A trifunctional probe for generation of fluorogenic glycan-photocrosslinker conjugates.

Abstract

Interactions between cell surface glycans and lectins mediate vital biological processes, yet their characterization is hindered by the low affinity of these binding events. While photoaffinity labeling can capture these interactions, traditional custom probes often demand tedious synthesis, are limited to simple glycans, and lack versatility. To overcome these limitations, we report a trifunctional scaffold enabling modular assembly of glycan probes. This scaffold integrates orthogonal sites for: (i) efficient late-stage ligation of native oligosaccharides via an N-alkoxy-amine, preserving glycan structure; (ii) flexible amide coupling of various photocrosslinkers, including a recently developed fluorogenic azidocoumarin for traceable labeling; and (iii) conjugation to reporter tags (e.g., biotin) or multivalent carriers through a carboxylic acid motif. We demonstrate the scaffold's utility by synthesizing probes bearing various fucosylated glycans. Probes incorporating the fluorogenic photocrosslinker achieved specific, light-induced labeling of the model lectin BambL. The platform's adaptability was further confirmed by generating monovalent biotinylated probes displaying the photoactive glycan. This modular strategy offers a practical solution to rapidly construct advanced chemical probes, facilitating the investigation of complex glycan recognition events in diverse biological systems.