Responsive Contrast Agents: Synthesis and Characterization of a Tunable Series of pH-Sensitive Near-Infrared Pentamethines

The demand for responsive dyes in optical imaging is high to achieve a better signal-to-noise ratio and, more specifically, to visualize acidic compartments of the endocytic pathway. Herein, we present a new synthetic route, with a step-by-step synthesis of water-soluble pH-sensitive cyanine dyes exhibiting pKa values in the region of physiological pH, as confirmed by absorption and fluorescence spectra. Moreover, modification of pKa values was achieved by two different substitution patterns, creating tunable pH-sensitive dyes. We demonstrated the functionality of the pH-sensitive dyes and their suitability as contrast agents for cellular uptake studies by preparing dye-labeled cetuximab and transferrin conjugates. Sulfonated head chains increased water solubility and prevented the formation of dimers, even in the context of dye-labeled bioconjugates. Confocal microscopy images of living cells revealed their pH-responsiveness, as specific fluorescence signal enhancements were observed in acidic compartmen...