Exploring Structural-Photophysical Property Relationships in Mitochondria-Targeted Deep-Red/NIR-Emitting Coumarins

Organic fluorophores operating in the optical window of biological tissues, namely in the deep-red and near-infrared (NIR) region of the electromagnetic spectrum, offer several advantages for fluorescence bioimaging applications owing to the appealing features of long-wavelength light, such as deep tissue penetration, lack of toxicity, low scattering, and reduced interference with cellular autofluorescence. Among these, COUPY dyes based on non-conventional coumarin scaffolds display suitable photophysical properties and efficient cellular uptake, with a tendency to accumulate primarily in mitochondria, which renders them suitable probes for bioimaging purposes. In this study, we have explored how the photophysical properties and subcellular localization of COUPY fluorophores can be modulated through the modification of the coumarin backbone. While the introduction of a strong electron-withdrawing group, such as the trifluoromethyl group, at position 4 resulted in an exceptional photostability and a remarkable redshift in the absorption and emission maxima when combined with a julolidine ring replacing the N,N-dialkylaminobenzene moiety, the incorporation of a cyano group at position 3 dramatically reduced the brightness of the resulting fluorophore. Interestingly, confocal microscopy studies in living HeLa cells revealed that the 1,1,7,7-tetramethyl julolidine-containing derivatives accumulated in the mitochondria with much higher specificity. Overall, our results provide valuable insights for the design and optimization of new COUPY dyes operating in the deep-red/NIR region.