Interrogating the glycome using click chemistry
Mbua, Eric Ngalle
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The past several years has seen a rapid development of the bioorthogonal chemical reporter methodology for the labelling of glycoconjugates of living cells and whole organisms. We have shown that 4-dibenzocyclooctynol (DIBO), which can easily be synthesized, reacts exceptionally fast in the absence of a Cu (I) catalyst with azido-containing compounds to give stable triazoles. The use of several cell lines with known defects in glycoconjugate glycosylation validated DIBO as a reagent for determining relative quantities of cell surface glycoconjugate sialylation. Using a chemical reporter strategy in combination with pharmacological treatments, we demonstrated a disease-specific and previously unrecognized accumulation of a diverse set of glycoconjugates in NPC1 and NPC2 fibroblasts within endocytic compartments. The labeled vesicles did not co-localize with the cholesterol-laden compartments of NPC cells, and treatment of either NPC1 or NPC2 cells with cyclodextrin was effective in reducing cholesterol storage and also the endocytic accumulation of sialoglycoproteins. Alterations in protein glycosylation occur during development and progression of many diseases, hence glycomics and glycoproteomics have emerged as important tools in glycobiomarker discovery. An enrichment method is described to covalently capture azide-tagged O-GlcNAc proteins, aimed at facilitating mass spectrometric analysis. Furthermore, the modified DIBO-resin is found to be very specific and with efficient enrichment of over 96% of modified O-GlcNAc proteins. A novel phototriggered click strategy is described for activation of cyclopropenones to produce reactive dibenzocyclooctynes upon irradiation for metal-free ligation for labeling of living cells modified with azidocontaining cell surface saccharides. The cyclopropenone-based phototriggered click chemistry offers exciting opportunities to label living organisms in a temporally and spatially controlled manner. It was found, for the first time, that the substitution pattern of the dibenzylcyclooctynes influences subcellular location. A highly polar sulfated dibenzocyclooctynylamides (S-DIBO) was developed, and we showed that DIBO can enter cells, thereby labeling intra- and extracellular azido-modified glycoconjugates, whereas S-DIBO cannot pass the cell membrane and therefore is ideally suited for selective labeling of cell surface molecules. The ability to selectively label cell surface molecules will yield unique opportunities for glycomic analysis and the study of glycoprotein trafficking.