Novel folding-assisted thioesterification method for semi-synthetic study of homogeneous glycoproteins

概要

Preparation of glycoproteins having homogeneous oligosaccharides is essential approach for understanding the glycan function at molecular level. However, current mammalian cell expression system is usually not able to yield such homogeneous form. Moreover, total chemical synthesis using solid phase peptide synthesis (SPPS) is time-consuming. Semi-synthesis combining E. coli expression and chemical synthesis appears to be an efficient method toward homogeneous glycoproteins. However, the current methods for expressed peptide thioesterification, which is essential for semi-synthesis, is sometimes incompatible toward synthesis of complex sequence.

In this research project, we have been establishing a novel semi-synthetic strategy without using SPPS in order to obtain homogeneous glycoprotein within a few chemical conversion steps. IL-6 having N-glycan at Asn143 was chosen as target glycoprotein. In this strategy, the challenging expressed-peptide thioesterification of N-terminal segment consist of 141 residues was achieved by a newly developed foldingassisted thioesterification method. (figure 1) In this method, peptide hydrazide derivative was obtained as a versatile precursor that could be utilized for various ligation method. The glycopeptide was prepared by thioacid capture ligation using chemically synthesized asialylglycodipeptide and an expressed peptide. The final ligation step which afforded fulllength glycosylated IL-6 was examined using thioester, selenoester or thioacid derivative which were prepared via folding-assisted thieosterification. (figure 2)