Enantioselective CC Bond Ligation Using Recombinant Escherichia coli‐Whole‐Cell Biocatalysts

Abstract

AbstractThiamine diphosphate (ThDP)‐dependent enzymes like benzaldehyde lyase from Pseudomonas fluorescens (BAL) and benzoylformate decarboxylase from Pseudomonas putida (BFD) are versatile biocatalysts for the CC bond ligation of aldehydes to form enantiomerically pure 2‐hydroxy ketones. However, the large‐scale application of this enzyme class is often restricted by the required external addition of the expensive cofactor ThDP, as well as by the common use of dimethyl sulfoxide (DMSO) as a cosolvent, which leads to problems during the work‐up procedure. In the present paper we demonstrate that the addition of the excess cofactors, ThDP and magnesium ions (Mg2+), is not required when BAL or BFD are used in Escherichia coli resting cells. Furthermore, the combination of these resting cells with a biphasic reaction medium [methyl tert‐butyl ether (MTBE)/aqueous buffer] allows an increase of the substrate concentration up to 1 M, and an efficient extractive work‐up. As a practical example, e.g., the synthesis of (R)‐2‐hydroxy‐3,3‐dimethoxy‐phenylpropanone from benzaldehyde and 2,2‐dimethoxyacetaldehyde was optimized, achieving an isolated yield of 78 %, and an enantiomeric excess of 98 % ee in 24 h when operating at a substrate concentration of 0.4 M. The described reaction system in a biphasic medium is suitable for a wide range of aldehydes as substrates. The biphasic reaction medium minimizes also the formation of by‐products, which were observed when this reaction was performed in the conventional DMSO/buffer system.