Metabolism of diazirine-modified N-acetylmannosamine analogs to photocrosslinking sialosides

摘要

Terminal sialic acid residues often mediate the interactions of cell surface glycoconjugates. Sialic acid-dependent interactions typically exhibit rapid dissociation rates, precluding the use of traditional biological techniques for complex isolation. To stabilize these transient interactions, we employ a targeted photocrosslinking approach in which a diazirine photocrosslinker is incorporated into cell surface sialylated glycoconjugates through the use of metabolic oligosaccharide engineering. We describe three diazirine-modified N-acetylmannosamine (ManNAc) analogs in which the length of the linker between the pyranose ring and the diazirine was varied. These analogs were each metabolized to their respective sialic acid counterparts, which were added to both glycoproteins and glycolipids. Diazirine-modified sialic acid analogs could be incorporated into both α2–3 and α2–6 linkages. Upon exposure to UV irradiation, diazirine-modified glycoconjugates were covalently crosslinked to their interaction partners. We demonstrate that all three diazirine-modified analogs were capable of competing with endogeneous sialic acid, albeit to varying degrees. We found that larger analogs were less efficiently metabolized, yet could still function as effective crosslinkers. Notably, the addition of the diazirine substituent interferes with metabolism of ManNAc analogs to glycans other than sialosides, providing fidelity to selectively incorporate the crosslinker into sialylated molecules. These compounds are non-toxic and display only minimal growth inhibition at the concentrations required for crosslinking studies. This report provides essential information for the deployment of photocrosslinking analogs to capture and study ephemeral, yet essential, sialic acid-mediated interactions.