Longer fatty acid-protected GalNAz enables efficient labeling of proteins in living cells with minimized S -glyco modification.

Metabolic glycoengineering provides a powerful tool to label proteins with chemical tags for cell imaging and protein enrichment. The structures of per- O -acetylation on unnatural sugars facilitate membrane permeability and increase cellular uptake and are widely used for metabolic glycan labeling. However, unexpected S -glyco modification was discovered via a non-enzymatic reaction with protein cysteines, which was initially conducted with the hydrolysis of anomeric acetate by esterase. Herein, we synthesized a series of GalNAz derivatives that were protected with various lengths of short-chain fatty acid, including acetate, propionate, butyrate, valerate and pivalate, to detect differences in labeling efficiencies and occurrence of S -glyco modification. Our results demonstrate that all the GalNAz derivatives could effectively label proteins in HeLa cells, except the pivalate group. Of note, But 4 GalNAz exhibited excellent labeling abilities compared with Ac 4 GalNAz from the results for western blot, flow cytometry and confocal laser scanning microscopy. Moreover, the results for the S -glyco-modification assay by western blot and chemoproteomic analysis indicated that But 4 GalNAz generated negligible unexpected labeling signals compared to Ac 4 GalNAz. Our study uncovers the distinct labeling efficiency of different protected groups on unnatural sugars, which provides an alternative strategy to explore novel glycan probes.