Utilization of Proteases from Aspergillus Species for Peptide Synthesis via the Kinetically Controlled Approach

We examined Aspergillus melleus protease (Amano protease P) and A. oryzae protease (Amano protease A) as catalysts for peptide bond formation via the kinetically controlled approach. As the coupling efficiency was only moderate, even with a good amino acid substrate as the carboxyl component, in acetonitrile as a solvent (with or without a small amount of added water) that we had mainly employed previously in α-chymotrypsin catalyzed couplings, other solvent systems were sought. In 1,1,1,3,3,3-hexafluoro-2-propanol-DMF (1:1) without added water, these Aspergillus proteases were found to remain active for a long period of time and to be utilizable for peptide synthesis when the carbamoylmethyl ester was employed as the acyl donor, though the coupling efficiencies were dependent rather largely on the combination of the amino acid residues at the coupling site. The superiority of the carbamoylmethyl ester to conventional esters, for example the methyl ester, was once again established. Furthermore, some segment condensations were also achieved by the same procedure.