Chiral alcohol biocatalysts

In summary, ketoreductases have emerged as valuable catalysts for asymmetric ketone reductions and are preparing to enter the mainstream of synthetic chemistry of chiral alcohols. These biocatalysts are used in three forms wild-type whole-cell microorganism, recombinant... 

Unlike the syntheses known hitherto, by applying these new biocatalysts it is possible to produce chiral alcohols with optical purities of >99% under moderate reaction conditions in an aqueous milieu. 

Matsuyama, A. Yamamoto, H. and Kobayashi, Y. Practical Apphcation of Recombinant Whole-cell Biocatalysts for the Manufacturing of Pharmaceutical Intermediates such as Chiral Alcohols. Org. Process Res. Dev. 2002, 6, 558-561. 

The chemoenzymatic synthesis of chiral alcohols is a field of major interest within biocatalytic asymmetric conversions. A convenient access to secondary highly enan-tiomerically enriched alcohols is the usage of alcohol dehydrogenases (ADHs) (ketoreductases) for the stereoselective reduction of prochiral ketones. Here, as in many other cases in asymmetric catalysis, enzymes are not always only an alternative to chemical possibilities, but are rather complementary. Albeit biocatalysts might sometimes seem to be more environmentally friendly, asymmetric ketone reduction... 

Yeast reductions have provided the synthetic organic chemist with highly versatile methods to prepare chiral alcohols from prochiral ketones of which Saccharomyces cerevisiae (baker s yeast) is the most commonly used biocatalyst. In addition to prochiral ketone reductions, hundreds of... 

Chiral alcohols are some of the most important chiral building blocks for the production of pharmaceuticals. The creation of chiral alcohols through the asymmetric reduction of prochiral carbonyl compounds using biocatalysts, such as microbial cells and commercially available oxidoreductases, has been... 

Based on the (/ )-specific ADH from L. kefir, a recombinant E. coli strain was constructed as a whole-cell biocatalyst, and co-expressed GDH was used for regeneration of NADPH [157]. These designer cells were applied for the reduction of 4-fluoroacetophenone to the corresponding optically active (/ )-4-fluorophe-nylethan-l-ol at 0.5 M educt concentration [158]. After a reaction time of 23 h, a conversion of >95% has been achieved, and the purified isolated chiral alcohol showed an ee value of >99% (87% yield). (S)-p-Halohydrins were obtained with this whole-cell catalyst by means of an enantioselective reduction of the corresponding ketones with both high conversions of >95% and enantioselectivities of >99% (Fig. 40). Base-induced cyclization of the [S-halohydrin led to enantiomeri-cally pure (S)-epoxides in high yield and enantiomeric purity (>99% ee) [159]. 

Although reductases play an important role in the in vivo synthesis of many chemicals (see flavour example in Fig. 7.11), little attention has been paid to this type of biocatalyst. In most cases whole microbial or plant cells are used to perform a bioreductive reaction due to the requirement for (expensive) cofactors. Typical examples include the reduction of certain double bonds in terpenes by plant cells [27,41], the reduction of Massoi lactone to R(+)-8-decalactone by Basidiomycetes and S. cerevisiae [28], and the baker s yeast-catalyzed reduction of ketones to (chiral) alcohols [42]. 

Although prochiral or chiral alcohols and carboxylic acid esters initially served as the primary classes of substrates, compounds susceptible to processing via these two routes now encompass diols, a- and 3-hydroxy acids, cyanohydrins, chlorohydrins, diesters, lactones, amines, diamines, amino alcohols, and a-and 3-amino acid derivatives. Gotor and Arroyo have reviewed the use of biocatalysts for the preparation of pharma-eeutical intermediates and fine ehemieals. Some specific examples are indieated below. 

For example, consider one of the simplest reduction processes, namely the reduction of a ketone to a secondary alcohol. Of course, this can be accomplished chemically using sodium borohydride. The same transformation can be achieved using baker s yeast, and one advantage of using the biocatalyst often can be seen immediately (i.e. optically active forms of chiral alcohols can be obtained). The reason is simple The bio-reduction... 

In this chapter, we describe development of novel enzymes including engineered PAR, LSADH, and P-keto ester reductase (KER) from P. citrinum key advances in tailoring biocatalysts by protein engineering and future aspect of them for improved bioreduction of ketones to synthesize various chiral alcohols. 

LSADH exhibits moderate activity in DMF-water and ethanol-water media, but low in 1-propanol-water medium (Figure 6.9d). However, E. coli biocatalyst expressing the LSADH exhibits sufficient activity to reduce various ketones in IPA-water system, as shown in Table 6.7. We determine that optimum I PA concentration of chiral alcohol production by free LSADH is around 10% (v/v) [26]. These results indicate that the effect of 1-propanol on LSADH should be more severe than that of I PA. Although the directed evolution of LSADH has not yet been performed, we think the increase of activity in 1-propanol/IPA would give a good result in further improvement of this enzyme as a biocatalyst. 

Table 6.7 Production of chiral alcohols from various ketones by coli biocatalysts [10].

Using traditional organic cosolvents, the bioreduction of para-bromo-2,2,2-trifluoroacetophenone 83 (Scheme 6.34) was limited to a maximum product concentration of 10 g/L due to substrate-induced deactivation of the R. eryth-ropolis (ADH RE) biocatalyst. However, by employing 10% (v/v) [BMP][NTf2], a water immiscible ionic liquid, more than 5 times the ketone was converted to the chiral alcohol in less than 24 h. The ionic liquid improved the initial reaction rate by more than four times in the presence of ionic liquid compared to an aqueous-only reaction. Moreover, the ionic liquid improved the stability of both the ADH RE and the GDH coenzyme compared to reactions with either organic cosolvents or aqueous buffer systems [59]. 

Alcohol oxidoreductases capable of oxidizing short chain polyols are useful biocatalysts in industrial production of chiral hydroxy esters, hydroxy adds, amino adds, and alcohols [83]. In a metagenomic study without enrichment, a total of 24 positive clones were obtained and tested for their substrate specifidty. To improve the detedion frequency, enrichment was performed using glycerol or 1,2-propanediol and further 24 positive clones were deteded in this study. 

The one-pot dynamic kinetic resolution (DKR) of ( )-l-phenylethanol lipase esterification in the presence of zeolite beta followed by saponification leads to (R)-l phenylethanol in 70 % isolated yield at a multi-gram scale. The DKR consists of two parallel reactions kinetic resolution by transesterification with an immobilized biocatalyst (lipase B from Candida antarctica) and in situ racemization over a zeolite beta (Si/Al = 150). With vinyl octanoate as the acyl donor, the desired ester of (R)-l-phenylethanol was obtained with a yield of 80 % and an ee of 98 %. The chiral secondary alcohol can be regenerated from the ester without loss of optical purity. The advantages of this method are that it uses a single liquid phase and both catalysts are solids which can be easily removed by filtration. This makes the method suitable for scale-up. The examples given here describe the multi-gram synthesis of (R)-l-phenylethyl octanoate and the hydrolysis of the ester to obtain pure (R)-l-phenylethanol. 

Enzymes are natural biocatalysts that are becoming increasingly popular tools in synthetic organic chemistry [1]. The major areas of exploration have involved the use of hydrolases, particularly esterases and lipases [2]. These enzymes are readily available, robust and inexpensive. The second most popular area of investigation has been the reduction of carbonyl compounds to chiral secondary alcohols using either dehydrogenases (with co-factors) or a whole-cell system such as bakers yeast [3]. 

Kroutil et al. have recently reported [18] an elegant one-pot oxidation/reduction sequence for the deracemization of a chiral secondary alcohol using a single biocatalyst. LyophiUzed cells of a Rhodococcus sp. CBS IVJ.Ti converted racemic 2-decanol into the (S)-enantiomer in 82% yield and 92% enantiomeric excess (e.e.). via a non-specific oxidation followed sequentially by an (S)-selective reduction (Scheme 6.5). Acetone was used as the hydrogen acceptor in the first step and isopropanol as the hydrogen donor in the second step. 

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