Toluene dioxygenase mediated oxidation of halogen-substituted benzoate esters

Article abstract of DOI:10.1039/c2ob25202c

A series of ortho-, meta-, and para- halogen-substituted methyl benzoate esters was subjected to enzymatic dihydroxylation via the whole-cell fermentation with E. coli JM109 (pDTG601A). Only ortho-substituted benzoates were metabolized. Methyl 2-fluorobenzoate yielded one diol regioselectively whereas methyl 2-chloro-, methyl 2-bromo- and methyl 2-iodobenzoates each yielded a mixture of regioisomers. Absolute stereochemistry was determined for all new metabolites. Computational analysis of these results and a possible rationale for the regioselectivity of the enzymatic dihydroxylation is advanced.

Full text of DOI:10.1039/c2ob25202c

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PAPER
Toluene dioxygenase mediated oxidation of halogen-substituted benzoate
esters†
Vladislav Semak,a Thomas A. Metcalf,a Mary Ann A. Endoma-Arias,a Pavel Machb and Tomas Hudlicky*a
Received 27th January 2012, Accepted 29th March 2012
DOI: 10.1039/c2ob25202c
A series of ortho-, meta-, and para- halogen-substituted methyl benzoate esters was subjected to
enzymatic dihydroxylation via the whole-cell fermentation with E. coli JM109 (pDTG601A). Only
ortho-substituted benzoates were metabolized. Methyl 2-fluorobenzoate yielded one diol regioselectively
whereas methyl 2-chloro-, methyl 2-bromo- and methyl 2-iodobenzoates each yielded a mixture of
regioisomers. Absolute stereochemistry was determined for all new metabolites. Computational analysis
of these results and a possible rationale for the regioselectivity of the enzymatic dihydroxylation is
advanced.
Introduction
The oxidation of aromatic compounds by toluene dioxygenase
(TDO) yields cis-dihydrodiols, over 400 of which are known.1
In 1968 Gibson isolated the first stable arene-cis-diol from a fer-
mentation of Pseudomonas putida grown in the presence of
para-chlorotoluene.2 He later developed a mutant strain that
lacked the requisite enzymes to process cis-dihydrodiols, allow-
Fig. 1 Microbial dihydroxylation of benzoate esters.
ing the accumulation of these metabolites in the cell broth.3
The genes encoding TDO were later cloned into a strain of
Escherichia coli producing the recombinant organism JM109
(pDTG601A), in which the protein synthesis is initiated by iso-
found that TDO oxidized methyl, ethyl, allyl, and propargyl ben-
zoates to their corresponding diols 2 in yields of about 1 g L−1
,
propylthiogalactose (IPTG) and thus no aromatic inducer is
required.4 This aspect greatly simplifies the identification of any
new metabolites as the diol derived from the inducer will be
absent from the product mixtures.
The first applications of cis-dihydrodiols in organic synthesis
were the synthesis of polyphenylene by the ICI group5 and the
preparation of pinitol from the meso-diol derived from benzene
accomplished by Ley some 20 years after Gibson’s disclosure.6
In 1988 our group published the formal synthesis of a prosta-
glandin from the diol derived from toluene.7 Since then, many
new metabolites have been discovered, and cis-dihydrodiols
have enjoyed widespread use in organic synthesis.8
whereas nPr, and iPr, esters were found to be poor substrates and
nBu, and tBu benzoate were not metabolized (Fig. 1). The diol
derived from the oxidation of ethyl benzoate was recently used
in several generations of synthesis of oseltamivir.10 In this dis-
closure, we report the metabolism of halogen-substituted methyl
benzoates, provide computational study and rationale for the
observed selectivities, and offer further applications for the use
of new metabolites.
Results and discussion
In 2009 we reported the microbial oxidation of several benzo-
In a recent paper9 we detailed the results of the microbial dihy-
droxylation of benzoate esters 1 and the application of dienediols
2 in the preparation of pseudo-sugars. To further extend the
applicability of diols derived from benzoate esters, we subjected
methyl halobenzoates to microbial dihydroxylation studies to
determine the effect of the halogen substituent on the outcome
of bio-oxidation. From the standpoint of synthesis, the halogen
group provides an additional reactive functionality that can be
further exploited in various radical or transition metal-catalyzed
coupling protocols for accessing new optically pure cis-diols not
ate esters 1 in order to probe the limits of substrate size.9 It was
aDepartment of Chemistry, Brock University, 500 Glenridge Ave,
St. Catharines, ON, Canada, L2S 3A1. E-mail: thudlicky@brocku.ca;
Fax: +1-905-984-4841; Tel: +1-905-688-5550×4956
bDepartment of Nuclear Physics and Biophysics, Faculty of
Mathematics, Physics and Informatics, Comenius University, Mlynská
dolina, 842 15 Bratislava, Slovakia.
†Electronic supplementary information (ESI) available. See DOI:
10.1039/c2ob25202c
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available by enzymatic dihydroxylation of the corresponding
aromatic substrates.
hydrogenation of the less hindered alkene catalyzed by rhodium
on carbon under a hydrogen atmosphere provided tetrahydro-
diols, which were protected as acetonides 8. Substitution of Br
or I with boronates followed by coupling with carbon, nitrogen,
or phosphorus nucleophiles afforded coupled products of type 9.
The coupling strategy employed by Boyd provides access to a
wide variety of optically pure intermediates. Similarly useful
would be an approach to diols derived from disubstituted aro-
matics especially ones containing functionalities of different
size, using the Charton steric parameter as an indicator of substi-
tuent size. Such substrates are processed according to a model
proposed by Boyd et al.8g,14 where the larger of the substituents
“directs” the oxidation.
The required methyl halobenzoate esters were prepared from
the corresponding commercially available halobenzoic acids by
treatment with sodium carbonate and dimethyl sulfate in
acetone. The methyl halobenzoate esters were incubated in Fern-
bach shake flasks (2 L) with E. coli JM109 (pDTG601A),
grown to an optimum optical density in a 15 L Biostat C fermen-
tor. After incubation for six hours, the broth was extracted with
1
EtOAc and analyzed by TLC and H NMR. If a new metabolite
was detected, preparative-scale fermentation was undertaken in a
15 L Biostat fermentor as previously described.11 We found that
meta- or para- substituted benzoate esters, unlike their dihalo- or
halo-alkyl counterparts, were not metabolized by TDO.
There are 15 known cis-dihydrodiols of type 11, derived from
o-, m-, and p-alkyl-/or halo-iodobenzenes, Fig. 2.12,15 In all
cases, the iodine atom “directed” the regiochemistry of
dihydroxylation.
The utility of the iodine atom as a directing group for bio-oxi-
dation and its subsequent reductive removal has been illustrated
in the work of Boyd and co-workers.12 A series of dihalogenated
arene substrates (Scheme 1) was oxidized using P. putida UV4.
Removal of iodine in the presence of bromine or fluorine was
achieved by means of catalytic hydrogenation as shown below
for the para isomer. The corresponding ortho and meta isomers
were also investigated. This study provided access to ent-halo
diols, which are not accessible from the corresponding haloben-
zenes using bacterial dioxygenases.
Fig. 2 Metabolites of iodobenzenes.
Nineteen cis-dihydrodiols derived from various isomers of
alkyl- or halo-benzoic acids have been reported; however, there
is no clear trend in regiochemical preferences of dihydroxylation.
Most of these substrates give ipso16 diols of type 14 (only a few
exceptions provide diols of type 13).17
There are, surprisingly, only seven metabolites derived from
various alkyl benzoate esters, as shown in Fig. 1.9,16a,18 In con-
trast to the number of metabolites available from the disubsti-
tuted arenes, disubstituted alkyl benzoate dihydroxylation has
not yet been reported even though the oxidation of substituted
benzoic acids is known (Fig. 3).16,17 We therefore decided to
pursue the current study to seek functionalized benzoate-derived
diols via subsequent coupling protocols for providing additional
building blocks not available directly by the enzymatic
dihydroxylation.
Scheme 1
Boyd and co-workers13 have recently demonstrated the versa-
tility of bromo- and iodobenzene-derived diols as starting
materials in the preparation of a wide array of other diols either
not accessible by fermentation or produced in low yields by bio-
oxidation (Scheme 2). The bio-oxidation of bromo- or iodoben-
zene 6 afforded the enantiomerically pure diols 7, and selective
Fig. 3 Metabolites of substituted benzoic acids.
Ortho-halogen substituted methyl benzoate esters 15 gave
diols of type 16 or 17, as shown in Table 1. Methyl 2-fluoro-
benzoate 15a furnished a single diol of type 16, whereas methyl
2-chloro-, methyl 2-bromo- and methyl 2-iodobenzoate gave
mixtures of 16 and 17. These observations are in agreement with
Boyd’s rules for predicting the regio- and stereochemistry of
dihydroxylation by TDO.8g,14
Scheme 2
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Table 1 Microbial dihydroxylation of methyl halobenzoate substrates
Starting material
Conversion (%)
49.6
Yield (g L−1
)
Productsa (ratio)
0.50
60.2
0.47
13.3
10.0
0.20
0.15
a Ratio was obtained using integration of 1H NMR peaks corresponding to olefinic signals in the crude mixture.
Scheme 3
Yields of diols derived from ortho-substituted benzoates are
given in Table 1, along with the ratios of regioisomers. The stab-
ility of diols 16 and 17 above was found to depend strongly on
the nature of the X group. The most electronegative substituent,
fluorine, confers the highest stability while the least electronega-
tive substituent, iodine, contributes to lower stability. In fact, the
diols 16d and 17c could not be isolated (these undergo a facile
dehydration to the corresponding phenols upon concentration in
the rotary evaporator, even at low temperatures) and hence were
characterized as the corresponding acetonide derivatives instead.
Even the acetonides had a limited stability at room temperature
and had to be stored at low temperatures.
This trend was reversed with substrate 15d where the iodine
atom directed the regiochemistry of the dihydroxylation.
With the exception of methyl 2-fluorobenzoate diol 16a, these
diols readily underwent dehydration at room temperature. They
are stable in crystalline form at −78 °C or in pH 8 phosphate
buffer at 0 °C. They are less stable than diols 2 derived from
non-halogenated alkyl benzoate substrates 1, for example,
methyl benzoate or ethyl benzoate.9
In order to determine the relative and absolute stereochemistry,
the new metabolites were protected as acetonides followed by
hydrogenation as shown below (Scheme 3). The products
obtained from diols 16 were matched with the fully hydrogen-
ated diol 19 derived from the known diol 29 obtained previously
from methyl benzoate. Similarly, diols 17 were converted to
For substrates 15a, 15b and 15c the major diol product was
the result of methyl carboxylate directing the dihydroxylation.
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ester 21, which was derived from known compound 2219 in two
steps. These experiments confirmed the relative as well as absol-
ute stereochemistry of the new metabolites as drawn.
The mechanism of the enzymatic dihydroxylation of arenes is
unknown. Various propositions have been advanced22 but none
has been proven by experiment. The original proposition by
Gibson23 for dioxetane intermediates is, to date, the only one
partially subjected to experimental investigation through studies
of the kinetic isotope effects in dehydrogenation to cathecols.24
We have briefly investigated mechanistic options involving
radical intermediates but have not arrived at any reasonable
mechanistic postulates.25 Other options include high oxidation
state iron peroxides, iron oxo species, and singlet oxygen
cycloadditons. Because the actual mechanism is not known, it is
logical that all possibilities are equivalent in merit until proven
otherwise. For this reason, the computational studies that follow
are based on the assumption that dioxetanes are the intermediates
in the reaction, whether formed by singlet oxygen cycloaddition
or internal redox reactions of triplet species. The results of these
computational studies follow.
Hydrogenation of acetonides 18a, 18b, 18c and 18d from type
16 diol resulted in a product that was identical in all properties to
ester 19, prepared from the known methyl benzoate diol 2. Simi-
larly, hydrogenation of acetonide 20b, 20c and 20d resulted in
the formation of ester 21 (except for the sign of optical rotation),
derived from methyl cyclohexenylcarboxylate ester 22. The start-
ing ester 22 was obtained in racemic form via Diels–Alder reac-
tion of butadiene sulfone and methyl acrylate in toluene at
130 °C performed for two days in a sealed tube. Resolution was
achieved using porcine pancreatic lipase (PPL) in phosphate
buffer at pH 7.0. Dihydroxylation of 22 followed by acetonide
formation afforded the desired standard compound cis (relative
stereochemistry of ester to acetonide) 21 as a 3 : 4 mixture with
its trans diastereomer (relative stereochemistry of ester to aceto-
nide), which was separated by silica gel column chromatography.
With respect to potential applications of diols derived from
halobenzoates, Porco and co-workers20 recently determined the
absolute stereochemistry of the natural product kibdelone C by
way of its total synthesis. A key intermediate, 24, was prepared
in 13 steps starting from commercially available material 23 as
shown below (Fig. 4). We have shown21 that diol 16d provided a
good choice for access to this key intermediate: two versus the
thirteen steps originally reported. Applications of the metabolites
derived from halobenzoates to the synthesis of additional kibde-
lone derivatives are now in progress with collaboration.
Methyl benzoates 15a–15d. Taking into account Boyd’s obser-
vations, we studied the geometry and electrostatic effects of
methyl benzoates 15a–15d. The resulting data were employed in
the calculation of energies of proposed dioxetane intermediates.
Two rotamers of benzoates 15a–15d were studied (Fig. 5).
In all cases except for 15a, the COOCH3 group is rotated out
of the plane of benzene ring. As seen from Table 2, this angle
varies from 17 to 31°, reducing π-interaction of COOCH3 group
with the aromatic ring. For benzoates 15a–15c, the anti-confor-
mer is more stable, but for iodine derivative 15d the syn arrange-
ment is preferred. Quantitatively, the stability difference between
syn and anti is significant only for 15a. In the case of Cl-, Br-
and I-substituted benzoates (15b–15d) the barrier for intercon-
version (1.3–1.5 kcal mol−1), is small (Table 2). Even one weak
hydrogen-bond formation can supply enough energy for a con-
formational change, and the COOCH3 group can easily adopt
conformation, as required by the active center of enzyme.
Electrostatic effects can also play a role in substrate docking.
Table 2 shows the atomic charges calculated by natural
Fig. 4 A short route for the preparation of Kibledone C intermediate
24.
Computational studies
Fig. 5 Rotamers of 15a–15b.
Table 2 Properties of 15a–15d
The Boyd empirical model, based on Charton steric parameters
(ν), cannot satisfactorily explain the observed regioselectivity in
TDO-catalysed cis-dihydroxylation of the ortho-substituted ben-
zoates 15a–15d. The size of the substituents, according to
Charton steric parameters, are as follows: COOMe (ν = 1.39) > I
(ν = 0.78) > Br (ν = 0.65) > Cl (ν = 0.55) > F (ν = 0.27). This
indicates that in all examples the COOMe should be the larger
stereodirecting group; however, in the case of iodine derivative
15d, the iodine atom is clearly dominant over the ester group.
As Boyd claims for the case of nonsymmetrical substituents,
other considerations, e.g., substituent length and conformation
in the vicinity of the active site, should also be taken into
consideration.8g,14
Out-of-
plane angle [kcal
[°]
Erelative
Barrier heights NPA charge
mol−1
]
[kcal mol−1
]
on X [e]
15a syn 0.0
anti 0.0
15b syn 25.5
anti 24.2
15c syn 23.0
anti 26.2
15d syn 17.4
anti 30.0
1.17
0.00
0.21
0.00
0.13
0.00
0.00
0.19
3.9
1.5
1.3
1.3
−0.355
−0.363
−0.005
−0.018
+0.088
+0.071
+0.216
+0.192
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population analysis (NPA)26 for halogen atoms in syn and anti
conformations. The charges of the oxygen atoms in the acetyl
group are negative in each case, but the charge of halogen is
variable. This can have a significant influence on the orientation
of the benzoate during docking in the active site of the enzyme.
This preference can be “matched” or “mismatched” with the pre-
ference based on the orientation effect of the functional group
(electronic or steric). As seen in Table 2, the charge on the
halogen is practically the same in either the syn and anti
conformation.
relative energies of these intermediates are related to barrier
heights for formation of diols (Hammond’s postulate) and thus
can serve as a rough guide to assess effect of different halogen
substituents.
It is not known whether singlet oxygen or another reactive
oxygen species (ROS) takes part in this reaction; however, our
calculations were based on oxygen in its ground (triplet) state as
we do not know the exact character of the reacting species
(singlet oxygen, superoxide anion, peroxy or hydroperoxy
radical, or any metal-bound species that may exist in the active
site). The proper theoretical description of singlet oxygen is tech-
nically more complicated, but for the present study only the rela-
tive preference for A and B in different halo-benzoates is
important. We also assume that the intermediate of the oxidation
is the dioxetane, as originally proposed by Gibson. This assump-
tion is valid for the comparisons made in the computational
study but may not accurately describe the actual intermediates in
the enzymatic reaction.
Formation of dioxetane intermediates: electronic effects
We wished to assess the combined orientational effect of X and
COOMe substituents on the specific type of reaction, i.e. for-
mation of dioxetane intermediate25a (see Scheme 4). Each
formed regioisomer of dioxetane intermediate can be in two rota-
meric forms, syn and anti. If the COOMe group is regiodirecting,
the intermediate must have the A type structure, if halogen is in
this role, the intermediate is of B type. Calculated reaction ener-
gies for formations of intermediates intA and intB in syn and
anti conformations by reaction of benzoate with triplet (ground
state) oxygen are listed in Table 3. The idea behind this is that
From the energy values in Table 3 we may conclude that the
most pronounced orientational effect exists in the case of 15a.
The energy difference between intA and intB is 3.4 kcal mol−1
.
In the case of 15d it drops to just 0.5 kcal mol−1. If there is
another orientational effect (steric, electrostatic etc.) that com-
petes with this preference for formation of intermediate intA,
Scheme 4
Table 3 Reaction energies for formation of intermediates intA (“actE A”) and intB (“actE B”)
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1
it can easily change this preference for iodine, but not for
fluorine. This effect will be very similar for Cl and Br substitu-
ents. If we would consider the reacting species to be singlet
oxygen (instead of triplet) in the lowest delta state, we can add
its experimental energy: +22.5 kcal mol−1 (relative to triplet O2)
to the left-hand side of reactions for the formation of intermedi-
ates A and B and thus reduce the reaction energy by this
amount. Resulting energies are then close to zero, indicating that
these reactions are now almost thermally neutral, i.e. reaction
589 nm. H and 13C spectra were recorded on a 300 MHz and
600 MHz Bruker spectrometer. All chemical shifts are referenced
to TMS or residual undeuterated solvent. Data of proton spectra
are reported as follows: chemical shift in ppm (multiplicity:
singlet (s), doublet (d), triplet (t), quartet (q) and multiplet (m)),
coupling constants [Hz], integration). Carbon spectra were
recorded with complete proton decoupling and the chemical
shifts are reported in ppm (δ) relative to solvent resonance as
internal standard. Mass spectra and high resolution mass spectra
were performed by the analytical division at Brock University.
enthalpy is ∼0 kcal mol−1
.
Computational details and methods
Conclusions
For calculations of molecular structures and energetics DFT
theory was used with B3LYP27 hybrid functional. This is an
obvious choice for organic molecules, because it supplies good
results and has been used with enough examples that a critical
assessment of its performance is available. What is less obvious
is the selection of appropriate basis sets, as we need basis set
which can describe, with comparable precision, all halogens,
including iodine. From relatively few possibilities we choose
SBKJC28 basis set with relativistic pseudopotentials, augmented
with 1p and 2d polarization functions.29 All reported energies
correspond to structures, fully optimized at the above level. All
calculations were done using GAUSSIAN03 program package.30
We have identified seven new metabolites from the TDO-cata-
lyzed dihydroxylation of o-halo benzoates. In general, benzoate
esters are converted to diols in significantly lower yields than
halobenzenes, and substituted benzoate esters are somewhat
reluctant substrates. However, all of these compounds are
accessible and can be prepared in multi-gram amounts by
fermentation.
Ab initio calculations were shown to be in good qualitative
agreement with the experimental results for microbial dihydroxy-
lation of ortho-substituted halo benzoates. The most pronounced
effect was observed in the case of fluorobenzoate 15a, where
TDO oxidation is regioselective. Practically no difference was
noted between chloro and bromo derivatives (15b and 15c) in
both the theoretical calculations and the experimental results.
Preference for the formation of B type diol 17d in the case of the
iodo derivative can be partially explained by Boyd’s rules. In
addition the positive charge (δ+) of iodine in 15d can influence
the orientation of the benzoate during docking to the active site
of the enzyme.
The new metabolites will find widespread use in the prep-
aration of optically pure diols not otherwise available from enzy-
matic oxidation of the corresponding arenes. The short
preparation of the intermediate in the kibledone C synthesis
lends credence to the above investigation. Although the precise
mechanism of the enzymatic dihydroxylation remains unsolved,
there is an increase in the power of prediction in investigations
of new arene substrates.
General procedure for acetonide formation
A catalytic amount of toluenesulfonic acid was added to a stirred
solution of diol (2 mmol) and dimethoxypropane (10 mmol) in
CH2Cl2 (10 mL). The reaction was monitored by TLC on silica
gel (1 : 1 EtOAc–hexanes). When all the starting material was
consumed, the reaction mixture was diluted with CH2Cl2
(10 mL), washed with 1.0 M NaOH (5 mL) and dried over anhy-
drous MgSO4. The filtrate was concentrated by rotary evapor-
ation and further purified by column chromatography on silica
gel (1 : 1 EtOAc–hexanes) to afford the acetonide as an oil
(80–95% yield).
General procedure for hydrogenation
To a solution of acetonide (0.20 mmol) in MeOH (1 mL) was
added PtO2 (catalytic, 10% w/w) and NEt3 (0.20 mmol). The
flask was evacuated and filled with H2 at atmospheric pressure.
After the reaction was judged complete by TLC (8–12 h), the
suspension was filtered through Celite; concentrated in the rotary
evaporator and purified by column chromatography on silica gel
using mixture of hexanes–EtOAc 4 : 1) as eluent. The eluent was
concentrated to give an oil (50–61% yield).
Experimental section
Inoculum was obtained from viable cells stored −78 °C in cryo-
vials. They were grown in suitable media as previously
described.11 Substrate was fed in 1 g increments over the course
of ∼3 h with metabolites being harvested in the usual manner.
All non-aqueous reactions were conducted in an argon atmos-
phere using standard Schlenk techniques for the exclusion of
moisture and air. Methylene chloride was distilled from calcium
hydride, THF and toluene were dried over sodium/benzophe-
none. Analytical thin layer chromatography was performed on
Silicycle 60 A° 250 mm TLC plates with F-254 indicator. Flash
column chromatography was performed using Kieselgel 60
(230–400 mesh). Melting points were recorded on a Hoover
Unimelt apparatus and are uncorrected. IR spectra were obtained
on a Perkin-Elmer One FT-IR spectrometer. Optical rotation was
measured on a Perkin-Elmer 341 polarimeter at a wavelength of
(5S,6R)-Methyl 2-fluoro-5,6-dihydroxycyclohexa-1,3-dienecar-
boxylate (16a). mp 74–76 °C (EtOAc); [α]2D0 +73.2 (c 1.05,
MeOH); Rf = 0.15 (1 : 1 hexanes–ethyl acetate); IR (film) 3558,
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3025, 1694, 1439, 1401, 1040 cm−1 1H NMR (600 MHz,
;
(5S,6R)-Methyl 2-chloro-5,6-dihydroxycyclohexa-1,3-dienecar-
boxylate (17b). [α]D20 = +86.6 (c 1.0, CHCl3); Rf = 0.25
(1 : 1 hexanes–ethyl acetate); IR (KBr) 3422, 2959, 1721, 1578,
CDCl3) δ 6.33 (m, 1H), 5.94 (ddd, J = 10.2, 8.3, 2.6 Hz, 1H),
4.71 (t, J = 6.2 Hz, 1H), 4.55 (m, 1H), 3.83 (s, 3H), 3.17 (brs,
1H), 3.09 (brs, 1H) ppm; 13C NMR (150 MHz, CDCl3) δ 166.0
(d, J = 2.2 Hz), 163.2 (d, J = 281.0 Hz), 143.1 (d, J = 12.1 Hz),
119.6 (d, J = 36.2 Hz), 106.2 (d, J = 2.2 Hz), 69.06 (s), 67.0 (d,
J = 6.6 Hz), 52.2 (s) ppm; 19F NMR (282 MHz, CDCl3) δ −92.6
(s) ppm; MS (EI) m/z (%): 188 (15), 133 (44), 119 (49), 102
(100), 91 (37), 90 (46), 86 (28), 74 (16), 46 (27); HRMS (EI)
calcd for C8H9FO4 (M+): 188.0485; found: 188.0484; MS
1
1444, 1270, 758 cm−1; H NMR (600 MHz, CDCl3) δ 6.35 (d,
J = 9.8 Hz, 1H), 6.03 (dd, J = 9.8, 3.0 Hz, 1H), 4.44 (m, 1H),
4.31 (d, J = 6.0 Hz, 1H), 3.83 (s, 3H), 2.50 (vbrs, 2H) ppm;
13C NMR (150 MHz, CDCl3) δ 164.77, 140.46, 127.77, 125.09,
124.00, 72.47, 67.36, 52.34 ppm; MS (EI) m/z (%) [M–H2O]+:
188 (15), 186 (43), 157 (32), 155 (100), 99 (14); HRMS (EI)
calcd for C8H9ClO4–H2O [M − H2O]+: 188.0084; found:
188.0077.
+
(FAB) m/z (%): 189 (24) [M + H]+, 188 (16) [M] , 171 (100),
139 (27), 59 (16); HRMS (FAB) calcd for C8H9FO4 (M+):
188.0485; found: 188.0479; Anal. calcd for C8H9FO4: C, 51.07;
H, 4.82; found: C, 51.18; H, 4.76.
(3aR,7aS)-Methyl 5-fluoro-2,2-dimethyl-3a,7a-dihydrobenzo
[d][1,3]dioxole-4-carboxylate (18a). [α]2D0 +23.8 (c 1.2, CHCl3);
Rf = 0.67 (1 : 1 hexanes–ethyl acetate); IR (film) ν 3020, 2932,
2254, 1706, 1613 cm−1; 1H NMR (600 MHz, CDCl3) δ 6.25 (m,
1H), 5.98 (m, 1H), 5.11 (t, J = 7.5 Hz, 1H), 4.87 (m, 1H), 3.86
(s, 3H), 1.46 (s, 3H), 1.42 (s, 3H) ppm; 13C NMR (150 MHz,
CDCl3) δ 164.9, 163.7, 161.9, 138.1, 119.8, 119.5, 106.4, 103.9,
72.0, 70.9, 52.2, 26.8, 25.2, 1.03 ppm; MS (EI) m/z (%): 228
(1.7), 213, (80.0), 197 (11.3), 181 (10.8), 171 (49.8), 139 (42.0),
127 (20.8); HRMS (EI) calcd for C11H13FO4: 228.0798; found:
228.0807.
(3S,4S)-Methyl 2-chloro-3,4-dihydroxycyclohexa-1,5-dienecar-
boxylate (16b). mp 107–109 °C (pentene–ethyl acetate); [α]D20
=
+36.06 (c 1.0, CHCl3); Rf = 0.18 (1 : 1 hexanes–ethyl acetate);
1
IR (KBr) 3398, 3459, 1698, 1317, 1057, 762 cm−1; H NMR
(300 MHz, CDCl3) δ 6.18 (ddd, J = 10.0, 2.4, 1.2 Hz, 1H), 6.01
(dd, J = 10.0, 2.4 Hz, 1H), 4.64 (ddd, J = 6.0, 4.7, 1.2 Hz, 1H),
4.52 (ddt, J = 8.7, 6.0, 2.4 Hz, 1H), 3.86 (s, 3H), 2.76 (d, J =
9.6 Hz, 1H), 2.60 (d, J = 4.7 Hz, 1H) ppm; 13C NMR
(150 MHz, CDCl3) δ 166.32, 138.78, 138.52, 127.50, 123.79,
68.56, 67.72, 52.27 ppm; MS (EI) m/z (%) [M]+: 204 (14), 173
(23), 172 (54), 155 (50), 146 (32), 145 (36), 144 (100), 143
(80), 139 (27), 99 (27), 81 (41), 53 (25), 51 (21); HRMS (EI)
calcd for C8H9ClO4: 204.0189; found: 204. 0190.
(3aR,7aS)-Methyl 5-chloro-2,2-dimethyl-3a,7a-dihydrobenzo
[d][1,3]dioxole-4-carboxylate (18b). [α]2D0 +141.4 (c 1.5,
CH2Cl2); Rf = 0.80 (1 : 1 hexanes–ethyl acetate); IR (film)
ν 2989, 2952, 1726, 1644, 1587 cm−1 1H NMR (300 MHz,
;
CDCl3) δ 6.08 (m, 2H), 5.07 (d, J = 8.1 Hz, 1H), 4.79 (dd, J =
8.1, 2.1 Hz, 1H), 3.86 (s, 3H), 1.43 (s, 3H), 1.40 (s, 3H) ppm;
13C NMR (75 MHz, CDCl3) δ 165.4, 137.0, 132.5, 127.5,
122.1, 106.5, 71.6, 71.0, 52.2, 26.7, 25.3 ppm; MS (EI) m/z (%):
244 (2.3), 231, (16.4), 229 (49.6), 213 (11.3), 197 (14.0), 187
(50.1), 186 (17.9), 157 (17.9), 155 (49.4); HRMS (EI) calcd for
C11H13ClO4: 244.0495; found: 244.00502.
(5S,6R)-Methyl
2-bromo-5,6-dihydroxycyclohexa-1,3-diene-
carboxylate (16c). mp 106–109 °C (CHCl3); [α]2D0 = +29.40 (c
1.0, DCM); Rf = 0.18 (1 : 1 hexanes–ethyl acetate); IR (KBr)
3402, 1703, 1437, 1314, 1234, 1048 cm−1; 1H NMR (600 MHz,
CDCl3) δ 6.17 (dd, J = 10.0, 2.5 Hz, 1H), 6.04 (ddd, J = 10.0,
2.5, 1.3 Hz, 1H), 4.57 (m, 1H), 4.49 (m, 1H), 3.85 (s, 3H), 3.00
(brd, J = 7.9 Hz, 1H), 2.97 (brs, 1H) ppm; 13C NMR (150 MHz,
CDCl3) δ 166.61, 137.54, 129.97, 128.04, 127.31, 68.37, 68.14,
52.28 ppm; MS (EI) m/z (%): 248 (9), 218 (38), 216 (47), 190
(82), 189 (53), 188 (85), 187 (48), 109 (71), 108 (31), 81 (100),
65 (79), 59 (45), 53 (54); HRMS (EI) calcd for C8H9BrO4 (M+):
247.9684; found: 247.9679.
(3aR,7aS)-Methyl-5-bromo-2,2-dimethyl-3a,7a-dihydrobenzo
[d][1,3]dioxole-4-carboxylate (18c). [α]2D0 +285.8 (c 1.1,
CH2Cl2); Rf = 0.72 (1 : 1 hexanes–ethyl acetate); IR (film)
ν 2988, 2951, 1725, 1639, 1582, 1434 cm−1 1H NMR
;
(300 MHz, CDCl3) δ 6.17 (d, J = 0.72 Hz, 1H), 5.94 (dd, J =
9.87, 3.36 Hz, 1H), 4.99 (d, J = 8.04 Hz, 1H), 4.75 (m, 1H),
3.82 (s, 3H), 1.38 (s, 3H), 1.35 (s, 3H) ppm; 13C NMR
(75 MHz, CDCl3) δ 165.8, 131.3, 129.9, 125.8, 125.7, 106.5,
This journal is © The Royal Society of Chemistry 2012
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(3aS,7aS)-Methyl 4-iodo-2,2-dimethyl-3a,7a-dihydrobenzo[d]
[1,3]dioxole-5-carboxylate (20d). [α]2D0 = +32.4 (c 3.3, CH2Cl2);
Rf = 0.35 (4 : 1 hexanes–ethyl acetate); IR (film) 3444, 2987,
72.1, 70.6, 52.2, 26.7, 25.2 ppm; MS (EI) m/z (%): 275 (24),
273 (25), 233 (41), 231 (45), 201 (28), 199 (28), 108 (33);
HRMS (EI) calcd for C11H13BrO4: 287.9997; found: 287.9994.
2951, 1730, 1372, 1243, 1062 cm−1 1H NMR (300 MHz,
;
CDCl3) δ 6.22 (d, J = 9.9 Hz, 1H), 6.10 (dd, J = 9.9, 4.2 Hz,
1H), 4.83 (d, J = 8.1 Hz, 1H), 4.63 (dd, J = 8.1, 4.2 Hz, 1H),
3.85 (s, 3H), 1.45 (s, 3H), 1.43 (s, 3H) ppm; 13C NMR
(75 MHz, CDCl3) δ 166.2, 133.2, 124.9, 123.8, 107.9, 106.6,
79.6, 71.0, 52.4, 26.8, 25.2 ppm; MS (EI) m/z (%): 337 (6.7),
336 (41.0), 319 (10.7), 279 (89.8), 278 (97.6), 247 (44.5), 231
(17.8), 220 (12.3), 152 (100.0), 151 (48.4), 127 (13.6); HRMS
(EI) calcd for C11H13IO4 (M+): 335.9864; found: 335.9859.
(3aR,4R,7aS)-Methyl
2,2-dimethylhexahydrobenzo[d][1,3]
dioxole-4-carboxylate (19). [α]2D0 +10.4 (c 1.2, CH2Cl2); Rf =
0.29 (6 : 1 hexanes–ethyl acetate); IR (film) ν 2987, 2950,
1742 cm−1; 1H NMR (300 MHz, CDCl3) δ 4.55 (dd, J = 5.4 Hz,
J = 3.6 Hz, 1H), 4.19 (dt, J = 7.8 Hz, J = 5.4 Hz, 1H), 3.74 (s,
3H), 2.62 (m, 1H), 1.77 (m, 5H), 1.50 (m, 1H), 1.35 (s, 3H)
ppm; 13C NMR (75 MHz, CDCl3) δ 172.9, 108.5, 74.1, 73.3,
51.9, 43.0, 27.8, 27.7, 25.8, 20.1, 19.4 ppm; MS (EI) m/z (%):
199 (100), 157 (17), 139 (10), 125 (58), 97 (29), 79 (59);
HRMS (EI) calcd for C11H18O4: 214.1205; found: 214.1201.
(3aR,5R,7aS)-Methyl
2,2-dimethylhexahydrobenzo[d][1,3]
dioxole-5-carboxylate (21). [α]2D0 +32.8 (c 4.4, CH2Cl2); Rf =
0.27 (4 : 1 hexanes–ethyl acetate); IR (film) ν 3453, 2987, 2952,
1731, 1638, 1436 cm−1; 1H NMR (300 MHz, CDCl3) δ 4.17 (m,
1H), 4.10 (m, 1H), 3.70 (s, 3H), 2.25 (m, 2H), 2.10 (m, 1H)
1.77 (m, 4H), 1.60 (m, 2H), 1,52 (s, 3H),1.35 (s, 3H) ppm;
13C NMR (75 MHz, CDCl3) δ 175.2, 108.3, 73.7, 72.4, 51.8,
39.4, 31.5, 28.3, 26.3, 26.1, 22.6 ppm; MS (EI) m/z (%): 199
(100), 125 (13), 97 (30), 78 (52), 69 (11); HRMS (EI) calcd for
C11H18O4: 199.0973; found: 199.0970.
(3aS,7aS)-Methyl 4-chloro-2,2-dimethyl-3a,7a-dihydrobenzo
[d][1,3]dioxole-5-carboxylate (20b). [α]2D0 +92.6 (c 1.0, CH2Cl2);
Rf = 0.80 (1 : 1 hexanes–ethyl acetate); IR (film) ν 2990, 2953,
1735, 1654, 1584, 1436, 1374, 1249 cm−1; 1H NMR (300 MHz,
CDCl3) δ 6.32 (d, J = 9.9 Hz, 1H), 5.97 (dd, J = 9.9, 3.6 Hz,
1H), 4.74 (dd, J = 8.4, 3.6 Hz, 1H), 4.68 (d, J = 8.4 Hz, 1H),
3.81 (s, 3H), 1.41 (s, 3H), 1.39 (s, 3H) ppm; 13C NMR
(75 MHz, CDCl3) δ 165.0, 137.9, 124.5, 123.8, 123.5, 107.1,
75.8, 71.8, 52.3, 26.7, 25.0 ppm; MS (EI) m/z (%): 244 (20),
213 (20), 229 (35), 186 (60), 155 (40), 59 (60), 43(100); HRMS
(EI) calcd for C11H13ClO4: 244.0495; found: 244.05024.
(S)-Methyl cyclohex-3-enecarboxylate(22)
Preparation of racemic 22. A solution of butadiene sulfone
(8.0 g, 64.4 mmol), methyl acrylate (3.7 g, 42.8 mmol) and
hydroquinone (100 mg, catalytic), in toluene (100 mL) was
heated to 110 °C in sealed tube for 2 d. The reaction mixture
was allowed to reach room temperature and was concentrated
using rotary evaporation of afford a dark brown viscous residue.
The residue was chromatographed on silica gel using hexanes as
eluent to afford the racemic ester 22 as a colorless oil (4.55 g,
75.6% yield).
(3aS,7aS)-Methyl 4-bromo-2,2-dimethyl-3a,7a-dihydrobenzo
[d][1,3]dioxole-5-carboxylate (20c). [α]2D0 +94.0 (c 0.2, CH2Cl2);
Rf = 0.72 (1 : 1 hexanes–ethyl acetate); IR (film) ν 3436, 2918,
1
1732, 1435 cm−1; H NMR (300 MHz, CDCl3) δ 6.29 (d, J =
Resolution of ester 22. A mixture of racemic ester 19 (500 mg,
3.57 mmol) and Porcine Pancreatic Lipase (50 mg) in 0.10 M
phosphate buffer (50 mL) was shaken in an orbital shaker at
20 °C. The pH was maintained at 7.0 by the addition of 0.10 M
NaOH. At the end of 6.5 h, the enzyme was filtered through
a plug of Celite. The filtrate was extracted with EtOAc (3 ×
20 mL). The organic extracts were combined and dried over
anhydrous Na2SO4. The filtrate was concentrated by rotary evap-
oration and further purified by column chromatography on silica
gel (6 : 1 hexanes–EtOAc) to afford ester 22 (148 mg, 29%
yield) as a yellowish oil. 89% ee [ α]2D0 −63.5 (c 1.0, CHCl3))
9.6 Hz, 1H), 6.08 (dd, J = 9.6, 3.9 Hz, 1H), 4.83 (d, 8.4 Hz,
1H), 4.74 (dd, J = 8.4, 3.9 Hz, 1H), 3.87 (s, 3H), 1.47 (s, 3H),
1.27 (s, 3H) ppm; 13C NMR (75 MHz, CDCl3) δ 165.7, 129.0,
127.9, 124.5, 123.7, 106.7, 76.8, 71.8, 52.4, 26.8, 25.1 ppm;
MS (EI) m/z (%): 288 (10.6), 233 (56.0), 232 (40.6), 231 (58.8),
230 (36.8), 201 (27.1), 199 (26.6), 189 (11.3), 187 (11.8), 185
(12.5), 183 (14.4); HRMS (EI) calcd for C11H13BrO4: 288.0002;
found: 287.9997.
1
lit19 [α]D20 −82.2 (c 1.0, CHCl3); H NMR (300 MHz, CDCl3)
δ 5.68 (s, 2H), 3.70 (s, 3H), 2.59 (m, 1H), 2.26 (m, 2H), 2.06
(m, 2H), 1.98 (m, 1H) 1.72 (m, 1H) ppm.
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pp. 130–139; (f) T. Hudlicky, in Green Chemistry. Designing Chemistry
for the Environment, ed. P. T. Anastas and T. C. Williamson, ACS Sym-
posium Series 626, American Chemical Society, Washington, D. C.,
1996, Ch. 14; (g) D. R. Boyd and G. N. Sheldrake, Nat. Prod. Rep.,
1998, 15, 309–324; (h) T. Hudlicky, D. Gonzales and D. T. Gibson,
Aldrichimica Acta, 1999, 32, 35–62; (i) D. R. Boyd, N. D. Sharma and
C. C. R. Allen, Curr. Opin. Biotechnol., 2001, 12, 564–573; ( j) D.
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(m) T. Hudlicky and J. W. Reed, Chem. Soc. Rev., 2009, 38, 3117–
3132.
Conversion of 22 to 21. A catalytic amount of OsO4 was added
to a stirred solution of ester 19 (100 mg, 0.72 mmol), N-methyl
morpholine N-oxide (82 mg, 0.72 mmol) in acetone–water
(2 mL/0.6 mL). The resulting solution was stirred for 1 h at
room temperature, and then it was quenched with 15% NaHSO3
solution (1 mL). The reaction mixture was diluted with EtOAc
(20 mL) and water (5 mL). The layers were separated and the
aqueous layer was extracted further with EtOAc (2 × 10 mL).
The combined organic extracts were dried over anhydrous
MgSO4. The filtrate was concentrated via rotary evaporation and
was used as crude in the next step.
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To the crude mixture of diol (from the previous step) dissolved
in CH2Cl2 (10 mL) was added dimethoxypropane (2.0 mL,
16.3 mmol) followed by a catalytic amount of pTsOH. The reac-
tion mixture was stirred at room temperature for 30 min. Then it
was diluted with 1 N NaOH (2 mL), the two layers were separ-
ated and the aqueous layer was further extracted with CH2Cl2
(2 × 5 mL). The organic layers were combined and dried over
anhydrous MgSO4. The filtrate was concentrated by rotary
evaporation and further purified by column chromatography on
silica gel (4 : 1 hexanes–EtOAc) to afford the desired product 21
(25 mg, 16.3% yield, over two steps) as a yellowish oil. 88% ee
[α]2D0 −25.1 (c 1.0, CH2Cl2)) compound 21 from 20b–20d
[α]2D0 +32.8 (c 4.4, CH2Cl2).
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Acknowledgements
The authors are grateful to the following agencies for financial
support of this work: Natural Sciences and Engineering Research
Council of Canada (NSERC) (Idea to Innovation and Discovery
Grants); Canada Research Chair Program, Canada Foundation
for Innovation (CFI), Research Corporation, TDC Research, Inc.,
TDC Research Foundation, and Brock University. Thanks are
also due to the Ministry of Education (Spain) for fellowships to
V.S. (AP2006-03468ESTANCIA-2010) and partial support of
Slovak grant agency VEGA (1/0524/11).
18 A. J. Blacker, R. J. Booth, G. M. Davies and J. K. Sutherland, J. Chem.
Soc., Perkin Trans. 1, 1995, 2861–2870.
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20 D. L. Sloman, J. W. Bacon and J. A. Porco, J. Am. Chem. Soc., 2011,
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4416 | Org. Biomol. Chem., 2012, 10, 4407–4416
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