Multiple site dioxygenase-catalysed cis-dihydroxylation of polycyclic azaarenes to yield a new class of bis-cis-diol metabolites

Article abstract of DOI:10.1039/a902427a

The enhanced stability of new mono-cis-dihydrodiol bacterial metabolites of tricyclic azaarenes has facilitated the dioxygenase-catalysed formation and isolation of the corresponding bis-cis-dihydrodiols (cis-tetraols) and a three step chemoenzymatic route to the derived arene oxide mammalian metabolites.

Full text of DOI:10.1039/a902427a

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Multiple site dioxygenase-catalysed cis-dihydroxylation of polycyclic azaarenes
to yield a new class of bis-cis-diol metabolites
Derek R. Boyd,*a Narain D. Sharma,a Jonathan G. Carroll,a Christopher C. R. Allen,b David A. Clarkeb and
David T. Gibsonc
a School of Chemistry, The Queen’s University of Belfast, Belfast, UK BT9 5AG. E-mail: dr.boyd@qub.ac.uk
b QUESTOR Centre, The Queen’s University of Belfast, Belfast, UK BT9 5AG
c Department of Microbiology and the Center for Biocatalysis and Bioprocessing, The University of Iowa Iowa City,
Iowa, 52242, USA
Received (in Cambridge, UK) 26th March 1999, Accepted 17th May 1999
The enhanced stability of new mono-cis-dihydrodiol bacte-
rial metabolites of tricyclic azaarenes has facilitated the
dioxygenase-catalysed formation and isolation of the corre-
sponding bis-cis-dihydrodiols (cis-tetraols) and a three step
chemoenzymatic route to the derived arene oxide mammal-
ian metabolites.
Other studies have shown that remote site oxidation of PAHs
can occur with animal liver enzymes to yield combinations of
phenol, epoxide and trans-diol derivatives in different benzene
rings.9–12 The small quantities of metabolites, e.g. bis-trans-
dihydrodiols, available from such monooxygenase-catalysed
(cytochrome-P450) oxidations9 are generally insufficient to
allow rigorous structural or stereochemical analysis.
Dioxygenase-catalysed oxidation of mono- and poly-cyclic
arenes by bacteria occurs widely in the environment.1–3
Dioxygenase enzymes catalyse monohydroxylation (at benzylic
and allylic centres), dihydroxylation (at alkene and arene
bonds), and a combination of both yielding triol bioproducts
(trihydroxylation of alkyl arenes). 1–3 cis-Dihydrodiol bio-
products are however very poor substrates for arene dioxyge-
nase enzymes and prior to this communication no report of
remote site bis-cis-dihydroxylation (tetrahydroxylation) has
appeared.
The biodegradation of polycyclic aromatic hydrocarbons
(PAHs) in eucaryotic systems, e.g. plants, animals and fungi,
has frequently been found to proceed via monooxygenase-
catalysed epoxidation followed by isomerisation to phenols or
epoxide hydrolase-catalysed hydrolysis to trans-dihydro-
diols.4,5 In a typical example the oxidation of acridine 1A using
rat liver enzymes yielded 2-hydroxyacridine and trans-dihy-
drodiol 6 via the arene oxide intermediate 5A (Scheme 1).6–8
cis-Dihydrodiol metabolites resulting from oxidation at the
5,6-bond of the bicyclic azaarenes quinoline,13 2-chloroquino-
line 14 and 2-methoxyquinoline14 were isolated using a mutant
strain (UV4) of the bacterium Pseudomonas putida (a source of
toluene dioxygenase). These bioproducts were found to be
remarkably stable in comparison with their carbocyclic ana-
logues, e.g. the 1,2-cis-dihydrodiol of naphthalene. On this
premise it was anticipated that the corresponding cis-dihy-
drodiol metabolites 2A and 2B, if formed from the tricyclic
azaarenes acridine 1A and phenazine 1B, would be much more
stable and consequently could prove to be valuable synthetic
intermediates.
Acridine 1A was biotransformed using a mutant strain of the
bacterium Sphingomonas yanoikuyae B8/36 (a source of
biphenyl dioxygenase, BPDO) following the reported proce-
dure.15 After removal of the bacterial cells the bioproducts were
then extracted with EtOAc to yield a relatively polar compound
(Rf 0.2, 5% MeOH in CHCl3) which was identified by spectral
methods (NMR, MS) and elemental microanalysis as dihy-
drodiol 2A. 1H NMR spectroscopy established that cis-
dihydroxylation had occurred exclusively at the 1,2-position
(J1,2 4.7 Hz). Reaction of cis-diol 2A ([a]D +72, MeOH) with
(R)-(+)- and (S)-(2)-2-(1-methoxyethyl)phenylboronic acid
(MPBA) yielded the boronate derivatives 4AR and 4AS
respectively. 1H NMR analyses of the boronates confirmed that
cis-diol 2A was enantiopure ( > 98% ee); the absolute config-
uration was determined as (1R,2S) by application of the
empirical 1H NMR rule earlier established for a series of MPBA
derivatives from other cis-dihydrodiol metabolites of
PAHs 15, 16 (Table 1). The (1R,2S) configuration for cis-diol 2A
OH
OH
X
X
N
OH
X
N
OH
_
_
O2 Biphenyl
O2
Biphenyl
dioxygenase
dioxygenase
N
HO
2A X = CH
2B X = N
1A X = CH
1B X = N
OH
3A X = CH
3B X = N
(R)-MPBA
or
(S)-MPBA
A
B
X = CH
X = N
OAc
COBr
Me
O2 Monooxygenase
X = CH Me
R
MeO
R =
H
Me
B
R
S
OAc
O
O
Br
X
O
NaOMe
MeO
H
R =
N
N
N
Me
4AR
and 4AS X = CH
5
7
4BR and 4BS X = N
Table 1 Yields, optical rotations and absolute configurations for metabo-
lites 2A, 2B, 3A and 3B obtained using S. yaniokuyae B8/36, and
derivatives 8, 9 and 5
Epoxide
H2O
hydrolase
OR
OR
OH
OR
N
OR
OH
1
2
1
2
[a]D/1021
6
7
5
6
RO
N
RO
N
N
Isolated
yield (%)
deg cm2 g21
(solvent)
Absolute
configuration
6
OR
OR
Compound
3B R = H
R = Ac
3A R = H
R = Ac
9
8
_
O
2A
2B
3A
3B
8
50–55
40
12
15
95
+72 (MeOH)
+102 (MeOH)
+266 (Pyridine)
+180 (MeOH)
+63 (CHCl3)
+83 (CHCl3)
+30 (CHCl3)
1R,2S
1R,2S
OR
OH
N
N
N
+
1R,2S,5R,6S
1R,2S,6R,7S
1R,2S,5R,6S
1R,2S,6R,7S
1R,2S
+
N
OR
OH
O
_
12
10 R = H
11 R = Ac
9
5
95
55
Scheme 1
Chem. Commun., 1999, 1201–1202
1201

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was independently confirmed by a stereochemical correlation
process involving oxidative degradation of the derived 1,2-dia-
cetoxy-1,2,3,4-tetrahydroacridine to give (2S,3S)-(2)-dimethyl
(2,3-diacetoxy)adipate of known configuration.17
also applicable to other relatively stable cis-dihydrodiol metab-
olites of bi- and tri-cyclic azaarenes, e.g. the cis-dihydrodiols of
2-chloroquinoline (5,6- and 7,8-).14 All arene oxide derivatives
of azaarenes (e.g. acridine 1,2-oxide 5) were found to
hydrolyse, under aqueous conditions, to the corresponding
trans-dihydrodiols (e.g. 6) by exclusive nucleophilic attack at
the allylic position.7,8 The cis-dihydrodiol 2A was also a minor
hydrolysis product of arene oxide 5.8
Recent studies of the bacterial metabolism of tetracyclic
arene substrates, each containing two bay regions (chrysene and
benzo[b]naphtho[2,1-d]thiophene), using S. yanoikuyae B8/36,
have shown the formation of relatively unstable bis-cis-
dihydrodiols as minor metabolites (0.3 and 3% yield, re-
spectively).21 Thus the new family of enantiopure arene tetraol
metabolites arising from sequential cis-dihydroxylation on the
arene Si+Si face is not confined to the linear azaarene series and
more examples are anticipated.
We thank the U.S. Public Health Service grant GM29909
from the National Institute of General Medical Sciences
(D. T. G.), the BBSRC (N. D. S) and the QUESTOR Centre
(C. C. R. A and D. T. C.), for financial support. We also wish to
acknowledge the valuable experimental assistance provided by
Eric Becker and Heiko Nieke (Fachhochschule Mannheim
under the EU Socrates Programme).
In later biotransformation studies of acridine 1A, total
removal of water from the centrifuged culture medium at 35–40
°C under reduced pressure, followed by extraction of the semi-
solid residue with EtOAc–MeOH (9+1) yielded a mixture of
cis-diol 2A and a more polar metabolite (Rf 0.15, 12% MeOH in
CHCl3) which was identified as the bis-cis diol 3A (Table 1) on
the basis of 1H NMR (COSY, NOE) and MS data and formation
of tetraacetate 8. The chirality of the bis-cis-diol 3A suggested
that it was formed by initial cis-dihydroxylation of acridine 1A
at the 1,2-bond on the Si+Si face of the molecule followed by
further cis-dihydroxylation at the 5,6-bond again on the Si+Si
face to yield the (1R,2S,5R,6S) enantiomer exclusively. Con-
firmation that the bis-cis-diol 3A had been derived from the
mono-cis-diol 2A was obtained by its addition as substrate to S.
yaniokuyae B8/36. The samples of bis-cis-diol 3A, isolated
from metabolism of either acridine 1A or the mono-cis-diol 2A,
were found to be indistinguishable.
Biotransformation of phenazine 1B with S. yaniokuyae B8/36
or Pseudomonas putida 9816/11 (a source of naphthalene
dioxygenase, NDO), and the normal extraction procedure
yielded, in both cases, a mono-cis-dihydrodiol (Rf 0.45, 10%
MeOH in CHCl3, 5% yield from NDO and 40% yield from
BPDO) which was identified as cis-1,2-dihydroxy-1,2-dihy-
drophenazine 2B from 1H NMR (J1,2 4.3 Hz) and MS analyses.
Formation of MPBA derivatives 4BR and 4BS of the mono-cis-
Notes and references
1 D. T. Gibson and V. Subramanian, in Microbial Degradation of
Organic Compounds, ed. D. T. Gibson, Marcel Dekker, New York,
1984, p. 181.
2 S. M. Resnick, K. Lee and D. T. Gibson, J. Ind. Microbiol., 1996, 17,
438.
3 D. R. Boyd and G. N. Sheldrake, Nat. Prod. Rep., 1998, 309.
4 D. R. Boyd and N. D. Sharma, Chem. Soc. Rev., 1996, 289.
5 S. C. Barr, N. Bowers, D. R. Boyd, N. D. Sharma, L. Hamilton,
R. A. S. McMordie and H. Dalton, J. Chem. Soc., Perkin Trans. 1, 1998,
3443.
6 K. D. McMurtrey and C. Welch, J. Liq. Chromatogr., 1986, 9, 2949.
7 D. R. Boyd, M. R. J. Dorrity, L. Hamilton, J. F. Malone and A. Smith,
J. Chem. Soc., Perkin Trans. 1, 1994, 2711.
8 D. R. Boyd, R. J. H. Davies, L. Hamilton, J. J. McCullough, J. F.
Malone, H. P. Porter, A. Smith, J. M. Carl, J. M. Sayer and D. M. Jerina,
J. Org. Chem., 1994, 59, 984.
9 K. L. Platt and I. Reischmann, Mol. Pharmacol., 1987, 32, 710.
10 D. R. Thakker, H. J. Yagi, M. Sayer, U. Kapur, W. Levin, R. L. Chang,
A. W. Wood, A. H. Conney and D. M. Jerina, J. Biol. Chem., 1984, 260,
11 249.
11 M. Boroujerdi, H. C. Kung, A. G. E. Wilson and M. W. Anderson,
Cancer Res., 1981, 41, 951.
12 H. Glatt, A. Seidel, O. Ribeiro, C. Kirkby, P. Hirom and F. Oesch,
Carcinogenesis, 1987, 8, 1621.
13 D. R. Boyd, N. D. Sharma, M. R. J. Dorrity, M. V. Hand, R. A. S.
McMordie, J. F. Malone, H. P. Porter, J. Chima, H. Dalton and G. N.
Sheldrake, J. Chem. Soc., Perkin Trans. 1, 1993, 1065.
14 D. R. Boyd, N. D. Sharma, J. G. Carroll, J. F. Malone and C. C. R. Allen,
Chem. Commun., 1998, 683.
15 D. R. Boyd, N. D. Sharma, R. Agarwal, S. M. Resnick, M. J. Schocken,
D. T. Gibson, J. M. Sayer, H. Yagi and D. M. Jerina, J. Chem. Soc.,
Perkin Trans. 1, 1997, 1715.
16 S. M. Resnick, D. S. Torok and D. T. Gibson, J. Org. Chem., 1995, 60,
3546.
17 D. R. Boyd, N. D. Sharma, R. Boyle, J. F. Malone, J. Chima, and H.
Dalton, Tetrahedron: Asymmetry, 1993, 4, 1307.
18 A. Albini, G. F. Bettinetti, E. Fasani and G. Minoli, J. Chem. Soc.,
Perkin Trans. 1, 1978, 299.
19 T. Hiroshi, S. Takashi, I. Masaur, Y.Hisashi and I. Yoshihiko, Agric.
Biol. Chem., 1988, 52, 301.
1
diol 2B and their H NMR analyses established that it was
enantiopure ( > 98% ee) and of (1R,2S) configuration from both
bacterial mutant strains. Application of the improved extraction
procedure (EtOAc–MeOH after removal of water from the
centrifuged bioextracts) led to the isolation of a mixture of
(1R,2S)-mono-cis-diol 2B with a second metabolite (Rf 0.12,
15% MeOH in CHCl3) from the S. yaniokuyae B8/36
biotransformation. This very polar bioproduct was identified as
the phenazine bis-cis-dihydrodiol 3B from NMR, MS and CD
spectral data and formation of tetraacetate 9; the structure was
confirmed by aromatisation (thermal dehydration) and acetyla-
tion of the resulting bis-phenol 10 to yield 1,6-diacetoxy-
phenazine 11.18
When (1R,2S)-mono-cis-diol 2B was added as substrate to S.
yaniokuyae the bis-cis-diol 3B was isolated as the sole
metabolite. The CD spectra of the bis-cis-diols 3A and 3B were
found to be very similar, as anticipated. Thus the absolute
configurations (1R,2S,5R,6S) and (1R,2S,6R,7S) were assigned
for metabolites 3A and 3B, respectively. 1,6-Dihydroxy-
phenazine 10, obtained by dehydration of the metabolite bis-cis-
dihydrodiol 3B, and the derived 1,6-dihydroxyphenazine
5,6-dioxide (iodinin) 12 have also been isolated from among a
range of phenazine antibiotics produced as secondary metabo-
lites in other bacterial systems.19
A further manifestation of the stability of the mono-cis-
dihydrodiol 2A became apparent from the reaction with
2-acetoxyisobutyryl bromide. It was anticipated that the
resulting product, 1-acetoxy-2-bromo-1,2-dihydroacridine 7,
would aromatise spontaneously. However, compound 7 proved
to be sufficiently stable to be isolated and identified by 1H NMR
analysis (crude yield ca. 80%) prior to treatment with NaOMe
to yield (1R,2S)-(+)-1,2-epoxy-1,2-dihydroacridine (acridine
1,2-oxide, 5). Thus the eucaryotic metabolite 5, derived from
acridine 1A, was obtained as a single enantiomer in two steps
with an overall yield of ca. 55% from the procaryotic metabolite
2A. This procedure compares favourably with our earlier
method for the synthesis of enantiopure acridine 1,2-oxide 5, an
eight step synthesis with an overall yield of 18%.4,8 It also
represents a significant improvement over an earlier five step
method for the synthesis of enantiopure arene oxides of PAHs
from the corresponding cis-dihydrodiols.20
20 D. R. Boyd, N. D. Sharma, R. Agarwal, N. A. Kerley, R. A. S.
McMordie, A. Smith, H. Dalton, A. J. Blacker and G. N. Sheldrake,
J. Chem. Soc., Chem. Commun., 1994, 1693.
21 D. R. Boyd, N. D. Sharma, F. Hempenstall, M. A. Kennedy, J. F.
Malone, C. C. R. Allen, S. Resnick and D. T. Gibson, J. Org. Chem.,
1999, in the press.
Preliminary studies have indicated that the two-step synthetic
procedure (2A?7? 5) used for the arene oxide synthesis is
Communication 9/02427A
1202
Chem. Commun., 1999, 1201–1202