Subtilisin and α-chymotrypsin catalyzed synthesis of peptides containing arginine and lysine p-nitroanilides as c-terminal moieties

p-Nitroanilides of N-acylated di-, tri- and tetrapeptides with C-terminal arginine or lysine residues have been obtained, as a rule with good yields, via acylation of arginine or lysine p-nitroanilides by methyl esters of respective N-acylated peptides, catalyzed by subtilisin or α-chymotrypsin. The synthesis might be performed by two routes-by reaction in water-organic solvent mixtures, catalyzed by dissolved enzyme, or by condensation of the components in organic solvents with low water content in the presence of any enzyme distributed over a silica support surface. The second approach seems to be preferable due to suppression of hydrolytic side reactions and improved stability of an enzyme. Subtilisin 72 is especially effective as a catalyst for the acylation of p-nitroanilides by N-protected tripeptide methyl esters-the derivatives capable of occupying the S 1, S 2 and S 3 subsites of its extended binding site. Even dipeptide esters with d-amino acids in P 2 position can be applied for p-nitroanilide acylation. The efficiency of α-chymotrypsin as a catalyst for peptide synthesis is more limited due to restricted specificity of this enzyme. Enzymatic acylation of argininge or lysine p-nitroanilides by peptide methyl esters give chromogenic substrates for proteinases with trypsin-like specificity.