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.     

Pepsin as a catalyst of peptide synthesis. Enzyme co-precipitation with emerging peptide products.

Pepsin successfully catalyzed the synthesis of several peptide derivatives from N-protected di- or tripeptides and amino acid or peptide esters or p-nitroanilides in dimethylformamide-water solutions at pH 4.6. An optimal substrates:pepsin ratio depended on the structure of starting peptides, especially their fit to the substrate binding sites of the enzyme. For hexapeptide Z-Ala-Ala-Phe-Leu-Ala-Ala-OCH3 formation, an equilibrium yield was attained at 1:3.10(5) enzyme-substrates ratio that indicated high efficiency of pepsin in synthesis reactions. In the course of the equilibrium peptide synthesis, pepsin gradually disappeared from the liquid phase due to its entrapment within a gel, formed by the hexapeptide product, while retaining its activity. The inclusion into the precipitate was not specific for pepsin, so far as inert proteins, lysozyme, ribonuclease A and carbonic anhydrase, when added to the reaction mixture, became also co-precipitated with the hexapeptide formed. It appears that co-precipitation of pepsin, an important factor limiting the enzyme efficiency, might be operative as well for other proteinases used to catalyze peptide synthesis.     

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.     

Agarose-chymotrypsin as a catalyst for peptide and amino acid ester synthesis in organic media

Summary Chymotrypsin immobilised within agarose gels by multi-point covalent attachment is a useful catalyst for peptide or amino acid ester synthesis in mainly organic media. Moist gel beads (optionally containing one reactant) may be suspended in organic phases based on ethyl acetate, 1,1,1-trichloroethane or pentan-3-one.     

alpha-Chymotrypsin in plastein synthesis: influence of substrate concentration on enzyme activity.

The role of alpha-chymotrypsin in the plastein reaction was studied using a peptic hydrolysate of albumin as substrate. Study of this reaction simultaneously by different methods showed that the plastein reaction is enzyme catalyzed and is highly dependent on environmental conditions. A gel permeation chromatography study of the plastein reaction showed simultaneous increases in the high- and low-molecular-weight oligopeptide fractions; a transpeptidation mechanism may be involved in the reaction. A study of the effect of substrate concentration on the plastein reaction catalyzed by alpha-chymotrypsin showed a profile with both hydrolytic and synthetic activities. This effect was also observed when the reaction course was followed by quantification of the free amino groups at different substrate concentrations, showing that a condensation mechanism is responsible for the synthetic activity when the substrate concentration is very high. These results have led us to conclude that the plastein reaction involves a transpeptidation and/or condensation mechanism, which is a function of the substrate concentration.     

Catechol as a nucleophilic catalyst of peptide bond formation.

The aminolysis of a mildly activated aminoacid ester, benzyloxycarbonyl-L-phenylalanine cyanomethyl ester, by glycine esters in the presence of catechol has been studied as a model of catalysis by RNA cis-vicinal-diol systems in protein biosynthesis. Catechol accelerated the aminolysis, especially in the presence of bases, probably by nucleophilic catalysis.     

MenD as a versatile catalyst for asymmetric synthesis

The thiamine diphosphate (ThDP)-dependent enzyme 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate synthase (MenD) from Escherichia coli K12, formerly known as SHCHC-synthase, catalyses the decarboxylation of α-ketoglutarate and the subsequent addition of the resulting succinyl-THDP to isochorismate. Here, the enzyme is tested for unphysiologial C–C bond-forming reactions.Condensation of α-ketoglutarate after decarboxylation to a broad range of aldehydes gave α-hydroxyketones with isolated yields from 26 to 87% and 94 to 98% ee for addition to aromatic aldehydes. MenD accepts a wide range of aldehydes as acceptor substrates to produce chiral α-hydroxyketones with conserved regioselectivity where the activated succinylsemialdehyde serves selectively as the donor. Regioselectivity is inverted only for condensation of α-ketoglutarate with pyruvate (activated acetaldehyde) as donor. Besides α-ketoglutarate, pyruvate and oxalacetate are accepted as donors in combination with benzaldehyde and 2-fluorobenzaldehyde as acceptors, however with decreased activity of C–C bond formation.The physiological 1,4-addition of α-ketoglutarate to isochorismate was investigated for acceptor substrate variability. (2S,3S)-2,3-Dihydroxy-2,3-dihydrobenzoate (2,3-CHD), which lacks the pyruvyl found in isochorismate, is converted to (5S,6S)-2-succinyl-5,6-dihydroxycyclohex-2-enecarboxylate. In contrast to the addition to carbonyls, the active site of MenD does appear to impose specific constraints on the acceptor substrate for 1,4-addition with α,β-unsaturated carboxylic acids.     

Enzymatic reactions in aqueous-organic media. IV. Chitin-alpha-chymotrypsin complex as a catalyst for amino acid esterification and peptide synthesis in organic solvents

Summary Chitin--chymotrypsin complex was found to be an effective catalyst for the esterification of N-acetyl-L-tryptophan in ethanol and for peptide synthesis from N-acetyl-L-tyrosine and glycinamide in acetonitrile. In the former reaction both initial rate of ester formation and equilibrium yield of the ester markedly increased by the complexation of chymotrypsin with chitin compared to free chymotrypsin.     

Comparison of methods for thermolysin-catalyzed peptide synthesis including a novel more active catalyst

This is a comparative study of the performance of thermolysin for enzymatic peptide synthesis by reversed hydrolysis in several different reaction systems. Z-Gln-Leu-NH(2) was synthesized in acetonitrile containing 5% water (with various catalyst preparation methods) as well as by the "solid-to-solid" and frozen aqueous methods. Reaction rates (values in nanomoles per minute per milligram) in acetonitrile depended significantly on the method of addition of enzyme: (a) direct suspension in the reaction mixture as freeze-dried powders gave 60 to 95; (b) addition as an aqueous solution, so that enzyme precipitates on mixing with acetonitrile, gave 230; (c) addition as an aqueous suspension gave a remarkable increase in reaction rates (up to 780); (d) immobilized enzymes (adsorbed at saturating loading on celite, silica, Amberlite XAD-7, or polypropylene, then dried by propanol rinsing) all gave <230. It is postulated that, starting with the enzyme already in the form of solid particles in aqueous buffer, there is a minimum chance of alteration of its optimal conformation during transfer to the organic medium. For solid-to-solid synthesis with 10% water content we found initial rates of 670 under optimized conditions. In frozen aqueous synthesis, rates were <10. Equilibrium yields were always around 60% in low water organic solvent, whereas they were found to >80% in the aqueous systems studied.     

Native and modified subtilisin 72 as a catalyst of peptide synthesis in media with low water content

The catalytic efficiencies of native subtilisin, its noncovalent complex with polyacrylic acid, and the subtilisin covalently immobilized in a cryogel of polyvinyl alcohol were studied in the reaction of peptide coupling in mixtures of organic solvents with a low water content in dependence on the medium composition, reaction time, and biocatalyst concentration. It was established that, in media with a DMF content > 80%, the synthase activity of modified subtilisins is higher than that of the native subtilisin. The use of N-acylpeptides with a free carboxyl group was found to be possible in organic solvents during the enzymatic synthesis catalyzed by both native and immobilized subtilisin. A series of tetrapeptide p-nitroanilides of the general formula Z-Ala-Ala-Xaa-Yaa-pNA (where Xaa is Leu, or Glu and Yaa is Phe or Asp) was obtained in the presence of immobilized enzyme in yields of 70-98% in DMF-MeCN without any activation of the carboxyl component and without protection of side ionogenic groups of polyfunctional amino acids.     

Acetophenone-based linker for solid-phase peptide synthesis.

A new and cost-effective linker for the generation of carboxylic acid end groups on Multipin supports (SynPhase crowns) has been developed. Synthesis of the linker was based on modification of grafted polystyrene (PS) crowns to generate a hydroxyethyl moiety which is acid labile in 10-20% trifluoroacetic acid (TFA) in dichloromethane (DCM). Solid-phase syntheses of model decapeptides using this linker are described.     

New hydrogenation catalyst: An advantageous method for the removal of hydrogenolysable protecting groups in peptide synthesis

Summary Removal of some commonly used protecting groups in peptide synthesis by catalytic transfer hydrogenation employing ammonium formate and magnesium is described. This method is equally competitive with other methods in deblocking most of the commonly used protecting groups in peptide synthesis.tert-Butyl derived and base labile protecting groups were completely stable under these conditions. The use of ammonium formate and magnesium makes this a rapid, low-cost alternative to palladium and reduces the work-up to a simple filtration and extraction operation.     

New hydrogenation catalyst: An advantageous method for the removal of hydrogenolysable protecting groups in peptide synthesis

Removal of some commonly used protecting groups in peptide synthesis by catalytic transfer hydrogenation employing ammonium formate and magnesium is described. This method is equally competitive with other methods in deblocking most of the commonly used protecting groups in peptide synthesis. tert-Butyl derived and base labile protecting groups were completely stable under these conditions. The use of ammonium formate and magnesium makes this a rapid, low-cost alternative to palladium and reduces the work-up to a simple filtration and extraction operation.     

Soft shell resins for solid-phase peptide synthesis.

Soft shell (SS) resins with a lightly or noncross-linked shell layer were prepared by reducing the amount of cross-linking agent, divinylbenzene (DVB), during seed suspension polymerization from polystyrene (PS) resin. These SS resins have a lower swelling volume than that produced by normal cross-linking. Despite its lower swelling, however, SS (10-00) resin, which consists of the 1% DVB-cross-linked core and the noncross-linked surface layer, showed higher efficiency in peptide synthesis compared with 1% DVB-PS resin and other SS resins.     

New tris-alkoxycarbonyl arginine derivatives for peptide synthesis.

alpha-Alkoxycarbonyl protected ornithines were treated with N,N'-[Z(2Cl)](2)-S-methylisothiourea and N,N'-[Z(2Br)](2)-S-methylisothiourea, N,N'-Z(2)-S-methylisothiourea and N,N'-Boc(2)-S-methylisothiourea to form N(alpha, omega, omega')-tris-alkoxycarbonyl arginines. Two of them, Boc-Arg-{omega,omega'-[Z(2Br)](2)}-OH and Boc-Arg-{omega,omega'-[Z(2Cl)](2)}-OH, were used for the synthesis of dermorphin fragments containing two or three arginine residues. Examination of the products by HPLC and ESI-MS revealed that the purity of the materials obtained with the use of the new derivatives was higher than that obtained in concurrent syntheses in which Boc-Arg(Tos) was used.     

Application of carboxypeptidase C for peptide synthesis.

Carboxypeptidase C partially purified from the flavedo of citrus fruit by a new, simple procedure was studied as a catalyst for peptide-bond formation. Dipeptides were obtained in high yields (80-95%) with Bz--Tyr--OEt as carboxyl-compound, and amino acid amides and amino acid alkylesters as nucleophiles. To characterize the synthesis reaction, a number of parameters such as pH, excess of the nucleophile, and the molarity of the buffer were evaluated. The yield of dipeptides depends on the side chain of the amino acid alkylester used as the carboxyl component as well as on the N-terminal protecting group. Esterase activity was minimal in the absence of a nucleophile, suggesting a modified mechanism for the synthesis reaction compared to other serine proteases. No secondary hydrolysis of the peptides formed was observed.