Biocatalytic reductions

Oxidoreductases play a central role in the metabolism and energy conversion of living cells. About 25% of the presently known enzymes are oxido reductases [104]. The classification of oxidoreductases is presented in Fig. 3.38. The groups of oxidases, monooxygenases and peroxidases - dealing with oxidations - will be described in Chapter 4. 

The vast majority of alcohol dehydrogenases require nicotanimide cofactors, such as nicotinamide adenine dinucleotide (NADH) and its respective phosphate NADPH. The structure of NAD/NADP is shown in Fig. 3.39. Hydrogen and two electrons are transferred from the reduced nicotinamide to the carbonyl group to effect a reduction of the substrate (see Fig. 3.39). 

The cofactors are relatively unstable molecules and expensive if used in stoichiometric amounts. In addition, they cannot be substituted by less expensive simple molecules. Since it is only the oxidation state of the cofactor which changes during the reaction it may be regenerated in situ by using a second redox reaction to allow it to re-enter the reaction cycle. Thus, the expensive cofactor is needed only in catalytic amounts, leading to a drastic reduction in costs 

However from the standpoint of green chemistry, the use of isolated enzymes (or dead whole cells) is highly preferred because it avoids the generation of copious amounts of biomass. It must be emphasized that the productivity of microbial conversions is usually low, since non-natural substrates are only tolerated at concentrations as low as 0.1-0.3% [106]. The large amount of biomass present in the reaction medium causes low overall yields and makes product recovery troublesome. Therefore the E-factors for whole cell processes can be extremely high. Moreover the use of wild-type cells often causes problems because an array of enzymes is present which can interfere in the reduction of a specific ketone (giving opposite selectivities). The use of recombinant techniques, however, which only express the desired enzyme can overcome this problem [108]. 

Much higher productivities can be obtained using isolated enzymes or cell extracts [118]. This approach is therefore highly preferred. Because of the importance of whole cell technology for biocatalytic reduction a few examples will be given. However the main part of this chapter will be devoted to industrial examples of bioreduction involving isolated enzymes and cofactor recycling. 

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Formate is one of the most representative hydrogen sources for the biocatalytic reduction because CO2 formed by the oxidation of formate is released easily from the reaction system [4]. For example, for the reduction of aromatic ketones by the... 

Since stereoselectivities of biocatalytic reductions are not always satisfactory, modification of biocatalysis are necessary for practical use. This section explains how to find, prepare, and modify the suitable biocatalysts, how to recycle the coenzyme, and how to improve productivity and enantioselectivity of the reactions. 

Biocatalytic reduction has been performed in nonaqueous solvents to improve the efficiency of the reaction. This section explains the use of organic solvent, supercritical fluids, and ionic liquid. 

Therefore, organic solvents have been widely used for biocatalytic reductions. An interesting example for stereochemical control by using organic solvents for... 

Organometallic aldehydes can be reduced enantioselectively with dehydrogenases. For example, optically active organometallic compounds having planar chiralities were obtained by biocatalytic reduction of racemic aldehydes with yeast [22c,d] or HLADH [22e] as shown in Figure 8.29. 

Development of new reduction systems that reduce sterically hindered compounds The reported examples of reduction of carbonyl compounds are usually for the substrates that can be easily reduced such as methyl ketones. Since the demand for reduction of various types of compounds is increasing, investigation of new biocatalytic reductions is required. Photosynthetic organisms are not investigated yet, and they may have new type of enzymes, which can reduce sterically hindered compounds. 

The biocatalytic reduction step B in synthetic route B demands more raw materials (mass index S , see equation (5.1)) and generates more waste (environmental factor , see equation (5.2)) as compared to reduction step C (Figure 5.1). Solvents used to perform the extraction of the product from the aqueous phase in reduction step B are denoted as auxiliaries in Figure 5.1. These solvents and the aqueous phase dominate the mass balances as well as the environmental scores in Figure 5.2 (M4, M8). 

Anderson, B.A., Hansen, M.M., Harkness, A.R. et al. (1995) Application of a practical biocatalytic reduction to an enantioselective synthesis of the 5H-2,3-benzodiazepine LY300164. Journal of the American Chemical Society, 117, 12358-12359. 

Kroutil, W., Mang, H., Edegger, K. and Faber, K. (2004) Recent advances in the biocatalytic reduction of ketones and oxidation of sec-alcohols. Current Opinion in Chemical Biology, 8 (2), 120-126. 

Van Deursen, R., Stampfer, W., Edegger, K. et al. (2004) Chemo- and stereo-selective biocatalytic reduction of a,/8-unsaturated ketones employing a chemo-tolerant ADH from Rhodococcus ruber DSM 44541. Journal of Molecular Catalysis B-Enzymatic, 31 (4-6), 159-163. 

Kaluzna, I.A., Feske, B.D., Wittayanan, W. et al. (2005) Stereoselective, biocatalytic reductions of alpha-chloro-beta-keto esters. The Journal of Organic Chemistry, 70 (1), 342-345. 

Wolberg, M., Hummel, W. and Muller, M. (2001) Biocatalytic reduction of beta,delta-diketo esters a highly stereoselective approach to all four stereoisomers of a chlorinated beta,delta-dihydroxy hexanoate. Chemistry -A European Journal, 7 (21), 4562-4571. 

In the approach followed in this invention [29], a biocatalytic agent converts the sulfur heterocycles into different molecules that do not exhibit the hydrophobic interactions. This is achieved by selectively cleaving carbon-sulfur bonds. The selectivity of the biocatalytic agent employed is limited to the carbon-sulfur bonds and no attack to the carbon-carbon skeleton was reported. Thus, it is expected that the proposed biocatalytic reduction of viscosity would not diminish the fuel value of the treated petroleum liquids. The biocatalyst employed consisted of the strain ATCC No. 53968 (see Section 20 and references therein), in an aqueous culture conventionally prepared by fermentation under aerobic conditions. The fermenting bioreactor is fed with a suitable nutrient medium, which comprises a conventional carbon source (dextrose and glycerol are recommended carbon sources. To confer maximal biocatalytic activity for the desired cleavage of organic C—S bonds, the bacteria was kept in a state of sulfur deprivation. 

In this section we review research on biocatalytic cathodes for oxidant reduction. The biocatalytic reduction of oxidants has only recently attracted renewed attention, with... 

Asymmetric Synthesis of (5)-Bis(trifluoromethyl)phenyiethanol by Biocatalytic Reduction of Bis(trifluoromethyl)acetophenone... 

The biocatalytic reduction of carboxylic acids to their respective aldehydes or alcohols is a relatively new biocatalytic process with the potential to replace conventional chemical processes that use toxic metal catalysts and noxious reagents. An enzyme known as carboxylic acid reductase (Car) from Nocardia sp. NRRL 5646 was cloned into Escherichia coli BL21(DE3). This E. coli based biocatalyst grows faster, expresses Car, and produces fewer side products than Nocardia. Although the enzyme itself can be used in small-scale reactions, whole E. coli cells containing Car and the natural cofactors ATP and NADPH, are easily used to reduce a wide range of carboxylic acids, conceivably at any scale. The biocatalytic reduction of vanillic acid to the commercially valuable product vanillin is used to illustrate the ease and efficiency of the recombinant Car E. coli reduction system." A comprehensive overview is given in Reference 6, and experimental details below are taken primarily from Reference 7. 

Venkitasubramanian, P., Daniels, L. and Rosazza, J.P.N., Biocatalytic reduction of carboxylic acids mechanism and application. In Biocatalysis in the Pharmaceutical and Biotechnology Industries, Patel, R. (ed). CRC Press LLC Boca Raton, FL, 2006, pp. 425-440. 

Scheme 6.2 Biocatalytic reduction of ketones with cofactor regeneration.

Simon, H., Bader, J., Guenther, H., Neumaim, S. and Thanos, J. (1985) Chiral compounds synthesized by biocatalytic reductions. Chem., Int. Ed. Engl., 24,... 

The Shimizu group investigated the potential of another structural type of sulfide organocatalyst, 5- and 6-membered cyclic sulfides [211]. These sulfides were prepared by biocatalytic reduction using baker s yeast. In particular the 5-... 

Nakamura K, Matsuda T (2006) Biocatalytic reduction of carbonyl groups. Curr Org Chem 10 1217-1246... 

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