1,6-Val₂GalNAz: An efficient metabolic chemical reporter for O-linked glycan labeling with minimal S-glyco...

Metabolic chemical reporters (MCRs) bearing bio-orthogonal handles, such as Ac₄GalNAz, are indispensable for glycan detection but notoriously generate non-specific, non-enzymatic S-linked glyco-conjugation, severely compromising specificity. Here, we developed a series of partially protected GalNAz derivatives with different chain lengths at the 1,6-positions to comprehensively evaluate and screen a potential MCR for glycan labeling detection. Our results demonstrated that 1,6-Val₂GalNAz enable to label glycoproteins at lower concentrations without generating unexpected S-glyco-modification. Moreover, the label signals from 1,6-Val₂GalNAz were mainly in the form of O-linked glycans. Of note, in live zebrafish, 1,6-Val₂GalNAz not only could robustly label glycoproteins in a time dependent manner, but also exhibited highly selective labeling ability in intestine and gill. Taken together, we concluded that 1,6-Val₂GalNAz was an efficient and selective MCR in metabolic labeling no matter in vitro and in vivo, providing a possibility to label sensitive cells at a lower and safety concentration.