The tripeptide Bz-Arg-Gly-Asp(-OMe)-OH was synthesized by enzymatic method. Bz-Arg-Gly-OEt was synthesized by trypsin in ethanol containing 0.1 M Tris/HCl buffer (pH 8.0), and then H-Asp(-OMe)(2) was incorporated into thr Bz-Arg-Gly-OEt using chymopapain in 0.25M CHES/NaOH buffer (pH = 9.0. EDTA 10 mM). The yield of Bz-Arg-Gly-OEt and Bz-Arg-Gly-Asp(-OMe)-OH were 80% and 70%: using 1M Bz-Arg-OEt and 0.5M Bz-Arg-Gly-OEt, respectively. For Br-Arg-Gly-OEt synthesis reaction at high concentrating of the substrates, the buffer content in ethanol was a key factor to determine the optimal reaction condition. Tn Bz-Arg-Gly-Asp(-OMe)-OH synthesis reaction, the yield was low in organic solvent due to various side products such as Bz-Arg-On, Bz-Arg-Gly-OH, and Bz-Are-Gly-Asp(-OMe)-Asp(-OMe)-OH, suggesting that chymopapain has a very broad substrate specificity of the S-1 site. The Br-Arg Gly-Asp(-OMe)-OH synthesis rate and its yield were dramatically elevated and the side reactions were reduced using only the CHES/NaOH buffer (pH = 9.0, EDTA 10 mM) as a reaction media. The final product Bz-Arg-Gly-Asp(-OMe)-OH was identified to bt formed via C-terminal hydrolysis of Bz-Arp-Gly-Asp(-OMe)(2) after the nucleophile. H-Asp(-OMe2), was added. (C) 2000 Elsevier Science Inc. All rights reserved.