Abstract: Based on a modular design strategy, this study developed a series of coumarin-chalcone-based reversible ratiometric fluorescent probes (Probes 1~3), using 7-aminocoumarin as the fluorophore and 2'-hydroxychalcone as the GSH reaction center. Different substituents (nitro, methoxy) were introduced at the 5-position of the chalcone phenyl ring as modulating modules to systematically tune the photophysical properties and reaction kinetics of the probes towards GSH. The results demonstrated that all probes exhibited a good ratiometric fluorescence response within the physiological concentration range of GSH (0~10 mM), with dissociation constants (Kd = 4.61~8.25 mM) suitable for intracellular GSH detection. Among them, Probe 1, by virtue of the synergistic electron-withdrawing effect of its nitro group and ketone carbonyl, not only displayed the highest sensitivity and the most significant ratiometric signal change, but also responded to GSH most rapidly, reaching equilibrium in approximately 5 minutes with a reaction rate constant of. 2.54 M-1s-1. Reversibility experiments confirmed that Probe 1 also possessed the most prominent reversible cycling characteristics. Fluorescence imaging studies further demonstrated the feasibility of ratiometric fluorescent imaging of GSH within live cells. The rational modular design in this work provides a high-performance tool for fluorescent imaging studies of dynamic GSH changes in living cells.

Key words: Fluorescent probe, Coumarin, Chalcone, Glutathione