カルボキシルエステラーゼ活性の高度な選択的検出のための比率測定型ナフタルイミド系蛍光プローブ

Carboxylesterase (CES) is a key Phase I metabolic enzyme, and the accurate assessment of its activity is essential for studies in drug metabolism, toxicology, and disease diagnosis. However, many existing fluorescent probes suffer from cross-reactivity with homologous enzymes such as acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). In this study, we designed carbamate as a CES-specific hydrolyzable moiety and conjugated it to the highly photostable naphthalimide fluorophore NIOH via either chlorinated or non-chlorinated self-immolative linkers, thereby developing two ratiometric fluorescent probes, NI-Cl and NI-W. Both probes exhibited negligible responses to AChE and BChE while enabling selective and sensitive ratiometric detection of CES. NI-Cl demonstrated an exceptionally low detection limit of 0.038 U/mL, and NI-W also showed strong performance with a detection limit of 0.048 U/mL. Importantly, NI-Cl successfully visualized endogenous CES activity in different live cells, providing a robust tool for elucidating the physiological and pathological roles of CES.