Fmoc-Based Synthesis of Peptide α-Thioesters Using an Aryl Hydrazine Support

Abstract

C-Terminal peptide thioesters are key intermediates in the synthesis/semisynthesis of proteins and of cyclic peptides by native chemical ligation. They are prepared by solid-phase peptide synthesis (SPPS) or biosynthetically by protein splicing techniques. Until recently, the chemical synthesis of C-terminal alpha-thioester peptides by SPPS was largely restricted to the use of Boc/Benzyl chemistry due to the poor stability of the thioester bond to the basic conditions required for the deprotection of the N(alpha)-Fmoc group. In the present work, we describe a new method for the SPPS of C-terminal thioesters using Fmoc/t-Bu chemistry. This method is based on the use of an aryl hydrazine linker, which is totally stable to conditions required for Fmoc-SPPS. When the peptide synthesis has been completed, activation of the linker is achieved by mild oxidation. This step converts the acyl hydrazine group into a highly reactive acyl diazene intermediate which reacts with an alpha-amino acid alkyl thioester (H-AA-SR) to yield the corresponding peptide alpha-thioester in good yield. This method has been successfully used to prepare a variety of peptide thioesters, cyclic peptides, and a fully functional Src homology 3 (SH3) protein domain.