A highly selective fluorescent ESIPT probe for the detection of Human carboxylesterase 2 and its biological applications

• A ratiometric fluorescent ESIPT probe for selective detection of human CE2 was developed for the first time.
• The probe can be selectively hydrolyzed by CE2 to release 3-hydroxylflavone which brings the remarkable changes in fluorescence spectrum.
• The probe can be used to efficiently detect the real activity of CE2 in biological samples.
• The probe also can monitor the real function of endogenous CE2 in living cells.

A new ratiometric florescence probe derived from 3-hydroxyflavone (3-HF) has been developed for selective and sensitive detection of human carboxylesterase 2 (CE2). The probe is designed by modulating the excited state intramolecular proton transfer (ESIPT) emission of 3-HF via introducing of 4-ethylbenzoyloxy group. Under physiological conditions, probe 1 displays satisfying stability with very low background signal, but it can be selectively hydrolyzed by CE2 to release free 3-HF which brings remarkable changes in fluorescence spectrum. Both reaction phenotyping and chemical inhibition assays demonstrate that probe 1 is highly selective for CE2 over other human hydrolases including carboxylesterase 1, cholinesterases and paraoxonases. Probe 1 has been applied successfully to measure the real activities of CE2 in human biological samples, as well as to screen CE2 inhibitors by using tissue preparations as the enzymes sources. Additionally, probe 1 is cell membrane permeable and can be used for cellular imaging of endogenous CE2 in living cells. All of these features make it possible to serve as a promising tool for exploring the individual differences in biological function of CE2, as well as for rapid screening of selective and potent inhibitors of CE2 for further clinical use.