One-step glycan photoaffinity probe synthesis enables identification of human milk oligosaccharide-interacting microbiota

Glycans are found on the surfaces of all living cells and engage in cell signaling through the binding of various molecular targets. However, chemical probes to detect and identify carbohydrate interactions remain difficult to produce, due in large part to the challenges of late-stage, site-specific functionalization of oligosaccharides. Here, we report the synthesis and application of a minimal bifunctional diazirine/alkyne linker utilizing an N,O-dialkylhydroxylamine (NODHA) moiety that can readily be installed on the anomeric position of unprotected sugars. Our linker functionalizes glycans with complete β-stereoselectivity, appends less mass than a single hexose unit, and maintains the closed-ring structure of the reducing end sugar. With this molecule, we generate glycan photoaffinity probes in a single step using unprotected human milk oligosaccharides (HMOs). We then validate binding selectivity of our compounds using lectins with known affinity towards our sugars. Finally, we demonstrate the utility of these glycan photoaffinity probes by performing photocrosslinking on a complex microbiota community and use cell sorting paired with metagenomic sequencing to identify potential new HMO-interacting microbes. Together, our work provides a one-step route to synthesize photoaffinity probes from unprotected, reducing glycans for interrogating carbohydrate interactions at the host-microbiota interface.