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.

2,7-Difluoropyronin B accumulates in cellular mitochondria, reacts with amines, and undergoes oxidation promoted by blue light to trigger mitochondrial depolarization.

Introduction

Mitochondria are dynamic rod-shaped organelles that biosynthesize cellular ATP.¹ This process requires a high negative potential (−120 to −180 mV) across inner mitochondrial membranes to couple electron transport to oxidative phosphorylation reactions.² Whereas mild depolarization of these membranes is associated with anti-aging effects,³ severe mitochondrial depolarization can lead to cellular dysfunction or death. Consequently, defects in mitochondria are linked to a wide range of pathologies including neurodegenerative disease, obesity, cardiomyopathy, and cancer.^4,5

To target mitochondria, a wide variety of small molecule probes have been employed.⁶ As shown in Fig. 1, lipophilic cations such as rhodamine 123 (1),⁷ pyronin B (2), and other related compounds^6,8 accumulate in polarized mitochondria, presumably due to electrophoresis across the inner membrane. Because rhodamines can be highly fluorescent, derivatives of these compounds have been extensively used to create cellular probes⁹ and sensors.¹⁰ In contrast, derivatives of pyronin B (2) have not been as extensively studied, but pyronins have been used for cell cycle analysis,¹¹ bioimaging,¹² and as sensors of pH,¹³ nerve agents,¹⁴ cellular peroxynitrite,¹⁵ heavy metals,¹⁶ ATP dynamics,¹⁷ proteases,¹⁸ and protein–protein interactions.¹⁹

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

Small molecules that uncouple the electron chain of mitochondria, or that otherwise depolarize mitochondrial membranes, provide important molecular probes with potential therapeutic applications.^20,21 Compounds that trigger photochemical depolarization of mitochondria have been used to study biological processes,^22,23 or sensitize cancer cells to chemotherapeutics.²⁴ However, existing photoactivatable probes of mitochondria rely on either UV-mediated cleavage of a nitrobenzyl group to release an uncoupler from a triphenylphosphonium salt²³ or produce reactive oxygen species by near-IR laser radiation of nanoparticles.²⁴ In an effort to provide more drug-like chemical tools to study mitochondria, we describe here the synthesis of novel analogues of pyronin B (2, Fig. 1). We identified 2,7-difluoropyonin B (3) as a unique probe that covalently modifies biological amines and rapidly depolarizes mitochondria upon irradiation of treated cells with visible blue light. The mechanism of this process is proposed to involve photochemical generation of reactive oxygen species (ROS) that oxidizes adducts of amines of key mitochondrial biomolecules.

Results and discussion

We investigated fluorinated analogues of pyronin B because fluorination is widely used to improve the properties of therapeutics and chemical probes.²⁵ This modification can profoundly affect drug metabolism, reactivity, and when incorporated into the fluorophores Oregon Green, Pacific Blue, and Pennsylvania Green, enhances photostability and fluorescence in acidic environments.^26,27 As shown in Scheme 1, pyronins 3, 4 and 8 were synthesized from the previously reported²⁸ 2,7-difluoroxanthone (7), whereas the azetidine analogues 5 and 6 were prepared from the known precursors 9–12.^28,29

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

Pyronin B (2) and its structurally similar N,N-dimethyl analogue (pyronin Y) are highly fluorescent.³⁰ To characterize the optical properties of analogues 2–8, we obtained absorbance and emission spectra in ethanol (Fig. 2). These studies revealed that modification of 2 with fluorine to yield 3 reduces fluorescence quantum yield by 40-fold (Φ(3) = 0.007) without substantially affecting other photophysical properties (Abs. λ_max(3) = 563 nm; Em. λ_max(3) = 586 nm). In contrast, the absorbance and fluorescence emission of aminopyronin 4 were blue shifted (Abs. λ_max(4) = 443 nm; Em. λ_max(4) = 531 nm) to provide a spectrally orthogonal fluorophore when excited with a 405 nm violet laser, although some excitation of 4 at 488 nm was also possible. The azetidine analogues 5 and 6 were spectrally similar to 2 but exhibited greater fluorescence quantum yields (Φ(5) = 0.63; Φ(6) = 0.27), consistent with prior studies of azetidine derivatives.^31,32

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).

To examine effects on living cells, the human cervical cancer cell line HeLa was treated with probes 1–6 and imaged by confocal laser scanning microscopy (Fig. S2†). Similar to rhodamine 123 (1) and pyronin B (2), 2,7-difluoropyronin B (3) and the pyronin azetidine (5) accumulated in rod-shaped organelles, consistent with localization in mitochondria. The specific localization of 3 and 5 in mitochondria was established by co-treatment with the spectrally orthogonal mitochondria probe MitoTracker Deep Red (MTDR, Fig. 3 and S3†). In contrast, the blue-shifted fluorescence of 4 was observed in both mitochondria and lysosomes as evidenced by colocalization with MTDR and LysoTracker red (Fig. S3†). Surprisingly, despite its high fluorescence in ethanol, 6 did not lead to appreciable cellular fluorescence, suggesting that altered reactivity towards cellular nucleophiles conferred by the strained azetidines abolishes fluorescence.

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.

In contrast to pyronins 2 and 4–6, the subcellular localization of 2,7-difluoropyronin B (3) was profoundly altered upon extended confocal imaging with a 488 nm laser. As shown in Fig. 3 (panels B and C), irradiation of cells treated with 3 at 488 nm for 60 s resulted in diffusion of fluorescence from mitochondria to the cytoplasm and nucleus, where fluorescence accumulated in nucleoli (Fig. 3). Because other pyronins have previously been shown to bind RNA in nucleoli of fixed cells that lack polarized mitochondria,^33,34 these results suggested that irradiation with visible blue light causes difluoropyronin B (3) to depolarize these organelles, diffuse into the cytoplasm, accumulate in the nucleolus, and bind RNA. To test this hypothesis, we treated cells with both 3 and CCCP, a small molecule uncoupler of oxidative phosphorylation that specifically depolarizes mitochondria. Confocal microscopy of cells treated with 3 (1 h, 10 μM), followed either by treatment with CCCP (10 μM, 5 min) or irradiation with the blue laser (60 s), showed that both of these conditions caused diffusion of fluorescence from mitochondria and accumulation of fluorescence in nucleoli (Fig. S4†). As a control, cells treated with pyronin B (2, 10 μM) were similarly affected by CCCP but not by blue light (Fig. S4†), further demonstrating the importance of the fluorine substituents of 3 for blue light-mediated depolarization of mitochondria. In contrast, the methylated analogue 8 accumulated in mitochondria, similar to 3, but it did not depolarize these organelles upon irradiation (Fig. S4†), demonstrating that 3 exhibits unique biological properties.

To investigate the mechanistic basis of mitochondrial depolarization mediated by irradiation of 3 with blue light, we examined the reactivity of 2, 3, 5, 6 with bovine serum albumin protein (BSA, Fig. 4) by fluorescence spectroscopy. All of these compounds were found to be stable alone at room temperature in pure water, but 3 uniquely reacted with BSA to form derivatives with a maximal absorbance at ∼439 nm and emission at ∼540 nm (excitation at 488 nm, Fig. 4, panels A and B). This emission profile at ∼540 nm corresponds closely to that of 9-ethyl amino difluoropyronin B (4) in EtOH (Fig. 2), suggesting the formation of aminopyronin derivatives. To investigate whether these derivatives are increased by blue light, solutions of BSA and 3 were additionally irradiated with a blue LED flashlight (490 nm, 10 min). As shown in Fig. 4 (panel B), emission at ∼540 nm was enhanced under these conditions. Excitation at 405 nm additionally revealed that 3 forms some difluoroxanthone 7 (Abs. λ_max,EtOH(7) = 389 nm; Em. λ_max,EtOH(7) = 452 nm)²⁸ in the presence of BSA, indicating hydrolysis of oxidized adducts to generate this product. To determine if this reactivity can be observed in cells, we compared living HeLa cells treated with 3 and 2 and generated fluorescence emission spectra by spectral scanning on a confocal laser scanning microscope. As shown in Fig. 5, excitation at 488 nm and 532 nm revealed that only the fluorinated derivative 3 is converted to 9-amino derivatives in living cells. Under these conditions, the low emission from 3 at 586 nm is likely due to the intensity of repeated laser irradiation required for spectral scanning, which promotes conversion of 3 to blue-shifted aminopyronins. Structurally related thiopyronins,^35–37 derived from attack by sulfhydryl groups, exhibit red-shifted emission spectra with λ_max above 600 nm, and these types of derivatives of 3 were not observed.

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.

The excitation of fluorophores typically generates reactive oxygen species (ROS).³⁸ To examine whether irradiation of 3 with blue light generates ROS that might promote conversion of amino adducts of biomolecules to aminopyronins, HeLa cells were treated with dihydroethidium (DHE). This non-fluorescent probe is oxidized by ROS to generate the highly fluorescent DNA-binding dye ethidium. Cells were treated with antimycin A as a positive ROS-promoting control, 2, and 3, and fluorescence of cellular nuclei quantified by image analysis of confocal micrographs. As shown in Fig. 5, antimycin A, 2, and 3 all increased basal levels of cellular ROS. However, irradiation of cells treated with 3 (2 μM) at 488 nm increased nuclear fluorescence by 1.5-fold after 1 min, and 2.7-fold after 2 min, but did not affect cells treated with 2 (2 μM). The magnitude of these results is similar to mitochondria-targeted coumarin derivatives that have been reported³⁹ to exhibit similar 2.5-fold increases in ROS in HeLa cells upon laser irradiation under normoxic conditions. These results indicate that treatment of cells with either 2 or 3 results in generation of ROS, but 3 uniquely enhances formation of additional ROS upon irradiation at 488 nm. This ROS is likely to further promote oxidation of amino adducts of 3 in mitochondria to generate 9-aminopyronin derivatives and profoundly affect the functions of biomolecules in these organelles.

We additionally assessed the stability of biological amino adducts derived from 3 by examining the efflux of probes 1–3 from living Jurkat lymphocytes. Cells were treated for 1 h, followed by washing with fresh media to allow efflux of small molecules, with subsequent analysis of cellular fluorescence by flow cytometry. As shown in Fig. 6, rhodamine 123 (1) is retained in mitochondria with a very long half-life of ∼17 h, whereas fluorescence upon treatment with both 2 (t_1/2 = 4 min) and 3 (t_1/2 = 5 min) was rapidly lost. Although 3 forms covalent aminopyronin adducts within cells, over 90% of its fluorescence could be washed away within 30 min (Fig. 6), indicating that it does not extensively form stable modifications with amines of proteins. This short half-life of 3 might also relate to its reaction with small molecule amines to form derivatives that undergo efflux upon washing. Alternatively, relatively rapid hydrolysis of the cellular aminopyronins visible by confocal microscopy to the less readily detected xanthone 7 might also contribute to loss of fluorescence upon removal of 3 from cells by washing. Partial localization of 4 in lysosomes may contribute to its extended half-life (t_1/2 = 60 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.

The cytotoxicity of 2–7 towards HeLa cells and Jurkat lymphocytes is shown in Fig. 7. Treatment of these cell lines for 48 h with these compounds followed by analysis of viability by flow cytometry revealed that difluoropyronin B (3) was ∼5-fold less toxic than pyronin B (2) in HeLa cells (IC₅₀(3) = 9 μM) and 10-fold less toxic than 2 in Jurkat cells (IC₅₀(3) = 1 μM). The hydrolysis product 7 was non-toxic to HeLa cells at concentrations below 30 μM (IC₅₀(7) = 51 μM), suggesting that hydrolysis of amino adducts derived from 3 to yield the essentially non-toxic small molecule 7 may contribute to the substantially lower toxicity of 3 compared to 2.

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%.

Conclusions

We propose that 2,7-difluoropyronin B (3) and blue light depolarize mitochondria through the mechanism shown in Fig. 8. As a lipophilic cation similar to pyronin B, 3 selectively accumulates in polarized mitochondria of living cells. However, the high electrophilicity conferred by the fluorines of 3 promotes rapid and reversible formation of adducts with amines in this organelle. Because mitochondria of mammalian cells are reported to exhibit elevated temperatures compared to the cytoplasm,⁴⁰ and have a matrix pH of ∼8.0,⁴¹ these conditions likely further promote the reactivity with amines in this subcellular environment. These amines could include reduced glutathione (GSH)^42,43 and/or spermine,^44,45 which are present in mitochondria at high concentrations (≥1 mM),⁴⁶ and are critical for maintenance of the mitochondrial membrane potential. Protein lysine residues or N-terminal amines could also represent subcellular targets of photochemical depolarization mediated by 3. We propose that the increase in cellular ROS mediated by treatment with 3 partially oxidizes amine adducts to aminopyronins. Subsequent excitation of unreacted 3 in mitochondria with blue light further increases ROS, enhances the formation of fluorescent aminopyronins, and drives photochemical depolarization. Consistent with this mechanism, blue light promotes reaction of GSH and spermine with 3 in pure water to form aminopyronins (and the hydrolysis product 7, Fig. S5†). Additionally, the 9-methyl analogue 8, which cannot be oxidized to form aminopyronins, does not depolarize mitochondria upon irradiation (Fig. S4†). The unique ability of 2,7-difluoropyronin B (3) to rapidly depolarize mitochondria upon irradiation with blue light offers a novel chemical tool for studies of mitochondrial biology.

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.

Conflicts of interest

There are no conflicts to declare.