New pH-Responsive Turn-On Xanthone Probes for Cellular Imaging in Strongly Acidic Media

Fluorescent probes are indispensable for cellular imaging and for monitoring pH-dependent biological processes. However, the majority of reported probes are effective only in near-neutral or mildly acidic ranges, with very few functioning reliably in strongly acidic environments (pH < 4). Designing water-soluble, biocompatible probes that can operate efficiently under such extreme conditions remains an important analytical challenge. To address this challenge, two pH-responsive fluorescent probes, Xpy and XpyF, based on a xanthone core, were designed and synthesized. Both probes exhibit excellent water solubility, low cytotoxicity, and strong turn-on fluorescence in acidic environments. The probes display distinct emission changes from blue at neutral to moderately acidic pH (3–7) to green at strongly acidic pH (1–2). Absolute quantum yield and TCSPC studies confirmed significant fluorescence enhancement and lifetime prolongation under acidic conditions, consistent with protonation-induced PET inhibition. Cellular imaging studies revealed efficient internalization and strong nuclear localization with high Pearson’s correlation coefficients. Importantly, while Xpy showed consistent nuclear staining across pH values, XpyF demonstrated a unique pH-dependent switch from cytoplasmic localization at pH 7.4 to nuclear accumulation at pH 4.3. Overall, Xpy and XpyF represent reliable nuclear-targeting sensors for strongly acidic media, illustrating how subtle structural modifications can yield distinct functional outcomes in bioimaging.