Many metabolic chemical reporters have been developed to identify O-GlcNAc. Metabolic chemical reporters are generally sugar analogues that bear an additional chemical moiety allowing for additional reactivity. For example, peracetylated GlcNAc (AcGlcNAz) is a cell-permeable azido sugar that is de-esterified intracellularly by esterases to GlcNAz and converted to UDP-GlcNAz in the hexosamine salvage pathway. UDP-GlcNAz can be utilized as a sugar donor by OGT to yield the O-GlcNAz modification. The presence of the azido sugar can then be visualized via alkyne-containing bioorthogonal chemical probes in an azide-alkyne cycloaddition reaction. These probes can incorporate easily identifiable tags such as the FLAG peptide, biotin, and dye molecules. Mass tags based on polyethylene glycol (PEG) have also been used to measure O-GlcNAc stoichiometry. Conjugation of 5 kDa PEG molecules leads to a mass shift for modified proteins - more heavily O-GlcNAcylated proteins will have multiple PEG molecules and thus migrate more slowly in gel electrophoresis. Other metabolic chemical reporters bearing azides or alkynes (generally at the 2 or 6 positions) have been reported. Instead of GlcNAc analogues, GalNAc analogues may be used as well as UDP-GalNAc is in equilibrium with UDP-GlcNAc in cells due to the action of UDP-galactose-4-epimerase (GALE). Treatment with AcGalNAz was found to result in enhanced labeling of O-GlcNAc relative to AcGlcNAz, possibly due to a bottleneck in UDP-GlcNAc pyrophosphorylase processing of GlcNAz-1-P to UDP-GlcNAz. AcGlcN-β-Ala-NBD-α-1-P(Ac-SATE), a metabolic chemical reporter that is processed intracellularly to a fluorophore-labeled UDP-GlcNAc analogue, has been shown to achieve one-step fluorescent labeling of O-GlcNAc in live cells. Metabolic labeling may also be used to identify binding partners of O-GlcNAcylated proteins. The N-acetyl group may be elongated to incorporate a diazirine moiety. Treatment of cells with peracetylated, phosphate-protected AcGlcNDAz-1-P(Ac-SATE) leads to modification of proteins with O-GlcNDAz. UV irradiation then induces photocrosslinking between proteins bearing the O'-GlcNDaz modification and interacting proteins.
Some issues have been identified with various metabolic chemical reporters, e.g., their use may inhibit the hexosamine biosynthetic pathway, they may not be recognized by OGA and therefore are not able to capture O-GlcNAc cycling, or they may be incorporated into glycosylation modifications besides O-GlcNAc as seen in secreted proteins. Metabolic chemical reporters with chemical handles at the N-acetyl position may also label acetylated proteins as the acetyl group may be hydrolyzed into acetate analogues that can be utilized for protein acetylation. Additionally, per-O-acetylated monosaccharides have been identified to react cysteines leading to artificial S-glycosylation. This occurs via an elimination-addition mechanism.