Ketoreductase KRED

In an effort to develop easy-to-use ketoreductase toolbox , we have surveyed the activity and enantioselectivity of a collection of ketoreductases (KRED) from various sources toward the reduction of a variety of ketones [90,91]. These studies served as a useful guideline for developing enzymatic processes for the production of optically pure chiral alcohols. For example, several chiral chlorohydrins of pharmaceutical importance were synthesized in both enantiomeric forms using the enzymes in this ketoreductase collection (Table 7.2) [92]. Further applications of this collection and other commercially available ketoreductases can be found in a recent review [9]. 

Biocatalysis is still an emerging field hence, some transformations are more established than others.Panke et alP have performed a survey of patent applications in the area of biocatalysis granted between the years 2000 and 2004. They found that although hydrolases, which perform hydrolyses and esterifications, still command widespread attention and remain the most utilized class of enzyme (Figure 1.5), significant focus has turned towards the use of biocatalysts with different activities and in particular alcohol dehydrogenases (ADHs) - also known as ketoreductases (KREDs) - used for asymmetric ketone reduction. 

There are basically two approaches to the synthesis of enantiomerically pure alcohols (i) kinetic resolution of the racemic alcohol using a hydrolase (lipase, esterase or protease) or (ii) reduction mediated by a ketoreductase (KRED). Both of these processes can be performed as a cascade process. The first approach can be performed as a dynamic kinetic resolution (DKR) by conducting an enzymatic transesterification in the presence of a redox metal [e.g. a Ru(ll) complex] to catalyze in situ racemization of the unreacted alcohol isomer [11] (Scheme 6.1). We shall not discuss this type of process in any detail here since it forms the subject of Chapter 1. 

Ketoreductases (KREDs) are dependent on nicotinamide cofactors NADH or NADPH. Due to the reaction mechanism, these rather costly cofactors are needed in stoichiometric amounts, disclosing an economic problem that has to be dealt with when using these enzymes. Many different possibilities for cofactor recycling have been established with three major approaches finding application in research and industry (Fig. 13). Further regeneration systems, such as electrochemical methods, are not discussed within this review [22-24, 37, 106-108],... 

In 2005, Kambourakis et al. reported the biocatalytic reduction of a-alkyl-1,3-diketones and a-alkyl-/l-ketoesters by employing isolated NADPH-dependent ketoreductases (KREDs). The corresponding optically pure single keto alcohols and hydroxy esters were obtained in quantitative yields (Scheme 3.9). The same group had previously reported the total synthesis of a new class of triterpene derivatives with anti-HIV activity, statin and statin analogues, based on a diastereoselective reduction of a 2-alkyl-substituted 3-ketoglutarate by a KRED. The results are summarised in Scheme 3.9. 

Biocatalytic Reduction with Ketoreductase KRED (KetoREDuctase)... 

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... 

Thanks to the availability of highly regio- and enantioselective reductive enzymes, namely, NADPH-dependent ketoreductases (KREDs), it has been recently demonstrated that consecutive reductions of substrates bearing two or more keto groups can be easily carried out in the same reaction vessel and without the isolation of the intermediates. 

Scheme 11.7 One-pot stereoselective synthesis of2-alkyl-l,3-diols by consecutive exploitation of selected ketoreductases (KRED).

In 2006, the group from Merck reported an interesting utilization of various commercially available ketoreductases (KREDs) for the reduction of a.p-unsaturated ketones 12 and 13 for the synthesis of chiral allylic alcohols 14 and IS [19]. This enzymatic reduction combined with a dynamic kinetic racemization proved to be a very powerful method for the production of optically pure product in excellent yield starting from a racemic ketone, as shown in Scheme 12.7. 

The regio- and stereoselective reductions of 2-substituted-l,3-diketones were extensively studied by Smonou and her coworkers [55]. In this work, the corresponding optically pure hydroxy ketones and diols were synthesized utilizing isolated NADPH-dependent ketoreductases (KRED). Although most... 

An enzymatic alternative for reducing ketone 79 was created by Codexis. A ketoreductase (KRED) was developed by directed evolution using high-throughput screens that mimicked the actual process conditions. Beneficial mutations obtained during each round were recombined and new mutations were introduced, guided by ProSAR. The productivity of the final enzyme was improved 2000-fold and stability was also substantially increased [109,110]. 

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