Enzymatic synthesis of peptides containing unnatural amino acids

Several proteases were studied as potential catalysts for the enzymatic synthesis of oligopeptides containing the unnatural amino acid allylglycine, the overall objective being the synthesis of a reactive tetrapeptide that could be chemically polymerized into a potentially biocompatible or biodegradable material. Commercially available enzymes were screened for esterase activity toward the methyl ester of the amino acid allylglycine (DL-AgOMe) to identify potential catalysts for dipeptide synthesis. Proteases from Aspergillus oryzae and Aspergillus sojae, pronase E and protease Nagarse synthesized the protected dipeptide Cbz-allylglycine-phenylalaninamide (Cbz-L-Ag-L-PheNH2) from Cbz-DL-AgOMe and L-PheNH2. However, the same enzymes were not able to catalyze the synthesis of Cbz-phenylalanine-allylglycine ethyl ester (Cbz-L-Phe-L-AgOEt). Thus, although these enzymes could use allylglycine as the acyl donor they could not employ it as the acyl acceptor in peptide synthesis. In contrast, chymotrypsin was able to use allylglycine ethyl ester (DL-AgOEt) as the acyl acceptor in the synthesis of Cbz-L-Phe-L-AgOEt, but was not able to synthesize Cbz-L-Ag-L-PheNH2. The two dipeptides, Cbz-allylglycine-phenylalanine and phenylalanine-allylglycine ethyl ester, served as substrates for the thermolysin-catalyzed synthesis of the tetrapeptide Cbz-L-Ag-L-Phe-L-Phe-L-AgOEt.