Fluorogenic Labeling Probe for the Imaging of Endogenous β-Galactosidase Activity in Cancer and Senescent Cells.

The sensitive detection of glycosidases in live cells is crucial to understanding their functional roles in disease progression. Here, we develop a fluorogenic labeling probe for β-galactosidase (β-Gal) based on a bright green-emitting fluorescent dye, fluorescein. Galactose was introduced to a fluoromethyl-substituted fluorescein derivative through a benzyl spacer, resulting in a quenched fluorescence due to spirocyclization of the dye. After removal of the galactosyl residue by β-Gal, an ∼210-fold enhanced green fluorescence (emission maximum at 524 nm) was detected, and the presence of other glycosidases and hydrolases did not produce false-positive signals. The probe was successfully used for imaging of the endogenous β-Gal activity in cancer and senescent cells, and the imaging results agree with the β-Gal expression level of the cells, as determined by Western blotting and polymerase chain reaction. Importantly, we demonstrated that upon hydrolysis of galactose, the fluoromethyl-substituted fluorescein derivative is covalently attached to adjacent proteins, both in solution and in live cells. This study offers a small-molecule probe for the sensitive monitoring of endogenous glycosidase activity.