The high selectivity of biocatalysis offers a valuable method for greener, more efficient production of enantiopure molecules. Operating immobilised enzymes in flow reactors can improve the productivity and handling of biocatalysts, and using H2 gas to drive redox enzymes bridges the gap to more traditional metal‐catalysed hydrogenation chemistry. Herein, we describe examples of H2‐driven heterogeneous biocatalysis in flow employing enzymes immobilised on a carbon nanotube column, achieving near‐quantitative conversion in <5 min residence time. Cofactor recycling is carried out in‐situ using H2 gas as a clean reductant, in a completely atom‐efficient process. The flow system is demonstrated for cofactor conversion, reductive amination and ketone reduction, and then extended to biocatalytic deuteration for the selective production of isotopically labelled chemicals.
The O 2-tolerant NAD +-reducing hydrogenase (SH) from Ralstonia eutropha (Cupriavidus necator) has already been applied in vitro and in vivo for H 2-driven NADH recycling in coupled enzymatic reactions with various NADH-dependent oxidoreductases. To expand the scope for application in NADPH-dependent biocatalysis, we introduced changes in the NAD +-binding pocket of the enzyme by rational mutagenesis, and generated a variant with significantly higher affinity for NADP + than for the natural substrate NAD + , while retaining native O 2-tolerance. The applicability of the SH variant in H 2-driven NADPH supply was demonstrated by the full conversion of 2-methyl-1-pyrroline into a single enantiomer of 2-methylpyrrolidine catalysed by a stereoselective imine reductase. In an even more challenging reaction, the SH supported a cytochrome P450 monooxygenase for the oxidation of octane under safe H 2 /O 2 mixtures. Thus, the redesigned SH represents a versatile platform for atomefficient, H 2-driven cofactor recycling in biotransformations involving NADPH-dependent oxidoreductases.
We demonstrate a recycling system for synthetic nicotinamide cofactor analogues using a soluble hydrogenase with turnover numbers of >1000 for reduction of the cofactor analogues by H2. Coupling this system...
Biocatalysis offers many advantages for selective isotopic labelling of valuable small molecules, such as the deuterated amino acids utilised in protein NMR. Until recently, applications of biocatalytic deuteration systems have been restricted by their requirement for a supply of super-stoichiometric quantities of a specifically labelled 2H-pre-cursor, which can be both costly to purchase and complex to prepare. Overcoming this hurdle, we have demonstrated a novel and easy to use H2-driven biocatalytic platform for the incorporation of 2H-atoms across a number of molecular functional groups. By combining the biocatalytic deuteration catalyst with enzymes capable of reductive amination, we synthesised a suite of multiply isotopically labelled amino acids from low-cost isotopic precursors, such as 2H2O and 15NH4+. Notably, this strategy enables the introduction of a 15N-label, 2H-label, and chiral centre all in a single-step, and gives rise to amino acid isotopologues on a half gram scale for use directly in the preparation of isotopically labelled proteins. To demonstrate the applicability of the approach in the workflow of protein NMR chemists, we prepared L-[α-2H,15N, β-13C]-alanine and integrated it into a large (> 400 kDa) heat-shock protein, which was subsequently analysable by Methyl-TROSY techniques, revealing new structural information.
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