Product Distribution of Steady–State and Pulsed Electrochemical Regeneration of 1,4-NADH and Integration with Enzymatic Reaction

The direct electrochemical reduction of nicotinamide adenine dinucleotide (NAD⁺) results in various products, complicating the regeneration of the crucial 1,4-NADH cofactor for enzymatic reactions. Previous research primarily focused on steady–state polarization to examine potential impacts on product selectivity. However, this study explores the influence of dynamic conditions on the selectivity of NAD⁺ reduction products by comparing two dynamic profiles with steady-state conditions. Our findings reveal that the main products, including 1,4-NADH, several dimers, and ADP-ribose, remained consistent across all conditions. A minor by–product, 1,6-NADH, was also identified. The product distribution varied depending on the experimental conditions (steady state vs. dynamic) and the concentration of NAD⁺, with higher concentrations and overpotentials promoting dimerization. The optimal yield of 1,4-NADH was achieved under steady–state conditions with low overpotential and NAD⁺ concentrations. While dynamic conditions enhanced the 1,4-NADH yield at shorter reaction times, they also resulted in a significant amount of unidentified products. Furthermore, this study assessed the potential of using pulsed electrochemical regeneration of 1,4-NADH with enoate reductase (XenB) for cyclohexenone reduction.