Semi-synthesis of ICOS by utilizing novel thioesterification approach by peptide-SEA
In the semi-synthesis of ICOS, the Okamoto and Kajihara group applied peptide-Cys thioesterification by using SEA (Fig.16).[42] ICOS is a co-stimulator for the activation of T cells and has N-glycosylation sites in the extracellular domain. The sequence of ICOS consists of 106 amino acids including two biantennary sialyl oligosaccharide at Asn 89 and Asn110. A convergent synthetic strategy was established for glycosyl ICOS using a recombinant polypeptide thioesterification as a key reaction. They employed three peptide segments and two sequential NCL to obtain a full-length glycoprotein. ICOS segment 141 (1-62) was co-expressed with Small Ubiquitin-related Modifier (SUMO) and six His (His Tag) sequences by E. coli expression system. Thioesterification applied SEA was progressed convergently after cleavage of SUMO sequences by SUMO protease to afford peptide-SEA 145. Obtained peptide-SEA 145 was subjected to NCL with glycopeptide 146 prepared from glycopeptides thioester 142 (63-88) and cysteinyl peptide 143 (89-106) synthesized by a standard Boc SPPS protocol. In terms of glycopeptide-thioester 142, they employed Fmoc-SPPS. The resultant all segments was sequentially coupled by NCL to obtain a full-length glycopeptide of ICOS. Using stepwise dialysis conditions, folding process with redox reagents yielded folded glycosyl ICOS 147 (Fig.16). ELISA, CD and MS spectrum indicated that semi-synthesized ICOS correctly folded structure having β-sheet rich conformation. By using both chemical synthesis and semi-synthesis as appropriate, it might be possible to synthesize glycoproteins with larger molecular weights
