Investigation of relationships between metabolic chemical reporter structures and S-glyco-modification effects

The approach of metabolic chemical reporters (MCRs) for labeling proteins has been widely used in the past several decades. Nevertheless, artificial side reaction generated with fully protected MCRs, termed S-glyco-modification, occurs with cysteine residues through base-promoted β-elimination and Michael addition, leading to false positives in the proteomic identification. Therefore, next generation of MCRs, including partially protected strategy and modifications on the backbone of monosaccharides, have emerged to improve the labeling efficiency. In this paper, we prepared fifteen kinds of unnatural monosaccharides to investigate the relationships of structures and S-glyco-modification labeling. Our results demonstrated that Ac₄GlcNAz and Ac₄GalNAz exhibited the most remarkable labeling effects among the detected compounds. Of note, Ac₄ManNAz, Ac₄6AzGlucose and Ac₄6AzGalactose containing similar structures but did not show similar robust signals as them. Moreover, other modifications on the 1-, 2-, 3-, 4- and 6-site indicated minimal side reactions of S-glyco-modification, raising a possibility that subtle modifications of monosaccharide substrate may alter its role in the process of biosynthesis, for example, by change of electronegativity or enhancement of steric hindrance effects. In conclusion, our discoveries provide a new avenue to choose appropriate probe for selective label proteins in vitro and in vivo without undesired S-glyco-modification.