Elsevier

Tetrahedron

Volume 60, Issue 3, 12 January 2004, Pages 633-640
Tetrahedron

Preparative asymmetric reduction of ketones in a biphasic medium with an (S)-alcohol dehydrogenase under in situ-cofactor-recycling with a formate dehydrogenase

https://doi.org/10.1016/j.tet.2003.11.066Get rights and content

Abstract

The substrate range of a novel recombinant (S)-alcohol dehydrogenase from Rhodococcus erythropolis is described. In addition, an enzyme-compatible biphasic reaction medium for the asymmetric biocatalytic reduction of ketones with in situ-cofactor regeneration has been developed. Thus, reductions of poorly water soluble ketones in the presence of the alcohol dehydrogenase from R. erythropolis and a formate dehydrogenase from Candida boidinii can be carried out at higher substrate concentrations of 10–200 mM. The resulting (S)-alcohols were formed with moderate to good conversion rates, and with up to >99% ee.

An enzyme-compatible biphasic reaction media for the asymmetric reduction of ketones with in situ-cofactor regeneration has been developed. As enzymes, a novel recombinant (S)-alcohol dehydrogenase from Rhodococcus erythropolis and a formate dehydrogenase were used.

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Introduction

The enzymatic asymmetric reduction with an in situ-NADH-cofactor regeneration using a formate dehydrogenase (FDH) from Candida boidinii (Scheme 1, part (a)) is an interesting technology for the production of optically active alcohols which are valuable specialty chemicals.1 A related process, namely the enzymatic reductive amination by means of an enzyme-coupled in situ-cofactor regeneration according to Scheme 1, part (b) has been shown to be technically feasible, and is a powerful tool for the preparation of enantiomerically pure α-amino acids. This process runs on the ton scale in the industrial synthesis of l-tert-leucine (Scheme 1, part (b)).2 The extension of this attractive enzymatic concept3., 4. towards the large scale synthesis of optically active alcohols5., 6., 7. is highly desirable but still some drawbacks exist.

Among the main limitations are the lacks of isolated NAD-dependent ADHs commercially available on a technical scale, and the lack of suitable reaction media, which guarantee a high ketone solubility. Thus, reactions are usually carried out at a substrate concentration in the range of 5–10 mM or below due to the low solubility of hydrophobic ketones in water. The presence of an organic solvent could improve the solubility of poorly water-soluble ketones, but generally causes significant enzyme deactivation. In particular, this is known for the formate dehydrogenase from C. boidinii8 which is sensitive to organic solvents.9 The best solution so far is represented by a continuous process with an enzyme-membrane reactor.10 Albeit good space–time yields in the range of 60–104 g/(L d) can be obtained,10a,b the reaction is limited by the solubility of the ketone in water, which is often below 5–10 mM. Thus, the development of alternative reaction concepts for the asymmetric reduction with isolated enzymes still is a challenge, as well as the access to NAD-dependent ADHs, which cover a broad substrate range. They preferably should be cloned and overexpressed in Escherichia coli under conditions showing a high production potential.

In this contribution, we report the investigation of the synthetic scope of the recently developed (S)-ADH from Rhodococcus erythropolis overexpressed in E. coli, as well as the first example of an asymmetric enzymatic reduction of poorly water soluble ketones including an in situ-recycling of the cofactor NADH with a FDH, which runs in the ‘direct’ presence of an organic solvent at high substrate concentrations.11

Section snippets

The scope of a new (S)-alcohol dehydrogenase from R. erythropolis

We recently developed an E. coli expression system for the NAD-dependent (S)-alcohol dehydrogenase from R. erythropolis,3k since previous studies12 revealed that this enzyme possess a potential for general applicability. This new alcohol dehydrogenase (ADH) isolated from R. erythropolis shows a tetrameric structure, it has a molecular weight of 36,206 kDa per subunit, and belongs to the group of zinc-containing medium-chain ADHs.3., 11. The developed expression in E. coli is efficient and has a

Experimental

The measurements of the enzymatic activity were carried out using the spectrophotometer JASCO V-530 UV/VIS. The solution of the (S)-alcohol dehydrogenase from R. erythropolis (overexpressed in E. coli) was prepared according to Refs. 3., 11.. The solution of the formate dehydrogenase from C. boidinii (mutant: C23S, C262A; overexpressed in E. coli) was prepared according to Ref. 8. The ketones, cofactors, sodium formate and the solvents are commercially available and were used in the activity

Acknowledgements

This work was supported by the Bundesministerium für Bildung und Forschung (Biotechnologie 2000—Nachhaltige BioProduktion; Project: ‘Entwicklung eines biokatalytischen und nachhaltigen Verfahrens zur industriellen Herstellung enantiomerenreiner Amine und Alkohole unter besonderer Berücksichtigung der Atomökonomie’).

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