Stereoselective cis-dihydroxylation of azulene and related non-aromatic polyenes

Article abstract of DOI:10.1016/S0957-4166(98)00161-X

Dioxygenase-catalysed dihydroxylation of azulene and related non- aromatic polyenes has been found to yield enantiopure chiral cis-diols of synthetic potential.

Full text of DOI:10.1016/S0957-4166(98)00161-X

TETRAHEDRON:
ASYMMETRY
Pergamon
Tetrahedron: Asymmetry 9 (1998) 1831–1834
Stereoselective cis-dihydroxylation of azulene and related
non-aromatic polyenes
Nigel I. Bowers,a,b Derek R. Boyd,a,∗ Narain D. Sharma,a Martina A. Kennedy,a
Gary N. Sheldrake a and Howard Dalton c
aSchool of Chemistry, The Queen’s University of Belfast, Belfast BT9 5AG, UK
bQUESTOR Centre, The Queen’s University of Belfast, Belfast BT9 5AG, UK
cDepartment of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK
Received 10 March 1998; accepted 21 April 1998
Abstract
Dioxygenase-catalysed dihydroxylation of azulene and related non-aromatic polyenes has been found to yield
enantiopure chiral cis-diols of synthetic potential. © 1998 Elsevier Science Ltd. All rights reserved.
Highly functionalised enantiopure compounds containing carbocyclic rings of differing sizes are useful
synthons for natural product synthesis. Although there have been many reports on the isolation and
application of cis-dihydrodiol metabolites of substituted benzenes, over the past decade,1 very little
work has been reported on the metabolism of non-aromatic polyenes. In this paper, we report the
biotransformation of azulene and conjugated monocyclic polyenes, containing 5–8 carbon atoms, to
produce metabolites for use in future synthetic studies.
cis-Dihydroxylation of the bicyclic arene naphthalene, 1, to yield the corresponding cis-dihydrodiol
enantiomer, 2, has been reported using dioxygenase enzymes.2–4 A similar cis-dihydroxylation of the
isoelectronic bicyclic hydrocarbon azulene, 3, has not been previously observed. The present study of
azulene, 3, and a range of related dienes 12, 14, 16, 18 and triene substrates 7 and 10 of differing ring sizes
is part of a wider programme to assess the value of dioxygenase enzymes as biocatalysts for oxidation
reactions of non-aromatic substrates.5,6
∗
Corresponding author. E-mail: DR.BOYD@QUB.AC.UK
0957-4166/98/$19.00 © 1998 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(98)00161-X

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Virtually all examples of cis-dihydroxylation of alkenes, reported using dioxygenases, were aryl
substituted alkenes.5–7 Evidence of cis-dihydroxylation and/or allylic monohydroxylation was initially
sought using whole cell preparations of the bacterium Pseudomonas putida UV4, a source of toluene
dioxygenase (TDO), and polyene substrates.
Azulene, 3, having less aromatic character (resonance energy ca. 45 kcal mol−1) than naphthalene 1
(resonance energy ca. 61 kcal mol−1), also has more options for regioselective cis-dihydroxylation. Thus,
while naphthalene, 1, yielded a single cis-dihydrodiol bioproduct, 2, azulene, 3, could, in principle, yield
three cis-diols due to dihydroxylation at the 1,2-, 4,5-, or 6,7-bonds.
Biotransformation of azulene, 3, using intact cells of P. putida UV4 under reported conditions8 was
found to be regioselective and gave a single metabolite (ca. 20% isolated yield). Based upon chromato-
graphic (TLC, PLC, HPLC), spectral (1H-COSY, NOE and 13C-NMR, IR, MS), microanalytical, and
chiroptical ([α]25 −107, MeOH) data the bioproduct was identified as the cis-diol 4. Metabolite 4 proved
D
to be relatively stable in neutral aqueous solution. cis-Dihydrodiol 2, obtained from naphthalene, 1, was
found to decompose under acidic conditions at a faster rate (×6) compared with cis-diol metabolite 4.
Purification by PLC (silica gel) and recrystallisation yielded compound 4 as a pure product which slowly
decomposed in air at ambient temperature to yield an intractable polymeric material. This behaviour
is consistent with a fulvene structure. Further confirmation of the cis-diol diene (fulvene) structure of
compound 4, was obtained by formation of a mixture of stable stereoisomeric cycloadducts 5 when N-
phenyl maleimide was used as a dienophile. The mixture of cycloadducts was separated by preparative
HPLC (Phenomenex Primesphere 5 C-18, MeOH/H20). Conversion of the major cycloadduct, separated
from the diastereomeric mixture 5, to the corresponding cyclic boronate derivatives, 6, formed using
(+)-(R)- and (−)-(S)-2-(1-methoxyethyl)-phenyl boronic acid (MPBA),9 indicated that the enantiomeric
excess (ee) value of the cis-diol metabolite 4 was ≥98%. The novelty of this oxidation lies in the
concomitant loss of aromaticity in both rings. Previous dioxygenase-catalysed cis-dihydroxylations of
polycyclic arenes, e.g. naphthalene, 1 involved only the loss of aromaticity in one ring.
Azulene 3 may be considered as two cyclic sub-units, i.e. an electron deficient cyclohexatriene ring and
an electron rich cyclopentadiene or fulvene ring. It is noteworthy that TDO-catalysed cis-dihydroxylation
of 3 occurs exclusively at the more electron deficient 4,5-double bond and ring (cycloheptatriene).

N. I. Bowers et al. / Tetrahedron: Asymmetry 9 (1998) 1831–1834
1833
Table 1
Chiroptical data for cis-diols 8, 11, 13, 15, 17 and 19
In order to gain further information on the regioselectivity and facial selectivity of TDO-catalysed cis-
dihydroxylation of azulene, 3, the related conjugated triene structures cycloheptatriene, 7, and dimethyl
fulvene, 10, and also the conjugated cyclic dienes, 12, 14, 16 and 18, were added as substrates to cultures
of P. putida UV4. The isolated yields (ca. 30%) of chiral cis-diol bioproducts have not been optimised.
The specific rotations [α]25, and ee values obtained are listed and compared with the literature [α]25
D
D
values using identical solvents10 in Table 1.
TDO-catalysed cis-dihydroxylation of cycloheptatriene, 7, yielded a mixture of chiral diol 8 and
achiral diol 9 in the ratio 2:1. The cis-dihydrodiol derivative 13 from cyclopentadiene 12 was an exception
in this series in having a lower ee value (20%). The absolute configurations of the cis-diol enantiomers
8, 11, 13, 15 and 17, synthesised by the Sharpless asymmetric dihydroxylation method10 (AD-mix-β)
or obtained by the TDO-catalysed method were identical (1R,2S) based upon the reported10 absolute
configurations. Enantiomeric excesses of cis-diols obtained using TDO (4, 8, 11, 15, 17 and 19) were
≥98%, while those synthesised using AD-mix-β10 (8, 11, 13, 15, 17 and 19) were in the range 5–40%.
When the naphthalene dioxygenase (NDO) enzyme system, present in the P. putida NCIMB 8859 and
9816/11 strains, was used as a biocatalyst with azulene 3 or cyclopentadiene 12, no cis-diol product was
isolated. However using the NDO system with substrates 7, 10, 14, 16 and 18 gave the corresponding cis-
dihydrodiols 8, 11, 15, 17 and 19 of identical ee values (≥98%) and absolute configurations, but having
lower yields compared to those obtained using TDO. The NDO biocatalyst, present in P. putida strain
NCIMB 9816/11, was found to be more regioselective than the TDO strain when cycloheptatriene 7 was
used as a substrate; it yielded only the chiral cis-diol 8. Based on the (1R,2S) configuration, assigned to
the cis-diol bioproducts 8, 11, 13, 15 and 17, the configurations of the other metabolites 4 and 19 are
assumed to be analogous, i.e. (4R,5S) and (1R,2S) respectively.
No evidence of TDO-catalysed monohydroxylation was observed at the allylic methylene groups in di-

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N. I. Bowers et al. / Tetrahedron: Asymmetry 9 (1998) 1831–1834
enes 12, 14, 16 and 18 or triene 7. Similarly when triene 10 was used as substrate only cis-dihydroxylation
was observed. Low yields (<5%) of both allylic hydroxylation and alkene cis-dihydroxylation products
were recorded with cyclohexene substrates using the TDO system.7,11 Preliminary biotransformation
data, obtained using acyclic diene substrates (methyl and ethyl substituted butadienes), has confirmed
1
that dioxygenase-catalysed dihydroxylation also occurs in acyclic polyene substrates. H-NMR and
GC–MS analyses indicate that the TDO system catalyses dihydroxylation in a regioselective manner,
e.g. monosubstituted alkenes being preferred over trisubstituted alkenes and trans-disubstituted being
the least attractive type of alkene substrate. The volatility of the butadiene substrates resulted in lower
isolated yields.
In summary, the present findings, allied to the recent report of the formation of cis-diol 11 of
unspecified enantiopurity and absolute configuration from the biotransformation of triene 10 using
isopropylbenzene 2,3-dioxygenase,12 suggest that dioxygenase enzymes of different types can catalyse
the cis-dihydroxylation of cyclic as well as acyclic polyene substrates. The biotransformation route to
cis-diol derivatives of polyenes offers, in terms of stereoselectivity and regioselectivity, advantages over
currently available catalytic asymmetric osmylation methods. The availability of enantiopure cis-diol
bioproducts 4, 8, 11, 15, 17 and 19 in a single enzyme-catalysed step now allows their potential as chiral
precursors in synthesis to be explored and synthetic studies to this end are currently in progress in our
laboratories.
We thank BBSRC (NDS), DENI (NIB), ESF (MAK), and the TDP (NIB) for support of this
programme, Professor R. A. More O’Ferrall (University College Dublin) for stability studies on the
cis-diol 4 and Professor D. T. Gibson (University of Iowa) for provision of the 9816/11 strain of P.
putida.
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