Bio‐electrocatalytic Alkene Reduction Using Ene‐Reductases with Methyl Viologen as Electron Mediator

Abstract Asymmetric hydrogenation of alkene moieties is important for the synthesis of chiral molecules, but achieving high stereoselectivity remains a challenge. Biocatalysis using ene‐reductases (EReds) offers a viable solution. However, the need for NAD(P)H cofactors limits large‐scale applications. Here, we explored an electrochemical alternative for recycling flavin‐containing EReds using methyl viologen as a mediator. For this, we built a bio‐electrocatalytic setup with an H‐type glass reactor cell, proton exchange membrane, and carbon cloth electrode. Experimental results confirm the mediator's electrochemical reduction and enzymatic consumption. Optimization showed increased product concentration at longer reaction times with better reproducibility within 4–6 h. We tested two enzymes, Pentaerythritol Tetranitrate Reductase (PETNR) and the Thermostable Old Yellow Enzyme (TOYE), using different alkene substrates. TOYE showed higher productivity for the reduction of 2‐cyclohexen‐1‐one (1.20 mM h −1 ), 2‐methyl‐2‐cyclohexen‐1‐one (1.40 mM h −1 ) and 2‐methyl‐2‐pentanal (0.40 mM h −1 ), with enantiomeric excesses ranging from 11 % to 99 %. PETNR outperformed TOYE in terms of enantioselectivity for the reduction of 2‐methyl‐2‐pentanal (ee 59 % ± 7 % ( S )). Notably, TOYE achieved promising results also in reducing ketoisophorone, a challenging substrate, with similar enantiomeric excess compared to published values using NADH.