Epoxide hydrolase enantioselective hydrolysis

Fig. 13. Mammalian epoxide hydrolase. Enantioselective hydrolysis of racemic epoxides. [160—...

G. Bellucci, C. Chiappe, F. Marioni, M. Benetti, Regio- and Enantioselectivity of the Cytosolic Epoxide Hydrolase-Catalysed Hydrolysis of Racemic Monosubstituted Alkyloxiranes ,./. Chem. Soc., Perkin Trans. 1 1991, 361 - 363 G. Bellucci, C. Chiappe, L. Conti, F. Marioni, G. Pierini, Substrate Enantioselection in the Microsomal Epoxide Hydrolase Catalyzed Hydrolysis of Monosubstituted Oxiranes. Effects of Branching of Alkyl Chains ,./. Org. Chem. 1989, 54, 5978 - 5983. 

Fig. 18. Yeast epoxide hydrolase. Enantioselective hydrolyses of aromatic [194] and aliphatic [194, 195] epoxide using whole cells of Rhodotorula glutinis. Stereoselective hydrolysis of cyclopentene oxide [194]...

G. Bellucci, C. Chiappe, F. Marioni, Enantioselectivity of the Enzymatic Hydrolysis of Cyclohexene Oxide and ( )-l-Methylcyclohexene Oxide A Comparison between Microsomal and Cytosolic Epoxide Hydrolases , J. Chem. Soc., Perkin Trans. 1 1989, 2369 -2373. 

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 . 

The first two reported studies concern the epoxide hydrolase from Aspergillus niger (ANEH) 95,96). The enzyme had previously been purified to homogeneity, the gene cloned and expressed in E. coli, and the catalytic hydrolysis of epoxides optimized to high substrate concentrations. Initial attempts were made to enhance the enantioselectivity of the ANEH-catalyzed hydrolytic kinetic resolution of glycidyl phenyl ether (rac-33). The WT leads to an E value of only 4.6 in favor of (5)-34 96). 

Petri, A., Marconcini, P. and Salvadori, P. (2005) Efficient immobilization of epoxide hydrolase onto silica gel and use in the enantioselective hydrolysis of racemic para-nitrostyrene oxide. J. Mol. Catal. B Enzymatic, 32, 219. 

Epoxide is an important intermediate for various bioactive compounds, so the demand for the chiral epoxide is increasing. Epoxide hydrolase can hydrolyze epoxide enantioselectively (Figure 20).21 For example, Aspergillus niger was used for the hydrolysis of carvone epoxide (Figure 20(a)).2 11 In the reaction of styrene oxide, the... 

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. 

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). 

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... 

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]. 

Enantioselective Hydrolysis of Racemic 1- 2, 3 -Dihydro Benzofb]Furan-4 -yl -1,2-Oxirane. Epoxide hydrolase catalyzes the enantioselective hydrolysis of an epoxide to the corresponding enantiomerically enriched diol and unreacted epoxide (22,23). The (5)-epoxide (12) is a key intermediate in the synthesis of a number of prospective drug candidates (24). The enantiospecific hydrolysis of the racemic 1- 2, 3 -dihydro benzo[b]furan-4 -yl -l,2-oxirane (13) to the corresponding (R)-diol (14) and unreacted -epoxide (12) (Fig. 4A) was demonstrated by Goswami et aL (25). Among cultures evaluated, two Aspergillus niger strains (SC 16310, SC 16311)... 

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]. 

Enzymic hydrolysis of the racemic mixture of fl-D-ribo-spoxide (68) and its L-enantiomer (69) using microsomal epoxide hydrolase ( MEH ) was found to be enantioselective, but not regiospecific. Thus the D-epoxide gave the D-arabino- and D xylo-diols (70) and (71) in an 88 12 ratio (Scheme 14), while the L-epoxide (69) remained unreacted In reviewing earlier work on... 

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