Dioxygenase-catalysed oxidation of disubstituted benzene substrates: Benzylic monohydroxylation versus aryl cis-dihydroxylation and the meta effect

Article abstract of DOI:10.1039/b608417f

Biotransformations of a series of ortho-, meta- and para-substituted ethylbenzene and propylbenzene substrates have been carried out, using Pseudomonas putida UV4, a source of toluene dioxygenase (TDO). The ortho- and para-substituted alkylbenzene substrates yielded, exclusively, the corresponding enantiopure cis-dihydrodiols of the same absolute configuration. However, the meta isomers, generally, gave benzylic alcohol bioproducts, in addition to the cis-dihydrodiols (the meta effect). The benzylic alcohols were of identical (R) absolute configuration but enantiomeric excess values were variable. The similar (2R) absolute configurations of the cis-dihydrodiols are consistent with both the ethyl and propyl groups having dominant stereodirecting effects over the other substituents. The model used earlier, to predict the regio- and stereo-chemistry of cis-dihydrodiol bioproducts derived from substituted benzene substrates has been refined, to take account of non-symmetric subsituents like ethyl or propyl groups. The formation of benzylic hydroxylation products, from meta-substituted benzene substrates, without further cis-dihydroxylation to yield triols provides a further example of the meta effect during toluene dioxygenase-catalysed oxidations. The Royal Society of Chemistry 2006.

Full text of DOI:10.1039/b608417f

View Article Online / Journal Homepage / Table of Contents for this issue
PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Dioxygenase-catalysed oxidation of disubstituted benzene substrates: benzylic
monohydroxylation versus aryl cis-dihydroxylation and the meta effect†
Derek R. Boyd,*a,b Narain D. Sharma,a,b Nigel I. Bowers,a Howard Dalton,c Mark D. Garrett,a
John S. Harrisona and Gary N. Sheldrake*a
Received 14th June 2006, Accepted 5th July 2006
First published as an Advance Article on the web 31st July 2006
DOI: 10.1039/b608417f
Biotransformations of a series of ortho-, meta- and para-substituted ethylbenzene and propylbenzene
substrates have been carried out, using Pseudomonas putida UV4, a source of toluene dioxygenase
(TDO). The ortho- and para-substituted alkylbenzene substrates yielded, exclusively, the corresponding
enantiopure cis-dihydrodiols of the same absolute configuration. However, the meta isomers, generally,
gave benzylic alcohol bioproducts, in addition to the cis-dihydrodiols (the meta effect). The benzylic
alcohols were of identical (R) absolute configuration but enantiomeric excess values were variable. The
similar (2R) absolute configurations of the cis-dihydrodiols are consistent with both the ethyl and
propyl groups having dominant stereodirecting effects over the other substituents. The model used
earlier, to predict the regio- and stereo-chemistry of cis-dihydrodiol bioproducts derived from
substituted benzene substrates has been refined, to take account of non-symmetric subsituents like ethyl
or propyl groups. The formation of benzylic hydroxylation products, from meta-substituted benzene
substrates, without further cis-dihydroxylation to yield triols provides a further example of the meta
effect during toluene dioxygenase-catalysed oxidations.
Introduction
Bacterial aryl ring dihydroxylating dioxygenase enzymes (dioxyge-
nases) have been widely recognised for their ability to catalyse the
introduction of two oxygen atoms (cis-dihydrodiol formation) to
substrates containing a benzene ring in a regio- and stereo-selective
manner. The formation of cis-dihydrodiol metabolites B, from
arenes A, was initially studied using mutant strains of bacteria
(mainly Pseudomonas) which allow these initial bioproducts and
other metabolites to accumulate (Scheme 1). Since the first report
of the isolation and identification of a cis-dihydrodiol metabolite
B, i.e. from the parent benzene ring A,1 in excess of three hundred
examples from substituted benzene substrates have now been
reported. The majority of these cis-dihydrodiols B are enantiopure
and are being widely used as synthetic precursors.2–12
Scheme 1
While dioxygenases, present in whole cells of selected mutants,
e.g. Pseudomonas putida UV4 (a source of toluene dioxygenase,
TDO), can catalyse arene cis-dihydroxylations (A → B, Scheme 1),
it has recently been found that they can also catalyse other types
of oxidations (e.g. A → C, D → E, F → G; Scheme 1 and
Scheme 2). In view of the increasing importance of cis-dihydrodiols
as synthetic precursors it was considered important to rationalise
the factors which allow a prediction to be made for the preferred
TDO-catalysed oxidation pathway.
Scheme 2
TDO-catalysed insertion of a single oxygen atom into a C–H
bond of an alkyl-substituted benzene ring (benzylic hydroxylation)
has also been observed, particularly in benzo-fused bicyclic
substrates where the benzylic alcohol products were readily
isolated.13–18
Although dioxygenase-catalysed benzylic hydroxylation of
alkyl-substituted benzene substrates A has been observed, prior
to the current study, in virtually all cases, isolation of the
benzylic alcohols C has proved to be elusive due to their being:
aSchool of Chemistry and Chemical Engineering, The Queen’s University
of Belfast, Belfast, UK BT9 5AG. E-mail: dr.boyd@qub.ac.uk; Tel: +44 28
9097 4421
bCenTACat, The Queen’s University of Belfast, Belfast, UK BT9 5AG
cDepartment of Biological Sciences, University of Warwick, Coventry, UK
CV4 7AL
† Electronic supplementary information (ESI) available: 1H-NMR spec-
troscopic data for cis-dihydrodiol metabolites 2B–12B, 14B–17B and 19B.
See DOI: 10.1039/b608417f
This journal is
The Royal Society of Chemistry 2006
Org. Biomol. Chem., 2006, 4, 3343–3349 | 3343
©

View Article Online
(a) excellent substrates (e.g. R = Me, Et, Pr) which are immediately
oxidised to the corresponding triols H (R = Me, Et, Pr), or (b)
unstable intermediates (R = CN, SMe, OAc) that spontaneously
decompose to benzaldehyde I (Scheme 2).18 In addition to arene
cis-dihydroxylation, dioxygenase enzymes have also been found
to catalyse the dihydroxylation of acyclic and cyclic conjugated
alkenes D to give the corresponding alkene diols E,19–32 and
monosulfoxidation of alkyl aryl sulfides F to give monosulfoxides
G 33–43 (Scheme 1).
Scheme 4
During the course of earlier studies from these laboratories,
using P. putida UV4 as a source of TDO, it was observed
that cis-dihydroxylation of disubstituted benzene substrates A
(R and Rꢀ = H) was slower, and yields of the corresponding
cis-dihydrodiols B were generally lower (Scheme 3).32,43,44 This
was particularly evident from combinatorial biotransformation
studies where the relative rates of cis-dihydroxylation of meta-
disubstituted benzene substrates A (R and Rꢀ = H) were lower
compared with monosubstituted benzenes A (Rꢀ = H) and ortho-
or para-disubstituted benzenes (R and Rꢀ = H, Scheme 3).
contrast with aliphatic hydroxylation of C-3 alkyl substrates J to
yield, exclusively, alcohols K. Alkyl substitution at C-2 resulted
in both alkyl and aryl hydroxylations, further demonstrating that
the regioselectivity of the TDO enzyme, during arene oxidations,
is dependent on the substitution pattern.
As a consequence of the reactivity of the transient benzylic
alcohol intermediates C (Scheme 2), very few examples of isolable
alcohols have been found from benzylic hydroxylation of alkyl-
substituted benzene substrates; the current study is primarily
focussed on this objective. In this context, the factors which
determine the nature of TDO-catalysed regioselectivity and stere-
oselectivity, observed during oxidation of disubstituted benzene
substrates A which contain an alkyl group (R = Et or Pr, Scheme 3
and Table 1), have now been examined to determine: (i) the relative
stereodirecting effects of the alkyl groups, (ii) if the arene cis-
dihydroxylation path to yield cis-dihydrodiols B is inhibited when
a meta substituent is present (a meta effect) and (iii) the preferred
stereochemistry of the benzylic alcohol metabolites C (Scheme 3).
=
Similarly, meta-substituted styrene substrates (A, R = CH CH2)
often yielded more of the exocyclic alkene diol E during TDO-
catalysed biotransformations (Scheme 3).32 The structural features
=
of substituted styrenes (A, R = CH CH2), which determine the
preference of TDO for dihydroxylation of either the arene ring
(to give a cis-dihydrodiol B) or the alkene group (to give an
alkene diol E), have also been determined.32 Furthermore, meta-
substituted phenyl methyl sulfides (A, R = SMe, meta Rꢀ = F,
Cl) gave sulfoxides (G, meta Rꢀ = F, Cl) exclusively rather than
cis-dihydrodiols B (R = SMe, meta Rꢀ = F, Cl) which were formed
when ortho and para substituents were present (Scheme 3). Thus,
the question of selectivity of this enzyme, present in P. putida UV4
whole cell systems, for either aryl rings or sulfur atoms has already
been addressed;43 all the results were consistent with the presence
of a meta effect.
Results and discussion
Results obtained from biotransformations of substituted alkyl-
benzene substrates A (R = H, Me, Et and Pr, Scheme 2), using P.
putida UV4, had shown that the TDO enzyme exhibits a marked
preference for oxidation of the arene ring to give the corresponding
cis-dihydrodiols B rather than the methyl or methylene group.
Thus, toluene A (R = H) gave only a trace of benzyl alcohol
C (1–2% yield; R = H). When other alkyl groups were present
in substrate A (R = Me; Et, or Pr), the proportion of attack at
the benzylic position could only be estimated from the isolated
yields of triols H [R = Me (5%), Et (26%), Pr (9%)] formed by
rapid oxidation of the corresponding benzyl alcohols C which
were generally undetected (Scheme 2).
Based on the observations that Et and Pr groups were more
likely to undergo benzylic hydroxylation than a Me group, albeit
in modest yield, using monosubstituted alkylbenzene substrates,
a systematic study of the TDO-catalysed oxidation of a series
of substituted ethylbenzene and propylbenzene substrates was
undertaken (Table 1). The structures of the bioproducts were
confirmed by spectroscopic comparisons with authentic samples,
Scheme 3
Recent oxidation studies45 of alkyl-substituted pyridine sub-
strates J, using P. putida UV4, also showed evidence of both
monohydroxylation of the alkyl group (equivalent to benzylic
hydroxylation) to yield alcohol K or monohydroxylation of
the electron-poor pyridine ring to yield hydroxypyridines M
(Scheme 4). Aromatic hydroxylation of the pyridine ring presum-
ably occurs via cis-dihydroxylation, to give unstable dihydrodiols
L (or a regioisomer), followed by dehydration. Aromatic mono-
hydroxylation of the aza-arene rings J to yield bioproducts M,
exclusively, when the alkyl group was at C-4 showed a marked
1
MS and H-NMR analyses (chemical shift, coupling constant,
NOE), and elemental microanalyses. For consistency of absolute
configuration assignment of the bioproducts, the carbon atom
bearing the Et or Pr groups was designated as C-1 for the disub-
stituted benzene substrates A and the benzyl alcohol products C
(Table 1). The 2-substituted alkylbenzene substrates (2A–5A, 14A)
and 4-substituted benzene substrates (6A–9A, 15A–17A) were
found to give the corresponding cis-dihydrodiols (2B–9B, 14B–
17B) exclusively. As expected, the isolated yields were generally
3344 | Org. Biomol. Chem., 2006, 4, 3343–3349
This journal is
The Royal Society of Chemistry 2006
©

View Article Online
Table 1 Relative yields, ee values and absolute configurations of cis-dihydrodiol and benzyl alcohol bioproducts
Arene
R
cis-Dihydrodiol
Benzyl alcohol
Yield (%)a
Rꢀ
Yield (%)a
Ee
Abs. config.
Ee
Abs. config.
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Et
Et
Et
Et
Et
Et
Et
2-H
2-F
2-Cl
2-Br
2-I
4-F
4-Cl
4-Br
4-I
3-F
3-Cl
3-Br
2-H
2-F
4-F
4-Cl
4-Br
3-F
3Cl
1A
2A
1B
2B
8
100
100
100
100
100
100
100
100
60, 10
14
>98
>98
>98
>98
>98
>98
>98
>98
>98
>98
>98
>98
>98
>98
>98
>98
>98
1S,2R b,c
1S,2R
1S,2R
1S,2R
1S,2R
1R,2R
1R,2R
1R,2R
1R,2R
1S,2R
1S,2R
1S,2R
1S,2R
1S,2R
1R,2R
1R,2R
1R,2R
1S,2R
1S,2R
1H
2C
92
0
0
0
0
0
0
0
0
40, 90
86
66
63 b,c
0
0
0
0
100
32
>98
1S,2R1ꢀR b,c
3A
3B
3C
4A
4B
4C
5A
5B
5C
6A
6B
6C
7A
7B
7C
8A
8B
8C
9A
9B
9C
10A
11A
12A
13A
14A
15A
16A
17A
18A
19A
10B
11B
12B
13B
14B
15B
16B
17B
18B
19B
10C
11C
12C
13H
14C
15C
16C
17C
18C
19C
2
6
18
R
R
34
R
37 b,c
100
100
100
100
0
1S,2R 1ꢀR
94
46
R
R
68
>98
a Relative yields of bioproducts B and C. b Ref. 18. c Isolated as a triol H.
low (2–50%) and no evidence of benzylic monohydroxylation or
trihydroxylation was observed. These cis-dihydrodiol metabolites
were all found to be present as single enantiomers (>98% ee),
by employing reported methods for enantiopurity determina-
The (2R) absolute configuration, assigned to each of the cis-
dihydrodiols 6B–9B and 15B–17B is consistent with the prediction
made on the basis of a simple model that had been proposed
earlier44 to account for the preferred absolute configurations of cis-
dihydrodiol metabolites (TDO enzyme present in P. putida UV4)
of 1,4-disubstituted benzene substrates (Scheme 5). This model
has also been used, recently, to rationalise both the regio- and
the stereo-selectivity of TDO-catalysed cis-dihydroxylation of 1,2-
and 1,3-disubstituted benzene substrates.7,8,10–12
1
tion, e.g. H-NMR analysis of diastereoisomeric boronate esters
formed using (R)- and (S)-2-(1-methoxyethyl)benzeneboronic acid
1
(MEBBA).18,29,32,37,43,46 In the H-NMR spectra of the boronates,
the d values for the CMe and OMe groups, obtained using
perdeuteriochloroform solvent and the (−)-(S)-boronic acid, were
all shifted downfield (CMe) and upfield (OMe) relative to the
signals obtained using (+)-(R)-boronic acid. On this basis, all the
cis-dihydrodiols were tentatively assigned a (2R) configuration.
This conclusion was supported by circular dichroism (CD)
spectroscopy and stereochemical correlation with related cis-
dihydrodiols of known configurations, e.g. those derived from 4-
substituted toluenes. With the exception of cis-dihydrodiol 2B,
all the cis-dihydrodiols showed a positive CD absorption at
longer wavelength (268–282 nm), in common with cis-dihydrodiol
metabolites of monosubstituted alkylbenzene substrates having
a (2R) configuration.47 Although cis-dihydrodiol 2B did not
show any CD absorption in the longer wavelength region, a
very similar CD spectrum was obtained for the corresponding
cis-dihydrodiol metabolite from 2-fluorotoluene whose absolute
configuration was established in an unequivocal manner (X-ray
crystallography) to possess a 2R configuration. Thus, by analyses
of 1H-NMR spectra (MEBBA derivatives) and CD spectroscopy
data (cis-dihydrodiol), the absolute configuration of each of
the cis-dihydrodiols 2B–12B, 14B–17B and 19B was found to
be (2R) (see ESI†, Table S1, for the 1H-NMR data of these
compounds).
Scheme 5
The model assumes a stereopreference for configuration B over
the enantiomeric configuration Bꢀ, based on the relative differences
in size between large (L) and small (S) conformationally inde-
pendent atoms or substituents, using standard steric parameter
values, e.g. the Charton steric parameter (t).48 Thus, the dominant
stereodirecting effect of the larger (L) atom or group was found to
follow the sequence CF3 (t 0.90) > I (t 0.78) > Br (t 0.65) > Cl
(t 0.55) ≈ Me (t 0.52) > F (t 0.27) > H (t 0.00) with decreasing
enantiopurity values as the size difference between the atoms or
groups became smaller. To accomodate substituents whose size
will be conformationally dependent, the predictive model has
recently been refined. A thiomethoxide group (SMe), having a
This journal is
The Royal Society of Chemistry 2006
Org. Biomol. Chem., 2006, 4, 3343–3349 | 3345
©

View Article Online
smaller t value (0.60) than a CF3 group or the halogen atoms (Cl,
Br and I), was found to have a stronger stereodirecting effect than
the atoms (H, F, Cl, Br, I) or groups (Me, CF3) studied earlier.43
The results shown in Table 1 indicate that: (a) the conformationally
dependent Et (t 0.56) and Pr (t 0.68) groups, present in the 1,4-
disubstituted benzene substrates 6A–9A and 15A–17A, exert a
stronger stereodirecting effect than the atoms (I, Br, Cl, F) or a
Me group (Pr > Et > I > Br > Cl ≈ Me > F > H), (b) the
dominant stereodirecting effect of the conformationally dependent
tetrahedral groups SMe, Et and Pr is determined by the preferred
conformation adopted within the active site rather than their t
values.
The cis-dihydrodiol metabolites 2B–5B and 14B from 1,2-
disubstituted benzene substrates 2A–5A and 14A (Table 1) were
all found be enantiopure (>98% ee) and of the same (2R) absolute
configuration. The dihydroxylation of the arene bond proximate to
the dominant stereodirecting group (L) was strongly favoured (no
evidence of the alternative regioisomers Bꢀ was obtained). These
results are again consistent with the conclusion that these alkyl
groups (L = Et and Pr) are stronger stereodirecting groups than
the halogen atoms (S = F, Cl, Br and I; Scheme 6).
chemistry of isolated bioproducts has contributed in refining the
predictive models (Schemes 5 and 6).
The benzylic alcohol products 10C–12C, 18C and 19C, obtained
from meta-substituted benzene substrates 10A–12A, 18A and 19A,
were formed in very low yields (1–6%). A wide range of ee
values (2–94%) was observed, using chiral stationary phase HPLC
analysis (Method B; CSPHPLC, Daicel OB column); in each
case an identical (R) absolute configuration was found, based on
optical rotation measurements and comparison with the literature
data. It was assumed that the variable ee values recorded could
be the result of asymmetric synthesis and/or kinetic resolution.
It is noteworthy that none of the ortho- or para-substituted
alkylbenzene substrates were found to be oxidised via benzylic
hydroxylation. Furthermore, in contrast with the benzylic alcohol
bioproducts 1C and 13C (formed initially from ethylbenzene
1A and propylbenzene 13A but rapidly converted, via TDO-
catalysed cis-dihydroxylation, to the corresponding triols 1H and
13H) none of the benzylic alcohols 10C–12C, 18C or 19C were
found to be oxidised to the corresponding triols. This observation
further supports the view that meta-substituted benzene substrates
generally show a preference for TDO-catalysed oxidation of
a peripheral substituent (benzylic hydroxylation, sulfoxidation
and alkene dihydroxylation) rather than an aryl group and that
TDO-catalysed cis-dihydroxylation is slower for 1,3-disubstituted
benzene substrates due to the meta effect. From the results
presented, it may be assumed that a substituent at a meta-postion
of a benzene substrate does not allow binding and catalysis of
arene cis-dihydroxylation at the TDO active site to occur as
readily as peripheral oxidation pathways. Our unpublished results
on the biotransformation (P. putida UV4) of ortho-, meta- and
para-xylene substrates show that the corresponding monobenzylic
alcohols are the only isolable bioproducts formed (6–13% yields).
This may be due to the slower rate of cis-dihydroxylation of the
disubstituted benzene rings allied to the availability (statistical
factor) of six benzylic C–H bonds.
Scheme 6
The cis-dihydrodiol metabolites B of 1,3-disubstituted benzenes,
obtained during our current studies, were also formed exclusively
(no evidence of Bꢀ) and found to be enantiopure and of identical
absolute configuration (Scheme 6). These cis-dihydrodiols were
again more difficult to form compared with the corresponding
diols from 1,2- or 1,4-disubstituted benzene substrates. Thus, no
evidence of cis-dihydrodiol 18B was found from biotransforma-
tion of the 1,3-disubstituted benzene 18A. However, during the
biotransformation of 1,3-disubstituted benzenes 10A–12A and
19A, cis-dihydroxylation did occur but exclusively on the arene
bond proximate to the Et and Pr groups to yield cis-dihydrodiols
10B–12B and 19B rather than the alternative regioisomers 10Bꢀ–
12Bꢀ and 19Bꢀ. This marked regioselectivity of TDO-catalysed
cis-dihydroxylation of 1,2- and 1,3-disubstituted benzenes further
supports our conclusion that the stereodirecting effects decrease
in the sequence Pr > Et > CF3 > I > Br > Cl ≈ Me > F > H.
The unoptimised isolated yields of cis-dihydrodiols 2B–12B,
14B–17B and 19B, obtained from substituted ethylbenzenes (2A–
12A, 3–55% yields) and propylbenzenes (14A–17A and 19A, 2–
40% yields), were generally much lower than the corresponding
monosubstituted benzene substrates (ethylbenzene 1A, 65% yield
including triol; propylbenzene 13A, 41% yield including triol).
Nevertheless, the determination of the preferred stereo- and regio-
Conclusion
The biotransformation of a series of substituted ethylbenzene
and propylbenzene substrates has yielded enantiopure (2R) cis-
dihydrodiols via TDO-catalysed dihydroxylation. The stereoselec-
tivity of this oxidation was rationalised, using a refined predictive
model where the effective size of conformationally dependent
substituents, e.g. Et and Pr groups, was found to have a dom-
inant stereodirecting influence. Meta-substituted ethylbenzene
and propylbenzene substrates were also found to yield benzylic
alcohol bioproducts rather than the corresponding triols; this was
consistent with earlier evidence of a meta effect.
Experimental
1H-NMR spectra were recorded at 300 MHz (Bruker Avance
DPX-300) and at 500 MHz (Bruker Avance DRX-500) in CDCl3
solvent, unless stated otherwise. Chemical shifts (d) are reported
in ppm relative to SiMe4 and coupling constants (J) are given in
Hz. Mass spectra were recorded at 70 eV on a VG Autospec Mass
Spectrometer, using a heated inlet system. Accurate molecular
weights were determined by the peak matching method with
perfluorokerosene as standard. Elemental microanalyses were
3346 | Org. Biomol. Chem., 2006, 4, 3343–3349
This journal is
The Royal Society of Chemistry 2006
©

View Article Online
obtained on a Perkin-Elmer 2400 CHN microanalyser. Circular
dichroism spectra were recorded on a Jasco J-720 instrument
in acetonitrile solvent. CSPHPLC analysis was carried out on
a Shimadzu LC-6A liquid chromatograph (Daicel OB column)
connected to Hewlett Packard diode array detector. Optical
rotation ([a]D) measurements were performed on a Perkin-
Elmer polarimeter at ambient temperature temperature (20 ◦C)
and are expressed in units of 10−1 deg cm2 g−1. Ethylbenzene
2A–12A and propylbenzene 14A–19A substrates were prepared
from commercially available substituted styrene and alkyl phenyl
ketones, using literature methods. The corresponding benzylic
alcohol metabolites 10C–12C, 18C, 19C were found to have
similar 1H-NMR spectral characteristics to those reported in the
literature.
Shake flask (<0.5 g) biotransformations were carried out
using P. putida UV4 under reported conditions.32 cis-Dihydrodiol
2B–12B, 14B–17B, 19B and benzyl alcohol bioproducts 10C–
12C, 18C, 19C obtained after bioconversion of the correspond-
ing substrates 2A–12A, 14A–19A were separated and purified
by preparative layer chromatography (PLC) (silica gel, 50%
EtOAc in hexane). The enantiomeric excess (ee) values of cis-
dihydrodiols 2B–12B, 14B–19B were determined via forma-
tion of the corresponding diastereoisomeric boronate esters us-
ing R- and S-2-(1-methoxyethyl)benzeneboronic acids followed
by 1H-NMR analysis of the diastereoisomeric composition
(Method A).46
100), 139 (M+ − I, 17); (Found: M+ 265.9804; C8H11IO2 requires
265.9804); >98% ee (Method A); CD k 236 nm (De 2.55), k 283 nm
(De 1.55).
cis-(1R,2R)-1,2-Dihydroxy-3-ethyl-6-fluorocyclohexa-3,5-diene
6B. From substrate 6A (0.140 g, 55%); mp 80–81 ◦C (from
hexane); [a]D +147 (c 0.4, MeOH); m/z (EI) (trimethylsilyl
derivative from BSTFA) 302 (M+, 35%), 191 (25), 147 (45), 73
(Me3Si, 100); (Found: M+ 158.0741; C8H11FO2 requires 158.0743);
>98% ee (Method A); CD k 209 nm (De 0.06), 264 nm (De 3.43).
cis-(1R,2R)-1,2-Dihydroxy-6-chloro-3-ethylcyclohexa-3,5-diene
7B. From substrate 7A (0.037 g, 8%); mp 90–91 ◦C (from
CH2Cl2–hexane); [a]D +92 (c 0.4, MeOH); (Found: C 55.1, H 6.8;
C8H11ClO2 requires C 55.2, H 6.4%); m/z (EI) 174 (M+, 46%), 156
(31), 93 (100); >98% ee (Method A); CD k 217 nm (De 1.451), k
282 nm (De 0.386).
cis-(1R,2R)-1,2-Dihydroxy-6-bromo-3-ethylcyclohexa-3,5-diene
8B. From substrate 8A (0.057 g, 32%); mp 116–118 ◦C (from
CHCl3); [a]D +27 (c 0.4, MeOH); m/z (EI) (trimethylsilyl derivative
from BSTFA) 364 [(81Br)M+, 10%], 362 [(79Br)M+, 10], 283 (M+ −
Br, 72), 73 (Me3Si, 100); (Found: M+ 217.9952; C8H1179BrO2
requires 217.9942); >98% ee (Method A); CD k 218 nm (De 2.87),
283 nm (De 0.253).
cis-(1R,2R)-1,2-Dihydroxy-3-ethyl-6-iodocyclohexa-3,5-diene
9B. From substrate 9A (0.145 g, 51%); mp 42–44 ◦C (from
CHCl3); [a]D +1.7 (c 0.7, MeOH); m/z (EI) 266 (M+, 45%), 248
(M+ − H2O, 50), 121 (100); (Found: M+ 265.9804; C8H11IO2
requires 265.9804); >98% ee (Method A).
The absolute configurations were determined by 1H-NMR
analysis of the chemical shift values of chiral boronate derivatives,
and comparison of CD spectra of cis-dihydrodiols. The ee values of
benzylic alcohol metabolites 10C–13C, 18C, 19C were determined
by CSPHPLC analyses using a Daicel OB column (hexane–
isopropanol, 95 : 5, Method B), and comparison of the sign of
specific optical rotations with the literature values.
cis-(1S,2R)-1,2-Dihydroxy-3-ethyl-5-fluorocyclohexa-3,5-diene
10B. From substrate 10A (0.029 g, 6%), mp 58–60 ◦C (from
CH2Cl2–hexane); [a]D +99 (c 0.4, MeOH); (Found: C 61.1, H 7.2;
C8H11FO2 requires C 60.7, H 7.0%); m/z (EI) 258 (M+, 64%), 140
(74); >98% ee (Method A); CD k 212 nm (De −4.222), 279 nm (De
0.872).
cis-Dihydrodiol bioproducts 2B–12B, 14B–17B, 19B
cis-(1S,2R)-1,2-Dihydroxy-3-ethyl-4-fluorocyclohexa-3,5-diene
2B. From substrate 2A (0.025 g, 10%); mp 59–61 ◦C (from
hexane); [a]D −6.2 (c 0.45, MeOH); m/z (EI) (trimethylsilyl deriva-
tive from BSTFA) 302 (M+, 90%), 73 (Me3Si, 100); (Found: M+
158.0745; C8H11FO2 requires 158.0743); >98% ee (Method A);
CD k 206 nm (De 0.527), 258 nm (De −0.909).
cis-(1S,2R)-1,2-Dihydroxy-5-chloro-3-ethylcyclohexa-3,5-diene
11B. From substrate 11A (0.023 g, 5%); mp 48–50 ◦C (from
CH2Cl2–hexane), [a]D +13 (c 0.8, MeOH); (Found: C 55.0, H 6.0;
C8H11ClO2 requires C 55.2, H 6.4%); m/z (EI) 174 (M+, 4%), 156
(63), 141 (100); >98% ee (Method A); CD k 209 nm (De −3.855),
274 nm (De 1.238).
cis-(1S,2R)-1,2-Dihydroxy-4-chloro-3-ethylcyclohexa-3,5-diene
3B. From substrate 3A (0.032 g, 7%); mp 57–59 ◦C (from
CH2Cl2–hexane); [a]D +81 (c 0.4, MeOH); (Found: C 55.3, H 6.1;
C8H11ClO2 requires C 55.2, H 6.4%); m/z (EI) 174 (M+, 32%), 156
(26), 93 (100); >98% ee (Method A); CD k 209 nm (De 2.434),
279 nm (De 0.872).
cis-(1S,2R)-1,2-Dihydroxy-5-bromo-3-ethylcyclohexa-3,5-diene
12B. From substrate 12A (0.014 g, 3%); mp 54–56 ◦C (from
CH2Cl2–hexane); [a]D −10 (c 0.6, MeOH); (Found: C 44.1, H 5.5;
C8H11BrO2 requires C 43.9, H 5.1%); m/z (EI) 200 (M+, 96%), 185
(100); >98% ee (Method A).
cis-(1S,2R)-1,2-Dihydroxy-4-fluoro-3-propylcyclohexa-3,5-diene
14B. From substrate 14A (0.080 g, 21%); mp 75–76 ◦C (from
CH2Cl2–hexane), [a]D +42 (c 1.1, MeOH); (Found: M+, 172.2006;
C9H13FO2 requires M+, 172.2060); m/z (EI) 172 (M+, 61%), 154
(18), 125 (60), 97 (100); >98% ee (Method A); CD k 206 nm (De
1.028), 288 nm (De 0.147).
cis-(1S,2R)-1,2-Dihydroxy-4-bromo-3-ethylcyclohexa-3,5-diene
4B. From substrate 4A (0.031 g, 18%); mp 71–72 ◦C (from
hexane); [a]D +47 (c 0.4, MeOH); m/z (EI) (trimethylsilyl deriva-
tive from BSTFA) 364 [(81Br)M+, 20%], 362 [(79Br)M+, 18], 283
(M+ − Br, 30), 73 (Me3Si, 100); (Found: M+ 217.9934; C8H1179BrO2
requires 217.9942); >98% ee (Method A); CD k 211nm (De 1.36),
276 nm (De 0.701).
cis-(1R,2R)-1,2-Dihydroxy-6-fluoro-3-propylcyclohexa-3,5-diene
15B. From substrate 15A (0.055 g, 18%), mp 66–67 ◦C (from
CH2Cl2–hexane); [a]D +120 (c 1.1, CHCl3); (Found: C 62.3, H
7.3; C9H13FO2 requires C 62.8, H 7.6%); m/z (EI) 172 (M+, 44%),
cis-(1S,2R)-1,2-Dihydroxy-3-ethyl-4-iodocyclohexa-3,5-diene 5B.
◦
From substrate 5A (0.132 g, 46%); mp 92–94 C (from CHCl3);
[a]D +71 (c 0.7, MeOH); m/z (EI) 266 (M+, 15%), 248 (M+ − H2O,
This journal is
The Royal Society of Chemistry 2006
Org. Biomol. Chem., 2006, 4, 3343–3349 | 3347
©

View Article Online
154 (20), 125 (68), 112 (100); >98% ee (Method A); CD k 214 nm
(De −5.136), 272 nm (De 2.84).
cis-(1R,2R)-1,2-Dihydroxy-6-chloro-3-propylcyclohexa-3,5-diene,
16B. From substrate 16A (0.312 g, 32%); mp 91–92 ◦C (from
hexane); [a]D +45 (c 0.8, CHCl3); (Found: C 57.3, H 6.8;
C9H13ClO2 requires C 57.3, H 6.95%); >98% ee (Method A); CD
k 218 nm (De 1.235), 276 nm (De 0.572).
7 D. R. Boyd and G. N. Sheldrake, Nat. Prod. Rep., 1998, 15, 309.
8 T. Hudlicky, D. Gonzalez and D. T. Gibson, Aldrichimica Acta, 1999,
32, 35.
9 D. T. Gibson and R. E. Parales, Curr. Opin. Biotechnol., 2000, 11, 236.
10 D. R. Boyd, N. D. Sharma and C. C. R. Allen, Curr. Opin. Biotechnol.,
2001, 12, 564.
11 R. A. Johnson, Org. React., 2004, 63, 117.
12 D. R. Boyd and T. Bugg, Org. Biomol. Chem., 2006, 4, 181.
13 D. R. Boyd, N. D. Sharma, N. I. Bowers, R. Boyle, J. S. Harrison, T.
Bugg, K. Lee and D. T. Gibson, Org. Biomol. Chem., 2003, 1, 1298.
14 D. R. Boyd, N. D. Sharma, P. J. Stevenson, J. Chima, D. J. Gray and
H. Dalton, Tetrahedron Lett., 1991, 32, 3887.
cis-(1R,2R)-1,2-Dihydroxy-6-bromo-3-propylcyclohexa-3,5-diene
17B. From substrate 17A (0.281 g, 40%); mp 93 ◦C (from
hexane); [a]D +68 (c 0.6, MeOH); (Found: C 46.2, H 5.5;
C9H13BrO2 requires C 46.4, H 5.6%); m/z (EI) 234 (M+, 4%), 232
(4), 216 (39), 214 (40), 187 (98), 185 (100); >98% ee (Method A);
CD k 215 nm (De 2.228), 277 nm (De 0.943).
15 J. M. Brand, D. L. Cruden, G. J. Zylstra and D. T. Gibson, Appl.
Environ. Microbiol., 1992, 58, 3407.
16 D. R. Boyd, N. D. Sharma, N. I. Bowers, P. A. Goodrich, M. R.
Groocock, A. J. Blacker, D. A. Clarke, T. Howard and H. Dalton,
Tetrahedron: Asymmetry, 1996, 7, 1559.
17 N. I. Bowers, D. R. Boyd, N. D. Sharma, P. A. Goodrich, M. R.
Groocock, A. J. Blacker, P. Goode and H. Dalton, J. Chem. Soc., Perkin
Trans. 1, 1999, 1453.
18 D. R. Boyd, N. D. Sharma, N. I. Bowers, J. Duffy, J. S. Harrison and
H. Dalton, J. Chem. Soc., Perkin Trans. 1, 2000, 1345.
19 L. P. Wackett, L. D. Kwart and D. T. Gibson, Biochemistry, 1988, 27,
1367.
20 R. A. S. McMordie, D. R. Boyd, N. D. Sharma, H. Dalton, P. Williams
and R. O. Jenkins, J. Chem. Soc., Chem. Commun., 1989, 339.
21 D. R. Boyd, M. R. J. Dorrity, J. F. Malone, R. A. S. McMordie, N. D.
Sharma, H. Dalton and P. J. Williams, J. Chem. Soc., Perkin Trans. 1,
1990, 489.
cis-(1S,2R)-1,2-Dihydroxy-5-chloro-3-propylcyclohexa-3,5-diene
19B. From substrate 19A (0.014 g, 2%); mp 61–63 ◦C (from
CHCl3); [a]D +42 (c 1.2, CHCl3); (Found: M+, 188.0602;
C9H13ClO2 requires M+, 188.0604); m/z (EI) 190 (M+, 20%), 188
(55), 172 (65), 170 (78), 143 (100), 141 (8); >98% ee (Method A);
CD k 211 nm (De −4.458), 268 nm (De 2.021).
Benzylic alcohol bioproducts 10C–12C, 18C, 19C
22 T. Hudlicky, H. Luna, G. Barbieri and L. D. Kwart, J. Am. Chem. Soc.,
All benzylic alcohol products were found to be spectroscopically
identical with literature data.
1988, 110, 4735.
23 T. Hudlicky, E. E. Boros and C. H. Boros, Tetrahedron: Asymmetry,
1993, 4, 1365.
(R)-1-(3-Fluorophenyl)ethanol 10C. From substrate 10A
(0.017 g, 4%); [a]D +0.9 (c 0.8, MeOH) (lit.49 [a]D +38.5, MeOH);
2% ee (Method B).
24 K. Konigsberger and T. Hudlicky, Tetrahedron: Asymmetry, 1993, 4,
2469.
25 D. T. Gibson, S. M. Resnick, K. Lee, J. M. Brand, D. S. Torok, L. P.
Wackett, M. J. Schocken and B. E. Haigler, J. Bacteriol., 1995, 177,
2615.
26 D. S. Torok, S. M. Resnick, J. M. Brand, D. L. Cruden and D. T.
Gibson, J. Bacteriol., 1995, 177, 5799.
27 D. R. Boyd, N. D. Sharma, N. A. Kerley, R. A. S. McMordie, G. N.
Sheldrake, P. Williams and H. Dalton, J. Chem. Soc., Perkin Trans. 1,
1996, 67.
(R)-1-(3-Chlorophenyl)ethanol 11C. From substrate 11A
(0.012 g, 6%); [a]D +1.1 (c 0.8, MeOH) (lit.50 [a]D +38.6, MeOH);
6% ee (Method B).
(R)-1-(3-Bromophenyl)ethanol 12C. From substrate 12A
(0.012 g, 6%); [a]D +4.8 (c 0.8, MeOH) (lit.51 [a]D +29, MeOH);
18% ee (Method B).
28 S. M. Resnick; and D. T. Gibson, Appl. Environ. Microbiol., 1996, 62,
1364.
29 D. R. Boyd, N. D. Sharma, B. Byrne, M. V. Hand, J. F. Malone, G. N.
Sheldrake, J. Blacker and H. Dalton, J. Chem. Soc., Perkin Trans. 1,
1998, 1935.
30 N. I. Bowers, D. R. Boyd, N. D. Sharma, M. A. Kennedy,
G. N. Sheldrake and H. Dalton, Tetrahedron: Asymmetry, 1998, 9,
1831.
(R)-1-(3-Fluorophenyl)propanol 18C. From substrate 18A
(0.010 g, 3%); [a]D +24.8 (c 0.36, CHCl3); 94% ee (Method B).
(R)-1-(3-Chlorophenyl)propanol 19C (see ref. 52). From sub-
strate 12A (0.007 g, 1%); [a]D +7.0 (c 0.2, CHCl3) (lit.52 [a]D +31,
MeOH); 46% ee (Method B).
31 D. R. Boyd, D. Clarke, M. C. Cleij, J. T. G. Hamilton and G. N.
Sheldrake, Monatsh. Chem., 2000, 131, 673.
32 D. R. Boyd, N. D. Sharma, I. N. Brannigan, M. R. Groocock, J. F.
Malone, G. McConville and C. C. R. Allen, Adv. Synth. Catal., 2005,
347, 1081.
Acknowledgements
33 J. R. Cashman, L. D. Olsen, D. R. Boyd, R. A. S. McMordie, R. Dunlop
and H. Dalton, J. Am. Chem. Soc., 1992, 114, 8772.
34 C. C. R. Allen, D. R. Boyd, H. Dalton, N. D. Sharma, S. A. Haughey,
R. A. S. McMordie, B. T. McMurray, G. N. Sheldrake and K. Sproule,
J. Chem. Soc., Chem. Commun., 1995, 119.
We wish to thank the BBSRC and CenTACat (N. D. S.), the
European Science Fund (J. S. H.), and the Department of
Education NI (N. I. B.) for funding.
35 K. Lee, J. M. Brand and D. T. Gibson, Biochem. Biophys. Res. Commun.,
1995, 212, 9.
36 D. R. Boyd, N. D. Sharma, S. A. Haughey, M. A. Kennedy, B. T.
McMurray, G. N. Sheldrake, C. C. R. Allen, H. Dalton and K. Sproule,
J. Chem. Soc., Perkin Trans. 1, 1998, 1929.
37 D. R. Boyd, N. D. Sharma, S. A. Haughey, J. F. Malone, A. King, B. T.
McMurray, R. Holt and H. Dalton, J. Chem. Soc., Perkin Trans. 1,
2001, 3288.
38 D. R. Boyd, N. D. Sharma, B. E. Byrne, S. D. Shepherd, V. Ljubez,
C. C. R. Allen, L. A. Kulakov, M. L. Larkin and H. Dalton, Chem.
Commun., 2002, 1914.
References
1 D. T. Gibson, J. R. Koch and R. E. Kallio, Biochemistry, 1968, 7, 2653.
2 D. A. Widdowson, D. W. Ribbons and S. D. Thomas, Janssen Chim.
Acta, 1990, 8, 3.
3 H. J. Carless, Tetrahedron: Asymmetry, 1992, 3, 795.
4 G. N. Sheldrake, in Chirality in Industry, eds. A. N. Collins,
G. N. Sheldrake and J. Crosby, J. Wiley, Chichester, 1992, ch. 6,
p. 128.
5 S. M. Brown and T. Hudlicky, in Organic Synthesis: Theory and
Applications, JAI Press Inc., Greenwich, 1993, p. 113.
6 S. M. Resnick, K. Lee and D. T. Gibson, J. Ind. Microbiol. Biotechnol.,
1996, 17, 438.
39 D. R. Boyd, N. D. Sharma, S. A. Haughey, J. F. Malone, B. T.
McMurray, G. N. Sheldrake, C. C. R. Allen and H. Dalton, Chem.
Commun., 1996, 2363.
3348 | Org. Biomol. Chem., 2006, 4, 3343–3349
This journal is
The Royal Society of Chemistry 2006
©

View Article Online
40 D. R. Boyd, N. D. Sharma, N. Gunaratne, S. A. Haughey, M. A.
Kennedy, J. F. Malone, C. C. R. Allen and H. Dalton, Org. Biomol.
Chem., 2003, 1, 984.
41 D. R. Boyd, N. D. Sharma, S. Haughey, M. A. Kennedy, J. F. Malone,
S. Shepherd, C. C. R. Allen and H. Dalton, Tetrahedron, 2004, 60,
549.
45 M. D. Garrett, R. Scott, G. N. Sheldrake, H. Dalton and P. Goode,
Org. Biomol. Chem., 2006, 4, 2710.
46 S. M. Resnick, D. S. Torok and D. T. Gibson, J. Org. Chem., 1995, 60,
3546.
47 D. R. Boyd, A. Drake, J. Gawronski, M. Kwit, J. F. Malone and N. D.
Sharma, J. Am. Chem. Soc., 2005, 127, 4308.
42 D. R. Boyd, N. D. Sharma, A. W. T. King, S. D. Shepherd, C. C. R.
Allen, R. A. Holt, H. R. Luckarift and H. Dalton, Org. Biomol. Chem.,
2004, 2, 554.
43 D. R. Boyd, N. D. Sharma, A. King, B. Byrne, S. A. Haughey,
M. A. Kennedy and C. C. R. Allen, Org. Biomol. Chem., 2004, 2,
2530.
44 D. R. Boyd, N. D. Sharma, M. V. Hand, M. R. Groocock, N. A. Kerley,
H. Dalton, J. Chima, G. N. Sheldrake and C. C. R. Allen, J. Chem. Soc.,
Chem. Commun., 1993, 974.
48 M. B. Smith and J. March, in March’s Advanced Organic Chemistry:
Reactions, Mechanism and Structure, John Wiley, New York, 5th edn.,
2001, p. 374.
49 K. Naemura, M. Murata, R. Tanaka, M. Yano, K. Hirose and Y. Tobe,
Tetrahedron: Asymmetry, 1996, 7, 3285.
50 S. Ogawa and N. Furukawa, J. Org. Chem., 1991, 56, 5723.
51 C.-P. Chen, K. Prasad and O. Repic, Tetrahedron Lett., 1991, 32, 7175.
52 A. J. Bustillo, J. Aleu, R. Hernandez-Galan and I. G. Collado,
Tetrahedron: Asymmetry, 2002, 13, 1681.
This journal is
The Royal Society of Chemistry 2006
Org. Biomol. Chem., 2006, 4, 3343–3349 | 3349
©