Semisynthesis of a Homogeneous Glycoprotein Using Chemical Transformation of Peptides to Thioester Surrogates.

Semisynthesis using recombinant polypeptides as building blocks is a powerful approach for the preparation of proteins with a variety of modifications such as glycosylation. The activation of the C terminus of recombinant peptides is a key step for coupling peptide building blocks and preparing a full-length polypeptide of a target protein. This article reports two chemical approaches for transformation of the C terminus of recombinant polypeptides to thioester surrogates. The first approach relies on efficient substitution of the C-terminal Cys residue with bis(2-sulfanylethyl)amine (SEA) to yield peptide-thioester surrogates. The second approach employs a native tripeptide, cysteinyl-glycyl-cysteine (CGC), to yield peptide-thioesters via a process mediated by a thioester surrogate. Both chemical transformation methods employ native peptide sequences and were thereby successfully applied to recombinant polypeptides. As a consequence, we succeeded in the semisynthesis of a glycosylated form of inducible T cell costimulator (ICOS) for the first time.

Effects of N-Glycans on Glycoprotein Folding and Protein Dynamics.

This chapter describes the folding of synthetic homogeneous glycosylpolypeptides into glycoproteins depending on the position and number of glycosylation sites and oligosaccharide structures. To evaluate the role of oligosaccharides in protein folding, we synthesized small glycoprotein models, homogeneous misfolded glycoproteins, and erythropoietins. In addition to these chemical syntheses, this chapter introduces a unique method for 15N-labeling of synthetic glycoproteins to enable structural analysis. Based on experimental results, it can be suggested that N-glycans stabilize the structure of glycoproteins.