Efficient Substitution Reaction from Cysteine to the Serine Residue of Glycosylated Polypeptide: Repetitive Peptide Segment Ligation Strategy and the Synthesis of Glycosylated Tetracontapeptide Having Acid Labile Sialyl-TN Antigens

This paper reports the synthesis of a 40-residue glycopeptide having two antigenic sialyl-T-N (NeuAc-alpha-(2,6)-GalNAc-Thr) residues in the MUCl sequence. This target glycopeptide is a tandem repeat form of 20-residue glycopeptides. For the synthesis of this large molecule, native chemical ligation (NCL) at the serine site was used ((NCLSer)-N-Cys). The concept of (NCLSer)-N-Cys relies on the following: (1) conventional NCL between peptide-a-thioester and the cysteine residue of another peptide segment; (2) methylation of the thiol that was used for NCL; (3) acidic CNBr conversion of the cysteine residue to the serine residue forming an O-ester linkage; and (4) an O- to N-acyl shift to couple the two glycopeptides through a native amide bond. To synthesize glycopeptide having an acid-labile sugar moiety, a 20-residue glycopeptide-alpha-thioester and 20-residue glycopeptide having a cysteine residue at the N-terminal were synthesized by solid phase glycopeptide synthesis, and then coupled by (NCLSer)-N-Cys. As the result of extensive investigation, CNBr activation with an additional acid (trifluoroacetic acid) was found to be essential to obtain good reactivity and yield, and this condition afforded a tandem repeat form of 40-residue sialylglycopeptide having two sialyl-TN residues. In addition to this, it was demonstrated that the cysteine thiol protected by the acetoamidomethyl (Acm) group did not react with the CNBr reagent, and therefore (NCLSer)-N-Cys can be used for repetitive native chemical ligation in the presence of a protecting N-terminal cysteine residue with an Acm group.