An activity-based near-infrared (NIR) fluorogenic probe for labeling lysosomal β-hexosaminidase in cells

Lysosomal β-hexosaminidase (β-Hex) plays a key role in glycan degradation, and its dysregulation is closely associated with various human diseases. Owing to its physiological and pathological significance, accurate detection of lysosomal β-Hex in cells is crucial for both fundamental research and potential therapeutic applications. In this study, we synthesized and evaluated a series of activity-based near-infrared (NIR) fluorogenic probes for β-Hex. These probes consisted of a sugar moiety as the recognition element, which was connected to a NIR fluorophore either directly or through a self-immolative linker, a carbamate leaving group for enzyme capture, and a clickable alkyne tag for enzyme isolation. Among the probes tested, βGalNAc-Bn-NIR(CM)-AY, in which βGalNAc was conjugated via a self-immolative linker to the carbamate-appended NIR fluorophore, demonstrated selective and efficient labeling of β-Hex. After overnight dialysis, the activity of the enzyme labeled with βGalNAc-Bn-NIR(CM)-AY was not restored, and the fluorescence of the labeled enzyme remained unchanged. These findings indicate that the probe labels the enzyme through a stable covalent linkage to inhibit its activity. In cell-based studies, this probe enabled fluorescence imaging of β-Hex within lysosomes (Pearson coefficient with LysoTracker = 0.9), and its fluorescence was retained after washing for 1 h, unlike a control probe (βGalNAc-Bn-NIR(Cl)-AY) lacking the covalent labeling ability. Furthermore, the probe allowed for the isolation of labeled β-Hex using affinity chromatography. Thus, βGalNAc-Bn-NIR(CM)-AY has proven to be an effective tool for the long-term fluorescence imaging and selective isolation of lysosomal β-Hex in live cells.