Metabolic Labeling and Imaging of Native Saccharomyces Cerevisiae Glycan by Using N-Acetylglucosamine-6-Phosphate Analogs

Abstract

N-acetylglucosamine (GlcNAc) is an essential component of fungal glycans, which are found in N-linked glycans, chitin polysaccharides in the cell wall, glycolipids, and other structures. Saccharomyces cerevisiae, a widely used fungal model organism, plays a significant role in glycoengineering studies. However, suitable tools for investigating its endogenous glycan synthesis are still lacking. Metabolic glycan labeling (MGL) has emerged as a powerful technique for tracking the fate of monosaccharides and offers a strategic approach to glycan research. Nevertheless, the absence of efficient transporters and kinases for GlcNAc analogs, such as N-azidoacetylglucosamine (GlcNAz), hinders direct glycan labeling with these probes without the construction of an exogenous pathway. In this study, we demonstrate that this challenge can potentially be addressed by directly utilizing probes based on monosaccharide derivatives downstream of the salvage pathway. We synthesized N-azidoacetylglucosamine-6-phosphate analogs (GlcNAz-Ps) with appropriate hydrophobic modifications and showed that these compounds can be incorporated metabolically into S. cerevisiae glycans in a concentration- and time-dependent manner. Further investigations utilizing specific inhibitors and glycosidases indicated that the probes could be integrated into the N-linked glycans and chitin, demonstrating their potential for use in imaging and glycoproteomics studies of Saccharomyces cerevisiae glycans.