Redox-Dependent Lipophilicity of Phenazine Metabolites Is Modulated by Intramolecular Hydrogen Bonds and Controls...

Phenazines are redox-active microbial metabolites produced and secreted in diverse ecological contexts from soils to chronic infections. Phenazines can function variously as antibiotics, extracellular electron shuttles, and nutrient scavengers. Key to understanding the impact of these functions is a robust expectation of phenazine retention or diffusion in a given context. But predicting phenazine fate and transport is difficult because of the chemical complexity of their local microenvironments. To address this challenge, we measured the octanol-water distribution coefficient (LogD) as a proxy for the lipophilicity of three naturally occurring phenazines produced by the opportunistic pathogen Pseudomonas aeruginosa: phenazine-1-carboxylic acid, phenazine-1-carboxamide, and pyocyanin. We investigated the behavior of both oxidized and reduced forms of these phenazines across broad ionic strength and pH conditions. While the ionic context exerts only small effects, the pH and redox state contribute strongly and independently to changes in phenazine lipophilicity. The pH trends are expected, but the observed redox dependence is generally missed by existing lipophilicity calculation methods. Additional experiments and density functional theory modeling of phenazines in their reduced and oxidized forms reveal that intramolecular hydrogen bonding contributes significantly to the increased lipophilicity of reduced phenazines that possess H-bond-accepting substituents in the 1-position, a phenomenon likely generalizable to other redox-active natural products with comparable intramolecular hydrogen-bonding frameworks. These results explain phenazine behavior in a biological context: redox state alone significantly alters retention of pyocyanin in planktonic P. aeruginosa cells, with the reduced species being predominantly retained by membranes.