Cis-Dihydroxylation of Tricyclic Arenes and Heteroarenes Catalyzed by Toluene Dioxygenase: A Molecular Docking Study and Experimental Validation

Article abstract of DOI:10.1002/adsc.201900147

Molecular docking studies of toluene dioxygenase led to the prediction that angular and lateral cis-dihydroxylation of tricyclic arene and heteroarene substrates could occur. Biotransformations of biphenylene, dibenzofuran, carbazole and dibenzothiophene, using Pseudomonas putida UV4 whole cells, expressing toluene dioxygenase, confirmed that both angular and lateral cis-dihydroxylation had occurred in the predicted regioselective and stereoselective manner. The toluene dioxygenase-catalysed (Pseudomonas putida UV4) biotransformation of dibenzofuran was optimized, to produce 1,2-dihydrodibenzofuran-1,2-diol as the major metabolite in excellent yield. 2-Hydroxydibenzofuran, resulting from dehydration of 1,2-dihydrodibenzofuran-1,2-diol, was also found to undergo cis- dihydroxylation to give a very minor cis-dihydrodiol metabolite. The enantiopurity (>98% ee) and (1R,2S) absolute configuration of the major dibenzofuran cis -dihydrodiol was rigorously established by catalytic hydrogenation and formation of methoxy(trifluoromethyl)phenylacetate derivatives and by X-ray crystallography of an epoxide derivative. Biotransformation of carbazole yielded anthranilic acid as the major metabolite and was consistent with angular cis-dihydroxylation. Synthesis of a cis- diol epoxide derivative showed that the main cis-dihydrodiol metabolite of dibenzofuran has potential in the chemoenzymatic synthesis of natural products. (Figure presented.).

Full text of DOI:10.1002/adsc.201900147

Title: cis-Dihydroxylation of Tricyclic Arenes and Heteroarenes
Catalyzed by Toluene Dioxygenase: A Molecular Docking Study
and Experimental Validation
Authors: Derek Boyd, N Sharma, Ian Brannigan, Christopher
McGivern, Peter Nockemann, Paul Stevenson, Colin
McRoberts, Patrick Hoering, and C Allen
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To be cited as: Adv. Synth. Catal. 10.1002/adsc.201900147
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10.1002/adsc.201900147
Advanced Synthesis & Catalysis
VERY IMPORTANT PUBLICATION
FULL PAPER
DOI: 10.1002/adsc.201((will be filled in by the editorial staff))
cis-Dihydroxylation of Tricyclic Arenes and Heteroarenes
Catalyzed by Toluene Dioxygenase: A Molecular Docking Study
and Experimental Validation
Derek R. Boyd,*a Narain D. Sharma,a Ian N. Brannigan,a Christopher J. McGivern,a
Peter Nockemann,a Paul J. Stevenson,a Colin McRoberts,b Patrick Hoeringc and
Christopher C. R. Allen*c
a School of Chemistry and Chemical Engineering, Queen’s University of Belfast, Belfast,
BT95AG, UK. 02890974421 E-mail: dr.boyd@qub.ac.uk, c.allen@qub.ac.uk.
b Agri-food and Biosciences Institute for Northern Ireland, Belfast, BT95PX, UK,
c School of Biological Sciences, Queen’s University of Belfast, Belfast, BT9 7BL, UK
Received: ((will be filled in by the editorial staff))
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201######.((Please
delete if not appropriate))
dibenzofuran cis -dihydrodiol was rigorously established by
catalytic hydrogenation and formation of dimethoxy phenyl
trifluoromethyl acetate derivatives and by X-ray
crystallography of an epoxide derivative. Biotransformation
of carbazole yielded anthranilic acid as the major
metabolite and was consistent with angular cis-
dihydroxylation. Synthesis of a cis- diol epoxide derivative
showed that the main cis-dihydrodiol metabolite of
dibenzofuran has potential in the chemoenzymatic synthesis
of natural products.
Abstract: Molecular docking studies of toluene
dioxygenase led to the prediction that angular and lateral cis-
dihydroxylation of tricyclic arene and heteroarene substrates
could
occur.
Biotransformations
of
biphenylene,
dibenzofuran, carbazole and dibenzothiophene, using
Pseudomonas putida UV4 whole cells expressing toluene
dioxygenase, confirmed that both angular and lateral cis-
dihydroxylation had occurred in the predicted regioselective
and stereoselective manner.
The toluene dioxygenase-catalysed (Pseudomonas putida
UV4) biotransformation of dibenzofuran was optimized, to Keywords: Tricyclic heteroarene biotransformations;
produce 1,2-dihydrodibenzofuran-1,2-diol as the major
metabolite in excellent yield. 2-Hydroxydibenzofuran,
resulting from dehydration of 1,2-dihydrodibenzofuran-1,2-
diol, was also found to undergo cis- dihydroxylation to give
a very minor cis-dihydrodiol metabolite. The enantiopurity
(>98% ee) and (1R,2S) absolute configuration of the major
molecular docking; toluene dioxygenase; lateral/angular
cis-dihydroxylation; dibenzofuran cis-dihydrodiol;
chemoenzymatic synthesis
dioxygenase (NDO), which mostly accept only
bicyclic and tricyclic arenes, and biphenyl
Introduction
dioxygenase
(BPDO)
which
catalyse
cis-
Ring hydroxylating dioxygenase enzymes,
having the ability to catalyse arene cis-
dihydroxylations, are available with a range of
binding site capacities, able to accommodate both
mono- and poly-cyclic arenes.[1a-f] Among
dioxygenases with smaller active sites are benzoate
dioxygenase (BZDO) which accepts benzoic acid
substrates, and toluene dioxygenase (TDO), which
generally accommodate only monocyclic and bicyclic
arenes. Larger binding sites are found in naphthalene
dihydroxylation of polycyclic arenes containing up to
five fused rings. NDO and BPDO are largely unable
to catalyse cis-dihydroxylation of monocyclic arenes,
exceptions being larger biaryl substrates, e.g. 3-
phenylphenol 1a.[2] Dioxygenase enzymes BZDO,
TDO, NDO and BPDO have all been employed in
chemoenzymatic synthesis, but to date most arene
cis-dihydrodiol metabolites, used as chiral synthons,
have been formed with monocyclic substrates and
TDO as biocatalyst. [1a-f]
1
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10.1002/adsc.201900147
Advanced Synthesis & Catalysis
Several molecular docking studies of TDO
with monocyclic aromatic substrates, have recently
been used to successfully match the resulting in silico
predictions
with
reported
benzylic
monohydroxylation and arene cis-dihydroxylation
[3a-e]
results.
Thus, AutoDock 4.2 molecular docking
studies, of substituted phenol and aniline substrates
with TDO, led to predictions about: (a) their preferred
orientations during cis-dihydroxylation and (b) the
resulting regioselectivity and stereoselectivity. These
expectations were later confirmed by experimental
results.[3c,3e] This approach was applied to the TDO
docking results obtained using 3-hydroxybiphenyl 1a
as substrate, where the corresponding metabolites 2a
and 3a were predicted to be the major and minor
products respectively, resulting from lateral cis-
dihydroxylation.[2] Biotransformation (P. putida UV4
mutant strain expressing TDO) of substrate 1a,
yielded metabolites (5S,6R)-2a and (2R,3S)-3a with
the predicted regio- and stereo-chemistry (Scheme 1).
Scheme 1. cis-Dihydroxylation of 3-hydroxybiphenyl 1a
to give cis-diols 2a and 3a.
Scheme 2. Dioxygenase (DO)-catalysed cis-
dihydroxylation of arenes 1b-1e to give cis-dihydrodiols
2b-2e, 3c-3e, 4c-4e, phenols 5c, 5e and sulfoxide 5d.
TDO-catalyzed regio- and stereo-selective
cis-dihydroxylations of bicyclic arenes, (e.g.
naphthalene) and heteroarenes (e.g. quinoline),
predicted by AutoDock Vina molecular docking
(unpublished
data),
matched
with
earlier
experimental results.[1a-c,4] Similar TDO-catalyzed
cis-dihydroxylations of tricyclic arenes and
heteroarenes, to our knowledge, have not been
reported. Thus, the current study, using the AutoDock
Vina program, was undertaken to find out if: (i) the
larger tricyclic arene substrates, e.g. biphenylene 1b,
dibenzofuran 1c, carbazole 1e and dibenzothiophene
1d would be accommodated by the TDO active site,
(ii) the experimental cis-dihydroxylation results,
obtained using TDO and NDO enzymes expressed by
mutant Pseudomonas strains, would match those
predicted by the molecular docking results (Scheme
2) and (iii) supporting evidence could be obtained for
the postulated role of transient metabolites leading to
the formation of phenols and other metabolites from
tricylic arenes and heteroarenes.
Results and Discussion
The biotransformation (P. putida UV4 whole
cells, expressing TDO) of biphenylene 1b had
resulted in cis-dihydroxylation at a ring junction
(angular) bond to give only cis-dihydrodiol 2b.[5a,5b]
Conversely, the biotransformation of tricyclic
heteroarenes 1c-1e by mutant strains of P. putida
9816/11 and P. fluorescens TTC1(NCIMB 40605),
both expressing NDO, resulted in lateral cis-
dihydroxylations to yield cis-diols 2c-2e.[6a-d]
Molecular docking studies were conducted to
compare with these reported biotransformation results
and to predict if similar results would be found using
TDO with potential substrates 1c-1e.
TDO docking of biphenylene 1b: Prior to the earlier
reports,
using
biphenylene
and
1,2-
dihydrobenzocyclobutene as substrates, TDO-
catalysed cis-hydroxylation at an angular position, in
a substituted monocyclic or bicyclic arene substrate
was unprecedented.[5a,5b] The cis-dihydroxylation (P.
2
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10.1002/adsc.201900147
Advanced Synthesis & Catalysis
Figure 1. Resonance structures of biphenylene 1b.
putida UV4) of biphenylene 1b was thus unusual,
since the attack occurred exclusively at an angular
bond (1,8b), to give (1S,8bR)-dihydrodiol 2b (66%
isolated yield) as the only isolated metabolite
(Scheme 2).
The opposite enantiomer (1R,8bS)-2b was
isolated later when biphenylene 1b was used as
substrate for a different Pseudomonas strain (C250),
expressing carbazole 1,9a-dioxygenase (CARD).[6b]
These reports provide a very rare example of
enantiocomplementarity
during
dioxygenase-
Biphenylene 1b, having two fused benzene
rings and with four possible canonical (resonance)
structures (4, 6, 8, 12), was classified as a
tricyclic arene substrate with some anti-aromatic
character (Figure 1).[7] Electron density is more
localized within the biphenylene ring and the 4
catalysed formation of arene cis-dihydrodiols. The
good agreement between the biotransformation result
and in silico TDO binding orientation of biphenylene
1b provided an incentive to extend the docking of
tricyclic heteroarenes 1c-1e, in the quest for further
evidence of lateral or angular cis-dihydroxylation.
(cyclobutadiene)
resonance
structure
being
disfavoured. Thus, its aromaticity was markedly
reduced compared with the tricyclic arenes 1c-1e and
was estimated as only 1.59 times that of benzene.[7]
TDO-catalysed angular cis-dihydroxylation, to yield
cis-dihydrodiol 2b, would therefore be more favoured
by the 6, 8 and 12 canonical structures.
1b1
Using Autodock Vina, the energetically most
favourable binding orientation of biphenylene 1b (-
6.6 kcal·mol-1, 1b1) within the active site of TDO is
shown in Figure 2. This orientation appeared to be
mainly due to: (i) attractive interactions of the
substrate with the orthogonal phenyl group of Phe-
216 and with the imidazole group of His-222 (edge-
to-face bonding) and (ii) the proximate hydrophobic
amino acids Ile-276, Leu-272, Ile-324, Val-309, Leu-
272 Phe-352 (van der Waals interactions). It would
also result in an exclusive preference for angular
rather than lateral cis-dihydroxylation and is matched
by the isolation of the remarkably stable enantiopure
cis-diol (1S, 8bR)-2b (> 98% ee), as the only detected
metabolite using P. putida UV4.[5a,5b]
).
Figure 2: In silico docking orientation of biphenylene 1b
with TDO. 1b1: -6.6 kcal·mol-1.
(ii) TDO docking of dibenzofuran 1c:
Molecular docking of dibenzofuran 1c with TDO
(Figure 3) led to the prediction of two preferred
orientations (1c1 and 1c2), which appeared to be very
similar to the preferred orientation of biphenylene 1b
(Figure
2).
1c1
1c2
Phe-372
Phe-366
His-222
Ala-223
Glu-215
Phe-216
Ile-276
Leu-272
Ile-324
Leu-321
Val-309
His-311
Figure 3. In silico docking orientations of dibenzofuran 1c with TDO. 1c1: -5.7 kcal·mol-1; 1c2: -5.0 kcal·mol-1.
3
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10.1002/adsc.201900147
Advanced Synthesis & Catalysis
Lateral cis-dihydroxylation was predicted to
heterocyclic rings.[10a] Recently, evidence for the
edge- to-face interactions between benzene and furan
or thiophene rings has been reported. [10b, 10c] To date
little evidence for edge-to-face bonding between
benzene and imidazole rings appears to have been
been reported. However, the preferred binding
orientations of dibenzofuran 1c with TDO shown in
Figure 3, are consistent with edge-to-face interactions
between the benzene ring, closer to dioxygen and the
phenyl ring of Phe-216, and also with the imidazole
ring of His-222.
occur preferentially at the 1,2-bond, to yield the
(1R,2S) enantiomer 2c, based on the higher binding
energy (-5.7 kcal·mol-1, 1c1). This docking study of
dibenzofuran 1c also predicted a less favoured
alternative orientation, based on the lower binding
energy (-5.0 kcal·mol-1, 1c2). However, with closer
proximity between the 4,4a-bond of substrate and
dioxygen (3.2 Å) could result in angular docking,
leading to the formation of a transient cis-dihydrodiol
metabolite 4c, ring opening to a catechol and further
biodegradation products. No docking evidence was
found to predict lateral cis-dihydroxylation (using
TDO) at the 3,4-bond of substrate 1c, to yield cis-
dihydrodiol 3c. This was presumed to be due to the
larger distance of the 3,4-bond from the bound
dioxygen molecule.
The differences in stereo- and regio-
selectivity between TDO and NDO can be explained
by the differing volumes of their active sites. The
overall number and distances between amino acids is
larger in NDO (ca. 20 amino acids, 11-15 Å) than in
TDO (17 amino acids, ca. 8-13 Å) (Figure SI 1).
Furthermore, CASTp calculations suggested that the
volume of the NDO active site (92.987) is 130%
larger than the TDO active site (40.140).[9c] The larger
capacity of the NDO active site, would allow both
binding orientations to facilitate formation of the
isolated cis-dihydrodiols 2c and 3c while the smaller
dimensions of the TDO active site, would favour
only the formation of cis-dihydrodiol 2c.
Biotransformations of dibenzofuran 1c
(catalyzed by ring hydroxylating dioxygenases with
larger active sites compared to TDO) yielded both
cis-dihydrodiols 2c and 3c along with other
metabolites, which were consistent with the initial
formation of transient angular cis-dihydrodiol 4c.[8a-8c]
Both NDO-catalysed biotransformations (P. putida
9816-11 and P. fluorescens TTC1 mutant strains) of
dibenzofuran 1c gave metabolites cis-dihydrodiol 2c
(60%) and cis-dihydrodiol 3c (40%).[6c,6d]
(iii) TDO-docking of carbazole 1e: The
hydrophobic and edge-to-face interactions and
preferred TDO binding orientation of carbazole 1e
(Figure 5, 1e1, -5.3 kcal·mol-1) are very similar to that
shown for the predicted lateral cis-dihydroxylation of
dibenzofuran 1c (Figure 3, 1c1, -5.7 kcal·mol-1) and
would lead to the formation of (4R,3S)-cis-
dihydrodiol 2e. NDO-catalyzed biotransformation of
carbazole 1e had been reported to give phenol 5e as
the only isolated metabolite.[6a] NDO-docking
orientations are shown in Figure SI 2. It was
postulated that phenol 5e could result from
dehydration of the unstable metabolite cis-
dihydrodiol 2e. Although a lower binding energy was
found for this TDO docking orientation (1e2, -4.6
kcal·mol-1), the shorter distances between proximate
dioxygen and 3,4-bond of substrate (3.1 Å) could
facilitate the angular cis-dihydroxylation of carbazole
1e, yielding the transient metabolite 4e with a
(1S,9aR) configuration. The formation of metabolite
4e was proposed during the CARDO-catalysed
biotransformation of carbazole 1e; it was assumed to
undergo further metabolism and ring opening, to
Recent computational modelling studies of
NDO with arene substrates have been reported.[9a,9b]
The current docking study of dibenzofuran 1c with
NDO, using Autodock Vina, produced two
orientations leading to the prediction that cis-
dihydroxylation and higher binding energies would
result in the formation of metabolites (1R,2S)-2c
(Figure 4, 1c3, -8.8 kcal·mol-1) and (3S,4S)-3c (Figure
4, 1c4, -8.5 kcal·mol-1). The predicted relative ratio
and absolute configurations of these major and minor
metabolites were in accord with the isolated
bioproducts 2c and 3c.[6c,6d]
The primary interactions leading to this
orientation of substrate 1c (Figure 3, 1c1 and 1c2)
were very similar to those found with biphenylene 1b
(Figure 2). Thus, one benzene ring was observed to
interact with the proximate hydrophobic amino acids
(Ile-276, Leu-272, Ile-324, Val-309, Leu-272 Phe-
352). Edge-to face interactions between phenyl rings
have been widely reported in reviews, but much less
information is available on similar interactions
between phenyl groups and five membered
yield a range of products including anthranilic acid.[8a,
8d]
4
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10.1002/adsc.201900147
Advanced Synthesis & Catalysis
1c3
1c4
His-208 Asn-201
His-213 Val-209
Asp-205
Trp-359
Leu-227
Ala-206
Phe-202
Phe-352
Val-260
Phe-224
Leu-307
Asn-297
Ser-310
His-295
Leu-253
Figure 4: In silico docking orientations of dibenzofuran 1c within the active site of NDO.
1c3: -8.8 kcal·mol-1; 1c4: -8.5 kcal·mol-1.
1e1
1e2
Figure
5.
In
silico
docking orientations
of carbazole
within
the
active
site
of TDO.
1e1: -5.3 kcal·mol-1; 1e2: -4.6 kcal·mol-1.
However, the low free binding energy (1d2, -2.9
kcal·mol-1) associated with this orientation would not
facilitate the formation of sulfoxide 5d or the
transient angular cis-dihydrodiol 4d. It is noteworthy
that while transient angular cis-diols 4c and 4e were
postulated as initially formed intermediates during
CARDO-catalysed dihydroxylation of dibenzofuran
1c and carbazole 1e, formation of the corresponding
(iv) TDO docking of dibenzothiophene 1d:
Docking of dibenzothiophene 1d with TDO (Figure
6) showed one favoured orientation (1d1, -5.0
kcal·mol-1) that was similar to those found using
biphenylene 1b (Figure 2), dibenzofuran 1c (Figure
3) and carbazole 1e (Figure 5). These orientations
were consistent with lateral cis-dihydroxylation
occurring, preferentially, at the 1,2-bond to form the
major metabolite (1R,2S)-2d enantiomer. The sulfur
atom in dibenzothiophene 1d was closer to the
nearest oxygen atom (3.3 Å) within the TDO active
site (Figure 6, 1d2) compared with NDO (4.0 Å,
Figure SI 3, 1d3) leading to the prediction that
sulfoxidation might occur more readily with TDO.
angular cis-diol 4d of dibenzothiophene 1d was not
[8a]
proposed.
NDO docking of dibenzothiophene
1d (Figure SI 3) led to the prediction that cis-
dihydrodiols 2d and 3d would be equally preferred
metabolites based on binding energies (1d3, -8.5
kcal·mol-1; 1d4, -8.5 kcal·mol-1). In practice, cis-
5
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10.1002/adsc.201900147
Advanced Synthesis & Catalysis
dihydrodiol 2d was the main metabolite (85-95%),
along with cis-dihydrodiol 3d (<5%) and sulfoxide
5d (<15%) as minor products from the NDO-
catalysed oxidation of dibenzothiophene 1.[6c,6d]
1d1
1d2
Figure 6: In silico docking orientations of dibenzothiophene 1d with TDO. 1d1: -5.0 kcal·mol-1; 1d2: -2.9 kcal·mol-1.
(Scheme 3). cis-Dihydrodiol 2c was the only
metabolite detected using E. coli TOP10109 (pCL-
41) and E. coli 1/210LK recombinant cells, both
expressing TDO. While lower yields were obtained
with these recombinant strains, they provided further
evidence that TDO was the biocatalyst responsible
for the cis-dihydroxylation of this tricyclic arene.
(v)
TDO-catalyzed
biotransformation
of
dibenzofuran 1c: Medium-scale biotransformations
(P.putida UV4) of dibenzofuran 1c (400-500 mg/L)
were conducted in 2 litre baffled flasks, employing a
slight modification of reported conditions.[2,3c,5b] At
pH below 7.5, very little evidence of cis-dihydrodiol
2c formation was observed (Scheme 3). Maintaining
the pH within the range 7.5-8.5 significantly reduced
the dehydration of metabolite 2c, to yield mainly
phenol 5c and enabled yields of up to 90% (LC-MS
analysis). Metabolite 2c was initially isolated in an
average yield of ca. 40%, but using an extended
biotransformation period (48 h) at pH 8.0 and a
careful workup procedure an optimal isolated yield of
85% was achieved.
Neither enantiopurity values nor absolute
configurations were established for metabolites
isolated during the NDO-catalysed (P. fluorescens
TTC1) biotransformation studies of dibenzofuran
1c.[6d] cis-Dihydrodiols 2c and 3c obtained using P.
putida 9816-11 were found to have >95% ee, by
treatment
with
(S)-
and
(R)-2(1-
1
methoxyethyl)phenylboronic acid (MPBA) and H-
NMR analysis of the resulting diastereomeric
boronate derivatives. A consistent trend in directional
chemical shifts of Me and MeO signals for MPBA
During
earlier
NDO-catalysed
biotransformations of dibenzofuran 1c at pH 7.0-7.3,
a range of metabolites was formed.[6b,6d] Among the
mixture, that proved difficult to separate, were cis-
dihydrodiols 2c and 3c and the corresponding
phenols, resulting from their partial dehydration. In
contrast to the very stable cis-diol metabolite 2b,
formed from biphenylene 1b, cis-dihydrodiol 2c was
found to be more susceptible to dehydration at ca. pH
7, yielding mainly 2-hydroxydibenzofuran 5c and 1-
hydroxydibenzofuran 8c as a very minor product
derivatives,
of
other
cis-diol
boronate
diastereoisomers, of known absolute configurations,
was also observed for cis- diols 2c and 3c; on this
basis, metabolite 2c was assigned a (1R,2S)
configuration, although no optical rotation values
([α]D) were reported for metabolites 2c and 3c. [6b] The
MPBA method was generally found to
6
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10.1002/adsc.201900147
Advanced Synthesis & Catalysis
Scheme 3. Reactions of cis-dihydrodiol 2c to yield cis-tetrahydrodiol 6c, diMTPA esters 7c(S), 7c(R), bromohydrin 9c and
diol epoxide 10c.
be very useful in assigning both ee values and
absolute configurations to most arene cis-
dihydrodiols.[11a]
cis-Dihydrodiol 2c was initially detected by
phase LC-MS analysis of the
reverse
biotransformation supernatant aqueous solution and
was identified by a large peak eluting after 18 min. In
some of the repeat biotransformations of
dibenzofuran 1c, evidence of a very minor metabolite
cis-Dihydrodiol metabolite 2c isolated during the
current study, was hydrogenated (Pd/C, MeOH) to
yield cis-tetrahydrodiol 6c (90 % yield). Reaction
with (+) and (-)-MTPA chloride gave the
corresponding diastereomeric (+)-7c (R) diMTPA
ester- and (-)-7c (S) - diMTPA ester (Scheme 3).
NMR analysis of the diMTPA esters showed that the
metabolite was enantiopure. This method had been
used earlier to determine both ee values and absolute
+
eluting after 12 min., with a mass of [M+NH4 ] =
236.09150, was also observed (SI: Figure 17). Since
2-hydroxydibenzofuran 5c is readily produced by
partial decomposition of cis-dihydrodiol 2c, during
the biotransformation, the possibility that this very
minor bioproduct could be 8-hydroxydibenzofuran-
1,2-diol 11c (calculated mass: 236.09173), was
considered (Scheme 4). Biotransformation of phenol
5c, to yield the possible metabolite 11c, resulted in a
significant proportion of the substrate being
recovered, however, LC-MS analysis of the
supernatant again detected the presence of this
metabolite in very low yield.
configurations of other cis-diol metabolites, derived
[11b]
from polycyclic arenes.
The diMTPA method
showed that the cis-dihydrodiol 2c ([α]D +217) was
enantiopure (>98% ee) and was assigned a (1R,2S)
configuration.
As part of a preliminary study of the potential of cis-
dihydrodiol 2c as a chiral synthon in natural product
synthesis, it was considered appropriate to establish
its absolute configuration by X-ray crystallography.
cis-Diol epoxide derivative 10c was synthesized in
two steps from cis-dihydrodiol 2c, via trans
bromohydrin intermediate 9c (Scheme 3). X-ray
crystallographic analysis of a suitable crystal (from
Et2O-hexane) of epoxide 10c established its absolute
configuration as (1aS,2R,8R,8aR) (Figure 7). The
(1R,2S) configuration for cis-dihydrodiol 2c,
presumed by the consistent trends in chemical shifts
of specific signals in NMR spectra of the MPBA and
1
The H NMR (400 MHz) spectrum of a late eluting
impure fraction, collected during the purification
(column
chromatography)
of
the
crude
biotransformed material obtained from dibenzofuran
1c, showed most of the characteristic cis-dihydrodiol
signals:  = 4.88 (1H, d, J = 6.9, 2.5 Hz, H-1), 5.96
(1H, ddd, J = 10.3, 2.5, 1.3 Hz, H-4), 6.43 (1H, dd, J
= 10.0, 2.8 Hz, H-3), which were close to but distinct
from those observed for the major cis-dihydrodiol
2c. Attempts to purify it further by PLC resulted in its
decomposition. In an effort to obtain more evidence
for the formation of hydroxydiol 11c, molecular
docking of phenol 5c with TDO was conducted
(Figure SI 4). The results indicated a preferred
orientation that would lead to the formation of
phenolic cis-dihydrodiol 11c with an identical
absolute configuration to that found in dibenzofuran
cis-dihydrodiol 2c, but with a low free binding energy
(-3.1 kcal·mol-1). Based on the limited LC-MS and
NMR data available an molecular docking
diMTPA
derivatives,
was
therefore
confirmed.[6c,11a,11b]
Figure 7. X-ray crystal structure of diol epoxide 10c.
7
This article is protected by copyright. All rights reserved.

10.1002/adsc.201900147
Advanced Synthesis & Catalysis
Scheme 4: Possible metabolic pathways during TDO - catalyzed biotransformations of dibenzofuran 1c using P. putida
UV4.
evidence, the possibility that biotransformation of
dibenzofuran 1c can occasionally yield both cis-
dihydrodiols 2c and 11c, via the metabolic pathway
shown in Scheme 4, cannot be excluded. The TDO-
catalysed formation of cis-dihydrodiols from
monocyclic phenols, e.g. compounds 2a and 3a from
substrate 1a was established earlier.[2, 3c, 3e]
crystalline product whose NMR spectrum confirmed
that the major metabolite was anthranilic acid 8e. The
isolation of bioproduct 8e and detection of 3-
hydroxycarbazole 5e (Scheme 5), were consistent
with the predicted formation of the corresponding
transient cis-diols 2e and 4e, via the other undetected
intermediates 6e and 7e (SI: Figure 2, 1e1, 2e2). The
isolation of cis-diol 2b and anthranilic acid 8e from
biotransformations of biphenylene 1b and carbazole
1e respectively, provided support for in silico-
predicted angular cis-dihydroxylations being
catalysed by TDO.
(vi) TDO-catalyzed biotransformation of
carbazole 1e: Biotransformations of carbazole 1e
had been reported using, P. putida 9816/11 (NDO
source), E. coli JM109(pKK223-3) (NDO source)
and Sphingobium yaniokuyae B8/36 (BPDO
source).[6a] No metabolites were identified from the
attempted biotransformation of carbazole 1e using P.
putida F39/D (TDO source), but NDO- and BPDO-
catalysed oxidations resulted in the formation of a
single metabolite, which was identified as 3-
hydroxycarbazole 5e.[6a] While the possibility of a
direct enzymatic monooxygenation was not excluded,
an alternative lateral cis-dihydroxylation mechanism
was proposed, through the formation of an unstable
cis-dihydrodiol 2e, followed by its spontaneous
dehydration (Scheme 5). A transient angular cis-
dihydrodiol metabolite 4e, resulting from a CARDO-
(vii) TDO-catalyzed biotransformation of
dibenzothiophene 1d: cis-Dihydrodiols, 2d, 3d and
sulfoxide 5d were identified as the major NDO-
catalysed metabolites of dibenzothiophene 1d, using
P. putida 9816-11, E. coli JM109(DE3)(pDTG141)
and P. fluorescens TTC1 cells. The enantiopurity
(>95%) of cis-diol 2d was determined by formation
of chiral boronate derivatives. [6c,6d]
While neither ee value nor absolute
configuration of the minor cis-dihydrodiol metabolite
3d (>5% yield) was reported, results from the NDO
docking study of dibenzothiophene 1d (SI Figure: 3
1d3, 1d4) suggest that metabolite 3d should be
enantiopure and have a (3S,4S) configuration.[6c,6d]
catalysed
(Pseudomonas
sp.CA10)
cis-
dihydroxylation of carbazole 1e was postulated to
account for the formation of anthranilic acid 8e as a
metabolite formed via catechol 6e and ketocarboxylic
acid 7e intermediates.[8a]
A small-scale (50 mg) biotransformation (P.
putida UV4) of dibenzothiophene 1d, under the
optimized conditions developed for dibenzofuran 1c,
was carried out. LC-MS analysis of the aqueous
supernatant showed the presence of a single
metabolite, which was identified as cis-dihydrodiol
2d at a retention time of 19.1 min. The limited
quantity of metabolite 2d available, following PLC
As expected, repeat biotransformations (P.
putida UV4) of carbazole 1e showed no evidence of
the predicted transient cis-dihydrodiol 2e. GC-MS
analysis of the crude extract, did however indicate
that anthranilic acid 8e was the major identifiable
metabolite. 3-Hydroxycarbazole 5e was also detected
but only as a very minor dehydration product (SI:
Figure 16), presumably derived from the unstable cis-
dihydrodiol 2e. Extraction (EtOAc) of bioproducts of
carbazole 1e, followed by PLC purification, yielded a
1
purification, was just sufficient to record H NMR
13
and C NMR spectra, high resolution MS data and a
crude optical rotation value. Comparison with the
literature data, based on the consistent trend in
chemical shifts observed for chiral
8
This article is protected by copyright. All rights reserved.

10.1002/adsc.201900147
Advanced Synthesis & Catalysis
Scheme 5: Possible metabolic pathways during TDO - catalyzed biotransformations of carbazole 1e using P. putida UV4.
boronates formed using (R) and (S) MPBA, was used
to assign the (1R,2S) absolute configuration to
to dioxygen within the active site can affect both the
binding and arene cis-dihydroxylation, rendering the
lateral or angular bond more reactive.
metabolite (+)-cis-dihydrodiol 2d.[6c]
As this
method, provided the correct absolute configuration
to the dibenzofuran cis-dihydrodiol (+)-2c during the
current study, the (+)-(1R,2S)-dihydrodiol 2d
assignment it was again presumed to be correct.
Conclusion
Application of the molecular docking
program to the TDO-catalysed cis-dihydroxylation of
the tricyclic arene substrates (1b-1e), led to the
prediction of preferred structures and absolute
configurations of isolated lateral (2c, 2d) and angular
(2b) cis-dihydrodiols and transient cis-dihydrodiols
(2e, 4e). In silico predicted results from TDO- and
NDO-catalysed cis-dihydroxylation of substrates 1c-
1e, were compared with literature results obtained
using NDO and new experimental results obtained
using TDO. The P. putida UV4 strain expressing
TDO, was selected for biotransformations to confirm
the
cis-Dihydrodiol 2d was isolated in an unoptimized
yield of 16% using TDO while a yield of 44% was
reported using NDO.[6c] It was noteworthy that a
much higher yield of cis-dihydrodiol 2c (>85%) was
obtained during the current programme. The
increased water solubility of the dibenzofuran 1c (3.1
mg L-1) compared with dibenzothiophene 1d (1.5 mg
L-1) could be an important factor in the higher yield
of metabolite 2c. Furthermore, as cis-dihydrodiol 2d
was the only detected metabolite, the task of
separation of metabolites encountered using NDO
was not required.[6c,6d] The predicted stereochemistry
from molecular docking of dibenzothiophene 1d with
TDO was in agreement with the assigned absolute
configuration of the cis-dihydrodiol 2d (Figure 5).
Dioxygenase-catalysed sulfoxidation of thiophenes
was reported using both TDO and NDO enzymes.[12]
Sulfoxide 5d was also found as a minor metabolite of
dibenzothiophene 1d in one of the studies using
NDO.[6c,6d] The very low free binding energy
associated with the preferred orientation of
dibenzothiophene 1d with TDO (Figure 6, 1d2 ), may
be an important factor in sulfoxide 5d not being
detected
during
the
TDO-catalyzed
biotransformation.
Scheme 6: Structures of secondary metabolites 3f, 4f, 3g,
4g, 13c and 14c obtained by chemoenzymatic synthesis
from cis-dihydrodiol metabolites 2f, 2g, 2c and tricyclic
arene substrates 1f, 1g and 1c.
Several reasons could account for in silico-
predicted metabolites not being observed
experimentally. These include product instability due
to decomposition, further metabolisation, alternative
oxidation pathways, toxicity and water-solubility. In
the case of TDO, the structure and physical properties
of the substrate, its free binding energy and proximity
Autodock Vina docking predictions. This resulted in
the isolation of the relatively stable cis-dihydrodiols
2b, 2c and 2d, and identification of metabolites 5e
and 8e, derived from the unstable carbazole cis-diols
9
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10.1002/adsc.201900147
Advanced Synthesis & Catalysis
analyses. Preparative layer chromatography (PLC)
separations of metabolites were carried out on glass plates
(20 cm x 20 cm) coated with Merck Keiselgel PF254/366
silica gel (21 g silica gel in 62 mL water).
2e and 4e respectively. The direct formation of stable
angular cis-diol 2b, and anthranilic acid 8e, via the
unstable angular cis-diol 4e, were in accord with in
silico predictions. These results also provide support
for the view that TDO could be responsible for both
lateral and angular cis-dihydroxylation of tricyclic
arenes.
Molecular Docking and in silico analysis
The protein models were obtained from the protein data
bank. TDO pdb id: 3en1; NDO pdb id: 1o7n. The crystal
structure of TDO does not contain bound dioxygen.
Dioxygen was inserted into the 3en1 model by
superimposing the iron centre of NDO, including the
aspartate and the two histidine residues, onto the iron
centre of TDO, using the “super” function of PyMOL (The
PyMOL Molecular Graphics System, Version 2.2.0
Schrödinger, LLC.). The dioxygen was extracted from the
superposition and combined with the 3en1 model.
The TDO-catalysed biotransformation of
dibenzofuran 1c was optimized, to obtain cis-
dihydrodiol 2c as the main metabolite in enantiopure
form and good yield. The (1R,2S) absolute
configuration of cis-dihydrodiol 2c was confirmed by
a combination of chemoenzymatic synthesis with
NMR analysis of chiral boronate and diMTPA esters
and X-ray crystallographic analysis of the derived
tricyclic (1aS,2R,8R,8aR)-diol epoxide 9c.
Molecular docking was performed using the AutoDock
Vina suite, version 1.1.2.[4] The docking site was set to
include all amino acids within 6 Å of the substrate.
TDO: center_x = 46.46, center_y = 120.49, center_z =
201.877; size_x = site_y = size_z = 16.
To date nearly all cis-dihydrodiols used in the
chemoenzymatic synthesis of natural products have
been derived from monocyclic arenes.[1f] The BPDO-
catalysed cis-dihydroxylation of acridine (1f) and
dictamnine (1g) was among the few examples where
tricyclic arene cis-dihydrodiols (2f and 2g) were used
NDO: center_x = 13.375, center_y = 49.781, center_z =
82.071; size_x = 20, size_y = 18, size_z = 18.
All docking was performed with an exhaustiveness of 100.
Autodock Vina automatically grouped similar orientations
and produced each result with a respective calculated
binding energy. Each docking yielded several possible
orientations, which were examined critically. The
orientations were then examined for their viability as a
substrate based on their calculated binding energy,
orientation and interaction/distance to the nearest residues.
in the chemoenzymatic
synthesis of secondary
metabolites including alkaloids (Scheme 6).[13] The
bacterial cis-dihydrodiol metabolites 2f and 2g were
used in the chemoenzymatic synthesis of (i)
mammalian
metabolites,
e.g.
arene
oxide
intermediate 3f and isolated trans-dihydrodiol 4f and
(ii) plant metabolites, e.g. intermediate arene oxide
3g and isolated furoquinoline alkaloid 4g. The
chemoenzymatic synthesis of the tricyclic fungal
metabolites, e.g. Ribisin A 13c and Ribisin B 14c
The volume of the active site was calculated using the
Computed Atlas of Surface Topography of proteins
(CASTp) 3.0.[9c] Submission of the whole proteins resulted
in calculated binding pockets larger than intended.
Therefore, the amino acids and iron centre of the active site
were submitted as a separate file.
from
a
monocyclic bacterial cis-dihydrodiol
metabolite of bromobenzene was reported.[14a-c]
Utilizing tricyclic cis-dihydrodiol 2c as a chiral
synthon, the chemoenzymatic synthesis of Ribisins
13c and 14c, is currently in progress.
Job IDs:
NDO full: j_5c76b7f48839b (active site: pocket 3)
NDO active site with water: j_5c76becff050b
NDO active site without water: j_5c76bea6a2319
TDO full: j_5c6d8d0808b18 (active site: pocker 6)
TDO active site: j_5c77a83860d0a
Experimental Section
NMR spectra were recorded on Bruker Avance 400,
General Electric QE-500, and Bruker AV-600 instruments.
Chemical shifts ( ) are reported in ppm relative to SiMe4
and coupling constants (J) are given in Hertz (Hz). Optical
rotations ([α]D) measurements were carried out on a
Perkin-Elmer 214 polarimeter. LC-TOF-MS analyses were
conducted on an Agilent 1100 series HPLC coupled to an
Agilent 6510 Q-TOF and a reverse phase column (Agilent
Eclipse Plus C18, 5 mm, 150 x 2.1 mm). Mass spectra (ES)
were recorded on an LCT Premier Mass Spectrometer.
Accurate molecular weights were obtained by the peak
matching method, using heptacosafluorotributylamine as
the standard reference and were accurate to within ±5x10-6
a.m.u. Melting points were recorded in degrees Celsius
using a Stuart SMP10 melting point apparatus. Merck
Kieselgel 60F254 analytical plates were used for TLC
Microbiological Methods
All biotransformations were performed using P. putida
UV4 whole cells, unless mentioned otherwise. The P.
putida UV4 bacteria were initially streaked on Luria-
Bertani medium (LB) plates and incubated for 24 h at 30ºC.
Individual colonies were then transferred to MSM plates
containing indole (0.0012% w/v 1 mM). Single colonies
were transferred to two minimal salt medium (MSM) agar
plates with 0.2% sodium pyruvate, one containing 1 mM
indole. Colonies on the indole containing plate were
visually selected for activity after 24 h of incubation at
30ºC, based on indigo (blue colour) being produced.
10
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10.1002/adsc.201900147
Advanced Synthesis & Catalysis
Cultures grown on the MSM plates were consistently
checked for TDO activity and absence of dehydrogenase
activity, using a 100 mL MSM culture, and 50 µL of
chlorobenzene. Formation of the cis-dihydrodiol
metabolite from chlorobenzene was followed by measuring
the UV absorbance at 295 nm.
(100 MHz, CDCl3):  = 155.4, 155.2, 127.3, 123.9, 122.9,
118.9, 113.7, 111.1, 69.4, 68.8, 26.2, 21.2. LRMS (EI): m/z
= 204 (M+, 10), 186 (56), 121 (100).
(1R,2S)-1,2-Di-[(2R)-2-methoxy-2-phenyl-2-
trifluoromethylacetoxyl]-1,2,3,4-
tetrahydrodibenzofuran 7c(R)
Biotransformations were carried out using MSM cultures
(500 mL, pH 7.5), containing 10 g/L glucose, and were
inoculated in 2000 mL baffled Erlenmeyer flasks with an
active colony from the MSM plate not containing indole.
The cultures were grown overnight by shaking at 140 rpm,
30oC. The tricyclic arenes (500 mg/L) and carbon sources
(4 g/L) were added to each 500 mL of culture medium in 2
L baffled shake flasks, which were incubated at 30oC, on
an orbital shaker (140 rpm) for 24 - 48 h and maintaining
the pH at 8. Samples were taken every 24 h and
immediately frozen for LC-MS analysis. The crude bio-
mixture was centrifuged (7000 rpm, 15 min, small scale)
and the aqueous supernatant solution was then decanted off.
The aqueous solution was concentrated in vacuo,
maintaining the water bath temperature at 40ºC. The
residue was extracted with ethyl acetate (3 x 100 mL) and
concentrated in vacuo to yield the crude products.
Diesterification of cis-tetrahydrodiol 6c with (+)-MTPA
chloride/pyridine yielded the diMTPA ester 7c(R) as a
colourless oil, which was purified by PLC (15% ether in
hexane, []D: +39.5 (c
0.7, CHCl3); (Found: M+
636.15813, C32H26O7F6 calcd. for 636.15827); 1H NMR
(500 MHz, CDCl3):  = 2.27 (1H, m, 3-H), 2.46 (1H, m,
3-H), 2.99 (2H, m, 4-H), 3.27 (3H, s, OMe), 3.54 (3H, s,
OMe), 5.57 (1H, m, 2-H), 6.50 (1H, d, J = 2.8 Hz, 1-H),
7.11 (2H, m, ArH), 7.22-7.31 (7H, m, ArH), 7.39-7.45 (3H,
m, ArH), 7.55 (1H, m, ArH) and 7.62 (1H, m, ArH);
LRMS (EI): m/z = 636 (M+, 35), 403 (56), 170 (100).
(1R,2S)-1,2-Di-[(2S)-2-methoxy-2-phenyl-2-
trifluoromethylacetoxyl]-1,2,3,4-
tetrahydrodibenzofuran 7c(S)
Colourless oil; []D: -33.7 (c 0.9, CHCl3); (Found: M+
Biotransformation of dibenzofuran 1c to give (1R,2S)-
1
636.15854, C32H26O7F6 calc. for 636.15827); H NMR
1,2-dihydrodibenzofuran-1,2-diol 2c [6c,6d]
(500 MHz, CDCl3):  = 2.15-2.26 (2H, m, 3-H), 2.98 (2H,
m, 4-H), 3.28 (3H, s, OMe), 3.57 (3H, s, OMe), 5.59 (1H,
m, 2-H), 6.68 (1H, d, J = 3.4 Hz, 1-H), 7.04 (2H, m, ArH),
7.21-7.46 (10H, m, ArH) and 7.51 (2H, m, ArH); LRMS
(EI): m/z = 636 (M+, 48), 403 (64), 170 (100).
cis-Dihydrodiol 2c was isolated as a metabolite from a
biotransformation of dibenzofuran 1c (250 mg, 1.49 mmol)
by extraction (3 x 100 mL EtOAc). The extract was
concentrated in vacuo to give metabolite 2c as a white
solid (250 mg, 84%); m. p. 96-98C (EtOAc / hexane); Rf
= 0.25 (30% EtOAc in hexane); [α]D: +217 (c 0.6, CHCl3);
(Found: C, 71.8, H 5.2%; C12H10O3 requires C, 71.3; H,
5.0%); HRMS (ES): (M+Na)+: 225.05222, calcd. for
Biotransformation of dibenzothiophene 1d to give
(1R,2S)-1,2-Dihydrodibenzothiophene-1,2-diol 2d [6c,6d]
cis-Diol 2d was isolated as a metabolite from a small-scale
biotransformation of dibenzothiophene 1d (50 mg, 0.27
mmol) by extraction (3 x 100 mL EtOAc). The extract was
concentrated in vacuo and the concentrate purified by PLC
to give cis-diol 2d as a white solid (9.4 mg, 16%). Rf =
0.25 (30% EtOAc in hexane); [α]D: +51 (c 0.2, MeOH);
HRMS (ES): [M+Na]+: 241.0296, C12H10O2SNa calcd. for
241.0299; 1H NMR (600 MHz, CDCl3):  = 4.68 (1H, dt, J
= 5.4, 2.6 Hz, 2-H), 4.99 (1H, dd, J = 5.9, 0.7 Hz, 1-H),
6.06 (1H, ddd, J = 9.5, 2.7, 0.7 Hz, 4-H), 6.53 (1H, dd, J =
9.7, 2.5 Hz, 3-H), 7.33 (1H, dt, J = 7.2, 1.2 Hz, 8-H), 7.42
(1H, dt, J = 7.2, 1.0 Hz, 7-H), 7.82 (1H, d, J = 8.1 Hz, 6-H),
7.91 (1H, d, J = 8.1 Hz, 9-H); 13C NMR (150 MHz,
CDCl3):  = 139.7, 137.9, 135.8, 132.2, 129.2, 125.0,
124.7, 122.8, 121.4, 120.7, 70.6, 65.5.
1
C12H10O3Na: 225.052058; H NMR (400 MHz, CDCl3): 
= 1.92 (1H, br s, OH), 2.78 (1H, br s, OH), 4.67 (1H, b m,
2-H), 4.96 (1H, d, J = 6.3 Hz, 1-H), 6.05 (1H, ddd, J =
10.0, 2.5, 0.7 Hz, 4-H), 6.51 (1H, dd, J = 10.0, 2.3 Hz, 3-
H), 7.27 (2H, m, 7-H and 8-H), 7.62 (1H, m, 9-H), 7.45
(1H, m, 6-H); 13C NMR (100 MHz, CDCl3):  = 155.1,
152.0, 134.3, 126.5, 124.6, 123.6, 119.2, 117.4, 113.5,
111.7, 70.8, 65.1; LRMS (EI): m/z = 202 (26),184 (100).
(1R,2S)-1,2-Dihydroxy-1,2,3,4-tetrahydrodibenzofuran
6c
A solution of cis-dihydrodiol metabolite 2c (100 mg, 5
mmol), in MeOH (15 mL) containing 10% Pd/C (5 mg)
was stirred (3 h) in an atmosphere of hydrogen under
normal pressure. The catalyst was filtered off, the filtrate
concentrated under reduced pressure, and the crude
product crystallized from CHCl3 to furnish the
hydrogenated tetrahydrodiol 6c as a white powder (91 mg,
90%); m. p. 96-97C (CHCl3/hexane); []D: +37.3 (c 0.7,
CHCl3); (Found: C, 70.8, H, 6.3%; C12H12O3 requires C,
Biotransformation of carbazole 1e to give anthranilic
acid 8e and 2-hydroxycarbazole 5e[6a]
A small scale biotransformation of carbazole 1e (50 mg,
0.30 mmol) followed by EtOAc extraction of the aqueous
supernatant on concentration gave the crude bioproduct
mixture. GC-MS analysis (Agilent Technologies Ultra 2 12
m x 0.2 mm i.d. x 0.33 μm column) of the crude extract
showed the presence of a major peak (9.5 min.) which was
identified as that of anthranilic acid 8e (MW 137), by
comparison with an authentic standard and also formation
1
70.6, H, 5.9%); H NMR (500 MHz, CDCl3):  = 2.05
(1H, m, 3-H), 2.17 (1H, m, 3´-H), 2.80 (1H, m, 4-H), 2.94
(1H, dt J = 17.0, 5.2 Hz, 4´-H), 4.07 (1H, dt, J = 9.9, 3.4
Hz, 2-H), 4.98 (1H, d, J = 3.9 Hz, 1-H), 7.27-7.34 (2H, m,
ArH), 7.43 (1H, m, ArH), 7.62 (1H, m, ArH); 13C NMR
11
This article is protected by copyright. All rights reserved.

10.1002/adsc.201900147
Advanced Synthesis & Catalysis
of the trimethylsilyl derivative. A very minor peak (16.6
min.) was identified as 3-hydroxy carbazole 5e (MW 183:
m/z 183, 154, 127, 92, 77); its mass fragmentation pattern
was identical to that reported in the literature. PLC
purification (35% EtOAc in hexane, Rf = 0.25) of the crude
mixture yielded (6.4 mg) pure crystalline anthranilic acid
8e.
Crystal Data: C12H10O4, M = 218.2: orthorhombic, space
group P212121 (no.19), a =5.56920(10), b = 7.35790(10),
c = 23.3201(5) Å, V = 955.60(3) Å3, Z = 4, T = 100.0(2) K,
Dcalc = 1.361 g/cm3,
10979 reflections measured
(12.616° ≤ 2Θ ≤ 144.426°), 1878 unique (Rint = 0.0236,
Rsigma = 0.0138) which were used in all calculations. The
final R1 was 0.0395 (I > 2σ(I)) and wR2 was 0.0962 (all
data).
(1R,2R,3S,4S)-3-Bromo-1,2,3,4-
tetrahydrodibenzo[b,d]furan-1,2,4-triol 9c
Acknowledgements
To a solution cis-dihydrodiol 2c (27 mg, 0.13 mmol) in
THF-H2O (4:1, 3 mL) was added NBS (40 mg, 0.22 mmol)
and the mixture was stirred overnight at room temperature.
After addition of Na2S2O5 (10 mg), the mixture was stirred
for 10 min., diluted with EtOAc (10 mL) and washed with
brine (5 mL). The organic phase was separated, dried
(MgSO4), and concentrated in vacuo. The crude material,
was purified by flash column chromatography (silica gel,
30% EtOAc in hexane) to give bromohydrin 9c as a purple
solid (27 mg, 68%); Rf = 0.22 (40% EtOAc in hexane);
[α]D: -33 (c 0.83, MeOH); 1H NMR (400 MHz, CD3OD): 
= 3.91 (1H, ddd, J = 10.3, 3.9, 0.9 Hz, 2-H), 4.35 (1H, ddd,
J = 10.3, 7.6, 0.7 Hz, 3-H), 4.96 (1H, d, J = 7.6 Hz, 4-H),
5.05 (1H, d, J = 3.9 Hz, 1-H), 7.26 (1H, dt, J = 7.6, 0.9 Hz,
8-H), 7.32 (1H, dt, J = 7.5, 1.0 Hz, 7-H), 7.48 (1H, dm, J =
8.0 Hz, 6-H), 7.65 (1H, dm, J = 7.5 Hz, 9-H); 13C NMR
(150 MHz, CD3OD):  = 157.3, 154.3, 128.1, 126.2, 124.4,
121.2, 116.7, 112.6, 73.5, 71.6, 65.2, 59.1.
We thank the EPSRC (CMG) and the Leverhume Trust (to
PH) for postgraduate Studentships and Dr. J. F. Malone
for helpful discussion.
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(1aS,2R,8R,8aR)-1a,2,8,8a-Tetrahydro-
benzo[b]oxireno[2,3-f]benzofuran-2,8-diol 10c
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Sodium methoxide (10 mg, 185 µmol) was added to a
solution of bromohydrin 9c (20 mg, 67 µmol) in THF (3
mL) and the reaction mixture was stirred (3 h) at room
temperature. The salts were removed by gravity filtration
and the filtrate was concentrated in vacuo. Purification of
the residue by flash column chromatography (silica gel,
50% EtOAc in hexane) gave epoxide 10c as a white
crystalline solid (8 mg, 55%); Rf = 0.2 (60% EtOAc in
hexane); HRMS (ES): [M+NH4]+: 236.0868, C12H14NO4
1
calcd. for 236.0923; H NMR (400 MHz, CD3OD):  =
[4]
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O. Trott, A. J. Olsen, J. Comput. Chem., 2010, 31,
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8-H), 7.21 (1H, dt, J = 7.6, 1.3 Hz, 6-H), 7.27 (1H, dt, J =
7.5, 1.5 Hz, 5-H), 7.45 (1H, d, J = 8.0 Hz, 4-H), 7.74(1H,
dd, J = 7.4, 1.2 Hz, 7-H); 13C NMR (100 MHz, CD3OD): 
= 157.0, 150.8, 128.4, 125.4, 123.8, 122.0, 114.3, 112.1,
64.9, 63.5, 57.6, 56.9; IR (KBr) νmax/cm-1 3995, 2956, 2927,
2853, 1781, 1596, 1354, 1181, 1228, 1051.
455-461.
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Crystal data collection was performed on a Rigaku Single
Crystal X-ray diffractometer, equipped with cryostat.
Measurements were carried out at 100 K using a Cu-Κα,
monochromator, λ = 1.54184 Å. Using Olex2, the structure
was solved with the ShelXT structure solution program
using Intrinsic Phasing and further refined with the ShelXL
refinement package using least squares minimisation.
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12
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10.1002/adsc.201900147
Advanced Synthesis & Catalysis
[8]
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D. R. Boyd, N. D. Sharma, B. McMurray, S. A.
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D. R. Boyd, N. D. Sharma, J. G, Carroll, P. L.
Loke, C. R. O’Dowd, C. C. R. Allen, RSC Adv.
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1494. ; c) D. Escudero, C. Estarellas, A. Frontera,
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[15]
CCDC 1894810 contains the supplementary
crystallographic data for this compound that can
be obtained free of charge from the Cambridge
Crystallographic Data Centre via www.ccde.cam.
ac.uk/data.
[11]
a) D. R. Boyd, N. D. Sharma, P. A. Goodrich, J. F.
Malone, G. McConville, J. S. Harrison, P. J.
Stevenson, C. C. R Allen Chirality, 2018, 30, 5-
13
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Advanced Synthesis & Catalysis
FULL PAPER
cis-Dihydroxylation of Tricyclic Arenes and
Heteroarenes Catalyzed by Toluene
Dioxygenase: A Molecular Docking Study
and Experimental Validation
Adv. Synth. Catal. Year, Volume, Page – Page
Derek R. Boyd, Narain D. Sharma, Ian N.
Brannigan, Christopher J. McGivern, Peter
Nockemann, Paul J. Stevenson, Colin
McRoberts, Patrick Hoering and Christopher
C. R. Allen
14
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