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

ABSTRACT

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, But4GalNAz exhibited excellent labeling abilities compared with Ac4GalNAz 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 But4GalNAz generated negligible unexpected labeling signals compared to Ac4GalNAz. Our study uncovers the distinct labeling efficiency of different protected groups on unnatural sugars, which provides an alternative strategy to explore novel glycan probes.