[Nonspecific trypsin substrates in the enzymatic synthesis of peptides]. / Nespetsificheskie substraty tripsina v fermentativnom sinteze peptidov.

Trypsin--catalyzed coupling of peptide segments in aqueous medium was studied. Carboxamido methyl esters (Cam esters) of peptides were used as acyl donors. Peptide segments involved in the coupling do not contain positively charged residues (Lys, Arg). Hydrophobic amino acids (except isoleucine and valine) are preferable as C-terminals in the peptide Cam esters used for the reaction. The coupling cannot take place if glutamic or aspartic acids occupy positions 1 or 2 in the amine component. Threefold excess of Cam ester provides a high yield of the coupling product. The coupling is free of secondary hydrolysis. A series of water soluble peptides (from hepta- to tetradecapeptides) were synthesized by this method.

Protease-catalyzed peptide synthesis using inverse substrates: the synthesis of Pro-Xaa-bonds by trypsin.

Benzyloxycarbonyl-L-proline p-guanidinophenyl ester is an "inverse substrate" for trypsin; i.e., the cationic center is included in the leaving group instead of being in the acyl moiety. This substrate can be used in trypsin-catalyzed acyl-transfer reactions leading to the synthesis of Pro-Xaa peptide bonds. The reaction proceeds about 20 times slower than reaction with similar alanine-containing substrates, but the ratio between synthesis and hydrolysis is more favorable. The investigation of a series of nucleophiles led to information about the specificity of the process. Nucleophiles differing only in the P(1)'-position show an increasing acyl transfer efficiency in the order Phe < Gly < Ley < Ser < Ala < lle. C terminal elongation of the nucleophiles is of minor influence on their efficiency. The formation of an H bond between the acyl-enzyme and the nucleophile seems to play an important role in the aminolysis of the acyl-enzyme.

Protease-catalyzed peptide synthesis using inverse substrates: the influence of reaction conditions on the trypsin acyl transfer efficiency.

Benzyloxycarbonyl-L-alanine p-guanidinophenyl ester behaves as a trypsin "inverse substrate," i.e., a cationic center is included in the leaving group instead of being in the acyl moiety. Using this substrate as an acyl donor, trypsin catalyzes the synthesis of peptide bonds that cannot be split by this enzyme. An optimal acyl transfer efficiency was achieved between pH 8 and 9 at 30 degrees C.The addition of as much as 50% cosolvent was shown to be of minor influence on the acyl transfer efficiency, whereas the reaction velocity decreases by more than one order of magnitude. The efficiency of H-Leu-NH(2) and H-Val-NH(2) in deacylation is almost the same for "inverse" and normal type substrates.

New approaches to peptide synthesis with the help of trypsin.

Picolyl esters of amino acids are suitable inverse substrates for trypsin. These esters can be used for peptide synthesis catalyzed by trypsin. Study of hydrolysis of non-specific substrates for trypsin permits to conclude, that hydrogen band in P'2 subsite is very important for their reactivity. Carboxamido methyl esters (CAM-esters) are the most suitable substrates for trypsin catalyzed peptide synthesis. Using CAM-esters and trypsin several di-,tri- and tetra-peptides were synthesized with good yields.

Thio-alpha-amino acids (S-acids) as a carboxyl component in peptide synthesis catalysed by papain.

Peptide synthesis catalysed by papain was studied using thio-alpha-amino acids (S-acids) as a carboxyl component. It was found, for example, that with Z-AlaSH (pK 2.70) the maximal yield of the peptide Z-AlaValNH2 was obtained at pH 8-8.5. A two-fold excess of Z-AlaSH furnished peptides with yields close to 100%. Thio-amino acids with bulky side groups, for example, Z-IleSH, Z-Asp(OBu')SH, gave peptides with a low yield. Papain interacts with Z-AlaSH better than do bromelain or ficin.

[Synthesis of tetracontapeptide--a hypothetical evolutionary precursor of calcium-binding proteins. I. Synthesis of (1-9), (10-14), (15-20), (21-26), (29-33), (34-40) fragments]. / Sintez tetrakontapeptida--gipoteticheskogo évoliutsionnogo predshestvennika kal'tsiisviazyvaiushchikh belkov. I. Sintez fragmentov (1-9), (10-14), (15-20), (21-26), (29-33), (34-40).

Fragments (1-9), (10-14), (15-20), (21-26), (29-33) and (34-40) of a tetracontapeptide hypothetical ancestor of calcium-binding proteins were synthesised with the use of pentafluorophenyl esters. Formation of a succinimide derivative was detected during synthesis of fragment (15-20) containing Asp(OBzl)-Gly sequence. To avoid this side process, tert-butylprotecting group was used instead of benzyl group. alpha-Carboxyls of C-terminal amino acids were protected by phenacyl group.

[Synthesis of leucine- and methionine-enkephalin using papain]. / Sintez leitsin- i metioninénkefalina s ispol'zovaniem papaina.

Thiol protease papain has been used for synthesis of leucine-and methionineenkephalin from methyl esters of N-protected amino acids. The synthesis was carried out in basic medium, minimizing the hazard of the secondary peptide hydrolysis. The reaction products remain in solution during the whole process. The yields at the final stage of the synthesis were 89% (leucineenkephalin) and 79% (methionineenkephalin).

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.