Electrocatalytic NAD+ reduction via hydrogen atom-coupled electron transfer

Nicotinamide adenine dinucleotide cofactor (NAD(P)H) is regarded as an important energy carrier and charge transfer mediator. Enzyme-catalyzed NADPH production in natural photosynthesis proceeds via a hydride transfer mechanism. Selective and effective regeneration of NAD(P)H from its oxidized form by artificial catalysts remains challenging due to the formation of byproducts. Herein, electrocatalytic NADH regeneration and the reaction mechanism on metal and carbon electrodes are studied. We find that the selectivity of bioactive 1,4-NADH is relatively high on Cu, Fe, and Co electrodes without forming commonly reported NAD2 byproducts. In contrast, more NAD2 side product is formed with the carbon electrode. ADP-ribose is confirmed to be a side product caused by the fragmentation reaction of NAD+. Based on H/D isotope effects and electron paramagnetic resonance analysis, it is proposed that the formation of NADH on these metal electrodes proceeds via a hydrogen atom-coupled electron transfer (HadCET) mechanism, in contrast to the direct electron-transfer and NAD˙ radical pathway on carbon electrodes, which leads to more by-product, NAD2. This work sheds light on the mechanism of electrocatalytic NADH regeneration, which is different from biocatalysis.


Chemicals and materials
All chemicals were analytical grade and were used as purchased without further purification.

Preparation and pre-treatment of electrodes
Cu, Co, Fe, Ni foams were cleaned by ultrasonication in ethanol for 15 min, then sealed by 704 silicone sealant exposing an area of 1 cm 2 . Before electrochemical measurements, they were cleaned by ultrasonication in 1 M HCl for 5 min to remove the native oxide layer and then rinsed with pure water and dried with Ar stream.
The Ru electrode was prepared by depositing Ru on the Ni foam substrate. A cleaned Ni foam was immersed in de-aerated 0.1 M HCl containing 0.01 M RuCl3 for 3 h.
The Pt electrode was cleaned by ultrasonication in 3 M H2SO4 for 30 min before use and then rinsed with pure water and dried with Ar stream. Then, it was sealed by 704 silicone sealant exposing an area of 1 cm 2 .
The Ag electrode was cleaned by ultrasonication in ethanol for 15 min, then sealed by 704 silicone sealant exposing an area of 1 cm 2 . Before electrochemical measurements, it was cleaned by ultrasonication in 3 M H2SO4 and then rinsed with pure water and dried with Ar stream.

Electrochemical measurements
All electrochemical measurements were conducted in a three-electrode setup with Pt counter electrode (2 cm × 0.5 cm) and Ag/AgCl (with saturated KCl) reference electrode. All potentials were controlled by a potentiostat (CHI 660e) without iR compensation.
The NADH regeneration reactions were conducted by controlled-potential electrolysis in an H-type cell containing 0.1 M sodium phosphate electrolyte, and the catholyte is 12 mL electrolyte with an initial NAD + concentration of 1 mM with a Nafion-117 membrane. Electrolytes were pre-saturated and bubbled with Ar under magnetic stir during measurements. Cu foam and other electrodes were used as work electrodes for NAD + reduction at applied potentials either -1.01 V, -1.11 V or -1.21 V vs. Ag/AgCl.

Quantification of various NAD + reduction products
The concentration of NADH was quantitatively determined by UV-visible light absorption and 1 HNMR measurements. Firstly, NADH was produced in 12 mL 0.
where A0 is the initial absorbance prior to enzymatic catalysis reaction, At is the final absorbance after enzymatic reaction and Ae is the absorbance of α-ketoglutaric acid, (NH4)2SO4 and GDH. 3 For the cases of metal electrodes, there was no NAD2 product. So the amount of 1,6-NADH was quantitatively determined from absorbance after enzymatic reaction，At (equation (2)), and then ADP-ribose amount was obtained by the difference of the initial amount of NAD + (which was all converted after the reaction) and amount of NADH (1,4-NADH + 1,6-NADH) produced, as equation (3) shows. C1,6-NADH = (At -Ae) / ɛ1,6-NADH (2) CADP-ribose = CNAD + -C1,4-NADH -C1,  For the case of a carbon felt, the amount of 1,4-NADH was determined according to equation (1) as mentioned above. The amount of 1,6-NADH was determined from the ratio of 1,4-NADH and 1,6-NADH based on its 1 HNMR data according to equation (4). Then the light absorption of 1,6-NADH (A1,6-NADH) was calculated according to equation (5). The light absorption of NAD2 can be calculated by subtracting the A1,6-NADH from At, and then the concentration of NAD2 can be obtained according to equation (6). And then ADP-ribose amount was obtained by the difference of the initial amount of NAD + and the total amount of other products, equation (7).

NMR measurements
The NAD + reduction products of Cu, Fe, Co foams and carbon felt were analyzed by 1 HNMR (Bruker 400 MHz) by adding 0.2 mL D2O into 0.6 mL electrolyte after reaction. The NAD + reduction products of Co foam were also analyzed by 1 HNMR (Bruker 700 MHz) by adding 0.2 mL D2O into 0.6 mL electrolyte after reaction.

EPR measurements
The existence of hydrogen radical (H·) was verified by EPR measurements (Bruker A200). The X-band EPR was measured at 9.325GHz and room temperature. The electrolysis was conducted by controlled-potential electrolysis in an H-type cell containing 0.1 M sodium phosphate electrolyte, and the catholyte is 6 mL electrolyte with a Nafion-117 membrane. Electrolytes were pre-saturated and bubbled with Ar under magnetic stir during measurements. Metal and carbon electrodes were used as work electrodes.
The electrocatalysis was run at -1.01 V vs. Ag/AgCl for 5 min to enable equilibrium between H· formation and quenching. Then 60 μL DMPO was injected to electrolyte near the working electrode. After 10 min electrolysis, 1 mL electrolyte was frozen in liquid nitrogen and stored under -20 o C before EPR measurement.

H/D isotope experiment
In H/D isotope experiment, the electrolysis was conducted by controlled-potential electrolysis in an H-type cell containing 0.1 M sodium phosphate H2O or D2O electrolyte, and the catholyte is 12 mL electrolyte with a Nafion-117 membrane. Electrolytes were pre-saturated and bubbled with Ar under magnetic stir during measurements. A Cu foam (1 cm 2 ) was used as the work electrode.
In activity and selectivity experiments, 100 μL electrolyte after the reaction was extracted from the cell and diluted to 2.5 mL for UV-vis determination every 15 min.
In KIE experiments, 100 μL electrolyte after the reaction electrolyte was extracted from the cell and diluted to 2.5 mL for UV-vis determination every 5 min.