ENZYMATIC CONDENSATION OF CHOLECYSTOKININ CCK-8(4-6) AND CCK-8(7-8) PEPTIDE-FRAGMENTS IN ORGANIC MEDIA

The kinetically controlled condensation reaction of Z-Gly-Trp-Met-OR1 (R-1: Et, Al, Cam) and H-Asp-(OR2)-Phe-NH2 (R-2: H, Bu-t) catalyzed by alpha-chymotrypsin deposited onto polyamide in organic media was stud ied. The effect of the drying process of the enzyme-support preparatio n, substrate concentrations, reaction medium, acyl donor, and nucleoph ile structure on both enzymatic activity and pentapeptide yield was in vestigated. The immobilized preparation directly equilibrated at a(w) = 0.113, gave higher enzymatic activities than dried with vacuum first , and then equilibrated at a(w) = 0.113. The addition of triethylamine to the reaction medium increased dramatically the enzymatic activity. However, the pentapeptide yield was affected neither by the drying pr ocedure nor by the addition of triethylamine. The donor ester Z-Gly-Tr p-Met-OAI gave initial reaction rates 2.6 times higher than the conven tional ethyl ester derivative but rendered similar yields. The best re sults were obtained using Z-Gly-Trp-Met-OCam as acyl-donor eater; 80% yield and initial reaction rates 4 times higher than the ethyl ester d erivative. In all cases, acetonitrile containing Tris-HCl 50 mM pH 9 b uffer (0.5% v/v) and triethylamine (0.5% v/v) was found to be the best reaction system. Under these conditions, it was possible to use the n ucleophile H-Asp-Phe-NH2 with beta-unprotected aspartic acid residue. In this case, 50% yield was obtained, but economic considerations coul d lead to select it as nucleophile. Finally, the fragment condensation reaction was carried out at gram scale, obtaining a 39% yield which i ncluded the reaction, removal of protecting groups and purification st eps. (C) 1997 John Wiley & Sons, Inc.