Tools and tactics to define specificity of metabolic chemical reporters

Metabolic chemical reporters (MCRs) provide easily accessible means to study glycans in their native environments. However, because monosaccharide precursors are shared by many glycosylation pathways, selective incorporation has been difficult to attain. Here, a strategy for defining the selectivity and enzymatic incorporation of an MCR is presented. Performing β-elimination to interrogate O-linked sugars and using commercially available glycosidases and glycosyltransferase inhibitors, we probed the specificity of widely used azide (Ac₄GalNAz) and alkyne (Ac₄GalNAlk and Ac₄GlcNAlk) sugar derivatives. Following the outlined strategy, we provide a semiquantitative assessment of the specific and non-specific incorporation of this bioorthogonal sugar (Ac₄GalNAz) into numerous N- and O-linked glycosylation pathways. This approach should be generally applicable to other MCRs to define the extent of incorporation into the various glycan species.