Fluorination-enhanced ESIPT probes based on 3-hydroxyflavone for cysteine detection in hydrophobic environments and living cells.

Excited-state intramolecular proton transfer (ESIPT) fluorophores, particularly 3-hydroxyflavone derivatives, are broadly utilized in fluorescent probe design due to their distinct photophysical properties. However, their practical applications are often hindered by inherent limitations. Herein, we report a strategy via site-specific fluorination at the 2-phenyl position of the 3-hydroxyflavone scaffold to improve their photophysical performance. Two fluorinated ESIPT fluorophores (PHF and PFF) were synthesized and further functionalized with an acrylate recognition moiety to afford the turn-on fluorescent probes PHFA and PFFA for cysteine (Cys) detection. Compared to the non-fluorinated analogue PHHA, the fluorinated probes exhibited enlarged Stokes shifts (up to 188 nm), lowered pKa values, and enhanced photostability. Both probes demonstrated rapid, highly sensitive, and selective fluorescence responses toward Cys over homocysteine, glutathione, and other biologically relevant species. Notably, the fluorinated probe PHFA enabled successful confocal imaging of both endogenous and exogenous Cys in live HepG2 cells with low cytotoxicity. Furthermore, the fluorophore PHF displayed pronounced fluorescence enhancement within the hydrophobic cavity of bovine serum albumin (BSA), extending its utility to protein-related microenvironments. This work highlights strategic fluorination as an effective approach for modulating the photophysical and recognition properties of ESIPT probes, offering a reliable tool for Cys sensing in complex biological systems.