Activity-Based Dicyanoisophorone Derivatives: Fluorogenic Toolbox Enables Direct Visualization and Monitoring of Esterase Activity in Tumor Models

The visualization and spatiotemporal monitoring of endogenous esterase activity are crucial for clinical diagnostics and treatment of liver diseases. Our research adopts a novel substrate hydrolysis-enzymatic activity (SHEA) approach using dicyanoisophorone-based fluorogenic ester substrates DCIP-R (R = R1–R6) to evaluate esterase preferences on diverse substrate libraries. Esterase-mediated hydrolysis yielded fluorescent DCIP–OH with a nanomolar detection limit in vitro. These probes effectively monitor ester hydrolysis kinetics with a turnover number of 4.73 s–1 and catalytic efficiency (kcat/Km) of 106 M–1 s–1 (DCIP-R1). Comparative studies utilizing two-photon imaging have indicated that substrates containing alkyl groups (DCIP-R1) as recognition elements exhibit enhanced enzymatic cleavage compared to those containing phenyl substitution on alkyl chains (DCIP-R4). Time-dependent variations in endogenous esterase levels were tracked in healthy and liver tumor models, especially in diethylnitrosamine (DEN)–induced tumors and HepG2-transplanted liver tumors. Overall, fluorescence signal quantifications demonstrated the excellent proficiency of DCIP-R1 in detecting esterase activity both in vitro and in vivo, showing promising potential for biomedical applications.