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

Abstract

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 MUC1 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 ((Cys)NCL(Ser)). The concept of (Cys)NCL(Ser) relies on the following: (1) conventional NCL between peptide-alpha-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 (Cys)NCL(Ser). 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-T(N) 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 (Cys)NCL(Ser) can be used for repetitive native chemical ligation in the presence of a protecting N-terminal cysteine residue with an Acm group.