Chem. Res. Toxicol. 1994, 7, 336-343
336
Stereoselectivity of in Vitro Isoprene Metabolism Dorothee Wistuba,**tKlaus Weigand,? and Hans Peter$ Znstitut fur Organische Chemie der Uniuersitat, Auf der Morgenstelle 18,0-72076 Tiibingen, Federal Republic of Germany, and Institut fur Arbeitsphysiologie an der Universitat, Ardeystrasse 67, 0-44139 Dortmund, Federal Republic of Germany Received June 11, 1993"
The stereoselectivity of the in vitro conversion of isoprene by liver enzymes of rats and mice was determined. Isoprene was epoxidized by cytochrome P450 of rats and mice to 2-isopropenyloxirane and 2-methyl-2-vinyloxirane with slight but different product enantioselectivity. Only with mouse liver microsomes was a distinct regioselectivity observed. Both monooxiranes were further epoxidized to 2-methyl-2,2/-bioxirane with substrate enantioselectivity, product diastereoselectivity, and with product enantioselectivity. The epoxide hydrolase-catalyzed hydrolysis with rat and mouse liver microsomes occurs with substrate enantioselectivity. A better kinetic resolution was found for 2-isopropenyloxirane than for 2-methyl-2-viny€oxirane. While 2(R)-isopropenyloxirane was conjugated preferentially with glutathione, catalyzed by glutathione S-transferase, no enantiomer differentiation takes place in the case of 2-methyl2-vinyloxirane.
Introduction With a worldwide production of more than 1.3 million tons per year ( I ) , isoprene (2-methyl-l,&butadiene) represents an important product of the petrochemical industry. This technical significance underlines the relevance of the investigation of the isoprene metabolites in mammals. As shown in Scheme 1,isoprene is epoxidized by cytochrome P450-dependent monooxygenases to the isomeric monooxiranes 2-isopropenyloxirane (1) and 2-methyl-2-vinyloxirane (2) (2, 3). Del Monte et al. reported that only 2-isopropenyloxirane(1) can be further epoxidized to the mutagenic isoprene diepoxide 2-methyl2,2'-bioxirane 5 (2). Both monooxiranes 1 and 2 are converted to the vicinal diols 3 and 4, catalyzed by epoxide hydrolase and conjugated with glutathione, catalyzed by glutathione S-transferase. Like most enzymes, cytochrome P450, epoxide hydrolase, and glutathione S-transferase may function as inherently chiral catalysts. Thus, the cytochrome P450catalyzed epoxidation of a series of aliphatic prochiral alkenes occurs with product enantioselectivity (prochiral recognition) (4). The conversion of alkyl-substituted oxiranes by epoxide hydrolase to form vicinal diols or by glutathione S-transferase to form glutathione conjugates occurs with substrate enantioselectivity (chiralrecognition, kinetic resolution) (5-8). The striking differences in the biological activities between oxirane enantiomers [e.g., benzo[alpyrene 7,8-oxide (91,phenyloxirane (IO)]underline the importanceof studies devoted to the determination of enantioselectivities in the formation and transformation of epoxides catalyzed by enzymes. The present paper reports on stereochemical and mechanistic aspects in the in vitro metabolism of isoprene with enzymes of rats and mice. * Correspondence should be addressed to this author at Auf der Morgenstelle 18, D-72076Tfibingen, Germany. Tel: 07071-296260. + Institut far Organische Chemie der Universikit. Institut ffir Arbeitsphysiologie an der Universikit. @Abstractpublished in Advance ACS Abstracts, April 1, 1994.
Scheme 1. Metabolism of Isoprene. Glutothioneconjugate
1
lCSH A
@
Y
''edt9,-
Isoprene
< OH
GST
I
,OH
5
CSH
CST 4
Clutathioneconjugote
GST = glutathione S-transferase;GSH = glutathione; P450 = cytochrome P450.
Materials and Methods Caution! The following chemicals are hazardous and should be handled carefully: 2-isopropenyloxirane, 2-methyl-2-vinyloxirane, and 2-methyl-2,2'-bioxirane. Materials. Glutathione, gluathione S-transferase, isocitrate, isocitrate dehydrogenase, and (trichloromethy1)oxirane were purchased from Sigma (Taufkirchen, FRG). Isoprene was purchased from Aldrich (Steinheim, FRG) and NADP from Boehringer (Mannheim,FRG). (2R)-2-Methyl-3-butene-1,2-diol was kindly provided by Professor Ernest L. Eliel (University of North Carolina, Chapel Hill, NC). 2-Isopropenyloxirane (1) was synthesized according to refs 11and 12. Methyl-2-vinyloxirane (2) was prepared as previously described (13). 2-Methyl-2,2'-bioxirane (5). m-Chloroperbenzoicacid (27.2 g, 126 mmol) was dissolved in 300 mL of dry dichloromethane, and 6 mL (60 mmol) of isoprene was added at 0 "C. The reaction mixture was allowed to stand for 14 days at 4 "C and 24 h at -20 O C . The solution was filtered and washed twice with 5 % sodium
0893-228~/94/2707-0336$04.50/0 0 1994 American Chemical Society
Stereoselectivity of Isoprene Metabolism sulfite and saturated sodium carbonate and then dried with anhydrous magnesium sulfate. The solvent was removed, and the residue was distilled in vacuo. The diastereomeric ratio [(2R,2'R) and (2S,2'S):(2S,2'R) and (2R,2'S) 1:1.6] wasdetermined by GLC (seecomplexationgaschromatography).Yield 1g(17%); bp72 OC/12 mmHg; W-NMR (CDCls) 6 (ppm) 16.64,17.25,44.29, 51.24,51.51,52.78,53.19,54.48,55.31; 1H-NMR (CDCl3) 6 (ppm) 1.29 (1.8H, s), 1.36 (3H, s), 2.60 (m), 2.70 (m), 2.86 (m), 2.94 (m); MS, m/z 39 (85), 41 (701, 43 (65), 55 (loo), 57, 69, 71, 99. 3-Methyl-3-butene-1,$-diol (3) and 2-met hyl-3-butene-1,2diol (4) were prepared by acid-catalyzed (3 N HC1) hydrolysis from the corresponding oxiranes (1 and 2). Preparation of 2(S)-Isopropenyloxirane (14). (2R)-1,2Isopropylidenebutane-l,2,3-triol(9).(2R)-1,2-Isopropylideneglyceraldehyde (8) (10 g, 77 mmol), prepared according to ref 14, was slowly added to a stirred solution of methyl lithium (1.6 M in diethyl ether) in 50 mL of dry diethyl ether at -60 "C under a nitrogen atmosphere. The reaction mixture was allowed to warm up to room temperature and then poured into an ice-saturated NH&l solution. The ether layer was separated, and the aqueous layer was extracted twice with 50 mL of ethyl acetate. The combined organic solutionwas dried with anhydrous K2CO3 and concentrated in vacuo. The residue was distilled. Yield 8.8 g (78%);bp 83-84 "C/20 mmHg; 1H-NMR (CDCls) 6 (ppm) 1.20 (3H, d), 1.36 (3H, s), 1.43 (3H, s), 2.65 (lH, s), 3.95 (4H, m); W-NMR (CDC13) 6 (ppm) 18.5, 25.1, 26.4, 64.9, 66.9, 79.5, 109.0; MS, m/z 43 (loo), 59, 71, 101 (60), 131. (2R)-1,2-Isopropylidene-1,2-dihydroxy-2-butanone (10) (15). Dimethyl sulfoxide (20.9mL) in 60 mL of dichloromethane was added to a stirred solution of oxalyl chloride (12.3 mL, 135 mmol) in 300 mL of dry dichloromethane under a nitrogen atmosphere at -60 "C. 9 (18 g, 123.3 mmol) was slowly added, and the mixture was stirred for 15 min. Then triethylamine (85 mL, 620 mmol) was added dropwise, and the reaction mixture was allowed to warm up to room temperature and was poured into ice-water (600 mL). The organic layer was separated, and the aqueous layer was extracted with dichloromethane. The combined organic solution was dried with anhydrous magnesium sulfate and concentrated in vacuo. The residue was distilled. Yield 16.4 g (92.3%);bp 67-68 "C/12 mmHg; lH-NMR (CDCl3) 6 (ppm) 1.40 (3H, a), 1.49 (3H, s), 2.25 (3H, s), 4.00 (lH, dd), 4.19 (lH, t),4.42 (lH, dd); 13C-NMR (CDCls) 6 (ppm) 25.0,26.0,66.4, 80.5,lll.O; IR (cm-l) 2990 (s), 2940 (m),2900 (m), 1740 (vs), 1450 (w), 1420 (w), 1380 (s), 1370 (s), 1360 (s), 1260 (s), 1210 (s), 1150 (s), 1060 (vs), 845 (8); MS, m/z 43 (1001, 51,61,73,99, 101, 144; (c = 2.1; benzene). [ a ] 2 3+65.07 ~ (2~-1,2-Isopropylidene-3-methyl-3-butene-l,2-diol ( 11). Anhydrous methyltriphenylphosphoniumbromide (39.65 g, 11.0 mmol) was slowly added to a solution of sodium bis(trimethy1sily1)amide in THF (1N) under a nitrogen atmosphere at room temperature. The reaction mixture was stirred for 30 min at room temperature and heated under reflux for 1h. The solvent was evaporated and hexamethylsilazane formed was removed in vacuo (1mmHg) at 100 "C. The yellow residue was dissolved in 100 mL of THF. 10 (16 g, 11.1 mmol) in 50 mL of THF was added to the solution at -10 OC. After addition, the solution was allowed to warm up to room temperature and poured into 200 mL of ice-water. The organic layer was extracted three times with diethyl ether and dried with anhydrous magnesium sulfate. The solution was concentrated in vacuo, and the residue was distilled. Yield 10.6 g (67%);bp 58-61 "C/36 mmHg; lH-NMR (CDC13 6 (ppm) 1.33 (3H, s), 1.38 (3H, s), 1.66 (3H, s), 3.57 (lH, dd), 4.02 (lH, dd), 4.45 (lH, dd), 4.83 (lH, s), 4.99 (lH, 8); '3CNMR (CDCls) 6 (ppm) 17.4, 25.6, 26.3, 68.4, 79.3, 109.3, 112.4, 142.5; IR (cm-l) 3080 (w), 2990 (s), 2940 (m), 2880 (m), 1650 (m), 1450 (m), 1370 (81, 1250 (s), 1210 (s), 1160 (s), 1060 (s), 900 (m), 850 (s), 790 (8); MS, mlz 43 (loo), 72 (70), 86,97, 113, 127 (85), 142;ee = 91% (determined by complexation gas chromatography); [(u]23~ +13.7 (c = 1.42; benzene). (2S)-3-Methyl-3-butene-l,2-diol(l2).2 mL HCl(3 N) was added to a solution of 11 (10 g, 70.4 mmol) in 70 mL of ethanol. The mixture was heated under reflux for 1h. After cooling to
Chem. Res. Toxicol., Vol. 7,No. 3, 1994 337 room temperature, the solution was neutralized with KHCO3, filtered, and concentrated. The residue was dissolved in 20 mL of methanol, and 50 mL of diethyl ether was added. This caused the precipitation of inorganic salts. After filtration and evaporation of the solvent, the residue was chromatographed on silica (1:ln-hexanelethylacetate). The combinedproduct was purified by Kugelrohr distillation. Yield 4.65 g (65%); 'H-NMR (CDCls) 6 (ppm) 1.73 (3H, s), 3.52 (lH, dd), 3.68 (lH, dd), 3.73 (lH, s), 4.16 (lH, d), 4.93 (lH, a), 5.04 (lH, 5); W-NMR (CDCls)6 (ppm) 18.9,65.3, 75.7, 111.9, 144.1; IR (cm-'1 3350 (vs), 2950 (s), 2880 (s), 1659 (m), 1450 (e), 1320 (m), 1070 (s), 1020 (s), 900 (s), 830 (m); MS, m/z 41,43,71 (loo), 84,102; ee = 89.3% (determined by inclusion gas chromatography, see enantiomer analysis); [(u]23D +13.96 (C 2.95; CH2C12). (2S)S-Methyl- 1-[(ptoluenesulfony1)oxy 1-3-buten-2-01 (13). To a solution of 3.5 g (34.3 mmol) of 12 in 35 mL of dry pyridine was added a solution of p-toluenesulfonyl chloride (6.54 g, 34.3 mmol) in 40 mL of dry pyridine at 0 "C. The mixture was stored in the refrigerator for 12 h and then poured into 100 mL of icewater. The aqueous layer was extracted four times with 50 mL of diethyl ether. The combined organic solution was washed five times with 40 mL of H2S04 (2 N), followed by saturated NaHCOs solution and brine, and then dried over magnesium sulfate. The concentration gave an oil, which crystallized at 4 "C. Yield 7.8 g (88.6%);mp 43-44 "C; lH-NMR (CDCl3) 6 (ppm) 1.68 (3H, s), 2.45 (3H, s), 3.95 (lH, dd), 4.08 (lH, d), 4.10 (lH, d), 4.30 (lH, t),4.95 (lH, s), 5.05 (lH, e), 7.35 (2H, d), 7.81 (2H, d); "C-NMR (CDCls)6 (ppm) 18.57,21.66,72.44,72.94,113.67,127.97,129.95, 132.73, 142.07, 145.10; IR (cm-l) 3500 (s), 3080 (w), 2980 (m), 2920 (m), 1650 (m), 1600 (s), 1595 (m), 1450 (s), 1360 (e), 1220 (w), 1180 (s), 1090 (a), 1040 (w), 1020 (w), 970 (s), 900 (m), 820 (s), 780 (s), 700 (w); MS, mlz 41,43, 55, 65, 71 (loo), 84, 91, 92, 107, 155, 172, 226; [(u]=~+12.6 (C = 3.0; CDCls). 2(S)-Isopropenyloxirane (14). 13 (3.2 g, 12.47 mmol) was quickly added to a vigorously stirred solution of 3 g of KOH in 5 mL of triethylene glycol at room temperature. The oil bath was heated to 130-140 "C, and 14 was collected in a -70 "C trap at reduced pressure (100 mmHg). Redistillation from calcium hydride afforded a colorless liquid. Yield0.43 g (41%); 'H-NMR (CDC13)6 (ppm) 1.63 (3H, s), 2.73 (lH, dd), 2.87 (lH, dd), 3.37 (lH, 5), 5.03 (lH, s), 5.17 (lH, s); 13C-NMR (CDCls) 6 (ppm) 16.09,46.70, 54.44, 114.55, 141.38; MS, mlz 39(100), 41. 50, 53, 55, 56,69,83,84; ee = 88.2% (determined by complexation gas chromatography on manganese(I1)bis 13-(heptafluorobutanoy1)(1R)amphoratel; [(Y]=D+49.4 (C = 2.1; CH2C12). (2R)-2-Methyl-2-vinyloxirane (15). 15 was prepared from (2R)-2-methyl-3-butene-l,2-diol according to 14. (2$,2'R)- and (2R,2'R)-2-Methyl-2,2'-bioxirane.Epoxidation of 2(S)-isopropenyloxiranewith m-chloroperbenzoicacid in dichloromethane (16)(2-h reaction time) yielded a mixture of (2S,2'R)- and (W1,2'R)-2-methy1-2,2'-bioxirane. The crude product was analyzed by GLC (see enantiomer analysis). (2.632'5)- and (2$,2'R)-2-Methyl-2,2'-bioxirane. Epoxidation of (2R)-2-methyl-2,2'-bioxiranewith m-chloroperbenzoicacid in dichloromethane (16)(the reaction mixture was allowed to stand for 10days in the refrigerator) yielded a mixture of (2S,2'S)and (2S,2'R)-2-methyl-2,2'-bioxirane.The crude product was analyzed by GLC (see enantiomer analysis). Enantiomer Analysis. Complexation Gas Chromatography (17). The enantiomers of 2-isopropenyloxirane (1) and 2-methyl-2-vinyloxirane(2) were separated on a 35 m X 0.2 mm glass capillary column coated with manganese(I1) bisI(3-heptafluorobutanoyl)-(1R)-camphoratel in SE 30 (dimethylpolysiloxane) at 30 "C. 2-Methyl-2,2'-bioxirane (5) was separated on a 25 m X 0.25 mm fused silica capillary column coated with nickel(11)bis [(3-heptafluorobutanoyl)-(lS)-10-ethylenecamphorate]in SE 30 at 90 OC. The diol acetonides (dioxolanes)were separated on a 25 m X 0.25 mm fused silica capillary column coated with nickel(I1)bis[(3-heptafluorobutanoyl)-(1R,2S)-pinan-4-onatelin SE 30 (18). Carrier gas: high-purity grade N2; detector: FID (flame ionization detector).
Wistuba et al.
338 Chem. Res. Toxicol., Vol. 7, No. 3, 1994 Inclusion Gas Chromatography (19). The enantiomers of (3) and 2-methyl-3underivatized 3-methyl-3-butene-l,2-diol butene-1,2-diol (4) were separated on a 25 m X 0.25 mm fused silica capillary column coated with 10% heptakis(2,3,6-tri-Omethyl)-8-cyclodextrin in OV 1701 at 70 or 80 OC. Carrier gas: high-purity grade Hz; detector: FID (flame ionization detector). Liver Microsomes. Male F-344 rata and B6C3F1 mice were obtained from the Lippesche Versuchstierzucht (Exertal, Germany). Preparation of hepatic microsomes from these animals and determination of cytochromeP450 were performed according to Remmer et al. (20). Incubations. Microsomal Epoxidation of Isoprene. The reaction mixture (0.5 mL), containing rat or mouse liver microsomes (1nmol of cytochrome P450), 0.15 M phosphate buffer (pH 7.4), NADP (10-3 M), isocitrate dehydrogenase (0.1 IU), isocitrate (8 X 10-9 M), MgClz (5 X 10-3 M), and 2-(trichloromethy1)oxirane (6 X 10-9 M) (to inhibit epoxide hydrolase), was incubated with isoprene (10 pmol) for 130 min at 37 OC. The percentage of the monooxirane enantiomers formed by the enzymatic isopreneepoxidationwere determined by complexation gas chromatography by the head-space technique (21). Control experiments ensured that the enantiomeric composition in the gas phase corresponded to that in solution. Within an incubation time of 130 min a repeated screening of the enantiomer composition of the oxiranes 1 and 2 by complexation gas chromatography at intervals of 20 min was carried out. Monooxiranes 1 and 2 are diastereomers; thus control experiments were performed to determine the correction factor of the diasteromeric ratio in the gas and in the liquid phase. For this, the reaction mixture was cooled with liquid nitrogen of above 5 “C and extracted with ethyl acetate or ethyl ether. The diastereomeric compositionof 1and 2 determined by GC analysis was compared at the gas phase before extraction and the organic phase after extraction. To prove that 2-(trichloromethyl)oxirane is an effective inhibitor of epoxide hydrolase, the incubation was carried out with the monooxirane 1or 2 instead of the substrate isoprene and without the NADPH-regenerating system (under these conditions no 2-methyl-2,2’-bioxiranewas formed). The amount and the enantiomeric composition of the monooxirane 1remained constant during the incubation time of 130 min. The decrease of monooxirane 2 corresponded to the spontaneous hydrolysis of 2 in phosphate buffer (pH 7.4) and was the same for both oxirane enantiomers. Microsomal Epoxidationof the Monooxiranes 1 and 2. It was carried out in analogy to the microsomal epoxidation of isoprene, but with a reaction mixture of lo00 or 1500p L and with monooxirane 1or 2 (10 pmol) instead of isoprene and for 45 min at 37 O C . The enzymatic epoxidation of the oxiranes was stopped by cooling at 0 OC. The reaction mixture was extracted with diethyl ether, and the enantiomeric composition of 2-methyl2,2’-bioxirane (5) was determined by complexation gas chromatography. To ensure that the 2-(trichloromethyl)oxirane acted as an efficient inhibitor, the incubation was carried out with 2-methyl-2,2’-bioxirane(5) instead of the substrate isoprene. The amount and the diastereomeric and enantiomeric composition of 5 remained constant during the incubation time of 45 min. Hydrolysis of the Monooxiranes 1 and 2. The reaction mixture (0.5 mL for the oxirane determination, and 1mL for the diol determination), containing rat or mouse liver microsomes (1 mg of protein/mL) and 0.15 M phosphate buffer (pH 7.41, was incubated 5 min at 37 “C, and then oxirane (4 mM) and standard (acetone, 4 mM) were added. The percentages of unchanged oxirane enantiomers were determined by complexation gas chromatography via the head-space technique. For GLC determination of the diols formed, the reaction mixture was cooled with liquid nitrogen at about 5 “C and extracted with diethyl ether. After concentration, the solution was analyzed by inclusion gas chromatography. Conjugation of the Monooxiranes 1 and 2 with Glutathione. The reaction mixture (0.5 mL) containing glutathione S-transferase (0.75 mg of protein/mL), glutathione (4 mM), and 0.1 M phosphate buffer (pH 6.5) was incubated 5 min at 37 OC,
r
0
l
I
5
I
I
10
I
I
15
I
20
min
Figure 1. Gas chromatographic enantiomer separation of 2-isopropenyloxirane (1) and 2-methyl-2-vinyloxirane (2) on a 35 m X 0.2 mm glass capillary column coated with 0.1 M manganese(I1)bis [3-(heptafluorobutanoyl)-(lR)-camphorate] in SE 30 at 30 OC; carrier gas, 0.8 bar Nz. and then oxirane (4 mM) and standard (acetone, 4 mM) were added. The percentages of unchanged oxirane enantiomers were determined by complexation gas chromatography via the headspace technique. The amount of unchanged substrates or the metabolites formed refer to the mean of at least three different incubations (overall deviation 0.5-2 % ). The curves shown in Figures 3-9 were formed by the arithmetic mean of 3-5 single curves.
Results and Discussion The stereochemical course of the three metabolic pathways, (a) epoxidation of isoprene and the monooxiranes 1 and 2, (b) hydrolysis of the monooxiranes 1 and 2, and (c) conjugation of the monooxiranes withglutathione (see Scheme 11, was investigated. As a convenient analytical tool, complexation gas chromatography and inclusion gas chromatography, which enable time-dependent enantiomer screening of oxiranes and diols in the nanogram range, were employed for the determination of the enantiomeric excess (ee) (see Figures 1and 2) and for the absolute configuration. Using these methods to determine the absolute configuration of the metabolites, the synthesis of reference substances with unequivocal stereochemistries was necessary. 2(S)-Isopropenyloxirane was prepared in five steps from D-mannitol via (2s)-3methyl-3-butene-l,2-diol (see Scheme 2). (2RI-P-Methyl2-vinyloxirane was synthesizedfrom 2(R)-methyl-3-butene1,2-diol. The epoxidation of 2(S)-isopropenyloxiranewith m-chloroperbenozicacid led to the diastereomeric (2S,2’R)The (2R,2’R)- and and (W1,2’R)-2-methyl-2,2’-bioxiranes. (2S,2’S)-isomers result from the epoxidation reaction of (2R)-2-methyl-2-vinyloxirane. Microsomal Epoxidation of Isoprene. Isoprene possesses two prochiral carbon-carbon double bonds. Thus, the epoxidation catalyzed by cytochrome P450dependent monooxygenases can occur with regioselectivity and/or product enantioselectivity (prochiral recognition) being determined by the preferential orientation of the oxygen attack at the enantiotopic double bonds.
Chem. Res. Toxicol., Vol. 7, No. 3, 1994 339
Stereoselectivity of Isoprene Metabolism
2
OH
OH
20
I 0
5
I
I
10
15
w
80
100
120
niiii
Figure 3. 2-Isopropenyloxirane and 2-methy1-2-vhyloxirane formed by epoxidation of isoprene with mouse liver microsomes [e, (2S)-2-methyl-2-vinyloxirane; m, (M)-2-methyl-2-vinyloxirane; A,2(S)-isopropenyloxirane;A,2(R)-isopropenyloxiranel.
OH
OH
LO
b 20
25
min
Figure 2. Gas chromatographic enantiomer separation of
3-methyl-3-butene-l,2-diol(3) and 2-methyl-3-butene-l,2-diol(4) on a 25 m x 0.25 mm fused silica capillary column coated with permethyl-@-cyclodextrinin OV 1701; carrier gas, 1 bar Hz; temperature, 60 "C (8-min isotherm), 60-90 O C (2 OC/min).
Scheme 2. Synthesis of 2(S)-Isopropenyloxirane
6
\
7
8
9
10
11
12
As has been demonstrated previously (211,the correct determination of regioselectivity or product enantioselectivity in the epoxidation reaction requires the complete inhibition of epoxide hydrolase, since the catalytic hydrolysisof many oxiranes represents an efficient competing enantioselective process (5). The epoxide 24trichloromethy1)oxiranehas been employed as an efficient inhibitor of epoxide hydrolase (22), and it was found that in ita presence the amount and the enantiomeric composition of both the monooxiranes 1 and 2 and the 2-methyl-2,2/bioxirane 5 remain constant during the incubation time. Hence, it can be concluded that the formation of the oxiranes is induced asymmetrically and that the enanti-
20
LO
60
80
100
120
iiiiii
Figure 4. 2-Isopropenyloxirane and 2-methyl-2-vinyloxirane formed by epoxidation of isoprene with rat liver microsomes [@,
(2Sj-2-methyl-2-vinyloxirane;(M)-2-methyl-2-vinyloxirane; A, 2(S)-isopropenyloxirane; A 2(R)-isopropenyloxirane].
omeric excess (ee) observed is not due to the kinetic resolution of racemic oxiranes by epoxide hydrolase (22). Contrary to 2-isopropenyloxirane and 2-methyl-2,2/-bioxirane (5), 2-methyl-2-vinyloxirane possesses a very high reactivity toward water and, thus, a half-life of only 75 min at pH 7.4 (2).This spontaneous hydrolysis, being a racemic process (both enantiomers react at the same rate) and competing with the enzymatic formation of 2-methyl2-vinyloxirane (2), leads to problems in the quantitative determination of the regioselectivity and product enantioselectivity for this substrate. The epoxidation of isoprene with mouse liver microsomes occurs with preferential oxygen attack a t the more crowded, disubstituted carbon-carbon double bond. After an incubation time of approximately 90 min, the concentration of oxiranes formed became constant, reflecting a balance between the rate of enzymatic formation and the spontaneoushydrolysis of 2-methyl-2-vinyloxirane (2); at steady state the ratio of 2-isopropenyloxirane (1) and 2-methyl-2-vinyloxirane (2) was 1:2.6 (see Figure 3). While 2-methyl-2-vinyloxirane (2) was formed with slight product enantioselectivity [preferential formation of the (2S)-enantiomer, ee = 6.9 f 1.0%I, 24sopropenyloxirane (1)wasformednearlyracemic(ee= 2.1 f 1.0%)(seeFigure 3). A remarkable species dependence of the microsomal regio- and enantioselective epoxidation of isoprene by cytochrome P450-dependent monooxygenases is observed when mouse and rat are compared. As shown in Figure 4,the epoxidation with rat liver microsomes leads to similar amounts of 2-isopropenyloxirane (1) and 2-methyl-2vinyloxirane (2). After approximately 40 min the amount of 2-methyl-2-vinyloxirane(2) decreases, caused by spon-
340 Chem. Res. Toxicol., Vol. 7, No. 3, 1994
Wistuba et al.
Scheme 3. Substrate Enantioselectivity (i) = (k1+ kz)vs (k3+ kd);Product Diastereoselectivity (ii) = kl vs kz and k3 vs 4; Product Enantioselectivity (iii) = k1 vs k3 and kz vs k4
R
Table 1. The Enantiomeric and Diastereomeric Composition of 2-Methyl-2f’-bioxirane (5) Formed by Epoxidation of Racemic 2-Isopropenyloxirane(1) and of Racemic 2-Methyl-2-vinyloxirane(2) with Rat and Mouse Liver Microsomes. substrate 2-methvl-2.2’-bioxirane rat (76 mouse (5%)
2 si
2s
’re
42R,2‘S 17:2S,2‘S
46.6 f 1.0 37.0 f 0.3
11.8 f 0.7 17.1 f 0.3
2R
si
2!%
“?&
0
+R fow/
R
b
a
2S,2’R
- 1 % 2S,2‘S
2R,2‘S
4
R’
2s
re
7
%2s7
47 0
2R,2’R
taneous hydrolysis. In this casethe spontaneous hydrolysis occurs at a higher rate as the formation of the oxirane. The formation of 2-methyl-2-vinyloxirane (2) occurs in analogy to mouse with a slight product enantioselectivity. Contrary to mouse, 2-isopropenyloxirane (1) was formed with a distinctly higher enantiomeric excess (ee = 35.6 f 1.0%). Always the @)-enantiomer was produced preferentially. This result is in agreement with the observed product enantioselectivity in the epoxidation reaction of similar small aliphatic alkenes such as propene, 1-butene, l,&butadiene, and 2-methyl-1-butenewith rat and mouse liver microsomes (4). While the two carbon-carbon double bonds of prochiral isoprene possess enantiotopic (23) faces, the remaining double bond of the chiral monooxiranes 1 and 2 possess diastereotopic (23) faces (see Scheme 3). In both monooxiranes 1and 2 two elements of (pro)stereogenicity (24) are inherently combined: (1)prochirality (re, si) and (2) chirality ( R , S). It was considered worthwhile to study their interdependence in the epoxidation reaction with liver microsomes of rat and mouse. The results with 2-isopropenyloxirane (1)and 2-methyl-2-vinyloxirane (2) are compared in Table 1. Three stereoselective processes (25, 26) may be considered (see Scheme 3): (i) substrate enantioselectivity (“chiral recognition” by enantiomer differentiation); (ii) product diastereoselectivity (diastereoface differentiation of an individual alkene enantiomer); and (iii) product enantioselectivity (“prochiral recognition” by external enantioface differentiation of the respective alkene enantiomers). The enzymatic epoxidation of the racemic chiral monooxiranes represents a competitive process between the enantiomers. In the case of 2-isopropenyloxirane (l), Table 1reveals only a slight substrate enantioselectivity (i) in favor of the (2R)-configurated oxirane on incubation with both rat or mouse liver microsomes. However, there is a distinct and different product diastereoselectivity (ii) in the rat and mouse microsomal epoxidation of the individual enantiomers of 2-isopropenyloxirane(1). While the epoxidation of 2(R)-isopropenyloxirane occurs preferentially a t the re face, the (2S)-enantiomer will be attacked by oxygen preferentially from the si face. A better discrimination between the diastereotopic faces in 2(R)isopropenyloxirane [si:re= 1:3.95 (rat) and 1:2.16 (mouse)l as compared with those of the (2S)-enantiomer [re:si = 1:1.66 (rat) and 1:1.72 (mouse)] was observed.
re
2 2S,2’R
26.4 f 1.0 29.0 & 1.4
2 2R,2’R
15.9 f 0.8 16.9 f 0.9
42S,2‘R 22S,2’S
44.0 f 1.7 32.6 f 1.7 11.3 f 0.6 14.8 f 0.8
2R si
2 2R,2’S
21.9 k 1.5 39.4 f 1.6
2 2R.2’R
22.8 f 0.4 13.2 f 0.8
2s
+
a Substrate enantioselectivity, (kl + k2) vs ( k ~ kd); product diastereoselectivity,&I vs k2 and k3 vs k,; product enantioselectivity, kl vs k3 and ki vs kr.
The results contained in Table 1may also be discussed in terms of product enantioselectivity (iii) whereby the differentiation of the enzyme between the externally enantiotopic faces ( 2 3 , 2 7 ) of the carbon-carbon double bond of 2-isopropenyloxirane (1)is considered. For both species, rat and mouse, a distinct but different product enantioselectivity was observed. The enantiomeric pair (2R,2’S)l(2S,2’R)’ was formed by epoxidation of the re face of the 2(R)-isopropenyloxirane and the si face of the (BS)-enantiomerwith an enantiomeric excess (ee)of 27.7 7% with rat liver microsomes and of 12.1 % with mouse liver microsomes in favor of the (2R,2’S)-enantiomer. (2S,2’S)and (2R,2’R)-2-methyl-2,2’-bioxirane was formed either racemic (mouse) or with an enantiomeric excess of ee = 14.8% (rat) of the (2R,2’R)-enantiomer. Contrary to the results of del Monte et al. (21, we find that 2-methyl-2vinyloxirane (2) was further epoxidized to 2-methyl-2,2’bioxirane (5) by both rat and mouse liver microsomes (see Figure 5 and Table 1). Inspection of Table 1 reveals differences in the substrate enantioselectivity (i) between the two species rat and mouse. While, in the case of rat, (2R)-2-methyl-2-vinyloxiranewas epoxidized preferentially, a slight preference of the (2S)-2-methyl-2-vinyloxirane has been observed in the mouse microsomal epoxidation. The rat microsomal epoxidation of (2R1-2-methyl2-vinyloxiraneoccurswith product diastereoselectivity (ii) in favor of the re face (si:re = 1:3.89). On the contrary, the enzyme cannot differentiatebetween the diastereotopic face of the (2S)-enantiomer. In analogy to 2-isopropenyloxirane (l),the mouse microsomal epoxidation occurs with distinct product diastereoselectivity. The re face was 1 The carbon atom C-2 of the substrate isopropenyloxirane becomes C-2’ of the product 2-methyl-2,2’-bioxirane because of the formal change of the carbon atom numbering cawed by the IUPAC rule. Because of the formal change in the descriptor caused by the priority rule of Cahn, Ingold, and Prelog the 2R (or 2s) stereochemistry of the substrate isopropenyloxirane becomes 2‘s (or 2’R) of the product 2-methyl-2,2’bioxirane and the 2 s (or 2R) stereochemistry of the substrate 2-methyl2-vinyloxiranebecomes2R (or 2s)of the product 2-methyl-2,2’-bioxirane9
Chem. Res. Toxicol., Vol. 7, No.3, 1994 341
Stereoselectivity of Isoprene Metabolism
50
103
1so
200
250
iiiiii
Figure 6. Hydrolysis of racemic 2-ieopropenyloxiranewith rat (A,2R; A, 2s) and mouse (0,2R; 0, 2s) liver microsomes. plllol
2.0
1.5
1.0
0.5
2
B 20
LO
60
80
100
iiiiii
Figure 7. Hydrolysis of racemic 2-methyl-2-vinyloxiranewith rat (e, 2R; A,2s) and mouse (W, 2R; 0 , 2 s ) liver microsomes [ 0 , spontaneous hydrolysis in phosphate buffer (pH 7.4)].
1
4
i -P
,
-
0 ’ 5 ’ bmbr Figure 5. 2-Methyl-2,2‘-bioxirane (5) formed by epoxidation of racemic isopropenyloxirane (A) and of 2-methyl-2-vinyloxirane (B) catalyzed by cytochrome P450 of rat liver [l, (2R,2’S)-2methyl-2,2’-bioxirane; 2, (2S,2‘R)-2-methyl-2,2‘-bioxirane; 3, (2S,2’S)-2-methyl-2,2’-bioxirane; 4, (2R,2’R)-2-methyl-2,2‘-bioxirane] .
epoxidized preferentially in the case of (2R)-2-methyl-2vinyloxirane (si:re = 1:2.20) and the si face in the case of the (2S)-enantiomer (re:si = 1:2.98). In comparison to 2-isopropenyloxirane (l),a higher product enantioselectivity (iii) in the rat microsomal epoxidation of 2-methyl2-vinyloxirane (2) was found. (2S,2’R)- and (2R,2’R)-2methyl-2,2’-bioxirane were formed with an enantiomeric excess of ee = 33.5 % and 8.6 76, respectively. The mouse microsomal epoxidation of 2-methyl-2-vinyloxirane (2) leads for both enantiomeric pairs of the diastereomer 2-methyl-2,2’-bioxiranesto a small enantiomeric excess [(2R,2’S): ee = 9.4%; (2S,2’S): ee = 5.7%]. Thus, an inverse sense of product enantioselectivity has been observed. Summarizing these results, it was found that
the oxygen attack occurs preferentially at the re face of the (2R)-enantiomerduring the rat and mouse microsomal epoxidation of 2-isopropenyloxirane and during the rat microsomal epoxidationof 2-methyl-2-vinyloxirane.Only in the case of mouse microsomal epoxidation of 2-methyl2-vinyloxirane was the si face of the (2S)-enantiomer epoxidized preferentially (see Table 1). Hydrolysis of the Monooxiranes. The microsomal epoxide hydrolase catalyzes the in vitro hydrolysis of 2-isopropenyloxirane (1)and 2-methyl-2-vinyloxirane (2) to form 3-methyl-3-butene-l,2-diol (3) or 2-methyl-3butene-1,2-diol(4), respectively. A time-dependent analysis of the chiral substrates during the enzymatic reaction will reveal the propensity of the enzyme system to differentiate between the oxirane enantiomers. Figure 6 shows that the hydrolysis of racemic 2-isopropenyloxirane (1) with mouse liver microsomes occurs with substrate enantioselectivity, whereby the (R)-enantiomer was consumed preferentially. In the case of the more reactive 2-methyl-Zvinyloxirane (2) a superposition of the spontaneous (racemic process) and the enzymatic (enantioselective process) hydrolysis was found (see Figure 7). Contrary to 2-isopropenyloxirane(I), the isomeric 2-methyl-Qvinyloxirane (2) was hydrolyzed only with slight substrate enantioselectivity and under slight preference of the (2S)-enantiomer (see Figure 7). The decrease of the substrate enantioselectivity but not the change of the sense of enantioselectivity can be rationalized by the coincidence of the spontaneous and the enzymatic hy-
342 Chem. Res. Toxicol., Vol. 7, No. 3, 1994
Wistuba et al.
Table 2. Enantiomeric Ratio of 3-Methyl-3-butene-1.2-diol Formed by Hydrolysis of Racemic 2-Isopropenyloxirane with Rat Liver Microsomes 3-methyl-33-methyl-3butene-l,2-diol incubation butene- 1,2-diol incubation time (min) 2S(%) 2R (%I time (min) 2 s (%) 2R (%) 50 40.9 59.1 200 54.4 45.6 100 52.8 47.2 1320 54.9 45.1 150 53.6 46.4
drolysis. Analogous results were found in the microsomal hydrolysis of 2-methyl-2-vinyloxirane (2), when rat and mouse are compared. For both species the (LS)-enantiomer was hydrolyzed preferentiallyand the reaction occurs with slight substrate enantioselectivity. On the contrary, remarkable species differences were observed in the hydrolysis of 2-isopropenyloxirane (1). (2R)-Isopropenyloxirane was consumed preferentially in the first stage of the reaction (see Figure 6). With an increasingincubation time the hydrolysis rate of the (2s)-enantiomer increases and, after approximately 40% conversion, becomes faster than the hydrolysis rate of the (2R)-enantiomer. Similar results were found in the hydrolysis of tert-butyloxirane (7,6), phenyloxirane (28),and @-nitropheny1)oxirane(29) and were explained with inhibitory effects of the (2R)enantiomers, possessing higher affinity for the epoxide hydrolase active site toward the (2S)-enantiomer. As previously shown for a series of monoalkyl-substithe ring opening of 2-isopropenyloxirane tuted oxiranes (7), (1) and 2-methyl-2-vinyloxirane (2) 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 comparing the time-dependent course of the excess enantiomers of the unchanged substrate and the diol formed. In agreement with previously investigated alkylsubstituted oxiranes (7,8), racemic 2-isopropenyloxirane (1) and racemic 2-methyl-2-vinyloxirane (2) form racemic diols after complete consumption,catalyzed by mouse liver microsomes. The same effect was observed only for 2-methyl-2-vinyloxirane(2) with rat liver microsomes but not for 2-isopropenyloxirane(1). After complete substrate consumption, the diol formed, 3-methyl-3-butene-l,2-diol, was produced with an enantiomeric excess of approximately 10% (see Table 2). This effect can be explained by different regioselectivities in the ring opening of the 2(R)- and the 2(S)-isopropenyloxirane. Conjugationwith Glutathione. Both monooxiranes 1 and 2 are conjugated in vitro with glutathione (Y-Lglutamyl-L-cysteinylglycine) catalyzed by glutathione Stransferase of rat. The metabolic transformation of 24sopropenyloxirane (1) occurs with substrate enantioselectivity whereby the (2RI-enantiomerwas conjugated preferentially (see Figure 8) in analogy to the hydrolysis catalyzed by epoxide hydrolase. Similar chiral-recognition phenomena with the same sign of enantioselectivity were observed in a series of small, sterically less hindered oxiranes such as methyloxirane, ethyloxirane, and vinyloxirane (5). Glutathione and glutathione S-transferase are inherently chiral. Thus, the occurrence of substrate enantioselectivity can arise from both the chirality of the enzyme glutathione S-transferase and/or the molecular configuration of the conjugation partner L,L-glutathione. In the absence of the enzyme, no reaction between 2-isopropenyloxirane (1) and glutathione takes place. On the contrary, the more reactive 2-methyl-2-vinyloxirane
L
-L.
20
LO
60
80
loo
120
min
Figure 8. Conjugation of racemic 2-isopropenyloxirane(1) with glutathione, catalyzed by glutathione S-transferase [A,(a)and A, (2S)-2-isopropenyloxirane]. pn1ol 2.01
1.5
b.,
Figure 9. Conjugation of racemic 2-methyl-2-vinyloxiranewith glutathione ( 0 , spontaneous hydrolysis of 2-methyl-2-vinyloxirane; 0,conjugationof 2-methyl-2-vinyloxiranewith glutathione; 0 and m, conjugation of 2-methyl-2-vinyloxiranewith glutathione catalyzed by glutathione S-transferase). (2), having a short half-life at pH 6.5, is conjugated with glutathione in the absence of glutathione S-transferase but without substrate enantioselectivity. The presence of glutathione S-transferase increases the rate of conjugation, but likewise no kinetic resolution occurs (see Figure 9).
Conclusion In the epoxidation reaction of isoprene catalyzed by cytochrome P450, we found that the (SI-monooxiranes were formed preferentially. In analogy to a series of small alkyl-substituted oxiranes (51, the favored formed (SIconfigurated monooxiranes were preferentially detoxified under catalysis of the epoxide hydrolase. On the contrary, (R)-isopropenyloxirane,formed in a smaller amount than the (S)-isopropenyloxirane,was hydrolyzed preferentially by mouse liver microsomes, so that at least under in vitro conditions an enrichment of the (5')-configurated monooxirane occurs.
Acknowledgment. The authors thank Professor h e a t L. Eliel (University of North Carolina, Chapel Hill, NC) for his generous supply of (2R)-2-methyl-&butene-l,2-
Stereoselectivity of Isoprene Metabolism
diol. The support of this work by the "Deutsche Forschungsgemeinschaft" and "Fonds der chemischen Industrie" is gratefully acknowledged.
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