Enantioselective oxidoreductases

For reduction of acetylenic ketones, two oxidoreductases were used [25]. Lactobacillus brevis alcohol dehydrogenase (LBADH) gave the (R)-alcohols and Candida parapsilosis carbonyl reductase (CPCR) afforded the (S)-isomer, both in good yield and excellent enantioselectivity. By changing the steric demand of the substituents, the enantiomeric excess values can be adjusted and even the configurations of the products can be altered (Figure 8.34). 

Schubert, T., Hummel, W., Kula, M.-R. and Muller, M. (2001) Enantioselective synthesis of both enantiomers of various propargylic alcohols by use of two oxidoreductases. European Journal of Organic Chemistry, (22), 4181—4187. 

Reduction of l-(chloro or bromo) -3-butyn-2-one (27e,f) with recLBADH affords enantiopure R-alcohols 28e,f, resulting in an interesting switch of the enantioselectivity of the enzymatic reduction. As the enantiomers (S) -28e,f can be obtained by recLBADH-catalyzed reduction of 27b-27d and subsequent removal of the si-lyl-protecting group, this enzyme offers unique access to a pair of enantiomers via the same oxidoreductase. Due to the high volatility of the substrates (27e,f) these transformations were only performed on an analytical scale. 

Biocatalysts, mainly hydrolytic enzymes and oxidoreductases, have been used for organic reactions due to their excellent enantioselectivities and environmentally friendliness.1 Typical enzymatic reactions used for the organic synthesis are shown in Figure 1. Especially, hydrolytic enzymes for kinetic resolutions of racemates have been utilized widely because of their high stabilities, wide substrate specificities, lack of cofactor requirements and high availabilities. 

Most of the enzymes show extremely strict chiral recognitions, and only one of the enantiomers can be the substrate of the enzyme. For example, chymotrypsin incorporates L-peptides only to the enzyme-substrate binding site to form enzyme-substrate complex, so it shows very high enantioselectivity (Figure 3 (a)). Oxidoreductases also form the enzyme-substrate complex of only one enantiomer, so enantioselectivities are high when isolated enzymes are used for reactions instead of whole cells containing both (R)- and (.S )-specific enzymes, which leads to overall low enantioselectivities. 

Numerous oxidoreductases enable highly chemo-, regio-, and enantioselective oxy-functionalization of readily available petrochemicals, xenobiotics, and larger molecules such as steroids under mild conditions (ambient temperature and pressure with water as the solvent). 

Capillary gas chromatographic determination of optical purities, investigation of the conversion of potential precursors, and characterization of enzymes catalyzing these reactions were applied to study the biogenesis of chiral volatiles in plants and microorganisms. Major pineapple constituents are present as mixtures of enantiomers. Reductions, chain elongation, and hydration were shown to be involved in the biosynthesis of hydroxy acid esters and lactones. Reduction of methyl ketones and subsequent enantioselective metabolization by Penicillium citrinum were studied as model reactions to rationalize ratios of enantiomers of secondary alcohols in natural systems. The formation of optically pure enantiomers of aliphatic secondary alcohols and hydroxy acid esters using oxidoreductases from baker s yeast was demonstrated. 

Oxidoreductases Catalyzing the Enantioselective Reduction of Oxoacid Esters in Baker s Yeast... 

By means of streptomycin sulfate treatment, Sephadex G-25 filtration, DEAE-Sepharose CL-6B chromatography, Sephadex G-150 filtration, and hydroxyapatite chromatography we succeeded in isolating and purifying two NADPH-dependent oxidoreductases from enzyme extracts of Saccharomvces cerevisiae. which catalyze the enantioselective reduction of 3-oxoacid esters to (S)- and (R)-3-hydroxyacid esters (11). 

The synthetic application of redox enzymes (oxidoreductases), especially in the field of enantioselective reductions and chemo-, regio-, and stereoselective oxidations, is very important [7,8] and covers a wide range of processes, as listed ... 

Reduced selectivity in bakers yeast mediated transformations is most likely due to the presence of several alcohol dehydrogenases possessing opposite enantioselectivity. Stewart et al. have successfully cloned 49 different oxidoreductases from Saccharomyces cerevisiae [47]. They could show that the identification of specific alcohol dehydrogenases will result in access to chiral alcohols in high optical purity. Moreover, both enantiomers are accessible sometimes using enzymes from one parental strain [48]. 

The types of enzymes used by organic chemists vary widely and include such well-known biocataiysts as lipases, esterases, oxidoreductases, oxinitrilases, transferases and aldolases [4]. An example which illustrates the industrial application of a lipase concerns the kinetic resolution of a chiral epoxy ester used as the key intermediate in the synthesis of the calcium antagonist Diltiazem, a major therapeutic in the treatment of high blood pressure [6] (Fig. 1). In developing the industrial process for the production of this drug, many different lipases were screened, but only the bacterial lipase from Serratia marescens showed both a sufficiently high activity and enantioselectivity. The intermediate is produced industrially on a scale of 50 tons/year. 

Together with enantioselective hydrolysis/acylation reactions, enantioselective ketone reductions dominate biocatalytic reactions in the pharma industry [10], In addition, oxidases [11] have found synthetic applications, such as in enantioselective Baeyer-Villiger reactions [12] catalyzed by, for example, cyclohexanone monooxygenase (EC 1.14.13) or in the TEMPO-mediated oxidation of primary alcohols to aldehydes, catalyzed by laccases [13]. Hence, the class of oxidoreductases is receiving increased attention in the field of biocatalysis. Traditionally they have been perceived as difficult due to cofactor requirements etc, but recent examples with immobilization and cofactor regeneration seem to prove the opposite. 

The reaction mechanism is quite similar to the one of VAO and also includes an intermediate, the para-quinone methide. Like VAO, 4-cresol-oxidoreductase also exhibits a high enantioselectivity for (S)-l-(4 -hydroxyphenyl)alkylalcohols1591. 

Ethylphenol oxidoreductase from Pseudomonas putida JD1 is structurally almost identical to 4-cresol oxidoreductase, but catalyzes the hydroxylation of para-alkylphe-nols with longer aliphatic chains (Table 16.3-12). The hydroxylation reactions enantioselectively produce (-R)-alcohols 64> 65l The regeneration properties of this enzyme are quite similar to 4-cresol oxidoreductase1611. 

Oxidoreductases are of special interest for the enantioselective reduction of prochiral ketones [49]. Formate dehydrogenase from Candida boidinii was found to be stable and active in mixtures of [MMIM][MeS04] with buffer. The apphcation of alcohol dehydrogenases for enzyme-catalyzed reactions in the presence of water-miscible ionic liquids could make use of another advantage of these solvents they increase the solubility of hydrophobic compounds in aqueous systems. By addition... 

The Wittig synthesis has also been successfully combined with oxidoreductase-catalyzed biotransformations in one-pot processes ranning in aqueous media, as demonstrated recently by the Groger and Hummel groups [49, 50]. The first example consists of an enantioselective two-step one-pot synthesis of allylic alcohols of type 61, which is based on an initial Wittig synthesis of the stabilized yhde 59 with aldehydes 54 and in situ reduction of the formed a,p-unsaturated ketones with an alcohol dehydrogenase (ADH) [49]. The desired allylic alcohols 61 were formed in up to 90% conversion and with excellent enantioselectivity (>99% ee) [49]. This one-pot process based on the use of an (S)-enantioselective ADH is shown in Scheme 19.20. Instead of C=0 double bond reduction, the C=C double bond can be reduced as well selectively when using an ene reductase instead of an ADH, and... 

The synthetic value of S. cerevisiae is increased by the fact that its oxidoreductase(s) will accept haloketones as substrates, reducing them with reasonable enantioselectivity to the (R) enantiomer. (Note that the stereochemical outcome is the same, despite the apparent inversion due to the CIP rules.)... 

The most practical method that is used in the industrial synthesis of esomeprazole involves titanium-catalyzed oxidation with an alkyl hydroperoxide, and a dialkyltartrate as chiral ligand, in an organic solvent such as dichloromethane. A variety of oxidoreductases are known to catalyze the enantioselective oxidation of prochiral sulfides, usually as whole-cell biotransformations in aqueous media, but no simple metal complexes have been shown to be effective in water and the development of practical systems employing aqueous hydrogen peroxide as the primary oxidant is still an important challenge. In this context it is worth mentioning the enantioselective sulfoxidation of prochiral sulfoxides catalyzed by the semisynthetic peroxidase, vanadium-phytase, in an aqueous medium. 

The biocatalytic counterpart for this transformation is done by the alcohol dehydrogenases [ADHs, EC 1.1.1.x., also called ketoreductases (KREDs) or carbonyl reductases (CRs)], which are able to perform stereoselective carbonyl reductions or enantioselective alcohol oxidations [5-8]. These enzymes are probably the most employed oxidoreductases and make use of a nicotinamide cofactor such as NADH or NADPH to transfer electrons into and from the target substrate. Depending on their substrate scope, ADHs can be divided into primary alcohol dehydrogenases, preferentially reducing aldehydes, and secondary alcohol dehydrogenases that have... 

Sonoike, S., Itakura, T, Kitamura, M., and Aoki, S. (2012) One-pot chemoenzymatic synthesis of chiral 1,3-diols using an enantioselective aldol reaction with chiral Zn complex catalysts and enzymatic reduction using oxidoreductases with cofactor regeneration. Chem. Asian J., 7 (1), 64-74. 

So far, some oxidoreductase-catalyzed reactions have been examined in reversed micelles. The enzyme activity and stability that depend largely on the microemulsion composition, mostly the water-to-stirfactant ratio (ivo), are often comparable to values in aqueous media. Orlich et al. reported the application of reverse micelles for ADH-catalyzed reduction of less water-soluble ketones in an FDHcontained water, cyclohexane, and Marlipal 013-16 as the surfactant. The reaction rate of ADH for the reduction of 2-heptanone in reverse micellar medium was increased up to 12 times compared to aqueous medium [84], The improved enzymes stability was observed at optimal Wq. Finally, it was possible to perform successful semibatch experiments reducing 2-butanone with full conversion and enantioselectivity [85]. 

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