The vital roles of metals in nutrients and medicines are not accessible to purely organic compounds.

Among the palette of previously described fluorescent organic molecules, coumarins are ideal candidates for developing cellular and molecular imaging tools due to their high cell permeability and minimal perturbation of living systems. However, blue-to-cyan fluorescence emission is usually difficult in in vivo applications due to the inherent toxicity and poor tissue penetration of short visible light wavelengths. Here, we introduce a new family of coumarin-based fluorophores, nicknamed COUPY, with promising photophysical properties, including emission in the far-red/near-infrared (NIR) region, large Stokes shifts, high photostability, and excellent brightness. COUPY fluorophores were efficiently synthesized in only three linear synthetic steps from commercially available precursors, with the N-alkylation of a pyridine moiety being the key step at the end of the synthetic route, as it allows for the tuning of the photophysical properties of the resulting dye. Owing to their low molecular weights, COUPY dyes show excellent cell permeability and accumulate selectively in nucleoli and/or mitochondria of HeLa cells, as their far-red/NIR fluorescence emission is easily detected at a concentration as low as 0.5 μM after an incubation of only 20 min. We anticipate that these coumarin scaffolds will open a way to the development of novel coumarin-based far-red to NIR emitting fluorophores with potential applications for organelle imaging and biomolecule labeling.

We report the synthesis and photochemical properties of a series of dicyanocoumarinylmethyl (DEAdcCM)‐ and dicyanocoumarinylethyl (DEAdcCE)‐based photocages of carboxylic acids and amines with absorption maximum around 500 nm. Photolysis studies with green light have demonstrated that the structure of the coumarin chromophore as well as the nature of the leaving group and the type of bond to be photocleaved (ester or carbamate) have a strong influence on the rate and efficiency of the uncaging process. These experimental observations were also supported by DFT calculations. Such differences in deprotection kinetics have been exploited to sequentially photolyze two dicyanocoumarin‐caged model compounds (e.g., benzoic acid and ethylamine), and open the way to increasing the number of functional levels that can be addressed with light in a single system, particularly when combining dicyanocoumarin caging groups with other photocleavable protecting groups, which remain intact under green light irradiation.

Replacement of electron-donating N,N-dialkyl groups with three- or four-membered cyclic amines (e.g., aziridine and azetidine, respectively) has been described as a promising approach to improve some of the drawbacks of conventional fluorophores, including low fluorescent quantum yields (Φ) in polar solvents. In this work, we have explored the influence of azetidinyl substitution on nonconventional coumarin-based COUPY dyes. Two azetidine-containing scaffolds were first synthesized in four linear synthetic steps and easily transformed into far-red/NIR-emitting fluorophores through N-alkylation of the pyridine moiety. Azetidine introduction in COUPY dyes resulted in enlarged Stokes' shifts with respect to the N,N-dialkylamino-containing parent dyes, but the Φ were not significantly modified in aqueous media, which is in contrast with previously reported observations in other fluorophores. However, azetidinyl substitution led to an unprecedented improvement in the photostability of COUPY dyes, and high cell permeability was retained since the fluorophores accumulated selectively in mitochondria and nucleoli of HeLa cells. Overall, our results provide valuable insights for the design and optimization of novel fluorophores operating in the far-red/NIR region, since we have demonstrated that three important parameters (Stokes' shifts, Φ, and photostability) cannot be always simultaneously addressed by simply replacing a N,N-dialkylamino group with azetidine, at least in nonconventional coumarin-based fluorophores.