Carbonyl reductases, substrates

Baker s yeast has been widely used for the reduction of ketones. The substrate specificity and enantioselectivity of the carbonyl reductase from baker s yeast, which is known to catalyze the reduction of P-keto ester to L-hydroxyester (L2-enzyme) [15], was investigated, and the enzyme was found to reduce chloro-, acetoxy ketones with high enantioselectivity (Figure 8.32) [24aj. 

Ema, T., Yagasaki, H., Okita, N. et al. (2006) Asymmetric reduction of ketones using recombinant E. coli cells that produce a versatile carbonyl reductase with high enantioselectivity and broad substrate specificity. Tetrahedron, 62 (26), 6143-6149. 

Zhu, D., Yang, Y., Buynak, J.D. and Hua, L. (2006) Stereoselective ketone reduction by a carbonyl reductase from Sporobolomyces salmonicolor. Substrate specificity, enantioselectivity and enzyme—substrate docking studies. Organic and Biomolecular Chemistry, 4 (14), 2690-2695. 

Table 3. Substrate specificities of aldehyde reductase (AR) of S. salmonicolor and carbonyl reductase (CR) of C. magnoliae...

In a photometric assay NADP(H)-dependent LBADH (see above) [9] and NAD(H) -dependent Candida parapsilosis carbonyl reductase (CPCR) [40] were identified as suitable catalysts accepting a broad range of ynones as substrates. Both enzymes catalyze the reduction of various aryl alkynones 21 with high enantioselectivity and efficiency (Scheme 2.2.7.13) [41]. 

Carbonyl reduction is a metabolic pathway widely distributed in nature. Many endogenous substances, such as prostaglandins, biogenic amines, and steroids, together with xenobiotic chemicals of several varieties, are transformed to the corresponding alcohols before further metabolism and elimination. Carbonyl reduction in several continuous cell lines was investigated using metyrapone as a substrate ketone. Quercitrin was reported to inhibit carbonyl reductase. 

Table 5. Comparison of substrate specificities and stereospecificities of the carbonyl reductases from various micro-organisms...

What are the bottelnecks for bioreduction The drawbacks of a bioreduction process involving whole cells of microoganisms can be summarized i) Microbial strains possessing both carbonyl reductase activity and cofactor (NAD(P)H)-regenerating activity are necessary to obtain a highmolar yield, because a stoichiometric amount of cofactor is required for substrate reduc-... 

This bioreduction system is applicable to the production of many other useful chiral alcohols by replacing the carbonyl reductase gene with that of another appropriate enzyme for carbonyl reduction (Fig. 19.6). A good library of microbial carbonyl reductases with different substrate and stereospecifici-... 

Carbonyl reductase plays a role in reducing a variety of aliphatic, alicyclic, and aromatic aldehydes and ketones, including endogenous ketosteroids and/ or prostanoids. This assay allows quantitation of both enantiomers formed from the substrate. 

There is complete reduction of a p- or o-quinone to the corresponding hydroquinone or catechol, respectively. In the human liver, carbonyl reductase may play a role in the reduction of some quinones. Catechols are primary substrates for catechol o-me-thyl transferase, but also undergo sulfation. However, for the antitumor quinones, mitomycin C, adriamycin, and daunomycin, two-electron reduction serves as an efficient bioactivation mechanism, elegantly affirming the concept of bioreductive alkylation for the preferential bioactivation of antitumor prodrugs with oxygen deficient tumors. 

A carbonyl reductase isolated form Rhodococcus erythropolis accepts a broad range of substrates, including a variety of compounds useful for synthetic chemistry, as shown in Table 15-11 2S1. Reduction of all the carbonyl compounds tested yielded (S)-configured hydroxyl compounds with high enantioselectivities. 

As a new option, for the bioconversion of poorly soluble substrates the classical EMR-concept can be extended to an Emulsion Membrane Reactor , comprising a separate chamber for emulsification (with a hydrophilic ultrafiltration membrane), an EMR-Ioop with a normal ultrafiltration module, and a circulation pump. This approach has been successfully demonstrated for the enzymatic reduction of poorly soluble ketones [107]. Using this device, e.g., for the enantioselective reduction of 2-octanone to (S)-2-octanol (e.e. >99.5%) with a carbonyl reductase from Candida parapsilosis under NADH-regeneration with FDH/for-mate, the total turnover number was increased by a factor 9 as compared with the classical EMR. 

Substrate specificities can be broad and overlapping both among CYP family members and between CYPs and FMOs, and since metabolic transformations are often sequential (e.g. aliphatic hydroxylation being followed by oxidation by alcohol dehydrogenase, further oxidation to the acid, etc.), many enzymes and many metabolites can be involved in processing a single drug. Only a few of the most important enzymes involved in Phase I transformations have been mentioned here. For these and many others (monoamine oxidase, xanthine oxidase, etc.), further information can be found in the previously cited reviews. Bear in mind too that not all Phase I reactions are oxidative enzymes like carbonyl reductases are important in metabolism as well. 

Xu, G.C., Yu, H.L, Zhang, X.Y., and Xu, J. H. (2012) Access to optically active aryl halohydrins using a substrate-tolerant carbonyl reductase discovered from Kluyveromyces thermotolerans. ACS Catal., 2, 2566-2571. 

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... 

In a very recent example, 4-tert-butylcydohexanone, a precursor of woody acetate, a perfume for cosmetics, was reduced with several commercial carbonyl reductases using 2-propanol to recycle the nicotinamide cofactor. Furthermore, a screening of organic cosolvents was done finding that methyl tert-butyl ether (MTBE) was very appropriate to dissolve this hydrophobic substrate. The reaction was done with 0.5 kg of substrate, with a concentration of 300 g/1 and at 45 °C, obtaining the cis-alcohol (25, Figure 4.4) with complete diastereoselectivity and 91% isolated yield [50]. 

Other remarkable stepwise transformations make use of the combination of several biocatalysts to obtain valuable compounds. In particular, a sequential process employing a recombinant enoate reductase (ER) and a carbonyl reductase has been used for the stereoselective redudion of both enantiomers of carvone to dihydrocarveol (Scheme 4.21) [72]. In the first step, hydrogenation of the endocydic C=C double bond of (R)- or (S)-carvone was achieved by ER from Lactobacillus casd (LacER). In the second step, wild-type or mutant reductases from Candida magnolia (CMCR) and Sporobolomyces sahnoni-color (SSCR) were employed, respectively. First, experiments were performed in a sequential manner and later the one-pot synthesis was tested with a substrate concentration of 0.1 M, obtaining the final compound with excellent conversions and de. 

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