Isoprene Monoepoxide Biotransformation
Chem. Res. Toxicol., Vol. 13, No. 9, 2000 833
129.9, 139.9, 145.0. Compound 10 (ee ) 95%, determined by
(consisting of 0.5 mM NADP+, 5 mM glucose 6-phosphate, and
0.5 unit/mL glucose-6-phosphate dehydrogenase), and TCPO (1
mM) to inhibit the mEH were initiated by the addition of 4 or
5 (30 mM). After 60 min, a saturating amount of NaCl was
added to precipitate the microsomal proteins and the reaction
mixtures were lyophilized. The solid phase was then dissolved
in ethyl acetate (2 mL), filtered, and analyzed by GC (NPGS
column, 120 °C) after addition of appropriate amounts of an
ethyl acetate stock solution of cycloheptanediol as an internal
standard.
1
GC). H NMR (CDCl3): δ 1.68 (s, 3H), 2.45 (s, 3H), 3.95 (dd, J
) 7.5 and 10.2 Hz, 1H), 4.10 (dd, J ) 3.4 and 10.2 Hz, 1H), 4.30
(dd, J ) 3.4 and 7.5 Hz, 1H), 4.95 (s, 1H), 5.05 (s, 1H), 7.35 (d,
AB system, 2H), 7.80 (d, AB system, 2H). 13C NMR (CDCl3): δ
18.5, 21.6, 72.34, 72.78, 113.58, 127.9, 129.9, 132.5, 141.9, 145.0.
2(S)-Isop r op en yloxir a n e (3). To a solution of 10 (50 mg)
in CH2Cl2 (2 mL) were added 50 mg of KOH and crown-18-ether
(5 mg). The mixture, stirred at room temperature for 18 h, was
filtered, and the solvent was evaporated to give (S)-3 (ee ) 95%,
determined by GC).
2(R)-Met h yl-2-vin yloxir a n e a n d 2(S)-Met h yl-2-vin yl-
oxir a n e (2). The 2:1 mixture of the two enantiomers of 2 was
prepared from the corresponding 2:1 mixture of 2(S)-2-methyl-
1-[p-(toluenesulfonyl)oxy]-3-butene-1,2-diol and 2(R)-2-methyl-
1-[p-(toluenesulfonyl)oxy]-3-butene-1,2-diol like 3. The crude
product was analyzed by GC.
(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 2 a n d 3.
Aliquots (10 µL) of an ethanolic stock solution of (()-2 or (()-3
were added to 1 mL of a microsomal preparation (control or PB-
induced) containing 2 or 4 mg of protein/mL in a such way to
obtain a substrate concentration of 5, 10, or 20 mM, and the
reaction mixtures were incubated at 37 °C. At prefixed times
(10, 25, 50, and 80 min), a saturating amount of NaCl was added
to precipitate the microsomal proteins, and the incubation
mixtures, after addition of a proper amount of cycloheptanol as
an internal standard, were analyzed directly by GC (Carbowax
column, 120 °C) to determine the diol yields.
(2S,2′R)- a n d (2R,2′R)-2-Meth yl-2,2′-bioxir a n e (6). Epoxi-
dation of 2(S)-isopropenyloxirane with m-chloroperbenzoic acid
in CH2Cl2 (2 h) gave a 6:4 mixture of (2S,2′R)- and (2R,2′R)-2-
methyl-2,2′-bioxirane (6). The crude product was analyzed by
GC.
Mixtu r e of Diep oxid es 6 En r ich ed in (2R,2′S)- a n d
(2R,2′R)-2-Meth yl-2,2′-bioxir a n e (6). Epoxidation of the 2:1
mixture of 2(R)-methyl-2-vinyloxirane and 2(S)-methyl-2-vinyl-
oxirane with m-chloroperbenzoic acid in CH2Cl2 gave a mixture
of the four epoxides enriched in the (2R,2′S)- and (2R,2′R)-
diastereoisomers. The crude product was analyzed by GC.
(2R,3R)- a n d (2R,3S)-3,4-Ep oxy-3-m eth yl-1,2-d iol (8). The
mixture of (2R,3R)- and (2R,3S)-8 was prepared from 2(S)-3-
methyl-3-butene-1,2-diol (5) like 8.
Mixtu r e of Ep oxyd iols 7 En r ich ed in (2R,3S)- a n d
(2R,3R)-3,4-Ep oxy-2-m eth yl-1,2-d iol (7). The mixture of ep-
oxydiols 7 enriched in the diastereoisomers (2R,3S)- and (2R,3R)-7
was prepared from 2(S)-2-methyl-3-butene-1,2-diol (4) like 7.
An im a ls a n d Micr osom a l P r ep a r a tion s. Male Sprague-
Dawley rats were purchased from Charles River. They were
treated for 3 days with PB ip (80 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).
The enantiomer ratios and the absolute configurations of the
formed diols were determined, after extraction with ethyl
acetate (5 × 1 mL) by GC on a chiral 30 m Chiraldex G-TA
(ASTEC) column (helium flow of 50 KPa, and with an evaporator
and detector set at 200 °C, at 85 °C) by comparison of the
retention times with those of samples of 2(S)-3-methyl-3-butene-
1,2-diol [(S)-5], 2(S)-2-methyl-3-butene-1,2-diol [(S)-4], and 2(R)-
2-methyl-3-butene-1,2-diol [(R)-4] obtained as reported above.
(4) 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 Diep oxid es 6. Aliquots
(10 µL) of an ethanolic stock solution of a 60:40 mixture of (()-
erythro-6 and (()-threo-6 were added to 1 mL of a microsomal
preparation (control or PB-induced) containing 2.5 or 5 mg of
protein/mL in a such way to obtain a substrate concentration
of 10, 25, or 50 mM, and the reaction mixtures were incubated
at 37 °C and pH 7.4. At prefixed times (30 min and 1, 2, 4, and
6 h), a saturating amount of NaCl was added to precipitate the
microsomal proteins, and the incubation mixtures, after addition
of a proper amount of cycloheptanediol as an internal standard,
were analyzed directly by GC (NPGS column, 120 °C) to
determine the epoxydiol yields.
En zym a tic In cu ba tion s. (1) Deter m in a tion of th e Cyto-
ch r om 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 in th e Oxid a tion of Mon oep oxid es 2 a n d
3. Incubation mixtures (2 mL) containing 100 mM potassium
phosphate buffer (pH 7.4), 4 mg of hepatic microsomal proteins,
a NADPH-generating system (consisting of 0.5 mM NADP+, 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 initiated by the addition of a proper
amount of 2 or 3. 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 (Carbowax column, 90 °C) after addition of
appropriate amounts of cycloheptanone as an internal standard.
The substrate concentrations ranged from 0.1 to 20 mM.
The enantiomeric composition of 6 was determined by GC
using a 30 m Chiraldex G-TA (ASTEC) column, with a helium
flow of 50 KPa, and with an evaporator and detector set at 200
°C, under the following conditions: 32 °C for 12 min, at a rate
of 10 °C/min, 55 °C for 4 min, at a rate of 10 °C/min, and 75 °C
for 22 min.
The enantiomer ratio and the absolute configurations of the
formed epoxydiols 7 and 8 at 20% conversion (6 h) were
determined, after dehydration and extraction with ethyl acetate,
by GC on a chiral 30 m Chiraldex G-TA (ASTEC) column
(helium flow of 50 KPa, and with an evaporator and detector
set at 200 °C, at 90 °C) by comparison of the retention times
with those of samples of erythro-(2R,3S)-3,4-epoxy-2-methyl-1,2-
diol and threo-(2R,3R)-3,4-epoxy-2-methyl-1,2-diol obtained in
excess by epoxidation of a 2:1 mixture of (S)-2-methyl-3-butene-
1,2-diol and (R)-2-methyl-3-butene-1,2-diol, as reported above.
The erythro and threo isomers have been identified, on the same
column, on the basis of the ratio (around 6:4) between the two
pairs of enantiomers corresponding to the ratio determined by
NMR.
(5) Eva lu a tion of th e m EH-Ca ta lyzed Hyd r olysis of
E p oxyd iols 7 a n d 8. Aliquots (10 µL) of an ethanolic stock
solution of mixtures of (()-erythro-7 and (()-threo-7 or (()-
erythro-8 and (()-threo-8 were added to 2 mL of microsomal
preparation (control or PB-induced) containing 2.5 or 5 mg of
protein/mL in a such way to obtain a substrate concentration
of 50 mM, and the reaction mixtures were incubated at 37 °C
and pH 7.4. At prefixed times (at 30 min and 1, 2, 4, and 6 h),
a saturating amount of NaCl was added to precipitate the
microsomal proteins, and the incubation mixtures, after addition
of a proper amount of cycloheptanediol as an internal standard,
were analyzed directly by GC (NPGS column, 120 °C) to
determine the epoxydiol yields. The epoxydiols 7 or 8 were
always quantitatively recovered, showing that these compounds
were not substrates for the mEH.
The absolute configurations of the excess enantiomers of 6
were determined by comparison of the retention times of the
two enantiomers of each isomer with those of (2S,2′R)-, (2R,2′R)-,
and (2R,2′S)-6 obtained as reported above.
(2) Deter m in a tion of th e Cytoch r om e P 450 Activity in
th e Oxid a tion of Diols 4 a n d 5. Incubation mixtures (10 mL)
containing 100 mM potassium phosphate buffer (pH 7.4), 2 mg
of hepatic microsomal proteins, a NADPH-generating system