Electrochemical Activation of Galactose Oxidase: Mechanistic Studies and Synthetic Applications

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.

Electrochemical Recycling of Adenosine Triphosphate in Biocatalytic Reaction Cascades

“…18,19 The use of a redox mediator that shuttles electrons between the electrode and the enzyme active site can alleviate some limitations. 20,21 Cyclic voltammetry (CV) of PO in the presence of substrate shows no redox event (Fig. S8), confirming that direct electron transfer between the electrode and the enzyme is not observed under these conditions.…”

On the Mediated Electron Transfer of Immobilized Galactose Oxidase for Biotechnological Applications

“…As a result, the implementation of biocatalytic transformations is currently of high relevance in the fine chemical and pharmaceutical industries. [4] Galactose oxidase (GalOx) is a mononuclear copper enzyme that catalyzes the oxidation of primary alcohols to aldehydes using molecular oxygen as the natural electron acceptor. The active site comprises a tyrosine radical and a Cu 2 + metal center as distinct one-electron acceptors.…”

“…Consequently, GalOx has become an industrially relevant enzyme applied in biocatalytic transformations for the synthesis of diverse products of interest [1,2] and has been widely explored in synthetic manufacturing, including the pharmaceutical industry. [4,15] Furthermore, GalOx is an attractive enzyme for biotechnological applications, as it also catalyzes the oxidation of biorenewable platform products such as glycerol [3] and 5hydroxymethylfurfural (HMF). [16] In contrast with other strategies for the catalytic conversion of these products, [17] biologically driven catalytic transformations are inherently more selective and constitute a renewable alternative with the possibility to operate at lower applied potentials, under mild pH values, and ambient temperature conditions.…”

“…Thus, technological applications of GalOx conventionally involve the addition of large amounts of catalase for catalyzing the dismutation of H 2 O 2 into H 2 O and O 2 . [1,3,4,15,16,18] Moreover, the Michaelis constant (K M ) for O 2 as substrate required for the regeneration of the enzyme active site has been estimated to be above 3 mM. [2,8] As this value is higher than the solubility of O 2 in aqueous solutions under ambient conditions, the reaction rate of conversion is highly affected by small variations in O 2 concentrations.…”

“…In particular, the use of enzymes for performing catalytic conversions offers several advantages, enabling environmentally friendly and highly selective transformations under mild and aqueous conditions. As a result, the implementation of biocatalytic transformations is currently of high relevance in the fine chemical and pharmaceutical industries [4] …”