Halogen Effects-Induced Bright D-π-A Fluorophore as Scaffold for NIR Fluorogenic Probes with High Contrast.

Endowing fluorogenic probes with ultrahigh contrast is essential to increasing the accuracy of fluorescence sensing and imaging. Phenolate-based D-π-A fluorophores (A-DOH) belong to a big family of fluorophores and have attracted increasing attention in fluorogenic probe design. However, the intrinsic dilemma of weak intracellular emission of traditional A-DOH fluorophores resulted in low contrast during live cell imaging. Herein, we present a general and robust approach to preparing novel A-DOH fluorophores with bright NIR fluorescence in living cells based on the unique halogen effects. The reported chlorinated A-DOH fluorophore (A1-2ClOH) has an extremely strong fluorescence in an aqueous solution of pH 7.4 and living cells, which is 194 and 30 times higher than that of the traditional halogen-free analogue (A1-OH), respectively. We systematically investigated and demonstrated that the distinct -I and +M halogen effects, which led to a drastic decrease in the pKa value and a significant enhancement in the fluorescence quantum yield, respectively, should be responsible for the tremendous fluorescence enhancement. The flexible phenol caging chemistry allows one to prepare multiple NIR fluorogenic probes based on the A1-2ClOH scaffold with high contrast for live cell imaging of a variety of analytes by introducing a corresponding triggering moiety. Moreover, the conjugated azide group of A1-2ClOH enables the integration of more functions as desired through a facile click reaction. A fluorogenic probe (mitoProbe-PN) was synthesized as a paradigm by equipping the A1-2ClOH scaffold with a mitochondria-targeting moiety and a peroxynitrite-responsive triggering group and demonstrated specific high-contrast fluorescence imaging of endogenous OONO- in mitochondria of living macrophages.