Electrochemical Activation of Galactose Oxidase: Mechanistic Studies and Synthetic Applications
Shaoguang Zhang, Serge Ruccolo, Anna Fryszkowska, Artis Klapars, Nicholas Marshall, Neil A. Strotman
Abstract
The enzyme galactose oxidase (GOase) is a copper radical oxidase that catalyzes the aerobic oxidation of primary alcohols to the aldehydes and has been utilized to that end in large-scale pharmaceutical processes. To maintain its catalytic activity and ensure high substrate conversion, GOase needs to be continuously (re)activated by 1e– oxidation of the constantly formed out-of-cycle species (GOasesemi) to the catalytically active state (GOaseox). In this work, we report an electrochemical activation method for GOase that replaces the previously used expensive horseradish peroxidase activator in a GOase-catalyzed oxidation reaction. First, the formation of GOaseox of a specifically engineered variant via nonenzymatic oxidation of GOasesemi was studied by UV–vis spectroscopy. Second, electrochemical oxidation of GOase by mediators was studied using cyclic voltammetry. The electron-transfer rates between GOase and various mediators at different pH values were determined, showing a dependence on both the redox potential of the mediator and the pH. This observation suggests that the oxidation of GOase by mediators at pH 7–9 likely occurs via a concerted proton-coupled electron-transfer (PCET) mechanism under anaerobic conditions. Finally, this electrochemical GOase activation method was successfully applied to the development of a bioelectrocatalytic GOase-mediated aerobic oxidation of benzyl alcohol derivatives, cinnamyl alcohol, and aliphatic polyols, including the desymmetrizing oxidation of 2-ethynylglycerol, a key step in the biocatalytic cascade used to prepare the promising HIV therapeutic islatravir.