Microbial epoxidation alkenes

Once the problems of product toxicity were surmounted by sophisticated process engineering, microbial epoxidation of alkenes became also feasible on an industrial scale [1153, 1154]. The latter was achieved by using organic-aqueous two-phase systems or by evaporation for volatile epoxides. For instance, the epoxy-phosphonic acid derivative fosfomycin [1155], whose enantiospecific synthesis by classical methods would have been extremely difficult, was obtained by a microbial epoxidation of the corresponding olefinic substrate using Penicillium spinulosum. 

The most intensively studied microbial epoxidizing agent is the co-hydroxylase system of Pseudomonas oleovorans [1156,1157]. It consists of three protein components rubredoxin, NADH-dependent rubredoxin reductase and an co-hydroxylase (a sensitive nonheme iron protein). It catalyzes not only the hydroxylation of aliphatic C-H bonds, but also the epoxidation of alkenes [1158, 1159]. The following rules can be formulated for epoxidations using Pseudomonas oleovorans (Scheme 2.155). 

Optically active epoxides are useful chiral synthons in the phamaceutical synthesis of prostaglandins. Microbial epoxidation of olefinic compounds was first demonstrated by van der Linden [241]. Subsequently, May et al. [242] demonstrated the epoxidation of alkenes in addition to hydroxylation of alkanes by an m-hydroxylase system. Oxidation of alk-l-enes in the range C6-C12, a,(o-dienes from C6-C12, alkyl benzene, and allyl ettiers were demonstrated using an co-hydroxylase enzyme system from Pseudomonas oleovorans. i -Epoxy compounds in greater than 75% e.e. were produced by epoxidation re tions using the co-hydroxylase system [243,244]. The epoxidation system from Nocardia cor-allina is very versatile, has broad substrate specificity, and reacts with unfunctionalized aliphatic as well as aromatic olefins to produce i -epoxides [245,246]. 

Prichanont, S., Leak, D.J. and Stuckey, D.C. (1998) Alkene monooxygenase-catalyzed whole cell epoxidation in a two-liquid phase system. Enzyme and Microbial Technology, 22 (6), 471 479. 

Enzymic asynmietric epoxidation of alkenes may be performed by pure monooxygenases. However, due to practical problems such as need of cofactors, microbial oxidation with whole cells has been more widely used for the purpose. One great disadvantage however, is the toxicity of epoxides towards living cells. 

In spite of the considerable value of epoxide hydrolases for fine chemical synthesis, it was only recently that a detailed search for epoxide hydrolases from microbial sources was undertaken by the groups of Furstoss185, 901 and Faber123, 79, 911, bearing in mind that the use of microbial enzymes allows an (almost) unlimited supply of biocatalyst. The screening was based along the following considerations on the one hand, the catabolism of alkenes often implies the hydrolysis of an epoxide inter-... 

For the enantioselective preparations of chiral synthons, the most interesting oxidations are the hydroxylations of unactivated saturated carbons or carbon-carbon double bonds in alkene and arene systems, together with the oxidative transformations of various chemical functions. Of special interest is the enzymatic generation of enantiopure epoxides. This can be achieved by epoxidation of double bonds with cytochrome P450 mono-oxygenases, w-hydroxylases, or biotransformation with whole micro-organisms. Alternative approaches include the microbial reduction of a-haloketones, or the use of haloperoxi-dases and halohydrine epoxidases [128]. The enantioselective hydrolysis of several types of epoxides can be achieved with epoxide hydrolases (a relatively new class of enzymes). These enzymes give access to enantiopure epoxides and chiral diols by enantioselective hydrolysis of racemic epoxides or by stereoselective hydrolysis of meso-epoxides [128,129]. 

Biocatalytic asymmetric epoxidation of alkenes catalyzed by monooxygenases cannot be performed on a preparative scale with isolated enzymes due to their complex nature and their dependence on a redox cofactor, such as NAD(P)H. Thus, whole microbial cells are used instead. Although the toxic effects of the epoxide formed, and its further (undesired) metabolism by the cells catalyzed by epoxide hydrolases (Sect. 2.1.5), can be reduced by employing biphasic media, this method is not trivial and requires bioengineering skills [1151]. Alternatively, the aUcene itself can constitute the organic phase into which the product is removed, away from the cells. However, the bulk apolar phase tends to damage the cell membranes, which reduces and eventually abolishes all enzyme activity [1152]. 

Epoxidation of Alkenes. Due to the fact that the asymmetric epoxidation of alkenes using monooxygenase systems is impeded by the requirement for NADPH-recycling and the toxicity of epoxides to microbial cells, the use of H202-depending peroxidases represents a valuable alternative. 

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