Enzyme-Catalysed Synthesis of Cyclohex-2-en-1-one cis-Diols from Substituted Phenols, Anilines and Derived 4-Hydroxycyclohex-2-en-1-ones

Article abstract of DOI:10.1002/adsc.201700711

Toluene dioxygenase-catalysed cis-dihydroxylations of substituted aniline and phenol substrates, with a Pseudomonas putida UV4 mutant strain and an Escherichia coli pCL-4t recombinant strain, yielded identical arene cis-dihydrodiols, which were isolated as the preferred cyclohex-2-en-1-one cis-diol tautomers. These cis-diol metabolites were predicted by preliminary molecular docking studies, of anilines and phenols, at the active site of toluene dioxygenase. Further biotransformations of cyclohex-2-en-1-one cis-diol and hydroquinone metabolites, using Pseudomonas putida UV4 whole cells, were found to yield 4-hydroxycyclohex-2-en-1-ones as a new type of phenol bioproduct. Multistep pathways, involving ene reductase- and carbonyl reductase-catalysed reactions, were proposed to account for the production of 4-hydroxycyclohex-2-en-1-one metabolites. Evidence for the phenol hydrate tautomers of 4-hydroxycyclohex-2-en-1-one metabolites was shown by formation of the corresponding trimethylsilyl ether derivatives. (Figure presented.).

Full text of DOI:10.1002/adsc.201700711

Title: Enzyme-Catalysed Synthesis of Cyclohex-2-en-1-one cis-Diols
and 4-Hydroxycyclohex-2-en-1-ones from Substituted Phenols
and Anilines
Authors: Derek Boyd, N Sharma, Peter Mcintyre, Paul Stevenson,
Colin McRoberts, Amit Gohill, Patrick Hoering, and C Allen
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To be cited as: Adv. Synth. Catal. 10.1002/adsc.201700711
Link to VoR: http://dx.doi.org/10.1002/adsc.201700711

10.1002/adsc.201700711
Advanced Synthesis & Catalysis
FULL PAPER
DOI: 10.1002/adsc.201((will be filled in by the editorial staff))
Enzyme-Catalysed Synthesis of Cyclohex-2-en-1-one cis-Diols
from Substituted Phenols, Anilines and Derived 4-
Hydroxycyclohex-2-en-1-ones
Derek R. Boyd,a,* Narain D. Sharma,a Peter B. A. McIntyre,a Paul J. Stevenson,a,* W.
Colin McRoberts,b Amit Gohil,c Patrick Hoeringc and Christopher C. R. Allenc
a School of Chemistry and Chemical Engineering, Queen’s University of Belfast, Belfast,
BT9 5AG, UK. 44 2890974421 E-mail: dr.boyd@qub.ac.uk or p.stevenson@qub.ac.uk
b Agri-food and Biosciences Institute for Northern Ireland, Belfast, BT9 5PX, 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))
bioproduct. Multistep pathways, involving ene reductase-
Abstract:
Toluene
dioxygenase-catalysed
cis-
and carbonyl reductase-catalysed reactions, were proposed
to account for the production of 4-hydroxycyclohex-2-en-1-
one metabolites. Evidence for the phenol hydrate tautomers
of 4-hydroxycyclohex-2-en-1-one metabolites was shown
by formation of the corresponding trimethysilylether
derivatives.
dihydroxylations of substituted aniline and phenol substrates,
with a Pseudomonas putida UV4 mutant strain and an
Escherichia coli pCL-4t recombinant strain, yielded identical
arene cis-dihydrodiols, which were isolated as the preferred
cyclohex-2-en-1-one cis-diol tautomers. These cis-diol
metabolites were predicted by preliminary molecular
docking studies, of anilines and phenols, at the active site of
toluene dioxygenase. Further biotransformations of
cyclohex-2-en-1-one cis-diol and hydroquinone metabolites,
using Pseudomonas putida UV4 whole cells, were found to
yield 4-hydroxycyclohex-2-en-1-ones as a new type of
phenol
Keywords: aniline biotransformations; cyclohex-2-en-1-
one cis-diols; 4-hydroxycyclohex-2-en-1-ones; phenol
hydrates.
identified using, mainly, Pseudomonas putida mutant
and Echerichia coli recombinant strains, expressing
ring-hydroxylating dioxygenases.[2b-m] Synthetic
applications of enantiopure substituted benzene cis-
dihydrodiols 2, obtained as toluene dioxygenase
(TDO)-catalysed cis- dihydroxylation products, from
monosubstituted benzene substrates 1, continue to be
widely reported, despite their limited stability.[2b-m]
Dehydration of most cis-dihydrodiols 2, to yield a
mixture of meta- and ortho-phenols 3 and 4, can
occur at ambient temperature. The rate of
dehydration, and ratio of phenol isomers, depend on
the type of
Introduction
Enzyme-catalysed formation of phenol metabolites,
from aromatic substrates, can proceed directly [1a] or
indirectly, via transient intermediates. Phenols have
been obtained indirectly by: (a) monooxygenase- or
peroxygenase-catalysed arene epoxidation and
spontaneous isomerization of arene oxide-oxepine
intermediates[1b-e] or (b) dioxygenase-catalysed cis-
dihydroxylation of arenes[2a-m] and dehydration of cis-
dihydrodiol intermediates.[3a,b] Since their discovery
by Gibson et al in 1968,[2a] more than four hundred
cis-dihydrodiol metabolites of substituted monocyclic
and polycyclic arenes have been isolated and
1
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10.1002/adsc.201700711
Advanced Synthesis & Catalysis
Scheme 1. Tandem TDO-catalysed cis-dihydroxylation of monosubstituted benzenes (1) and phenols (3, 4) to yield cis-
dihydrodiols (2, 5, 7) and cyclohex-2-en-1-one cis-diol keto-tautomers (6, 8).
substituent and pH of solution (Scheme 1).[3a,b]
diphenylamine- and toluene-dioxygenases, have been
reported to catalyse the oxidation of aniline
substrates.[6d, f-i] Although cis-diol metabolites were
often postulated as intermediates leading to the
formation of catechol, hydroquinone and phenol
metabolites of anilines,[6c-j] to date none have been
detected or isolated. Thus, TDO-catalysed cis-
dihydroxylation, at the 1,2- and 2,3-bonds of 4-
chloroaniline, was proposed (using P. putida T57) as
a possible initial step in the formation of both
`Since phenols are widely distributed in the
environment, as natural products, arene metabolites
and environmental pollutants, their biodegradation
pathways have been extensively studied.[4a-g] Bacterial
metabolism of phenols often results in a wider range
of metabolites, compared with most non-phenolic
aromatic substrates. Recent biotransformation results
of TDO-catalysed oxidations of phenols, e.g. 3 and 4,
using P. putida UV4, showed that, in addition to the
expected, catechol and hydroquinone metabolites, the
corresponding cyclohex-2-en-1-one cis-diols 6a-d, 8a
and 8b were also isolated, as the preferred keto
tautomers of the initially formed enolic cis-
dihydrodiols 5a-d, 7a and 7b. The dihydroxylation
was regio- and stereo-selective and cyclohex-2-en-1-
one cis-diols 6a-d, 8a and 8b were often found to be
the major isolated metabolites. More than twenty
members of this new cis-diol family have now been
isolated.[5a-e]
catechol
and
phenol
metabolites.[6i]
Biotransformations using other substituted anilines
and bacterial strains, were also found to yield
catechol and hydroquinone metabolites.[6a,c] In silico
molecular docking studies, on aniline substrates, were
thus conducted to: (a) predict the most favourable
structures of expected metabolites and (b) compare
the substrate docking results with the experimental
results of TDO-catalysed cis-dihydroxylation of
aniline substrates.
The biodegradation pathways, for anilines,
have also been studied, due to their presence in the
environment, as a result of the partial combustion of
tobacco and automotive fuels, the application of
pesticides / herbicides and the production of
pharmaceuticals, dyestuffs and textiles.[6a-j] Many
aniline derivatives are known to be genotoxic and
cytotoxic, severely inhibiting cell growth in soil
bacteria and slowing their mineralization.[6a,g]
Results and Discussion
P. putida UV4 biotransformations of aniline and
phenol substrates, to yield cyclohex-2-en-1-one cis-
diols and 4-hydroxycyclohex-2-en-1-ones
(i) Molecular docking of meta-substituted aniline
substrates 9a-d with TDO
Recent molecular docking studies, of the meta
substituted phenols 3a and 3b, at the active site of
TDO,[5f] were based on a comparison with an X-ray
crystal structure of TDO and docked toluene
substrate.[5g] These studies of TDO, without dioxygen
incorporation (3EN1M model), provided preferred
orientations of phenol substrates 3a and 3b (Scheme
Similar to the metabolism of electron-rich
phenols,[5a-e] ring hydroxylating dioxygenase-
catalysed biotransformations of electron-rich anilines
(using Pseudomonads and other bacterial species)
have also been reported, to yield catechol and
hydroquinone metabolites.[6a-j] Aniline-, biphenyl-
2
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10.1002/adsc.201700711
Advanced Synthesis & Catalysis
Scheme 2. TDO-catalysed cis-dihydroxylation of meta-anilines 9 to yield cis-diols 6, cis-triols 12 and catechols 13.
1) required for production of the corresponding enol
cis-dihydrodiols 5a (12% docking iterations) and 5b
(66% docking iterations). Catechols 13a and 13b
(Scheme 2) were also predicted (54% docking
iterations from phenol 3a and 34% from 3b).
the detection of cis-diol 2 (R=NHPh), it was
postulated that formation of the major metabolite,
phenol 4 (R= NHPh), had resulted from dehydration
of this transient intermediate.[6f]
The qualitative nature of docking results
recorded may not quantitatively reflect the
experimental results (using P. putida UV4 whole
cells), due to further metabolism by the co-induced
enzymes. Apart from this caveat, the 3EN1M and
3EN1M-O2 models, employed for TDO docking
studies[5f] of phenol substrates 3, were found to be
useful predictors of the preferred regiochemistry and
stereochemistry of cis-diol metabolites. These models
have now been applied to cis-dihydroxylation of
aniline substrates 9a-d (Scheme 2). From analysis of
the data collected, it was predicted that the NH-
imines 11a-d would be the preferred tautomers of the
initially formed enamine cis-diols 10a-d. It was also
assumed that: (i) this type of imine would readily
hydrolyse, during the biotransformation, to yield the
corresponding cyclohex-2-en-1-one cis-diols 6a-d
and (ii) as observed,[5c] the formation of cis-triols 12,
via carbonyl reductase (CRED)-catalysed reduction
of the ketone group in cis-diols 6, would occur using
P. putida UV4 whole cells. The 3EN1M and
3EN1M-O2 model docking studies of anilines 9a-d
with TDO were also expected to provide evidence of
preferred substrate orientations, leading to the
formation of catechols 13a-d, via alternative types of
transient aniline cis-diol intermediates 14a-d,
resulting from cis-dihydroxylation at the 1,2- (ipso-
ortho-) bond, as was proposed.[6c, 6e-i]
Docking of phenols 3a and 3b, with
dioxygen incorporated TDO (3EN1M-O2 model), led
to the predicted formation of catechols (54% docking
iterations from 3a, 34% from 3b), but not of cis-
dihydrodiols 5a and 5b. Further biotransformation (P.
putida UV4) of catechols 13, by a catechol
dioxygenase-catalysed ring-opening process and
other enzyme-catalysed reactions, gave a range of
carboxylic acid metabolites. The formation of
catechols, as arene metabolites, was also reported to
inhibit the TDO activity, therefore reducing cis-
dihydrodiol yields.[6k]
The predicted, and isolated, cyclohex-2-en-1-
one tautomers, derived from phenols, e.g. 6a and 6b,
were single enantiomers, having an (S) absolute
configuration at C-5. The main attractive interactions,
at the TDO active site, involved: (i) hydrogen
bonding of the phenol OH group with the C=O group
of Gln-215 and the imidazole ring of His-311, (ii) van
der Waals interactions of the hydrophobic Me group
of the phenol with the proximate alkyl (Ala-223, Val-
309, Leu-321, Ile-324) and aryl (Phe-366) groups.
It was speculated that similar binding of
phenols with His-311 and Gln-215, at the TDO active
site, might also apply to aniline substrate
interactions.[5f] An earlier in silico molecular binding
model for diphenylamine 1 (R=NHPh, Scheme 1), at
the active site of biphenyl dioxygenase (BPDO,) led
to the prediction that cis-dihydroxylation would yield
aniline cis-dihydrodiol intermediate 2 (R=NHPh).[6e]
Although BPDO-catalysed cis-dihydroxylation of
substituted aniline 1 (R=NHPh)[6d] did not result in
cis-Dihydroxylation, at the 4,5-bond and %
formation of enamine cis-diols 10a-d, was predicted
(Table 1), from TDO docking orientations (using
3EN1M model), for meta substituted anilines 9a (Fig.
1A, 47%), 9b (Fig. 1B, 90%), 9c (Fig. 1C, 80%) and
9d (Fig. 1D, 100%). The substrate binding, according
3
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10.1002/adsc.201700711
Advanced Synthesis & Catalysis
Table 1. Predicted, using the 3EN1M model of TDO, initial metabolites 11a-d, catechols 13a-c and isolated cis-
diol 6a-d products of 3-substituted aniline substrates 9a-d.
c
e
Figure Predicteda Productb
%
ΔGd
-5.08
-5.00
-5.27
-5.14
-5.14
-5.07
-6.13
KD
Substrate
9a 3-Methoxyaniline
1A
1B
1C
1D
47
26
90
4
190.24
216.49
137.66
169.39
171.53
191.97
31.88
11a
13a
11b
13b
11c
13c
11d
6a
6b
6c
6d
9b 3-Methylaniline
9c 3-Trifluoromethylaniline
80
20
100
9d 3-Iodoaniline
a
b
Predicted metabolite; Detected product; c Orientation occurrence; d Binding energy (kJ/mol);e Dissociation constant
(µM)
Figure 1. Molecular docking of meta substituted anilines 9a-d (Figs. 1A-1D) at the active site of TDO.
1A
1B
Asp219
Gln215
Phe372
Ala223
Phe216
Phe366
His311
Ile324
Val309 Leu321
1C
1D
to the in silico docking, was facilitated by two major
interactions: (i) van-der- Waals interaction of Ala-
223, Val-309, Leu-321, Ile-324, Phe-366 and Phe-
372 amino acid residues with the substituents (Me,
OMe, CF3, I) and (ii) H-bonding of the NH2 group to
Gln 215, and sometimes to His-311, in a similar
manner to the docking of phenols.[5f]
4
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10.1002/adsc.201700711
Advanced Synthesis & Catalysis
The docking experiments (3EN1M model) of
The first objective of the study was to
provide experimental evidence for TDO-catalysed
cis-diol formation from aniline substrates, but the
presence of other metabolites extended our interest
into exploring the complete metabolic profile of
anilines with the P. putida biocatalyst. A CRED
enzyme, present in P putida UV4, was previously
found to catalyse the reduction of the ketone group of
anilines 9a-d also led to the prediction that catechols
13a-c might also be formed, via a minor pathway (4-
26% docking orientations), by dihydroxylation at the
1,2-position, to yield intermediate cis-diols 14a-c.
Employing the 3EN1M-O2 model of TDO, catechols
13a-d were predicted to be the major metabolites (91-
100% docking orientations), without evidence of
orientations of anilines 9a-d, leading to the
metabolite
6c,
to
yield
(1R,2S,4R)-6-
corresponding
cis-diols
10a-d
(Supporting
(trifluoromethyl)cyclohex-5-ene-1,2,4-triol 12c as a
major bioproduct.[5c] A similar result was obtained
with aniline substrate 9c when metabolites cis-diol 6c
and triol 12c were identified by LC-TOFMS analysis.
Information Figs. S1-S4). These results (3EN1M-O2
model) were similar to those found earlier for phenol
substrates 3a and 3b, where catechols 13a and 13b
were predicted to be the major metabolites (54 and
100% docking orientations) without evidence for the
formation of cis-diols 5a and 5b.[5f]
Catechols were identified as aniline
metabolites[6a-i] and molecular docking (3EN1M and
3EN1M-O2 models) experiments of TDO also
suggested their formation from anilines 9a-d (Table
1). LC-TOFMS analysis did not show direct evidence
of catechol metabolites 13a-d, but indirect evidence,
for the formation of catechol metabolite 9a, was
observed by the formation of a carboxylic acid
metabolite, whose molecular weight was consistent
with structure 15a, formed by catechol dioxygenase-
catalysed ring opening and reductase-catalysed
reduction (Fig. 2). Similar ring-opened metabolites
15 (R = Me, CF3) were previously reported from the
corresponding phenols (3b and 3c) and catechols
Based on these predictive in silico docking
(3EN1M model) studies of TDO (Table 1),
experimental evidence was sought, for TDO-
catalysed cis-dihydroxylation of meta-anilines 9a-d,
by LC-TOFMS analysis of the crude biotransformed
culture medium. Aniline substrates 9a-d were added,
to P. putida UV4 cultures, under conditions similar to
those reported for the corresponding meta-phenols
3a-d.[5a-e] The cyclohex-2-en-1-one cis-diols 6a-d,
previously reported[5a-e] as phenol metabolites
(Scheme 1), were also detected as aniline metabolites
(Scheme 2), in accord with the predictions from in
silico studies. The documented high cytotoxicity of
anilines[6a,e] required a ten-fold reduction of the
substrate concentration (0.05 mg/mL), for total
conversion. Phenols 3a-d and corresponding anilines
9a-d, applied in the same low concentrations,
produced comparable yields of cyclohexenone cis-
diols 6a-d, which were identified by comparison
(LC/TOFMS and GC-MS) with authentic samples. A
sample of cis-diol 6a (ca. 6 mg) was also isolated by
PLC, from the partial biotransformation using a
higher concentration of aniline 9a; its structure,
absolute configuration (4S,5S) and enantiopurity
(>98% ee) was found to be identical with the
metabolite derived from phenol 3a. From this result,
combined with the in silico docking studies (Figs.
1A-D), it was predicted that the (S)-absolute
configuration at the C-5 position and ee value (>98%)
of cis-diol metabolites 6b-d, derived from the
corresponding aniline substrates 9b-d, would be
identical to those obtained from phenols 3b-d.
(13b
and
13c).[5c]
GC-MS
analysis
of
trimethylsilylated samples, prepared from freeze-
dried aliquots collected during TDO-catalysed
dihydroxylation of anilines 9a and 9c, showed the
presence of disilylated cis-diol 6a and 6c and derived
hydroquinones 16a and 16c respectively, but no
catechols were detected. The difficulty encountered
in the detection of catechol metabolites 13a-d was
probably due to: (i) the activity of a catechol
dioxygenase enzyme present in P. putida UV4 and
(ii) the low yields of all metabolites resulting from
the cytotoxicity of aniline substrates.
Biotransformations of anilines 9a-d, with
the recombinant strain, E. coli pCL-4t (expressing
TDO), and LC-TOFMS analysis of the
biotransformed aqueous material, again showed the
presence
of
5
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10.1002/adsc.201700711
Advanced Synthesis & Catalysis
Figure 2. Structures of metabolites 15-18 and 25.
]
1).[5e The mother liquors from this recrystallization
cyclohexenone cis-diol metabolites 6a-d. The
reduced tolerance of E. coli cells, to the toxic aniline
substrates, gave lower yields, compared with those
found using P. putida UV4 cells. However, the E.
coli pCL-4t biotransformation studies did provide
evidence that TDO, rather than other types of
dioxygenase, was responsible for the formation of
cis-diols 6a-d.
contained
a
mixture of unidentified minor
metabolites, which were examined further during this
study. Column chromatography of the mixture
yielded a new minor metabolite (1% isolated yield),
which was structurally identified as 4-hydroxy-3-
methoxycyclohex-2-en-1-one 19a ([]D -31.9).
A chemoenzymatic synthesis of metabolite
19a, starting from 2-bromo-2-cyclohexen-1-one 20,
established (4S) as its absolute configuration (Scheme
3). Step (i) employed a CRED-catalysed reduction of
the ketone group of 2-bromo-2-cyclohexen-1-one 20,
to give the enantiopure synthetic precursor
cyclohexenol 21S ([]D -80.3, 88%).[5h] Further
chemical steps involved, hydroxyl group protection
The biotransformation and molecular
docking results, recorded for aniline substrates 9a-d,
were consistent with a metabolic pathway via TDO-
catalysed formation of enamine cis-dihydrodiols 10a-
d, tautomerisation to the preferred NH-imine cis-diols
11a-d, and rapid hydrolysis to yield cyclohex-2-en-1-
one cis-diols 6a-d (Scheme 2). Thus, the family of
cyclohex-2-en-1-one cis-diol metabolites is formed,
from both substituted phenols and anilines, by TDO-
catalysed cis-dihydroxylation. The molecular docking
results can also be used to rationalise the reported
formation of catechol and hydroquinone metabolites
of aniline substrates,[6a-j] via ring hydroxylating
dioxygenase catalysis.
(ii, 21S
29%), nucleophilic substitution (iv, 23S 24S, 69%)
and deprotection (v, 24S 19aS, 62%, Scheme 3).
22S, 94%), allylic oxidation (iii, 22S 23S,
This synthetic sample of compound 19aS (>98% ee)
had a higher optical rotation ([]D -48.8), compared
with the corresponding metabolite (19aS) derived
from phenol 3a. The lower enantiopurity (65% ee) of
the minor metabolite, 4-hydroxy-3-methoxycyclohex-
2-en-1-one 19aS, compared to the major metabolite,
(4S,5S)-3-methoxycyclohex-2-ene-1-one 6a (>98%
ee), was of mechanistic relevance, in the context of
biosynthetic pathways from 3-methoxyphenol 3a
(Scheme 4), which will be discussed in Section (iii).
(ii) Biotransformations of phenol substrates, to
yield 4-hydroxycyclohex-2-en-1-ones
Earlier larger scale biotransformations of meta- and
ortho-phenols 3 and 4, showed a wide range of
metabolite types, including cyclohexenone cis-diols 6
and 8, cyclohexene cis-triols 12, catechols 13, α-
hydroxycarboxylic acids 15, hydroquinones 16,
cyclohexanone cis-diol isomers 17cis and 17trans and
1,2,4-trihydroxycyclohexanes 18[5a-e] (Scheme 1 and
Fig. 2). Several minor metabolites of methoxyphenols
3a and 4a, however, remained unidentified;[5e] their
structures, absolute configurations and metabolic
pathways for their formation are presented in this
section.
A previous biotransformation (glucose as
carbon source) of 2-methoxyphenol 4a (96 g),
followed by column chromatography, resulted in a
separable mixture of isomeric cyclohexanone cis-
diols, (2S,3S,4S)-17acis (13% isolated yield) and
(2R,3S,4S) 17atrans (1% isolated yield).[5e] Metabolites
17acis and 17atrans were formed via an ene reductase
(ERED)-catalysed reduction of the initial bioproduct,
cyclohex-2-en-1-one cis diol 8a (Scheme 1). Using
LC-TOFMS and GC-MS analyses, the relative ratios
of metabolites from guaiacol 4a were found to vary
widely, during time course studies of the
biotransformations, depending on the choice of
carbon
Recrystallization of the crude mixture of
metabolites,
obtained
from
an
earlier
biotransformation of 3-methoxyphenol 3a (96 g),
with glucose as carbon source, yielded cis-diol 6a as
the major component (38% isolated yield, Scheme
6
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10.1002/adsc.201700711
Advanced Synthesis & Catalysis
Scheme 3. Chemoenzymatic synthesis of 4-hydroxy-3-methoxycyclohex-2-en-1-one 19aS from 2-bromo-2-cyclohexen-1-
one 20.
source (glucose or pyruvate) and TDO source (P.
putida or E. coli). Other metabolites from phenol 4a
were identified as cyclohex-2-en-1-one cis diol 8a,
hydroquinone 16a, catechol 13a and its α-
hydroxycarboxylic acid derivative 15a.
Iodocyclohex-2-en-1-one cis-diol 6d, a major
metabolite of 3-iodophenol 3d,[5a,c] was isolated in
variable yields (30-70%), along with other
metabolites including 4-hydroxycyclohex-2-en-1-one
19eR. LC-TOFMS analysis of the biotransformed
aqueous culture medium, detected the presence of
cyclohexanone cis-diol 26e and GC-MS analysis of
the EtOAc concentrate, after trimethylsilylation with
N-methyl-N-(trimethylsilyl)trifluoroacetamide
(MSTFA), showed that iodocyclohexene cis-triol
12d, cyclohexane cis-triol 18e, iodocatechol 13d,
iodohydroquinone 16d and cyclohexene cis-triol 12
(R=H) were also present as minor metabolites
(Scheme 2 and 4, and Fig. 2).
Column chromatography fractions, from the
earlier study,[5e] that appeared to be an inseparable
mixture of two unidentified isomeric metabolites of
phenol 4a were retained for further examination.
During the current study, this mixture, was finally
separated by careful multiple elution PLC. The minor
isomer (2% isolated yield) was indistinguishable
from 4-hydroxy-3-methoxycyclohex-2-en-1-one 19aS
derived from 3-methoxyphenol 3a. The structure and
absolute configuration of the major isomer (18%
isolated yield, [α]D -29), was identified as (4S)-4-
hydroxy-2-methoxycyclohex-2-en-1-one 25aS. This
metabolite was also isolated as a dehydration product
A preliminary biotransformation of ortho-
cresol 4b, resulted in the isolation of (4S,5R,6S)-4,5-
dihydroxy-6-methylcyclohex-2-en-1-one 8b, as the
only identified metabolite (1% yield).[5a] Repeated
metabolism studies of phenol 4b, revealed that in
addition to cis-diol 8b, four other minor metabolites
were present. Catechol 13b and hydroquinone 16b
were identified by trimethylsilylation of a small
portion of the crude freeze-dried extract, followed by
GC-MS analysis of the products and comparison with
authentic samples.
of
(2S,3S,4S)-3,4-dihydroxy-2-
methoxycyclohexanone 17acis (Scheme 4).
A repeat biotransformation of guaiacol 4a
again resulted in the formation of chiral metabolites
17acis, 25aS and 19aS, but in a different ratio based on
isolated yields, i.e. 11%, 3%, <1% respectively. This
prompted a time course biotransformation study of
methoxyphenol substrates 3a and 4a, which showed
increases in the relative yields of 4-hydroxy-3-
methoxycyclohex-2-en-1-ones 19aS and 25aS
respectively, with glucose, rather than pyruvate, as a
carbon source and during the later stages (>8 h) of the
biotransformations.
Time course LC-TOFMS analysis, of the
crude culture medium from a biotransformation of
ortho-cresol 4b, indicated that, in addition to 8b, a
very minor bioproduct was formed, which rapidly
metabolized further. This early eluting bioproduct,
was tentatively identified as cyclohexanone cis-diol
17bcis (Scheme 4). Its identity was confirmed by
catalytic hydrogenation (Pd/C, MeOH) of metabolite
8b, to yield an identical sample of (2S,3R,4S)-3,4-
dihydroxy-2-methylcyclohexanone 17bcis. Work up
of the biotransformed material[5a] and separation of
the crude mixture, by column chromatography
followed by PLC of early eluting fractions, gave a
new metabolite, which was identified as 4-hydroxy-2-
methylcyclohex-2-en-1-one 25bS ([α]D - 49.0). The
opposite enantiomer, 25bR, ([α]D + 46.7), had been
synthesised earlier by an alternative route, involving
manganese acetate-mediated acetoxylation and
lipase-catalysed ester hydrolysis.[7]
The
unexpected
discovery
of
4-
hydroxymethoxycyclohex-2-en-1-ones 19aS and
25aS, metabolites of phenol substrates 3a and 4a,
allied to the earlier isolation of compound 19eR, as a
minor metabolite of 3-iodophenol 3d,[5a] raised the
possibility that compounds 19aS, 25aS and 19eR,
could be the first members of a new family of phenol
metabolites. To investigate the possible metabolic
pathways, leading to the formation of 4-
hydroxycyclohex-2-en-1-ones 19aS, 19eR and 25aS,
repeat biotransformations of 3-iodophenol 3d and
ortho-cresol 4b were conducted (Scheme 4).
7
This article is protected by copyright. All rights reserved.

10.1002/adsc.201700711
Advanced Synthesis & Catalysis
via a similar metabolic pathway (4b
25bS).
8b
17bcis
(iii) Biosynthetic pathways for the formation of 4-
hydroxycyclohex-2-en-1-one - metabolites from
phenols
While the formation of three 4-
hydroxycyclohex-2-en-1-ones (19eR, 25aS and 25bS),
from the corresponding phenol substrates (3d, 4a and
4b), resulted from a common biosynthetic sequence,
this pathway would not result in the formation of
metabolite 19aS from phenol 3a. Furthermore, the
higher enantiopurity (>98% ee), of bioproducts 19eR,
25aS and 25bS from phenols 3d, 4a and 4b,
contrasted with the lower ee value of metabolite 19aS
(ca. 65% ee, from phenol 3a) found earlier. This
indicates the probability of a different mechanism
being involved in the formation of 4-
hydroxycyclohex-2-en-1-one 19aS from phenols 3a
and 4a.
The metabolic sequences, to account for the
formation of the 4-hydroxycyclohex-2-en-1-one
metabolites 19eR, 19aS, 25aS and 25bS, as minor
products from the corresponding phenol substrates
(3d, 3a, 4a and 4b), via the initial cyclohexenone cis-
diol metabolites 6d, 6e, 6a, 8a and 8b, are presented
in Scheme 4.
It is proposed that, during the P. putida UV4
biotransformation of 3-iodophenol 3d, an ERED-
catalysed ene reduction of cyclohexenone cis-diol 6d,
followed by a dehydrohalogenation of the resulting
cyclohexanone cis-diol could occur, to give the
transient parent cyclohex-2-en-1-one cis-diol 6e
(Scheme 4). Intermediate 6e was not detected,
possibly due to its further rapid ERED-catalysed ene
reduction to form the detected transient metabolite
26e. CRED-catalysed reduction of metabolite 26e
yielded the cis-triol metabolite 18e (Fig. 2, R = H),
while its facile dehydration also gave 4-cyclohex-2-
ene-1-one 19eR. A precedent for this type of reductive
With cyclohexenone cis-diol 6a as substrate,
4-hydroxycyclohexenone 19aS was identified as the
main metabolite with hydroquinone 16a as a minor
product resulting from the dehydration. Since
hydroquinone 16a also being formed by the
dehydration of cyclohexenone cis-diol 8a, its possible
role as an intermediate during formation of 4-
hydroxycyclohex-2-en-1-one 19aS was examined.
The biotransformation pathways of phenols 3a and
4a, with hydroquinone 16a as an intermediate, were
dehalogenation mechanism, (6d
6e) by the
ERED-catalysed reduction-spontaneous β-elimination
of β-halo-α,β-unsaturated carboxylic esters, has been
reported.[8] Other examples of ERED-catalysed ene
reductions, of α,β-unsaturated ketones, e.g.
metabolites 8a→17acis and 8b→17bcis, have been
found during biotransformations[5e] (Scheme 4).
postulated to proceed in five steps (3a
6a
16a
16a
27a
27a
28a
28a
19aS and 4a
8a
19aS) as shown in Scheme 4. Further
confirmation of these metabolic sequences was
obtained by the biotransformation of hydroquinone
16a as substrate to yield metabolite 4-
hydroxycyclohex-2-en-1-one 19aS.
Further evidence of the metabolic sequence
(3d
6d
6e
26e
19eR), involving both
TDO and ERED enzymes, was found when cyclohex-
2-en-1-one cis-diol 6e ([α]D - 217), obtained by
hydrogenolysis of metabolite 6d, was added as
substrate. 4-Hydroxycyclohex-2-en-1-one 19eR and
triol 18e were the only identified metabolites.
Enantiopure 4-hydroxycyclohex-2-en-1-one 19eR,
([α]D +110) synthesised by an alternative
chemoenzymatic route using lipase enzymes, has
been utilized as a chiral precursor in synthesis.[9a,b]
It is postulated that the oxidation of
hydroquinone 16a, to benzoquinone intermediate
27a, could have resulted from either a non-enzymatic
autoxidation or peroxidase activity in P. putida UV4
cells, followed by an ERED-catalysed reduction of
benzoquinone 27a (Scheme 4). The transient
intermediate, cyclohex-2-ene-1,4-dione 28a, could
either tautomerize back to hydroquinone 16a or
undergo an asymmetric CRED-catalysed ketone
reduction. This could account for metabolite 19aS
having a lower enantiopurity (65% ee) compared with
the other 4-hydroxycyclohex-2-en-1-ones 19eR, 25aS
and 25bS or cyclo cis-diol precursors 6a and 8a
(>98% ee).
Biosynthetic sequences, involving TDO-
catalysed cis-dihydroxylation of phenols 4a and 4b,
to yield enantiopure cyclohex-2-en-1-one cis-diols
followed by an ERED-catalysed reduction / β-
elimination mechanism, are shown in Scheme 4. The
metabolic pathway proposed for the formation of 4-
hydroxycyclohex-2-en-1-one 25aS from phenol 4a,
Fungal metabolism of benzoquinone 29, with
cultures of Phanerochaete chryosporium,[10] to form
4-hydroxycyclohex-2-enone 30 (Fig. 3), provides a
(4a
8a
17acis
25aS), was supported by
results obtained using (2S,3S,4S)-cyclohexanone cis-
diol 17a as substrate; compound 25aS was the only
bioproduct formed. It was presumed that the
biotransformation of phenol 4b, to yield 4-
hydroxycyclohex-2-en-1-one 25bS, would also occur
precedent for the metabolic sequence (27a
28a
19aS). Dehydration of cyclohexenone cis-diols 6a
and 8a, to give hydroquinone 16a, and of
cyclohexanone cis-diols 17a, 17b and 26e, to form 4 -
8
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10.1002/adsc.201700711
Advanced Synthesis & Catalysis
hydroxycyclohex-2-en-1-ones 25aS, 25bS and 19eR
respectively, could occur during biotransformations,
putida UV4 cultures, is among the few reports of
arene to arene hydrate biotransformations (Scheme 1,
Fig. 3).[11b]
via chemocatalysis or
a
dehydratase-catalysed
process.
Metabolite 31, a highly unstable compound,
with a propensity to rapidly dehydrate back to
substrate 1 (R = COMe), was only identified as an
iron tricarbonyl complex.[11b] To study the stability of
monocyclic arene hydrates, racemic samples of
(iv) Biotransformations of monocylic arenes, to
yield arene hydrates
The hydration of conjugated and non-conjugated
alkene bonds, catalysed by hydratase or hydrolyase
enzymes, e.g. aconitase, fumarase and crotonase, is a
common step in primary metabolism.[11a] There are
very few reported examples of enzymatic hydrations
of arenes, to form the corresponding arene hydrates.
The formation of arene hydrate metabolite 31 from
acetophenone substrate 1 (R = COMe) using P.
Scheme 4. Metabolic pathways for the formation of 4-hydroxycyclohex-2-en-1-ones 19eR, 19aS, 25aS and 25bS,
from phenols 3d, 3a, 4a and 4b respectively.
Figure 3. Structures of compounds 29-36.
i
t
compounds 32 (R = Me, Et, Pr, Bu), and 33 (R =
CO2Me and Ph), were synthesised from 3-substituted
1,4-cyclohexadienes,[11c] and enantiopure arene
hydrates 33 (R = F, Cl, Br, CF3) and 34 (R = Br) from
the corresponding cis-dihydrodiol metabolites 2.[11d]
Kinetic studies of the acid-catalysed dehydration of
these arene hydrates showed that they aromatized
9
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10.1002/adsc.201700711
Advanced Synthesis & Catalysis
much faster (3.7x102 - 6.9x 104 fold) than cis-
dihydrodiols 2.[11c]
Molecular docking results, of four aniline substrates
with TDO, led to the prediction that in common with
phenols, cis-dihydroxylation of anilines could occur,
to yield cis-diols and catechols. The premise was
confirmed by the detection and isolation, of identical
cyclohex-2-en-1-one cis-diol metabolites, from the
corresponding meta-phenols and meta-anilines. The
initial formation of enamines and the NH-imine
tautomers, followed by their rapid hydrolysis, could
account for the formation of cyclohex-2-en-1-one cis-
diols in low yields from the anilines. Although
catechols had been found earlier as aniline
metabolites, no direct evidence was found in the
study, possibly due to further metabolism by catechol
dioxygenase.
The monocyclic arene hydrate 35, an
unstable intermediate, formed during the biosynthesis
of the antibiotic bacilysin, was obtained by the
enzymatic decarboxylation of prephenate, using an E.
coli recombinant strain, expressing phenate
decarboxylase.[12a-c] Arene hydrate 35 was found to
undergo a slow non-enzymatic, or a rapid enzyme-
catalysed isomerization, to yield the more stable
vinylogous 4-hydroxycyclohexenone metabolite 36.
Intermediate 35 appears to be among the very few,
isolated and fully characterized, monocyclic arene
hydrate metabolites.
Phenol metabolites, 4-hydroxycyclohex-2-en-
1-one 19eR, 19aS, 25aS and 25bS, identified during the
study could, in principle, equilibrate with the
corresponding phenol hydrate tautomers 19e'R, 19a'S,
25a'S and 25b'S; in practice only the keto tautomers
were observed by NMR spectroscopy. Similarly, the
methoxycyclohexenone cis-diol 6a, metabolite of 3-
methoxyphenol 3a, showed no evidence of enol
tautomer 5a, from proton NMR analysis (Scheme 1).
Trimethylsilylation of cis-diol 6a with MSTFA, and
GC-MS analysis of the product, showed a major peak
(95%) with ([M]+ m/z 302), indicating formation of
the diTMS derivative.[5e] The molecular ion ([M]+ m/z
374), corresponding to the minor peak (5%), was
from a triTMS derivative and provided indirect
evidence of the elusive enol tautomer 5a. Similar
treatment of 4-hydroxycyclohex-2-en-1-ones 19eR
and 19aS with MSTFA, and GC-MS analyses of the
products showed the formation of monoTMS
derivatives 19e'R and 19a'S (major peaks). The minor
peaks were attributed to the diTMS derivatives of the
undetected phenol hydrate tautomers 19e'R and 19a'S.
Biotransformation of cyclohex-2-en-1-one
cis-diols, resulted in the formation of a new range of
minor metabolites, which were identified as 4-
hydroxycyclohex-2-en-1-ones. Their structures and
absolute configurations were determined by
chemoenzymatic synthesis and stereochemical
correlation; these new phenol metabolites have
considerable synthetic potential.
Although single step hydratase or hydrolase
activity has been reported for alkenes, we are
unaware of similar activity with arene substrates.
Multistep
metabolic
pathways,
involving
cyclohexenone cis-diol, cyclohexanone cis-diol and
hydroquinone intermediates, are now proposed to
explain the formation of 4-hydroxycyclohex-2-en-1-
ones as a new type of phenol metabolite. Direct
evidence for the presence of enol tautomers (arene
hydrates) of 4-hydroxycyclohexenones was not
found; indirect evidence was obtained following
trimethylsilylation and GC-MS analyses.
The lack of evidence, for monocyclic arene
hydrates of similar structure to metabolite 31,[11b]
during the biotransformations of substituted benzene
substrates by P. putida UV4 or other microbial
systems could be due to the absence of suitable
hydrolase / hydratase enzymes or rapid dehydration
and reformation of the arene substrates.[11c] Under
similar biotransformation conditions, the TDO-
catalysed cis-dihydroxylation of arene hydrate 33 (R
Experimental Section
Experimental Details
NMR spectra were recorded on Bruker Avance-400, DPX-
300 and DRX-500 instruments. Chemical shifts (δ) are
reported in ppm relative to SiMe4, and coupling constants
(J) are given in Hz. IR spectra were recorded on a Perkin-
Elmer 983G spectrometer. Optical rotation ([α]D)
measurements were carried out on a Perkin-Elmer 214
polarimeter. LC-TOFMS analyses were conducted using
an Agilent 1100 series HPLC coupled to an Agilent 6510
Q-TOF (Agilent Technologies, USA) and a reverse phase
column (Agilent Eclipse Plus C18, 5 mm, 150 x 2.1 mm)
under reported conditions.[5e] GC-MS analysis of
metabolites was carried after silylation using MSTFA and
an Agilent Technologies 6890N gas chromatograph linked
to a 5973 mass selective detector and Agilent Technologies
HP Ultra 2 column as reported.[5e] For TLC analysis,
=
CF3) yielded the opposite enantiomer of
cyclohexene triol 12c,[11d] and thus revealed an
alternative metabolic pathway for monocyclic arene
hydrates. The results presented herein demonstrate
that the more stable keto tautomers of arene hydrates
can be obtained from phenols using P. putida UV4.
Conclusion
10
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10.1002/adsc.201700711
Advanced Synthesis & Catalysis
Merck Kieselgel 60F254 analytical plates were used and
PLC separation of metabolites was carried out using glass
plates (20 cm x 20 cm) coated with Merck Keiselgel
PF254/366 silica gel. Column chromatography was
performed on Merck Keiselgel type 60 (250-400 mesh).
genetic algorithm with a population size of 150 each,
terminating after 2 500 000 energy evaluations or 27 000
generations (whichever occurs first; standard settings). The
docking results were analysed with ADT, to obtain
docking coordinates, calculated binding energy (in kJ) and
calculated dissociation constant KD (in µM). All docking
studies were performed as rigid docking, keeping all
positions, orientations and protonation states of all amino
acid atoms locked in place. The 100 docking orientations
were automatically analysed by ADT and divided into
orientationally and energetically similar groups. The
orientation with the highest binding energy of each group
was saved as representative for each group. The
conformation of toluene, in the crystal structure, was used
as a reference, to establish and confirm the viability of the
Phenols 3a-d, 4a, 4b, anilines 9a-d, catechols 13a, 13b,
hydroquinones 15a, 15b and methoxybenzoquinone 27a
were purchased from Sigma-Aldrich Co. Authentic
samples of cyclohex-2-en-1-one cis-diols 6a-d and
cyclohexene cis-triol 12c were available from earlier
studies.[5a-e] Commercially obtained anilines 9a-d showed
no detectable traces of phenols 4a-d on GC-MS analysis,
prior to their use as substrates. Biotransformations were
carried out using P. putida UV4 cells, unless mentioned
otherwise.
docking procedure and parameters, as
a
similar
conformation was obtained from docking with ADT.
Molecular modelling
3D visualization
Substrate docking studies were performed according to an
earlier procedure.[5f] The required in silico models of
substrates were created in .pdb-format with UCSF Chimera
1.10.2 (https://www.cgl.ucsf.edu/chimera/). In silico
dockings were performed with AutoDock suite 4.2
(autodock4, autogrid4). The Graphical User Interface
(GUI), including python scripts for ligand and receptor
preparation, was part of AutoDock Tools 1.5.6. AutoDock
suite and AutoDock tools (ADT) are provided by the
Scripps Research Institute (http://autodock.scripps.edu/).[13]
The TDO crystal structure was accessed from the Protein
Data Bank (PDB code 3en1, resolution of 3.2 Å). The raw
3en1 crystal structure .pdb-file of TDO includes a docked
toluene structure in the active site, which was removed
with UCSF chimera 1.10.2 prior to docking. The resulting
model was called 3EN1M. Ligand and receptor were then
prepared in accordance with the ADT tutorial
(http://autodock.scripps.edu/faqs-help/tutorial/using-
autodock-4-with autodocktools/2012_ADTtut.pdf) utilising
the ‘prepare_receptor4.py’ python script included in ADT,
missing atoms were repaired, hydrogens and Gasteiger
charges added, and non-polar hydrogens merged. The
resulting .pdbqt-file of the crystal structure was used in all
docking calculations. .pdb-files of substrates were
automatically converted to the required .pdbqt-format by
ADT. The docking grid was adjusted to include all amino
acids within 5 Å of toluene in the crystal structure: Gln215,
Phe216, Asp219, Met220, His222, Ala223, His228,
Val309, His311, Leu321, Ile324, Phe366, Phe376.
The amino acids of the TDO active site were visualized
with PyMol 1.7.4.5, from the Protein Data Bank (PDB)
3EN1 file coordinates. The docking results were imported
into PyMol, from the respective .pdb files created with
AutoDock4 and AutoDock Tools. Measurements between
atoms were calculated with PyMol’s incorporated
measurement tool.
Biotransformation of anilines 9a-d
Initial small scale biotransformations of anilines 9a-d and
LC-TOFMS analyses of metabolites were conducted,
under conditions similar to those reported for phenol
substrates.[5a-e] However, due to the increased toxicity of
anilines 9a-d, compared with phenol substrates 3a-d, a
significant proportion of residual aniline substrates were
consistently present along with cyclohexenone cis-diol
metabolites 6a-d. To achieve complete conversions, lower
aniline concentrations were used (ca. 0.05 mg/ml).
Cyclohexenone cis-diol metabolites 6a-d and cis-triol 12c
were readily identified as aniline metabolites, by
comparison of LC-TOFMS data with the authentic
samples. As cyclohexenone cis-diols 6a-d and cis-triol 12c
had been fully characterised [5a-e], only the LC-TOFMS
retention times (min.) and accurate mass values were
recorded for aniline substrates 9a-d.
3-Methoxycyclohex-2-en-1-one cis-diol 6a.[5d] 3.43 min.,
HRMS: [M+H]+ 159.0646, calcd. for C7H11O4 159.0652.
PyMol was used to incorporate dioxygen into the 3EN1M
model, by superimposing the iron complex (Fe, His222,
His228, and Asp376) of TDO (pdb code: 3en1) with that of
NDO (pdb code: 1o7m), and copying the dioxygen
positions to 3EN1M. The resulting model was called
3EN1M-O2.
3-Methylcyclohex-2-en-1-one cis-diol 6b.[5a] 4.85 min.,
HRMS: [M+H]+ 143.0698, calcd. for C7H11O3 143.0703.
3-Trifluoromethylcyclohex-2-en-1-one cis-diol 6c.[5c]
10.93 min., HRMS: [M+H]+ 197.0417, calcd. for
C7H8O3F3 197.0420.
Docking resolution = 0.247 Å; xsize = 40; xoffset = 12.833;
ysize = 60; yoffset = -4.472; zsize = 72; zoffset = 3.917. The grid
parameter file (.gpf) was used to create the grid map files
(.glg) using autogrid4. The search protocol for docking
used the internal default docking parameters of AutoDock
4.2, starting the ligand at a random location. The docking
was set to be performed as 100 runs of the Lamarckian
3-Iodocyclohex-2-en-1-one cis-diol 6d.[5a] 10.19 min.,
HRMS: [M+H]+ 254.9502, calcd. for C6H8O3I 254.9513.
6-Trifluoromethylcyclohex-5-ene cis-triol 12c.[5c] 9.65
min., HRMS: [M+NH4]+ 216.0831, calcd. for C7H13NO3F3
216.0847.
11
This article is protected by copyright. All rights reserved.

10.1002/adsc.201700711
Advanced Synthesis & Catalysis
Cyclohex-2-en-1-one cis-diol metabolite 6a (ca. 6 mg) was
isolated from a biotransformation of aniline 9a (1.25 g in 4
L culture medium). Purification, by column chromatograph
(hexane → EtOAc), of the crude product obtained after
usual work up followed by PLC (50% EtOAc in hexane),
of selected combined fractions, gave metabolite 6a. The
metabolite was found to have identical spectroscopic
(NMR) and chiroptical ([α]D) properties to an authentic
sample derived from phenol 3a. No catechol metabolites
MHz) δ = 29.5, 34.0, 56.2, 65.8, 102.1, 176.2, 198.6; IR
(film) νmax/cm-1 3389, 2945, 1630, 1609, 1231.
(b) Chemoenzymatic synthesis of (S)-4-hydroxy-3-
methoxycyclohex-2-en-1-one 19aS
(S)-2-Bromo-2-cyclohexen-1-ol 21S. This compound was
available as
biotransformation
a
colourless oil from an earlier
of 2-bromo-2-cyclohex-2-en-1-one
[5h]
[6a-e]
20, [α]D - 80.3 (c 1.77, CHCl3), ee 99.8% (chiral GC
analysis).
were detected (GC-MS analysis) in the crude bio-
extracts of aniline substrates 9a-d.
(S)-(2-Bromocyclohex-2-enyloxy)-tert-
Biotransformation of phenols 3a, 3d, 4a and 4b and
synthesis of (-)-(S)-4-hydroxy-3-methoxycyclohex-2-en-
1-one 19aS
butyldimethylsilane
22S.[15]
tert-Butyldimethylsilyl
trifluoromethanesulfonate (280 μl, 1.21 mmol) was added
to a solution of alcohol 21S (200 mg, 1.10 mmol),
maintained at 0°C in dry CH2Cl2 (10 mL) containing
triethylamine (240 μl, 1.69 mmol). The reaction mixture
was stirred (2 h) at 0°C, allowed to warm to room
temperature and then ice (20 g) and CH2Cl2 (20 mL) were
added to it. After thoroughly mixing the reaction mixture
by shaking, the organic layer was separated, washed with
brine (15 mL), dried (Na2SO4), and concentrated to give a
yellow oil. It was purified by flash chromatography
(hexane) to yield the TBS ether 22S as a colourless oil (310
Large scale biotransformations of phenols 3a and 4a, using
whole cell cultures of P. putida UV4 with glucose as a
carbon source, were reported.[5e] Pooled column fractions,
from that investigation containing unidentified metabolites,
were re-examined during the current study. Time course
study of small scale biotransformations of phenols 3a, 3d,
4a and 4b and anilines 9a-d, were conducted under similar
conditions, using glucose or pyruvate as carbon sources.[5a-
e]
Chiral metabolites, 4-hydroxycyclohex-2-en-1-ones 19a,
1
mg, 94%); Rf 0.4 (hexane); [α]D - 82.9 (c 0.8, CHCl3); H
19e, 25a and 25b, cyclohexenone cis-diols 6a and 6d,
cyclohexanone cis-diols 17a, 17b and 26e, and cis-triol
12c were detected directly in the crude aqueous culture
medium by LC-TOFMS analyses, prior to their isolation.
Catechols 13a-d, hydroquinones 16a-d were identified by
GC-MS analyses of the crude bio-extracts, after EtOAc
extraction and trimethylsilylation (MSTFA) of the dried
concentrates.
NMR (400 MHz) δ = 0.11 (3 H, s, SiMe), 0.17 (3 H, s,
SiMe), 0.92 (9 H, s, CMe3), 1.40 (1 H, m, H-5), 1.55-1.68
(3 H, m, H-5', H-6, H-6'), 1.83 (1 H, m, H-4), 1.95 (1 H, m,
H-4'), 4.02 (1 H, m, H-1), 5.97 (1 H, dd, J = 4.7, 3.7 Hz,
H-3); 13C NMR (100 MHz,) δ = - 4.5, - 4.3, 17.4, 18.3,
26.0 (3C), 27.9, 33.9, 70.8, 126.0, 132.1; IR (film):
νmax/cm-1 2949, 2930, 1644, 1252, 1093.
(S)-3-Bromo-4-(tert-butyldimethylsilyloxy)cyclohex-2-
en-1-one 23S. A solution of tert-butyl hydroperoxide in
water (6 M, 0.63 mL, 3.78 mmol) was added dropwise into
a mixture of TBS ether 22S (200 mg, 0.68 mmol), K2CO3
(47 mg, 0.34 mmol), diacetoxyiodobenzene (670 mg, 2.1
mmol) and butyl butyrate (1.5 mL) maintained at 0°C. The
reaction mixture was stirred (0°C, 8 h), diluted with a
mixture of 20% ether in hexane (10 mL), filtered
(diatomaceous earth), and the filtrate concentrated to give a
crude yellow oil. It was purified by column
chromatography (5% Et2O in hexane) to give enone 23S as
a colourless oil (60 mg, 29%); Rf 0.28 (5% Et2O in
hexane); [α]D + 41.5, (c 0.5, CHCl3); HRMS: (LC-
TOFMS) [M+H]+ 305.0560, calcd. for C12H22O2SiBr
(a) 4-Hydroxy-3-methoxycyclohex-2-en-1-one 19aS
new metabolite of 3-methoxyphenol 3a
a
In the large scale biotransformation of phenol 3a, the
major metabolite, cyclohex-2-en-1-one cis-diol 6a (45 g,
38% yield), was isolated by crystallization from the crude
extract.[5e] GC-MS analysis of the retained combined
fractions showed that catechol 13a and hydroquinone 14a
were also present among a mixture of unidentified
metabolites. One very minor metabolite was identified as
2-hydroxy-6-methoxy-6-oxohexanoic acid 15a by LC-
TOFMS analysis: [M+H]+ 177.0758, calcd. for C7H13O5
177.0758; [M+Na]+ 199.0577, calcd. for C7H12O5Na
199.0582; [M+NH4]+ 194.1018, calcd. for C7H16NO5
194.1028. LC-TOFMS analysis of the combined fractions
indicated that another unidentified metabolite was present;
its molecular weight was consistent with structure 19aS.
Separation of the combined fractions by careful column
chromatography (hexane →50% EtOAc in hexane) gave a
pure sample of metabolite 19aS.
1
305.05670; H NMR (400 MHz) δ = 0.15 (3 H, s, SiMe),
0.20 (3 H, s, SiMe), 0.93 (9 H, s, CMe3), 2.07 (1 H, dddd, J
= 13.7, 7.8, 6.0, 4.6 Hz, H-5), 2.25 (1 H, dddd, J = 13.7,
9.1, 4.6, 4.0, H-5' Hz), 2.38 (1 H, ddd, J = 16.9, 7.8, 4.6
Hz, H-6), 2.67 (1 H, ddd, J = 16.9, 9.2, 4.6 Hz, H-6'), 4.50
(1 H, dd, J = 6.0, 4.0 Hz, H-4), 6.44 (1 H, s, H-2); 13C
NMR (100 MHz) δ = - 4.6, - 4.4, 18.3, 25.8 (3C), 32.0,
33.4, 71.0, 132.8, 152.6, 196.0; IR (film) νmax/cm-1 2954,
1689, 1610, 1471.
(4S)-4-Hydroxy-3-methoxycyclohex-2-en-1-one 19aS.[14]
Colourless oil (1.2 g, 1%); Rf 0.15 (50% EtOAc in
hexane); [α]D - 31.9 (c 1.0, CHCl3); HRMS: (TOF-LCMS)
1
[M+H]+ 143.0709, calcd. for C7H11O3 143.0708; H NMR
(S)-4-(tert-Butyldimethylsilyloxy)-3-methoxycyclohex-2-
en-1-one 24S. To a solution of enone 23S (40 mg, 0.13
mmol) in MeOH (2 mL) was added K2CO3 (35 mg, 0.26
mmol), and the mixture kept at room temperature (2 h)
without stirring. The reaction mixture was filtered, the
(400 MHz) δ = 2.01 (1 H, ddddd, J =12.5, 10.1, 7.9, 4.3,
0.6 Hz, H-5), 2.25-2.37 (2 H, m, H-5', H-6), 2.59 (1 H, m,
H-6'), 2.65 (1 H, br s, OH), 3.76 (3 H, s, Me), 4.47 (1 H,
dd, J =8.4, 5.0 Hz, H-4), 5.34 (1 H, s, H-2), 13C NMR (100
12
This article is protected by copyright. All rights reserved.

10.1002/adsc.201700711
Advanced Synthesis & Catalysis
filtrate carefully concentrated in vacuo, and the light
yellow volatile oil obtained was purified by PLC (10%
Et2O in pentane) to furnish methoxy compound 24S as a
colourless oil (23 mg, 69%); Rf 0.2 (5% Et2O in pentane);
HRMS: (ES) [M+H]+ 257.1571, calcd. for C13H25O3Si
0.6, H-3); 13C NMR (100 MHz) δ = 32.6, 35.2, 55.5, 66.5,
118.8, 151.4, 194.0. Addition of (2S, 3S, 4S)-3,4-
dihydroxy-2-methoxycyclohexanone 17acis as substrate to
P. putida UV4, resulted in the formation of metabolite 4-
hydroxy-2-methoxycyclohex-2-enone 25aS and provided
confirmation of its (4S) absolute configuration by
stereochemical correlation.
1
257.1573; H NMR (400 MHz) δ = 0.08 (3 H, s, SiMe),
0.11 (3 H, s, SiMe), 0.89 (9 H, s, CMe3), 1.95-2.14 (2 H,
m, H-5, H-5'), 2.29 (1 H, ddd, J = 16.7, 5.8, 4.4 Hz, H-6),
2.67 (1 H, ddd, J = 16.7, 10.1, 5.0 Hz, H-6'), 3.70 (3 H, s,
OMe), 4.32 (1 H, dd, J = 5.0, 4.1 Hz, H-4), 5.27 (1 H, s, H-
2); 13C NMR (100 MHz) δ = - 5.0, - 4.6, 18.3, 25.8 (3C),
31.0, 32.9, 55.8, 67.2, 102.2, 176.9, 199.3; LRMS: (EI):
217 (100), 257 (5), 304 (60), 445 (50), 1065 (15); IR (film)
νmax /cm-1 2955, 2930, 1670, 1618, 1226, 834.
(d) New metabolites of 2-methylphenol (o-cresol) 4b
Biotransformation (P.putida UV4) of o-cresol 4b was
found
to
yield
(4S,5R,6S)-4,5-dihydroxy-6-
methylcyclohex-2-en-1-one 8b as the only identified
metabolite.[5a] Repeat biotransformation of phenol 4b,
under similar conditions, and GC-MS analysis of a small
portion of the crude concentrate, after extraction with
EtOAc and silylation, showed that catechol 13b and
hydroquinone 16b were present among the minor
metabolites. LC-TOFMS analysis (4.31 min.) and
comparison with an authentic sample, confirmed that
metabolite 8b was the main component: [M+H]+ 143.0702,
calcd. for C7H11O3 143.0708 and [M+Na]+ 165.0516, calcd.
for C7H10O3Na 165.0528. LC-TOFMS analysis also
indicated the presence of 4-hydroxy-3-methylcyclohex-2-
en-1-one 25bS (11.98 min) and cyclohexanone cis-diol
17bcis (3.9 min.) as minor metabolites. PLC purification
(50% EtOAc in hexane) yielded 4-hydroxy-3-
methylcyclohex-2-en-1-one 25bS; the structure and
absolute configuration were established by comparison
with the literature data of its opposite enantiomer.
(S)-4-Hydroxy-3-methoxycyclohex-2-en-1-one 19aS. A
THF solution of tetrabutylammonium fluoride (1 M, 118
µl, 0.12 mmol) was added to a solution of compound 24S
(20 mg, 0.08 mmol) in THF (4 mL) at 0°C. The reaction
mixture was stirred and allowed to warm slowly (2 h) to
10°C. The concentrated reaction mixture was purified by
PLC (50 % EtOAc in hexane) to give hydroxyenone 19aS
as a colourless oil (7 mg, 62%); Rf 0.15 (50% EtOAc in
hexane); [α]D - 48.8 (c 0.53, CHCl3). The synthetic sample
of hydroxyenone 19aS was found to be identical with the
enzymatically formed metabolite 19aS.
(c) New metabolites of 2-methoxyphenol 4a
LC-TOFMS analysis of the aqueous bio-extract, obtained
from the biotransformation of 2-methoxyphenol substrate
4a, showed the presence following metabolites:
cyclohexanone cis-diols 17acis and 17atrans, cyclohex-2-en-
1-one cis diol 8a, hydroquinone 16a, catechol 13a and its
hydroxycarboxylic acid derivative 15a, and some
(4S)-Hydroxy-2-methylcyclohex-2-en-1-one 25bS. Light
yellow oil (23 mg, 0.56 % yield); Rf 0.20 (50% EtOAc in
hexane); [α]D - 49.0 (c 0.7, CHCl3), (Lit.[7] Ent. [α]D + 46.7,
CHCl3); HRMS: (EI) M+ 126.0685, calcd. for C7H10O2
126.0681; 1H NMR (400 MHz) δ = 1.79 (3 H, s, Me), 1.95
(1 H, m, H-5), 2.37 (2 H, m, H-4, H-5), 2.62 (1 H, m, H-6),
2.8 (1 H, br s, OH), 4.55 (1 H, m, H-4), 6.73 (1 H, m, H-3);
13C NMR (100 MHz) δ = 15.9, 33.1, 35.9, 66.9, 135.9,
148.4, 199.9.
unidentified
bioproducts.[5e]
The
large-scale
biotransformation of 2-methoxyphenol 4a and purification
by column chromatography (hexane → EtOAc) had
yielded (2S, 3S, 4S)-cis-diol 17acis (2.5 g) and (2R, 3S, 4S)-
trans-diol 17atrans (0.25 g).[5e] LC-TOFMS analysis of
unidentified pooled chromatography fractions (2.6 g,
eluent 50% EtOAc in hexane), indicated it to be a mixture
(9:1) of two isomeric compounds ([M+H]+ 143), which
could not be separated. This retained concentrated mixture
was re-examined during the study. A pure sample of the
major isomer, separated by multiple elution PLC (2.5%
MeOH in CHCl3), was identified as 4-hydroxy-2-
methoxycyclohex-2-en-1-one 25aS. The minor isomer of
the mixture was found to be indistinguishable from
(2S,3R,4S)-3,4-Dihydroxy-2-methylcyclohexanone
17bcis. A solution of 2-methylcyclohex-2-en-1-one cis-diol
8b (5 mg), in methanol (1 mL) containing 10% Pd/C (ca. 1
mg), was stirred overnight at room temperature, under
hydrogen atmosphere and normal pressure. The catalyst
was filtered off, the filtrate concentrated and the product
purified by PLC (80% EtOAc in hexane), to give the
hydrogenated cyclohexanone cis-diol 17bcis (4.2 mg) as a
colourless oil; Rf 0.32 (75% EtOAc in hexane); [α]D + 1.2
(c 0.35, MeOH); LC-TOFMS: 3.9 min. [M+H]+ 145.0859,
calcd. for C7H13O3 145.0865; [M+NH4]+ 162.1125, calcd.
for C7H16NO3 162.1130; [M+Na]+ 167.0679, calcd. for
metabolite
4-hydroxy-3-methoxycyclohex-2-en-1-one
19aS, which was derived from 3-methoxyphenol 3a.
(4S)-4-Hydroxy-2-methoxycyclohex-2-en-1-one
25aS.
1
C7H12O3Na 167.0684; H NMR (400 MHz) δ = 1.14 (1 H,
Light yellow oil, Rf 0.26 (5% MeOH in CHCl3); [α]D - 29.0
(c 1.1, CHCl3); HRMS: (LC-TOFMS) [M+H]+ 143.0703,
calcd. for C7H11O3 143.0708; [M+Na]+ 165.05216, calcd.
for C7H10O3Na 165.0528; [M+K]+ 181.0261, calcd. for
C7H10O3K 181.0267; [M+H - H2O]+ 125.0523, calcd. for
d, J = 6.9 Hz, Me), 1.63 (2 H, br s, 2x OH), 2.03-2.20 (2 H,
m, H-5, H-5'), 2.33-2.41 (2 H, m, H-6, H-6'), 2.57 (1 H, qd,
J = 6.9, 2.6 Hz, H-2), 4.14 (1 H, m, H-4), 4.17 (1 H, ddd, J
= 11.0, 5.3, 2.6 Hz, H-3); 13C NMR (100 MHz) δ = 10.8,
28.4, 38.0, 47.0, 70.8, 76.9, 209.8. The sample of
compound 17bcis obtained by hydrogenation showed
identical LC-TOFMS data to that of metabolite 17bcis.
1
C7H8O2 125.0524; H NMR (400 MHz) δ = 1.94 (1 H,
dddd, J = 12.6, 11.0, 8.1, 4.5 Hz, H-5), 2.28 (1 H, m, H-5'),
2.40 (1 H, ddd, J = 17.0, 11.1, 4.6 Hz, H-6), 2.56 (1 H, bs,
OH), 2.68 (1 H, ddd, J = 17.0, 6.3, 4.5 Hz, H-6'), 3.60 (3
H, s, OMe), 4.68 (1 H, m, H-4), 5.82 (1 H, dd, J = 3.5 Hz,
13
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10.1002/adsc.201700711
Advanced Synthesis & Catalysis
S. Gronert, S. C. L. Kammerlin, J. R. Keeffe, R. A. More
O’Ferrall, J. Am. Chem. Soc., 2012, 134, 14056-14069.
(e) GC-MS analysis of diTMS derivatives of 4-
hydroxycyclohex-2-en-1-ones 19eR and 19aS and triTMS
derivatives of phenol hydrates (cyclohexa-1,5-diene-1,4-
diols) 19e'R and 19a'S
[4]
a)
J.
C.
Spain,
D.T.Gibson,
Appl.Environ.Microbiol.,1988, 54, 1399-1404; b) J. C
Spain, G. J. Zylstyra, C. K. Blake, D. T. Gibson, Appl. and
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Trimethylsilylation of metabolites 19eR and 19aS with
MSTFA, and GC-MS analyses of the silyl derivatives,
showed two peaks in each case. The major peaks were due
to the monoTMS derivatives of the keto tautomers 19eR
(5.05 min., 88%, [M+H]+, m/z = 184) and 19aS (8.61 min.,
81%, [M+H]+, m/z = 214). The minor peaks (6.40 min.,
12%, [M+H]+, m/z = 256) and (8.69 min., 19%, [M+H]+,
m/z = 286) were consistent with triTMS derivatives of the
corresponding enol tautomers 19e'R and 19a'S.
Acknowledgements
We thank the Department of Education and Learning,
Northern Ireland (to PABMcI) and the Leverhume Trust
(to PH) for postgraduate Studentships.
[5] a) D. R. Boyd, N. D. Sharma, J. F. Malone, C. C. R.
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15
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Advanced Synthesis & Catalysis
FULL PAPER
Enzyme-Catalysed Synthesis of Cyclohex-2-
en-1-one cis-Diols from Substituted Phenols,
Anilines and Derived 4-Hydroxycyclohex-2-
en-1-ones
Adv. Synth. Catal. Year, Volume, Page – Page
Derek R. Boyd,* Narain D. Sharma, Peter B. A.
McIntyre, Paul J. Stevenson,* W. Colin
McRoberts, Amit Gohil, Patrick Hoering and
Christopher C. R. Allen
16
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