Bringing biocatalytic deuteration into the toolbox of asymmetric isotopic labelling techniques

Abstract

Enzymes dependent on nicotinamide cofactors are important components of the expanding range of asymmetric synthetic techniques. New challenges in asymmetric catalysis are arising in the field of deuterium labelling, where compounds bearing deuterium (²H) atoms at chiral centres are becoming increasingly desirable targets for pharmaceutical and analytical chemists. However, utilisation of NADH-dependent enzymes for ²H-labelling is not straightforward, owing to difficulties in supplying a suitably isotopically-labelled cofactor ([4-²H]-NADH). Here we report on a strategy that combines a clean reductant (H₂) with a cheap source of ²H-atoms (²H₂O) to generate and recycle [4-²H]-NADH. By coupling [4-²H]-NADH-recycling to an array of C=O, C=N, and C=C bond reductases, we demonstrate asymmetric deuteration across a range of organic molecules under ambient conditions with near-perfect chemo-, stereo- and isotopic selectivity. We demonstrate the synthetic utility of the system by applying it in the isolation of the heavy drug (1S,3'R)-[2',2',3'-²H₃]-solifenacin fumarate on a preparative scale.

Fig. 1. Methods for introduction of deuterium into organic molecules and drug candidates.

State-of-the-art approaches for carbon–hydrogen or carbon–halogen hydrogen isotope exchange; these methods use either ²H₂ or ²H₂O as the deuterium source. The biocatalytic reductive deuteration strategy demonstrated in this work uses ²H₂O as the deuterium source and H₂ as a clean reductant.

Fig. 2. Biocatalytic reductive deuteration via recycling of [4-²H]-NADH.

a Biocatalytic reductive deuteration requires a supply of [4-²H]-NADH. b Conventional [4-²H]-NADH-recycling strategies are driven by a sacrificial deuterated reductant. c Schematic representation of the heterogeneous biocatalytic system demonstrated in this work for H₂-driven generation of the labelled cofactor [4-²H]-NADH, with ²H₂O supplying the deuterium atoms. For complete structures of the cofactors, see Supplementary Fig. 1 and for details of the heterogeneous biocatalyst, see Supplementary Methods.

Fig. 3. Scope of asymmetric biocatalytic reductive deuteration.

Typical reaction conditions: 0.5 mL, 5 mM reagent, 0.5 mM NAD⁺, ²H₂O (98 %), Tris-²HCl (p²H 8.0, 100 mM), 1–5 vol.% ²H₆-DMSO, 16 h, 20 ^oC, 1 bar H₂ and shaking at 500 r.p.m. Catalysts: Excess NADH-dependent reductase, 400 μg carbon (160 pmol hydrogenase, 260 pmol NAD⁺ reductase). For reductive amination reactions: 25 mM NH₄Cl. *All transformations were fully selective for the reduction of the desired functional group except 6c, for which a known enzymatic side reaction yielded an extra product (see Supplementary Fig. 47 and associated discussion). Full experimental and analytical details are provided in Supplementary Methods.

Fig. 4. Scalable biocatalytic deuteration for preparation of heavy solifenacin fumarate.

a Preparation of trideuterated (R)-3-quinuclidinol via biocatalytic reductive deuteration and b incorporation into the pharmaceutical compound (1S,3′R)-solifenacin fumarate. In each case, the mass spectroscopy traces show the expected +3 shift in m/z values of the isolated product (lower plot) relative to their natural abundance standards (upper plot). Full experimental and analytical details are provided in Supplementary Methods.