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

Abstract

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.

Fig. 1. Structures of known (1, 2, 7) and novel (3–6, 8) molecular probes.

Scheme 1. Synthesis of molecular probes 3–6 and 8. Cationic products were isolated as TFA salts.

Fig. 2. (A) Optical spectra and photophysical properties in EtOH. Absorbance spectra (Abs.) were generated at 5 μM (2–6, 8) or 100 μM (7). Emission spectra (Em.) were collected at 25 nM (2, 5, 6), 1 μM (7), 2.5 μM (3, 8), and 2 μM (4).

Fig. 3. (A) Confocal and DIC micrographs of HeLa cells treated with difluoropyronin B (3, 10 μM, 1 h) followed by MitoTracker Deep Red (MTDR, 100 nM, 4 min). (B and C) Confocal and DIC micrographs of HeLa cells treated with 3 before (B) and after (C) irradiation at 488 nm for 60 s, showing blue-light mediated redistribution of fluorescence from mitochondria to the cytoplasm and nucleoli. Scale bars = 25 microns.

Fig. 4. Fluorescence emission spectra of pyronins (10 μM) after treatment with bovine serum albumin (BSA, 100 μM) for 1 h at 22 °C in pure water. Only 3 forms 9-aminopyronins as evidenced by the emission peak at ∼535 nm. In panel B, +hv indicates that solutions were subsequently irradiated with blue light (490 nm, LED flashlight, 10 min). Values are normalized to the maximal emission observed upon excitation at the wavelengths shown.

Fig. 5. (A) Fluorescence emission spectra of pyronins 2 and 3 (2 μM) in mitochondria of living HeLa cells. Emission profiles were acquired by spectral scanning with a Leica SPE confocal microscope. Values are normalized to the maximal emission observed upon excitation at 532 nm (for 2) or 488 nm (for 3). (B) Effects of 2 and 3 and blue light on ROS in living HeLa cells. Cells were treated with 2 (2 μM), 3 (2 μM), or the ROS-promoting positive control antimycin A (20 μM) for 1 h followed by dihydroethidium (DHE, 5 μM) for 1 h prior to quantification of fluorescence of nuclei by confocal microscopy (N ≥ 10, mean ± SEM, 63× objective). +hv = irradiation at 488 nm on a confocal microscope for 1 or 2 min.

Fig. 6. Fluorescence of probes 1–7 in Jurkat lymphocytes after washing with probe-free medium as quantified by flow cytometry. Cells were treated with probes (10 μM) for 1 h (37 °C). At each time point shown, cells were washed with fresh complete media lacking the probe prior to analysis of cellular fluorescence (Ex. 405 nm or 488 nm) by flow cytometry. Data was fit to an exponential one-phase decay model (GraphPad Prism 9). Standard errors are ±∼5 min. The short half-life of 3 suggests that covalent adducts with biomolecules in cells are reversible or transient.

Fig. 7. Cytotoxicity of 2–7 towards HeLa cells (A) and Jurkat lymphocytes (B) after 48 h quantified by flow cytometry. Standard errors in IC₅₀ values are ∼10%.

Fig. 8. Proposed mechanism of photochemical depolarization of mitochondria. Pyronin 3 accumulates in these organelles, forms reversible adducts of amines of mitochondrial biomolecules, and generates ROS that promotes some oxidation to fluorescent 9-aminopyronins. Irradiation of these cells with blue light triggers an additional burst of ROS that further converts mitochondrial amines to fluorescent 9-aminopyronins, causing organelle dysfunction that results in depolarization.