Species-selective activation of a β-galactosidase fluorogenic probe

Fluorogenic probes capable of distinguishing enzyme activity between species are crucial for precise detection in complex biological environments. Herein, we present 5-(Pentafluorobenzoylamino) Fluorescein di-β-D-galactopyranoside (PFB-FDG), a substrate selectively activated by human β-galactosidase (β-gal) without cross-reactivity with bacterial β-gal. We tested PFB-FDG under various control conditions using purified enzymes and live cells to demonstrate that it does not activate in the presence of bacterial β-gal. Moreover, molecular docking studies suggest that the selectivity results from steric hindrance within the smaller binding pocket of bacterial β-gal, in contrast to the larger binding pocket of the human enzyme. This species-selective probe represents a major advancement, with its unique properties providing a valuable tool for future studies on human-bacterial interactions.