Acid-catalyzed enzymatic hydrolysis of 1-methylcyclohexene oxide

Article abstract of DOI:10.1016/S0957-4166(99)00449-8

Limonene-1,2-epoxide hydrolase from Rhodococcus erythropolis DCL14, an enzyme involved in the limonene metabolism of this microorganism, catalyzes the enantioselective hydrolysis of 1-methylcyclohexene oxide. (1R,2S)-1- Methylcyclohexene oxide was the preferred substrate and it was mainly hydrolyzed to (1S,2S)-1-methylcyclohexane-1,2-diol, while (1S,2R)-1- methylcyclohexene oxide was converted more slowly and mainly yielded (1R,2R)- 1-methylcyclohexane-1,2-diol. The reaction proceeded with a high regioselectivity (C1:C2, 85:15). H₂¹⁸O-labelling experiments confirmed that the nucleophile was mainly incorporated at the most substituted carbon atom, suggesting that limonene-1,2-epoxide hydrolase uses an acid-catalyzed enzyme mechanism.

Full text of DOI:10.1016/S0957-4166(99)00449-8

Pergamon
Tetrahedron: Asymmetry 10 (1999) 4225–4230
Acid-catalyzed enzymatic hydrolysis of 1-methylcyclohexene
oxide
a,∗,†
a
a,b,‡
Mariët J. van der Werf,
Jan A. M. de Bont and Henk J. Swarts
a
Division of Industrial Microbiology, Department of Food Technology and Nutritional Sciences, Wageningen University, PO
Box 8129, 6700 EV Wageningen, The Netherlands
Laboratory of Organic Chemistry, Department of Biomolecular Sciences, Wageningen University, PO Box 8026, 6700 EG
Wageningen, The Netherlands
b
Received 27 September 1999; accepted 8 October 1999
Abstract
Limonene-1,2-epoxide hydrolase from Rhodococcus erythropolis DCL14, an enzyme involved in the limo-
nene metabolism of this microorganism, catalyzes the enantioselective hydrolysis of 1-methylcyclohexene oxide.
1R,2S)-1-Methylcyclohexene oxide was the preferred substrate and it was mainly hydrolyzed to (1S,2S)-1-
(
methylcyclohexane-1,2-diol, while (1S,2R)-1-methylcyclohexene oxide was converted more slowly and mainly
yielded (1R,2R)-1-methylcyclohexane-1,2-diol. The reaction proceeded with a high regioselectivity (C1:C2,
8
5:15). H218O-labelling experiments confirmed that the nucleophile was mainly incorporated at the most sub-
stituted carbon atom, suggesting that limonene-1,2-epoxide hydrolase uses an acid-catalyzed enzyme mechanism.
1999 Elsevier Science Ltd. All rights reserved.
©
1. Introduction
Enantiopure epoxides are versatile synthons in the total synthesis of more complex biologically
active compounds. Both chemical1,2 and biological methods for the production of these epoxides are
2,3
available, and kinetic resolutions using epoxide hydrolases (EHs) have been studied as these enzymes
4,5
are stable and cofactor-independent.
Hydrolytic epoxide cleavage is either: (i) a base-catalyzed nucleophilic event, without apparent
activation of the epoxide ring; or (ii) an acid-catalyzed event, implying an electrophilic activation of
the epoxide ring concerted with the nucleophilic attack. Base-catalyzed hydrolysis is by nucleophilic
∗
†
Corresponding author. Tel: +31-30-6944071; Fax: +31-30-6944466; e-mail: vanderwerf@voeding.tno.nl
Present address: TNO-Voeding, Department of Applied Microbiology and Gene Technology, PO Box 360, 3700 AJ Zeist,
The Netherlands.
‡
Present address: IPO-DLO, PO Box 9060, 6700 GW Wageningen, The Netherlands.
0957-4166/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(99)00449-8

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6
attack predominantly at the less substituted or less hindered carbon atom, while under acidic conditions
nucleophilic attack mainly takes place at the more substituted carbon atom, resulting in a stabilization of
7
the carbocation formed in the transition state. Both mechanisms result in a retention of configuration at
the carbon atom at which the nucleophilic attack does not take place.
Remarkably, the many EHs from various sources, ranging from humans to bacteria, form a homo-
geneous group of enzymes belonging to the α,β-hydrolase fold superfamily that are structurally and
8,9
mechanistically similar. They all show a base-catalyzed mechanism, resulting in the trans-addition of
water at the less sterically hindered or less substituted carbon atom.10
Recently, we described the novel limonene-1,2-epoxide hydrolase (LEH) from Rhodococcus erythro-
polis DCL14, which does not belong to the α,β-hydrolase fold EHs.1
1,12
In this report we describe the
regioselectivity of the LEH-catalyzed conversion of 1-methylcyclohexene oxide.
2. Results
1
-Methylcyclohexene oxide 1 is a good substrate for LEH.11 The enzyme preferentially hydrolyzed
(1R,2S)-1 mainly into (1S,2S)-1-methylcyclohexane-1,2-diol 2, while (1S,2R)-1 was converted more
slowly and mainly yielded (1R,2R)-diol-2 (Scheme 1, Fig. 1). From the data presented in Fig. 1, a
regioselectivity of C1:C2 (85:15) was calculated. Both enzymatic reactions proceeded with retention
of configuration at the less substituted carbon atom, indicating that nucleophilic attack occurred at the
more substituted carbon atom (Scheme 1).
Scheme 1. Epoxide hydrolase-catalyzed hydrolysis of 1-methylcyclohexene oxide 1 into trans-1-methylcyclohexane-1,2-diol 2
To confirm that the C–O bond of the oxirane ring was cleaved at the tertiary carbon atom, the LEH-
catalyzed hydrolysis of epoxide-1 was performed in 18O-labelled water. Samples were taken after 35
min, when (1R,2S)-1 is completely converted with almost no conversion of the (1S,2R)-1 enantiomer,
and after 5 h, when both enantiomers were almost completely converted (Fig. 1). The products were
analyzed by GC–MS using a chiral stationary phase. The mass peak (m/z 132) of the product formed in
1
8
O-labelled water was 2 mass units higher than that of unlabelled product (m/z 130). The position of
18
the O-label was determined by analysis of the fragmentation pattern, and is based on the initial loss
of water from the secondary alcohol, which is much more favourable than loss of water from tertiary
alcohols. The mass spectrum of the main product [(1S,2S)-2] after 35 min of reaction showed an initial
loss of unlabelled water (m/z 114), indicating that the O-label was inserted at the tertiary carbon atom.
The minor product, (1R,2R)-2, contained the O-label mainly at the secondary alcohol, as indicated by
a loss of labelled water (m/z 112). The formation of the latter compound can be explained by the fact
13
18
18

M. J. van der Werf et al. / Tetrahedron: Asymmetry 10 (1999) 4225–4230
4227
Figure 1. Conversion of 1-methylcyclohexene oxide 1 into trans-1-methylcyclohexane-1,2-diol 2 by purified LEH from R.
erythropolis DCL14. ♦, (1R,2S)-1; ꢀ, (1S,2R)-1; ꢁ, (1S,2S)-2; ꢂ, (1R,2R)-2
that the LEH-catalyzed conversion of (1R,2S)-1 is not absolutely regioselective, and by the much higher
reaction rate of LEH with (1R,2S)-1 than with (1S,2R)-1. Therefore, most of the (1R,2R)-2 formed after
35 min originates from (1R,2S)-1 (Fig. 1). In contrast, after 5 h of incubation most of the label in (1R,2R)-
2
was situated at the tertiary carbon atom, as now the (1S,2R)-epoxide-1 has been the main substrate for
18
the formation of (1R,2R)-2. These results with O-water are in agreement with those described in Fig. 1.
Chemically, 1-methylcyclohexene oxide is rapidly hydrolyzed under acid conditions, while under basic
18
conditions hydrolysis proceeded very slowly if at all. The H SO -catalyzed hydrolysis of 1 in O-
2
4
labelled water confirmed that under acid conditions nucleophilic attack indeed takes place at the tertiary
carbon atom, as was concluded from the initial loss of unlabelled water (m/z 114) from the racemic
trans-1-methylcyclohexane-1,2-diol products formed.
3. Discussion
1-Methylcyclohexene oxide 1 contains both a secondary and tertiary carbon atom in the oxirane ring,
and the EH-catalyzed hydrolysis of this compound gives an indication about the reaction mechanism used
by the enzyme. The presently known α,β-hydrolase fold EHs convert 1 with inversion of configuration
at the less substituted carbon atom (Scheme 1).4
,14,15
This indicates that nucleophilic attack takes place
at the less substituted carbon atom, which is in agreement with the base-catalyzed mechanism of α,β-
hydrolase fold EHs.
The data obtained in this study show that, in contrast to the α,β-fold EHs, LEH converts 1 by
nucleophilic attack with a very strong preference for the more substituted carbon atom. There are two
possible explanations for this phenomenon: (i) LEH uses an acid-catalyzed reaction mechanism; or (ii)
LEH uses a base-catalyzed reaction mechanism, but due to the geometry of the active site the attack of the
nucleophile has to occur at the more hindered carbon atom. Previously, we obtained indications that LEH
uses a reaction mechanism that differs from the α,β-hydrolase fold EHs as: (i) it has a pH-profile which
is very different from that of the α,β-hydrolase fold EHs; (ii) it contains none of the highly conserved
motifs of the catalytic triad of the α,β-hydrolase fold EHs; and (iii) its natural substrate, limonene-
,2-epoxide, is only significantly hydrolyzed chemically under acid conditions (as is epoxide 1).1
1,12
1

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All these findings strongly suggest that LEH, in contrast to the α,β-hydrolase fold EHs, uses an acid-
catalyzed reaction mechanism. Previously, an acid-catalyzed mechanism for the enzymatic hydrolysis of
epoxides has been suggested in a few isolated instances.1
6–19
However, in all these studies crude enzyme
preparations or whole cells were used, and consequently did not lead to conclusive results. This is the
first time that an acid-catalyzed mechanism has been implicated as the mechanism for an EH purified
to homogeneity. This is yet another characteristic of LEH, besides its previously described physical,
11,12
biochemical and structural properties,
that clearly sets LEH apart from the hitherto described EHs.
4. Experimental
4.1. General
Racemic 1-methylcyclohexene oxide 1 was prepared from 1-methyl-1-cyclohexene with m-
20 18
chloroperoxybenzoic acid as the catalyst.
O-water (95% isotopic enrichment) was obtained from
Aldrich. LEH was purified from the cell extract of cells of Rhodococcus erythropolis DCL14 grown on
11
limonene as described previously.
Chromatographic analyses were carried out using a Chrompack CP9000 gas chromatograph equipped
with an FID detector. The enantiomers were analyzed on a chiral fused silica cyclodextrin capillary
α-DEX 120 column (30 m length, 0.25 mm internal diameter, 0.25 µm film thickness; Supelco,
Zwijndrecht, The Netherlands). Analyses were performed using N (1.0 ml/min) as carrier gas (injector
2
200°C, detector 250°C). The temperature program was as follows: 7 min at 70°C, from 70 to 110°C
at 10°C/min, then 15 min at 110°C. Chiral GC–MS analyses were carried out on an HP5973 MSD gas
chromatograph equipped with the same α-DEX 120 column and temperature program. Carrier gas He
(1.0 ml/min), injector 220°C. Electron Impact MS data were obtained at 70 eV. Optical rotations were
measured with a Perkin–Elmer model 241 polarimeter.
4.2. Reactions
The reaction mixtures (2 ml) consisted of 50 mM potassium phosphate buffer (pH 7.0), 5 mM 1-
methylcyclohexene oxide 1 and 4.5 µg/ml purified LEH (30°C) in 15 ml vials fitted with Teflon Mininert
valves (Supelco Inc.) preventing evaporation of 1. The vials were placed in a water bath (30°C) and, after
different time intervals, a vial was removed from the water bath and the reaction was terminated by the
addition of 1 ml ethyl acetate. The vials were vigorously shaken to enable quantitative extraction of 1 and
2
. The ethyl acetate layer was pipetted in a microcentrifuge tube and centrifuged (3 min, 15 000×g) to
achieve separation of the two layers. Subsequently 1 µl of the ethyl acetate layer was analyzed by chiral
GC.
The reaction mixtures for the incubation with 18O-water consisted of 250 µl 18O-H O, 10 µl of a 500
2
mM potassium phosphate buffer (pH 7.0), 5 µl of a purified LEH solution (0.67 mg/ml) and 2 µl of a
1-methylcyclohexene oxide:acetone (1:9) mixture in 4 ml vials fitted with Teflon Mininert valves. After
30 min and 5 h of incubation at 30°C, the reaction was terminated by the addition of 250 µl of ethyl
acetate, and treated as described above. Subsequently, 1 µl of the ethyl acetate layer was analyzed by
chiral GC–MS.
The reaction mixtures for chemical acid- or base-catalyzed hydrolysis of 1-methylcyclohexene oxide 1
1
8
consisted of 100 µl H O or O-H O, 2 µl 2N H SO or 4N NaOH and 1 µl 1-methylcyclohexene oxide
2
2
2
4
1
in 4 ml vials fitted with Teflon Mininert valves. After 60 min at room temperature, the reaction was

M. J. van der Werf et al. / Tetrahedron: Asymmetry 10 (1999) 4225–4230
4229
terminated by the addition of 100 µl of ethyl acetate, and 1 µl of the ethyl acetate phase was analyzed by
chiral GC–MS.
4.3. Product identification
The enantiomers of 1-methylcyclohexene oxide 1 and 1-methylcyclohexane-1,2-diol 2 were identified
by determining the optical rotation of the remaining epoxide isomer and the formed diol15 at 55%
conversion. The reaction medium was first extracted with hexane for selective extraction of epoxide 1, and
was subsequently extracted with ethyl acetate for quantitative extraction of the diol product. The positive
[
α] for both the ‘enriched’ epoxide 1 and the diol 2 at 55% conversion established the configurations
D
14,15
as depicted in Fig. 1.
their GC data with those of the α,β-hydrolase fold EH-catalyzed hydrolysis of 1.
Moreover, the configurations of the compounds were confirmed by comparing
4
4.4. (1R,2S)-1-Methylcyclohexene oxide 1
+
MS: m/z 112 [M ] (2), 111 (5), 97 (100), 83 (10), 69 (25), 55 (41), 43 (84); t =7.80 min (α-DEX 120,
R
temperature program).
4.5. (1S,2R)-1-Methylcyclohexene oxide 1
MS: same as for (1R,2S)-1; t =7.98 min (α-DEX 120, temperature program).
R
4.6. (1S,2S)-1-Methylcyclohexane-1,2-diol 2
+
+
MS: m/z 130 [M ] (8), 112 [M −H O] (22), 97 (25), 84 (15), 71 (100), 58 (38), 43 (68); t =22.72 min
2
R
(α-DEX 120, temperature program).
4.7. (1R,2R)-1-Methylcyclohexane-1,2-diol 2
MS: same as for (1S,2S)-2; t =23.07 min (α-DEX 120, temperature program).
R
4
.8. On secondary carbon atom 18O-labelled 1-methylcyclohexane-1,2-diol 2
MS: m/z 132 [M ], 112 [M −H218O], and the other major fragmentation peaks (97, 84, 71, 58, 43)
+
+
were the same as those in unlabelled 1-methylcyclohexane-1,2-diol 2.
4
.9. On tertiary carbon atom 18O-labelled 1-methylcyclohexane-1,2-diol 2
+
+
MS: m/z 132 [M ], 114 [M −H O], and the other major fragmentation peaks (99, 86, 73, 60, 45) were
2
2
higher than those in unlabelled 1-methylcyclohexane-1,2-diol 2.
Acknowledgements
This work was supported by grant BIO4-CT95-0049 from the European Community. We thank Martin
de Wit and Carel Weijers (Division of Industrial Microbiology, Wageningen University) for technical
assistance and the synthesis of 1-methylcyclohexene oxide, respectively.

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