Isolated enzymes, asymmetric oxidation

A successful case study for asymmetric nitrogen oxidation was reported for a series of (hetero)aromatic tertiary amines. High diastereoselectivity was observed for the enzyme-mediated oxidation of S-(—)-nicotine by isolated CHMOAdneto to give the corresponding ds-N-oxide [215]. The stereoselectivity of this biooxidation was complementary to the product obtained by flavin M O (FM O) from human li ver (trows-selective [216]) as well as unspecific oxidations by FMOs from porcine and guinea pig liver. 

Apart from the asymmetric metal catalysis, enantioselective Baeyer-Villiger oxidations mediated by enzymes have been known for some time [32,33,34]. Both whole-cell cultures and isolated enzymes, usually flavin-dependent monooxygenases, can be used to oxidize ketones enantioselectively. For future improvements in the asymmetric Baeyer-VilHger oxidation the use of chiral Lewis acids in combination with an appropriate oxidant seems worthy of intensive investigation. 

Reduction with isolated enzymes avoids difficulties associated with diffusion limitations and also avoids the presence of many different enzymes, present in the whole cell, which can cause side reactions or reduced enantioselectivity. The main drawback, however, is the instability of the isolated enzyme and the requirement for added co-factor NAD(H) or NADP(H), which are the oxidized (or reduced) forms of nicotinamide adenine diphosphate or its 2 -phosphate derivative. These co-factors are expensive, but can be used as catalysts in the presence of a co-reductant such as formate ion HCOO or an alcohol (e.g. isopropanol or ethanol). The reduction of ketones occurs by transfer of hydride from the C-4 position of the dihydropyridine ring of NADH or NADPH (7.105). Only one of the two hydrogen atoms is transferred and this process occurs within the active site of the enzyme to promote asymmetric reduction. 

Recently, the enzyme was expressed in baker s yeast and used for the asymmetric oxidation of alkyl cyclohexanones. The use of designer baker s yeast combined the advantages of using purified enzymes (single catalytic species, no overmetabolism) with the benefits of whole-cell reactions (experimentally simple, no cofactor regeneration necessary). The enantioselectivities observed with the recombinant enzyme were the same as those with the original, isolated enzyme. 

Based on the above observations, the employment of chiral metal catalysts for the asymmetric BV oxidation is still underdeveloped for practical appUcations on the scale of natural products and drugs synthesis. Only biocatalytic BV oxidations performed with isolated enzymes or whole cells containing cyclohexanone monooxygenases (CHMOs) or BV monooxygenases (BVMOs) shows practical conversions as weU as enantioselectivity above 95% ee with cyclohexanones as substrates. 

It also has to be stressed that many useful redox enzymes for asymmetric oxidations are not commercially available as isolated enzymes—alcohol dehydrogenases are the main exception here— but have to be isolated from the wild-type organism or produced in recombinant form. For that reason, applied biocatalysis is a multidisciplinary field where the expertise of biologists, chemists, and engineers is required. 

The asymmetric hydrolysis of (exo,exo)-7-oxabicyclo[2.2.1]heptane-2,3-dimethanol, diacetate ester (37) to the corresponding chiral monoacetate ester (38) (Fig. 12B) has been demonstrated with lipases [61]. Lipase PS-30 from P. cepacia was most effective in asymmetric hydrolysis to obtain the desired enantiomer of monoacetate ester. The reaction yield of 75 M% and e.e. of >99% were obtained when the reaction was conducted in a biphasic system with 10% toluene at 5 g/liter of the substrate. Lipase PS-30 was immobilized on Accurel PP and the immobilized enzyme was reused (5 cycles) without loss of enzyme activity, productivity, or e.e. of product (38). The reaction process was scaled up to 80 liters (400 g of substrate) and monoacetate ester (38) was isolated in 80 M% yield with 99.3% e.e. The product was isolated in 99.5% chemical purity. The chiral monoacetate ester (38) was oxidized to its corresponding aldehyde and subsequently hydrolyzed to give chiral lactol (33) (Fig. 12B). The chiral lactol (33) obtained by this enzymatic process was used in chemoenzymatic synthesis of thromboxane A2 antagonist (35). 

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