as pmax). Particular attention was given to the analysis of the effects of the initial concentrations and kinetic constants on pmax. Computer analysis demonstrated that at a given ratio of initial S and N concentrations pmax is affected only by the ratio of the second order rate constants for enzymatic hydrolysis of S and P (alpha) and the ratio of rate constants for an attack of the acyl-enzyme intermediate by nucleophile and water (beta). These conclusions apply regardless of the existence of enzyme forms other than a free enzyme and an acyl-enzyme intermediate. Thus, the kinetically controlled maximum yield of peptide (pmax) can be calculated a priori from the values of alpha and beta which can be readily evaluated from the reference data. Simple explicit expressions were obtained, allowing fairly accurate prediction of pmax for a broad spectrum of S and N initial concentrations.     

Enzymatic reactions in aqueous-organic media. VIII. Medium effect and nucleophile specificity in subtilisin-catalyzed peptide synthesis in organic solvents

Summary Subtilisin Carlsberg and subtilisin BPN' (nagarse) catalyze peptide bond formation from aromatic amino acid esters and glycinamide in hydrophilic organic solvents. The activities of subtilisin and product compositions are different in several organic solvents; reactions in acetonitrile, tetrahydrofuran, and propylene carbonate gave the peptide in excellent yields, while in N,N-dimethylformamide and methanol the enzyme activity was largely retarded. The yield of the peptide is also dependent on water content in the reaction solutions. Optimum water contents are in the range from 3 to 7 %. The reaction is strongly specific for glycinamide as an amine component, and amides of alanine, valine, and leucine gave the corresponding peptides in poor yields.     

Acyl group transfer by proteases forming acyl-enzyme intermediate: kinetic model analysis

A kinetic model of peptide synthesis via transfer of the acyl moiety from activated derivatives of amino acids (S) to nucleophiles (N) catalyzed by proteases forming an acyl-enzyme intermediate has been analyzed. The kinetic model takes into account the subsequent enzymatic hydrolysis of synthesized peptide (P), and so the kinetic curve for this compound shows a maximum (denoted as p(max)). Particular stress is placed on analyzing the effects of initial concentrations and of kinetic constants on the value of p(max).The analysis has demonstrated that at a given ratio of initial S and N concentrations, p(max) is affected only by (i) the ratio of the second-order rate constants for enzymatic hydrolysis of S and P(alpha) and (ii) the ratio of rate constants for an attack of the acyl-enzyme intermediate by the nucleophile and water (beta). These conclusions apply regardless of the existence of linear inhibition by the components of the reaction mixture. Thus, the kinetically controlled maximum yield of peptide (p(max)) can be calculated a priori from values of alpha and beta that can be estimated experimentally or from reference data. Simple analytical expressions were obtained, allowing a fairly accurate prediction of p(max) for a broad spectrum of S and N initial concentrations.     

Native and modified subtilisin Karlsberg as an effective catalyst of peptide bond formation in organic media

The activity and stability of native subtilisin Karlsberg and subtilisin 72 and their complexes with sodium dodecyl sulfate (SDS) in organic solvents were studied. The kinetic constants of the hydrolysis of specific chromogenic peptide substrates Z- ALA-Ala-Leu-pNA and Glp-Ala-Ala-Leu-pNA by the subtilisins were determined. It was found that the subtilisin Karlsberg complex with SDS in anhydrous organic solvents is an effective catalyst of peptide synthesis with multifunctional amino acids in positions P1 and P'1 (Glu, Arg, and Asp) containing unprotected side ionogenic groups.     

Dipeptide derivative synthesis catalyzed by Pseudomonas aeruginosa elastase.

Pseudomonas aeruginosa elastase was used to synthesize various N-protected dipeptide amides. The identity of the products was confirmed by FAB(+)-MS. After recrystallization, the yield of their synthesis was calculated, their purity was checked by RP-HPLC and their melting point was measured. With regard to the hydrolysis, it is well-established that the enzyme prefers hydrophobic amino acids in P'1 position and it has a wide specificity for the P1 position. This specificity was demonstrated to be quite unchanged when comparing the initial rates of peptide bond formation between different carboxyl donors (Z-aa) and nucleophiles (aa-NH2). The elastase, but not the thermolysin, was notably able to incorporate tyrosine and tryptophan in P'1 position. Furthermore, synthesis initial rates were at least 100 times faster with the elastase. To overcome the problematic condensation of some amino acids during chemical peptide synthesis, it has been previously suggested that enzymatic steps can combine with a chemical strategy. We demonstrated that the elastase readily synthesizes dipeptide derivatives containing various usual N-protecting groups. It was especially able to condense phenylalaninamide to Fmoc- and Boc-alanine. Increasing interest in peptides containing unnatural amino acids led us to try the elastase-catalyzed synthesis of Z-dipeptide amides including those amino acids in the P1 position. A synthesis was demonstrated with alphaAbu, Nle, Nva and Phg.     

Engineering subtilisin and its substrates for efficient ligation of peptide bonds in aqueous solution

Protein engineering techniques were used to construct a derivative of the serine protease subtilisin that ligates peptides efficiently in water. The subtilisin double mutant in which the catalytic Ser221 was converted to Cys (S221C) and Pro225 converted to Ala (P225A) has 10-fold higher peptide ligase activity and at least 100-fold lower amidase activity than the singly mutated thiolsubtilisin (S221C) that was previously shown to have some peptide ligase activity [Nakatsuka, T., Sasaki, T., & Kaiser, E.T. (1987) J. Am. Chem. Soc. 109, 3808-3810]. A 1.5-A X-ray crystal structure of an oxidized derivative of the double mutant (S221C/P225A) supports the protein design strategy in showing that the P225A mutation partly relieves the steric crowding expected from the S221C substitution, thus accounting for its improved catalytic efficiency. Stable and synthetically reasonable alkyl ester peptide substrates were prepared that rapidly acylate the S221C/P225A enzyme, and aminolysis of the resulting thioacyl-enzyme intermediate by various peptides is strongly preferred over hydrolysis. The efficiency of aminolysis is relatively insensitive to the sequence of the first two residues in the acyl acceptor peptide whose alpha-amino group attacks the thioacyl-enzyme. To obtain greater flexibility in the choice of coupling sites, a set of three additional peptide ligases were engineered by introducing mutations into the parent ligase (S221C/P225A) that were previously shown to change the specificity of subtilisin for the residue nearest the acyl bond (the P1 residue). The specificity properties of the parent ligase and derivatives of it paralleled those of wild type and corresponding specificity variants.(ABSTRACT TRUNCATED AT 250 WORDS)     

Trypsin-specific acyl-4-guanidinophenyl esters for alpha-chymotrypsin-catalysed reactions computational predictions, hydrolyses, and peptide bond formation.

The function of acyl-4-guanidinophenyl esters as substrate mimetics for the serine protease alpha-chymotrypsin was investigated by protein-ligand docking, hydrolysis, and acyl transfer experiments. On the basis of protein-ligand docking studies, the binding and hydrolysis properties of these artificial substrates were estimated. The predictions of the rational approach were confirmed by steady-state hydrolysis studies on 4-guanidinophenyl esters derived from coded amino acids (which alpha-chymotrypsin is not specific for), noncoded amino acids, and even simple carboxylic acid moieties. Enzymatic peptide syntheses qualify these esters as suitable acyl donors for the coupling of acyl components far from the natural enzyme specificity, thus considerably expanding the synthetic utility of alpha-chymotrypsin.     

Characterization Of The S' Subsite Specificity Of Trypsin

The S' subsite specificity of bovine trypsin has been studied by partitioning of o-nitrophenylsulfenyl-L-arginyl-trypsin (formed using o-nitrophenylsulfenyl-L-arginine alkyl esters as acyl donors) between various amino acid-derived nucleophiles and water. The data obtained from spectrophotometric measurements confirmed a preference of trypsin for arginine residues in the P'₁-position, which is less marked but quite similar to that of chymotrypsin. The amides of leucine, phenylalanine, methionine, threonine, lysine and valine are better for synthesis than the corresponding methyl esters, and show a moderate nucleophile efficiency, decreasing in that order. Amides of acidic amino acids and D-leucine were ineffective in forming the peptide bond, whereas norvaline amide and dipeptide amides lead to increased aminolysis.     

Enzymatic peptide synthesis by the recombinant proline-specific endopeptidase from Flavobacterium meningosepticum and its mutationally altered Cys-556 variant

Proline-specific endopeptidase (PSE) (EC 3.4.21.26) was investigated for its potential as a catalyst in peptide synthesis. Using an activated peptide ester or a peptide amide as the acyl component, the enzyme catalyzed kinetically controlled aminolysis and transpeptidation respectively, with various amino acid amides as acyl acceptors. To a certain extent the nucleophile preference reflected the amino acid preference in the S₁-position of the enzyme in peptide hydrolysis: the highest fractions of aminolysis were obtained using amino acid amides with hydrophobic side-chains (e.g. Leu-NH₂, Phe-NH₂). PSE also catalyzed the thermodynamically controlled condensation of short peptides with a free carboxyterminus and various amino acid amides. This enabled us to examine the acceptance of different acyl components in the substrate-binding site of the enzyme with regard to their amino acid composition: In the S₁ position proline was clearly favored, but alanine was also accepted, whereas the S₂ subsite accepted various amino acids rather unspecifically. Since PSE was shown to be extremely sensitive against water-miscible organic solvents, an alternative approach was used to increase yields in enzymatic peptide synthesis: a derivative of PSE in which the catalytic Ser-556 is converted to a Cys was constructed by protein engineering. This mutant (PSE_cys) exhibited a dramatically increased peptide ligase activity in aqueous solution.