The tetrapeptide Bz-Arg-Gly-Asp-Ser-NH2 (Bz-RGDS-NH2) as a precursor of cellular adhesion motif of RGDS was synthesized by a combination of chemical and enzymatic methods in this study for the first time. First of all; the precursor tripeptide Gly-Asp-Ser-NHZ (GDS-NHz) was synthesized by a novel chemical method in four steps including chloroacetylation of L-aspartic acid, synthesis of chloroacetyl L-aspartic acid anhydride, synthesis of CICH(2)COAsp-SerOMe and ammonolysis of CICH(2)COAsp-SerOMe. Secondly, trypsin-catalyzed synthesis of Bz-RGDS-NH2 using Bz-Arg-OEt (Bz-R-OEt) as the acyl donor and GDS-NH2 as the nucleophile under kinetic control in low-water organic media was carried out. The reaction conditions optimized are pH 8.0, 30 degrees C, 14h, in ethanol/Tris-HCl buffer system (97:3, v/v). The yield of Bz-RGDS NH2 is 68.3 1.74% for pre-treated trypsin prepared by dissolving trypsin powder in 0.1 M Tris-HCl buffer (pH 8.0) and lyophilizing for 24 h in a freeze-drier with -55 degrees C, 4 x 10(-4)-1 x 10(-2) mbar. Compared to trypsin powder, pre-treated trypsin (pH memory) displays higher catalytic activity and stability in low-water organic media, the yield at pH 8.0 increased by 7%. The secondary hydrolysis of the tetrapeptide product did not take place in this low-water organic medium. (c) 2006 Elsevier Inc. All rights reserved.