Enantioselective hydrolysis with epoxide hydrolases

Biocatal3 ic resolution of racemic glycidyl phenyl ether using BmEH under modulated conditions. 

The bioeatalytic resolution of epoxides has mainly been performed in single aqueous buffer systems. In our laboratory, BrriEH has been proved to be very useful for ehiral synthesis of (f )-GPE on a preparative scale. More importantly, this enzyme exhibited a complementary enantiospecificity as compared with those deseribed so far, affording the unreacted epoxide in (S)-configuration which is the solely useful enantiomer for synthesis of bioactive p-blockers. In a single aqueous phase, however, most epoxides including GPE may be spontaneously hydrolyzed into vicinal diols without any enantiospecificity. In addition, only at a very low concentration can epoxides be dissolved in aqueous media. The instability and low solubility of epoxides in the aqueous phase may result in a remarkable decrease in the yield of kinetic resolutions, thus limiting the application of these resolution processes on a practical scale. 

The partitioning behavior of the epoxide (substrate) and the diol (product) between the two phases was first investigated. As a result, both the epoxide and the diol were mostly dissolved in the organic phase when chloroform, dodecanol, or n-decane was used. Moreover, dodecanol and n-decane easily formed emulsions with the buffer (potassium phosphate or KPB), which were diffieult to separate. When using n-hexane, n-octane, or isooctane as the organic solvent, the epoxide partitioned mainly in the organic phase while the diol dissolved mainly in the aqueous phase. The partition difference 

The seeond eriterion for the selection of a proper organic solvent was its effeets on the enzyme activity and stability. Seven organic solvents were tested, ehloroform, cyclohexane, n-hexane, n-octane, isooctane, dodecanol, and n-deeane, with logP values of 2.0, 3.2, 3.5, 4.5, 4.5, 5.0, and 5.6, respee-tively. The logP of a solvent, the logarithm of the partition coefficient of the solvent in a standard mixture of 1-octanol and water, is a parameter often used for predieting its biocompatibility, and is usually more indicative of the dissolved solvent. 

The choice of a phase volume ratio for the two-phase bioconversion was made after examining the modulating effects of this parameter on the enzyme activity, stability, and enantiospecificity. The volume fraction of the organic phase in a two-liquid phase system may modulate the biohydrolysis of GPE in two ways. Firstly, different phase volume fractions may cause different interfacial areas due to altered degrees of emulsification under the same mixing condition, which may change the mass-transfer rate of the substrate to the aqueous phase as well as that of the product to the organic phase. Secondly, 

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Microreactor technology offers the possibility to combine synthesis and analysis on one microfluidic chip. A combination of enantioselective biocatalysis and on-chip analysis has recently been reported by Beider et al. [424]. The combination of very fast separations (<1 s) of enantiomers using microchip electrophoresis with enantioselective catalysis allows high-throughput screening of enantioselective catalysts. Various epoxide-hydrolase mutants were screened for the hydrolysis of a specific epoxide to the diol product with direct on-chip analysis of the enantiomeric excess (Scheme 4.112). 

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]. 

Benzyloxy-2-methylpropane-l,2-diol, a desymmetrized form of 2-methylpropane-1,2,3-triol with its terminal hydroxy being protected as a benzyl ether, was prepared using the B. subtilis epoxide hydrolase-catalyzed enantioselective hydrolysis of the racemic benzyloxymethyl-l-methyloxirane readily available from methallyl chloride and benzyl alcohol. The preparation of the racemic epoxide, a key intermediate, was described in Procedures 1 and 2 (Sections 5.6.1 and 5.6.2), its overall yield being 78 %. The combined yield of enantiomerically pure (7 )-3-benzyloxy-2-methylpropane-l,2-diol was 74 % from ( )-benzyloxymethyl-l-methyloxirane, as described in Procedures 3-5 (Sections 5.6.3 and 5.6.5), with the overall procedures leading to the biocatalytic dihydroxylation of benzyl methallyl ether . 

Active hits were found for every type of substrate screened, including those for which other known microbial epoxide hydrolases were ineffective. For example, hydrolysis of m-stilbenc oxide was not successful with several microbial EHs tested previously.4243 By contrast, several of our new enzymes actively hydrolyzed this substrate and exhibited excellent enantioselectivities (>99% ee). It is important to note that these enzymes were found to be capable of selectively hydrolyzing a wide range of mc.vo-cpoxidcs, including cyclic and acyclic alkyl- and aryl-substituted substrates. 

Similar results were described by Mamouhdian and Michael, who isolated 18 bacterial strains able to produce optically enriched epoxides with excellent ee s (up to 98%) [104, 105]. However, in the case of trflns-(2J ,3J )-epoxybutane, it was shown that the enantiomeric enrichment is in fact due to a second-step enantioselective hydrolysis of the epoxide, which is first produced in racemic form. This, interestingly, is an unexpected example of the possible use of microbial epoxide hydrolases for the synthesis of enantiopure epoxides (see below). 

Scheme 2.15 gives some examples of the use of epoxide hydrolases in organic synthesis. Entries 1 to 3 are kinetic resolutions. Note that in Entry 1 the hydrolytic product is obtained in high e.e., whereas in Entry 2 it is the epoxide that has the highest e.e. In the first case, the reaction was stopped at 18% conversion, whereas in the second case hydrolysis was carried to 70% completion. The example in Entry 3 has a very high E (> 100) and both the unreacted epoxide and diol are obtained with high e.e. at 50% conversion. Entry 4 shows successive use of two separate EH reactions having complementary enantioselectivity to achieve nearly complete... 

The maximum aetivity of the isolated enzyme was observed at 30 °C and pH 6.5 in a buffer system with 5% (v/v) DMSO as a eosolvent. The enzyme was very stable at pH 7.5 and retained full activity after ineubation at 40 °C for 6 h. Interestingly, when the eosolvent DMSO was replaced by an emulsifier (Tween-80, 0.5% w/v) as an alternative modulator to disperse the water-in-soluble substrate, the apparent activity of the epoxide hydrolase significantly increased by 1.8-fold, while the optimum temperature shifted from 30 to 40 °C and the half-life of the enzyme at 50 °C increased by 2.5 times (Figure 2.5). The enzymatic hydrolysis of rac-PGE was highly enantioselective, with an B-value (enantiomeric ratio) of 69.3 in the Tween-80 emulsion system, which is obviously superior than that (41.2) observed in the DMSO-modulated system. ... 

Epoxide hydrolase has emerged as an important enzyme for the asymmetric synthesis of enantiopure epoxides and diols [24]. The hydrolase HXN-200 has been shown to catalyse the enantioselective hydrolysis of meso epoxides to give optically active diols (Scheme 4.14) [25]. A related group of enzymes is the haloalkane dehalogenases that display epoxide hydrolase activity with nucleophiles other than water (Scheme 4.15) [26]. 

This process only becomes possible when both enantiomers are converted by two independent enantioselective reactions to the same enantiomeric product. Both pathways must exhibit an opposite sense of enantioselectivity. For example, as shown in Scheme 5.58, whole-cell microbial transformation of a racemic epoxide using two different organisms, each harbouring a hydrolase that performs the enantioselective hydrolysis of the epoxide ring (with opposite stereocontrol), to give a single enantiomeric 1,2-diol as the sole product in high yield with excellent enantiomeric excess [148]. 

S. Wu, A. Li, Y.S. Chin, Z. Li, Enantioselective hydrolysis of racemic and mesu-epoxides with recombinant Escherichia coli expressing epoxide hydrolase from Sphingomonas sp. FlXN-200 preparation of epoxides and vicinal diols in high ee and high concentration, ACS Catal. 3... 

Chang, D., Wang, Z., Heringa, M.R, Wirthner, R., Witholt, B. and Li, Z. (2003) Highly enantioselective hydrolysis of alicyclic meso-epoxides with a bacterial epoxide hydrolase from Sphingomonas sp. HXN-200 simple synthesis of alicyclic vicinal frans-diols. Chem. Commun., 960-961. 

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