Fluorescent labeling of lysosomal α-L-fucosidase using an activity-based near-infrared (NIR) fluorogenic probe

Lysosomal α-L-fucosidase catalyzes the degradation of fucosylated glycoconjugates and is implicated in multiple pathological events. Although conventional fluorogenic substrates have been utilized to detect the glycosidase in cells, they are inappropriate for long-term imaging owing to the diffusion of the fluorophores released upon enzymatic cleavage. On the other hand, existing activity-based probes are useful for capturing the target enzyme, but they cannot be readily used for live-cell fluorescence imaging.

We developed αFuc-Bn-NIR(CM)-AY, an activity-based near-infrared (NIR) fluorogenic probe for selective fluorescence imaging and covalent labeling of active α-L-fucosidase. The probe comprised (1) an α-L-fucose recognition unit linked to a hemicyanine-derived NIR fluorophore through a self-immolative scaffold, (2) a carbamate-based reactive moiety appended to the fluorophore, and (3) a terminal alkyne bioorthogonal handle. The probe functioned as a mechanism-based, irreversible inhibitor that covalently labeled and inactivated active α-L-fucosidase upon fluorescence activation. In addition, αFuc-Bn-NIR(CM)-AY showed high selectivity toward α-L-fucosidase and enabled robust fluorescent labeling of the enzyme. Moreover, the probe allowed detection of the enzyme at concentrations as low as 20 nM, and the labeling was markedly reduced by an α-L-fucosidase inhibitor. In live cells, enzyme-mediated activation produced persistent lysosome-associated fluorescence with minimal cytotoxicity. The terminal alkyne further facilitated click chemistry-mediated biotinylation and affinity enrichment of labeled lysosomal α-L-fucosidase from treated cells.

αFuc-Bn-NIR(CM)-AY integrates fluorescence activation, covalent enzyme labeling, and affinity-based enrichment within a single NIR platform, overcoming the diffusion-associated limitation of conventional fluorogenic substrates and the imaging limitation of existing activity-based probes. It provides a useful tool for prolonged imaging and covalent capture of active lysosomal α-L-fucosidase.