A dual-emission near-infrared fluorogenic probe for sensing Catechol-O- methyltransferase activity from in vitro to in vivo.

Human catechol-O-methyltransferase (COMT, EC 2.1.1.6) plays a key role in neurotransmitter signaling and metabolism in the central nervous system. Optical sensing of COMT activity provides a powerful approach to study its biological functions, however, existing fluorescent probes are limited by short emission wavelengths and low sensitivity. Herein, we present TCFC, the first dual-emission near-infrared (NIR) fluorogenic substrate for COMT, rationally designed via a structure-modulated strategy. Under physiological conditions, TCFC was rapidly O-methylated by COMT to yield product 8-MTCFC, which exhibited strong fluorescence at 620 nm and 695 nm upon excitation at 470 nm and 610 nm, respectively. The dual-emission property of TCFC enhanced detection sensitivity and versatility, enabling accurate assessment of COMT activity across individuals and species while supporting high-throughput screening of potential COMT inhibitors. Moreover, the NIR emission of TCFC allowed in situ visualization of COMT activity in living cells and various brain regions of rats with high spatiotemporal resolution, and revealed a reduction of COMT activity in the hippocampal region of a mouse model of Alzheimer's disease, offering insights into its role in neurodegeneration. Collectively, TCFC proved to be a powerful NIR fluorogenic substrate for monitoring COMT activity in complex biological systems, enabling mechanistic studies and high-throughput screening of potential inhibitors.