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Asymmetic Microsomal Epoxidation of Simple Prochiral Olefins.

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[3] 1 (10.9 mmol) in a stream of dry Nzis passed into a stirred solution of
LiOCMe, (30.5 mmol) in 40 mL n-pentane at -60°C. The coolant is removed and the mixture gently warmed up to room temperature and stirred for 12 h. The solvent is then carefully removed, resulting in formation of a yellow, glass-like solid, which is taken up in n-pentane and
cooled to -35°C. The solvent is slowly removed at this temperature.
Crystals of 5 , which can be separated manually, are included in the residual solid. 5 decomposes immediately at room temperature in vacuo,
but under N 2 is stable for a few hours. If they are imbedded into KelF
wax, the crystals can be stored for several weeks.
[4] LiOCMe?: Space group C2, Cm or C2/m, a=2963, b=1767, c = 1032
p m ; p= 93”: K. Huml, Czech. J . Phys. E l 5 (1965) 699.
I51 Siemens AED-2 single crystal diffractometer, MoKaradiation, computer: Data General Eclipse S-250, all programs from G.M.S. 5 , space
group C2/m,
a=2263.7(3), b = 1938.6(4), c = 1329.1(2) pm;
p= 124.83(1)”, V=4.783 x lo9 pm’; 1933 independent reflections with
~ I ~=].I9
F>~u((F); R=0.167, R,=0.136, W ~ ’ = U ~ ( F , ) + O . O O I I Fp..I.
g/cm’. Further details of the crystal structure investigation can be obtained from the Fachinformationszentrum Energie Physik Mathematik,
D-7514 Eggenstein-Leopoldshafen 2, by quoting the depository number
CSD 51065, the names of the authors, and the journal citation.
161 R. Huisgen, W. Mack, Chem. Ber. Y3 (1960) 332.
171 H. Schmidbaur, A. Schier, U. Schubert, Chem. Ber. 116 (1983) 1938.
181 H. Koster, D. Thoennes, E. Weiss, J. Organomer. Chem. 160 (1978) 1.
[9] D. Barr, W. Clegg, R. E. Mulvey, R. Snaith, J. Chem. SOC.Chem. Commun. 1984. 79.
1101 M. F. Lappert, M. J. Slade, A. Singh, J. Am. Chem. SOC.105 (1983)
[Ill H. Hope, P. P. Power, J . Am. Chem. SOC.I05 (1983) 5320.
[12] R. Amstutz, W. B. Schweizer, D. Seebach, J. D. Dunitz, Helu. Chim. Acta
64 (1981) 2617.
(131 G. W. Klumpp, P. J. A. Genrink, A. L. Spek, A. J. M. Duisenberg, J.
Chem. SOC.Chem. Commun. 1983, 814.
[14] D. Barr, W. Clegg, R. E. Mulvey, R. Snaith, J. Chem. SOC.Chem. Commun. 1984, 226.
[IS] W. Clegg, R. Snaith, H. M. M. Shearer, K. Wade, G. Whitehead, J.
Chem. SOC.Dalton Trans. 1983, 1309.
[I61 R. Zerger, W. Rhine, G. Stucky, J . Am. Chem. SOC.96 (1974) 6048.
[17] W. H. Ilsley, T. F. Schaaf, M. D. Click, J. P. Oliver, J. A m . Chem. SOC.
102 (1980) 3769.
alized, prochiral olefins 1; the extent of product enantioselectivity is found to depend critically on the structure of
the olefin. In order to determine rapidly and precisely the
enantiomeric excess and absolute configuration of the oxiranes 2 formed, complexation gas chromatography was
used@].By means of this method, even highly volatile oxiranes which are difficult to isolate can be analyzed reliably
on the nanogram scale by head-space analysis of incubation mixtures without recourse to derivatization procedures. In this way, the enantioselective conversion can be
monitored continuously without requiring to interrupt the
reaction; this is a prerequisite for distinguishing between
product and substrate selectivity.
We studied the product enantioselectivity of the enzyme-catalyzed aerobic epoxidation of prochiral olefins 1
to oxiranes 2 by liver microsomes of male, phenobarbitalinduced Wistar rats. The results are summarized in Table
ll7]. Figure 1 shows a representative complexation gas
Table 1. Microsomd~enantioselective epoxidation of simple olefins [7]. The
formula refers to the enantiomers of (2R)-configuration (2e: (3R)-configuration.
Enantiomer ratio [a]
e x . [%I [bl
43 (2R.3R) : 57 (2S,3S)
: o
39 ( R )
: 61 ( S )
48 (2R,3R) : 52 (2S.3S)
48 (2R,3S) : 52 (2S.3R)
40-tl.4 [c]
40-t 1.0
40+ 1.4
14+ 1.0
22 f 4.0
Asymmetric Microsomal Epoxidation
of Simple Prochiral Olefins**
By Volker Schurig* and Dorothee Wistuba
As highly selective catalysts, enzymes can distinguish
not only between enantiotopic groups and faces of prochiral molecules (product enantioselectivity) but also between
the enantiomers of racemic mixtures (substrate enantioselectivity)“’.
In the metabolism of xenobiotics having aromatic or
olefinic double bonds in the endoplasmic reticulum of
liver cells, reactive oxiranes (epoxides) are formed enantioselectively. Thus, cytochrome P-450 dependent monooxygenases epoxidize aromatic polycyclic hydrocarbons with
high enantioselectivity[*]. The asymmetric epoxidation of
prochiral olefins (styrene, I-octene) is also known131.The
reactive oxiranes formed often exhibit a high mutagenic
and carcinogenic potential which frequently depends on
the enantiomer composition and the absolute configurationL41. Until now, the methods used to determine the enantiomeric purity in studies of enantioselective epoxidations
have been time consuming and imprecise[51.
We have found that in microsomal epoxidations monooxygenases can differentiate between the enantiotopic
faces of the double bond of small, aliphatic, nonfunction[*] Prof. Dr. V. Schurig, Dipl.-Chem. 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. We thank Prof. Dr. A . Wendel, Tiibingen, for supplying the microsomes.
0 Verlag Chemie GmbH. 0-6940 Weinheim, 1984
[a] Absolute configurations were determined by coinjecting oxiranes of
known chirality (K. Hintzer, dissertation, Universitat Tiibingen 1983). The
absolute configuration of vinyloxirdne 2c was assigned using the “quadrant
rule” [6]. [b] The error limits refer to the reproducibility o f a minimum of
seven incubations with one microsome preparation. [c] When racemic methyloxirane 2a was incubated with 2-(trichloromethyl)oxirane-inhibited microsomes the e.e. did not change by O + _ 1% in the gas phase. This therefore ensures that the e x . values determined by complexation gas chromatography
are not altered by enantioselective associations in the incubation medium
(e.g. by chiral cell membranes).
The highest enantioselectivity of the cytochrome P-450
dependent monooxygenases was observed in the epoxidation of olefins with terminal double bonds ( l a , b, c, f).
Neither extension of the length of the carbon chain nor alteration of its degree of hybridization has an effect on the
enantiomeric composition. In contrast, the enantioselectivity of the enzyme system is markedly influenced by the degree of substitution of the olefinic double bond. Thus, the
enantiomeric excess decreases noticeably along the series
methyloxirane 2a >trans-2,3-dimethyloxirane2d > trimethyloxirane 2e (=zero). The (S)-enantiomer is always
formed preferably. The molecular geometry of the olefin 1
also influences the enantioselective behavior of monooxygenases. Comparison of the microsomal epoxidation of
isomeric pentenes indicates that the enantiomeric excess is
higher for the 2,2-disubstituted oxirane 2f than for the cis/
trans-2,3-disubstituted isomers 2h and 2g. These cis/transoxiranes exhibit only a slight (equal) enantiomeric excess.
0570-0833/84/1010-0796 %! 02.50/0
Angew. Chem. Int. Ed. Engl. 23 (1984) No. 10
Biochem. Biophys. Res. Commun. 106 (1982) 602, and literature cited
[31 T. Watabe, N. Ozawa, A. Hiratsuka, Biochem. Pharmacol. 32 (1983) 777;
L. P. C. Delbressine, P. J . van Bladeren, F. L. M. Smeets, F. Seutter-Berlage, Xenobiotica I 1 (1981) 589; P. R. Ortiz de Montellano, B. L. K. Mangold, C. Wheeler, K. L. Kunze, N. 0. Reich, J . Biol. Chem. 258 (1983)
141 W. Levin, R. L. Chang, A. W. Wood, H. Yagi, D. R. Thakker, D. M. Jerina, A. H. Conney, Cancer Res. 44 (1984) 929, and literature cited therein.
[S] The enantiomeric purity is given ah enantiomeric excess e.e. [ e x .= 100
[6] V. Schurig, W. Biirkle, J. Am. Chem. Soc. 104 (1982) 7573; V. Schurig in J.
D. Morrison: Asymmetric Synthesis. Vol. I , Academic Press, New York
1983, p. 59.
171 The incubation mixture of 0.5 rnL total volume contains microsomes (1.5
mg p r o t e i d m l ) , 0.15 M phosphate buffer pH 7.4, NADP’ (lo-’ M), isocitrate dehydrogenase (0.1 I.U.), (2)-isocitrate (8 x
M), 2-(trichloroM) and olefin 1
methy1)oxirane ( 4 x 10-’-9 x lo-’ M), MgClz (5 x
(3-13 pmol). Temperature: 37°C. Incubation time: 30 min.
181 The racemic oxiranes are incubated with microsomes (1.5 mg protein/
mL) in 0.15 M phosphate buffer (pH 7.4) at 37°C; the enzymatic hydrolysis is studied by complexation gas chromatography. The incubation time
(maximum 2 h) is a function of the substrate.
191 If, for example, in the microsomal epoxidation of 1,3-butadiene to vinyloxirane the inhibition of the epoxide hydrolase is omitted, a racemic
product is feigned when both formation and hydrolysis of the (S)-enantiomer are preferred; H. M. Bolt, G . Schmiedel, J. G. Filser, H. P. Rolzhauser, K. Lieser, D. Wistuba, V. Schurig, J . Cancer Res. Clin. Oncol. 106
(1983) 112.
t [min]
Fig. I. Detection of the enantioselective microsomal epoxidation of 1,3-butadiene (=“diolefin”) lc to vinyloxirane Ze. First enantiomeric pair: 2-(trichloromethy1)oxirane (inhibitor). Second enantiomeric pair: vinyloxirane
(ex.: 40% (S)). Column: 40 m x 0.25 mm deactivated glass capillary coated
with 0.125 m nickel bis(3-heptafluorobutyryl-(l R)-camphorate) 161 in OV 101.
70”C, 2 bar Nz. imp. =impurity.
The oxiranes 2 occur in “steady-state” concentrations
since they are hydrolyzed to diols by the epoxide hydrolase present in the microsomal fraction. The continuous
determination of the enantiomeric composition of the oxiranes indicates that this secondary reaction also proceeds
asymmetrically (“kinetic resolution”). In 2a -h the @)-enantiomer reacts more rapidly than the (R)-enantiomer[*I.
Informative data with respect to the product enantioselectivity of the oxirane can therefore be obtained only by effective inhibition of the epoxide hydr~lase[’~.
For this purpose, 2-(trich1oromethyl)oxirane has been used as an inhibitor. In the complexation gas chromatogram, the enantiomers of the inhibitor are eluted before the oxiranes 2a h (cf. also Fig. 1). If the enantiomer composition of the oxiranes formed is determined by complexation gas chromatography as a function of the incubation time, a constant
value is found (enantioselective formation reaction) provided the inhibitor is present in excess ; thereafter, hydrolysis (enantioselective reaction) of the oxiranes commences.
These results underline the necessity of continuously monitoring both the enantiomeric excess and the concentration
of the oxirane during incubation. The method of complexation gas chromatography used here for the first time has
therefore proved to be a reliable probe for the investigation of asymmetric enzymatic reactions.
Received: May 11, 1984 [Z 830 IE]
German version: Angew. Chem. 96 (1984) 808
[ I ] A notable example is the enantioselective biosynthesis of only one lanosterol enantiomer from prochiral squalene via 2.3-squalene epoxide.
121 P. J. van Bladeren, R. N. Armstrong, D. Cobb, D. R. Thakker, D. E.
Ryan, P. E. Thomas, N. D. Sharma, D. R. Boyd, W. Levin, D. M. Jerina,
Angew. Chem. Int. Ed. Engl. 23 (1984) No. 10
Easy Rotation about the
Exocyclic Carbon-Carbon Bond in
Lithium a-Aminoenolates: A Comparison**
By Gernot Boche*, Ferdinand Bosold, and Robert Eiben
In the a-aminoenolates (“amide enolates”) 1, 2, and 3
we found for the first time an easy rotation about the exocyclic CC double bond“’ in a compound of this sort.
The 100-MHz ‘H-NMR spectra of lithium dimethylamino(cycloheptatrieny1idene) methoxide 1 are temperaturedependent[’]. At 39”C, the signals of H’ and H6 (6=5.17
and 4.93; an assignment was not made) are noticeably separated, whereas at 70°C they coalesce due to rapid rotation
about the C7Cs bond, and at 83 “C split into a doublet.
A free activation enthalpy of AG’ (70°C) = 17.5 f0.5
kcal/mol is calculated for the barrier to rotation in 1. The
barrier for rotation about C7Cxin the dihydro analogue 2
was determined in a similar way (AG+ (25°C) 14.0k0.5
kcal/mol). The rotation about C5C6 in 3, on the other
hand, could not be frozen-in even at -106”C, i.e.
AGC (- 106”C)<8 kcal/m01[~,~].
In 2, the negative charge is stabilized in the cross-conjugated pentadienide moiety, in 3, in the cyclopentadienide
moiety. The energetic advantage of the cyclic 6n-delocalization (“aromaticity”) in 3 explains the low barrier to ro[*] Prof. Dr. G. Boche, F. Bosold, Dipl.-Chem. R. Eiben
Fachbereich Chemie der Universitat
Hans-Meerwein-Strasse, D-3550 Marburg (FRG)
This work was supported by the Fonds der Chemischen Industrie
0 Verlag Chemie GmbH, 0-6940 Weinheirn, 1984
0570-0833/84/1010-0797 $ 02.50/0
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simple, asymmetic, prochiral, olefin, epoxidation, microsomal
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