Activity-Based\nDicyanoisophorone Derivatives: Fluorogenic\nToolbox Enables Direct Visualization and Monitoring of Esterase Activity\nin Tumor Models

The visualization and spatiotemporal monitoring of endogenous\nesterase\nactivity are crucial for clinical diagnostics and treatment of liver\ndiseases. Our research adopts a novel substrate hydrolysis-enzymatic\nactivity (SHEA) approach using dicyanoisophorone-based fluorogenic\nester substrates <b>DCIP-R (R = R1–R6</b>) to evaluate\nesterase preferences on diverse substrate libraries. Esterase-mediated\nhydrolysis yielded fluorescent <b>DCIP–OH</b> with a\nnanomolar detection limit <i>in vitro</i>. These probes\neffectively monitor ester hydrolysis kinetics with a turnover number\nof 4.73 s<sup>–1</sup> and catalytic efficiency (<i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub>) of 10<sup>6</sup> M<sup>–1</sup> s<sup>–1</sup> (<b>DCIP-R1</b>). Comparative\nstudies utilizing two-photon imaging have indicated that substrates\ncontaining alkyl groups (<b>DCIP-R1</b>) as recognition elements\nexhibit enhanced enzymatic cleavage compared to those containing phenyl\nsubstitution on alkyl chains (<b>DCIP-R4</b>). Time-dependent\nvariations in endogenous esterase levels were tracked in healthy and\nliver tumor models, especially in diethylnitrosamine (DEN)–induced\ntumors and HepG2-transplanted liver tumors. Overall, fluorescence\nsignal quantifications demonstrated the excellent proficiency of <b>DCIP-R1</b> in detecting esterase activity both <i>in vitro</i> and <i>in vivo</i>, showing promising potential for biomedical\napplications.