Hybrid Chemo-, Bio-, and Electrocatalysis for Atom-Efficient Deuteration of Cofactors in Heavy Water

Abstract

Deuterium-labeled nicotinamide cofactors such as [4-²H]-NADH can be used as mechanistic probes in biological redox processes and offer a route to the synthesis of selectively [²H] labeled chemicals via biocatalytic reductive deuteration. Atom-efficient routes to the formation and recycling of [4-²H]-NADH are therefore highly desirable but require careful design in order to alleviate the requirement for [²H]-labeled reducing agents. In this work, we explore a suite of electrode or hydrogen gas driven catalyst systems for the generation of [4-²H]-NADH and consider their use for driving reductive deuteration reactions. Catalysts are evaluated for their chemoselectivity, stereoselectivity, and isotopic selectivity, and it is shown that inclusion of an electronically coupled NAD⁺-reducing enzyme delivers considerable advantages over purely metal based systems, yielding exclusively [4S-²H]-NADH. We further demonstrate the applicability of these types of [4S-²H]-NADH recycling systems for driving reductive deuteration reactions, regardless of the facioselectivity of the coupled enzyme.

Scheme 1. Biocatalytic Deuteration via Formation of the Deuterium-Labeled Cofactor [4-²H]-NADH: (A) [4-²H]-NADH May Be Used to Prepare Specifically [²H] Labeled Organic Products by Enzymatic Reductive Deuteration; (B) Atom-Efficient Routes to NAD⁺ Reduction (Driven by H₂ Gas or an Electrode) Can Be Used in ²H₂O to Generate [4-²H]-NADH

For complete structures of the cofactors see Figure S1 in the Supporting Information.

Figure 1

The suite of heterogeneous catalysts investigated here for electrode or ^xH₂-driven generation of the labeled cofactor [4-²H]-NADH. For the electrode-driven approaches (top), either Electro-Enzymatic (NAD⁺ reductase on carbon on electrode) or Electro-Chemo (Pt/C on electrode) systems were used. For the ^xH₂-driven methods (bottom), each catalyst comprises a site for H₂ oxidation and a site for NAD⁺ reduction. The Bio system combines a hydrogenase (green square) and NAD⁺ reductase (purple rectangle), the Chemo-Bio system combines Pt (gray circle) and a NAD⁺ reductase, and the Chemo system utilizes just Pt for both half-reactions (all immobilized on a conductive carbon support).

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).

Figure 3

The isotopic selectivity of H₂-driven NADH generation from NAD⁺ by Bio, Chemo-Bio, and Chemo catalysts in the presence of ^xH₂O and ^xH₂. Analogous experiments were performed in varying mixtures of ¹H₂O and ²H₂O, under either (left) ¹H₂ or (right) ²H₂ gas. Lines of best fit through data for the Bio system are shown (green line: slope = 0.94, R² = 0.98 for each). The percent isotopic selectivity toward ²H incorporation into the [4]-position of NADH was determined using ¹H NMR spectroscopy as described in Section S.1.2.2 in the Supporting Information. Experimental details are provided in Section S.1.2.1 in the Supporting Information.

Figure 4

Understanding %²H incorporation of enzymes with opposing facioselectivity. (A) When ADH enzymes with opposite facioselectivity are supplied with [4S-²H]-NADH, ^xH^– is removed from either the S or R face of the cofactor and transferred to the substrate (acetophenone). This leads to generation of either a ²H-labeled product or an unlabeled product (with the ²H label remaining on the oxidized cofactor). (B) If access to the [S-²H]-labeled alcohol is required, the cofactor can be recycled in situ. The first cofactor turnover leads to unlabeled product (i), and further cofactor turnovers proceed via [4-²H₂]-NADH and lead to labeled product (ii). (C) The Bio system was used to supply labeled cofactor to (S)-ADH with a range of cofactor/substrate ratios, and the resulting solutions were analyzed by ¹H NMR spectroscopy (see Figure S11 in the Supporting Information). Increasing the cofactor turnover number was found to increase ²H incorporation into the phenylethanol product, tending toward 100%. Reactions were conducted on a 0.5 mL scale in ²H₅-Tris-²HCl (100 mM, p²H 8.0) with 400 μg of the Bio system as the catalyst and an excess (500 μg) of (S)-ADH being shaken under a steady stream of H₂ gas at 20 °C. In all of the reactions the initial loading of acetophenone was kept constant at 10 mM, with 2 vol % ²H₆-DMSO as a cosolvent. The starting concentration of NAD⁺ was then varied from 0.1 to 25 mM to give varying turnover numbers, with all reactions giving conversions >70% after 16 h.