Stereo- and regioselectivity in the P450-catalyzed oxidative tandem difunctionalization of 1-methylcyclohexene

Article abstract of DOI:10.1016/j.tet.2013.04.132

The selective partial oxidation of small non-functionalized molecules using biocatalysis based on P450 monooxygenases is known to be difficult due to the expected poor regio- and stereoselectivity, but in this study it was nevertheless attempted. 1-Methyl

Full text of DOI:10.1016/j.tet.2013.04.132

Tetrahedron 69 (2013) 5306e5311
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Stereo- and regioselectivity in the P450-catalyzed oxidative tandem
difunctionalization of 1-methylcyclohexene
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Gheorghe-Doru Roiban a , Ruben Agudo y, Manfred T. Reetz a
*
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Department of Chemistry, Philipps-Universitat Marburg, Hans-Meerwein Str., 35032 Marburg, Germany
Max-Planck-Institut fur Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mulheim an der Ruhr, Germany
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a r t i c l e i n f o
a b s t r a c t
Article history:
The selective partial oxidation of small non-functionalized molecules using biocatalysis based on P450
monooxygenases is known to be difficult due to the expected poor regio- and stereoselectivity, but in this
study it was nevertheless attempted. 1-Methylcyclohexene was subjected to oxygen-mediated bio-
catalytic oxidation using P450-BM3 as the catalyst. Both oxidative hydroxylation and epoxidation were
observed, in some cases leading to hydroxy epoxides with high diastereo- and enantioselectivity espe-
cially when employing BM3 mutants.
Received 13 February 2013
Received in revised form 25 April 2013
Accepted 29 April 2013
Available online 6 May 2013
Keywords:
Ó 2013 Elsevier Ltd. All rights reserved.
Enantioselectivity
CH-activation
P450
Monooxygenase
Diastereoselectivity
1. Introduction
ensures regio- and stereoselective radical H-abstraction followed by
rapid CeO bond formation. In these and other studies, P450-BM3 is
Transition metal catalyzed chemo-, regio-, and stereoselective ox-
idative functionalization of simple and complex organic compounds
continues to be an area of intense interest, CH-activationwith selective
introduction of hydroxy groups being a prominent example.1 Alter-
natively, oxidative hydroxylation can be catalyzed bycytochrome P450
enzymes,2 which are also capable of olefin epoxidation. In these bio-
catalytic reactions oxygen (air) serves as the oxidant. These ubiquitous
enzymes are Feeheme-dependent with very large binding pockets,
catalyzing, forexample, the regio- and stereoselective hydroxylation of
precursors of terpenes, alkaloids, and steroids at late stages of bio-
synthesis. When ‘foreign’ lipophilic compounds enter organisms,
P450-catalyzed detoxication occurs by way of solubilization as a result
of hydroxylation, which need not be regio- or stereoselective.
often used,6 because it contains a covalently fused NADPH-domain
and has proven to be a viable catalyst for a number of trans-
formations. More recently, directed evolution has also been applied
to considerably smaller molecules, which also bear functional
groups, 1-cyclohexene carboxylic acid ester 1 being an example for
which two different P450-BM3 mutants were evolved leading to
regioselective hydroxylation with optional formation of (R)-2 and
(S)-2, respectively.7 Another impressive example is a P450-BM3
mutant, which hydroxylates N-benzyl pyrrolidine regioselectively
at the 3-position leading to the S-configurated alcohol.8 It is likely
that H-bonds and/or electrostatic as well as hydrophobic effects
contribute to the formation of active poses of these relatively small
molecules in the large P450-BM3 binding pocket.
When a P450 catalyst fails to be selective in the partial oxidation
of a substrate of practical interest in synthetic organic chemistry or
biotechnology, the wild-type (WT) can in principle be tuned by the
methods of directed evolution.3,4 However, the control of both
regio- and stereoselectivity is a particularly difficult goal, which has
not been achieved until recently using steroids as model com-
pounds.5 The mutations introduced by this form of protein engi-
neering cause the substrate to be bound in an appropriate pose
above the catalytically active high-spin hemeeFe]O species, which
* Corresponding author. Tel.: þ49 (0) 6421 28 25500; fax: þ49 (0) 6421 28
25620; e-mail address: reetz@mpi-muelheim.mpg.de (M.T. Reetz).
y
Equal contribution.
0040-4020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2013.04.132

G.-D. Roiban et al. / Tetrahedron 69 (2013) 5306e5311
5307
It can be surmised that the difficulty in controlling both regio-
and stereoselective CH-activating hydroxylation increases when
subjecting small substrates devoid of functional groups to P450-
catalysis. In the complete absence of functional groups capable of
undergoing hydrogen bonding or electrostatic effects, various cat-
alytically active poses are likely leading to many different oxidation
products. Indeed, in protein engineering attempts to control both
regio- and stereoselectivity of P450 enzymes as catalysts in the
hydroxylation of such structurally simple alkanes as n-pentane or
n-hexane have not been very successful to date.9 The selective
oxidative functionalization of 1-methylcyclohexene (3) using P450-
catalysis has not been attempted thus far, but it is also likely to be
a difficult task. The only functional group in this substrate is the
olefinic double bond, which is less likely to undergo specific
binding modes. Indeed, one can anticipate several active poses,
including those that lead to epoxidation. Tandem oxidation leading
to hydroxyl-epoxides is also possible, which would be of particular
synthetic interest.
specifically evolved for the regio- and enantioselective hydroxyl-
ation of the more functionalized substrate 1. Using this strategy we
hoped to gain information for future directed evolution optimiza-
tion, especially in the quest to control selectivity in tandem
difunctionalization.
2. Results and discussion
In initial experiments, WT P450-BM3 was used as the bio-
catalyst in the oxidation of substrate 3 under different reaction
conditions (variation of temperature, buffer pH, reaction time as
well as NADPþ concentration). In order to measure P450 activity,
several small scale hydroxylation experiments as well as medium
and higher scale experiments were conducted (final experimental
conditions are listed in the Experimental section). Reactions per-
formed with resting cells proved to be cleaner than those using
crude lysate. Furthermore, resting cells do not need the addition of
exogenous NADPþ, since similar conversion values and selectivities
were found using concentrations, which ranged from 0 to 0.5 mM
of NADPþ. All reaction products were first identified by GCeMS and
subsequently fully characterized by comparison with authentic
samples (Fig. 1, top).
It can be seen that the product mixture is the result of a fairly
indiscriminate oxidation process, regio- and stereoselectivity be-
ing poor. In addition to unselective oxidation, subsequent re-
actions occur to a small degree under the reaction conditions,
namely rearrangement of allylic alcohol 4 to 8 and ring-opening
hydrolysis of epoxide 5 with formation of trans-10 (see also
Table 1). Thus, biocatalysis using WT P450-BM3 in the oxidation
The present study constitutes an exploratory investigation of
using P450-BM3 as a catalyst in the partial oxidation of substrate 3.
The initial goal was to obtain a complete fingerprint of all oxidation
products, including stereochemical assignments. Should this oxi-
dation turn out to be unselective with formation of many different
products, as expected, we planned to test some of the best mutants
Fig. 1. GC chromatograms of the oxidation products of 1-methylcyclohexene (3) catalyzed by WT P450-BM3 (top) and mutant F87I/A328V (down) using resting cells (3 h of reaction
time). GC conditions: 30 m DB1, inner diameter 0.25 mm; pressure: 0.5 bar H2; injector: 230 ꢀC; oven: temperature gradient: 50 ꢀC isothermic 10 min, then from 50 to 205 ꢀC with
12 ꢀC/min, FID detector: 350 ꢀC.

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G.-D. Roiban et al. / Tetrahedron 69 (2013) 5306e5311
Table 1
Performance of WT P450-BM3 and mutant F87I/A328V here serving as catalysts in the oxidative hydroxylation of substrates 3, rac-4, and rac-5a
Entry Substrate Mutation
Products (conversion (%)/configuration)
Substrate
Conversion Time
concentration (%)
(%)
(h)
4
5
6
7
8b
9
10c 11
12
13
1
2
3
3
WT
16/32(S) 34/25(1R,2S) 13/29(S)
5
17 4/cisþtrans (60:40)
<1 4/cisþtrans
1/cis
5
38 mM
>97
3
3
(77:23)
d
F87I/A328V 32/56(R) 21/22(1R,2S) 10/0
10 19 4/trans (1S,2R,6R)
(98% ee)
<1
d
4
38 mM
>97
3
4
rac-4
rac-4
WT
F87I/A328V
d
d
d
d
d
d
22 42 36/cisþtrans (15:85)
39 37 24/trans (1S,2R,6R)
(72% ee)
d
d
d
d
d
d
d
d
13 mM
13 mM
28
54
21
21
5
rac-5
rac-5
WT
d
d
d
d
d
4/cis
52 44/cisþtrans
(97:3)
76 8/cis
d
d
d
d
13 mM
13 mM
46
9
21
3d
6
F87I/A328V
d
d
d
d
d
16/cis
Values obtained from average of atleast three independent experiments performed with resting cells using NADPþ 50
Alcohol was racemic in all the cases.
Only the trans-diastereoisomer detected in all cases.
mM.
a
b
c
d
Reaction time of 21 h leads to high amount of impurities.
of 1-methylcyclohexene 3 is quite different from the analogous
reaction of 1-cyclohexene carboxylic acid ethyl ester (1). The latter
substrate resulted in the formation of (R)-2 (34% ee) and other
regioisomeric alcohols in a ratio of 84:16, but epoxides were not
formed. Since epoxidation is an electrophilic process, the
electron-poor olefinic bond in this substrate is not epoxidized.
These results are in accord with the observation that P450-BM3-
catalyzed oxidation of unsubstituted cyclohexene results primar-
ily in epoxidation (85%) in addition to allylic alcohols (15%).10
The reaction pathways of 1-methylcyclohexene (3) were
established by performing additional experiments using WT P450-
BM3 as the catalyst and racemic 3-methylcyclohex-2-en-1-ol (rac-
4) and 1-methyl-1,2-epoxycyclohexane (rac-5) as substrates
(Scheme 1). These experiments led to six products, namely 7, 8, and
cis- and trans-9 for the alcohol 4 and cis-9, trans-10, and cis- and
trans-11, respectively, in the case of epoxide 5, which had been
observed in the initial oxidation of substrate 3.
Scheme 1. Products obtained as a result of WT P450-BM3 catalyzed oxidation of
substrates rac-4 and rac-5.
Scheme 2. Primary and secondary oxidative processes in the reaction of 1-
methylcyclohexene (3) using WT P450-BM3 as catalyst, secondary rearrangements,
and hydrolyzes being included in the description concerning the origin of all products.
As before, the products were identified by comparison with
authentic samples. The results allow a complete reaction scheme to
be established, which defines the origin of all products (Scheme 2).
Due to the lack of selectivity of WT P450-BM3 as a catalyst in the
model reaction, several mutants previously evolved for the regio-
and stereoselective allylic hydroxylation of the ester 1 were tested
in the oxidation of substrate 3 (see Experimental section for the
complete data). BM3 mutant F87I/A328V proved to be considerably
more selective than WT, the GC chromatogram of the crude product
mixture indicating fewer peaks (Fig. 1, bottom). Analysis of the data
shows that regio- and stereoselectivity in the biotransformation of
3 have improved to a notable extent relative to the results of the
oxidation using WT BM3 (Table 1). Especially diastereo- and
enantioselectivity in the formation of the hydroxy-epoxide trans-9
are high (98% ee in favor of the (1S,2R,6R)-stereoisomer). Although
conversion to this product is still low, the result is remarkable, and
also of potential synthetic interest. Indeed, the preparation of trans-
configurated diastereoisomers of this type starting from
substituted olefins is known to be tedious and occurs with lower
selectivities, generally cis-products being favored.11
In small scale experiments performed with mutant F87I/A328V,
enantioselectivity of trans-9 reached 97e99% ee. However, upon

G.-D. Roiban et al. / Tetrahedron 69 (2013) 5306e5311
5309
up-scaling, enantioselectivity decreases to 81% ee. Similar effects
have been observed in other systems.7 It is interesting to note that
trans-9 is formed only via alcohol 4. When rac-5 was subjected to
oxidation using mutant F87I/A328V, the only diastereoisomer
detected was cis-9 (Table 1, entry 5). Moreover, when rac-4 was
used as the starting material in a reaction catalyzed by mutant F87I/
A328V, epoxide trans-9 was obtained with a moderate enantiose-
lectivity (72% ee in favor of the (1S,2R,6R)-stereoisomer). This
suggests that only the (R)-enantiomer of the alcohol is the pre-
ferred substrate in the next oxidation step, while the (S)-enantio-
mer is oxidized to ketone 7. Furthermore, control measurements
using alcohol rac-4 and mutant F87I/A328V showed that the
starting material remains racemic, suggesting a double kinetic
resolution: the initially formed alcohol (R)-4 formed reacts further
to form trans-9, while alcohol (S)-4 is oxidized to ketone 7.
Reaction 3/trans-9 was up-scaled employing mutant F87I/
A328V and using 480 mg (5 mmol) of starting material, pure
product being successfully isolated following column chromatog-
raphy (24 mg).
biohydroxylation, aliquots were resuspended in 480
m
L lysis buffer
[phosphate buffer (pH 7e8, 100 mM), lysozyme (14 mg/mL), and
DNAse I (6 U/mL)] and incubated at 37 ꢀC during 20 min at 700
rpm in a thermomixer. After this time, tubes were centrifuged at
10 000 rpm at 4 ꢀC for 15 min to pull down the cellular debris.
Supernatant (c.a. 500 mL) was transferred to a new 1.5 mL tube,
containing 500
mL reaction buffer [phosphate buffer (pH 7e8,
100 mM), NADPþ (50e1000
mM) and glucose (100 mM). Reaction
started after addition of 1
mL of compound 3 (8 mM final con-
centration, 8
20 h at 700 rpm in a thermomixer. After incubation time, 700
reaction was extracted with ethyl acetate (700 L) and the organic
m
mol)]. Samples were incubated at 25 ꢀCe37 ꢀC for
mL of
m
layer subjected to GC analysis. Best results were achieved
using buffer pH 7.4, NADPþ 500
temperature.
m
M, and 25 ꢀC as incubation
Different P450-BM3 mutants were tested for biohydroxylation
of compound 3 (single mutants F81R, A82L, A82M, F87D, F87G,
F87I, L181E, I263G, and double mutants F87I/A328V, F87V/A328N,
F87V/A328V, I263G/A328S). Individual BOU730 colonies harbor-
ing mutated plasmids were inoculated in 1 mL of LB with kan
3. Conclusions and perspectives
(50
preculture was transferred to 15 mL of TB with kan (50
Cultures were grown as above, and an aliquot of 10 mL was pel-
leted and resuspended in 430 L lysis buffer [phosphate buffer
m
g/mL). After 5 h of incubation at 37 ꢀC with shaking, this
g/mL).
m
We have identified all products arising from the P450-BM3
catalyzed oxidation of 1-methylcyclohexene (3), including the as-
signment of absolute and relative stereochemistry. No less than
a dozen oxidation products were formed in this biocatalytic pro-
cess, conversion to epoxy alcohols being of special synthetic in-
terest. Unfortunately, such tandem bifunctionalization using WT
P450-BM3 occurred only to a minor extent, stereoselectivity also
being poor. However, the use of a P450-BM3 mutant previously
evolved for the highly regio- and stereoselective oxidative hy-
droxylation of 1-cyclohexene carboxylic acid methyl ester (1)
showed an intriguing degree of selectivity in this bifunctionaliza-
tion to provide the trans epoxy alcohol with high diastereo- and
enantioselectivity, although with low conversion. These findings
set the stage for future directed evolution work in the quest to
optimize tandem bifunctionalization of olefin 3 and of related
substrates.
m
(pH 7.4, 100 mM), lysozyme (14 mg/mL), and DNAse I (6 U/mL)]
and incubated at 37 ꢀC during 20 min at 700 rpm. Tubes were
centrifuged at 10 000 rpm at 4 ꢀC for 20 min and 425
mL of su-
pernatant were mixed with glucose (100 mM final concentration),
NADPþ (500 mM final concentration), and compound 3 (33 mM
final concentration, 17
20 h with 700 rpm in a thermomixer. Reaction volume was
extracted with 500 L of ethyl acetate and subjected to GC anal-
m
mol). Samples were incubated at 37 ꢀC for
m
ysis. Best results (enantio- and stereoselectivities) were obtained
using mutant F87I/A328V.
4.1.3. Medium scale biohydroxylation using P450 mutants. Biohy-
droxylation reaction with best mutant F87I/A328V was performed
using resting cells or lysate. An Erlenmeyer flask (100 mL) con-
taining LB (15 mL) and kan (50 mg/mL) was inoculated with a colony
4. Experimental section
4.1. Molecular biology
from BOU730 cells expressing corresponding P450-BM3 mutant
and incubated 5 h at 37 ꢀC with shaking. An aliquot of this pre-
culture (5 mL) was inoculated into TB (50 mL) containing kan
(50 m
g/mL) and allowed to grow at 30 ꢀC until O.D. of 0.8e0.9 at
4.1.1. Reagents. Escherichia coli BOU730 cells used are described
elsewhere.7 Electro-competent cells were prepared in-house
according to standard protocols.12 DNAse I and lysozyme were
obtained from AppliChem. NADPþ was purchased from Calbio-
chem. LuriaeBertani (LB) medium as a powder mixture, kanamycin
600 nm was reached, then IPTG was added to a final concentration
of 0.2 mM. Culture was grown at 30 ꢀC during 20 h with gentle
agitation and cells were pelleted by centrifugation (6 min, 4000
rpm at 4 ꢀC). For experiments performed with lysate cells, pellets
were resuspended in 10 mL of lysis buffer [phosphate buffer (pH
7.4, 100 mM), lysozyme (14 mg/mL), and DNAse I (6 U/mL)], in-
cubated at 37 ꢀC 45 min and centrifuged 20 min at 4000 rpm. Su-
pernatants (c.a. 9 mL) were mixed with glucose (100 mM final
(kan) and carbenicillin were obtained from Roth. iso-Propyl-b-D-
thiogalactoside (IPTG) was purchased from Fermentas. Terrific
Broth TB medium contained yeast extract (24 g/L), peptone (12 g/L),
glycerol (4 mL/L), KH2PO4 (17 mM), and K2HPO4 (72 mM).
concentration), NADPþ [0e100
and compound 3 (38 mM, 380
mL (0e0.5 mM final concentration)],
mmol). For experiments carried out
4.1.2. Small scale biohydroxylation using P450 mutants. Initially, WT
P450-BM3 was tested for optimizing biohydroxylation reaction
conditions of compound 3. For small scale reactions, an individual
BOU730 colony harboring plasmid pRSF-P450-BM37 was in-
with resting cells, pellets were resuspended in reaction buffer
[phosphate buffer (pH 7.4, 100 mM)], NADPþ [0e100
L (0e0.5 mM
final concentration)], glucose (100 mM), and compound 3 (38 mM,
380 mol). Reactions were carried out in 100 mL flasks with tight
closure using rubber caps at 25 ꢀC, for 3e21 h with mild agitation.
At different time points, 700 L of reaction mixtures were extracted
with 700
m
m
oculated in 5 mL of LB with kan (50
at 37 ꢀC with shaking, this preculture was transferred to 50 mL of
TB with kan (50
g/mL). Cultures were grown at 30 ꢀC until O.D. of
mg/mL). After 5 h of incubation
m
m
mL ethyl acetate and subjected to GC analysis. Resting cells
0.8e0.9 at 600 nm was reached, then IPTG was added to a final
concentration of 0.2 mM and the cultures were allowed to grow at
30 ꢀC with vigorous agitation for 20 h. Cells were collected
by dividing the culture in 1 mL aliquots, which were centrifuged
at 10 000 rpm for 6 min at room temperature, supernatant dis-
carded and pellets stored at ꢁ20 ꢀC for subsequent use. For
did not need addition of exogenous NADPþ (similar results were
found adding either 0 or 0.5 mM of NADPþ) to perform the reaction.
In contrast, this was necessary when using lysate cells. Results
shown in Table 1 belong to experiments performed with resting
cells using NADPþ 50
mM and incubated for 21 h. Reactions with
rac-4 and rac-5 were carried out using the protocol described above

5310
G.-D. Roiban et al. / Tetrahedron 69 (2013) 5306e5311
for resting cells employing NADPþ 50
(13 mM final concentration) of starting material in each case.
m
M and adding 130
m
mol
en-1-ol (4) purchased from SigmaeAldrich its analytical data cor-
responds to the published ones.16 The absolute configuration was
assigned by optical rotation comparison with an authentic sample
described by Okamura and Wu.17 Chiral GC conditions: 25 m IVA-
4.1.4. High scale biohydroxylation using P450 mutants. Reaction
between methylcyclohexene (3) and P450 mutant F87I/A328V was
up-scaled according to the published protocol.7 Briefly, an Erlen-
DEX 1, inner diameter 0.25 mm film thickness 0.15 mm; pressure:
0.5 bar H2; injector: 220 ꢀC; oven: 35 min isothermic at 75 ꢀC; FID
meyer flask (100 mL) containing LB (20 mL) and kan (50
mg/mL) was
detector: 320 ꢀC; (S)-4 13.55 min, (R)-4 17.00 min.
inoculated with a colony from BOU730 cells expressing P450 mu-
tant F87I/A328V and incubated for 6 h at 37 ꢀC with shaking. This
preculture was inoculated into TB (500 mL) containing kan (50 mg/
4.2.3. Absolute configuration determination of 1,2-epoxy-1-methyl
cyclohexane (5). A single colony of E. coli BL-21 Gold (DE3) colony
harboring plasmid pETLEH18 (limonene epoxide hydrolase) was
picked and inoculated in 10 mL of TB containing carbenicillin
mL) and allowed to grow at 30 ꢀC until O.D. of 0.8e0.9 at 600 nm
was reached, then IPTG was added to a final concentration of
0.2 mM and the culture grown at 30 ꢀC during 16e20 h with gentle
agitation. Cells were pelleted by centrifugation (15 min, 4000
rpm at 4 ꢀC), and the pellet was resuspended in 100 mL of reaction
(50 ng/
incubation, this preculture was added to 100 mL of TB containing
carbenicillin (50
g/mL) and 0.5% lactose, and incubated at 28 ꢀC for
m
L) and 0.5% lactose and incubated over night at 28 ꢀC. After
m
buffer phosphate buffer (pH 7.4, 100 mM), NADPþ (300
mM), and
20 h with gentle agitation. Expression of LEH was confirmed by
SDS-PAGE analysis (Data not shown). After incubation, 10 mL of
culture were pelleted by centrifugation (15 min, 4000 rpm at room
temperature) then pellet was resuspended in 1 mL of lysis buffer
[phosphate buffer (pH 7.4, 100 mM), lysozyme (14 mg/mL), and
DNAse I (6 U/mL)] and incubated at 37 ꢀC during 20 min at 700
rpm in a thermomixer. The sample was centrifuged at 10 000 rpm
at 4 ꢀC for 30 min to pull down the cellular debris. Supernatant (c.a.
glucose (100 mM). The mixture was transferred to a 500 mL
Erlenmeyer, and compound 3 (480 mg, 50 mM final concentration,
5 mmol) was added to start reaction, which was carried out at 25 ꢀC
for 24 h with mild agitation. After completion, the reaction mixture
was extracted with ethyl acetate, organic phase dried, concen-
trated, and subjected to column chromatography.
4.2. Chemistry
1 mL) was transferred to a new 1.5 mL tube and 3 mL of rac-5 were
added (27 mM final concentration). Reaction was incubated at 30 ꢀC
4.2.1. General remarks. Compounds 1-methylcyclohexene, 3-
methylcyclohex-2-en-1-one, 3-methylcyclohex-2-en-1-ol, trans-1-
methylcyclohexane-1,2-diol, and all other GC controls were pur-
chased from Acros, SigmaeAldrich and Alfa and used without fur-
ther purification. NMR spectra were recorded on a Bruker Avance
300 or DRX 400 (1H: 300 MHz or 400 MHz, 13C: 75 MHz or
101 MHz) spectrometer using TMS as internal standard (d¼0).
High-resolution mass spectra recorded in APCI mode were per-
formed on a ThermoScientific LTQ-FT spectrometer. Conversion and
enantiomeric excess were determined by achiral and chiral gas
chromatography. Analytical thin layer chromatography was per-
formed on Merck silica gel 60 F254q while for column chroma-
tography Merck silica gel 60 (230e400 mesh ASTM) was used.
Determination of the relative configuration was performed after
comparison with commercial available racemic samples. Racemic
1,2-epoxy-1-methylcyclohexane (5) was prepared by oxidation
with m-CPBA and its analytical data correspond to those published
in the literature.13 Absolute configuration of epoxide (5) was de-
termined after reaction with limonene epoxide hydrolase (LEH)
(see below). Racemic 8 was obtained following the same synthetic
route as that reported by O’Brien et al.14 Its spectroscopic data were
found to be identical with the ones described in the literature.15 A
racemic mixture of cis-9 [(1S,2S,6R)-9 and its enantiomer
(1R,2R,6S)-9] was obtained by m-CPBA oxidation of commercially
available alcohol rac-4. Its spectroscopic data were found to be
identical with ones described in literature.11a GC analyses showed
for 5 min at 700 rpm in a thermomixer. At different reaction times,
aliquots of 100 mL were harvested, extracted with 100 mL of ethyl
acetate, and subjected to chiral GC analysis for monitoring reaction
progress. Enantiomers assignment was based on the fact that LEH
digests enantiomer (1R,2S)-5 faster than enantiomer (1S,2R)-5.18
Chiral GC conditions: 25 m IVADEX 1, inner diameter 0.25 mm,
film thickness 0.15 m
m; pressure: 0.4 bar H2; injector: 220 ꢀC; oven:
temperature ramp 40 ꢀCe60 ꢀC with 1 ꢀC/min ramp, 25 min; FID
detector: 320 ꢀC; (1S,2R)-5 14.64 min, (1R,2S)-5 15.16 min.
4.2.4. Synthesis of 2-methylcyclohex-2-en-1-ol (S)-6. The reaction
of 1-methylcyclohexene catalyzed by mutant F87I/A328V was up-
scaled as described above. After extraction with ethyl acetate and
concentration, the crude reaction was purified by column chroma-
tography (ethyl acetate/petroleum ether 1:4) to afford compound 2-
methylcyclohex-2-en-1-ol (S)-6, which contained traces of alcohol
5. Its structure was proven after comparison of its analytical data
(1H, 13C NMR) with those reported by Craig et al.19 Absolute con-
figuration was assigned by optical rotation comparison with an
authentic sample, according to Craig et al.19 Chiral GC conditions:
25 m IVADEX 1, inner diameter 0.25 mm film thickness 0.15 mm;
pressure: 0.8 bar H2; injector: 220 ꢀC; oven: 70 min isothermic at
40 ꢀC; FID detector: 320 ꢀC; (R)-6 59.18 min, (S)-6 63.48 min.
4.2.5. Synthesis of 3-methyl-trans-2,3-epoxycyclohexan-1-ol trans-
(1S,2R,6R)-9. The reaction of 1-methylcyclohexene catalyzed by
mutant F87I/A328V was up-scaled as described above using
480 mg of methylcyclohexene. After extraction and concentration,
the crude reaction was purified by column chromatography (ethyl
acetate/petroleum ether 1:1) to afford 3-methyl-trans-2,3-
epoxycyclohexan-1-ol trans-(1S,2R,6R)-9 (24 mg, 4% yield). Its
spectroscopic data were found to be identical with the ones de-
scribed in the literature.21 (Rf¼0.43, ethyl acetate/petroleum ether
a
ratio of 97:3 (cis-9/trans-9). A racemic mixture of cis-12
[(1S,2R,6S)-12 and its enantiomer (1R,2S,6R)-12] and trans-12
[(1R,2R,6R)-12 and its enantiomer (1S,2S,6S)-12] were obtained by
oxidation with m-CPBA using as starting material rac-8 (97:3; cis-
12/trans-12). A racemic mixture of cis-11 [(1S,2S,6R)-11 and its
enantiomer (1R,2R,6S)-11] and trans-11 [(1R,2S,6S)-11 and its en-
antiomer (1S,2R,6R)-11] were obtained by oxidation with m-CPBA
using as starting material 6 (85:15; cis-11/trans-11).
1:1); 1H NMR (300 MHz, CDCl3)
d
¼4.00 (t, 3J¼7.2 Hz, 1H), 2.92 (s,
1H), 2.00e1.82 (m, 2H), 1.80e1.62 (m, 2H), 1.59e1.06 (m, 2H), 1.34
4.2.2. Synthesis of 3-methylcyclohex-2-en-1-ol (R)-4. Reaction of 1-
methylcyclohexene catalyzed by mutant F87I/A328V was up-scaled
as described above. After extraction with ethyl acetate and con-
centration, the crude reaction mixture was subjected to column
chromatography to afford compound (R)-4 (56% ee). The compound
was identified after comparison with racemic 3-methylcyclohex-2-
(s, 3H) ppm; 13C NMR (75 MHz, CDCl3)
d¼67.01, 63.47, 58.90, 30.25,
29.70, 23.45, 15.89 ppm; HRMS (APCIþ) calcd for C7H13O2 [MþH]þ:
129.0910; found: 129.0911; (1S,2R,6R)-9, 97e99% ee (small scale),
81% ee (high scale). The absolute configuration was determined
after a sample of (R)-5 (56% ee) was reacted with m-CPBA (GC vial,
CH2Cl2, room temperature, 24 h) to afford a mixture of cis and

G.-D. Roiban et al. / Tetrahedron 69 (2013) 5306e5311
5311
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trans-diastereoisomers (87/13, cis/trans) according to GCeMS
analysis. After chiral GC comparison with trans-9 the major peak
of the trans-diastereoisomer was assigned to (1S,2R,6R)-9 while
the minor peak of the trans-diastereoisomer was assigned to
(1R,2S,6S)-9. Chiral GC conditions: 25 m Lipodex G, inner diameter
0.25 mm; pressure: 0.5 bar H2; injector: 230 ꢀC; oven: temperature
gradient from 60 ꢀC to 100 ꢀC with 1 ꢀC/min ramp then from 100 ꢀC
to 220 ꢀC 12 ꢀC/min; FID detector: 350 ꢀC; (1S,2R,6R)-9 32.89 min,
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€
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ethyl acetate and concentration, the crude reaction was purified by
column chromatography (ethyl acetate/petroleum ether 1:1) to
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found to be identical with ones described in literature.20 In addi-
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methylcyclohexane-1,2-diol. Relative configuration of trans-10
was further confirmed after comparison with cis-10, sample ob-
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literature protocols.20
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Acknowledgements
Financial support by the Max-Planck-Society and the Arthur C.
Cope Foundation is gratefully acknowledged. We thank Stephanie
Dehn and Corinna Heidgen for GC analyses and Jonas Schwaben for
help with optical rotation measurements.
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