Electrochemical Activation of Galactose Oxidase: Mechanistic Studies and Synthetic Applications

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 (GOase(semi)) to the catalytically active state (GOase(ox)). 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 GOase(ox) of a specifically engineered variant via nonenzymatic oxidation of GOase(semi) 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.

Automated summary

An electrochemical activation method for galactose oxidase (GOase) was developed to replace the expensive horseradish peroxidase activator in large-scale pharmaceutical processes. The study showed that the electron-transfer rates between GOase and various mediators depend on both the redox potential of the mediator and the pH. The electrochemical GOase activation method was successfully applied to the aerobic oxidation of benzyl alcohol derivatives and other compounds, including a key step in the biocatalytic cascade for the HIV therapeutic islatravir.