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.
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.
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 catalysed by papain was studied using thio‐α‐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(OBut)SH, gave peptides with a low yield. Papain interacts with Z‐AlaSH better than do bromelain or ficin.
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