Figure 2.

Selectivity of electrode and H₂-driven catalyst systems for generation of [²H]-labeled NADH. (A) Distribution of products formed by each of the Electro-Enzymatic, Electro-Chemo, Bio, Chemo-Bio, and Chemo- catalysts, studied under comparable conditions. The three catalysts containing an NAD⁺ reductase unit were found to be selective for a single product: [4S-²H]-NADH. Reaction mixtures contained 4 mM NAD⁺ in ²H₂O (p²H 8.0), and the products were analyzed using HPLC and ¹H NMR (Figures S5 and S6 in the Supporting Information). For electrode-driven experiments, the electrode was poised at −560 mV vs SHE. For H₂-driven reactions the catalysts were operated in H₂-saturated solution (2 bar of H₂). (B) Experiments to determine the relationship between the enzyme/metal mass ratio and the chemoselectivity, isotopic selectivity, and stereoselectivity of the Chemo-Bio catalyst for making [4S-²H]-NADH from NAD⁺ under ¹H₂ gas in ²H₂O. Comparisons of selectivity were all made at similar levels of conversion (90–95%). (i) Chemoselectivity (as measured by HPLC): in converting NAD⁺ to 1,4-NADH, the formation of a biologically inactive side product (1,6-NADH) by Pt/C catalysts was increasingly suppressed by the addition of NAD⁺ reductase. (ii) Isotopic selectivity and stereoselectivity (as measured by ¹H NMR): of the 1,4-NADH formed, a distribution of isotopomers was observed consisting of [4R-²H]-NADH, [4S-²H]-NADH, and unlabeled (no ²H) NADH. Again, increasing the NAD⁺ reductase loading on the Pt/C catalyst led to the formation of exclusively [4S-²H]-NADH. Note: (*) The Electro-Chemo system was also operated at more negative potentials, giving rise to NADH and biologically inactive forms (see Section S.2.1 in the Supporting Information).