New families of enantiopure cyclohexenone cis-diol, o-quinol dimer and hydrate metabolites from dioxygenase-catalysed dihydroxylation of phenols

Article abstract of DOI:10.1039/b905940g

Toluene dioxygenase-catalysed cis-dihydroxylation of phenols has led to the discovery of new enantiopure cyclohexenone cis-diol, o-quinol dimer and phenol hydrate metabolites having synthetic potential.

Full text of DOI:10.1039/b905940g

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New families of enantiopure cyclohexenone cis-diol, o-quinol dimer
and hydrate metabolites from dioxygenase-catalysed dihydroxylation
of phenolsw
Derek R. Boyd,*a Narain D. Sharma,a John F. Malonea and Christopher C. R. Allenb
Received (in Cambridge, UK) 25th March 2009, Accepted 22nd April 2009
First published as an Advance Article on the web 13th May 2009
DOI: 10.1039/b905940g
Toluene dioxygenase-catalysed cis-dihydroxylation of phenols
has led to the discovery of new enantiopure cyclohexenone cis-
diol, o-quinol dimer and phenol hydrate metabolites having
synthetic potential.
the major bioproduct (95% relative yield) along with catechol
2d (1% relative yield). PLC purification of the bioproduct
mixture also yielded the cyclohexenone cis-diol 6dR ([a]D +94,
4% relative yield) as a new metabolite (Scheme 2).
The assumption that both compounds 2d and 6dR were
derived from p-xylenol 1d was confirmed by addition of
substrate 1d and isolation of 6dR as the major bioproduct
(17% yield) with catechol 2d (9% yield) as the minor
metabolite. In addition, one more minor metabolite, 11d, of
lower polarity than metabolite 6dR was also isolated. The
structure and absolute configuration of enone cis-diol 6dR
was assigned on the basis of spectroscopic data and an
X-ray crystal structure analysis of the monocamphanate
(Camph.) derivative 7d ([a]D +39, Fig. 1, CCDC 721025)
formed by reacting with (ꢀ)-(S) camphanic chloride in
pyridine solution. Metabolite (+)-6dR (and thus tautomer 5dR)
was enantiopure ( Z 98% ee) and had the (4S,5R,6S) absolute
configuration.
Oxygenase-catalysed hydroxylation of aromatic rings, to yield
phenols, catechols and hydroquinones, is often the initial step
in their biodegradation.1a–e Bacterial metabolism of phenols
1a–e to yield the corresponding catechols 2a–e and/or hydro-
quinones 3a–d has thus been reported earlier using toluene
(TDO),1a–c naphthalene (NDO),1d o-xylene (XDO),1e and
toluene–biphenyl hybrid dioxygenases (TDO–BPDO) as
biocatalysts (Scheme 1). cis-Dihydrodiols (e.g. 4e1b and 5d1e)
have been proposed as transient intermediates in these oxida-
tions of phenols (e.g. 1e and 1d, respectively) to yield the
corresponding catechol (e.g. 2e) and hydroquinone (e.g. 3d).1d,e
Neither of these cis-dihydrodiol types (4 and 5) have
previously been observed during phenol biotransformations.
The first arene cis-dihydrodiol metabolite (cis-3,5-
cyclohexadien-1,2-diol B from benzene A, R = R0 = H)
was isolated in 1968.2a Over the past decade 4400 members
of this arene-derived cis-dihydrodiol family have been
identified.3a–f Monosubstituted benzene cis-dihydrodiol
metabolites (B, R a H, R0 = H) have since attracted
considerable interest as synthetic precursors of phenols2b
and many other derivatives.3a–f The stability of arene cis-
dihydrodiols B was variable, with some undergoing auto-
catalytic dehydration to the corresponding phenols and thus
loss of chirality.2b Thus, the cis-dihydrodiol metabolite B
of p-xylene A (R = R0 = Me), obtained using Pseudomomas
putida 39/D, a source of TDO, was found to dehydrate in situ
to yield p-xylenol 1d.1a Escherichia coli recombinant strains
containing XDO or TDO/BPDO also gave phenol 1d
The third metabolite of p-xylenol 1d, purified by PLC
(7% yield), was identified as o-quinol dimer 11d (R00 = H,
[a]D +62). NMR spectroscopic analysis and an X-ray crystal
structure analysis of the corresponding monocamphanate 12d
(R = R0 = Me, R00 = Camph., Fig. 2, CCDC 721026) showed
that the dimer 11d was enantiopure (Z 98% ee) and had the
(1R,2S,3R,7S,8S,10R) absolute configuration (Scheme 2).
A possible biosynthetic pathway to metabolite 11d is shown
in Scheme 2. This could involve (i) TDO-catalysed asymmetric
oxidative dearomatisation of phenol 1d to yield the transient
cis-dihydrodiol 8d and the cyclohexenone cis-diol tautomer 9d,
(ii) dehydration to yield the transient o-quinol 10d, (iii)
Diels–Alder cyclodimerisation of compound 10d to give the
o-quinol dimer 11d as a new bacterial metabolite. Dimer 11d
or hydroquinone 3d as bioproducts of p-xylene
(R = R0 = Me) (Scheme 1).1e
A
In the current study, using P. putida UV4 (a source of TDO)
and p-xylene A (R = R0 = Me) as substrate, the previously
reported cis-dihydrodiol (B, R = R0 = Me)1a was isolated as
a School of Chemistry and Chemical Engineering, Queen’s University
Belfast, Belfast, UK BT9 5AG. E-mail: dr.boyd@qub.ac.uk;
Fax: +44 (0)28 90975418; Tel: +44 (0)28 90975419
b School of Biological Sciences, Queen’s University Belfast, Belfast,
UK BT9 5AG
w Electronic supplementary information (ESI) available: Full experi-
mental procedures and characterization data for all new compounds.
CCDC 721024–721026. For ESI and crystallographic data in CIF or
other electronic format see DOI: 10.1039/b905940g
Scheme 1 Dioxygenase-catalysed formation of phenols 1, catechols 2
and hydroquinones 3 via cis-dihydrodiols.
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Biotransformation (P. putida UV4) of the less substituted
phenols 1a–c and p-cresol was also examined. While hydro-
quinone 3a and catechol 2a were formed from phenol 1a, no
cyclohexenone cis-diol products were isolated from it or
p-cresol (Schemes 1 and 2). However, o-cresol 1b gave the
corresponding enone cis-diol 6bR ([a]D +223), as a very minor
metabolite (1% yield), among a number of other, as yet
unidentified, bioproducts. Circular dichroism (CD) spectro-
scopy indicated that bioproduct 6bR had an identical absolute
configuration (4S,5R,6S) to cyclohexenone cis-diol 6dR.
m-Cresol 1c proved to be a better substrate than o-cresol 1b
and yielded the corresponding enone cis-diol 6cS (15% yield,
[a]D ꢀ115) and catechol 2c (4% yield, Scheme 1). The CD
spectrum of the m-cresol metabolite 6cS was consistent with it
having the opposite absolute configuration (4R,5S) relative to
enone cis-diols 6bR and 6dR. The enantiopurity values for the
cyclohexenone cis-diols 6bR, 6cS and 6dR were confirmed
( Z 98% ee) using chiral boronate derivatives similar to those
reported earlier for substituted benzene cis-dihydrodiols B.5a–c
As the meta regioisomer 1c was the better substrate, further
studies were conducted on m-iodophenol 1f (Scheme 3) where
the resulting cyclohexanone cis-diol could, in principle,
undergo hydrogenolysis and coupling reactions. This indeed
proved to be a good substrate and yielded the stable enone
cis-diol 6fS ([a]D ꢀ38) as the major bioproduct in variable yield
(30–70%) with catechol 2f and several other minor metabolites
(o10%, Schemes 1 and 3). An anomalous dispersion X-ray
crystal structure analysis of compound 6fS (Fig. 3, CCDC
721024) showed that (i) the OH group proximate to the iodine
substituent adopted a pseudo-equatorial conformation, (ii) a
planar enone group was present, and (iii) it had a (4S,5S)
absolute configuration. The ee value ( Z 98%) was again
determined from its chiral boronate derivative.
Scheme
2 Dioxygenase-catalysed formation of cyclohexenone
cis-diols, and o-quinol dimers from phenols via cis-dihydrodiol
intermediates.
In marked contrast to the cis-dihydrodiol metabolites B
(Scheme 1) of iodobenzene A (R = I, R0 = H) and toluene A
(R = Me, R0 = H), which decomposed rapidly in the presence
of a trace of acid,2b cyclohexenone cis-diols 6b–d and 6f
(Scheme 2) were remarkably stable. Aromatisation however
occurred, with t1/2 values of several hours in 6 M perchloric
Fig. 1 X-Ray crystal structure of compound 7d.
Fig. 2 X-Ray crystal structure of compound 12d.
has recently been isolated as a minor product (7% yield,
[a]D +45.7) of chemical asymmetric oxidative dearomatisation/
dimerisation of 2,4-dimethylphenol.4a
Dimeric sesquiterpenes of similar structure to compound
11d, but with different alkyl substituents (R0), e.g. aquaticol,
have been isolated from plants and chemically synthesised.4a–c
It has been postulated4a–d that the biosynthetic pathway to
these plant metabolites also involves o-quinols as intermediates
in an enzyme-catalysed tandem phenol oxidation–cycloaddition
process. Our current results support this hypothesis.
Scheme 3 Reagents and conditions: (a) P. putida UV4/O2; (b) HClO4;
(c) H2, Pd–C; d) (Ph3P)4Pd, Bu3SnCN, THF; (e) Pd(OAc)2,
NaOAcꢂ3H2O, CO, MeOH.
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the corresponding phenols 1, have recently been detected
during these preliminary studies. This may be indicative of
the general applicability of these new pathways.
Further work, (i) to find other bacterial strains and
dioxygenases capable of producing these new phenol
metabolites, (ii) to optimise the yields of cyclohexenone
cis-diols, and (iii) to utilise them as chiral synthons, is
currently in progress.
Fig. 3 X-Ray crystal structure of compound 6fS.
acid at room temperature, to yield the corresponding hydro-
quinones e.g. 3f (Scheme 3). While the enol 5fS is the initial
metabolite of phenol 1f, during the formation of cyclo-
hexenone cis-diol tautomer 6fS, and possibly also during its
aromatisation to yield hydroquinone 3f, it was not detected.
Furthermore, treatment of cyclohexenone cis-diol 6fS with
CH2N2 showed no evidence of enol methylation, and attempts
We thank Prof. R. A. More O’Ferrall and Dr S. N. Rao for
preliminary kinetic results, Dr J. T. G. Hamilton (AFBI) for
valuable LC-MS analytical data. Funding (to NDS) was
provided by CenTACat and SFI (Grant No. 04/IN3/B581).
Notes and references
to form
a cycloadduct from the diene tautomer 5fS
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with 4-phenyl-1,2,4-triazoline-3,5-dione (dienophile) were
unsuccessful.
Hydrogenolysis of enone cis-diol 6fS yielded the
unsubstituted derivative (4R,5S)-6aS (81% yield, [a]D ꢀ217),
a tautomer of cis-dihydrodiol 5aS and a potential metabolite
of phenol 1a (Scheme 3). The enantiomer of 6aS was
synthesised earlier as an intermediate during the chemical
oxidation of quinic acid to hydroquinone 3a.6 Replacement
of the iodine atom in cyclohexenone cis-diol 6fS with other
substituents (e.g. CN and CO2Me) was achieved under similar
conditions to those used on the cis-dihydrodiol of iodobenzene
B (R = I, R0 = H).7a,b Thus, using Stille coupling conditions
(Bu3SnCN, [Ph3P]4Pd), enone cis-diol (4R,5S)-6gS (48% yield,
([a]D ꢀ136) was obtained and Pd-catalysed carbonylation
produced compound (4R,5S)-6hS (66% yield, [a]D ꢀ48,
Scheme 3).
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Cyclohexenone cis-diol 6aS proved to be an excellent sub-
strate for P. putida UV4, yielding two major bioproducts
(13 and 14). (4R)-4-Hydroxy-2-cyclohexenone 138a–c
([a]D +104, 23% yield), also found as one of the minor
metabolites of phenol 1f, is an arene hydrate (keto tautomer)
of phenol 1a. Arene hydrates of acetophenone8d and
naphthalene8e have been reported earlier as metabolites using
P. putida UV4. The other metabolite of compound 6aS was
(1R,2S,4S)-1,2,4-trihydroxycyclohexane 14 ([a]D ꢀ17, 42%
yield). Compounds 138a–c and 14,9a,b hitherto unknown as
phenol metabolites, may have been formed, from the parent
enone cis-diol 6aS, via reductase-catalysed alkene hydro-
genation followed by dehydration or ketone reduction,
respectively. Evidence of both ketone reduction and alkene
hydrogenation of an enone group has been observed earlier
during biotransformations with P. putida UV4.
Preliminary LC-MS and NMR spectroscopy analyses of the
crude mixtures of phenol bioproducts have revealed that the
TDO enzyme, when expressed in an E. coli recombinant strain,
also catalysed the formation of many cyclohexenone cis-diols
as the only identified metabolites. It is probable that other
bacterial strains containing TDO (e.g. P. putida 39D) will also
yield cyclohexenone cis-diols. Further members of the
cyclohexenone cis-diol 6, the o-quinol dimer 11, and phenol
hydrate families 13, formed by bacterial biotransformation of
9 (a) C. Colas, B. Quiclet-Sire, J. Cleophax, J. M. Delaumeny,
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