Trialkyl(vinyl)phosphonium Chlorophenol Derivatives as Potent Mitochondrial Uncouplers and Antibacterial Agents

Abstract

Trialkyl phosphonium derivatives of vinyl-substituted p-chlorophenol were synthesized here by a recently developed method of preparing quaternary phosphonium salts from phosphine oxides using Grignard reagents. All the derivatives with a number (n) of carbon atoms in phosphonium alkyl substituents varying from 4 to 7 showed pronounced uncoupling activity in isolated rat liver mitochondria at micromolar concentrations, with a tripentyl derivative being the most effective both in accelerating respiration and causing membrane potential collapse, as well as in provoking mitochondrial swelling in a potassium-acetate medium. Remarkably, the trialkyl phosphonium derivatives with n from 4 to 7 also proved to be rather potent antibacterial agents. Methylation of the chlorophenol hydroxyl group suppressed the effects of P₅₅₅ and P₄₄₄ on the respiration and membrane potential of mitochondria but not those of P₆₆₆, thereby suggesting a mechanistic difference in the mitochondrial uncoupling by these derivatives, which was predominantly protonophoric (carrier-like) in the case of P₅₅₅ and P₄₄₄ but detergent-like with P₆₆₆. The latter was confirmed by the carboxyfluorescein leakage assay on model liposomal membranes.

Scheme 1. Synthesis of the Phosphonium Salts

Figure 1

(A) Crystal packing of P₃₃₃, view along the c axis. (B) ORTEP representation of P₃₃₃ showing 50% probability thermal ellipsoids. C atoms, gray; N atoms; blue; O atoms, red.

Figure 2

(A) Stimulation of respiration of rat liver mitochondria by P₅₅₅ and P₅₅₅-OMe. Numbers on the curves correspond to the respiration rates in nmol O₂/min/mg of protein. The medium was supplemented with 2 μM rotenone. Additions where indicated: 5 mM succinate, 6 μM P₅₅₅, 6 μM P₅₅₅-OMe, and 30 μM DNP. (B) Dose dependence of stimulation of RLM respiration by P₅₅₅ (closed triangles) and P₅₅₅-OMe (open circles) with succinate as a substrate. (C) Histogram of the concentrations of P₃₃₃–P₈₈₈ corresponding to half-maximal respiration rates (mean ± S.D., n = 4). For other conditions, see the Experimental Section.

Figure 3

(A) Effect of P₄₄₄ (black curve) and P₄₄₄-OMe (red curve) on the mitochondrial membrane potential estimated by absorbance changes of the potential-sensitive dye safranine O (15 μM). Panels (B) and (C) showing similar data for P₅₅₅/P₅₅₅-OMe and P₆₆₆/P₆₆₆-OMe, respectively. Addition of 2 μM (first arrow) and 4 μM (second arrow) and successive additions of 8 μM of the compounds are marked by arrows. Measurements of the membrane potential were performed in the incubation medium (see the Experimental Section) with 5 mM succinate and 2 μM rotenone.

Figure 4

(A,B) Swelling of rat liver mitochondria induced by P₃₃₃–P₈₈₈ (10 μM) in the potassium acetate medium. Swelling by 300 nM CCCP is shown in panel (A). (C) Swelling at t = 50 s. The data points represent mean ± SD of three independent experiments.

Figure 5

(A) Carboxyfluorescein (CF) leakage from liposomes induced by P₃₃₃–P₈₈₈. The concentration of the compounds was 3 μM. CF-loaded liposomes (100 mM CF inside) were formed from DPhPC. The concentration of the liposome lipid was 10 μg/mL. The buffer was 10 mM Tris and 100 mM KCl, pH = 7.5. (B) CF leakage at t = 100 s. The data points represent mean ± SD of three independent experiments.

Scheme 2. Scheme of Protonophoric Activity of P₅₅₅ in Mitochondria (Left Side) and the Formation of Defects in Lipid Bilayer Integrity by P₆₆₆ (Right Side), Enabling Permeability for Protons and Potassium Ions

Figure 6

Dose-dependent effect of P₃₃₃–P₈₈₈ on the growth kinetics of Bacillus subtilis assessed with a 96-well Multiskan FC microplate reader with the absorbance at 620 nm at 30 °C. The data points represent mean ± SD of at least three experiments.

Figure 7

Hemolytic activity of P₅₅₅ and P₅₅₅-OMe estimated as an efflux of hemoglobin from human red blood cells during 1 h at 37 °C. The data points represent mean ± SD of at least three experiments.