The Small Molecule 2-Azido-2-deoxy-glucose Is a Metabolic Chemical Reporter of O-GlcNAc Modifications in Mammalian Cells, Revealing an Unexpected Promiscuity of O-GlcNAc Transferase

Abstract

Glycans can be directly labeled using unnatural monosaccharide analogs, termed metabolic chemical reporters (MCRs). These compounds enable the secondary visualization and identification of glycoproteins by taking advantage of bioorthogonal reactions. Most widely used MCRs have azides or alkynes at the 2-N-acetyl position but are not selective for one class of glycoprotein over others. To address this limitation, we are exploring additional MCRs that have bioorthogonal functionality at other positions. Here, we report the characterization of 2-azido-2-deoxy-glucose (2AzGlc). We find that 2AzGlc selectively labels intracellular O-GlcNAc modifications, which further supports a somewhat unexpected, structural flexibility in this pathway. In contrast to the endogenous modification N-acetyl-glucosamine (GlcNAc), we find that 2AzGlc is not dynamically removed from protein substrates and that treatment with higher concentrations of per-acetylated 2AzGlc is toxic to cells. Finally, we demonstrate that this toxicity is an inherent property of the small-molecule, as removal of the 6-acetyl-group renders the corresponding reporter nontoxic but still results in protein labeling.

Azide‐ and Alkyne‐Bearing Metabolic Chemical Reporters of Glycosylation Show Structure‐Dependent Feedback Inhibition of the Hexosamine Biosynthetic Pathway

“…Furthermore, this suggests that treating cells with these MCRs will have little effect on the endogenous biosynthesis of UDP‐GlcNAc through the HBP. Finally, we tested the most recent MCRs developed by our lab: UDP‐6AzGlcNAc and UDP‐2AzGlc, which are selective for labeling O‐GlcNAc modifications, and UDP‐6AzGlc, which we recently showed might read out on intracellular O‐glucose modifications (Figure B) . In the case of UDP‐6AzGlcNAc, we saw inhibition in a similar range as UDP‐GlcNAc, thus suggesting that the 6‐azido‐6‐deoxy substitution does not interfere with binding of the UDP‐sugar to the allosteric site.…”

Development of IsoTaG, a Chemical Glycoproteomics Technique for Profiling Intact N- and O-Glycopeptides from Whole Cell Proteomes

“…In general, the spectral counts were predicted by Western blot data obtained prior to MS analysis, and appears to reflect general trends in the ability to metabolically label different cell lines. 46 …”

Metabolic Chemical Reporters of Glycans Exhibit Cell‐Type‐Selective Metabolism and Glycoprotein Labeling

“…We were interested in addressing the selectively of MCRs, with the goal of developing reporters that could be used for the analysis of a single class of glycoproteins. We recently demonstrated that changes to the structure of the MCRs could yield reporters that are highly selective for O‐GlcNAcylation, including 6‐azido‐6‐deoxy‐ N ‐acetyl‐glucosamine (6AzGlcNAc) and 2‐azido‐2‐deoxy‐glucose (2AzGlc) . Here, we explored the relationship between MCR metabolism, leading to MCR donor sugar accumulation, and protein incorporation levels.…”