CHIRALITY 417S184 (1992)

7/29/2019 CHIRALITY 417S184 (1992)

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CHIRALITY 417S184 (1992)

Enantio- and Regioselectivity in theEpoxide-Hydrolase-CatalyzedR ing Opening of Simple Aliphatic Oxiranes:Part I: Monoalkylsubstituted OxiranesD. WISTUBA AND V. SCHURIGInstitut fur Organische Chemie der Universitat, Tubingen, Germany

ABSTRACT The in vitro conversion of chiral aliphatic monoalkylsubstituted oxiranesinto 12-diols catalyzed by epoxide hydrolase of rat liver microsomes occurs with substrateenantioselectivity and regioselectivity. Substrate enantioselectivity is generally low, and has thesame sense, for methyloxirane, vinyloxirane, epichloro-, and epibromohydrin. In the hydrolysisof t-butyloxirane inhibitory effects are involved leading to a complex pattern of enantioselectivity.

All investigated monosubstituted aliphatic oxiranes are hydrolyzed with high regioselectivityby nucleophilic attack of water at the unsubstituted ring carbon atom. The enantiomericexcess of the unreacted oxirane substrates and the diol metabolites formed were determinedby complexation and inclusion gas chromatography.KEY WORDS: enantioselective hydrolysis, regioselective hydrolysis, epoxide hydrolase, subo1992 Wiley-Liss, Inc.strate enantioselectivityINTRODUCTIONOxiranes are reactive initial metabolites in the cytochromeP-450-catalyzed biotransformation of xenobiotics (alkenes andarenes), L2 possessing high alkylating potential. In the detoxicationof these electrophilic metabolites, microsomal epoxidehydrolase (mEH)4,5 and glutathione S-transferase (GST)6 playimportant 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 transformationis a chiral recognition process (kinetic resolution, substrateenantiosekctivity).The striking differences in the biological activities betweenoxirane enantiomers (e.g., benzo[a]pyrene 7,8-0xide,~ph enyloxiraneg)underline the importance of studies devoted to the determinationof enantioselectivities in the formation and transformationof epoxides catalyzed by enzymes.The oxirane hydrolysis catalyzed by mEH occurs by nucleophilicattack of water on the oxirane carbon atom to form

vicinal diols. 4,10 Studies with l80-labeled oxiranes or withH2 l80 have shown that the enzyme-catalyzed hydrolysis alsoproceeds with a high degree of regioselectivity favoring theattack by water on the less sterically hindered oxirane carbonatom. 11,12 mEH possesses both substrate enantioselectivity inthe 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], polycyclicaromatic oxiranes (e.g., benzo[aEpyrene-4,5-oxid1e8 ),phenyloxirane 19-21 and alkylsubstituted aliphatic oxiranes7,22), 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/

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_. JR Cyt. P-450[OIConjugationatGlutathioneScheme I. Mechanistic pathway of alkene-alkene oxide (oxiranebalkanediol 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 completesubstrate enantioselectivity and regioselectivity in the epoxide-hydrolase-catalyzed ring opening of the simplest chiralcis-dialkylsubstituted oxirane, i.e., cis-2-ethyl-3-methyloxirane.The present paper reports on stereochemical and mechanisticaspects of the epoxide-hydrolase-catalyzedh ydrolysis of simple aliphatic monosubstituted oxiranes using rat liver microsomes.

As a convenient analytical tool, complexation gasReceived 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 179chromatography, 24 enabled time-dependent measurements inthe nanogram concentration range for the determination of theenantiomeric excess and the absolute configuration of aliphaticoxiranes (without derivatization) and of the correspondingdiols (after derivatization with acetone or n-butylboronicacid 25). In addition, gas chromatographic analysis of underivatizeddiols was carried out on permethylated p-cyclodextrin. 26MATERIALS AND METHODSMaterialsMethyloxirane 1, vinyloxirane 2, t-butyloxirane 3, epichlorohydrin4, and epibromohydrin 5 were obtained from

Aldrich (Steinheim, F.R.G.). (R)-Methyloxirane was preparedfrom (S)-alanine,2 7 (S)-methyloxirane was prepared from@)-ethyl lactate, 28 (R)-t-butyloxirane was prepared from 1-hydroxy-3,3-dimethylbutan-2-onveia (2R)-3,3-dimethylbutane-12-diol,2 9 and (S)-vinyloxirane, (S)-epichlorohydrin3,1 and(R)-3-bromopropane-1,2-di0w1~er~e prepared from 2,3-isopropylidene-(R)-gyl ceraldehyd e. 33Chiral Gas ChromatographyComplexation gas chromatography on chiral nickel(I1)

bis-~helates~~The enantiomers of methyloxirane 1, vinyloxirane 2, t-butyloxirane3, epichloroh ydrin 4, and epibromohydrin 5 were separatedon a 25 m x 0.25 mm glass or fused silica capillarycolumn coated with nickel(I1) bis[(3-heptafluorobutanoyl)-(1R)-camphoratela in OV 101 or SE 30. The enantiomers of propane-1,2-diol,3-butene-1,2-di3o-lc,h loropropane-1,2-diola,n d 3-bromopropane-1,2-diolw ere separated after derivatization withacetone 25 on a 36 m x 0.25 mm glass capillary column or a 25m 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 acidzon a 40 m x 0.25 mm glass capillary column coated withnickel(I1) bis[(3-hepatfluorobutanoyl)-(1R)-camphorate] at 60-90°C (carrier gas: high-purity grade N2). The stereochemistryof the oxiranes and diols has been determined by coinjection of

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the optically active oxiranes or diols with known absoluteconfiguration on the respective chiral columns. 25,34Inclusion gas chromatography on pennethylatedp-cyclodextrin 35The enantiomers of underivatized 3,3-dimethylbutane-1,2-diol were separated on a 25 m x 0.25 mm fused silica capillarycolumn coated with 10% heptakis(2,3,6-tri-O-methyl-P-cyclodextrin)in OV 1701 at 90°C (carrier gas: H2).T he stereochemistry

has been determined by coinjection of (2R)-3,3-dimethylbutane-l,2-diol.Liver MicrosomesRat 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. 36IncubationsThe 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, pH7.4 was incubated 5 min at 37”C, than oxirane [methyloxirane1, 4 or 2 mM, respectively; (R)- and (S)-methyloxirane, 2 mM;vinyloxirane 2,4 mM; t-butyloxirane 3,4 mM, epichlorohydrin4, 15 mM, epibromohydrin 5, 10 or 15 mM, respectively], andstandard (acetone or 3-methylbutan-2-one) were added.For the GLC determination of diols the reaction mixture wascooled with liquid nitrogen (to freeze the aqueous phase) andextracted six times with diethyl ether. The organic layer wasdried over Na2SO4. After evaporation of the organic phase, theresidue was dissolved in 500 11 acetone and 1 mg p-toluenesulfonic acid was added to form diol acetonides. The mixturewas allowed to stand for 20 min and was than cooled in an icebath and subsequently 500 pl n-hexane and 1 ml water wereadded. After vigorous shaking, the organic phase was separated,dried over Na2S04, and analyzed by complexation gaschromatography. For derivatization with n-butylboronic acid,the residue obtained after extraction and evaporation wasredissolved in 500 11 diethyl ether and 1 mg n-butylboronicacid 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 mixturewas used.The chemical yield of 3-bromopropane-1,2-diol was determinedby the method of enantiomer labeZ~ng.T~h~e- c~o~m bineddiethyl ether extracts (see above) were separated in two equalparts. A known amount of synthetic (2R)-3-bromopropane-1,2-diol (ee > 99.5%) was added to one-half. Both halves werederivatized with acetone (see above) and analyzed by GLC. Thecalculation of the diol amount was carried out according to

Blair et al.37The percentages of unreacted oxirane enantiomers were determinedduring the incubation in intervals of 520 min bycomplexation gas chromatography via the head-space technique7~39~a4n0d refers to the mean of at least six different

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incubations. Control experiments established that the enantiomericcomposition in the gas phase corresponded to that insolution. The percentages of the formed diol metabolite enantiomerswere determined by complexation gas chromatographyor by inclusion gas chromatography at least for three repeatedincubation experiments. All measurements of one sample werecarried out at least three times (overall deviation of the enantiomericcomposition for repeated incubation experiments:

O.!X%).RESULTS AND DISCUSSIONThe influence of different alkyl groups on the enantiosekctidyand regiosekctiuity in the epoxide-hydrolase-catalyzedh ydrolysisof five racemic monoalkylsubstituted oxiranes (methyloxirane1, vinyloxirane 2, t-butyloxirane 3,epichlorohydrin 4, and epibromohydrin 5) was investigated.1 2 3 4 5The time-dependent chiral analysis of the substrates duringthe enzymatic hydrolysis revealed the propensity of the en180WISTUBA AND SCHURIG

Incubatioutime: 10 minStandardr 20 min 30 minS40 min 50 minL , I - I0 4 8 0 4 8 0 4 8 0 4 8 0 4 8t tminl t Iminl t [minl t Iminl t IminlFig. 1. Analysis of the kinetic resolution of yac epichlorohydrin 4 catalyzed by mEH. Standard 3-methylbutan-2-one(25 m x 0.25 mm glass capillary column coated with 0.08 m Ni(I1) bis[(3-heptafluorobutanoyl)-(ZR)-camphoratei]n OV101,80"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 relativesterahemistry,* 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 resultswere 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 rabbitmEH and kinetic analysis of the diol formed, but they did notreport 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, ossessinga 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*42The enantioselective hydrolysis of t-butyloxirane (see Fig. 4)

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and of isopropenyloxirane is a new example of this phenomenon. As shown in Figure 4, methyloxirane 1 was hydrolyzedwithout 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 hydrolysisof the substrate employed as racemic mixture, or as theindividual (R)- or (S)-enantiomers. For vinyloxirane 2 also no

biphasic kinetic come of substrate hydrolysis was observedand, consequently, no competitive substrate inhibition occurred.The time dependence of the substrate conversion of epichloro-4 and epibromohydrin 5 showed, at a decreasing concentrationof the (R)-enantiomera, n increase of the reaction ratepnoloxirane50 60Incubation time [minl10 20 30 40Fig. 2. Substrate enantioselective hydrolysis of rac methyloxirane 1 (H)

and rac t-butyloxirane 3 (0)ca talyzed by mEH.ENANTIO- AND REGIOSELECTIVE OXIRANE HYDROLYSIS 181mEH than 1-3 and the medium substrate enantioselectivity isalmost identical for both. Thus, the kinetic resolution is notinfluenced by the nature of the halogen atom.For all oxiranes studied, the enantiomer possessing an alkylsubstituent right in front or left behind of the oxirane ringoriented in the paper plane with the oxygen atom on the topwas hydrolyzed preferentially. In the case of t-butyloxirane 3this observation applies only for the hydrolysis of the individualenantiomers and not for the racemate due to inhibitoryeffect of the (R)-enantiomer, possessing a higher affinity for themEH active site. Contrary to our results on 1-5, Ekllucci etal.,22 using rabbit liver microsomes, found a small positiveoptical 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 catalyzedby 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 separatelyand 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 competitiveinhibitory effect must be involved.While methyloxirane 1 and vinyloxirane 2 were hydrolyzedwith nearly the same, but low substrate enantiosehctivity, anearly 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. alsofound only a slight substrate enantioselectivity in the case of substituted oxiranes containing unbranched alkyl groups like

n-butyl or n-octyloxirane. The oxirane halides, epichlorohydrin4 and epibromohydrin 5 represent much better substrates for If the two enantiomers of a racemic substrate (oxirane) areconsumed at different rates (kinetic resolution), the enantiomericexcess of the formed metabolite (diol) is time-dependent

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and thus changes with the percent of conversion (see Table 1).In the case of propane-12-diol and 3-butene-1,2-diol, themetabolites of methyloxirane 1 and vinyloxirane 2, respectively,the excess enantiomers obtained during the enzymatichydrolysis were of the (S)-configurationB. y analogy, the formationof (R)-3-chloropropane-1,2-diol(,R )-3-bromopropane-l,2-diol, and (R)-3,3-dimethylbutane-1,2-di(osle e Fig. 5) occurspreferentially. After complete substrate conversion the corresponding

diols were obtained racemic in each case (seeTable 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 carbonatom (2) with inversion of configuration at (2).Retention C- (x) H,O H,O (y) - InversionFrom the results cited above, the ring opening of methyloxirane1, 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 mechanismwas corroborated by use of the enantiomerically pureoxiranes 1 4 and the determination of the absolute configurationof the diol formed, or by comparing the time-dependentcourse of the excess enantiomers of the unchanged substrateand the diol formed. The enantiomeric excess (ee) of the individualoxirane enantiomers at the beginning of the enzymaticconversion was not identical with the ee of the correspondingdiol formed after complete substrate consumption (see Table 2).182 WISTUBA AND SCHURIGTABLE 1. Time dependence of the enantiomeric composition of the diol metabolite during themEH-catalyzed hydrolysisSubstrate~ ~~EnantiomericcompositionSubstrate conc. Incubation timeMetabolite (mM) (min) ( R W ) (%YO)Methyloxirane 1 Propane-1,2-diol 4Vinyloxirane 2 3-Butene-1,2-diol 4t-Butyloxirane 3 3,3-Dimethyl-butane-1,2-diol 4

Epichlorohydrin 4 3-Chloropropane-1,2-diol 15Epibromohydrin 5 3-Bromopropane-1,2-diol 10153040601020307025

3545556510

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2030405060102030

4045.947.849.449.940.743.546.550.096.095.3

93.985.449.972.471.767.561.752.550.075.966.858.850.754.152.250.650.159.356.553.550.0

4.04.76.114.650.127.628.332.538.347.550.0

24.133.241.249.3Incubation

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timeI35 niinR50 min 70 min’hI _ .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 mEH-catalyzed hydrolysisof t-butyloxirane 3 (25 m x 0.25 mm fused silica capillary coated with permethylated P-cyclodextrin in OV 1701,ENANTIO- AND REGIOSELECTNE OXIRANE HYDROLYSIS 183TABLE 2. Comparison of the enantiomeric composition of some optical active oxiranes and thecorresponding 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-butylboronic 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.26This effect can be used as an alternative method to “0-labelingexperiments for the quantitative determination of the regzoselectiuityof the epoxide-hydrolase-catalyzed water attack onthe condition that no uncatalyzed hydrolysis takes place, completesubstrate 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 microsomesheated to 100”C, and determination of the amount of oxiranes by GLC using an internal standard, nonenzymatichydrolysis under the enzymatic incubation conditions was excluded.

If the investigated racemic monoalkyl substituted oxiranesform racemic diols after complete consumption, the ratioof water attack (x/y) (see Scheme 11) at the unsubstituted andthe 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 attacktakes place with high regioselectivity (for methyloxirane 1with 98 f 0.2%, vinyloxirane 2 with 96.3 f 0.3%, and for epichlorohydrin4 with 97.2 f 0.3%) at the unsubstituted ringcarbon atom. In the case of t-butyloxirane 3 a completelyregioselective behavior was found (see Table 2) [(R)-t-butyloxirane

shows an enantiomeric excess of 99.48fOo.10%a nd theformed 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 reportedin the mEH-catalyzed hydrolysis of some monoaryl- and

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pyridylsubstituted oxiranes. ’,12OHX @ HOCH, +#..,,,R.‘\ # (S)-diol 4‘0# 4 - .R H ‘\‘ .“‘.? /‘

J ,,q(S)-oxirone‘.(R)-oxirone(R)-diolSdiol = x . Sozirane+ Y . RorirencIldiol = X ‘ Rozimne + Y ’ SorirancScheme II. Stereochemical pathway of oxirane hydrolysis.CONCLUSIONSimple chiral monosubstituted aliphatic oxiranes are hydrolyzedwith modest substrate enantioselectiuily by mEH of rat

liver in vitro to 1,Zdiols. In the case of oxiranes with unbranchedalkyl substituents it was found that the preferentiallyhydrolyzed enantiomers possess the same relative stereochemistry.In contrast, t-butyloxirane shows an opposite sense of enantioselectivity. The ring opening of the oxiranes investigatedoccurs with high regioselectiuity (9&1000/,). Water attacktakes place at the sterically less hindered, unsubstituted oxiranecarbon atom.

ACKNOWLEDGMENTSThe authors wish to thank Professor Wendel (Konstanz,Germany) for the supply of microsomes. The support of thiswork by “Deutsche Forschungsgemeinschaft” and “Fonds der chemischen Industrie” is gratefully acknowledged.LITERATURE CITED1.2.3.4.5.6.7.

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Fjellstedt, T.A., Allen, R.H., Duncan, B.K., Jacoby, W.B. Enzymatic conjugationof epoxides with glutathione. J.Biol.Chem. 2483702-3707, 1973.Wistuba, D., Schurig, V. Complementary epoxide hydrolase- vs glutathionS-transferase-catalyzed kinetic resolution of simple aliphatic oxiranscompleteregio and enantioselective hydrolysis of cis-2-ethyl-3-methyloxirane.

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CHIRALITY 417S184 (1992)

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