Enzymatic enantioselective reduction of α-ketoesters by a thermostable 7α-hydroxysteroid dehydrogenase from Bacteroides fragilis (original) (raw)
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Journal of the American Chemical Society, 1986
The asymmetric reduction of aliphatic acyclic ketones (C4-Cl0 substrates) is efficiently achieved by using alcohol dehydrogenase from Thermoanaerobium brockii either as a homogeneous, heat-treated, cell-free extract or following immobilization on a solid support. Both methods are superior to the use of whole-cell fermentation. The experimental conditions for working with TBADH were studied and optimized in order to improve reaction rates and the optical purity of the product. An interesting substrate size-induced reversal of stereoselectivity was observed. The smaller substrates (methyl ethyl, methyl isopropyl, or methyl cyclopropyl ketones) are reduced to R alcohols, whereas the higher ketones form the S enantiomer.
Bioresources and Bioprocessing, 2019
Background: A thermostable alcohol dehydrogenase from Thermoanaerobacter brockii (TbSADH) has been repurposed to perform asymmetric reduction of a series of prochiral ketones with the formation of enantio-pure secondary alcohols, which are crucial chiral synthons needed in the preparation of various pharmaceuticals. However, it is incapable of asymmetric reduction when applied to bulky ketones. Recently, mutations at two key residues A85 and I86 were shown to be crucial for reshaping the substrate binding pocket. Increased flexibility of the active site loop appears to be beneficial in the directed evolution of TbSADH towards difficult-to-reduce ketones. Methods: Using the reported mutant A85G/I86A as template, double-code saturation mutagenesis (DCSM) was applied at selected residues lining the substrate binding pocket with a 2-membered reduced amino acid alphabet. Results and conclusions: The mutant A85G/I86A was first tested for activity in the reaction of the model substrate (4-chlorophenyl)-(pyridin-2-yl)methanone, which showed a total turnover number (TTN) of 3071. In order to further improve the turnovers, a small and smart mutant library covering a set of mutations at Q101, W110, L294, and C295 was created. Eventually, a triple-mutant A85G/I86A/Q101A was identified to be a superior catalyst that gave S-selective product with 99% ee and 6555 TTN. Docking computations explain the source of enhanced activity. Some of the best variants are also excellent catalysts in the reduction of other difficult-to-reduce ketones.
Applied microbiology and biotechnology, 2014
Enzyme-catalyzed enantioselective reductions of ketones and keto esters have become popular for the production of homochiral building blocks which are valuable synthons for the preparation of biologically active compounds at industrial scale. Among many kinds of biocatalysts, dehydrogenases/reductases from various microorganisms have been used to prepare optically pure enantiomers from carbonyl compounds. (S)-1-phenylethanol dehydrogenase (PEDH) was found in the denitrifying bacterium Aromatoleum aromaticum (strain EbN1) and belongs to the short-chain dehydrogenase/reductase family. It catalyzes the stereospecific oxidation of (S)-1-phenylethanol to acetophenone during anaerobic ethylbenzene mineralization, but also the reverse reaction, i.e., NADH-dependent enantioselective reduction of acetophenone to (S)-1-phenylethanol. In this work, we present the application of PEDH for asymmetric reduction of 42 prochiral ketones and 11 β-keto esters to enantiopure secondary alcohols. The hig...
Enantioselective microbial reduction of substituted acetophenones
Tetrahedron-asymmetry, 2004
The chiral intermediate (S)-1-(2 0 -bromo-4 0 -fluoro phenyl)ethanol 2 was prepared by the enantioselective microbial reduction of 2-bromo-4-fluoro acetophenone 1. Organisms from genus Candida, Hansenula, Pichia, Rhodotorula, Saccharomyces, Sphingomonas and Baker's yeast reduced 1 to 2 in >90% yield and 99% enantiomeric excess (ee). In an alternative approach, the enantioselective microbial reductions of methyl, ethyl, and tert-butyl 4-(2 0 -acetyl-5 0 -fluorophenyl) butanoates 3, 5, and 7, respectively, were demonstrated using strains of Candida and Pichia. Reaction yields of 40-53% and ee's of 90-99% were obtained for the corresponding (S)-hydroxy esters 4, 6, and 8. The reductase, which catalyzed the enantioselective reduction of ketoesters was purified to homogeneity from cell extracts of Pichia methanolica SC 13825. It was cloned and expressed in Escherichia coli with recombinant cultures used for the enantioselective reduction of keto methyl ester 3 to the corresponding (S)-hydroxy methyl ester 4. On a preparative scale, a reaction yield of 98% and an ee of 99% was obtained.
Tetrahedron, 2006
The substrate selectivity and stereoselectivity of a series of ketoreductases were evaluated toward the reduction of two sets of b-ketoesters. Both the structural variety at b-position and the substituent at a-position greatly affected the activity and stereoselectivity of these ketoreductases. For the first set of b-ketoesters, at least one ketoreductase was found that catalyzed the formation of either (D) or (L) enantiomer of b-hydroxyesters from each substrate with high optical purity, with the only exception of ethyl (D)-3-hydroxy-3-phenylpropionate. For the second set of b-ketoesters with a-substituents, the situation is more complex. More commonly, a ketoreductase was found that formed one of the four diastereomers in optically pure form, with only a few cases in which enzymes could be found that formed two or more of the diastereomers in high optical purity. The continued development of new, more diverse ketoreductases will create the capability to produce a wider range of single diastereomers of 2-substituted-3-hydroxy acids and their derivatives.
Advanced Synthesis & Catalysis, 2012
α-Alkyl-β-hydroxyesters were obtained via DKR employing purified or crude E. coli overexpressed alcohol dehydrogenases (ADHs). ADH-A from R. ruber, CPADH from C. parapsilosis and TesADH from T. ethanolicus afforded syn-(2R,3S) derivatives with very high selectivities for sterically not impeded ketones ('small-bulky' substrates), while ADHs from S. yanoikuyae (SyADH) and Ralstonia sp. (RasADH) could also accept bulkier ketoesters ('bulkybulky' substrates). SyADH also provided preferentially syn-(2R,3S) isomers and RasADH showed in some cases good selectivity towards the formation of anti-(2S,3S) derivatives. With anti-Prelog ADHs such as LBADH from L. brevis or LKADH from L. kefir, syn-(2S,3R) alcohols were obtained with high conversions and diastereomeric excess in some cases, especially with LBADH. Furthermore, due to the thermodynamically favoured reduction of these substrates, it was possible to employ just a minimal excess of 2propanol to obtain the final products with quantitative conversions.
ChemBioChem, 2020
Effective procedures for the synthesis of optically pure alcohols are highly valuable. A commonly employed method involves the biocatalytic reduction of prochiral ketones. This is typically achieved by using nicotinamide cofactor‐dependent reductases. In this work, we demonstrate that a rather unexplored class of enzymes can also be used for this. We used an F420‐dependent alcohol dehydrogenase (ADF) from Methanoculleus thermophilicus that was found to reduce various ketones to enantiopure alcohols. The respective (S) alcohols were obtained in excellent enantiopurity (>99 % ee). Furthermore, we discovered that the deazaflavoenzyme can be used as a self‐sufficient system by merely using a sacrificial cosubstrate (isopropanol) and a catalytic amount of cofactor F420 or the unnatural cofactor FOP to achieve full conversion. This study reveals that deazaflavoenzymes complement the biocatalytic toolbox for enantioselective ketone reductions.
Applied Microbiology and Biotechnology, 1990
A new alcohol dehydrogenase catalysing the enantioselective reduction of acetophenone to R ( + )phenylethanol was found in a strain of Lactobacillus kefir. A 70-fold enrichment of the enzyme with an overall yield of 76% was obtained in two steps. The addition of Mg 2+ ions was found to be necessary to prevent rapid deactivation. The enzyme depends essentially on N A D P H and was inactive when supplied with N A D H as the coenzyme. Important enzymological data of the dehydrogenase are: Km (acetophenone) 0.6 raM, K~ (NADPH) 0.14 mM, and a pH optimum for acetophenone reduction at 7.0. Addition of EDTA leads to complete deactivation of the enzyme activity. Added iodoacetamide or p-hydroxymercuribenzoate cause only slight inhibition, revealing that the active centre of the enzyme contains no essential SH-group. Besides acetophenone several other aromatic and long-chain aliphatic secondary ketones are substrates for this enzyme. Batch production of phenylethanol was examined using three different methods for the regeneration of N A D P H : glucose/glucose dehydrogenase, glucose-6-phosphate/glucose-6-phosphate dehydrogenase, and isopropanol.