Epoxide hydrolases enantioselectivities

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

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]...

Several reports regarding the directed evolution of enantioselective epoxide hydrolases (EHs) have appeared [23,57-59]. These enzymes constitute important catalysts in synthetic organic chemistry [4,60]. The first two reported studies concern the Aspergillus niger epoxide hydrolase (ANEH) [57,58]. Initial attempts were made to enhance the enantioselectivity of the AN E H -catalyzed hydrolytic kinetic resolution of glycidyl phenyl ether (rac-19). The WT leads to an Evalue of only 4.6 in favor of (S)-20 (see Scheme 2.4) [58]. 

Turning to enzymatic hydration, we see from the data in Table 10.1 that phenanthrene 9,10-oxide Fig. 10.10, 10.29) is an excellent substrate for epoxide hydrolase. Comparison of enzymatic hydration of the three isomeric phenanthrene oxides shows that the Vmax with the 9,10-oxide is greater than with the 1,2- or the 3,4-oxide the affinity was higher as well, as assessed by the tenfold lower Km value [90]. Furthermore, phenanthrene 9,10-oxide has a plane of symmetry and is, thus, an achiral molecule, but hydration gives rise to a chiral metabolite with high product enantioselectivity. Indeed, nucleophilic attack by epoxide hydrolase occurs at C(9) with inversion of configuration i.e., from below the oxirane ring as shown in Fig. 10.10) to yield the C-H9.S, 10.S )-9,10-dihydro-9,10-diol (10.30) [91],... 

The base-catalyzed hydration of 2-phenyloxirane involves nucleophilic attack preferentially at C(3) (0-C(3) cleavage), but with only partial regio-selectivity. Acid-catalyzed hydration is mainly by 0-C(2) cleavage. The hydration of 2-phenyloxirane catalyzed by epoxide hydrolase is characterized by its very high regioselectivity for the less-hindered, unsubstituted C(3) [175] [176], involving retention of configuration at C(2). In other words, (R)-and (5)-2-phenyloxirane are metabolized to (/ )- and (S)-l-phenylethane-l,2-diol (10.118), respectively. Substrate enantioselectivity was also character-... 

R. N. Armstrong, B. Kedzierski, W. Levin, D. M. Jerina, Enantioselectivity of Microsomal Epoxide Hydrolase toward Arene Oxide Substrates , J. Biol. Chem. 1981, 256, 4726 - 4733. 

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. 

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 . 

Over the past year, racemic 1,2-dialkyl epoxides were resolved enzymatically using soluble epoxide hydrolase (sEH), although the outcome of the reaction is characteristically substrate-dependent. In an example of the best enantioselection exhibited, epoxide 65 afforded the (Ji ,4i )-diol 66 upon treatment with sEH at pH 7.4. The course of these reactions is different from those in which the same substrates were treated with microsomal epoxide hydrolase <99T11589>. 

Schottler M, Boland W (1996) Biosynthesis of dodecano-4-lactone in ripening fruits Crmal role of an epoxide-hydrolase in enantioselective generation of aroma components of the nectarine ( Prunus persica var. nucipersica ) and the strawberry (Fragaria ananassa). Helv Chim Acta 79 1488... 

The MS assay has also been applied successfully in the directed evolution of enantioselective epoxide hydrolases acting as catalysts in the kinetic resolution of chiral epoxides [35]. Moreover, Diversa has recently employed the MS-based technique for desymmetrization of a prochiral dinitrile catalyzed by mutant nitrilases [36]. In this industrial application one of the nitrile moieties was labeled with 15N, which means that the two pseudo enantiomeric products differ by only one mass unit. 

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. 

Enantioselective epoxidation Epoxide hydrolase Solution phase [424]... 

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). 

Fig. 9. Iterative CASTing in the evolution of enantioselective epoxide hydrolases as catalysts in the hydrolytic kinetic resolution of rac-8...

Zha D, Wilensek S, Hermes M, Jaeger K-E, Reetz MT (2001) Complete reversal of enantioselectivity of an enzyme-catalyzed reaction by directed evolution. Chem Commun (Cambridge UK) 2664-2665 Zou JY, Hallberg BM, Bergfors T, Oesch F, Arand M, Mowbray SL, Jones TA (2000) Structure of Aspergillus niger epoxide hydrolase at 1.8 resolution Implications for the structure and function of the mammalian microsomal class of epoxide hydrolases. Structure (London) 8 111-122... 

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