Near-Infrared Ratiometric Hemicyanine Fluorescent Probes for Monitoring Mitochondrial pH Dynamics in Live Cells during Oxidative Stress and Hypoxia

Novel near-infrared ratiometric molecules (probes <b>A</b> and <b>B</b>) produced by linking formyl-functionalized xanthene and methoxybenzene moieties, respectively, onto a xanthene-hemicyanine framework are detailed. Probe <b>A</b> exhibited a primary absorption peak at 780 nm and a shoulder peak at 730 nm and exhibited fluorescence at 740 nm↓ (signifies a downward shift in intensity upon acidification) in a pH 9.3 buffer and 780 nm↑ at pH 2.8 under excitation at 700 nm. Probe <b>B</b> featured absorptions at 618 and 668 nm at pH 3.2 and at 717 nm at pH 8.6, and fluorescence at 693 nm↑ at pH 3.2 and at 739 nm↓ at pH 8.6, in mostly the red to near-IR region. The ratiometric changes in the intensity of the fluorescent absorptions were reversed between <b>A</b> and <b>B</b> upon acidification as indicated by the arrows. Theoretical calculations confirmed that there were slight changes in conformation between probes and the protonated molecules, suggesting that the changes in emission spectra were due mostly to conjugation effects. Calculations at the APFD/6-311+g(d,p) level with a solvent described by the polarizable continuum model resulted in p<i>K</i> _a values for <b>A</b> at 6.33 and <b>B</b> at 6.41, in good agreement with the experimentally determined value of 6.97 and an average of 6.40, respectively. The versatilities of the probes were demonstrated in various experimental contexts, including the effective detection of mitochondrial pH fluctuations. Live cell experiments involving exposure to different pH buffers in the presence of H⁺ ionophores, monitoring mitophagy processes during cell starvation, studying hypoxia induced by CoCl₂ treatment, and investigating responses to various oxidative stresses are detailed. Our findings highlight the potential of attaching xanthene and methoxybenzaldehyde groups onto xanthene-hemicyanine structures as versatile tools for monitoring pH changes in a variety of cellular environments and processes.