Dioxygenase-catalysed dihydroxylation of arene cis-dihydrodiols and acetonide derivatives: A new approach to the synthesis of enantiopure tetraoxygenated bioproducts from arenes

Article abstract of DOI:10.1039/b612191h

cis-Dihydrodiols of anthracene and benz[a]anthracene, and acetonide derivatives of the cis-dihydrodiols of benzene, fluorobenzene, biphenyl and phenanthrene have been identified as substrates for dioxygenase enzymes, yielding the corresponding enantiopure

Full text of DOI:10.1039/b612191h

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Dioxygenase-catalysed dihydroxylation of arene cis-dihydrodiols and
acetonide derivatives: a new approach to the synthesis of enantiopure
tetraoxygenated bioproducts from arenes
Derek R. Boyd,*a Narain D. Sharma,a Tayeb Belhocine,a John F. Malone,a Stuart McGregora and
Christopher C. R. Allenb
Received (in Cambridge, UK) 24th August 2006, Accepted 12th September 2006
First published as an Advance Article on the web 5th October 2006
DOI: 10.1039/b612191h
region, e.g. diols 2 and 6 from phenanthrene and chrysene
respectively. Conversely, no cis-dihydroxylation was observed at
the more accessible regions of highest electron density i.e. the
K-regions of phenanthrene (9,10 bond to give cis-diol 3) or
benz[a]anthracene (5,6 bond to give cis-diol 5). cis-Diols 1(R 5 H,
F, Ph), 2 and 4 are among the bacterial metabolites isolated earlier
and examined as substrates in the current study. The K-region cis-
dihydrodiols 3 and 5, synthesised by chemical dihydroxylation
(RuCl3/NaIO4) of phenanthrene and benz[a]anthracene, were also
tested. Using chrysene or cis-diol metabolite 6, as substrates for
S. yanoikuyae B8/36, revealed the presence of a second, less stable
bioproduct. This was identified as bis(cis-dihydrodiol) 9, the first
member of a new family of tetraol metabolites.2h It was assumed
that the presence of two favoured bay regions in chrysene was an
important factor in the formation of bis(cis-diol) 9.
cis-Dihydrodiols of anthracene and benz[a]anthracene, and
acetonide derivatives of the cis-dihydrodiols of benzene,
fluorobenzene, biphenyl and phenanthrene have been identified
as substrates for dioxygenase enzymes, yielding the correspond-
ing enantiopure arene bioproducts, bis(cis-dihydrodiol)s and
cis-diol acetonides respectively.
Mutant bacterial strains containing dioxygenases, e.g. toluene and
biphenyl dioxygenases (TDO and BPDO), but lacking in the
corresponding diol dehydrogenase enzymes, have been used to
produce cis-dihydrodiol metabolites (cis-diols), from substituted
monocyclic, e.g. 1 using TDO, and polycyclic arenes, e.g. 2, 4 and
6 with BPDO, which have in turn been used widely in synthesis.1a–f
Many arene cis-diol metabolites (.300) have been isolated,1a–f
often in relatively large quantities (10–100 g). However, until the
present, bis(cis-dihydrodiol) 9 was the sole representative member
of this family of PAH metabolites. As part of our quest to find
further members of this elusive family of metabolites, a series of
monocyclic cis-diols 1 (R 5 H, F, Ph) and the bicyclic cis-diol
from naphthalene, all obtained earlier in good yields using P. putida
UV4 (a source of TDO), were examined as substrates for the same
mutant strain. Similarly cis-dihydrodiols 2–6, derived from the
corresponding PAHs, were also tested as substrates for the same
mutant strain containing BPDO. No evidence of bis(cis-dihydro-
diol) formation was found using monocyclic cis-diols 1 (R 5 H, F,
Ph) or PAH cis-diols of naphthalene and phenanthrene (2 or 3).
The bis(cis-diol) metabolite 7 was, however, obtained from
further biotransformation of the non-K-region cis-diol of anthra-
cene 4, using S. yanoikuyae B8/36 under conditions reported
earlier.2f,2g Metabolite 7 was isolated by PLC (20% yield, [a]D
+136, MeOH, .98% ee). Based on spectroscopic (NMR, MS) and
stereochemical correlation studies, its absolute configuration was
assigned as (1R,2S,5R,6S). Under similar conditions, the racemic
K-region cis-diol 5 yielded a bis(cis-dihydrodiol) metabolite 8 after
PLC purification (7% yield, [a]D +106, MeOH, .98% ee). The
recovered sample of cis-dihydrodiol 5 was found to be slightly
enriched in one enantiomer (60% recovered yield, [a]D 28, THF,
6% ee) whose (5R,6S) absolute configuration had been assigned
earlier.3 The exclusive BPDO-catalysed cis-dihydroxylation of the
(5S,6R) enantiomer of cis-diol 5, allied to spectroscopic studies of
the metabolite and its derivatives, allowed the (5S,6R,10S,11R)
absolute configuration to be assigned to bis(cis-dihydrodiol) 8. It is
noteworthy that all members of the PAH bis(cis-dihydrodiol)
BPDO-catalysed cis-dihydroxylations of polycyclic aromatic
hydrocarbons (PAHs), were mainly achieved using the B8/36
mutant strain of Sphingomonas yaniokuyae (originally described
as a Beijerinckia strain).2a–h The active site in this type of
dioxygenase was of sufficient size to accommodate larger PAH
substrates including anthracene,2a,2b phenanthrene,2a,2c benz[a]
anthracene,2d,2e and chrysene.2f–2h Thus, using S. yanoikuyae
B8/36 whole cells, it was possible to obtain cis-diol metabolites,
e.g. 2, 4 and 6 from the corresponding PAHs. Regioselectivity
during BPDO-catalysed arene cis-dihydroxylation was strongly
favoured at relatively hindered arene bonds proximate to a bay
aSchool of Chemistry and Chemical Engineering, Belfast, UK BT9 5AG.
E-mail: dr.boyd@qub.ac.uk; Fax: +44 (0) 2890 976524;
Tel: +44 (0) 2890 974421
bSchool of Biological Sciences, The Queen’s University of Belfast,
Belfast, UK BT9 5AG
4934 | Chem. Commun., 2006, 4934–4936
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family (tetraols 7–9) had similar relative and absolute configura-
tions; this resulted from the second cis-dihydroxylation occurring
on a benzene ring separated by one or two other rings from, and
syn to, the existing cis-diol moiety.
metabolites. Thus, TDO-catalysed dihydroxylation (P. putida
UV4) of fluorobenzene yielded a mixture of cis-diol enantiomers
1S and 1R (ca. 80 : 20, R 5 F). This mixture was converted into the
corresponding acetonide enantiomers 12S and 12R (ca. 80 : 20, 90%
yield). anti-cis-Dihydroxylation was found to occur exclusively on
one acetonide enantiomer 12R, to yield the cis-diol acetonide
(1R,2R,5S,6S)-13R (10% yield, [a]D +77, CHCl3, .98% ee,
Scheme 2) as the sole identified metabolite with a large proportion
of residual substrate. The yield of cis-diol acetonide 13R was much
lower than that of metabolite 11S. This was assumed to be due to
the restricted capacity of the TDO active site to accommodate
larger substrates.
The observation that metabolism of cis-diols 4 and 5 yields
bis(cis-diols) 7 and 8, but that cis-diols of benzene 1 (R 5 H),
substituted benzene 1 (R 5 F, Ph), naphthalene and phenanthrene
(2 and 3) fail to form this type of metabolite, merits explanation.
The relatively hydrophilic nature of the smaller cis-diol precursors
1 (R 5 H, F, Ph) and those from naphthalene could be an
important factor. These cis-diols may already be sufficiently water-
soluble to satisfy the mineralisation/detoxification requirements of
the bacterial cells. While the relative water solubilities of the cis-
diol substrates from PAHs are currently unknown, they may
parallel those of the parent PAHs whose values (mg L21) followed
the sequence naphthalene (31.7) . biphenyl (7.0) . phenanthrene
. (1.29) & anthracene (0.073) & benz[a]anthracene (0.014).4 On
this basis, the formation of the bis(cis-diols) of anthracene 7 and
benz[a]anthracene 8 in low yields may be due to the lower water
solubilities of cis-diol substrates 4 and 5. Further factors for failure
may include (i) the inability of TDO or BPDO to catalyse cis-
dihydroxylations on adjacent PAH rings, e.g. cis-diols of
naphthalene and phenanthrene 3, (ii) bay region steric congestion,
e.g. cis-diol 2 and (iii) instability of the bis(cis-diols).
The more stable acetonide derivative 14 of the (1S,2R)-cis-diol
of biphenyl 1 (R = Ph), was used as a substrate for the BPDO
enzyme (S. yanoikuyae B8/36). Recovered substrate (30% yield)
and a single cis-diol acetonide metabolite (1S,2R,19S,29R)-15 (20%
yield, [a]D +280, .98% ee) were obtained as a result of cis-
dihydroxylation of the phenyl ring. The enantiopurity and absolute
configuration were established by spectroscopic analyses of the
metabolite 15 and bis(acetonide) derivative 16 (90% yield, [a]D
+204, CHCl3, .98% ee), which showed C2 symmetry (Scheme 3).
Finally, having established that acetonide derivatives of cis-diols,
from the monocyclic arene series, can provide single enantiomer
tetraoxygenated bioproducts, using both TDO (11S and 13R) and
BPDO (15), the possibility of a similar process occurring in a
member of the corresponding PAH series was then investigated.
Acetonide 17, from the bay region (3S,4R)-cis-diol 2, was selected
as a substrate of BPDO due to its structural similarity to acetonide
14 and produced two cis-diol acetonides (18 and 19). These were
separated by HPLC (250 6 10 mm Primespher 5 C-18 column
and 20% MeCN in H2O as eluent).
Reduction of the hydrophilicity of the monocyclic cis-dihydro-
diols 1 (R 5 H, F, Ph), was achieved by protection as the
corresponding acetonide derivatives. The biotransformation
(P. putida UV4) of meso-acetonide 10, using reported conditions,5
resulted in an anti-cis-dihydroxylation to give (1S,2R,3S,4S)-cis-
diol acetonide 11S, (80% yield, [a]D +152, CHCl3, .98% ee) as the
sole metabolite (Scheme 1).
The minor metabolite was easily identified as (3S,4R,5R,6S)-cis-
diol 18 (3% yield, [a]D +130, CHCl3, .98% ee). Treatment with
DMP–TsOH yielded the (3S,4R,5R,6S)-bis(acetonide) 20 (90%
yield, [a]D +16, CHCl3). 1H-NMR analysis of compound 20
showed that it also had C2 symmetry. The formation of the minor
metabolite cis-diol acetonide 20 was remarkable, since it must have
resulted from BPDO-catalysed anti-cis-dihydroxylation in the
sterically hindered bay region.
Metabolite 11S is a valuable secondary synthon which is nor-
mally difficult to obtain. The other enantiomer of this acetonide,
11R, is available from osmylation and dehalogenation of the cis-
dihydrodiol acetonides of bromo- or iodobenzene (Scheme 1).
Compound 11R has been widely used as a precursor of pinitols,
conduritols and inositols and other natural products.1a–f In
addition to its potential use in the synthesis of the opposite
enantiomers of natural products, using literature procedures,
further applications are currently in progress as part of our
continuing programme into the synthesis of unnatural products
e.g. carbasugars.6
The absolute configuration of the major bioproduct 19 (15%
yield, [a]D +90, CHCl3) was established as (3S,4R,7S,8R) through a
stereochemical correlation sequence involving catalytic hydrogena-
tion of the alkene bonds (Pd/C, H2; 95% yield) followed by
treatment with DMP–TsOH to give the bis(acetonide) 21 (97%
The new concept of converting a cis-diol into the less
hydrophilic acetonide derivative, to enhance its acceptability as a
substrate, was then tested on two other monocyclic arene cis-diol
Scheme 2
Scheme 3
Scheme 1
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Fig. 1 Crystal structure of one of the two crystallographically indepen-
dent molecules of (+)-cis-(1S,2R)-1,2,3,4-tetrahydro-phenanthrene dicam-
phanate 22bcam
.
In conclusion, this communication provides preliminary evi-
dence of dioxygenase-catalysed cis-dihydroxylation to yield (i)
enantiopure bis(cis-diol) metabolites of PAHs (7, 8) at non-K
region and bay region positions using BPDO, (ii) enantiopure cis-
diol acetonides of monocyclic arenes (11S, 13R, 15) and tricyclic
PAHs (18, 19), using TDO and BPDO enzymes respectively.
We thank CenTACat and HEA NS Programme and Science
Foundation Ireland (NDS) and the European Social Fund (TB,
SMcG) for financial support. We also wish to acknowledge receipt
of the P. putida UV4 and S. yanoikuyae B8/36 strains from the
laboratories of Dr R. Holt (robert.holt@)npilpharma.co.uk) and
Prof. R. Parales (rparales@ucdavis.edu) respectively.
Scheme 4
yield, [a]D +72, CHCl3, Scheme 4). To assign the relative and
(3S,4R,7S,8R) absolute configuration of bis(acetonide) 21
(Scheme 4), a chemical resolution method for racemic cis-1,2,3,4-
tetrahydrodiol 22a–22b was developed. This was achieved by
fractional crystallisation of the corresponding dicamphanate esters
22acam and 22bcam formed from (1S)-camphanic chloride.
Diastereoisomer 22acam ([a]D 2140, CHCl3) was crystallised from
EtOH while a pure sample of the more soluble residual compound
22bcam ([a]D +106, CHCl3) was obtained by crystallisation from a
mixture (3 : 2) of EtOH–CHCl3. The absolute configuration of
dicamphanate 22bcam was established as (1S,2R) by X-ray
crystallography. It had been expected that the configuration would
be determined relative to the known configuration of the
camphanate groups but the fortuitous crystallisation of isomer
22bcam as the CHCl3 solvate led to the independent determination
of configuration as (1S,2R) from the anomalous X-ray scattering
of the solvent Cl atoms{ (Fig. 1).
Hydrolysis of dicamphanate 22acam gave the (1R,2S) enantio-
mer of the tetrahydro-phenanthrene cis-diol 22a ([a]D 2100,
CHCl3); it was then used as a substrate with S. yanoikuyae B8/36.
syn-cis-Dihydroxylation occurred at the pseudo bay region and the
(3S,4R,7S,8R) tetraol 23 was isolated as the sole metabolite (20%
yield, [a]D 2123, MeOH). On hydrogenation, followed by reaction
with DMP–TsOH, bis(acetonide) 21 (95% yield, [a]D +66, CHCl3),
of identical absolute configuration to that formed from metabolite
19, was obtained. Thus, the stereochemical correlation sequence
(Scheme 4) established the absolute configuration of bis(acetonide)
21 as (3S,4R,7S,8R) with .98% ee. The major metabolite, cis-diol
acetonide 19, therefore, should also have been formed via a
BPDO-catalysed syn-cis-dihydroxylation of acetonide 17. The
recovered sample, after addition of racemic cis-diol (22a–22b) as
substrate, was found to be enantioenriched in enantiomer 22b,
indicating that kinetic resolution had occurred during BPDO-
catalysed dihydroxylation.
Notes and references
{ CCDC reference number 618516. For crystallographic data in CIF or
other electronic format see DOI: 10.1039/b612191h
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4936 | Chem. Commun., 2006, 4934–4936
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