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Complementary Epoxide Hydrolase vs Glutathione S-Transferase-Catalyzed Kinetic Resolution of Simple Aliphatic OxiranesЦComplete Regio- and Enantioselective Hydrolysis of cis-2-Ethyl-3-methyloxirane.

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Complementary Epoxide Hydrolasevs Glutathione S-Transferase-Catalyzed Kinetic
Resolution of Simple Aliphatic OxiranesComplete Regio- and Enantioselective Hydrolysis
of cis-2-Ethy1-3-methyloxirane**
By Dorothee Wistuba and Volker Schurig*
Inspired by the fascinating stereoselectivity observed in
the biosynthesis of steroids (e.g. formation of enantiomerically pure lanosterol from achiral all-trans-squalene via
2,3-squalene oxide"]) we have investigated the enantioselectivity of the biotransformation of simple xenobiotic
oxiranes with microsomal and cytosolic rat liver enzymes.
R3
R'
zationf6b1from eryfhr0-(2S,3R)-2,3-pentanediol,~~"~is
eluted
as the first peak. The four configurationally isomeric 2,3pentanediols can be separated on nickel(rr) bis(3-heptafluorobutanoyl-( lR,2S)-4-pinan0nate)[~~I
(Fig. lb). Both
threo-(2S,3S)-2,3-pentanediol(prepared in nine steps from
(2R,3R)-tartaric acid via (2S,3S)-isopropylidenethreitol by
C, chain extensionf6") as well as erythro-(2S,3R)-2,3-~entanediol (prepared in six steps from (2S,3S)-allothreonine
via (2S,3S)-dihydroxybutyric acid by C , chain extension16'1) are eluted as second peak of each enantiomeric
pair (Fig. lb). The stereochemistry of the epoxide-hydrolase-catalyzed ring-opening of cis-2-ethyl-3-methyloxirane
follows from Figures l a and lc: only the (2R,3S)-configurated oxirane"] is biotransformed, and exclusively threo(2R,3R)-2,3-pentanediol is formed as metabolite by regioselective ring opening with Walden inversion at C-3."'
1
1
(I)
(Cytochrorne P-450)
Steroid biosynthesis~']
3
Epoxide
hydroloses
2
Glutathione S transferoses
Conjugation a t
giutathione
In the reaction sequences (a) and (b) enantioselectivity
can arise by epoxidation of an alkene possessing enantiotopic faces (''prochiral recognition", product enantioselectivity"]) and/or by kinetic resolution of the intermediary
oxirane 2 ("chiral recognition", substrate enantioselectivity13]). For the formation of highly enantiomerically-enriched products (ee>99%) at least one step, (I), or (11) and
(III), respectively, must be highly enantioselective. In the
case of incomplete asymmetric induction the product and
substrate enantioselectivities can either be mutually increased to high ee or compensated to lower ee. While the
microsomal epoxidation of cis-2-pentene in step (I) shows
no significant product enantioselectivity,'21 in the epoxidehydrolase-catalyzed ring-opening (11) of rac. cis-2-ethyl-3methyloxirane to give 2,3-pentanediol we found complete
substrate regioselectivity a n d substrate enantioselectivity
(ee > 99%) within the precision of the r n e a ~ u r e r n e n t . ~ ~ ~
Complexation gas chromatography, which enables timedependent measurements in the nanogram range, was employed for the determination of the enantiomeric excess
(ee) and the absolute configuration :Is1 cis-2-ethyl-3-methyloxirane is separated into the antipodes on nickel(i1) bis(3heptafluorobutanoyl-( IR)-camphorate)f6a1(Fig. la); the
(2R,3S)-enantiomer,"I which was prepared free of racemi-
Epoxide-hydrolases :
2R,3S (cis) [2S,3S (trans) > 2R.3R (trans)]> 2S,3R (cis) (no
reaction)
*
[*) Prof. Dr. V. Schurig, Dr. D. Wistuba
lnstitut fur Organische Chemie der Universitat
Auf der Morgenstelle 18, D-7400 Tiibingen (FRG)
[**I
This work was supported by the Deutsche Forschungsgemeinschaft and
the Fonds der Chemischen Industrie. Presented in part at the International Symposium o n Activation of Dioxygen Species and Homogeneous Catalytic Oxidations, Galzignano (Padua, Italy), June 25, 1984. Extract from the Dissertation by D. Wisruba, Universitat Tiibingen 1986.
1032
0 VCH Veriagsgesellschaft mbH. 0-6940 Weinheim. 1986
This result shows that substrate enantioselectivity, which
has been demonstrated for sterically hindered bicyclic
epoxides such as 3-terf-butyl- 1,2-epoxycyclohexane,"
already occurs in the case of the smallest aliphatic cis-oxirane consisting of oppositely configurated carbon atoms.
Since practically no product enantioselectivity is observed
in step (I),[21 the enantiomer (2S,3R)-2-ethyl-3-methyloxirane being inactive towards epoxide-hydrolases must be
detoxified via another pathway (vide infra) in the in-vivornetabolization of cis-2-pentene.
In the case of trans-2-ethyl-3-methyloxirane,which is
formed from trans-2-pentene without appreciable product
enantioselectivity (I),f21no complete substrate enantioselectivity is observed during the hydrolytic ring-opening (11):
although the trans-(2S,3S) enantiomer, which has been
chemically correlated with threo-(2S,3S)-pentanediol,l6"'is
preferentially metabolized (significantly slower, however,
than the cis-(2R,3S) isomer), the trans-(2R,3R) antipode (in
contrast to the inactive cis-(2S,3R) isomer) is also a substrate, even if a poor one, for epoxide-hydrolases."] erythro-(2R,3S)-2,3-Pentanediol,whose enantiomeric composition is turn-over-dependent (ee= 58% after 2 h), is preferentially formed as metabolite of rac. trans-2-ethyl-3-methyloxirane.
A complementary behavior in regard to substrate diastereoselectivity (cis-trans geometry of the oxirane ring)
and substrate enantioselectivity with epoxide-hydrolases
appears during conjugation of the four configurationally
isomeric 2-ethyl-3-methyloxiranes at glutathione (Y-(L)glutamyl-(L)-cysteinyl-glycine) catalyzed by glutathione Stransferases (111). The rate of the catalytic ring-opening of
the oxiranes decreases in the order:
Conjugation at glutathione catalyzed by glutathione Stransferases (Fig. 2a):
2R,3R (trans)
slow)
[2S,3R (cis) > 2R.3S (cis)]> 2S,3S (/runs) (very
0570-0833/86/1111-1032 $ 02.50/0
Angew. Chem. Int. Ed. Engl. 25 (1986)
No. I 1
2R3R
us
i
a1
incubarion
tme
50mln
35 mcn
20 m i n
5 rnlil
2S3R
Standard
I_ic
I
cl
0
5
10 t w i n 1
-
0
5
2531
253R
2S3R
il
't,
10 t [minl
0
5
-
c
10 t
mim 0
5
10 t lminl
0
5
15
10
t l">i"l
Fig. I . a) Kinetic analysis of the enantiomeric composition of ruc. cis-2-ethyl-3-methyloxirane during enzymatic hydrolysis by rat liver microsomes. Standard:
3-methyl-2-butanone (25 m x 0.25 mm glass capillary coated with 0.08 m nickel(l1) bis(3-heptafluorobutanoyl-(lR)-camphorate) [6a] in OV 101, 80°C. 1.1 bar N2).
Incubation run (0.5 m L total volume): microsomes (1 mg protein/mL), 0.15 M phosphate buffer pH 7.4, ruc. cis-2-ethyl-3-methyloxirane (4 mM), 37°C. Complexation gas-chromatographic head-space analysis of the oxirane. b) Quantitative complexation gas chromatographic separation of the four configurationally isomeric
2,3-pentanediols as acetonides [6d]. c) threo-(2R,3R)-2,3-pentanediol, formed in the enzymatic hydrolysis of ruc. cis-2-ethyl-3-methyloxirane (36m x 0.25 mm glass
in O V 101, 70°C, I bar N2).Incubation run (cf. caption to Fig. la) (5 mL
capillary coated with 0.075 m nickel(!r) bis(3-heptafluorobutanoyl-(lR,2S)-4-pinanonate)
total volume): after cessation of reaction (- 195'C) extracted six times with diethyl ether (0°C). concentrated, and diol converted into acetonide [6d].
Thus, the metabolites of the enzymatic epoxidation of cis/
trans-2-pe11tene,'~' i.e. cis-(2S,3R)-2-ethyl-3-methyloxirane,
which is completely inactive toward epoxide-hydrolases, is
transformed by conjugation at glutathione, and frans(2S,3S)-2-ethyl-3-methyloxirane,
which is essentially inaccessible for conjugation, is a preferred substrate for epoxide- hydrolases.
The complementary chiral-recognition phenomena observed in the case of 2-ethyl-3-methyloxirane also occurs in
a series of smaller, sterically less-demanding oxiranes. It
follows from Table 1 that the S-configurated enantiomers
are preferentially metabolized in the biotransformation of
aliphatic oxiranes by epoxide-hydrolases while the R antipodes are preferentially metabolized by glutathione Stransferase-catalyzed conjugation at glutathione.
In the kinetic resolution of these substrates in uitro by
glutathione S-transferase-catalyzed conjugation at glutathione we found a considerable substrate enantioselectivity
that was dependent upon the degree of substitution and
geometry of the oxirane (Fig. 2). Monoalkyl-substituted
oxiranes 2 ( R ' = R2= R3 = H, R4= CH3, CH2CI, C2H5,
CH2=CH2) of configuration R ( S for the potential mutagen (chloromethy1)oxirane = epichlorohydrin, cf. footnote [c] in Table 1) are conjugated significantly more rapidly than the antipodes at glutathione. Whereas (R)-ethyloxirane is completely conjugated after 30 minutes incubation time, the remaining (enantiomerically pure) S enantiomer was only consumed to the extent of 18%. 50% conversion is already observed after 5 min for the R-enanAngeu. Chem. I n ( . Ed. Engl. 2s (1986) No. 11
Table 1. Complementary in ui/ro substrate enantioselectivity of the biotransformation of racemic aliphatic oxiranes 2 during the hydrolysis to 3 catalyzed by epoxide-hydrolases and by the conjugation at glutathione catalyzed
by glutathione S-transferases [a,b].
Oxirane 2
Hydrolysis to 3
(epoxide-hydrolases)
Conjugation at
glutathione
(glutathione
S-transferases)
Methyloxirane
S > R
R > S
(Chloromethy1)oxirane (epiR > S [c]
S > R [c]
chlorohydrin)
S > R
R % S
S > R
R > S
Ethyloxirane
2R.3R > 2 S . X
Vinyloxirane
2S.3S > 2R,3R
truns-2,3-Dimethyloxirane
2S.3S > 2R,3R
2R,3R 2S.3.S
rruns-2-Ethyl-3-methyloxirane 2R.3S > 2S.3R (inactive) 2S.3R > 2R.3S
cis-2-Ethyl-3-methyloxirane
S> R
R> S
Trimethyloxirane
*
~
~
[a] Assignment of the absolute configuration of synthetic oxiranes of known
chirality (K. Hintzer, Dissertation, Universitat Tiibingen 1983. Vinyloxirane:
R. J. Crawford, S. B. Lutener, R. D. Cockroft, Can. J . Chem. 54 (1976) 3364).
[b] S > R means: the S-enantiomer reacts more rapidly than the R-antipode.
[c] The chloro-substitution leads to a formal reversal of the descriptors R and
S.
tiomer of the cytotoxic 1,3-butadiene metabolite vinyloxirar~e,[~]
and only after 25 min in the case of the S antipode
2
(Fig.
2b).
Dialkyl-substituted
trans-oxiranes
( R 2 = R3= H, R ' = R 4 = C H 3 and R ' =CH3, R4=C2H5)and
trimethyloxirane 2 ( R ' = H , R 2 = R 3 = R 4 = C H 3 ) with R
configuration are likewise conjugated more rapidly than
0 VCH VerlugsgesellschajrmbH, 0-6940 Weinheim, I986
0570-0833/86/III1-I033$ 02.5#/0
I033
10-2
pmol
10-2
a)
p mot
b)
Oxirani
Oxiranc
25
25
2535
5
20
2R35
2c
253R
15
15
\
10
10
5
5
4
20
40
60
80
-.
A
-
A
100
.
r
120
- A ~ R ~ R
7
140
Incubation time [ m i n i
20
40
60
80
Incubation time jrninl
Fig. 2. Kinetic resolution by conjugation at glutathione catalyzed by glutathione S-transferases at 37°C. Incubation run (0.5 m L total volume): glutathione Stransferases (SIGMA, D-8024 Deisenhofen) (0.75 mg protein/mL: origin: rat); glutathione (SIGMA) (4 mM); 0.1 M phosphate buffer pH 6.5; oxirane ( I mM),
(chloromethy1)oxirane (4 m M ) . Glutathione and glutathione S-transferases were pre-incubated in phosphate buffer for 5 min. Oxirane and standard (acetone or
3-methyl-2-butanone)were added as a solution in diethyl ether. The continuous determination of the oxirane enantiomers with respect to the standard was carried
out by head-space analysis by means of complexation gas chromatography 12, 6al. All incubations were repeated at least three times. a) cis- ( 0 )and trans2-Ethyl-3-methyloxirane (+);b) ethyloxirane ,).(
vinyloxirane (+)and trimethyloxirane (A).
the S antipodes (50% conversion in 85 min in the case of
trans-(2R,3R)-2,3-dimethyloxiraneand 10 min in the case
of trans-(2R,3R)-2-ethyl-3-methyloxirane,with less than
10% conversion in 20 min for (R)-trimethyloxirane and in
80 rnin for the S-antipode).
The occurrence of substrate enantioselectivity, arising
from the chirality of the glutathione S-transferases and/or
the configuration of the conjugation partner (t,L)-glutathione, has also been reported in the conjugation of phenyloxiranel"' and arene oxides."']
The chiral recognition phenomena found here for simple aliphatic oxiranes are of significance in the generalization of model concepts on the active center of the epoxidehydro lase^,"^' for continuing mechanistic investigations on
the conjugation at glutathione, and for the derivation of
structure-activity relationships, which go beyond the uniform treatment of the antipodes in case of racemic substrates.["I In conclusion, we have shown that the enzymatic
kinetic resolution proves to be an efficient pathway for access to enantiomerically highly-enriched products of metabolization (cf- introductionary remarks).
Received: April 28, 1986;
supplemented: August 21, 1986 [ Z 1752/1862 IE]
German version: Angew. Chem. 98 (1986) 1008
[I] J. Retey, J. A. Robinson: Srereuspecificiry in Urgonic Chemrsfry and Enzymology, Verlag Chemie, Weinheim 1982, p. 236.
1034
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[21 V. Schurig, D. Wistuba, Angew. Chem. 96 (1984) 808; Angew. Chem. Inr.
Ed. Engl. 23 (1984) 796.
131 I n the case of 2, R ' = R' R L =R4, product enantioselectivity can be effected by differentiation of enantiotopic groups of the achiral meso-substrate.
[41 High regioselectivity and insignificant enantioselectivity was found in
the case of cis-2-methyl-3-pentyloxirane:
R. P. Hanzlik, S. Heideman, D.
Smith, Biochem. Biophys. Rer. Cummicn. 82 (1978) 310.
[Sl The formal difference in the numbering of the C-atoms in the oxirane
and epoxyalkane nomenclature should be noted (e.g. cis-(2R,3S)-2ethyl-3-methyloxirane s cis-(ZS.3 R)-2,3-epoxypentane).
161 a)V. Schurig, W. Biirkle, J . Am. Chem. Soc. 104 (1982) 7573; b) V. Schurig, B. Koppenhoefer, W. Biirkle, J . Org. Chem 45 (1980) 538, and references cited therein; c) K. Hintzer, Dissertorion. Universitat Tubingen
1983; d) V. Schurig, D. Wistuba, Terrahedron Lett. 25 (1984) 5633.
[7] a) G. Bellucci, G. Berti, R. Bianchini, P. Cetera, E. Mastrorilli, J . Org.
Chem. 47(1982) 3105; b) G. Bellucci, G. Berti, M. Ferretti, E. Mastrorilli, L. Silvestri, ibid. 50 (1985) 1471.
(81 In the case of frans-2.3-dimethyloxirane,
the (2S,3S)-enantiorner is likewise hydrolyzed more rapidly than the (2R,3R)-antipode by epoxide hydrolases.
[9] H. M. Bolt, G . Schmiedel, J . G. Filser, H. P. Rolzhauser, K. Lieser, D.
Wistuba, V. Schurig, J . Cancer Res. Clin. Uncol. 106 (1983) 112.
[lo] T. Watabe, N. Ozawa, A. Hiratsuka, Biochem. Pharmacol. 32 (1983) 777;
cf. also T. Watabe, A. Hiratsuka, T. Tsurumori, ibrd. 33 (1984) 405 I ; Biochem Biophys. Res. Commun. 130 (1985) 65.
[I11 0. Hernandez, M. Walker, R. H. Cox, G. L. Foureman, B. R. Smith, J. R.
Bend, Biochem. Biophys Res. Commun. 96 (1980) 1494, C. Boehlert, R.
N. Armstrong, ibid. 121 (1984) 980.
[I21 F. Oesch, N. Kaubisch, D. M. Jerina, J. W. Daly, Biochemistry I0 (1971)
4858.
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simple, resolution, aliphatic, epoxide, hydrolases, methyloxirane, glutathione, enantioselectivity, kinetics, oxiranesцcomplete, ethyl, cis, catalyzed, regin, complementary, transferase, hydrolysis
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