Bipyridines as Mediating Artificial Cofactors to replace NAD(P)H

BPYMAC (Bipyridines as Mediating Artificial Cofactors to replace NAD(P)H) addressed a major limitation of enzyme-based manufacturing: many useful dehydrogenase enzymes require the natural cofactors NADH or NADPH to supply reducing equivalents. These cofactors are effective in biology but are costly, unstable and difficult to recycle efficiently in an industrial process. Their repeated addition also complicates product separation.
The project aimed to replace NAD(P)H with tailored viologen-based artificial cofactors. Viologens can be attached to a polymer, combined with an enzyme in a hydrated coating on an electrode and repeatedly reduced using electricity. The enzyme and artificial cofactor therefore remain confined at the electrode while the starting material and product remain in solution. This approach could simplify cofactor recycling, catalyst recovery and downstream processing.
Two contrasting application areas were selected as proof-of-concept tests: conversion of carbon dioxide towards valuable C₁ products and enantioselective synthesis of chiral pharmaceutical intermediates. Their broader purpose was to determine whether the artificial-cofactor concept could be transferred between different NAD(P)H-dependent enzymes. The pathway to impact was therefore to design a tunable polymer platform, validate it with more than one dehydrogenase, establish reactor-relevant performance and identify the chemical features and mechanism that govern successful NADH replacement.

BPYMAC synthesised and characterised viologen-functionalised polymers and integrated them with enzymes in crosslinked hydrogel films on carbon-cloth electrodes. Electrochemical methods were used to regenerate the reduced viologen during catalysis, while analytical methods quantified the products and verified that activity depended on the enzyme.
The first study used formate dehydrogenase to reduce carbon dioxide to formate without adding NADH. It compared the artificial-cofactor bioelectrode with conventional NADH-dependent biocatalysis and two NADH-regeneration approaches. The work also tested suppression of the reverse reaction, cofactor turnover, faradaic efficiency and the effect of increasing electrode area.
The second study transferred the platform to alcohol dehydrogenase for conversion of formaldehyde to methanol. A library of nine viologen polymers was compared to determine how redox potential, charge, hydration and polymer structure influence activity. Denatured-enzyme and non-viologen polymer controls were used to exclude non-enzymatic reduction. Experiments in deuterated water, supported by mass spectrometry and nuclear magnetic resonance, investigated how a one-electron viologen material can replace a cofactor that normally transfers a hydride.
The original complete carbon-dioxide-to-methanol cascade could not be assembled because commercial formaldehyde dehydrogenase became unavailable. Engineered enzymes were sought through another CSIC group but arrived too close to the end of the project for systematic testing. The required enantioselective alcohol dehydrogenases for the chiral case study could not be supplied in the small research quantity by the available EU suppliers; non-EU alternatives were identified, but delivery would have occurred after the action. The project therefore adapted its scientific focus to cross-enzyme transferability, structure-activity relationships and mechanism. The results were disseminated through a peer-reviewed open-access article, a public ADH preprint/revised manuscript and four conference presentations during the action.

The project demonstrated that electrode-regenerated viologen polymers can replace NADH in two distinct dehydrogenases. In the carbon dioxide study, the formate-dehydrogenase bioelectrode produced 43 times more formate than the NADH-only comparison and achieved a faradaic efficiency of 95.4%. The reverse formate-oxidation reaction was suppressed, and product formation increased when the electrode area was enlarged from 0.09 to 2.25 cm². These results provide direct evidence that the immobilised artificial-cofactor architecture has characteristics relevant to scale-up, efficient electron use and simpler downstream processing.
In the alcohol-dehydrogenase study, all nine viologen polymers supported methanol formation without NADH. An amino-functionalised viologen scaffold that had already worked with formate dehydrogenase also worked with alcohol dehydrogenase, directly demonstrating transferability. However, the best polymer for alcohol dehydrogenase was not simply the one with the most negative redox potential or the highest accessible redox-site density. Charge, hydration and compatibility between the polymer scaffold and the enzyme were equally or more important. Isotope experiments supported a mechanism in which the reduced polymer supplies electrons while hydrogen equivalents are supplied from the surrounding solution, rather than the polymer acting as a conventional hydride donor.
The main scientific impact is a shift from viewing viologens as generic low-potential electron carriers to treating them as tunable artificial cofactors whose molecular environment must be matched to each enzyme. The technological impact is an electrode-confined catalyst that combines enzyme immobilisation, cofactor localisation and electrical recycling. Potential future applications include carbon utilisation, multi-enzyme C₁ conversion, asymmetric synthesis and other NAD(P)H-dependent electrosyntheses.
The technology remains at laboratory proof-of-concept level. Further uptake requires access to the missing specialist enzymes, integration of the full cascade, testing with enantioselective enzymes, operational-lifetime and reuse studies, continuous-flow reactors, scale-up beyond geometric area, product-purification studies and techno-economic and environmental assessment. The results provide a strong basis for further collaborative research, future funding, industrial engagement and evaluation of intellectual-property opportunities once performance and durability data are sufficiently mature.

Periodic Reporting for period 1 - BPYMAC (Bipyridines as Mediating Artificial Cofactors to replace NAD(P)H)

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