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 ( 2 H) atoms at chiral centres are becoming increasingly desirable targets for pharmaceutical and analytical chemists. However, utilisation of NADH-dependent enzymes for 2 H-labelling is not straightforward, owing to difficulties in supplying a suitably isotopically-labelled cofactor ([4-2 H]-NADH). Here we report on a strategy that combines a clean reductant (H 2 ) with a cheap source of 2 H-atoms ( 2 H 2 O) to generate and recycle [4-2 H]-NADH. By coupling [4-2 H]-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'-2 H 3 ]solifenacin fumarate on a preparative scale.

Rapid, Heterogeneous Biocatalytic Hydrogenation and Deuteration in a Continuous Flow Reactor

“…Molecules bearing asymmetric deuterium ( 2 H) centers are desirable for medicinal and analytical applications, [33,34] but are synthetically difficult to achieve. We have identified H 2 ‐driven biocatalysis in heavy water, 2 H 2 O, as a new route to these targets [35] . The H 2 ‐driven system presented here allows for biocatalytic deuteration without the need to co‐feed expensive pre‐labelled compounds such as D‐[1‐ 2 H]‐glucose and [ 2 H 7 ]‐isopropanol, and without the need for 2 H 2 gas.…”

“…Enzymes used : Soluble hydrogenase (SH) from Ralstonia eutropha , alanine dehydrogenase (AlaDH) and quinuclidinone reductase from Agrobacterium tumefaciens ( At QR) were prepared in‐house [35] . Alcohol dehydrogenase (ADH105, Johnson Matthey, Cambridge, UK) was used as supplied.…”

“…We have identified H 2 -driven biocatalysis in heavy water, 2 H 2 O, as a new route to these targets. [35] The H 2 -driven system presented here allows for biocatalytic deuteration without the need to co-feed expensive pre-labelled compounds such as D-[1-2 H]-glucose and [ 2 H 7 ]-isopropanol, and without the need for 2 H 2 gas. Instead, by simply exchanging the buffer from H 2 O to 2 H 2 O, > 99 % conversion of 2 mM NAD + 7 to the deuterated reduced form [4SÀ 2 H]À NADH 8 was achieved in 2.2 min in the flow reactor, and with > 99 % isotopic selectivity.…”

Synthesis of [4S‐2H]NADH, [4R‐2H]NADH, [4‐2H2]NADH and [4‐2H]NAD+ cofactors through heterogeneous biocatalysis in heavy water

“…The heterogeneous biocatalyst (Biocat/C) comprised a NiFe‐hydrogenase (Hydrogenase 1 from Escherichia coli ) and an NAD + reductase (I64A variant of the soluble hydrogenase from Ralstonia eutropha ) co‐immobilised on carbon black particles (Black Pearls 2000 [BP2000], Cabot Corporation) according to published protocols 4 . In a typical experiment, 400 μg of carbon (supporting around 80‐140 μg each of hydrogenase and NAD + reductase) was used per 1 ml of reaction mixture.…”

“…Isotopic labelling of these molecules is a well‐established technique for probing enzymatic mechanisms, 1 exemplified by the work of JW Cornforth on the stereoselectivity of biocatalysed processes, for which he was awarded the 1975 Nobel Prize in Chemistry 2,3 . More recently, deuterated NADH has been shown to be a useful tool for the preparation of synthetically challenging asymmetric heavy drug analogues 4 …”