Small molecule fluorophores represent robust tools for the analysis of complex biological systems. Notably, near-infrared (NIR) fluorophores could enable deep-tissue imaging applications in multicellular organisms. Despite this promise, NIR fluorophores with improved physical properties for for imaging applications are still in huge demand. Thus, there is a need for chemistry-focused campaigns aimed at the rational improvement of NIR fluorophores. For example, methylene blue represents a promising NIR fluorophore scaffold but is plagued by relatively weak fluorescence brightness in aqueous solutions partly due to aggregation. We set out to further red-shift its excitation and emission while simultaneously addressing fluorescence quenching due to aggregation. To achieve this goal, we hypothesized that substitution of the bridging atom of methylene blue with a negatively charged sp<sup>3</sup> phosphinate functionality would substantially red-shift the excitation and emission as well as suppress fluorophore aggregation. Our results clearly demonstrate that the resulting phosphinate substituted thiazine dyes display a substantial red-shift in excitation and emission (up to 143 nm compared to the parent dye). Moreover, these dyes display virtually no aggregation in aqueous solution, yielding a 14-fold enhancement in fluorescence brightness under physiological conditions. The dyes represent the most red-shifted thiazines to date and their improved water solubility opens expanded applications in imaging. Together, these results validate our rational design hypothesis and provide novel, compact NIR fluorophores with potential utility in biological imaging experiments.

Rational development of next generation NIR fluorophores

ACS Spring 2022

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