Dodecyl and octyl esters of fluorescein as protonophores and uncouplers of oxidative phosphorylation in mitochondria at submicromolar concentrations

Abstract

In our search for fluorescent uncouplers of oxidative phosphorylation, three esters of fluorescein, n-butyl-, n-octyl-, and n-dodecyl-oxycarbonyl-fluorescein (C4-FL, C8-FL, C12-FL) were synthesized and characterized. With increasing liposomal lipid content, the long-chain alkyl derivatives of fluorescein (C8-FL, C12-FL and commercially available C18-FL), but not C4-FL and unsubstituted fluorescein, exhibited an increase in fluorescence polarization reflecting the dye binding to liposomes. C12-FL induced proton permeability in lipid membranes, while C4-FL was inactive. In contrast to C4-FL and C18-FL, C12-FL and C8-FL increased the respiration rate and decreased the membrane potential of isolated rat liver mitochondria with half-maximal effective concentrations of 700nM and 300nM, respectively. The effect of Cn-FL on the respiration correlated with that on proton permeability of the inner mitochondrial membrane, as measured by induction of mitochondria swelling in the potassium acetate medium. Binding of C8-FL to mitochondria depended on their energization, which was apparently associated with pH gradient generation across the inner mitochondrial membrane in the presence of a respiratory substrate. In wild-type yeast cells, C12-FL localized predominantly in plasma membrane, whereas in AD1-8 mutants lacking MDR pumps, it stained cytoplasmic organelles with some preference for mitochondria. Fluorescent uncouplers can be useful as a tool for determining their localization in a cell or distribution between different tissues in a living animal by fluorescent microscopy.

Mitochondria-Targeted Triphenylphosphonium-Based Compounds: Syntheses, Mechanisms of Action, and Therapeutic and Diagnostic Applications

“…444 Similar effects were observed previously for the dodecyl and octyl esters of fluorescein. 445 The fluorescent properties of mitoFluo enabled the study on the uptake of the probe from medium to mitochondria. The energization of mitochondria with succinate resulted in the probe uptake.…”

Fluorescein Derivatives as Antibacterial Agents Acting via Membrane Depolarization

“…To synthesize fluorescein decyl ester (C10-FL), we used the esterification of the carboxyl group with a C10 alkyl substituent (Scheme S2, SI). The synthesis was performed via the reaction between 1-bromodecane and the previously obtained Na salt of fluorescein, i.e., by using an approach developed earlier for the synthesis of alkylrhodamine [27] and alkylfluorescein [21] derivatives.…”

“…At present, there is a revival of interest in uncouplers because of the growing evidence of their broad-spectrum therapeutic efficacy [1][2][3][4][5][6][7][8][9], including antibacterial potency [10][11][12][13][14][15][16][17][18][19], which was actually found very early [20]. To this end, a series of esters of fluorescein were obtained and studied at our laboratory, manifesting themselves as rather effective mitochondrial uncouplers [21][22][23]. Amongst them, the most intriguing compound was fluorescein decyl(triphenyl)phosphonium ester (mitoFluo, Figure 1), which was able to accumulate in energized mitochondria due to a cationic triphenylphosphonium (TPP) moiety, which was readily monitored via its bright fluorescence [22], and which exhibited neuro-and nephroprotective properties [23].…”

SR4 Uncouples Mitochondrial Oxidative Phosphorylation, Modulates AMP-dependent Kinase (AMPK)-Mammalian Target of Rapamycin (mTOR) Signaling, and Inhibits Proliferation of HepG2 Hepatocarcinoma Cells

“…Mitochondrial swelling was measured in non-respiring mouse liver mitochondria incubated in buffered isotonic potassium acetate in the presence of valinomycin as described (37,38). Briefly, isolated liver mitochondria were added to 1 ml of isotonic acetate buffer (145 mM potassium acetate, 5 mM TrisHCl, 0.5 mM EDTA, 5 M valinomycin, and 1 M rotenone, pH 7.4) in a cuvette at a final concentration of 0.5 mg/ml mitochondrial protein.…”