CHIRALITY 417S184 (1992) Enantio- and Regioselectivity in the Epoxide-Hydrolase-CatalyzedR ing Opening of Simple Aliphatic Oxiranes: Part I: Monoalkylsubstituted

Oxiranes D. WISTUBA AND V. SCHURIG Institut fur Organische Chemie der Universitat, Tubingen, Germany ABSTRACT The in vitro conversion of chiral aliphatic monoalkylsubstituted oxiranes into 12-diols catalyzed by epoxide hydrolase of rat liver microsomes occurs with substrate enantioselectivity and regioselectivity. Substrate enantioselectivity is generally low, and has the same sense, for methyloxirane, vinyloxirane, epichloro-, and epibromohydrin. In the hydrolysis of t-butyloxirane inhibitory effects are involved leading to a complex pattern of enantioselectivity. All investigated monosubstituted aliphatic oxiranes are hydrolyzed with high regioselectivity by nucleophilic attack of water at the unsubstituted ring carbon atom. The enantiomeric excess of the unreacted oxirane substrates and the diol metabolites formed were determined by complexation and inclusion gas chromatography. KEY WORDS: enantioselective hydrolysis, regioselective hydrolysis, epoxide hydrolase, subo 1992 Wiley-Liss, Inc. strate enantioselectivity INTRODUCTION Oxiranes are reactive initial metabolites in the cytochrome P-450-catalyzed biotransformation of xenobiotics (alkenes and arenes), L2 possessing high alkylating potential. In the detoxication of these electrophilic metabolites, microsomal epoxide hydrolase (mEH)4,5 and glutathione S-transferase (GST)6 play important key roles. Like most enzymes, cytochrome P-450, mEH and GST may function as inherently chiral catalysts. While, according to Scheme I, the epoxidation of the alkene by cytochrome P-450 represents a prochiral recognition process (product enantioselectivity), the subsequent oxirane transformation is a chiral recognition process (kinetic resolution, substrate enantiosekctivity). The striking differences in the biological activities between oxirane enantiomers (e.g., benzo[a]pyrene 7,8-0xide,~ph enyloxiraneg) underline the importance of studies devoted to the determination of enantioselectivities in the formation and transformation of epoxides catalyzed by enzymes. The oxirane hydrolysis catalyzed by mEH occurs by nucleophilic attack of water on the oxirane carbon atom to form vicinal diols. 4,10 Studies with l80-labeled oxiranes or with H2 l80 have shown that the enzyme-catalyzed hydrolysis also proceeds with a high degree of regioselectivity favoring the attack by water on the less sterically hindered oxirane carbon atom. 11,12 mEH possesses both substrate enantioselectivity in the hydrolysis of racemic alicyclic oxiranes [e.g., (E/Z)-3-, or 4-t-butyl-1,2-epoxycyclohexane,1(3E~)1-44 ,5-dimethyl-1,2-epoxycyclohexane, l 5 (E/Z)-3-bromo-1,2-epoxycyclohexaln6e3l 7], polycyclic aromatic oxiranes (e.g., benzo[aEpyrene-4,5-oxid1e8 ), phenyloxirane 19-21 and alkylsubstituted aliphatic oxiranes 7,22), and product enantioselectivity in the hydrolysis of Presented at the Second International Symposium on Chiral Discrimination, May 27-31, 1991, Rome, Italy. @ 1992 Wiley-Liss, Inc. OH /

_. JR Cyt. P-450 [OI Conjugation at Glutathione Scheme I. Mechanistic pathway of alkene-alkene oxide (oxiranebalkane diol metabolism (only one enantiomer is shown). meso-substrates such as epoxycyclohexane l2 and cis-2,3-diphenyloxirane. 21,23 In a previous investigation we found a complete substrate enantioselectivity and regioselectivity in the epoxidehydrolase-catalyzed ring opening of the simplest chiral cis-dialkylsubstituted oxirane, i.e., cis-2-ethyl-3-methyloxirane. The present paper reports on stereochemical and mechanistic aspects of the epoxide-hydrolase-catalyzedh ydrolysis of simple aliphatic monosubstituted oxiranes using rat liver microsomes. As a convenient analytical tool, complexation gas Received for publication June 14, 1991; accepted January 2, 1992. Address reprint requests to Prof. Dr. V. Schurig, Auf der Morgenstelle 18, D-7400 Tubingen, Germany. ENANTIO- AND REGIOSELECTIVE OXIRANE HYDROLYSIS 179 chromatography, 24 enabled time-dependent measurements in the nanogram concentration range for the determination of the enantiomeric excess and the absolute configuration of aliphatic oxiranes (without derivatization) and of the corresponding diols (after derivatization with acetone or n-butylboronic acid 25). In addition, gas chromatographic analysis of underivatized diols was carried out on permethylated p-cyclodextrin. 26 MATERIALS AND METHODS Materials Methyloxirane 1, vinyloxirane 2, t-butyloxirane 3, epichlorohydrin 4, and epibromohydrin 5 were obtained from Aldrich (Steinheim, F.R.G.). (R)-Methyloxirane was prepared from (S)-alanine,2 7 (S)-methyloxirane was prepared from @)-ethyl lactate, 28 (R)-t-butyloxirane was prepared from 1hydroxy-3,3-dimethylbutan-2-onveia (2R)-3,3-dimethylbutane12-diol,2 9 and (S)-vinyloxirane, (S)-epichlorohydrin3,1 and (R)-3-bromopropane-1,2-di0w1~er~e prepared from 2,3-isopropy lidene-(R)-gyl ceraldehyd e. 33 Chiral Gas Chromatography Complexation gas chromatography on chiral nickel(I1) bis-~helates~~ The enantiomers of methyloxirane 1, vinyloxirane 2, t-butyloxirane 3, epichloroh ydrin 4, and epibromohydrin 5 were separated on a 25 m x 0.25 mm glass or fused silica capillary column coated with nickel(I1) bis[(3-heptafluorobutanoyl)-(1R)camphoratela in OV 101 or SE 30. The enantiomers of propane1,2-diol,3-butene-1,2-di3o-lc,h loropropane-1,2-diola,n d 3bromopropane-1,2-diolw ere separated after derivatization with acetone 25 on a 36 m x 0.25 mm glass capillary column or a 25 m x 0.25 mm fused silica capillary column coated with nickel(11) bis[(3-heptafluorobutanoyl)-(lR,5S)-pinan-4-0natien] ~O V 101 or SE 30 or after derivatization with n-butylboronic acidz on a 40 m x 0.25 mm glass capillary column coated with nickel(I1) bis[(3-hepatfluorobutanoyl)-(1R)-camphorate] at 6090C (carrier gas: high-purity grade N2). The stereochemistry of the oxiranes and diols has been determined by coinjection of

the optically active oxiranes or diols with known absolute configuration on the respective chiral columns. 25,34 Inclusion gas chromatography on pennethylated p-cyclodextrin 35 The enantiomers of underivatized 3,3-dimethylbutane-1,2diol were separated on a 25 m x 0.25 mm fused silica capillary column coated with 10% heptakis(2,3,6-tri-O-methyl-P-cyclodextrin) in OV 1701 at 90C (carrier gas: H2).T he stereochemistry has been determined by coinjection of (2R)-3,3-dimethylbutanel,2-diol. Liver Microsomes Rat liver microsomes were kindly provided by Prof. A. Wendel (Institute of Biology, University of Konstanz, Germany) with induction carried out as follows: phenobarbital (1 ml/mg) was administered in the drinking water of male Wistar rats for 5 days. The rats were sacrificed 1 day later and the liver microsomes were obtained as described previously. 36 Incubations The reaction mixture (0.5 ml when oxiranes were investigated and 5 ml when diols were screened) containing rat liver microsomes (1 mg protein/ml) and 0.15 M phosphate buffer, pH 7.4 was incubated 5 min at 37C, than oxirane [methyloxirane 1, 4 or 2 mM, respectively; (R)- and (S)-methyloxirane, 2 mM; vinyloxirane 2,4 mM; t-butyloxirane 3,4 mM, epichlorohydrin 4, 15 mM, epibromohydrin 5, 10 or 15 mM, respectively], and standard (acetone or 3-methylbutan-2-one) were added. For the GLC determination of diols the reaction mixture was cooled with liquid nitrogen (to freeze the aqueous phase) and extracted six times with diethyl ether. The organic layer was dried over Na2SO4. After evaporation of the organic phase, the residue was dissolved in 500 11 acetone and 1 mg p-toluene sulfonic acid was added to form diol acetonides. The mixture was allowed to stand for 20 min and was than cooled in an ice bath and subsequently 500 pl n-hexane and 1 ml water were added. After vigorous shaking, the organic phase was separated, dried over Na2S04, and analyzed by complexation gas chromatography. For derivatization with n-butylboronic acid, the residue obtained after extraction and evaporation was redissolved in 500 11 diethyl ether and 1 mg n-butylboronic acid was added. After standing for 10 min the solution was analyzed. For the enantiomer analysis of 3,3-dimethyl-butane-1,2-diol, the diethyl ether phase after extraction of the incubation mixture was used. The chemical yield of 3-bromopropane-1,2-diol was determined by the method of enantiomer labeZ~ng.T~h~e- c~o~m bined diethyl ether extracts (see above) were separated in two equal parts. A known amount of synthetic (2R)-3-bromopropane-1,2diol (ee > 99.5%) was added to one-half. Both halves were derivatized with acetone (see above) and analyzed by GLC. The calculation of the diol amount was carried out according to Blair et al.37 The percentages of unreacted oxirane enantiomers were determined during the incubation in intervals of 520 min by complexation gas chromatography via the head-space technique7~ 39~a4n0d refers to the mean of at least six different

incubations. Control experiments established that the enantiomeric composition in the gas phase corresponded to that in solution. The percentages of the formed diol metabolite enantiomers were determined by complexation gas chromatography or by inclusion gas chromatography at least for three repeated incubation experiments. All measurements of one sample were carried out at least three times (overall deviation of the enantiomeric composition for repeated incubation experiments: O.!X%). RESULTS AND DISCUSSION The influence of different alkyl groups on the enantiosekctidy and regiosekctiuity in the epoxide-hydrolase-catalyzedh ydrolysis of five racemic monoalkylsubstituted oxiranes (methyloxirane 1, vinyloxirane 2, t-butyloxirane 3, epichlorohydrin 4, and epibromohydrin 5) was investigated. 12345 The time-dependent chiral analysis of the substrates during the enzymatic hydrolysis revealed the propensity of the en180 WISTUBA AND SCHURIG Incubatiou time: 10 min Standard r 20 min 30 min S 40 min 50 min L,I-I 048048048048048 t tminl t Iminl t [minl t Iminl t Iminl Fig. 1. Analysis of the kinetic resolution of yac epichlorohydrin 4 catalyzed by mEH. Standard 3methylbutan-2-one (25 m x 0.25 mm glass capillary column coated with 0.08 m Ni(I1) bis[(3-heptafluorobutanoyl)(ZR)-camphoratei]n OV 101,80"C, 1 bar Nz. zyme system to discriminate between the oxirane enantiomers (substrate enuntiosekctivity, chid recognition, kinetic resolution). It was found that (S)-methyloxirane(, S)-vinyloxirane(,R )epichloro-,a nd (R)-epibromohydrinp, ossessing the same relative sterahemistry,* were preferentially hydrolyzed (see Figs. 1,2, and 3). In contrast, (R)-t-butyloxirane was consumed preferentially in the first stage of the reaction. After approx. 90% conversion of (R)-t-butyloxiraneh, owever, the hydrolysis of the (S)-enantiomer occurs at a faster rate, as that of the @)-enantiomer in the first part of the reaction (see Fig. 2). Similar results were found in the hydrolysis of isopropenyloxirane, 41 one of the primary metabolites of isoprene. In the case of t-butyloxirane, Bellucci et al. made the same observation by using rabbit mEH and kinetic analysis of the diol formed, but they did not report on the kinetic course of the unchanged oxirane enantiomers." Similar results were obtained in the hydrolysis of phenyloxiranem and of p-nitrophenyloxirane4 2 and was explained by inhibitory effects of the (R)-enantiomersp, ossessing a higher affinity for the mEH active site toward the (S)-enantiomer. All these substrates show a typical biphasic shapem of their kinetic profile during the enzymatic hydrolysis. 20,22*42 The enantioselective hydrolysis of t-butyloxirane (see Fig. 4)

and of isopropenyloxirane is a new example of this phenomenon. As shown in Figure 4, methyloxirane 1 was hydrolyzed without inhibition by its enantiomers. The kinetic course for *Formal change of the descriptor caused by the priority rule of Cahn, Ingold, and Prelog. the (R)- and (S)-enantiomersw as independent from the hydrolysis of the substrate employed as racemic mixture, or as the individual (R)- or (S)-enantiomers. For vinyloxirane 2 also no biphasic kinetic come of substrate hydrolysis was observed and, consequently, no competitive substrate inhibition occurred. The time dependence of the substrate conversion of epichloro4 and epibromohydrin 5 showed, at a decreasing concentration of the (R)-enantiomera, n increase of the reaction rate pnol oxirane 50 60 Incubation time [minl 10 20 30 40 Fig. 2. Substrate enantioselective hydrolysis of rac methyloxirane 1 (H) and rac t-butyloxirane 3 (0)ca talyzed by mEH. ENANTIO- AND REGIOSELECTIVE OXIRANE HYDROLYSIS 181 mEH than 1-3 and the medium substrate enantioselectivity is almost identical for both. Thus, the kinetic resolution is not influenced by the nature of the halogen atom. For all oxiranes studied, the enantiomer possessing an alkyl substituent right in front or left behind of the oxirane ring oriented in the paper plane with the oxygen atom on the top was hydrolyzed preferentially. In the case of t-butyloxirane 3 this observation applies only for the hydrolysis of the individual enantiomers and not for the racemate due to inhibitory effect of the (R)-enantiomer, possessing a higher affinity for the mEH active site. Contrary to our results on 1-5, Ekllucci et al.,22 using rabbit liver microsomes, found a small positive optical rotation for hexane-l,Zdiol, the metabolite of n-butyloxirane, which agrees with a preferential hydrolysis of the (R)-oxirane. Fig. 3. Substrate enantioselective hydrolysis of YUC epichlorohydrin 4 catalyzed by mEH. Fig. 4. Time course of the mEH-catalyzed ring opening of (R) (a)(,S -)( O), and YUC (0m)eth yloxirane 1 (0su m of the enantiomers, measured separately and coinciding with and 0)an d YUC t-butyloxirane (+). Substrate concentration: MC methyloxirane 1,2 mM; (S)-methyloxirane, 2 mM; (R)-methyloxirane, 2 mM; t-butyloxirane, 4 m"d, of the (S)-enantiomer (see Fig. 3). Accordingly, a weak competitive inhibitory effect must be involved. While methyloxirane 1 and vinyloxirane 2 were hydrolyzed with nearly the same, but low substrate enantiosehctivity, a nearly complete kinetic resolution [N 5% (R)- and N 95% (S)-t-butyloxirane] was found for t-butyloxirane at about 50% conversion. In agreement with our results, Ekllucci et al. also found only a slight substrate enantioselectivity in the case of substituted oxiranes containing unbranched alkyl groups like n-butyl or n-octyloxirane. The oxirane halides, epichlorohydrin 4 and epibromohydrin 5 represent much better substrates for If the two enantiomers of a racemic substrate (oxirane) are consumed at different rates (kinetic resolution), the enantiomeric excess of the formed metabolite (diol) is time-dependent

and thus changes with the percent of conversion (see Table 1). In the case of propane-12-diol and 3-butene-1,2-diol, the metabolites of methyloxirane 1 and vinyloxirane 2, respectively, the excess enantiomers obtained during the enzymatic hydrolysis were of the (S)-configurationB. y analogy, the formation of (R)-3-chloropropane-1,2-diol(,R )-3-bromopropane-l,2diol, and (R)-3,3-dimethylbutane-1,2-di(osle e Fig. 5) occurs preferentially. After complete substrate conversion the corresponding diols were obtained racemic in each case (see Table 1). Hydrolysis of monoalkylsubstituted oxiranes may proceed (x) at the unsubstituted ring carbon atom (3) with retention of configuration at (2) or 01) at the alkylsubstituted ring carbon atom (2) with inversion of configuration at (2). Retention C- (x) H,O H,O (y) - Inversion From the results cited above, the ring opening of methyloxirane 1, vinyloxirane 2, t-butyloxirane 3, epichlorohydrin 4, and epibromohydrin 5 occurs with retention of configuration. Thus, as expected, nucleophilic ring opening takes place at the less hindered, unsubstituted oxirane carbon atom. This mechanism was corroborated by use of the enantiomerically pure oxiranes 1 4 and the determination of the absolute configuration of the diol formed, or by comparing the time-dependent course of the excess enantiomers of the unchanged substrate and the diol formed. The enantiomeric excess (ee) of the individual oxirane enantiomers at the beginning of the enzymatic conversion was not identical with the ee of the corresponding diol formed after complete substrate consumption (see Table 2). 182 WISTUBA AND SCHURIG TABLE 1. Time dependence of the enantiomeric composition of the diol metabolite during the mEH-catalyzed hydrolysis Substrate ~ ~~ Enantiomeric composition Substrate conc. Incubation time Metabolite (mM) (min) ( R W ) (%YO) Methyloxirane 1 Propane-1,2-diol 4 Vinyloxirane 2 3-Butene-1,2-diol 4 t-Butyloxirane 3 3,3-Dimethyl-butane-1,2-diol 4 Epichlorohydrin 4 3-Chloropropane-1,2-diol 15 Epibromohydrin 5 3-Bromopropane-1,2-diol 10 15 30 40 60 10 20 30 70 25 35 45 55 65 10

20 30 40 50 60 10 20 30 40 45.9 47.8 49.4 49.9 40.7 43.5 46.5 50.0 96.0 95.3 93.9 85.4 49.9 72.4 71.7 67.5 61.7 52.5 50.0 75.9 66.8 58.8 50.7 54.1 52.2 50.6 50.1 59.3 56.5 53.5 50.0 4.0 4.7 6.1 14.6 50.1 27.6 28.3 32.5 38.3 47.5 50.0 24.1 33.2 41.2 49.3 Incubation

time I 35 niin R 50 min 70 min h I_. 0 5 10 rnin 0 5 10 rnin 0 5 10 min * Fig. 5. Analysis of the enantiomeric composition of 3,3-dimethylbutane-l,2-didoulr ing the mEHcatalyzed hydrolysis of t-butyloxirane 3 (25 m x 0.25 mm fused silica capillary coated with permethylated Pcyclodextrin in OV 1701, ENANTIO- AND REGIOSELECTNE OXIRANE HYDROLYSIS 183 TABLE 2. Comparison of the enantiomeric composition of some optical active oxiranes and the corresponding diols after complete substrate conversion Substrate (RX% ) (S)(/) product (RW) (.%YO) (R)-Methyloxirane 98.6 1.4 (ZR)-Propane-1,2-dioI 96.6 3.4 (S)-Methyloxirane 0.7 99.3 (ZS)-Propane-1,2-dioI 2.5 97.5 (R)-t-Butyloxirane 99.7 0.3 (ZR)-3,3-Dirnethylbutane-1,2-diol 99.8 0.2 (9-Vinyloxirane 1.1 98.9 (ZS)-3-Butene1-, 2-diol 4.8 95.2 (3-Epichlorohydrin 5.0 95.0 (2S)-3-Chloropropane-1,2-diol 7.5 92.5 The enantiomeric composition of the oxiranes was determined without derivatization, that of (2R)-an d (ZS)-propane-l,Z-dioI after derivatization with n-butyl boronic acid, and that of (ZS)-3-butene-l, Z-diol and (ZS)-3-chloropropane-l,2-diaoflte r derivatization with acetone by complexation gas chromatographyB and of underivatized (R)-3,3-dimethylbutane-l,Z-diboyl inclusion gas chromatography.26 This effect can be used as an alternative method to 0-labeling experiments for the quantitative determination of the regzoselectiuity of the epoxide-hydrolase-catalyzed water attack on the condition that no uncatalyzed hydrolysis takes place, complete substrate conversion is involved, and the (R)- and the (S)-oxirane enantiomer must be consumed with the same regioselectivity [exception: enantiomerically pure enantiomers (ee = l000/)]. By control experiments without microsomes, or with microsomes heated to 100C, and determination of the amount of oxiranes by GLC using an internal standard, nonenzymatic hydrolysis under the enzymatic incubation conditions was excluded. If the investigated racemic monoalkyl substituted oxiranes form racemic diols after complete consumption, the ratio of water attack (x/y) (see Scheme 11) at the unsubstituted and the alkylsubstituted ring carbon atom must be the same for the (R)- and the (S)-enantiomer. It follows from the results of Table 2 that the water attack takes place with high regioselectivity (for methyloxirane 1 with 98 f 0.2%, vinyloxirane 2 with 96.3 f 0.3%, and for epichlorohydrin 4 with 97.2 f 0.3%) at the unsubstituted ring carbon atom. In the case of t-butyloxirane 3 a completely regioselective behavior was found (see Table 2) [(R)-t-butyloxirane shows an enantiomeric excess of 99.48fOo.10%a nd the formed metabolite (2R)-3,3-dimethyl-butane-1,2-diaoln ee of 99.55 * O.lOo/o after complete substrate conversion]. Similar high degrees of regioselectivity (> 96%) were reported in the mEH-catalyzed hydrolysis of some monoaryl- and

pyridylsubstituted oxiranes. ,12 OH X @ HOCH, +#..,,, R .\ # (S)-diol 4 0 # 4 - .R H \ . .? / J ,,q (S)-oxirone . (R)-oxirone (R)-diol Sdiol = x . Sozirane+ Y . Rorirenc Ildiol = X Rozimne + Y Soriranc Scheme II. Stereochemical pathway of oxirane hydrolysis. CONCLUSION Simple chiral monosubstituted aliphatic oxiranes are hydrolyzed with modest substrate enantioselectiuily by mEH of rat liver in vitro to 1,Zdiols. In the case of oxiranes with unbranched alkyl substituents it was found that the preferentially hydrolyzed enantiomers possess the same relative stereochemistry. In contrast, t-butyloxirane shows an opposite sense of enantioselectivity. The ring opening of the oxiranes investigated occurs with high regioselectiuity (9&1000/,). Water attack takes place at the sterically less hindered, unsubstituted oxirane carbon atom. ACKNOWLEDGMENTS The authors wish to thank Professor Wendel (Konstanz, Germany) for the supply of microsomes. The support of this work by Deutsche Forschungsgemeinschaft and Fonds der chemischen Industrie is gratefully acknowledged. LITERATURE CITED 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. Maynert, E.W., Foreman, R.L., Watabe, T. Epoxides as obligatory intermediates in metabolism of olefins to glycols. J.Biol.Chem. 245:5234-5238, 1970. Jerina, D.M., Daly, J.W., Arene oxides: A new aspect of drug metabolism. Science 185:575-582, 1974. Ehrenberg, L., Hussain, S. Genetic toxicity of some important epoxides. Mut.Res. 86:l-113, 1981. Oesch, F. Mammalian epoxide hydrases: Inducible enzymes catalyzing the inactivation of carcinogenic and cytotoxic metabolites derived from aromatic and olefinic compounds. Xenobiotica 3305340, 1973. Seidegard, J., DePieme, J.W. Microsomal epoxide hydrolase. Properties, regulation and function. Biochim.Biophys.Acta 695251-270, 1983.

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