Deracemization of (±)-cis-dialkyl substituted oxides via enantioconvergent hydrolysis catalysed by microsomal epoxide hydrolase

Article abstract of DOI:10.1016/S0957-4166(97)00630-7

Both enantiomers of cis-(±)-2,3-epoxyheptane 1a, cis-3,4-epoxyheptane 1b, cis-3,4-epoxynonane 1c, cis-3,4-epoxynonane-1-ol 1d, and cis-1-methoxy-3,4-epoxynonane 1e undergo a highly stereoselective microsomal epoxide hydrolase catalysed hydration at the (S) carbon to give the corresponding threo (R,R)-diol at complete conversion. A total kinetic resolution of racemic epoxides is also obtained with 1a and 1e.

Full text of DOI:10.1016/S0957-4166(97)00630-7

Pergamon
Tetrahedron: Asymmetry 9 (1998) 341–350
Deracemization of (ꢀ)-cis-dialkyl substituted oxides via
enantioconvergent hydrolysis catalysed by microsomal epoxide
hydrolase
Cinzia Chiappe,∗ Antonio Cordoni, Giacomo Lo Moro and Consiglia Doriana Palese
Dipartimento di Chimica Bioorganica, via Bonanno 33, 56126 Pisa, Italy
Received 24 November 1997; accepted 11 December 1997
Abstract
Both enantiomers of cis-(ꢀ)-2,3-epoxyheptane 1a, cis-3,4-epoxyheptane 1b, cis-3,4-epoxynonane 1c, cis-
3,4-epoxynonane-1-ol 1d, and cis-1-methoxy-3,4-epoxynonane 1e undergo a highly stereoselective microsomal
epoxide hydrolase catalysed hydration at the (S) carbon to give the corresponding threo (R,R)-diol at complete
conversion. A total kinetic resolution of racemic epoxides is also obtained with 1a and 1e. © 1998 Elsevier Science
Ltd. All rights reserved.
1. Introduction
Epoxide hydrolases (EH) are important enzymes involved in the metabolism of a broad variety
of epoxides,1 many of which are mutagenic and/or carcinogenic, generally formed in vivo from the
Cytochrome P-450 catalysed biooxidation of compounds containing olefinic or aromatic functionalities.2
Widely studied for their biological implications1 and for the determination of their intimate mechanism
of action,3 these enzymes have been more recently investigated for their use as asymmetric biocatalysts.4
Stereochemical investigations, carried out extensively with mammalian microsomal epoxide hydrolase
(mEH), have demonstrated that the addition of water promoted by these enzymes, and occuring through
the formation of a diol monoester intermediate,3 often exhibits a remarkable substrate enantioselectivity
towards racemic epoxides,5 or product enantioselectivity with meso epoxides.6 In the latter case the
mEH catalysed oxirane ring opening generally occurs at an (S) oxirane carbon to give (R,R) diols
with high enantiomeric excesses.6 Furthermore, even if the reaction is often endowed with a very
high regioselectivity, the nucleophilic attack occuring at the less substituted or less hindered oxirane
carbon,7 it has been recently demonstrated that mEH catalyses the enantioconvergent transformation
∗
Corresponding author. E-mail: cinziac@farm.unipi.it
0957-4166/98/$19.00 © 1998 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(97)00630-7

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C. Chiappe et al. / Tetrahedron: Asymmetry 9 (1998) 341–350
of racemic cis-β-alkyl substituted styrene oxides8 and long chain cis-dialkyl substituted epoxides9 to
give, at complete conversion, the (R,R) threo diols, often with >90% enantiomeric excesses (e.e.). It is
noteworthy that a similar behaviour has been observed in the hydrolysis of (ꢀ)-disparlure by EH in gypsy
moth antennae,10 of cis-(ꢀ)-9,10-epoxystearic acid by soybean fatty acid EH,11 and, more recently, of
cis-(ꢀ)-2,3-epoxyheptane by Nocardia EH1-epoxide hydrolase.12
In order to verify the generality of this behaviour, the biotransformation of five cis-disubstituted epox-
ides, and in particular of cis-(ꢀ)-2,3-epoxyheptane 1a, cis-3,4-epoxyheptane 1b, cis-3,4-epoxynonane
1c, cis-3,4-epoxynonane-1-ol 1d, and cis-1-methoxy-3,4-epoxynonane 1e, has been investigated using a
rabbit liver microsomal preparation as the source of mEH.
2. Results and discussion
The ability of a rabbit liver microsomal preparation containing mEH to catalyse the hydrolysis of
the differently substituted epoxides 1a–e was preliminarly checked measuring the saturation velocities.
The epoxides (10–30 mM) were incubated with the microsomal preparation,13 diluted to a protein
concentration of 10–20 mg of protein/ml, at 37°C and pH 7.4. The reactions were stopped by extraction
of the products and the extracts were analysed by GLC.
The diol formation was linear with time and with the microsomal protein amount, and was independent
of the substrate concentration, indicating enzyme saturation. The saturation rates, expressed in nmol/(mg
protein×min), were: 1a, 3.1; 1b, 0.5; 1c, 0.3; 1d, 5.5 and 1e, 4.4.
The stereochemical behaviour of the biotransformation of 1a–e was determined by incubating the
epoxides under saturation conditions. The reactions were stopped at different times and the residual
epoxides and the formed diols were analyzed by GLC using a chiral column and/or by HPLC after
transformation of the diols into the corresponding MTPA esters. The results are reported in Table 1.
The absolute stereochemistry of diols 2a–e formed by enzymatic hydrolysis of the racemic substrates
was established on the basis of specific rotation and chemical correlation. The relationship between
specific rotation and absolute configuration was known for diols 2a and 2b,12,14 and this allowed us to
establish the (R,R) configuration for the dextrorotatory diols arising from the mEH catalyzed hydrolysis
of 1a and 1b. No information was available concerning the absolute configurations of diols (+)-2c–e.
The same (R,R) configuration was, therefore, attributed to diol (+)-2c by comparison of its retention
time on the chiral GLC column with that of a sample of (3S,4S)-(−)-2c independently synthesized from
trans-3-nonene by Sharpless dihydroxylation using AD-mix-α.15 It is known that with trans disubstituted
olefins it is possible, with reasonable certainty, to predict the face enantioselectivity by applying a simple
mnemonic rule.15 The same (R,R) absolute configuration was therefore attributed to diols (+)-2d and
(+)-2e by chemical correlation with that of 2c. Diol (+)-2d was transformed into diol 2c, through a
preliminary esterification of the primary hydroxy group using an equimolar amount of tosyl chloride,
followed by reduction with LiAlH4.

C. Chiappe et al. / Tetrahedron: Asymmetry 9 (1998) 341–350
343
Table 1
Enantiomeric excesses and absolute configurations of epoxides and diols obtained by mEH catalysed
hydrolysis of racemic substrates 1a–e
The chemical correlation between diols (+)-2d and (+)-2e was instead deduced from the comparison
of the specific rotations of 1,3,4-trimethoxynonane 3 arising from exhaustive methylation of both diols
using NaH and CH3I.
The absolute configuration of the residual enriched epoxides was determined in the cases of 1a by
reduction of the oxirane ring with LiAlH4 and co-injection of the resulting mixture of 2- and 3-heptanol
with a sample of commercial (R)-2-heptanol, after transformation into the corresponding trifluoroacetyl
derivatives.
Similarly, the absolute configuration of 1c was established, after reduction of the oxirane ring with
LiAlH4 followed by methylation of the OH group, by co-injection of the resulting mixture of 3-
and 4-methoxynonane with a sample of (R)-4-methoxynonane. This optically active compound was

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C. Chiappe et al. / Tetrahedron: Asymmetry 9 (1998) 341–350
prepared from commercial (S)-1-octyn-3-ol by treatment with BuLi and CH3I followed by the catalytic
hydrogenation of the triple bond. Experiments carried out with racemic 1a or 1c showed that in both cases
the four products, (+)- and (−)-2-heptanol and (+)- and (−)-3-heptanol, or (+)- and (−)-3-methoxynonane
and (+)- and (−)-4-methoxynonane, were completely separated on the chiral column.
The same configuration (3R,4S) was then attributed to the unreacted epoxide 1d by chemical
correlation. The enantiomerically enriched epoxide 1d was transformed into the corresponding p-
toluenesulfonate and after treatment with LiAlH4 and methylation of the OH group in a mixture of 3- and
4-methoxynonane. Finally, the absolute configuration of the enantiomerically pure 1e was determined by
comparison of its retention time on the chiral GLC column with that of a sample obtained from (3R,4S)-
1d by methylation of the OH group with CF3SO3CH3 in anhydrous methylene chloride.
All of the stereochemical results reported in Table 1 show that, in analogy to the mEH catalyzed
hydrolysis of cis aryl alkyl and long chain dialkyl substituted alkene oxides,8,9 and consistent with the
recently reported results about the biohydrolysis of racemic epoxides catalyzed by microbial EH12 (with
the exception of 1a) the oxirane ring opening of all the other substrates occurs stereoselectively at the
(S) configured carbons of both enantiomers to give at complete conversion the corresponding (R,R)
diols with very high enantiomeric excesses (80–98%). Furthermore, whereas the enzymatic hydrolysis
of epoxides 1a and 1e proceeds through a complete kinetic resolution giving >98% ee of both (2R,3S)-1a
and (3R,2S)-1e at 50% conversion, a lower substrate enantioselectivity, but still in favour of the (3R,4S)
enantiomer, was found with 1c and 1d, and minimal selection was observed with 1b.
Once more, the observed substrate and product enantioselectivity can be therefore rationalized on
the basis of the previously proposed topology of the active site, for which two hydrophobic pockets of
different shapes and sizes, situated at the right and left rear side of the epoxide binding site, able to
accommodate the two lipophilic syn substituents have been postulated.13 In agreement with this model,
both enantiomers of each epoxide can fit into the active site with the same orientation of the oxirane ring

C. Chiappe et al. / Tetrahedron: Asymmetry 9 (1998) 341–350
345
Fig. 1.
and with the two alkyl groups occupying opposite positions (Fig. 1). Furthermore, whereas in the case of
mono- 1,1-disubstituted or trisubstituted racemic epoxides, the observed regioselectivity can be explained
by a preferential nucleophilic attack on the least substituted, or sterically hindered carbon of the oxirane
ring, according to the catalytic mechanism,3 with cis-1,2-disubstituted epoxides in a large number of
cases no steric or electronic effects are able to differentiate the two oxirane centres. In these cases,
therefore, only the preference for the nucleophilic attack at the (S) configured chiral centre, exhibited in
the hydrolysis of meso compounds,6 determines the regio- and stereochemistry of the ring opening.
The high degree of recognition of the enantiotopic ring carbons therefore determines the product
enantioselectivity observed in the hydrolysis of epoxides 1b–e, while the stereochemical behaviour in
the hydrolysis of 1a can be rationalized by taking into account that the mEH catalyzed hydrolysis
of the (2S,3R) enantiomer occurs with complete regioselectivity, through the attack at the S oxirane
carbon bearing the less hindered substituent (methyl group), whereas that of the (2R,3S) enantiomer
is completely non-regioselective. In the oxirane ring opening of this enantiomer there is probably
competition between the stereocontrolled attack at the more hindered S configured carbon and the attack
at the methyl substituted (R) centre. Since the mEH hydrolysis of cis-β-methyl styrene oxide gave, at
complete conversion, a racemic diol by a regioselective oxirane ring opening at the methyl substituted
carbon,8 the data obtained in this work for 1a seems to indicate that, at variance with the phenyl group,
the n-butyl sustituent is not able to avoid ca. 50% of the reaction occurring through nucleophilic attack
at the adjacent carbon.
Finally, as far as the substrate enantioselectivity is concerned, the results obtained in this work are
consistent with those reported for other racemic epoxides, and point to the formation of a more stable
complex of the enzyme with the enantiomer bearing the bulkier lipophilic group on the right side when
the oxirane ring is oriented as reported in Figure 1. In agreement with this model, in the cases of 1a
the n-butyl group, but not the small methyl substituent, is able to give rise to a stronger binding into
the lipophilic pocket on the right side, determining the formation of a more stable enzyme substrate
complex for the enantiomer having an (R) configuration at the C(3) carbon. This determines an effect of
competitive inhibition by the 2S,3R epoxide on the hydrolysis of its enantiomer, which is responsible for
the observed kinetic resolution. On the other hand, in the case of 1b probably both the ethyl group of one
enantiomer and the n-propyl substituent of the other can bind to this pocket giving two enzyme–substrate
complexes of comparable stability. No competitive inhibition between enantiomers therefore operates in
this substrate, and no relevant kinetic resolution is observed. The ability of the enzyme to discriminate
between the two enantiomers arises again in the hydrolysis of 1c, where the sizes of the two substituents
are sufficiently different, and in those of 1d and 1e. Although in these latter substrates the ethyl group
presents a substituent, they probably have the more polar nature, due to the presence of a hydroxy and
methoxy group, respectively, which makes the interaction of the n-pentyl chain of the (3S,4R) enantiomer
with the lipophilic pocket on the right side relatively more important.
In conclusion, the results of this investigation not only give further information about the mechanism
of hydrolysis of mEH but also confirm that cis-disubstituted oxides are hydrolysed by microsomal
epoxide hydrolase to diols in an anti stereospecific way and, with the exception of the methyl derivatives,

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C. Chiappe et al. / Tetrahedron: Asymmetry 9 (1998) 341–350
in a stereoconvergent way to give practically optically pure threo-(R,R) diols at 100% hydrolysis.
Furthermore, depending on the relative sizes of the two substituents the reaction can occur with high
substrate enantioselection to give at 50% hydrolysis an enantiomerically pure residual epoxide and an
enantiomerically pure diol. This once again indicates that microsomal epoxide hydrolase can be useful
for small scale kinetic resolutions of epoxides and for the preparation of chiral vicinal diols, and that
it may became of much greater preparative utility when biotechnologically produced enzyme becomes
available in large amounts.
3. Experimental section
1
Optical rotations were measured with a Perkin–Elmer 241 polarimeter. The H and 13C NMR spectra
were registered in CDCl3 with a Bruker AC 200 instrument using TMS as the internal reference. HPLC
analyses were carried out with a Waters 600E apparatus equipped with a diode array detector. The
e.e.s of the diols 2d and 2e were determined after conversion into the diastereoisomeric MTPA esters
by HPLC using a Nitrile S5 column with hexane/2-propanol (99.5:0.5) as the eluant at a flow rate of
1 ml/min. The yields of recovered epoxides and formed diols and the e.e.s of epoxides 1a–e and of
diols 2a–c were obtained by GLC analysis using a Carlo Erba HRGC 5300 instrument equipped with a
20 m Chiraldex G-TA (ASTEC) column, evaporator and detector 245°C, helium flow 1 ml/min, at the
following temperatures: 1a–c 80°C; 1d 130°C; 1e 120°C. 2a, as bis(trifluoroacetyl)derivative 80°C; 2b
110°C; 2c 130°C; 2d, as trifluoroacetyl derivative 130°C.
3.1. Materials
Commercial cis-2-heptene (Aldrich, 97%), cis-3-heptene (Aldrich, 96%), and cis-3-nonen-1-ol
(Aldrich, 95%) were used without further purification. cis-3-Nonene was prepared from cis-3-nonen-
1-ol by tosylation of the hydroxy group followed by reduction with lithium aluminium hydride.
cis-1-Methoxy-3-nonene, was obtained by methylation according to the general alkylation procedure.16
cis-2,3-Epoxyheptane 1a, cis-3,4-epoxyheptane 1b, cis-3,4-epoxynonane 1c, cis-3,4-epoxynonane-1-
ol 1d, and cis-1-methoxy-3,4-epoxynonane 1e were synthesized by epoxidation of the corresponding
olefins with m-chloroperbenzoic acid and KF in dichloromethane for 24 h, followed by distillation.
1
1a H NMR δ ppm: 0.97 (t, 3H, J=7.30 Hz, CH3); 1.27 (d, 3H, J=5.60 Hz, CH3); 1.30–1.60 (m, 6H,
CH2); 2.93 (m, 1H, CH); 3.09 (m, 1H, CH). 13C NMR δ ppm: 13.04 (CH3); 13.89 (CH3); 22.49, 27.10,
28.50 (3CH2); 52.60 (CH); 57.12 (CH). 1b: oil, 1H NMR δ ppm: 0.97 (t, 3H, CH3); 1.05 (t, 3H, CH3);
1.40–1.60 (m, 6H, CH2); 2.90 (m, 2H, CH). 13C NMR δ ppm: 11.22 (CH3); 14.69 (CH3); 20.55, 21.70,
30.34 (3CH2); 57.95 (CH); 59.10 (CH). 1c: 1H NMR δ ppm: 0.82 (t, 3H, J=6.74 Hz, CH3); 0.96 (t, 3H,
J=7.50 Hz, CH3); 1.30 (m, 4H, CH2); 1.48 (m, 4H, CH2); 2.80 (m, 2H, CH). 13C NMR δ ppm: 10.40
1
(CH3); 13.80 (CH3); 20.92, 22.44, 26.10, 27.47, 31.58 (5CH2); 57.30 (CH); 58.30 (CH). 1d: H NMR
δ ppm: 0.80 (t, 3H, J=6.20 Hz, CH3); 1.20–1.50 (m, 6H, CH2); 1.57 (m, 2H, CH2); 1.74 (m, 2H, CH2);
2.83 (m, 1H, CH); 2.98 (m, 1H, CH); 3.69 (t, 2H, J=6.2 Hz, CH2OH). 13C NMR δ ppm: 14.40 (CH3);
1
23.00, 26.58, 28.27, 31.13, 32.10 (5CH2); 55.46 (CH); 57.55 (CH); 60.58 (CH2). 1e: H NMR δ ppm:
0.84 (t, 3H, J=6.80 Hz, CH3); 1.30 (m, 4H, CH2); 1.45 (m, 4H, CH2); 1.75 (m, 2H, CH2); 2.95 (m, 2H,
CH); 3.30 (s, 3H, OCH3); 3.47 (m, 2H, CH2OCH3). 13C NMR δ ppm: 14.50 (CH3); 23.12, 26.71, 28.40,
28.98, 32.24 (5CH2); 55.28 (CH); 57.66 (CH); 59.35 (CH2); 70.62 (OCH3).
threo-2,3-Heptanediol 2a, threo-3,4-heptanediol 2b, threo-3,4-nonanediol 2c, threo-1,3,4-nonanetriol
2d, and threo-1-methoxy-3,4-nonanediol 2e, were obtained by HClO4 (0.05 M) promoted hydrolysis of

C. Chiappe et al. / Tetrahedron: Asymmetry 9 (1998) 341–350
347
(ꢀ)-1a–e in 60:40 THF:H2O for 24 h. 2a: oil, 1H NMR δ ppm: 0.90 (t, 3H, J=6.6 Hz, CH3); 1.17 (d, 3H,
J=6.30 Hz, CH3); 1.20–1.50 (m, 6H, CH2); 3.3 (m, 1H, CH); 3.55 (m, 1H, CH). 13C NMR δ ppm: 13.85
(CH3); 19.24 (CH3); 22.59, 27.62, 32.79 (3CH2); 70.70 (CH); 75.99 (CH). 2b: oil, 1H NMR δ ppm: 0.97
(t, 3H, CH3); 0.9 (t, 3H, CH3); 1.2 (m, 2H, CH2); 1.40 (m, 4H, CH2); 3.24 (m, 1H, CH); 3.35 (m, 1H,
CH). 13C NMR δ ppm: 9.95 (CH3); 13.96 (CH3); 18.77, 26.22, 35.56 (3CH2); 73.74 (CH); 75.80 (CH).
2c: oil, 1H NMR δ ppm: 0.89 (t, 3H, J=6.4 Hz, CH3); 0.97 (t, 3H, J=7.50 Hz, CH3); 1.30 (m, 4H, CH2);
1.42–1.65 (m, 6H, CH2); 3.35 (m, 2H, CH). 13C NMR δ ppm: 10.67 (CH3); 14.63 (CH3); 23.21, 26.00,
1
26.9, 32.50, 34.05 (5CH2); 74.70 (CH); 76.45 (CH). 2d: oil, H NMR δ ppm: 0.89 (t, 3H, J=6.20 Hz,
CH3); 1.20–1.50 (m, 8H, CH2); 1.70 (m, 2H, CH2); 3.45 (m, 1H, CH); 3.63 (m, 1H, CH); 3.78 (t, 2H,
J=5.5 Hz, CH2OH). 13C NMR δ ppm: 13.95 (CH3); 22.55, 25.38, 31.80, 33.10, 35.15 (5CH2); 59.90
1
(CH2); 72.96 (CH); 74.55 (CH). 2e: oil, H NMR δ ppm: 0.89 (t, 3H, J=6.25 Hz, CH3); 1.30 (m, 4H,
CH2); 1.45 (m, 4H, CH2); 1.78 (m, 2H, CH2); 3.35 (s, 3H, OCH3); 3.42 (m, 2H, CH2OCH3); 3.60 (m,
2H, CH). 13C NMR δ ppm: 13.96 (CH3); 22.53, 25.36, 31.80, 33.08, 33.30 (5CH2); 58.69 (CH2); 70.58
(OCH3); 73.00 (CH); 74.25 (CH).
(3S,4S)-(−)-3,4-nonanediol was prepared from trans-3-nonene, using AD-mix α, according to the
1
standard procedure reported by Sharpless.15 [α]D=−30 (c=1, MeOH), H NMR δ ppm: 0.89 (t, 3H,
J=6.4 Hz, CH3); 0.97 (t, 3H, J=7.50 Hz, CH3); 1.30 (m, 4H, CH2); 1.42–1.65 (m, 6H, CH2); 3.35 (m,
2H, CH). 13C NMR δ ppm: 10.67 (CH3); 14.63 (CH3); 23.21, 26.00, 26.9, 32.50, 34.05 (5CH2); 74.70
(CH); 76.45 (CH).
3.1.1. (R)-(+)-4-Methoxynonane
(S)-(−)-octyn-3-ol (530 mg, 4.2 mmol) was dissolved in anhydrous THF (8 ml) and a solution of
BuLi (5 ml, 1.6 M in hexane) was added at 0°C. The reaction mixture was stirred for 3 h and then
iodomethane (0.7 ml, 8.5 mmol) was added. After a night at room temperature the solution was diluted
with dichloromethane, and washed with water. The organic phase was dried (MgSO4) and evaporated to
give a residue (630 mg) which was subjected to hydrogenation in ethyl acetate (30 ml) using Pd–C (100
mg) as catalyst. After 24 h at room temperature the reaction mixture was filtered on a Celite bed and the
solution was dried (MgSO4) and evaporated to give 870 mg of a crude product identified by NMR as
1
(R)-(+)-4-methoxynonane. [α]D=+2.57 (c=1, CHCl3). H NMR δ ppm: 0.85 (t, 3H, CH3); 0.89 (t, 3H,
CH3); 1.25–1.55 (m, 12H, CH2); 3.10 (m, 1H, CHOCH3); 3.30 (s, 3H, OCH3). 13C NMR δ ppm: 14.02
(CH3); 14.25 (CH3); 18.50; 22.65, 25.00, 32.10, 33.40, 35.70 (6CH2); 56.30 (OCH3); 80.78 (OCH3).
3.2. Enzymatic hydrolysis
3.2.1. Rates of mEH catalyzed hydrolysis of epoxides 1a–e
Aliquots (50 µl) of ethanolic stock solutions of (ꢀ)-1a–e were added to 2 ml of diluted microsomal
preparation13 containing 10 or 20 mg of protein/ml, in a such way as to obtain a 10, 20 or 30
mM final substrate concentration, and the mixtures were incubated with shaking at 37°C. After 10
and 20 min (or 20 and 40 min for 1c) the reactions were stopped by extraction with ethyl acetate
(2 ml) containing a proper amount of benzaldehyde as a standard. After centrifugation, the extracts
were analyzed by GLC for the quantification of the unreacted epoxides and the formed diols. The
diol formation was linear with time and protein concentration, and was independent of the subtrate
concentration, indicating enzyme saturation. Blank experiments, carried out under identical conditions
but using boiled microsomal preparations, showed that no spontaneous hydrolysis occurred under the
employed conditions. The average saturation rates in nmol/(mg protein×min) obtained for the various
substrates with the microsomal preparation were: 1a, 3.1; 1b, 0.5; 1c, 0.3; 1d, 5.5; 1e, 4.2.

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C. Chiappe et al. / Tetrahedron: Asymmetry 9 (1998) 341–350
3.2.2. Determination of enantiomeric excesses
Aliquots (20 µl) of 2 M ethanolic stock solutions of (ꢀ)-1a–e were added to 2 ml of microsomal
preparation containing 20 mg of protein/ml and the mixtures were incubated with shaking at 37°C. At
prefixed times the reactions were stopped by extraction with ethyl acetate containing a proper amount of
benzaldehyde as a standard for the quantification of the residue epoxide, and the extracts were analyzed
using the chiral column for the determination of the e.e.s of the residue epoxides and formed diols. At
least three determinations were made at each time. The average results are reported in Table 1.
3.2.3. Diol isolation
In order to determine the absolute configuration of the excess enantiomer of the formed diols, epoxides
1b–e as neat liquids (ca. 40–60 mg) were added to 20 ml of the microsomal preparation, containing 20
mg of protein/ml, and the reaction mixtures were incubated with shaking at 37°C for the time necessary to
obtain the proper conversion. The incubation mixtures were then stopped by extraction with ethyl acetate.
The organic phases were diluted to an exactly known volume and a proper amount of the standard was
added to an aliquot of these extracts in order to verify by GLC the conversion. The remaining parts of the
organic phases were evaporated in vacuo and the residues were chromatographed on silica gel columns
to give pure diols 2a–e. 2a, [α]D=+13.4 (c=1, EtOH)12; e.e. 56%; 2b, [α]D=+19.0 (c=1, CHCl3)14; 2c,
[α]D=+30.0 (c=1, MeOH); 2d, [α]D=+26 (c=1, MeOH); e.e. 90%; 2e, [α]D=+7.8 (c=1, MeOH), e.e.
80%.17
3.3. Determination of the absolute configuration
The absolute configuration of the excess enantiomer of the residue epoxides was determined on the
crude reaction mixtures, after evaluation of the enantiomeric excesses by GLC on a chiral colunm, as
reported below:
3.3.1. Epoxide 1a
LiAlH4 (20 mg) was added to a solution containing the enantiomerically pure (GLC) cis-2,3-
epoxyheptane (1a, 20 mg, 0.17 mmol) in anhydrous ethyl ether (2 ml) and the mixture was stirred at
room temperature for 3 h. After addition of ice the organic phase was washed with water and dried
(MgSO4) to give a mixture of 2- and 3-heptanol. The absolute configuration of 2-heptanol was shown to
be (R) by comparison of its emergence order with that of a sample of commercial (R)-2-heptanol.
3.3.2. Epoxide 1c
LiAlH4 (20 mg) was added to a solution containing the enantiomerically enriched (GLC) cis-3,4-
epoxyheptane (1c, 20 mg, 0.17 mmol) in anhydrous ethyl ether (2 ml) and the mixture was stirred at
room temperature for 3 h. After addition of ice the organic phase was washed with water and dried
(MgSO4) to give a mixture of 3- and 4-heptanol, which was subjected to methylation by treatment
with NaH and CH3I according to the general alkylation procedure.16 The absolute configuration of 4-
methoxynonane was shown to be (R) by comparison of its emergence order with that of a sample of
(R)-(+)-4-methoxynonane prepared as reported above.
3.3.3. Epoxide 1d
Tosyl chloride (40 mg) was added to a solution containing the enantiomerically enriched (GLC) cis-
3,4-epoxynonan-1-ol (1d, 30 mg) in pyridine (2 ml) and the mixture was stirred at room temperature.
After 3 h the reaction mixture was diluted with ice water, acidified with conc. HCl and extracted with

C. Chiappe et al. / Tetrahedron: Asymmetry 9 (1998) 341–350
349
ethyl ether. The organic phase was washed with saturated NaHCO3, dried (MgSO4), and evaporated
to give a crude product which was subjected to methylation by treatment with NaH and CH3I.16 The
absolute configuration of 4-methoxynonane was shown to be (R) by comparison of its emergence order
with that of a sample of (R)-(+)-4-methoxynonane.
3.3.4. Epoxide 1e
To a solution of the enantiomerically enriched (GLC) (3R,4S)-cis-3,4-epoxynonane-1-ol (1d, 70 mg)
in anhydrous dichloromethane (6 ml), containing 2,6-di-t-butyl-4-methylpyridine (1 mmol), methyl
triflate (0.6 mmol) was added under an argon atmosphere, and the mixture was refluxed for 2 h, and then
filtered. The solution was washed with 5% HCl and saturated NaHCO3, dried (MgSO4) and evaporated
to give a crude product, (3R,4S)-cis-1-methoxy-3,4-epoxynonane 1e, having the same retention time on
the chiral GLC column as a sample of pure 1e, arising from the partial mEH catalyzed kinetic hydrolysis
of (ꢀ)-1e.
The absolute configuration of the excess enantiomer of the formed diols was determined on the pure
product as reported below.
3.3.5. Diol 2d
Tosyl chloride (55 mg, 27 mmol) was added to a pyridine solution (2 ml) of the enantiomerically
enriched threo-(+)-1,3,4-nonanetriol, (2d, 40 mg) and the mixture was stirred at room temperature. After
3 h the reaction mixture was diluted with ice water, acidified with HCl and extracted with ethyl ether.
The organic phase was washed with saturated NaHCO3, dried (MgSO4), and evaporated to give a crude
product which was subjected to reduction by treatment with LiAlH4 (20 mg) in anhydrous ethyl ether
(2 ml). After 3 h at room temperature, ice was added to the reaction mixture and the organic phase was
separated, washed with water, dried (MgSO4) and evaporated to give the enantiomerically enriched diol
2c.
3.3.6. Diol 2e
The enantiomerically pure or enriched diols 2d and 2e (30 mg) were subjected to exhaustive methyl-
ation by treatment with NaH and CH3I according to the general alkylation procedure,16 to give in both
cases a crude product which was identified as (+)-1,3,4-trimethoxynonane. [α]D=+7.7 (c=1, CH3OH),
95% ee. 1H NMR δ ppm: 0.89 (t, 3H, J=6.4 Hz, CH3); 1.25 (m, 10H, CH2); 3.35 (s, 3H, OCH3); 3.40 (s,
3H, OCH3); 3.42 (s, 3H, OCH3); 3.46 (m, 2H, CH2OCH3); 3.75 (m, 2H, CH). 13C NMR δ ppm: 14.05
(CH3); 22.61, 25.66, 26.20, 29.70, 30.14; (5CH2); 58.40 (OCH3); 58.50 (OCH3); 58.70 (OCH3); 69.00
(CH2); 78.00 (CH); 81.70 (CH).
Acknowledgements
This work was supported in part by grants from Consiglio Nazionale delle Ricerche (CNR, Roma) and
Ministero dell’Università e della Ricerca Scientifica e Tecnologica (MURST, Roma).
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