Selective Engineering of Linkage‐Specific α2,6‐N‐Linked Sialoproteins Using Sydnone‐Modified Sialic Acid Bioorthogonal Reporters

Abstract

The metabolic oligosaccharide engineering (MOE) strategy using unnatural sialic acids has recently enabled the visualization of the sialome in living systems.However,MOE only reports on global sialylation and dissected information regarding subsets of sialosides is missing. Described here is the synthesis and utilization of sialic acids modified with asydnone reporter for the metabolic labeling of sialoconjugates.T he positioning of the reporter on the sugar significantly altered its metabolic fate.Further in vitro enzymatic assays revealed that the 9-modified neuraminic acid is preferentially accepted by the sialyltransferase ST6Gal-I over ST3Gal-IV,l eading to the favored incorporation of the reporter into linkage-specific a2,6-N-linked sialoproteins.T his sydnone sugar presents the possibility of investigating the roles of specific sialosides.

Expanding the Strain‐Promoted 1,3‐Dipolar Cycloaddition Arsenal for a More Selective Bioorthogonal Labeling in Living Cells

“…Introduction of the sydnone moiety onto sialic acid demonstrated that the chemical reporter significantly altered the metabolic fate of the studied sugar. While Neu5SydCl was not metabolically incorporated into cell‐surface sialoconjugates, Neu9NSydCl was well tolerated by the glyco‐biosynthetic machinery of the cell [39] . By employing an in vitro enzymatic‐ 1 H‐NMR coupled assay, we identified CMP‐sialic acid synthetase, the enzyme responsible for the activation of sialic acid into its cytidine monophosphate‐analogue, as the enzymatic roadblock for the biosynthesis of sialoglycans with the Neu5SydCl precursor [39] …”

“…This strategy implies that the unnatural sugar must traverse biosynthetic pathways, thus requiring the chemical reporter to be structurally minimally invasive [38] . Despite the many attractive features of employing azido‐sugars for the MOE, and notably its inconspicuousness, we recently showed that azides can be reduced into amines in biological systems by the action of endogenous thiols, potentially leading to the formation of undesired metabolites [39] . Having recently identified sydnones as highly stable chemical reporters for the imaging of modified‐proteins in complex cellular extracts, [34] as well as for the postsynthetic labeling of oligonucleotides in fixed cells, [35] we envisaged to utilize sydnones instead of azides for imaging complex glycans in living mammalian cells [39] .…”

“…Despite the many attractive features of employing azido‐sugars for the MOE, and notably its inconspicuousness, we recently showed that azides can be reduced into amines in biological systems by the action of endogenous thiols, potentially leading to the formation of undesired metabolites [39] . Having recently identified sydnones as highly stable chemical reporters for the imaging of modified‐proteins in complex cellular extracts, [34] as well as for the postsynthetic labeling of oligonucleotides in fixed cells, [35] we envisaged to utilize sydnones instead of azides for imaging complex glycans in living mammalian cells [39] . We focused our attention on the labeling of sialoconjugates since sialic acids are usually found at the nonreducing end of complex glycans and consequently are ideally placed for interacting with the cell's exogenous environment.…”

Design and synthesis of metabolic chemical reporters for the visualization and identification of glycoproteins

“…Chinoy et al synthesized sialic acid derivatives with sydnones on either the C5 or N -acetyl position, termed Neu5SydCl and Neu9NSydCl respectively. 49 Interestingly, Neu5SydCl was not metabolically efficiently transferred on to protein substrates while Neu9NSydCl showed robust labeling. In addition, Neu9NSydCl could only be tolerated a few STs indicating that Neu9NSydCl labeling could differentiate among various types of sialosides.…”

Sydnone Methides: Intermediates between Mesoionic Compounds and Mesoionic N‐Heterocyclic Olefins

“…Currently, mesoionic compounds experience a veritable renaissance. As examples, the role of sydnones to serve as masked, cyclic dipolarophiles in [2+3]‐cycloadditions [1] has been significantly expanded by copper‐catalyzed [2] or metal‐free strain‐promoted click ring closure reactions [3] and applications as biologically active compounds, [4] bioorthogonal reporters, [5] imaging reagents, [6] and fluorogenic compounds [7] . Furthermore, they can be used for the in vitro bioconjugation of purified proteins [8] .…”