Enantioselectivity in the Rabbit Liver Microsomal Biotransformation of Styrene and Phenylpropenes

The rabbit liver microsomal P-450 catalyzed oxidation of styrene (1a) and isomeric phenylpropenes, trans-1-phenylpropene (1b), cis-1-phenylpropene (1c) and 3-phenylpropene (1d), was investigated and the enantioselectivity of the epoxidation of the olefinic double bond was determined by checking the enantiomeric excesses of the corresponding first formed epoxides (2). These enantiomeric excesses were always modest, ranging between 7% of (1S,2S)-(2b) and 22% of (1R,2R)-(2c). In the case of (1d) a nonenantioselective hydroxylation at the benzylic-allylic C(3) was also oberved. The ratio between this hydroxylation and olefin epoxidation of (Id) was 1:2.     

Kinetics and stereochemistry of the microsomal epoxide hydrolase-catalyzed hydrolysis of cis-stilbene oxides

The microsomal epoxide hydrolase (mEH)-catalyzed hydrolysis of cis-4,4′–dimethylstilbene oxide ( 1a ), cis-4,4′-diethylstilbene oxide ( 1b ), cis-4,4′-diisopropylstilbene oxide ( 1c ), and cis-4,4′-dichlorostilbene oxide ( 1d ) have been investigated using rabbit liver microsomal preparations. The kinetic parameters, K_m and V_max, and the absolute stereochemistry of the reactions have been determined and compared with those of cis-stilbene oxide ( 1e ). All epoxides 1a – d are hydrolyzed by mEH with high product enantioselectivity to give (R,R)-(+)-diols with ee ≥ 90%. The presence of the substituents on the phenyl rings markedly reduces the rates of mEH catalyzed hydrolysis with respect to cis-stilbene oxide, by increasing K_m and reducing V_max in the cases of 1a , 1b , and 1d , or reducing only the V_max in the case of 1c . The very low V_max, together with a persistent ability to fit into the mEH active site, make all these epoxides, and particularly 1c , inhibitors of cis-stilbene oxide hydrolysis. The kinetic and stereochemical results are interpreted on the basis of the proposed topology of the mEH active site. © 1994 Wiley-Liss, Inc.     

Enantioselectivity in the Rabbit Liver Microsomal Biotransformation of Styrene and Phenylpropenes

The rabbit liver microsomal P-450 catalyzed oxidation of styrene (1a) and isomeric phenylpropenes, trans-1-phenylpropene (1b), cis-1-phenylpropene (1c) and 3-phenylpropene (1d), was investigated and the enantioselectivity of the epoxidation of the olefinic double bond was determined by checking the enantiomeric excesses of the corresponding first formed epoxides (2). These enantiomeric excesses were always modest, ranging between 7% of (1S,2S)-(2b) and 22% of (1R,2R)-(2c). In the case of (1d) a nonenantioselective hydroxylation at the benzylic-allylic C(3) was also oberved. The ratio between this hydroxylation and olefin epoxidation of (Id) was 1:2.     

Cloning,expression and enantioselective hydrolytic catalysis of a microsomal epoxide hydrolase from a marine fish, <Emphasis Type="Italic">Mugil cephalus</Emphasis>

The cDNA of a marine fish microsomal epoxide hydrolase (mEH) gene from Mugil cephalus was cloned by rapid amplification of cDNA ends (RACE) techniques. The homology model for the mEH of M. cephalus showed a characteristic structure of α/β-hydrolase-fold main domain with a lid domain over the active site. The characteristic catalytic triad, consisting of Asp(238), His(444), and Glu(417), was highly conserved. The cloned mEH gene was expressed in Escherichia coli and the recombinant mEH exhibited (R)-preferred hydrolysis activity toward racemic styrene oxide. We obtained enantiopure (S)-styrene oxide with a high enantiopurity of more than 99% enantiomeric excess and yield of 15.4% by batch kinetic resolution of 20 mM racemic styrene oxide.     

Activation of microsomal epoxide hydrolase by interaction with cytochromes P450: kinetic analysis of the association and substrate-specific activation of epoxide hydrolase function

The kinetics of the association between cytochrome P450 (P450) and microsomal epoxide hydrolase (mEH) was studied by means of resonant mirror based on the principle of surface plasmon resonance. The dissociation equilibrium constants (K(D)) for the affinity of P450 enzymes for mEH were estimated by resonant mirror using an optical biosensor cell covalently bound to rat mEH. Comparable K(D) values were obtained for CYP1A1 and 2B1, and these were greater by one order of magnitude than that for the CYP2C11. To clarify the influences of P450 enzymes on the catalytic activity of mEH, the hydrolyzing activity for styrene oxide and benzo(a)pyrene-7,8-oxide [B(a)P-oxide] was analyzed in the presence or absence of P450s. Styrene oxide hydrolysis was activated by all P450s including the CYP1A, 2B, 2C, and 3A subfamilies. In agreement with the association affinity determined by resonant mirror, CYP2C11 tends to have enhanced activity for styrene oxide hydrolysis. On the other hand, B(a)P-oxide hydrolysis was enhanced by only CYP2C11 while CYP1A1 and CYP2B1 had no effect. These results suggest that (1) many P450 enzymes associate nonspecifically with mEH, (2) the CYP2C11 plays a greater role in the association/activation of mEH and (3) the P450-mediated activation of mEH depends upon the substrate of mEH.     

Stereoselective Epoxidation of 4-Bromo-1-Butene and 3-Butene-1-Ol with Three Alkene-Utilizing Bacteria

Stereoselective epoxidation of 4-bromo-1-butene and 3-butene-1-ol was carried out with three alkene-utilizing bacteria: Mycobacterium LI, Mycobacterium E3 and Nocardia IP1. The enantiomeric compositions of 4-bromo-1,2-epoxybutane and 4-hydroxy-1,2-epoxybutane were determined by optical rotation and checked by ¹H NMR using a chiral shift reagent and by ¹⁹F NMR after reaction with the Mosher reagent. The presence of a bromine atom or a hydroxyl group induced some variations in the stereochemical course of the microbial epoxidation. The epoxides were produced predominantly in the 2R-form but their enantiomeric composition depended on both the microorganism used and on the substrate studied.     

Rabbit microsomal epoxide hydrolase: isolation and characterization of the xenobiotic metabolizing enzyme cDNA

Many endogenous and xenobiotic chemicals are metabolized to epoxides which may be enzymatically hydrated, via microsomal epoxide hydrolase (mEH), to less reactive dihydrodiol derivatives. On the basis of the reported rabbit mEH amino acid sequence [F. S. Heinemann and J. Ozols (1984) J. Biol. Chem. 259, 797-804], we constructed a 35 base oligonucleotide which was used to screen rabbit liver cDNA libraries. Overlapping rabbit mEH clones were isolated and the full-length cDNA sequence of 1653 bp was determined. The rabbit nucleotide sequence has a high degree of similarity (greater than 75%) with cDNA sequences reported for rat and human mEH. Northern blot analyses with fragments of the rabbit cDNA demonstrate that mEH messenger RNA (mRNA) is expressed constitutively in the liver and induced following exposure to phenobarbital or polychlorinated biphenyls. Constitutive expression of mEH mRNA is also observed in rabbit kidney, testes, and lung. Using benzo[alpha]pyrene-4,5-oxide as substrate, mEH enzymatic activity is shown to correlate closely with tissue levels of mEH mRNA. Southern blot analyses of rabbit DNA suggest that the mEH gene exists as a single copy per haploid genome. The mEH amino acid sequences of the human and rat were compared to that of the deduced rabbit protein in order to analyze the degree of conservation and hydropathy profiles in these species. This comparison permitted the formulation of a computer-assisted model of mammalian mEH as it may relate to the microsomal membrane.     

Development of fluorescent substrates for microsomal epoxide hydrolase and application to inhibition studies

The microsomal epoxide hydrolase (mEH) plays a significant role in the metabolism of numerous xenobiotics. In addition, it has a potential role in sexual development and bile acid transport, and it is associated with a number of diseases such as emphysema, spontaneous abortion, eclampsia, and several forms of cancer. Toward developing chemical tools to study the biological role of mEH, we designed and synthesized a series of absorbent and fluorescent substrates. The highest activity for both rat and human mEH was obtained with the fluorescent substrate cyano(6-methoxy-naphthalen-2-yl)methyl glycidyl carbonate (11). An in vitro inhibition assay using this substrate ranked a series of known inhibitors similarly to the assay that used radioactive cis-stilbene oxide but with a greater discrimination between inhibitors. These results demonstrate that the new fluorescence-based assay is a useful tool for the discovery of structure–activity relationships among mEH inhibitors. Furthermore, this substrate could also be used for the screening chemical library with high accuracy and with a Z′ value of approximately 0.7. This new assay permits a significant decrease in labor and cost and also offers the advantage of a continuous readout. However, it should not be used with crude enzyme preparations due to interfering reactions.     

Physico-chemical properties of the epoxide hydrolase from Rhodosporidium toruloides

Residue-specific chemical modification of amino acid residues of the microsomal epoxide hydrolase (mEH) from Rhodosporidium toruloides UOFS Y-0471 revealed that the enzyme is inactivated through modification of Asp/Glu and His residues, as well as through modification of Ser. Since Asp acts as the nucleophile, and Asp/Glu and His serve as charge relay partners in the catalytic triad of microsomal and soluble epoxide hydrolases during epoxide hydrolysis, inactivation of the enzyme by modification of the Asp/Glu and His residues agrees with the established reaction mechanism of these enzymes. However, the inactivation of the enzyme through modification of Ser residues is unexpected, suggesting that a Ser in the catalytic site is indispensable for substrate binding by analogy of the role of Ser residues in the related L-2-haloacid dehalogenases, as well as the ATPase and phosphatase enzymes. Co²⁺, Hg²⁺, Ag⁺, Mg²⁺ and Ca²⁺ inhibited enzyme activity and EDTA increased enzyme activity. The activation energy for inactivation of the enzyme was 167 kJ mol^–1. Kinetic constants for the enzyme could not be determined since unusual behaviour was displayed during hydrolysis of 1,2-epoxyoctane by the purified enzyme. Enantioselectivity w as strongly dependent on substrate concentration. When the substrate was added in concentrations ensuring two-phase conditions, the enantioselectivity was greatly enhanced. On the basis of these results, it is proposed that this enzyme acts at an interface, analogous to lipases.     

Microbial epoxide hydrolases for preparative biotransformations

Epoxide hydrolases from microbial sources are highly versatile biocatalysts for the asymmetric hydrolysis of epoxides on a preparative scale. Besides kinetic resolution, which furnishes the corresponding vicinal diol and remaining non-hydrolysed epoxide in nonracemic form, enantioconvergent processes are possible: these are highly attractive as they lead to the formation of a single enantiomeric diol from a racemic oxirane. The data accumulated over recent years reveal a common picture of the substrate structure selectivity pattern of microbial epoxide hydrolases and indicate that substrates of various structural types can be selectively hydrolysed with enzymes from certain microbial sources.     

Production of chiral epoxides by an ethene-utilisingMicrococcus sp.

Summary An ethene-utilising bacterium was isolated in pure culture from soil and was tentatively identified as aMicrococcus sp. The organism accumulated epoxyalkanes (0.2–13 mM) from internal, terminal, cyclic and aryl-substituted olefins and exhibited a substrate specificity which was different from that expected on the basis of the chemical reactivity pattern in peracid epoxidations. Epoxyalkanes were hydrolysed at a much slower rate than the epoxidation step which allowed them to accumulate. Ethene-grown cells catalysed the stereospecific formation of R-1,2-epoxypropane (enantiomeric excess: e.e.=96%), R-1,2-epoxybutane (e.e.=94%) andtrans-(2R,3R)-epoxybutane (e.e.=84%). An ethene monooxygenase was implicated in the production of chiral epoxides in cell-free extracts of the bacterium. The (2S,3S)-enantiomer of racemictrans-2,3-epoxybutane was stereoselectively hydrolysed to completion resulting in an enrichment in the (2R,3R)-enantiomer. Further hydrolysis of 1,2-epoxyalkanes (C₃-C₄), however, occurred via complete destruction of both stereoisomers.     

The 3,4-oxide is responsible for the DNA binding of benzo[ghi]perylene, a polycyclic aromatic hydrocarbon without a "classic" bay-region

The polycyclic aromatic hydrocarbon (PAH) benzo[ghi]perylene (BghiP) lacks a "classic" bay-region and is therefore unable to form vicinal dihydrodiol epoxides thought to be responsible for the genotoxicity of carcinogenic PAHs like benzo[a]pyrene. The bacterial mutagenicity of BghiP increases considerably after inhibition of the microsomal epoxide hydrolase (mEH) indicating arene oxides as genotoxic metabolites. Two K-region epoxides of BghiP, 3,4-epoxy-3,4-dihydro-BghiP (3,4-oxide) and 3,4,11,12-bisepoxy-3,4,11,12-tetrahydro-BghiP (3,4,11,12-bisoxide) identified in microsomal incubations of BghiP are weak bacterial mutagens in strain TA98 of Salmonella typhimurium with 5.5 and 1.5 his+-revertant colonies/nmol, respectively. After microsomal activation of BghiP in the presence of calf thymus DNA three DNA adducts were detected using 32P-postlabeling. The total DNA binding of 2.1 fmol/microg DNA, representing 7 adducts in 10(7) nucleotides, was raised 3.6-fold when mEH was inhibited indicating arene oxides as DNA binding metabolites. Co-chromatography revealed the identity between the main adduct of metabolically activated BghiP and the main adduct of the 3,4-oxide. DNA adducts of BghiP originating from the 3,4,11,12-bisoxide were not found. Therefore, a K-region epoxide is proposed to be responsible for the genotoxicity of BghiP and possibly of other PAHs without a "classic" bay-region.     

Biocatalysis of nitro substituted styrene oxides by non-conventional yeasts

Yeast strains (410) from more than 45 different genera were screened for the enantioselective hydrolysis of nitro substituted styrene oxides. These strains included 262 yeasts with known epoxides hydrolase activity for various other epoxides. Epoxide hydrolase activity for p-nitrostyrene oxide (pNSO) (177 strains) and m-nitrostyrene oxide (mNSO) (148 strains) was widespread in the yeasts, while activity for o-nitrostyrene oxide (oNSO) was less ubiquitous (22 strains). The strains that displayed enantioselectivity in the hydrolysis of one or more of the nitro substituted styrene oxides (35 strains) were also screened against styrene oxide (SO). Rhodosporidium toruloides UOFS Y-0471 displayed the highest enantioselectivity for pNSO (ee 55%, yield 35%) while Rhodotorula glutinis UOFS Y-0653 displayed the highest enantioselectivity for mNSO (ee >98%, yield 29%), oNSO (ee 39%, yield 19%) and SO (ee >98%, yield 19%). (R)-Styrene oxide was preferentially hydrolysed to the corresponding (R)-diol with retention of configuration at the stereogenic centre. In the case of the nitro substituted styrene oxides the absolute configurations of the remaining epoxides and the formed diols were not established.     

Molecular engineering of epoxide hydrolase and its application to asymmetric and enantioconvergent hydrolysis

Safety and regulatory issues favor increasing use of enantiopure compounds in pharmaceuticals. Enantiopure epoxides and diols are valuable intermediates in organic synthesis for the production of optically active pharmaceuticals. Enantiopure epoxide can be prepared using epoxide hydrolase (EH)-catalyzed asymmetric hydrolysis of its racemate. Enantioconvergent hydrolysis of racemic epoxides by EHs possessing complementary enantioselectivity and regioselectivity can lead to the formation of enantiopure vicinal diols with high yield. EHs are cofactor-independent and easy-to-use catalysts. EHs will attract much attention as commercial biocatalysts for the preparation of enantiopure epoxides and diols. In this paper, recent progress in molecular engineering of EHs is reviewed. Some examples and prospects of asymmetric and enantioconvergent hydrolysis reactions are discussed as supplements to molecular engineering to improve EH performance.     

Enantioselective epoxidation and carbon-carbon bond cleavage catalyzed by Coprinus cinereus peroxidase and myeloperoxidase

We demonstrate that myeloperoxidase (MPO) and Coprinus cinereus peroxidase (CiP) catalyze the enantioselective epoxidation of styrene and a number of substituted derivatives with a reasonable enantiomeric excess (up to 80%) and in a moderate yield. Three major differences with respect to the chloroperoxidase from Caldariomyces fumago (CPO) are observed in the reactivity of MPO and CiP toward styrene derivatives. First, in contrast to CPO, MPO and CiP produced the (S)-isomers of the epoxides in enantiomeric excess. Second, for MPO and CiP the H(2)O(2) had to be added very slowly (10 eq in 16 h) to prevent accumulation of catalytically inactive enzyme intermediates. Under these conditions, CPO hardly showed any epoxidizing activity; only with a high influx of H(2)O(2) (300 eq in 1.6 h) was epoxidation observed. Third, both MPO and CiP formed significant amounts of (substituted) benzaldehydes as side products as a consequence of C-alpha-C-beta bond cleavage of the styrene derivatives, whereas for CPO and cytochrome c peroxidase this activity is not observed. C-alpha-C-beta cleavage was the most prominent reaction catalyzed by CiP, whereas with MPO the relative amount of epoxide formed was higher. This is the first report of peroxidases catalyzing both epoxidation reactions and carbon-carbon bond cleavage. The results are discussed in terms of mechanisms involving ferryl oxygen transfer and electron transfer, respectively.     

Nonionic analogs of oligonucleotide duplexes. Calculations using a molecular mechanics methods of the effect of configuration at the asymmetrical phosphorus atom in phosphonic and triester derivatives of d(TpCH3)6, d(TpOEt)6 on the structure and stability of their complexes with dA6

Molecular mechanical calculations of the optimal structure and relative stability of duplexes of dA6 with non-ionic oligonucleotide analogs of the methylphosphonate d(TpCH3)6 and phosphotriester d(TpOEt)6 have been carried out. Duplexes of dA6 and non-ionic oligonucleotide analogs with Rp enantiomeric configuration of modified phosphorous groups in d(TpCH3) and Sp in d(TpOEt) turned out to be more stable than those with second enantiomeric configurations Sp and Rp, respectively. The main factors of energetic selectivity between Sp and Rp stereoisomers have been found to be steric strains and electrostatic interaction between asymmetric modified phosphate groups and the d-ribose 5' end. NOE generated distances between protons of alkyl substitute and d-ribose H2", H3' have been analysed. The results of the calculations are in agreement with experimental observations.     

Hepatic bile acid metabolism and expression of cytochrome P450 and related enzymes are altered in Bsep −/− mice

The bile salt export pump (BSEP/Bsep; gene symbol ABCB11/Abcb11) translocates bile salts across the hepatocyte canalicular membrane into bile in humans and mice. In humans, mutations in the ABCB11 gene cause a severe childhood liver disease known as progressive familial intrahepatic cholestasis type 2. Targeted inactivation of mouse Bsep produces milder persistent cholestasis due to detoxification of bile acids through hydroxylation and alternative transport pathways. The purpose of the present study was to determine whether functional expression of hepatic cytochrome P450 (CYP) and microsomal epoxide hydrolase (mEH) is altered by Bsep inactivation in mice and whether bile acids regulate CYP and mEH expression in Bsep ^?/? mice. CYP expression was determined by measuring protein levels of Cyp2b, Cyp2c and Cyp3a enzymes and CYP-mediated activities including lithocholic acid hydroxylation, testosterone hydroxylation and alkoxyresorufin O-dealkylation in hepatic microsomes prepared from female and male Bsep ^?/? mice fed a normal or cholic acid (CA)-enriched diet. The results indicated that hepatic lithocholic acid hydroxylation was catalyzed by Cyp3a/Cyp3a11 enzymes in Bsep ^?/? mice and that 3-ketocholanoic acid and murideoxycholic acid were major metabolites. CA feeding of Bsep ^?/? mice increased hepatic Cyp3a11 protein levels and Cyp3a11-mediated testosterone 2β-, 6β-, and 15β-hydroxylation activities, increased Cyp2b10 protein levels and Cyp2b10-mediated benzyloxyresorufin O-debenzylation activity, and elevated Cyp2c29 and mEH protein levels. We propose that bile acids upregulate expression of hepatic Cyp3a11, Cyp2b10, Cyp2c29 and mEH in Bsep ^?/? mice and that Cyp3a11 and multidrug resistance-1 P-glycoproteins (Mdr1a/1b) are vital components of two distinct pathways utilized by mouse hepatocytes to expel bile acids.     

Oxidation of propene and 1-butene byMethylococcus capsulatus andMethylosinus trichosporium

Summary Methane-grown cells ofMethylococcus capsulatus andMethylosinus trichosporium readily oxidized propene and various isomers of butene to their respective epoxides. When examined in a proton NMR spectrum using tris([3-trifluoromethylhydroxymethylene]-d-camphorato), europium III derivative as an optically active chemical shift reagent, the products propylene oxide and 1,2-epoxybutane were found to contain equal amounts of both isomers. Methane-grown cells of both bacteria had considerable levels of reducing equivalents to catalyze the epoxidation of gaseous olefins. Cells depleted of reductants catalyzed the oxidation in the presence of low levels of methanol or formaldehyde with a stoichiometry of about 2:1. The rates of epoxidation of propene and 1-butene in a continuous reactor were 2–3-times that of a batch-wise reaction; the epoxidation activity, however, was lost within 3 h. The inactivation was attributed to the reactivity of the accumulated epoxides in the reactor. Propene and 1-butene oxidation by both bacteria were drastically inhibited by the respective products. Thus, the major problem in the application of microorganisms for production of epoxides from gaseous olefins is the rapid separation of the reactive products.     

A high-performance liquid chromatographic method for the enantiomeric analysis of the enzymatically synthesized coenzyme A ester of 2-tetradecylglycidic acid

The enantiomeric composition of an enzymatically synthesized sample of the coenzyme A ester of 2-tetradecylglycidic acid (TDGA-CoA) was determined by the use of high-performance liquid chromatography with a chiral stationary phase. The stationary phase was commercially available and consisted of (R)-N-(3,5-dinitrobenzoyl)phenylglycine covalently bonded to aminopropyl silica gel. Analysis was performed using the phenacyl derivative of 2-tetradecylglycidic acid (TDGA), obtained by mild hydrolysis of the TDGA-CoA followed by reaction of the extracted TDGA with phenacyl chloride. Chromatography showed the enantiomeric purity of TDGA-CoA, synthesized in a rat liver microsomal enzyme mixture over a 2-h period, to be a 15.6:1 ratio of the R:S enantiomers (88% ee). The result demonstrates the steroselectivity of the long-chain fatty acid-coenzyme A synthetase for chiral fatty acid epoxide, TDGA.