A Search for Peptide Ligase: Cosolvent-Mediated Conversion of Proteases to Esterases for Irreversible Synthesis of Peptides'

Serine and cysteine proteases (trypsin, chymotrypsin, papain, subtilisin) in the presence of certain concentrations of water-miscible organic solvents express no amidase activities. The esterase activities, however, remain significant. These modified enzymes can be used as peptide ligases in a kinetically controlled approach (aminolysis) for the stepwise synthesis and fragment coupling of peptides, the products of which are free from secondary hydrolysis. Preparative syntheses of peptides containing both Dand L-amino acids and penicillin precursors containing 0-methyl-D-allothreonine and a-aminoadipic acid under such conditions are demonstrated. A comparative study of the enzymatic synthesis of Z-Phe-Leu-NH, in anhydrous DMF and in aqueous DMF (50% water, v/v) indicates that the rate in aqueous DMF is >10000 times faster. Chymotrypsin, subtilisin, and Streptomyces griseus protease are inactive toward Z-Phe-OMe + Leu-NH2 in anhydrous DMF. The utilization of proteases as catalysts in peptide synthesis began early this century2 and is still an area of active r e ~ e a r c h . ~ Some of the processes have been commercialized; of particular importance is the production of aspartame4 and human i n s u h 5 The advantages of enzymatic peptide synthesis are freedom from racemization, minimal activation and side-chain protection, mild reaction conditions, high regioand stereoselectivity, and enzyme immobilization allowing for catalyst recovery in large-scale processes. Further, the reactions can be carried out in a mixture of organic solvent and water, devoid of the problem of low solubility of protected peptides in the organic solvents employed in chemical synthesis. With these advantages, however, come the disadvantages that the amidase activity of proteases causes a secondary hydrolysis of the growing peptides and that the substrates of proteases are generally limited to natural L-amino acid derivat i v e ~ . ~ ~ ~ Two strategies are generally used in enzymatic peptide synthesis: One is a thermodynamically controlled synthesis (Le., a direct reversal of the catalytic hydrolysis of peptides; eq I) , and the other RCO2H + H2NR' RCONHR' + H20 (1 ) a