Abstract Recent progress in glycan research has been driven by widespread implementations of metabolic oligosaccharide engineering. Complementing existing approaches, we here introduce bifunctional, UV-crosslinkable and clickable N-acetylglucosamine and N-acetylgalactosamine analogues, which enable direct visualization of the intracellular probe distribution as well as distinguishing monomeric and macromolecule-bound fractions. Using this feature, we find that monomeric N-acetylmonosaccharides partition into RNA-rich nuclear compartments such as nuclear speckles and nucleoli. This suggests the existence of spatially separated N-acetylmonosccharide pools within the nucleoplasm. Taken together, bifunctional N-acetylmonosaccharide probes are a powerful discovery tool for probing intracellular localization of monosaccharides and glycosylated macromolecules.

Bifunctional Monosaccharides Preferentially Localize to Nuclear Subcompartments