Antifreeze glycoprotein (AFGP) is a mucin type O-glycoprotein consists of Ala-Thr-Ala triglycopeptide repeat where the Thr residue is glycosylated with a D-Galβ1-3-D-GalNAcα1. It has been suggested that the core O-GalNAc residue is involved in the event of antifreeze activity of AFGP. However the functional role that this simple sugar residue plays at the molecular level is still unclear.
To uncover the general function of O-GalNAc, we conducted total chemical synthesis of various length of homogeneous O-GalNAcylated AFGP. This synthesis was achieved using a peptidyl-N-pivaloylguanidine that is an “unreactive” peptide in peptide coupling reactions, but is interconvertible with a “reactive” peptide-α-thioester only by a simple treatment with thiol under buffer condition.
The unique switchable reactivity of peptidyl-N-pivaloylguanidine enabled us to perform an efficient sequential peptide coupling strategy. As a result, we have successfully synthesized various lengths of homogeneous O-GalNAcylated AFGP including 120 amino acids length having 40 O-GalNAcylation sites. Through the structural analysis by CD spectroscopy and evaluation of the antifreeze activity of the synthetic AFGP(GalNAc)s, it was revealed that O-GalNAcylation is an essential O-glycosylation for both structural and functional basis of AFGP to exhibit antifreeze activity.