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Published on: September 28, 2022
4-Formyl-N-Methylpyridinium-Mediated N-Terminal Cysteine Modification/Removal Facilitates One-Pot Multiplex Peptide
Bingcheng Wei1, Xinyao Wang2, Farong Ye3
1Center For Chemical Glycobiology, Shanghai Key Laboratory For Antibody-Drug Conjugates With Innovative Target, National Key Laboratory of Innovative Immunotherapy, Zhangjiang Institute for Advanced Study, School of Chemistry and Chemical Engineering, Shanghai Jiao Tong University, Shanghai, China.
None:
The chemical synthesis of proteins with site-specific modifications remains a fundamental challenge in chemical biology. One-pot peptide ligation strategies have emerged as powerful tools to enhance synthetic efficiency, primarily relying on N-terminal cysteine (Cys) protection. However, current Cys deprotection conditions require various reagents or pH adjustments during the reaction, rendering downstream processing cumbersome. Here, a visible-light-mediated deprotection strategy using 2-(N-methylpyridinium-4-yl)-thiazolidine (4-NMP-Thz) as a novel N-terminal Cys-protecting group is reported. This reaction, catalyzed by [Ru(bpy)3]Cl2 at physiological pH (6.0-8.0), enables smooth one-pot multi-segment peptide assembly. The strategy demonstrates complete orthogonality to native chemical ligation (NCL) and desulfurization conditions, eliminating the requirement for intermediate purification or pH adjustment. This methodology was used to facilitate an efficient one-pot synthesis of a 400-amino acid (aa) glycosylated MUC1 glycoprotein bearing 40 O-glycosyl modifications that is difficult to prepare using previously reported techniques. The 400-aa MUC1 significantly enhanced antigenic immunogenicity compared with shorter MUC1 glycopeptides. This streamlined approach establishes a robust platform for the construction of complex post-translationally modified proteins.
