Synthesis of a fluorinated pyronin that enables blue light to rapidly depolarize mitochondria

Fluorinated analogues of the fluorophore pyronin B were synthesized as a new class of amine-reactive drug-like small molecules. In water, 2,7-difluoropyronin B was found to reversibly react with primary amines to form covalent adducts. When this fluorinated analogue is added to proteins, these adducts undergo additional oxidation to yield fluorescent 9-aminopyronins. Irradiation with visible blue light enhances this oxidation step, providing a photochemical method to modify the biological properties of reactive amines. In living HeLa cells, 2,7-difluoropyronin B becomes localized in mitochondria, where it is partially transformed into fluorescent aminopyronins, as detected by spectral profiling confocal microscopy. Further excitation of these cells with the blue laser of a confocal microscope can depolarize mitochondria within seconds. This biological activity was only observed with 2,7-difluoropyronin B and was not detected with analogues such as pyronin B or 9-methyl-2,7-difluoropyronin B. This irradiation with blue light enhances the cellular production of reactive oxygen species (ROS), suggesting that increased ROS in mitochondria promotes the formation of aminopyronins that inactivate biomolecules critical for maintenance of mitochondrial membrane potential. The unique reactivity of 2,7-difluoropyronin B offers a novel tool for photochemical control of mitochondrial biology.

Automated summary

Fluorinated analogues of pyronin B, a fluorophore, were synthesized and shown to react reversibly with primary amines. These analogues undergo oxidation in the presence of proteins, yielding fluorescent pyronins, and this oxidation step can be enhanced by blue light irradiation. The fluorinated analogue 2,7-difluoropyronin B was found to localize in mitochondria in living HeLa cells and depolarize the mitochondria upon blue laser excitation. This photochemical control of mitochondrial biology is attributed to the enhanced cellular production of reactive oxygen species.