Alkyl-substituted phenylamino derivatives of 7-nitrobenz-2-oxa-1,3-diazole as uncouplers of oxidative phosphorylation and antibacterial agents: involvement of membrane proteins in the uncoupling action

Abstract

In search for new effective uncouplers of oxidative phosphorylation, we studied 4-aryl amino derivatives of a fluorescent group 7-nitrobenz-2-oxa-1,3-diazol (NBD). In our recent work (Denisov et al., Bioelectrochemistry, 2014), NBD-conjugated alkyl amines (NBD-Cn) were shown to exhibit uncoupling activity. It was concluded that despite a pKa value being about 10, the expected hindering of the uncoupling activity could be overcome by insertion of an alkyl chain. There is evidence in the literature that the introduction of an aryl substituent in the 4-amino NBD group shifts the pKa to neutral values. Here we report the data on the properties of a number of 4-arylamino derivatives of NBD, namely, alkylphenyl-amino-NBD (Cn-phenyl-NBD) with varying alkyl chain Cn. By measuring the electrical current across planar bilayer lipid membrane, the protonophoric activity of Cn-phenyl-NBD at neutral pH grew monotonously from C1- to C6-phenyl-NBD. All of these compounds increased the respiration rate and reduced the membrane potential of isolated rat liver mitochondria. Importantly, the uncoupling action of C6- and C4-phenyl-NBD was partially reversed by glutamate, diethyl pyrocarbonate (DEPC), 6-ketocholestanol, and carboxyatractyloside, thus pointing to the involvement of membrane proteins in the uncoupling activity of Cn-phenyl-NBD in mitochondria. The pronounced recoupling effect of DEPC, an inhibitor of an aspartate–glutamate carrier (AGC), and that of its substrates for the first time highlighted AGC participation in the action of potent uncouplers on mitochondria. C6-phenyl-NBD produced strong antimicrobial effect on Bacillus subtilis, which manifested itself in cell membrane depolarization and suppression of bacterial growth at submicromolar concentrations.