Abstract
Acidity plays an important role in numerous physiological and pathological events. Non-invasively monitoring pH dynamics would be valuable for understanding pathological processes and optimizing therapeutic strategies. Although numerous near-infrared (NIR) fluorophores have been developed to monitor acidification in vivo, the experimental results are difficult to verify at the molecular or cellular level using a fluorescence microscope or flow cytometer due to the lack of lasers with excitation wavelengths in the NIR wavelength range. This work presents a sequential condensation strategy for obtaining unsymmetrical pentamethine cyanines with fine-tuned pK (a) values and improved yields. These deep-red fluorophores with pH responsiveness can not only be used to monitor acidification in live cells using confocal microscopic imaging and flow cytometry, but they can also be used to non-invasively identify infected tissue with a low pH value in live mouse models. In addition, the acidity in infected tissue slices was verified under a conventional confocal microscope. Overall, this work demonstrates a new synthetic method with improved yields for unsymmetrical pentamethine cyanines that can report acidity. These pH-responsive deep-red fluorophores not only provide new tools for accessing pH-associated physiological and pathological events, but they can also help in understanding in vivo imaging results at the molecular or cellular level due to their detectability by multiple imaging instruments.