Stereochemical aspects in the 4-vinylcyclohexene biotransformation with rat liver microsomes and purified P450s. Monoepoxides and diols

Article abstract of DOI:10.1021/tx000255q

The stereochemical course of the biotransformation of 4-vinylcyclohexene (VCH, 1) by liver microsomes from male and female control and induced rats and purified rat P450 2B1 and 2E1 has been determined. The epoxidation of 1, catalyzed by male microsomes, occurs on both the endo- and exocyclic double bond to give four isomeric epoxides, cis-4-vinylcyclohexene 1,2-epoxide (2), trans-4-vinylcyclohexene 1,2-epoxide (3), (4R*,7S*)-4-vinylcyclohexene 7,8-epoxide (4), and (4R*,7R*)-4-vinylcyclohexene 7,8-epoxide (5). On the other hand, microsomes from female rats catalyzed primarily the endocyclic epoxidation. The stereoselectivity of this process was strongly dependent on gender and P450 induction. Only the phenobarbital and pyrazole, at lower levels, were able to enhance the epoxidation of 1 and mostly on the endocyclic double bond. Also, P450 2E1 and 2B1 in a reconstituted system were able to perform the epoxidation of 1 primarily on its endocyclic double bond. The metabolites, cis- and trans-4-vinylcyclohexene 1,2-epoxide (2 and 3, respectively) and the isomeric 4-vinylcyclohexene 7,8-epoxides (4 and 5), were rapidly biotransformed into the corresponding vicinal diols by mEH-catalyzed hydrolysis. The reaction of the endocyclic epoxides occurred with good substrate diastereo- and enantio-selectivity favoring the hydrolysis of epoxides (1S,2R,4S)-3 and (1R,2S,4S)-2 to give, before 50% conversion, selectively (1R,2R,4S)-diol (6). At variance, the hydrolysis of the exocyclic epoxides was characterized by a high level of substrate enantioselection associated with a very low, if any, level of substrate diastereoselection, the two epoxides, (4R,7S)-4 and (4R,7R)-5, being hydrolyzed practically with the same rate. On the basis of the major resistance to mEH hydrolysis, the endocyclic epoxides, (1R,2S,4R)-3 and (1S,2R,4R)-2, are expected to be further oxidized, in a stereochemical manner, to the specific mutagenic diepoxides which are thought to play a crucial role in VCH ovotoxicity. Thus, VCH ovotoxicity may be markedly affected by the reactivity of the diepoxidic stereoisomers formed and detoxicated.

Full text of DOI:10.1021/tx000255q

4
92
Chem. Res. Toxicol. 2001, 14, 492-499
Ster eoch em ica l Asp ects in th e 4-Vin ylcycloh exen e
Biotr a n sfor m a tion w ith Ra t Liver Micr osom es a n d
P u r ified P 450s. Mon oep oxid es a n d Diols
,
†
†
†
Cinzia Chiappe,* Antonietta De Rubertis, Massimiliano De Carlo,
‡
,‡
Giada Amato, and Pier Giovanni Gervasi*
Dipartimento di Chimica Bioorganica e Biofarmacia, Universit a` di Pisa, via Bonanno 33,
5
6126 Pisa, Italy, and Istituto di Mutagenesi e Differenziamento, area della Ricerca CNR,
Via G. Moruzzi, 56100 Pisa, Italy
Received December 18, 2000
The stereochemical course of the biotransformation of 4-vinylcyclohexene (VCH, 1) by liver
microsomes from male and female control and induced rats and purified rat P450 2B1 and
2
E1 has been determined. The epoxidation of 1, catalyzed by male microsomes, occurs on both
the endo- and exocyclic double bond to give four isomeric epoxides, cis-4-vinylcyclohexene 1,2-
epoxide (2), trans-4-vinylcyclohexene 1,2-epoxide (3), (4R*,7S*)-4-vinylcyclohexene 7,8-epoxide
(4), and (4R*,7R*)-4-vinylcyclohexene 7,8-epoxide (5). On the other hand, microsomes from
female rats catalyzed primarily the endocyclic epoxidation. The stereoselectivity of this process
was strongly dependent on gender and P450 induction. Only the phenobarbital and pyrazole,
at lower levels, were able to enhance the epoxidation of 1 and mostly on the endocyclic double
bond. Also, P450 2E1 and 2B1 in a reconstituted system were able to perform the epoxidation
of 1 primarily on its endocyclic double bond. The metabolites, cis- and trans-4-vinylcyclohexene
1
,2-epoxide (2 and 3, respectively) and the isomeric 4-vinylcyclohexene 7,8-epoxides (4 and 5),
were rapidly biotransformed into the corresponding vicinal diols by mEH-catalyzed hydrolysis.
The reaction of the endocyclic epoxides occurred with good substrate diastereo- and enantio-
selectivity favoring the hydrolysis of epoxides (1S,2R,4S)-3 and (1R,2S,4S)-2 to give, before
5
0% conversion, selectively (1R,2R,4S)-diol (6). At variance, the hydrolysis of the exocyclic
epoxides was characterized by a high level of substrate enantioselection associated with a very
low, if any, level of substrate diastereoselection, the two epoxides, (4R,7S)-4 and (4R,7R)-5,
being hydrolyzed practically with the same rate. On the basis of the major resistance to mEH
hydrolysis, the endocyclic epoxides, (1R,2S,4R)-3 and (1S,2R,4R)-2, are expected to be further
oxidized, in a stereochemical manner, to the specific mutagenic diepoxides which are thought
to play a crucial role in VCH ovotoxicity. Thus, VCH ovotoxicity may be markedly affected by
the reactivity of the diepoxidic stereoisomers formed and detoxicated.
In tr od u ction
hydrolysis to the corresponding vicinal diols (4-6). In
mouse, VCH was found to be metabolized to 1,2-VCHE
much faster than in rat both in vivo (3) and in vitro (7)
and that the hydrolysis of both the monoepoxides and
diepoxide is less efficient in mice than in rats (7). These
data taken together have suggested that the resistance
of the rat to ovarian tumor induction by VCH is due to
the inability of the rat to produce and mantain in the
blood a sufficient amount of diepoxide to cause oocyte
destruction and cancerogeneity. The differences in the
metabolic patterns of VCH in the two species are related
to a different substrate specificity and expression of the
orthologous enzymes implied and in particular to the
P450 2A and 2B enzymes, indicated to be the major
enzymes involved in the epoxidation(s) of VCH (8, 9).
4
-Vinylcyclohexene (VCH, 1) is an industrial compound
used as an intermediate in rubber industry and in
chemical manifactures and produced as a byproduct in
butadiene processing (1). Exposure to this compound is
of toxicological significance, as rodent studies have
pointed out that its toxic effects specifically target the
ovary (2, 3). It is noteworthy that ovarian neoplasms, as
well as follicular loss, are species dependent (3), they
1
occur in B6C3F mice but not in rats, and these variations
are most likely related to differences in the biotransfor-
mation of 1 to ovotoxic monoepoxides and to the di-
epoxide. The latter compound, which has been character-
ized as a direct mutagen and carcinogen (1), is thought
to be the ultimate ovitoxic metabolite of VCH (2).
-Vinylcyclohexene (1) is oxidized by P450 systems to
the isomeric monoepoxides 4-vinylcyclohexene 1,2-ep-
oxide (1,2-VCHE) and 4-vinylcyclohexene 7,8-epoxide
It is noteworthy that the metabolic picture related to
4
1
biotransformation is much more complicated than that
reported in Scheme 1. The metabolism studies, carried
out using 1 as a substrate, have not ever considered the
stereochemistry of the products arising from oxidation
and/or hydrolysis, but the stereochemical aspects in the
biotransformation processes of these compounds may be
important in determining their toxicity. In fact, it has
(
7,8-VCHE) (Scheme 1). Both epoxides are further oxi-
dized to the diepoxide (VCD) in competition with the
†
Universit a` di Pisa.
‡
Istituto di Mutagenesi e Differenziamento.
1
0.1021/tx000255q CCC: $20.00 © 2001 American Chemical Society
Published on Web 04/18/2001

Stereochemistry of 4-Vinylcyclohexene Biotransformation
Chem. Res. Toxicol., Vol. 14, No. 5, 2001 493
Sch em e 1
Ma ter ia ls. PB, Pyr, and â-NF were obtained from common
commercial sources. 4-Vinylcyclohexene (1) and (()-4-vinyl-
cyclohexene 1,2-epoxide (1:1 mixture of the cis and trans
isomers) (2 and 3, respectively) were purchased from Aldrich
Chemical Co. (Milwaukee, WI). TCPO was obtained from EGA-
Chemie (Steinheim-Albuch, Germany). (S)-4-Vinylcyclohexene
(1) was prepared as previously reported (12).
Sep a r a tion of th e 1:1 Mixtu r e of Ep oxid es 2 a n d 3. The
1
2
:1 mixture of epoxides 2 and 3 (3 g, 24 mmol) was dissolved in
0 mL of chloroform, and 5.8 g (29 mmol) of p-nitrobenzoyl
chloride (recrystallized from petroleum ether) was added. With
magnetic stirring, a stream of anhydrous HCl was slowly passed
into the solution over a period of 20 min. After 6 h at room
temperature, the solution was washed with water to remove the
excess acid and evaporated to give a residue which was taken
up in pyridine. A few chips of ice were added, after which the
mixture was extracted with ethyl ether and washed with 5%
3
HCl, and then with a diluted aqueous solution of NaHCO and
water. The combined organic solution was evaporated to give a
crude product (3.1 g) which was purified by column chromatog-
raphy using hexanes and ethyl acetate (8:2) as the eluent. The
fractions containing practically a sole product (trans-2-chloro-
cis-4-vinylcyclohexyl-p-nitrobenzoate) were collected and evapo-
rated: 1H NMR (CDCl
m, 2H), 5.25 (m, 1H), 5.85 (m, 1H), 8.25 (AA′BB′ system, 4
) δ 0.8-2.7 (m, 7H), 4.35 (m, 2H), 5.10
3
(
1
3
aromatic H); C NMR (CDCl
CH), 56.5 (CH), 62.2 (CH), 114 (CH
41 (CH).
To a solution of trans-2-chloro-cis-4-vinylcyclohexyl-p-nitro-
benzoate (150 mg) in methanol (10 mL) was added 150 mg of
Na CO dissolved in 0.1 mL of water. The mixture was refluxed
3
) δ 25.1 (CH
2 2
), 26.55 (CH ), 34.7
(
1
2
), 123.5 (2CH), 130.7 (2CH),
2
3
with stirring for 1 h, then diluted with water, and extracted
with pentane. The organic phase was dried and evaporated to
1
give a residue containing epoxide 2: H NMR (CDCl
2
3
) δ 0.8-
1
3
.0 (m, 7H), 3.05 (m, 2H), 4.85 (m, 2H), 5.5 (m, 1H); C NMR
CDCl ) δ 23.0 (CH ), 26.5 (CH ), 31.0 (CH ), 33.8 (CH), 51.2
CH), 52.7 (CH), 112.7 (CH ), 141.7 (CH).
The cis-epoxide 2 was identified by lithium aluminum hydride
reduction (13), which gives as the major product cis-4-vinyl-
) δ 1.1-1.8 (m, 8H), 2.4 (m, 1H),
.95 (m, 1H), 5.05 (m, 2H), 5.75 (m, 1H); 13C NMR (CDCl
) δ
6.9 (CH ), 32.5 (CH ), 40.8 (CH), 67.5 (CH), 112.9 (CH ), 143.6
(
(
3
2
2
2
2
been shown (10, 11) that the epoxidation of alkyl-
substituted olefins, catalyzed by P450s, may occur with
a stereo- and enantioselectivity which strongly depend
on the specificity and composition of the P450 enzymes,
while induction may favor the microsomal epoxide hy-
drolase (mEH) -catalyzed hydrolysis of epoxides, a proc-
ess which generally occurs with substrate and product
enantioselection.
1
cyclohexanol: H NMR (CDCl
3
3
2
3
2
2
2
(
CH).
(
()-4-Vin ylcycloh exen e 7,8-Ep oxid e (4) a n d (()-4-Vin yl-
1
cycloh exen e 7,8-Ep oxid e (5). 3-Cyclohexene-1-carboxalde-
hyde (3.3 g, 30 mmol) was slowly added to a stirred mixture of
trimethylsulfonium methyl sulfate (6.5 g, 34 mmol) and NaOH
(
7.5 g) in anhydrous dichloromethane (50 mL). After 5 h at room
temperature, water (25 mL) was added. The organic phase was
), and
Therefore, to gain insight into the stereochemical and
mechanistic aspect of the metabolism of 4-vinylcyclo-
hexene, as a first step, we investigated (i) the effect of
the pretreatement of rats with three P450 inducers,
phenobarbital (PB), pyrazole (Pyr), and â-naphthoflavone
separated, washed with water (2 × 15 mL), dried (MgSO
4
concentrated in vacuo. The residue was distilled to give a 1:1
1
mixture of epoxides 4 and 5: yield 3.25 g (86%); H NMR
(
CDCl
3
) δ 1.3-1.6 (m, 2H), 1.7-2.2 (m, 12H), 2.50 (dd, J ) 2.9
(â-NF), and the role of two purified P450s (2B1 and 2E1)
13
and 4.9 Hz, 2H), 2.78 (m, 4H), 5.6 (m, 4H); C NMR (CDCl
3
) δ
in the stereochemistry of 1 oxidation to the isomeric
endocyclic epoxides cis- and trans-4-vinylcyclohexene 1,2-
epoxide (2 and 3, respectively), as well as the isomeric
esocyclic epoxides (4R*,7S*)- and (4R*,7R*)-4-vinyl-
cyclohexene 7,8-epoxide (4 and 5, respectively), and (ii)
the mEH-catalyzed hydrolysis of epoxides 2 and 3, and
23.9 (CH ), 24.3 (CH ), 24.6 (CH ), 25.2 (CH ), 26.9 (CH ), 27.8
2
2
2
2
2
(CH
2
), 35.8 (CH), 36.2 (CH), 45.4 (CH
2
), 46.0 (CH
2
), 55.5 (CH),
55.9 (CH), 125.1 (CH), 125.4 (CH), 126.7 (CH), 127.1 (CH).
(()-4-Vin ylcycloh exen e 1,2-d iol (6) was prepared by acid-
catalyzed hydrolysis (1 N HClO
2
1
4
) of the 1:1 mixture of epoxides
and 3: yield 90%; H NMR (CDCl ) δ 1.4-2.0 (m, 6H), 2.5 (m,
H), 3.4 (m, 1H), 3.6 (m, 1H), 5.05 (d, J ) 16.5 Hz, 1H), 5.05 (d,
1
3
4
and 5, to the corresponding diols 6-8.
J ) 10.5 Hz, 1H), 5.85 (ddd, J ) 16.5, 10.5, and 5.5 Hz, 1H);
1
3
C NMR (CDCl
3 2 2 2
) δ 27.4 (CH ), 27.9 (CH ), 35.7 (CH ), 35.8 (CH),
Ma ter ia ls a n d Meth od s
70.7 (CH), 74.3 (CH), 113.7 (CH ), 127.1 (CH).
2
(
()-4-Vin ylcycloh exen e 7,8-d iol (7) a n d (()-4-vin ylcyclo-
h exen e 7,8-d iol (8) were prepared by acid-catalyzed hydrolysis
1 N HClO ) of the ca. 1:1 mixture of epoxides 4 and 5: yield
0%; H NMR (CDCl ) δ 1.5-2.1 (m, 14H), 3.4-4.0 (m, 6H), 5.65
m, 4H); 13C NMR (CDCl
) δ 24.4 (CH ), 24.8 (2CH ), 25.0 (CH ),
26.9 (CH ), 27.6 (CH ), 36.4 (CH), 36.5 (CH), 64.2 (CH ), 64.6
CH ), 75.7 (CH), 75.8 (CH), 125.5 (CH), 125.9 (CH), 126.7 (CH),
127.2 (CH).
Ca u tion : The following chemicals are hazardous and should
be handled carefully: 4-vinylcyclohexene, 4-vinylcyclohexene 1,2-
epoxide, and 4-vinylcyclohexene 7,8-epoxide.
(
9
(
4
1
3
3
2
2
2
1
2
2
2
Abbreviations: mEH, microsomal epoxide hydrolase; PB, pheno-
(
2
barbital; Pyr, pyrazole; â-NF, â-naphthoflavone; TCPO, trichloro-
propene oxide; GC, gas chromatography.

4
94 Chem. Res. Toxicol., Vol. 14, No. 5, 2001
1R,2S,4S)- a n d (1S,2R,4S)-4-Vin ylcycloh exen e 1,2-Ep -
Chiappe et al.
(
and the solvent was evaporated to give a 1:1 mixture of
(4R,7S)-4 and (4S,7S)-5. The crude product was analyzed by
GC, on a chiral 30 m Chiraldex G-TA (ASTEC) column at 70 °C
(helium flow of 50 kPa, with an evaporator and detector set at
200 °C).
oxid e. Epoxidation of (S)-4-vinyl-1-cyclohexene with m-chloro-
perbenzoic acid in dichloromethane (reaction for 2 days) yielded
a mixture of (1R,2S,4S)- and (1S,2R,4S)-4-vinyl-1-cyclohexene
1
,2-epoxide. The crude product was analyzed by GC on a 50 m
chiral CP-Cyclodex B (CHROMPACK) column at 85 °C (helium
flow of 50 kPa, with an evaporator and detector set at 200 °C).
(4S,7S)-4-Vin ylcycloh exen e 7,8-Ep oxid e (5). (4S,7S)-5
was synthesized from (4S,7S)-8 according to the procedure
reported above for the preparation of the diastereoisomeric
mixture of (4R,7S)-4 and (4S,7S)-5. ee ) 95% (determined by
GC under the conditions reported above).
(4S,7R)-4-Vin ylcycloh exen e 7,8-Ep oxid e (4). (4S,7R)-4
was synthesized from (4S,7R)-7 according to the procedure
reported above for the preparation of the diastereoisomeric
mixture of (4R,7S)-4 and (4S,7S)-5. ee ) 95% (determined by
GC under the conditions reported above).
(
1R,2R,4S)-4-Vin ylcycloh exen e 1,2-Diol (6). Acid-cata-
lyzed (1 N HClO ) hydrolysis of the 1:1 mixture of (1R,2S,4S)-
and (1S,2R,4S)-4-vinyl-1-cyclohexene 1,2-epoxide gave (1R,2R,4S)-
-vinylcyclohexene 1,2-diol (6). The crude product was analyzed
4
4
by GC, after transformation into the corresponding bis-tri-
fluoroacetyl derivatives, on a chiral 30 m Chiraldex G-TA
(ASTEC) column at 80 °C (helium flow of 50 kPa, with an
evaporator and detector set at 200 °C).
P r ep a r a tion of (4S,7S)- a n d (4R,7S)-4-Vin ylcycloh exen e
An im a l Micr osom a l P r ep a r a tion s a n d En zym es. Male
and female Sprague-Dawley rats were purchased from Charles
River. Male rats were treated for 3 days with PB ip (80 mg/kg
daily), â-NF (40 mg/kg daily), or Pyr (200 mg/kg daily), and
microsomes were obtained from the liver as previously described
(15). Microsomal protein concentrations were assayed by using
the method of Lowry et al. (16); the total P450 concentration
was measured according to the method of Omura and Sato (17).
P450 2B1, 2E1, and NADPH-cytochrome P450 reductase were
purified from rat liver as previously described (10).
7
,8-Diol. (1) P r otection of th e En d o Cyclic Dou ble Bon d .
To a solution of 1 (330 mg, 3 mmol) in 1,2-dichloroethane (15
mL) was added a solution of Br (480 mg, 3 mmol) in the same
2
solvent (15 mL), and the reaction mixture was stored in the dark
at room temperature until it was colorless. Then the reaction
mixture was washed with water, and the organic phase, dried
(
MgSO
dibromide 9 (800 mg).
2) Asym m etr ic Dih yd r oxyla tion . The crude product aris-
4
), was evaporated to give a crude mixture containing
(
ing from bromine addition to the endocyclic double bond (800
mg) was dihydroxylated using AD-mix R, according to the
standard procedure reported by the Sharpless group (14).
En zym a tic In cu ba tion s. (1) Deter m in a tion of th e P 450
Activity a n d of th e En a n tiom er ic Excesses of th e P r od -
u cts. Incubation mixtures (2 mL) containing 100 mM potassium
phosphate buffer (pH 7.4), 5 mg of hepatic microsomal proteins,
(
3) Dep r otection of th e En d ocyclic Dou ble Bon d . To an
+
ethanolic solution (8 mL) of diols 10 (300 mg) arising from
the NADPH-generating system, consisting of 0.5 mM NADP ,
asymmetric dihydroxylation were added 300 mg of Zn power
and 20 mg of ZnCl , and the reaction mixture was stirred at
2
room temperature for 2 days. Then the reaction mixture was
filtered and diluted with water, and the reaction products were
extracted three times with ethyl acetate. The organic layer was
5 mM glucose 6-phosphate, and 0.5 unit/mL glucose-6-phosphate
dehydrogenase, and trichloropropene oxide (TCPO, 1 mM), to
inhibit the mEH, were preincubated at 37 °C for 5 min, and
the reactions were initiated by the addition of a proper amount
of 1. The substrate concentration was 10 mM to saturate the
P450 enzymes (7). After 60 min, a saturating amount of NaCl
was added to precipitate the microsomal proteins. The reaction
products were extracted with ethyl acetate (2 × 5 mL) and
analyzed by GC after addition of appropriate amounts of
cyclohexanol as an internal standard. The enantiomeric com-
position of monoepoxides was determined by GC on a 50 m chiral
CP-Cyclodex B (CHROMPACK) column (helium flow of 50 kPa,
with an evaporator and detector set at 200 °C, at 85 °C) for 2
and 3 and on a chiral 30 m Chiraldex G-TA (ASTEC) column
(helium flow of 50 KPa, with an evaporator and detector set at
200 °C, at 70 °C) for 4 and 5.
dried (MgSO
pressure to give a practically 1:1 mixture of (4R,7S)- and
4S,7S)-4-vinylcyclohexene 7,8-diol (7 and 8). The crude product
4
), and the solvent was removed under reduced
(
was analyzed by NMR and GC, after transformation into the
corresponding bis-trifluoroacetyl derivatives, on a chiral 30 m
Chiraldex G-TA (ASTEC) column at 90 °C (helium flow of 50
kPa, with an evaporator and detector set at 200 °C).
P r ep a r a tion of (4S,7S)-4-Vin ylcycloh exen e 7,8-Diol (8).
(4S,7S)-8 was synthesized from (S)-4-vinylcyclohexene (1) ac-
cording to the procedure reported above for the preparation of
the diastereoisomeric mixture of (4R,7S)-7 and (4S,7S)-8. The
crude product was analyzed by GC, after transformation into
the corresponding bis-trifluoroacetyl derivatives, under the
conditions reported above.
The absolute configurations of the excess enantiomers of 2-5
were determined by comparison of the retention times of the
two enantiomers of each isomer with those of enantiomerically
pure compounds obtained as reported above.
P r ep a r a tion of (4S,7R)-4-Vin ylcycloh exen e 7,8-Diol (7).
(
4S,7R)-7 was synthesized from (S)-4-vinylcyclohexene (1) ac-
The epoxidations of 1 were also assessed in a reconstituted
system (0.5 mL) containing 0.1 nmol of purified rat 2B1, or 2E1,
0.3 nmol of P450 reductase, 30 µg of dilauroylphosphatidyl-
choline, and the substrate at a concentration of 10 mM. The
reactions were carried out at 37 °C for 30 min after the addition
of 1 mM NADPH, and the products were extracted and analyzed
as described above.
cording to the procedure reported above for the preparation of
the diastereoisomeric mixture of (4R,7S)-7 and (4S,7S)-8 using
AD-mix â. The crude product was analyzed by GC after
transformation into the corresponding bis-trifluoroacetyl deriva-
tives, under the conditions reported above.
P r ep a r a tion of (4S,7S)-4-Vin ylcycloh exen e 7,8-Ep oxid e
(
5) a n d (4R,7S)-4-Vin ylcycloh exen e 7,8-Ep oxid e (4). To 300
(2) Deter m in a tion of th e P r od u ct En a n tioselectivity of
th e m EH-Ca ta lyzed Hyd r olysis of Mon oep oxid es 2 a n d 3.
Aliquots (10 µL) of an ethanolic stock solution of the mixture of
(()-2 and (()-3 were added to 1 mL of a microsomal preparation
(control) containing 3 or 5 mg of protein/mL in a such way to
obtain a 20 mM substrate concentration, and the reaction
mixtures were incubated at 37 °C. At prefixed times (between
1 and 5 h), a saturating amount of NaCl was added to precipitate
the microsomal proteins and the incubation mixtures were
extracted with hexane (3 × 1 mL) to remove the unreacted
epoxides followed by a brief centrifugation to separate the
emulsion. Cyclohexanol was added as a standard to the com-
bined extracts, which were analyzed directly by GC on a 50 m
chiral CP-Cyclodex B (CHROMPACK) column (helium flow of
50 KPa, with an evaporator and detector set at 200 °C, at 85
mg (2 mmol) of a 1:1 mixture of (4R,7S)-7 and (4S,7S)-8 in 4
mL of dry pyridine was added a solution of p-toluenesulfonyl
chloride (420 mg, 2.2 mmol) in the same solvent (4 mL) at 0 °C.
The mixture was stored in the refrigerator for 12 h and then
diluted with 10 mL of an ice/water mixture. The aqueous layer
was extracted four times with 5 mL of CH
organic solution was washed five times with 5 mL of H
N), followed by a saturated NaHCO solution, dried (MgSO
2
Cl
2
. The combined
SO (2
),
2
4
3
4
and evaporated in vacuo to give a mixture of (4S,7S)-4-
vinylcyclohexene-8-[(p-toluenesulfonyl)oxy] 7,8-diol (11) and
(4R,7S)-4-vinylcyclohexene-8-[(p-toluenesulfonyl)oxy] 7,8-diol
(12). To the crude reaction mixture (400 mg), dissolved in CH Cl
(20 mL), were added 600 mg of KOH and 18-crown-6 (50 mg).
2
2
The mixture, stirred at room temperature for 18 h, was filtered,

Stereochemistry of 4-Vinylcyclohexene Biotransformation
C). The enantiomer ratios and the absolute configurations were
Chem. Res. Toxicol., Vol. 14, No. 5, 2001 495
°
determined for each couple of diastereoisomers by comparison
of the retention times with those of samples of enantiomerically
pure epoxides and diols obtained as reported above.
The incubation mixtures were then extracted with ethyl
acetate (5 × 1 mL), and the enantiomer ratios and the absolute
configurations of the formed diols were determined by GC on a
chiral 30 m Chiraldex G-TA (ASTEC) column (helium flow of
5
0 KPa, with an evaporator and detector set at 200 °C, at 80
°
C), after transformation into the corresponding bis-trifluoro-
acetyl derivatives, by comparison of the retention times with
those of a sample of pure (1R,2R,4S)-4-vinylcyclohexene 1,2-
diol (6) obtained as reported above. Blank experiments carried
out by boiling deactivated microsomal preparations show that
spontaneous hydrolysis does not contribute to diol formation
under the incubation conditions.
(
3) Deter m in a tion of th e P r od u ct En a n tioselectivity of
th e m EH-Ca ta lyzed Hyd r olysis of Mon oep oxid es 4 a n d 5.
Aliquots (10 µL) of an ethanolic stock solution of a 46:54 mixture
of (()-4 and (()-5 were added to 1 mL of a microsomal
preparation (control) containing 2 or 3 mg of protein/mL in a
such way to obtain a 20 mM substrate concentration, and the
reaction mixtures were incubated at 37 °C and pH 7.4. At
prefixed times (1, 2, 3, and 4 h), a saturating amount of NaCl
was added to precipitate the microsomal proteins and the
incubation mixtures were extracted with hexane (3 × 1 mL) to
remove the unreacted epoxides followed by a brief centrifugation
to separate the emulsion. Cyclohexanol was added as a standard
to the combined extracts, which were analyzed directly by GC
on a 30 m Chiraldex G-TA (ASTEC) column (helium flow of 50
KPa, with an evaporator and detector set at 200 °C, at 70 °C).
The enantiomer ratios and the absolute configurations were
determined for each couple of diastereoisomers by comparison
of the retention times with those of samples of pure (4S,7S)-4-
vinylcyclohexene 7,8-epoxide (5), (4R,7S)-4-vinylcyclohexene 7,8-
epoxide (4), and (4S,7R)-4-vinylcyclohexene 7,8-epoxide (4)
obtained as reported above.
F igu r e 1. (A) Gas chromatographic enantiomer separation of
(
1R,2S,4S)-2, (1S,2R,4R)-2, (1R,2S,4R)-3, and (1S,2R,4S)-3. (B)
Gas chromatographic enantiomer separation of (4R,7S)-4, (4S,7R)-
4, (4R,7R)-5, and (4S,7S)-5. Analysis conditions are outlined in
Materials and Methods.
Sch em e 2
The incubation mixtures were then extracted with ethyl
acetate (5 × 1 mL), and the enantiomer ratios and the absolute
configurations of the formed diols were determined by GC on a
GC 30 m Chiraldex G-TA (ASTEC) column (helium flow of 50
KPa, with an evaporator and detector set at 200 °C, at 90 °C),
after transformation into the corresponding bis-trifluoroacetyl
derivatives, by comparison of the retention times with those of
samples of (4R,7S)-4-vinylcyclohexene 7,8-diol (7), (4S,7S)-4-
vinylcyclohexene 7,8-diol (8), and (4S,7R)-4-vinylcyclohexene
ence substances with unequivocal stereochemistries was
necessary.
Syn th esis of Refer en ce VCH Meta bolites. The
separation of the 1:1 mixture of epoxides 2 and 3 was
achieved by transformation into the regioisomeric mix-
ture of the two corresponding chloro-p-nitrobenzoates,
which were purified by column chromatography, and
the isolated trans-2-chloro-cis-4-vinylcyclohexyl-p-nitro-
benzoate was again transformed into the epoxides 2 by
treatment with potassium carbonate in methanol. (S)-4-
Vinylcyclohexene was prepared from d-pantolacton via
an asymmetric Diels-Alder reaction, as previously re-
ported (12). The epoxidation of (S)-4-vinylcyclohexene
with m-chloroperbenzoic acid led to the diastereoisomeric
(1R,2S,4S)-2 and (1S,2R,4S)-3. Since the retention times
of the cis and trans isomers have been known, it has been
possible to attribute at each GC peak the absolute
stereochemistry. The diastereoisomeric mixture of ep-
oxides (1R,2S,4S)-2 and (1S,2R,4S)-3 was transformed
7
,8-diol (7) obtained as reported above. Blank experiments
carried out by boiling deactivated microsomal preparations show
that spontaneous hydrolysis does not contribute to diol forma-
tion under the incubation conditions.
Resu lts a n d Discu ssion
The stereochemical course of epoxidation of the endo
and exocyclic double bond of 4-vinylcyclohexene and of
the hydrolysis of the corresponding formed monoepoxides
2
and 3, and 4 and 5, was investigated using various
microsomal preparations from male and female rats and
two purified P450s (2B1 and 2E1). This investigation has
also been carried out with male rats since the level of
VCH oxidation on the exocyclic double bond with mi-
crosomes from female rats was found, in keeping with
previous authors (7), to be too low to allow a stereochem-
istry study. As a convenient analytical tool, inclusion gas
chromatography, which enables a time-dependent enan-
tiomer screening of epoxides and diols in the nanomole
range, was employed for the determination of the enan-
tiomeric ratios (Figure 1) and for the absolute configura-
tions. To use this method to determine the absolute
configuration of the metabolites, the synthesis of refer-
4
into the corresponding diol (1R,2R,4S)-6 by HClO -
catalyzed hydrolysis. A ca. 1:1 mixture of (4R,7S)-4-
vinylcyclohexene 7,8-epoxide (4) and (4S,7S)-4-vinyl-
cyclohexene 7,8-epoxide (5) was prepared in five steps
via a monotosyl derivative from the corresponding diol
mixture [(4R,7S)-7 and (4S,7S)-8], the latter being syn-
thesized by asymmetric Sharpless dihydroxylation using
AD-mix R of olefin 1, previously protected on the endo-
cyclic double bond by bromination (14) (see Scheme 2).

4
96 Chem. Res. Toxicol., Vol. 14, No. 5, 2001
Chiappe et al.
Ta ble 1. Ster eoch em ica l Cou r se for th e Oxid a tion on th e En d ocyclic Dou ble Bon d of 1 by Micr osom es fr om Ma le a n d
F em a le Con tr ol a n d Tr ea ted Ra ts a n d P u r ified P 450s (2B1 a n d 2E1)a
(
1R,2S,4S)-2 (b)c
(1S,2R,4R)-2 (d)c
(1R,2S,4R)-3 (c)c
(1S,2R,4S)-3 (a)c
[nmol (%)]
[nmol (%)]
[nmol (%)]
[nmol (%)]
CTR
CTR
PB
Pyr
â-NF
22 (31)
8 (17)
(<1)
26 (37)
27 (56)
125 (22)
52 (41)
27 (32)
12 (28)
30 (39)
23 (32)
10 (21)
125 (22)
23 (19)
23 (28)
5 (12)
b
3 (6)
85 (15)
29 (23)
12 (15)
16 (37)
6 (7)
232 (41)
21 (17)
21 (25)
10 (23)
32 (41)
2
2
E1
B1
10 (13)
a
Each value represents the mean of at least two determinations. b From female rats. c a-d refer to the pathways reported in Scheme
3
.
Ta ble 2. Ster eoch em ica l Cou r se for th e Oxid a tion on th e
Exocyclic Dou ble Bon d of 1 by Micr osom es fr om Ma le
Sch em e 3
a
Con tr ol a n d Tr ea ted Ra ts
(
4R,7S)-4 (d)b (4S,7R)-4 (b)b (4R,7R)-5 (c)b (4S,7S)-5 (a)b
[nmol (%)]
[nmol (%)]
[nmol (%)]
[nmol (%)]
CTR
PB
Pyr
41 (40)
27 (21)
17 (23)
(<5)
30 (30)
32 (25)
(<1)
15 (15)
19 (15)
40 (55)
19 (42)
15 (15)
51 (39)
16 (22)
18 (40)
â-NF
(<5)
a
Each value represents the mean of at least two determinations.
b
a-d refer to the pathways reported in Scheme 4.
The pure (4S,7S)-4 and the pure (4S,7R)-5, and the
corresponding diols (4S,7S)-8 and (4S,7R)-7, were pre-
pared following the same synthetic pathway starting
from pure (4S)-1 and using AD-mix R and AD-mix â in
the dihydroxylation step, respectively.
Sch em e 4
Ep oxid a tion of 4-Vin ylcycloh exen e 1 by Ra t Liver
Micr osom es. 4-Vinylcyclohexene possesses two prochiral
carbon-carbon double bonds and a chiral center. The
enzymatic epoxidation of the chiral diene represents a
competitive process between the enantiomers which can
occur with regioselectivity, and substrate and/or product
enantio- and diastereoselectivity. The correct determi-
nation of regio- and stereoselectivity in the epoxidation
reactions requires the complete inhibition of epoxide
hydrolase, since the enzymatic hydrolysis of the epoxides
may represent an efficient competing enantioselective
process. The stereochemical course of the double bond
oxidation of 1 in the corresponding endo- and exocyclic
epoxides 2 and 3, and 4 and 5, was investigated by
incubating 1 (10 mM) in the presence of TCPO (1 mM),
an efficient inhibitor of epoxide hydrolase (18), by using
liver microsomes obtained from untreated rats or rats
pretreated with PB, Pyr, and â-NF, classical inducers of
oxidized primarily the endocyclic double bond. With PB-
induced microsomes which give the highest yield in
oxidation products, the reaction is characterized by a
remarked regioselectivity favoring the formation of the
endocyclic epoxides. It is noteworthy that, while the PB-
induced microsomes and, to a lower extent, the Pyr-
induced microsomes, increase the total amount of endo-
cyclic double bond oxidation products, these inducers, as
well the â-NF-induced microsomes, practically do not
enhance the chemical yield of the exocyclic double bond
oxidation products. The stereochemical behavior strongly
depends on the specificity and composition of the P450
isoforms. It must be remarked that for each double bond
the stereochemical results, arising from the competition
among processes a-d (Schemes 3 and 4), may be ra-
tionalized considering three stereoselective processes:
substrate enantioselectivity, product diastereoselectivity,
and product enantioselectivity.
The endocyclic double bond oxidation proceeds with
control male microsomes with a moderate substrate
enantioselectivity (63:37), given by a + b versus c + d,
in favor of the (4S)-enantiomer, which is however in-
verted when induced microsomes are used. On the other
hand, the substrate enantioselectivity in the exocyclic
double bond oxidation with control male microsomes is
very low, 45:55, in favor of the (4R)-enantiomer, but
increases to 22:78 using Pyr-induced microsomes, while
a moderate selection 64:36 in favor of the (4S)-enantiomer
was found using PB-induced microsomes. Also, the
2
B1/2-3A, 2E1, and IA1/2, respectively (15). At prefixed
times (60 min), the reactions were stopped by adding a
saturating amount of NaCl, and the incubation mixtures
were extracted with ethyl acetate and analyzed by GC
on a chiral column, after addition of cyclohexanol as an
internal standard, to evaluate the chemical yields. The
use of the chiral column allowed evaluation of not only
the diastereoisomeric ratio of the formed epoxides but
also the enantiomeric ratio for each diastereoisomer and
the absolute configuration of the enantiomer in excess
(see Figure 1). The latter was evaluated by the compari-
son of the retention time of the formed products with
those of samples of known configuration prepared as
reported above.
Related to the stereochemical behavior of the epoxi-
dation of 1, the data reported in Tables 1 and 2 show
that both double bonds are oxidized by liver microsomes
from male rats, whereas microsomes from female rats

Stereochemistry of 4-Vinylcyclohexene Biotransformation
Chem. Res. Toxicol., Vol. 14, No. 5, 2001 497
product diastereoselectivity, given by a versus b and c
versus d, shows a behavior that is inducer- and gender-
dependent. The epoxidation of the endocyclic double bond
of (4R)-1 with male control microsomes occurs with an
extremely high face selectively to give exclusively trans-
epoxide 3, while that of the (4S)-enantiomer is practically
nonselective. With female control microsomes, the enan-
tiofacial selectivity in the endocyclic double bond oxida-
tion of both enantiomers (4R and 4S) is similar to that
observed with male microsomes but less pronounced. The
induction generally reduces the product diastereoselec-
tivity characterizing the epoxidation of the (4R)-enanti-
omer, while with PB-induced microsomes, a moderate
enantioselection in favor of the cis-epoxide 2 has been
observed. In the oxidation of (4S)-1, instead a moderate
effect has been found only in the case of â-NF-induced
microsomes. Related to the exocyclic double bond oxida-
tion, the use of control microsomes gives the four dia-
stereoisomeric epoxides with a moderate diastereoselec-
tivity in favor of (4S,7R)-4 and (4R,7S)-4 which however
is reversed, favoring epoxides (4S,7S)-5 or (4R,7R)-5, with
induced microsomes.
2E1 are similar to those obtained using PB- and Pyr-
induced microsomes, respectively, there are some differ-
ences which may be attributed to the involvement in the
oxidation process of other isoforms present in the mi-
crosomal preparations.
In conclusion, the oxidation data for 1 seem to indicate
that, as previously observed (10, 11) for the stereoselec-
tivity in the P450 oxidation of prochiral and chiral olefins,
besides 2B1 and 2E1, other isoforms including the major
ones expressed in male and female rats are involved in
the epoxidation process and the single enzymes catalyze
the reaction with a different stereoselectivity, substrate
enantioselectivity, product enantioselectivity, and prod-
uct diastereoselectivity. The different stereoselectivity
may imply a different chemical recognition which can be
determined by the active site architecture of the P450
isoenzymes able to give distinct interactions of the two
enantiomers of the substrate in the fundamental state
(affecting the binding) and/or in the active complex
(affecting kcat). However, at least concerning the product
diastereoselectivity, the possibility that the different
behavior may arise from the involvement of different iron
species (peroxo, hydroperoxo, or oxenoid), or the two
reactive states of the same active species, which can
perform the epoxidation also through different mecha-
nisms as recently suggested for alkyl hydroxylation (19),
cannot be excluded.
Finally, the results reported in Tables 1 and 2 may be
discussed in terms of product enantioselectivity, given
by a versus c and by b versus d, arising from the
differentiation between the two enantiotopic faces of the
double bond of the two enantiomers. When the formation
of cis-epoxide 2 is taken into account, male control
microsomes and female ones, although to a lesser extent,
showed a high product enantioselectivity in favor of
enantiomer (1R,2S,4S)-2. This selectivity markedly de-
creases with Pyr-induced microsomes and increases again
in favor of the cis-(1S,2R,4R)-2 enantiomer using PB- and
â-NF-induced microsomes. On the contrary, trans-epoxide
Hyd r olysis of Mon oep oxid es 2 a n d 3, a n d 4 a n d
5
3
. The P450-catalyzed oxidation of monoepoxides 2 and
, as well as 4 and 5, to the corresponding diepoxides
competed with the hydrolysis of the monoepoxides to the
corresponding vicinal diols (see Scheme 1), the latter
process of which is exclusively due to the enzymatic
reaction. Blank experiments carried out using heat-
deactivated microsomal preparations showed indeed that
the spontaneous hydrolysis can always be neglected. To
evaluate the stereoselectivity of the mEH-catalyzed hy-
drolysis of the monoepoxides, therefore, the experiments
were carried out by incubating the 50:50 mixture of the
two distereoisomeric endocyclic epoxides 2 and 3 (20
mM), or the 46:54 mixture of exocyclic epoxides 4 and 5,
with a microsomal preparation from control rats, con-
taining 2-3 mg of protein/mL, at 37 °C and pH 7.4. The
incubations were stopped at different times, and the
residual epoxides and the formed diols (after derivatiza-
tion) were identified by GC, after addition of cyclohexanol
as an internal standard, on a chiral column. The results
are reported in Tables 3 and 4.
3
is formed in a practically racemic way when the
incubations were carried out using male control mi-
crosomes but not female ones. When Pyr-induced mi-
crosomes were used, the reaction is characterized by a
moderate selectivity in favor of (1R,2S,4R)-3. Related to
the exocyclic double bond oxidation, the product enantio-
selectivity is low or completely absent when control or
â-NF-induced microsomes are used but increases with
all the other inducers. The oxidation with Pyr-induced
microsomes gives exclusively epoxide (4R,7S)-4 and
selectively (46% ee) epoxide (4R,7R)-5. At variance, PB-
induced microsomes give epoxide 4 without any product
enantioselection, while in the case of 5, the formation of
the (4S,7S)-enantiomer is favored.
It is noteworthy that all the monoepoxides were
substrates for mEH but the hydrolysis rate of epoxides
4 and 5 was 3-4 times higher than that of epoxides 2
and 3. Furthermore, the stereochemical results reported
in Table 3 show that the hydrolysis of the 1:1 mixture of
epoxides 2 and 3 proceeds with a high substrate dia-
stereo- and enantioselectivity, as well as with a high
product enantioselectivity. In agreement with the pre-
viuosly obtained results about the mEH-catalyzed hy-
drolysis of epoxy derivatives of cyclohexenes (20), the
hydrolysis takes place exclusively by diaxial opening of
the oxirane ring, and the two diaxial diol enantiomers,
(1R,2R,4S)-6 and (1S,2S,4R)-6, were the sole products
(Scheme 5). The four diastereoisomeric epoxides are
transformed into the corresponding diols with a different
rate, showing that, as observed in the mEH-catalyzed
hydrolysis of the stereoisomeric 4-tert-butyl-1,2-epoxy-
cyclohexanes (20), the helicity of the cyclohexane ring and
The stereoselectivity of the oxidation of 1 was also
evaluated by reconstituting the monooxygenase reaction
in the presence of DLPC, NADPH-cytochrome P450
reductase, purified rat liver 2B1 or 2E1, and NADPH.
Only epoxides 2 and 3, arising from the regioselective
oxidation of the endocyclic double bond, were extracted
from the reconstituted system of both P450 2B1 and 2E1
and analyzed by GC on the chiral column. It is of interest
to note also that 2E1, although to a lesser extent than
2
B1, is able to perform the oxidation on the endocyclic
double bond, in agreement with the results obtained with
Pyr-induced microsomes. The data, reported in Table 1,
show a high substrate enantioselectivity for the 2B1 but
not for the 2E1, in favor of the (4R)-enantiomer which is
associated with a high product enantioselectivity, at least
concerning the formation of cis-epoxide 2. It is noteworthy
that although the results obtained using purified 2B1 and

4
98 Chem. Res. Toxicol., Vol. 14, No. 5, 2001
Chiappe et al.
Ta ble 3. Tim e a n d Ster eoch em ica l Cou r se of th e m EH-Ca ta lyzed Hyd r olysis of a 50:50 Mixtu r e of En d ocyclic Ep oxid es 2
a n d 3a
residual epoxides (%)
(1S,2R,4R)-2 (1R,2S,4R)-3
25 25
formed diols (%)
time (h)
0
hydrolysis (%)
(1R,2S,4S)-2
(1S,2R,4S)-3
(1R,2R,4S)-6
(1S,2S,4R)-6
0
25
25
19
3
25
-
100
100
86
-
0
0
0
1
3
2
.5
6.5
17.5
63
25
25
11
0
25
25
25
5
17.5
12.5
0
14
50
1
95
0
0
50
a
Each value represents the mean of at least two determinations.
Ta ble 4. Tim e a n d Ster eoch em ica l Cou r se of th e m EH-Ca ta lyzed Hyd r olysis of a 46:54 Mixtu r e of Exocyclic Ep oxid es 4
a n d 5a
residual epoxides (%)
formed diols (%)
(4S,7R)-7 (4R,7R)-8
time (h)
hydrolysis (%)
(4R,7S)-4
(4S,7R)-4
(4R,7R)-5
(4S,7S)-5
(4R,7S)-7
(4S,7S)-8
0
1
2
6
0
9
41
99
23
23
20
1
23
19
7
27
22
9
27
27
22
0
-
-
-
-
-
-
14
27
44
34
23
55
43
27
9
0
0
23
a
Each value represents the mean of at least two determinations.
Sch em e 5
Sch em e 6
the orientation of the substituent (vinyl group) with
respect to the oxirane ring play an important role in
determining the substrate diastereoselectivity. In ac-
cordance with the hydrolysis of 4-tert-butyl-1,2-epoxy-
cyclohexanes, the first metabolized epoxide is trans-
Finally, related to the hydrolysis of the exoepoxides 4
and 5, the data reported in Table 4 show that, as
generally observed in the mEH-catalyzed hydrolysis of
monosubstituted epoxides (22), the nucleophilic attack
occurs regioselectively on the less substituted carbon to
give, with uncomplete hydrolysis, the corresponding diols
having at C(7) the same configuration of the transformed
epoxides. Furthermore, the reaction occurs with a high
level of substrate enantioselection, which previously was
observed exclusively in the mEH-catalyzed hydrolysis of
monosubstituted epoxides bearing branched bulky alkyl
groups, suggesting that the cyclohexane ring is able to
satisfy this requirement (Scheme 6). For both diastereo-
isomeric epoxides, the enantiomer which is preferentially
hydrolyzed is that having the (R)-configuration at the
oxirane carbon, i.e., the enantiomer bearing the cyclo-
hexenyl group in the proper position to interact with the
hypothesized mEH hydrophobic pocket (22) situated to
the right backside of the epoxide binding site when the
epoxide is oriented with the oxygen upward. This should
be the enantiomer which gives the more stable enzyme-
substrate complex, and acts as a competitive inhibitor
toward the other enantiomer. The mEH-catalyzed hy-
drolysis of the ca. 1:1 mixture of the two epoxides 4 and
(1S,2R,4S)-3 having the six-membered ring held in the
conformation of (3,4 M) elicity, which generally is pref-
erentially hydrolyzed (20, 21), the substituent on the
right when the epoxide is oriented with the oxygen
upward, and attack takes place on the (S)-carbon.
However, in the case of cis-epoxide 2, at variance with
the results mentioned above for 4-tert-butyl-1,2-epoxy-
cyclohexanes, nucleophilic attack preferentially occurs on
the (S)-carbon of the enantiomer having the (3,4 M)
conformation, showing that for this substrate these
features are more important than the disposition of the
substituent with respect the oxirane ring. This aspect
seems to determine the relative reactivities of the two
enantiomers of cis-4-tert-butyl-1,2-epoxycyclohexane. It
is however possible that the two substituents, which have
different steric requirements and different abilities to
interact with the mEH active site, may affect in a
different way the balance of the factors which determine
the substrate enantioselectivity.

Stereochemistry of 4-Vinylcyclohexene Biotransformation
proceeds however without substrate diastereoselection;
the two epoxides, (4S,7R)-4 and (4R,7R)-5, are hydrolyzed
practically with the same rate, showing that for these
substrates the stereochemistry at the carbon adjacent to
the oxirane ring does not affect the reaction selectivity.
Chem. Res. Toxicol., Vol. 14, No. 5, 2001 499
col. Appl. Pharmacol. 105, 372-381.
5
(
4) Smith, B. J ., Carter, D. E., and Sipes, I. G. (1990) Comparison of
the disposition and in vitro metabolism of 4-vinylcyclohexene in
the female mouse and rat. Toxicol. Appl. Pharmacol. 105, 364-
3
71.
(5) Carpenter, S. C., and Keller, D. A. (1994) Species differences in
metabolism of 4-vinylcyclohexene (VCH) and its epoxide metabo-
lites in vitro. Toxicologist 14, 338 (Abstract 1312).
Con clu sion s
(
6) Gervasi, P. G., Abboandandolo, A., Citti, L., and Turchi, G. (1980)
Microsomal 4-vinylcyclohexene monooxygenase and mutagenic
activity of metabolite intermediates. In Proceedings of the Inter-
national Conference of Industrial and Enviromental Xenobiotics:
Biotransformation and Pharmacokinetics (Gut, I., Ed.) pp 205-
In conclusion, these results show that the stereochem-
ical course of the biotransformation of 4-vinylcyclohexene
formulates a composite picture markedly affected by the
composition of different P450s present in the male and
female control or induced rat liver.
2
10, Springer-Verlag, Berlin.
7) Keller, D. A., Carpenter, S. C., Cagen, S. Z., and Reitman, F. A.
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(
(
The stereochemical composition of the primarily formed
metabolites, endo- and exocyclic monoepoxides 2 and 3,
and 4 and 5, strongly depends on the specificity and
composition of the P450 isoforms, but it also affected by
the substrate stereo- and enantioselectivity character-
izing the mEH-catalyzed hydrolysis of these substrates.
The hydrolysis of the endocyclic epoxides occurs with a
good substrate diastereo- and enantioselectivity favoring
the hydrolysis of epoxides (1S,2R,4S)-3 and (1R,2S,4S)-2
to give, before 50% conversion, selectively (1R,2R,4S)-
diol 6. At variance, the hydrolysis of the exocyclic
epoxides is characterized by a high level of substrate
enantioselection associated with very little, if any, sub-
strate distereoselection, and the two epoxides, (4S,7R)-4
and (4R,7R)-5, are hydrolyzed practically with the same
rate.
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of cytochrome P-450 enzymes after repaeted exposure to 4-vinyl-
1
cyclohexene in B6C3F0 mice. Drug. Metab. Dispos. 27, 281-287.
(
9) Smith, B. J ., Sipes, I. G., Stevens, J . C., and Halpert, J . R. (1990)
The biochemical basis for the species difference in the microsomal
4-vinyl-cyclohexene epoxidation between female mice and rats.
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10) Chiappe, C., De Rubertis, A., Amato, G., and Gervasi, P. G. (1998)
Stereochemistry of the biotransformation of 1-hexene and 2-meth-
yl-1-hexene with rat liver microsomes and purified P450s of rats
and humans. Chem. Res. Toxicol. 11, 1487-1493.
(11) Chiappe, C., De Rubertis, A., Tinagli, V., Amato, G., and Gervasi,
P. G. (2000) Stereochemical course of the biotransformation of
isoprene monoepoxides and of the corresponding diols with liver
microsomes from control and induced rats. Chem. Res. Toxicol.
(
1
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(
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4
major epoxides to be further oxidated to diepoxides. The
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selective manner. Thus, on the basis of the balance
between the formation and detoxication pathway and
their reactivity, specific diepoxidic stereoisomers may
account for the VCH ovotoxicity in a given species.
Work is underway to investigate the stereochemical
aspects of the second part of VCH biotransformation in
the rat. Next, the availability of the reference stereo-
isomers will allow a stereochemical study in mice and
humans, and by comparison, it will be possible to reveal
the toxicological significance deriving from the VCH
whole metabolism.
(
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(
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(
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(
18) Oesch, F., Kaubisch, N., J erina, D. M., and Daly, J . W. (1971)
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ation by cytochrome P450: Stepwise and effectively concerted
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(
Ack n ow led gm en t. This work was supported by
grants from Consiglio Nazionale delle Ricerche (CNR,
Rome, Italy) and Universit a` di Pisa.
8
977-8989.
(
20) Bellucci, G., Berti, G., Ingrosso, G., and Mastrorilli, E. (1980)
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3
(
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(
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