Sensing biothiols via thiol-chromene addition triggered fluorophore activation by intramolecular cyclization

Abstract

Biological thiols constitute a vital class of redox regulators essential for maintaining intracellular homeostasis. Herein, we engineered three innovative fluorescent probes (CCZU series) through a rational molecular design strategy combining thiol-chromene addition and intramolecular cyclization mechanisms for sensing biothiols. These probes feature tunable electron-withdrawing groups that enable selective recognition of biothiols via a cascade reaction process: initial Michael addition of thiols to the α,β-unsaturated ketone moiety triggers chromene ring-opening, followed by spontaneous intramolecular cyclization to yield highly fluorescent coumarin derivatives. Systematic probe evaluation identified CCZU-3 as an optimal candidate, demonstrating superior water solubility, rapid response time (~5 min), and remarkable fluorescence amplification (>180-fold) with nanomolar sensitivity. The probe's biological utility was validated through fluorescence imaging of biothiols in live cells and zebrafish. Significantly, CCZU-3 enabled fluorescent analysis of altered thiol content associated with redox imbalance in a diabetic cell models. This work establishes a novel sensing platform offering promising applications in disease diagnosis and redox-targeted therapeutic development.