Chemo-enzymatic synthesis of precursor tetrapeptide Bz–RGDS–NH 2 of cellular adhesion motif in low-water organic media

Abstract

The tetrapeptide Bz–Arg–Gly–Asp–Ser–NH 2 (Bz–RGDS–NH 2) 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–NH 2 (GDS–NH 2) 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 ClCH 2COAsp–SerOMe and ammonolysis of ClCH 2COAsp–SerOMe. Secondly, trypsin-catalyzed synthesis of Bz–RGDS–NH 2 using Bz–Arg–OEt (Bz–R–OEt) as the acyl donor and GDS–NH 2 as the nucleophile under kinetic control in low-water organic media was carried out. The reaction conditions optimized are pH 8.0, 30 °C, 14 h, in ethanol/Tris–HCl buffer system (97:3, v/v). The yield of Bz–RGDS–NH 2 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 °C, 4 × 10 −4–1 × 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.