A near-infrared naphthalimide-based fluorescent probe for real-time imaging of small-molecule thiols in living cells.

A novel near-infrared fluorescent probe, (E)-2-butyl-5-(2-(3-(dicyanomethylene)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)-1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-6-yl 2,4-dinitrobenzenesulfonate (NA-BH), was rationally designed and synthesized for the detection and monitoring of cysteine (Cys), homocysteine (Hcy) and hydrogen sulfide (H2S) dynamics in living systems. This probe exhibits a significant fluorescence enhancement response to small-molecule thiols (SMBs) Cys, Hcy and H2S, but only a weak response to glutathione (GSH). Given the higher intracellular abundance and stronger pathophysiological relevance of Cys, this study uses Cys as a representative to evaluate the probe's response mechanism, spectral performance, and live-cell imaging capability. The probe operates via photoinduced electron transfer and exhibits an emission maximum at 732 nm with a large Stokes shift of 123 nm. These optical properties facilitate deep tissue penetration and minimize interference from background autofluorescence. Detection of SMBs and H2S is achieved via a specific nucleophilic substitution reaction with the 2,4-dinitrobenzenesulfonyl group, resulting in a distinct fluorescence off-on response. NA-BH exhibits high sensitivity toward SMBs and H2S, accompanied by fast response and a low detection limit. Importantly, cell imaging experiments confirmed that the probe can dynamically distinguish and monitor the fluctuations of endogenous and exogenous small-molecule biothiol levels in living cells in real time. This probe provides a reliable and efficient molecular tool for advancing physiological and pathological research related to SMBs.