Regioselectivity and stereoselectivity of dioxygenase catalysed cis-dihydroxylation of mono- and tri-cyclic azaarene substrates

Article abstract of DOI:10.1039/b810235j

cis-Dihydrodiol metabolites were obtained from dioxygenase-catalysed asymmetric dihydroxylations of five monocyclic (azabiphenyl) and four tricyclic (azaphenanthrene) azaarene substrates. Enantiopurity values and absolute configuration assignments were determined using a combination of stereochemical correlation, X-ray crystallography and spectroscopy methods. The degree of regioselectivity found during cis-dihydroxylation of monocyclic azaarenes (2,3 bond >> 3,4 bond) and of tricyclic azaarenes (bay region > non-bay region bonds) was dependent on the type of dioxygenase used. The cis-dihydrodiol metabolite from an azaarene (3-phenylpyridine) was utilised in the chemoenzymatic synthesis of the corresponding trans-dihydrodiol. The 2008 Royal Society of Chemistry.

Full text of DOI:10.1039/b810235j

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Regioselectivity and stereoselectivity of dioxygenase catalysed
cis-dihydroxylation of mono- and tri-cyclic azaarene substrates†
Derek R. Boyd,*a Narain D. Sharma,a Gerard P. Coen,a Francis Hempenstall,a Vera Ljubez,a John F. Malone,a
Christopher C. R. Allenb and John T. G. Hamiltonb,c
Received 17th June 2008, Accepted 29th July 2008
First published as an Advance Article on the web 5th September 2008
DOI: 10.1039/b810235j
cis-Dihydrodiol metabolites were obtained from dioxygenase-catalysed asymmetric dihydroxylations of
five monocyclic (azabiphenyl) and four tricyclic (azaphenanthrene) azaarene substrates. Enantiopurity
values and absolute configuration assignments were determined using a combination of stereochemical
correlation, X-ray crystallography and spectroscopy methods. The degree of regioselectivity found
during cis-dihydroxylation of monocyclic azaarenes (2,3 bond >> 3,4 bond) and of tricyclic azaarenes
(bay region > non-bay region bonds) was dependent on the type of dioxygenase used. The
cis-dihydrodiol metabolite from an azaarene (3-phenylpyridine) was utilised in the chemoenzymatic
synthesis of the corresponding trans-dihydrodiol.
tative indole cis-dihydrodiol, being an unstable hemiaminal,
spontaneously dehydrated to yield indoxyl which in turn was
Introduction
autoxidised to yield indigo.18 By contrast, the electron-poor
pyridine ring has proved to be much more resistant to dioxygenase-
catalysed cis-dihydroxylation. Thus, TDO-catalysed oxidation
of substituents attached to a pyridine ring, e.g. sulfoxidation
of SR groups,19 monohydroxylation of alkyl groups,20 or cis-
dihydroxylation of benzofused rings,12–16 all occur more readily
than cis-dihydroxylation of a pyridine ring. Indirect evidence
for the TDO-catalysed formation of unstable cis-dihydrodiols as
minor metabolites of a pyridine ring was found when 2-chloro- and
2-methoxy quinolines were both converted to a 2-quinolone cis-
diol13 and hydroxypyridines were obtained from the corresponding
2- and 4-alkyl pyridines.20,21
As our earlier studies of the TDO-catalysed cis-dihydroxylation
of azaarenes were mainly focussed on bicyclic substrates, e.g.
quinoline, isoquinoline, quinoxaline and quinazoline rings,12,13 the
major emphasis of the current programme was on the regio- and
stereo-selectivity of dioxygenase-catalysed cis-dihydroxylation of
monocyclic azaarene analogues of biphenyl 1a (Scheme 1) and of
tricyclic azaarene analogues of phenanthrene 4a containing a bay
region (Scheme 2). These substrates were selected on the basis
of similar steric requirements, i.e. the coplanar conformations
of the monocyclic azaarenes 1b–1d and the planar tricyclic
azaarene analogues of phenanthrene 4a, e.g. benzo[h]quinoline 4b,
benzo[f ]quinoline 4c, phenanthridine 4d and benzo[c]cinnoline 4e.
It was, therefore, anticipated that all of these azaarenes would be
similarly accommodated at the BPDO active site.
The asymmetric dihydroxylation of carbocyclic arenes to yield
the corresponding cis-dihydrodiols, using ring hydroxylating
dioxygenase enzymes as biocatalysts, and their applications in
synthesis, have been widely reported.1–11 Most of the known arene
cis-dihydrodiols have been produced from substituted benzenes
(monocyclic arene substrates) and toluene dioxygenase (TDO)
as biocatalyst. Bicyclic and tricyclic arenes have been mainly
biotransformed to the corresponding cis-dihydrodiols, using naph-
thalene dioxygenase (NDO) and biphenyl dioxygenase (BPDO)
enzymes which have larger active sites. Tetracyclic and pentacyclic
arenes could only be accommodated by the dioxygenase BPDO
having the largest active site; the only acceptable monocyclic arene
substrates for NDO and BPDO dioxygenases appeared to be the
biaryl type e.g. biphenyl 1a.
The oxidative biotransformations of azaaromatic substrates
can, in principle, yield enantiopure bioproducts that are of
potential value as chiral building blocks in the synthesis of target
molecules.1–11 Surprisingly, few azaheterocyclic cis-dihydrodiols
have been isolated, stereochemically assigned and applied as chiral
precursors in synthesis. Different types of dioxygenase enzyme
(NDO, BPDO and phenanthrene dioxygenase, PDO) were used
as biocatalysts in earlier studies for the production of a small
number of cis-dihydrodiols from di-,12,13 tri-14,15 and tetra-cyclic16
azaarenes.
It was assumed that cis-dihydroxylation of an electron-rich
pyrrole ring, e.g. indole, occurred readily to give the corre-
sponding heterocyclic cis-dihydrodiol.17 Unfortunately the pu-
Supporting evidence for this presumption is now presented
through the biotransformation of the monosubstituted benzene
rings bearing pyridine (1b–1d), pyrrole (1e) or pyrazole (1f)
ring substituents, to yield the corresponding carbocyclic cis-
dihydrodiols 2b–2d as major metabolites and of fused benzene
rings in the tricyclic azaarenes 4b–4e to give mainly the corre-
sponding cis-dihydrodiols 5b–5e.
A further objective of this programme was to examine the
comparative effect of different types of dioxygenase enzyme
(TDO, NDO, and BPDO) on the regio- and stereo-selectivity
aSchool of Chemistry and Chemical Engineering, The Queen’s University
of Belfast, Belfast, BT9 5AG, UK. E-mail: dr.boyd@qub.ac.uk; Tel: +44
(0)28-90974421
bSchool of Biological Sciences, The Queen’s University of Belfast, Belfast,
BT9 5AG, UK
cAgri-food and Biosciences Institute for Northern Ireland, Newforge Lane,
Belfast, UK BT9 5PX
† CCDC reference numbers 69114 (2e) and 691142 (2g). For crystallo-
graphic data in CIF or other electronic format see DOI: 10.1039/b810235j
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Scheme 1
Scheme 2
of cis-dihydroxylation of monosubstituted benzene rings bear-
ing an azarene substituent or of benzene rings fused directly
to an azaarene ring. Evidence for the NDO-catalysed cis-
dihydroxylation of the 3,4-bond of azaarene 1b to yield the unusual
cis-dihydrodiol 3b, as a minor metabolite, has now been obtained.
Furthermore, a change in regioselectivity from the preferred bay
region has been observed using a site-directed mutant strain of
NDO (NDOF352V) and the tricyclic azarene substrates 4b and 4d
where the corresponding non-bay region cis-dihydrodiols 6b and
6d have been isolated as major metabolites. The final phase of this
study was designed to show how a typical azaarene cis-dihydrodiol
2c could be utilised in a synthesis of the corresponding trans-
dihydrodiol 17.
bioproduct (87%), but also at the 3,4 bond, to yield the abnormal
regioisomer 3a as a minor metabolite (13%, Scheme 1).22,23
Addition of the azabiaryls 2-phenyl- (1b), 3-phenyl- (1c) and
4-phenyl- (1d) pyridines to whole cells of a constitutive mutant
strain (UV4), of the bacterium Pseudomonas putida (expressing
TDO) gave the corresponding cis-dihydrodiols 2b–2d, only in low
yields (ca.: 1%, Scheme 1). The cis-dihydrodiol metabolite 2b of
2-phenylpyridine 1b had not been found during earlier studies
using TDO as biocatalyst.24 As the phenylpyridine substrates
1b–1d are of almost identical shape to the parent substrate
biphenyl 1a, it was assumed that they would be more acceptable
substrates for BPDO and would thus give higher yields of the
corresponding cis-dihydrodiols. This premise was confirmed by
their biotransformation using whole cells of an inducible mutant
strain (B8/36), of Sphingomomas yanoikuyae (expressing BPDO).
The corresponding cis-dihydrodiols 2b–2d, of identical structure
and absolute configuration to those found using TDO, were
isolated as the only identified bioproducts with yields in the range
of 31–59%.
The cis-dihydrodiols 2b ([a]D +173, MeOH; 59% yield), 2c ([a]D
+249, THF; 49% yield) and 2d ([a]D +181, THF; 31% yield) were
each shown to be single enantiomers (ee >98%) by formation of
the corresponding boronate esters using (+)-(R) and (-)-(S)-2-(1-
methoxyethyl)phenylboronic acid (MEBBA).1H-NMR analysis of
the MEBBA derivatives of cis-diol metabolites 2b–2d also allowed
their absolute configurations to be tentatively assigned as (1S,2R),
employing methods successfully used for other cis-dihydrodiol
Results and discussion
(a) Normal dioxygenase-catalysed cis-dihydroxylation at the 2,3
bond of the monosubstituted benzene substrates 1b–1f
At the beginning of this study, biphenyl 1a was assumed to be
unique among monosubstituted benzenes as the only acceptable
substrate for all three types of dioxygenases used (TDO, NDO
and BPDO). Each of these dioxygenases can catalyse oxidation
at the 2,3 bond of biphenyl 1a, to give enantiopure (1S,2R)-
cis-dihydrodiol 2a with yields increasing in accord with size of
the dioxygenase active site, i.e. in the sequence TDO < NDO
< BPDO.22,23 The NDO-catalysed biotransformation of biphenyl
1a was unusual, since cis-dihydroxylation occurred not only at
the expected 2,3 bond to yield cis-dihydrodiol 2a as the major
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metabolites.25,26 The absolute configuration of (+)-cis-dihydrodiol
2c was then rigorously established as (1S,2R) by hydrogenation of
the 5,6-bond to yield the cis-tetrahydrodiol 2g followed by X-ray
crystallographic analysis using the anomalous dispersion method
(Fig. 1). Compound 2g in the solid state showed the carbocyclic
ring having the half-chair conformation with the hydroxyl group
proximate to the pyridine ring being pseudoaxial. The torsion
angle along the inter-ring bond is +48◦. The (1S,2R) absolute
configurations, assigned to compounds 2b, 2c and 2d by the
MEBBA method, were supported by comparison of their very
similar circular dichroism (CD) spectra.
metabolites of monosubstituted27 and 1,4-disubstituted benzene
substrates.26 Similar preferred pseudoaxial conformations, re-
sulting from reduced steric interactions between substituents R
and proximate hydroxyl groups, would be expected to occur in
solution. The three independent molecules of 2e differed only
in the conformation of the pyrrole ring, with torsion angles of
+14◦, +29◦ and -30◦, respectively, along the inter-ring bond. This
is most likely due to crystal packing factors as each of the six
crystallographically independent hydroxyl groups is involved in
intermolecular hydrogen-bonding, as both a hydrogen donor and
a hydrogen acceptor. cis-Dihydrodiol metabolite 2f was found to be
enantiopure (ee >98%, MEBBA formation) and its CD spectrum
was consistent with a (1S,2R) absolute configuration.
An earlier study had shown that 2-phenylpyridine 1b, 1-
phenylpyrrole 1e and 1-phenylpyrazole 1f were substrates for
BPDO.24 One of the resulting cis-dihydrodiols, 2f, was not isolated
as it appeared to spontaneously decompose via dehydration to
yield the corresponding phenol bioproduct. Although the other
cis-dihydrodiols 2b and 2e were isolated, their optical rotations, ee
values and absolute configurations were not reported.24 While it is
probable that metabolites 2b and 2e isolated earlier have the same
absolute configurations as of those obtained during this study, in
the absence of chiroptical data from the previous study,24 it was
not possible to make a direct stereochemical comparison.
Fig. 1 X-Ray crystal structure of the cis-tetrahydrodiol 2g.
Biotransformations of 1-phenylpyrrole 1e and 1-phenylpyrazole
1f were carried out using P. putida UV4 to give the corresponding
cis-dihydrodiols 2e and 2f (Scheme 1). These were obtained in
higher yield (12%) compared with that of the phenyl pyridine
substrates 1b–1d (ca.: 1%) using P. putida UV4. This improved
cis-dihydroxylation of azaarenes 1e and 1f with P. putida UV4
could be due to their reduced size which allows them to fit more
easily into the smaller active site of the TDO enzyme. It is also
possible that dihydroxylation of the electron-rich azaarene rings
(cf.: the cis-dihydroxylation of the pyrrole ring of indole)17 may
have occurred with the resultant unstable water-soluble cis-diols,
or their ring-opened derivatives, not being isolated.
An X-ray crystal structure analysis of cis-dihydrodiol 2e ([a]D
+144, THF. Fig. 2), using the anomalous dispersion method,
showed the presence of three crystallographically independent
molecules in the solid state. All had the (1S,2R) configuration and
the same conformation for the carbocyclic ring, with a pseudoaxial
hydroxyl group adjacent to the pyrrole ring. This preference for
the hydroxyl group nearest to the bulky (azaarene) substituent in
compound 2e to adopt a pseudoaxial conformation in the solid
state has previously been observed for most other cis-dihydrodiol
(b) Normal dioxygenase-catalysed cis-dihydroxylation at a
bay-region bond of the azaphenanthrene substrates 4b–4e
BPDO-catalysed biotransformation of azaphenanthrenes, benzo-
[h]quinoline 4b, benzo[f ]quinoline 4c, phenanthridine 4d and
benzo[c]cinnoline 4e using S. yanoikuyae B8/36, was found in
each case, to yield the corresponding bay-region cis-dihydrodiol
as the only (or major) isolated bioproducts i.e. 5b, ([a]D +167,
MeOH; 50% yield), 5c ([a]D +153, MeOH; 67% yield), 5d ([a]D
+82, MeOH; 72% yield), and 5e ([a]D -280, pyridine; 62% yield)
(Scheme 2). The biotransformation of phenanthridine 4d, using
an E. coli recombinant strain expressing the PDO gene, had
earlier been reported to yield 5d as one of three metabolites.15
As expected, no evidence of cis-dihydroxylation was found in the
electron-poor pyridine rings in either this or the earlier study.15
Using S. yanoikuyae B8/36 expressing BPDO as biocatalyst,
benzo[h]quinoline substrate 4b was also transformed into the non-
bay-region cis-diol 6b, ([a]D +46, MeOH), as a minor metabolite
(13% yield) which was separated from the major metabolite 5b
by PLC. The pattern of a strong preference for BPDO-catalysed
cis-dihydroxylation at the bay region, observed earlier when
phenanthrene 4a was mainly biotransformed into cis-dihydrodiol
5a (>90%),28,29 was thus also evident in the azaphenanthrene series.
The structures and % ee values (>98%) of the cis-dihydrodiols 5b–
5e and 6b were determined by analysis of the 1H-NMR spectra of
the diols and their MEBBA derivatives.
In the absence of X-ray crystallographic data, a stereochemical
correlation sequence for cis-dihydrodiols 5b and 5d, involving
hydrogenation (5b → 7; 5d → 11), diacetylation (7 → 8; 11 →
12), oxidative cleavage (8 → 9; 12 → 9) and methylation (9 →
10) to yield dimethyl (2,3-diacetoxy)adipate 10 ([a]D +153) of
established (2S,3S) configuration, was used (Scheme 3). This
provided an unequivocal method for the determination of the
(9S,10R) absolute configuration and enantiopurity (>98% ee) of
Fig. 2 X-Ray crystal structure view one of the three independent
molecules of cis-dihydrodiol 2e.
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the corresponding boronate derivatives, was recently obtained
(Scheme 1).30,31 The minor cis-dihydrodiol metabolites 3a22,23
and 3h30,31 are therefore among the relatively few literature
examples of the dioxygenase-catalysed cis-dihydroxylation of
monosubstituted benzene substrates occurring at the 3,4-bond.
In view of the difficulty of obtaining these abnormal cis-
dihydrodiol regioisomers, e.g. 3h (R = Me) and 3i (R = F), from
dioxygenase-catalysed cis-dihydroxylation at the 3,4-bonds of
monosubstituted benzene substrates 1h (R = Me) and 1i (R = F),
new chemoenzymatic methods for their synthesis from the normal
isomers 2h (R = Me) and 2i (R = F) have now been developed.32
Recent studies of the large-scale (>100 g) production of the
normal cis-dihydrodiol metabolite (2i, R = F) of fluorobenzene (1i,
R = F) from our laboratories have produced the first example of
a TDO-catalysed oxidation at the 3,4 bond of a monosubstituted
benzene (unpublished data). This unusual (1S,2R)-cis-dihydrodiol
regioisomer (3i, R = F, [a]D -21, MeOH, ee 20%) (lit.32 [a]D -101,
c 0.5, MeOH, ee >98%) was only present as a minor metabolite
(< 3%) that was isolated along with the normal cis-dihydrodiol
(2i, R = F, >97%).
In order to investigate further the unusual regioselective cis-
dihydroxylation of the 3,4 bond in monosubstituted benzenes,
exemplified by the formation of cis-dihydrodiol 3a catalysed by
NDO,22,23 a comparative metabolism study was carried out, using
an inducible mutant strain of P. putida (9816/11, expressing
NDO) and the monocyclic azaarene 1b as substrate. Although
cis-dihydroxylation of substrate 1b using NDO again occurred
at the 2,3-bond to give diol 2b ([a]D +173, MeOH), the isolated
yield (25%) was lower than that found earlier using BPDO (59%).
1H-NMR analysis of the crude product mixture confirmed that
cis-dihydrodiol 2b was the dominant bioproduct (≥95% relative
yield). Several very weak signals in the relevant baseline section
Scheme 3
(+)-cis-diols 5b and 5d. It also confirmed the validity of the method
adopted earlier for determination of ee values where analysis of the
1H-NMR spectra of the corresponding MEBBA derivatives was
also used in the assignment of absolute configurations to cis-diols
5b–5e (9S,10R) and 6b (7R,8S). A modified Mosher’s method was
used earlier in assigning the (9S,10R) configuration to metabolite
5d but its optical rotation value was not reported.15
1
of the H-NMR spectrum of the crude extract suggested that a
(c) Abnormal dioxygenase-catalysed cis-dihydroxylation at the 3,4
bond of the monosubstituted benzene substrate 1b and at non-bay
region bonds in azaphenanthrenes 4b and 4d
second cis-dihydrodiol, could be present as a very minor (≤5%)
component.
More reliable evidence of the minor cis-dihydrodiol regioi-
somer of compound 2b was found by LC/MS analysis, using
reverse phase chromatography (aqueous MeOH as eluent) and
electrospray ionisation MS of the crude extract; it confirmed the
presence of a major peak (≥95%) with an identical retention time
(7.1 min) and an accurate mass corresponding to cis-dihydrodiol
2b ([M + H]+ 190.08546). However, a minor peak (≤ 5%) eluting
slightly later, at 7.8 min, with an almost identical mass ([M + H]+
190.08567) and fragmentation pattern as the earlier peak, sup-
ported the presence of a regioisomer. MS/MS analysis focussing
on the molecular ion at m/z 190 throughout the analysis period
provided further evidence of a regioisomeric cis-dihydrodiol. The
accurate mass data (172.07503, 144.07786, 78.03393) of the main
product ions, from fragmentation of the minor compound, were
found to be in accordance with those obtained from the major cis-
dihydrodiol 2b. When collision energy of 10 eV was used, the main
fragment ion observed for both the unknown minor metabolite
and compound 2b, was at m/z 172. This facile loss of a molecule
of water, in each case, through aromatisation is entirely consistent
with the structures of compounds 2b and a regioisomer, e.g. 3b,
where the hydroxyl groups are located on adjacent carbon atoms.
Based on the LC-MS results, the biotransformation was re-
peated several times to obtain a sufficient quantity of compound
3b for recording 1H-NMR (2D-COSY), EI MS, CD spectra and an
It is noteworthy that both TDO and BPDO enzymes catalysed
the cis-dihydroxylation of the biaryls 1a–1f, exclusively, at the 2,3
bond to yield the corresponding diols 2a–2f (Scheme 1). Similar
results have been reported for all other monosubstituted benzene
substrates using TDO as biocatalyst.1–11 Earlier studies22,23 showed
that cis-dihydroxylation of biphenyl 1a occurred mainly (87%) at
the 2,3 bond to yield (1S,2R)-cis-dihydrodiol 2a (ee >98%) as
the major bioproduct with P. putida (9816/4, a source of NDO).
However, a significant proportion (13%), of (1S,2R)-dihydrodiol
3a (ee >98%) was also isolated as a result of cis-dihydroxylation
occurring at the 3,4 bond (Scheme 1). Regioselectivity for the 3,4
bond became more marked when a site-directed mutant strain
of E. coli containing a modified form of NDO (NDOF352V) was
employed. Use of this strain, formed by a Phe-352-Val mutation,
occurring near the mononuclear non-heme iron atom in the a sub-
unit of the NDO active site, resulted in a remarkable change in both
regio- and stereo-selectivity. Thus, cis-dihydroxylation occurred
almost exclusively at the 3,4 bond (99%), to yield cis-dihydrodiol
3a with an excess of the opposite (1R,2S) configuration (ee 77%).23
Evidence for ortho-xylene dioxygenase-catalysed cis-
dihydroxylation of toluene 1h occurring at the 2,3 and 3,4
bonds to give cis-dihydrodiols 2h (R = Me, major) and 3h
(R = Me, minor) respectively, based on GC-MS analysis of
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optical rotation. The data collected finally provided unequivocal
evidence for the regioisomer structure 3b, resulting from cis-
dihydroxylation at the 3,4 bond of azaarene 1b. cis-Dihydrodiol
3b was found to be enantiopure (ee >98%) by formation and 1H-
NMR analysis of the corresponding MEBBA derivatives. The CD
spectrum of cis-dihydrodiol 3b confirmed that it had the same
(1S,2R) absolute configuration as for the other cis-dihydrodiols
2b, 2c and 2d.
The more sterically demanding tricyclic azaphenanthrenes 4b–
4e were not substrates for the TDO expressed in P. putida
UV4, but the larger active sites present in NDO and BPDO
were able to accommodate them. Thus, when P. putida 9816/11
(expressing NDO) was used, the corresponding bay-region cis-
dihydrodiols 5b–5e were isolated in low yields (7–17%). Using
S. yanoikuyae B8/36 (expressing BPDO), these cis-dihydrodiols
were obtained in higher yields (50–67%), with identical ee values
(>98%) and absolute configurations i.e. 9S,10R (5b–5e) (Scheme 2,
Table 1).
Despite the similar stereoselectivities, observed during NDO-
and BPDO-catalysed cis-dihydroxylations of azaphenanthrenes
4b–4e to yield mainly the corresponding cis-dihydrodiol metabo-
lites 5b–5e, some differences in regioselectivity were observed
(Scheme 2, Table 1). Benzo[h]quinoline 4b metabolism, using
NDO as biocatalyst, gave (9S,10R)-cis-diol 5b and (7R,8S) cis-
diol 6b (60:40) compared with BPDO (80:20). A marked increase in
regioselectivity for NDO-catalysed cis-dihydroxylation at the 7,8
bond in compound 4b was found when the site-directed mutant
The dramatic change in regioselectivity, obtained using the site-
directed mutant strain E. coli
(source of NDO-352V), is
NDO-F352V
clearly evident compared with a preference for cis-dihydroxylation
of the bay regions present in phenanthrene 4a → 5a, (90%),22,23
benzo[h]quinoline 4b → 5b, (60–80%) and phenanthridine 4d →
5d, (100%), when using NDO and BPDO (Table 1). This metabolic
profile was reversed using NDOF352V where a preference for the
non-bay regions was found using phenanthrene 4a → 6a (83%),22,23
benzo[h]quinoline 4b → 6b (100%) and phenanthridine 4d → 6d
(56%).
cis-Diols 5a, 6a, 5b, 6b, 5c, 5d and 5e, obtained using NDO
and BPDO, were consistently found to be enantiopure (>98% ee)
and of allylic (S) configuration. By contrast, the enantioselectivity
associated with cis-diols formed using NDOF352V, to give bay region
cis-diols produced of the same (S) configuration but with lower %
ee values e.g. 5a (95%)22,23 and 5d (51%) and non-bay region cis-
diols having the opposite allylic (R) configuration e.g. 6a (91%),22,23
6b (>98%) and 6d (84%) (Table 1). The structural changes induced
by mutation of the Phe-352 amino acid in NDO has recently
been shown to result in a different orientation of phenanthrene
4a and thus in an altered pattern of regio- and enantio-selectivity
of cis-dihydroxylation.33 A similar change in orientation could
also account for the observed change in regio- and enantio-
selectivity associated with NDOF352V-catalysed dihydroxylation of
the azaarenes 4b and 4d.
(d) Application of the azarene cis-dihydrodiol 2c in the
chemoenzymatic synthesis of the corresponding
trans-dihydrodiol 17
E. coli
strain was used. It gave the non-bay region cis-
NDO-F352V
diol 6b as the sole metabolite (100%) but in low yield (11%) and
of opposite (7S,8R) configuration (ee >98%) compared with the
normal NDO and BPDO enzymes. Similarly, biotransformation
of phenanthridine 4d, using NDO (P. putida 9816/11) or BPDO
(S. yanoikuyae B8/36), yielded the enantiopure (ee >98%) sole
metabolite (9S,10R)-cis-diol 5d as the result of cis-dihydroxylation
occurring exclusively at the bay-region (Scheme 2, Table 1).
When this biotransformation was repeated using the E. coli
NDO-F352V recombinant strain, the bay region cis-dihydrodiol 5d was
again formed (10% yield) having an identical (9S,10R) absolute
configuration but a much lower enantiopurity (51% ee). cis-
Dihydrodiol 6d was the major bioproduct (12% yield) having an
excess (84%) of the (3R,4S) enantiomer.
To date, few of the cis-dihydrodiol metabolites from azaarene sub-
strates have been employed in synthesis. However cis-dihydrodiols
from bicyclic azaarenes (e.g. quinoline) and tricyclic azaarenes
(e.g. acridine) have been used in the synthesis of arene ox-
ides and trans-dihydrodiols.11,14 cis-Dihydrodiol metabolites of 2-
chloroquinolines have been used as precursors of 2,2¢-bipyridine
ligands in our laboratories (reference 11 and unpublished data).
The synthetic potential of the monocyclic azaarene cis-dihydrodiol
2c was demonstrated by its conversion to the corresponding trans-
dihydrodiol 17. The six-step synthetic sequence involved (i) partial
hydrogenation (2c → 2g), (ii) Mitsunobu inversion (2g → 13),
Table 1 Relative (isolated) yields of cis-dihydrodiol products 5b–5e, 6b and 6d enantiomeric excess values (% ee), and absolute configurations (ab.con.)
Substrate
Enzyme
Relative (isolated) product yield, % ee, ab.con.
Relative (isolated) product yield, % ee, ab.con
4b
5b
6b
NDO
BPDO
NDOF352V
60 (16) >98, 9S,10R
80 (50) >98, 9S,10R
0
40 (10) >98, 7R,8S
20 (13) >98, 7R,8S
100 (11)a >98, 7S,8R
4c
4d
5c
NDO
BPDO
100 (17) >98, 9S,10R
100 (67) >98, 9S,10R
5d
0
0
6d
0
NDO
BPDO
NDOF352V
100 (7) >98, 9S,10R
100 (72)a >98, 9S,10R
44 (10) 51, 9S,10R
5e
100 (16) >98, 9S,10R
100 (62) >98, 9S,10R
0
56 (12) 84, 3R,4S
4e
NDO
BPDO
0
0
a
1H-NMR analysis of the crude extract showed traces of a further cis-dihydrodiol regioisomer that was neither isolated nor identified.
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(iii) diacetylation (13 → 14), (iv) allylic bromination (14 → 15),
(v) dehydrobromination (15 → 16) and (vi) hydrolysis (16 →
17) (Scheme 4). A similar method had recently been used to
synthesise trans-dihydrodiols from the corresponding arene cis-
dihydrodiols.34
Flash column chromatography and PLC were performed on
Merck Kieselgel type 60 (250–400 mesh) and PF254/366 respec-
tively. Merck Kieselgel type 60F254 analytical plates were used
for TLC. Liquid chromatography/mass spectrometry (LC/MS)
analyses were conducted using an Agilent 1100 series HPLC
coupled to an Agilent 6510 Q-TOF (Agilent Technologies, USA).
Separation was performed using a reverse phase column (Luna
C18 (2) 5 mm, 150 ¥ 2.0 mm, Phenomenex, UK) together with
the corresponding guard column (C18, 4 ¥ 2.0 mm, Phenomenex,
UK). The mobile phase consisted of 95% methanol in channel A,
and 5% methanol in channel B. The system was programmed to
perform an analysis cycle consisting of 30% A for 1 min, followed
by gradient elution from 30% to 95% A over a 11 min period,
hold at 95% A for 5 min, return to initial conditions over 5 min
and then hold these conditions for a further 5 min. The flow
rate was 0.20 ml min-1 and the injection volume was 5 ml. MS
experiments were carried out using ESI in positive ion mode with
the capillary voltage set at 4.0 kV. The desolvation gas was nitrogen
set at a◦flow rate of 11 L min-1 and maintained at a temperature
of 350 C. Collision energy values of 10, 20, 30 and 40 eV were
employed for MS/MS experiments and data were collected for
100 ms at each value.
The small scale (0.2–5.0 g) shake flask biotransformations
and bioproduct isolations were carried out using whole cells of
Scheme 4
P. putida UV4 (TDO), P. putida 9816/11 (NDO), E. coli
F352V
(NDOF352V) and S. yanoikuyae B8/36 (BPDO) as sources of the
dioxygenase enzymes, using methods described earlier.12,13,16,22,23,34
The bioproducts were isolated by repeated extraction with EtOAc.
The extracts were dried (Na2SO4), concentrated, and purified using
either PLC or flash column chromatography on silica gel. The ee
values of bioproducts were determined by analysis of the chemical
shift values of methoxyl signals from 1H-NMR spectra of (-)-(S)
and (+)-(R)-2-(1-methoxyethyl)benzeneboronate esters (MEBBA
esters).25,26
Conclusion
A strong preference for cis-dihydroxylation at the 2,3 bond of the
carbocyclic ring of substrates 1b–1f and at a bay region bond
of the tricyclic substrates 4b–4e was observed when NDO and
BPDO enzymes were used. Thus, enantiopure cis-dihydrodiols
2b–2f and 5b–5e were isolated with an allylic (S) configura-
tion, based on stereochemical correlation, X-ray crystallography,
circular dichroism and NMR spectroscopy methods. The first
example of a NDO-catalysed cis-dihydroxylation, occurring at
the 3,4 bond of a monosubstituted azaarene (2b) to yield the
corresponding (1S,2R)-cis-dihydrodiol (3b), was found. Both
regioselectivity and enantioselectivity were found to be reversed
using the modified NDO dioxygenase (NDOF352V); the non-bay
region cis-dihydrodiols 6b and 6d, having an excess of the allylic (R)
enantiomer, were found to be dominant. The general applicability
of a chemoenzymatic route, from monocyclic cis-dihydrodiols to
the corresponding trans-dihydrodiols has been demonstrated by
the conversion of azaarene cis-diol 2c to trans-diol 17.
(+)-(1S,2R)-1,2-Dihydroxy-3-(2¢-pyridyl)cyclohexa-3,5-diene 2b
Substrate 1b, S. yanoikuyae B8/36: colourless oil (0.72 g, 59%);
Rf 0.34 (75% ethyl acetate–hexane); [a]D +173 (c 1.2, MeOH)
(Found: M+, 189.0798. C11H11NO2 requires 189.0789); nmax (neat)
3353 cm-1 (O–H); dH (500 MHz, CDCl3) 4.43 (1 H, m, J1,2 6.2, H-
1), 4.83 (1 H, d, J2,1 6.2, H-2), 6.07–6.11 (2 H, m, J5,4 5.4, H-5, H-6),
6.58 (1 H, d, J4,5 5.4, H-4), 7.01 (1 H, dd, J4¢,3¢ 8.2, J4¢,5¢ 3.7, H-4¢),
7.54 (1 H, d, J3¢,4¢ 8.2, H-3¢), 7.61 (1 H, dd, J5¢,4¢ 3.7, J5¢,6¢ 3.1, H-5¢),
8.44 (1 H, d, J6¢,5¢ 3.1, H-6¢); dC (125 MHz, CDCl3) 67.42, 68.99,
119.96, 122.07, 123.64, 124.65, 131.57, 136.91, 136.18, 148.15,
157.16; m/z (EI) 189 (M+, 41%), 171 (18), 160 (100), 93 (11);
CD: De 5.0 (321 nm) De -2.0 (271 nm), De -8.6 (220 nm); ee >98%
(MEBBA).
Expermental
NMR (1H and 13C) spectra were recorded on Bruker Avance
DPX-300 and DPX-500 instruments and mass spectra were run
at 70 eV, on a VG Autospec Mass Spectrometer, using a heated
inlet system. Accurate molecular weights were determined by the
peak matching method, with perfluorokerosene as the standard.
Elemental microanalyses were carried out on a PerkinElmer 2400
CHN microanalyser. For optical rotation ([a]D) measurements (ca.
20 ◦C, 10-1 deg cm2 g-1), a PerkinElmer 341 polarimeter was used.
Electronic circular dichroism (ECD) spectra were recorded on a
Jasco J-720 instrument in acetonitrile solvent.
Substrate 1b, P. putida 9816/11: colourless oil (0.030 g, 25%);
>95% relative yield by LC-MS analysis; [a]D +170 (c 1.5, MeOH);
ee >98% (MEBBA).
(-)-(1S,2R)-1,2-Dihydroxy-4-(2¢-pyridyl)cyclohexa-3,5-diene 3b
Substrate 1b, P. putida 9816/11: light yellow oil (0.003 g, 2%); Rf
0.20 (5% MeOH–CHCl3); [a]D -28.0 (c 0.32, MeOH) (Found: M+,
189.0790. C11H11NO2 requires 189.0789); dH (500 MHz, CDCl3)
4.32 (1 H, m, H-1), 4.48 (1 H, m, H-2), 6.20 (1 H, dd, J6,1 4.3, J6,5
,
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9.7, H-6), 6.56 (1 H, m, J3,2 3.7, H-3), 6.74 (1 H, d, J5,6, 9.7, H-5),
7.21 (1 H, m, H-5¢), 7.51 (1 H, d, J6¢,5¢ 8, H-6¢), 7.70 (1 H, m, J5¢,6¢
8, J5¢,3¢ 1.8, H-5¢), 8.58 (1 H, dd, J3¢,4¢ 4.9, J3¢,5¢ 1.8, H-3¢); CD: De
2.05 (290 nm), De -4.42 (222 nm), De -1.66 (206 nm) and De -1.85
(204 nm); ee >98% (MEBBA).
isotropic vibration parameters. R1 = 0.074 for 3210 data with
Fo > 4s(Fo), 358 parameters, wR2 = 0.206 (all data), GoF =
-3
˚
1.03, Flack ¥ parameter = -0.07(18), Drmin,max = -0.25/0.31 e A .
CCDC reference number 691141.
(+)-(1S,2R)-1,2-Dihydroxy-3-(1¢-pyrazolyl)cyclohexa-3,5-diene 2f
(+)-(1S,2R)-1,2-Dihydroxy-3-(3¢-pyridyl)cyclohexa-3,5-diene 2c
Substrate 1f, P. putida UV4: colourless oil (0.028 g, 12%); [a]D
+144 (c 0.23, THF) (Found: M+, 178.0742. C9H10N2O2 requires
178.0747); nmax (neat) 3356 cm-1 (O–H); dH (500 MHz, CD3OD)
4.84 (1 H, ddd, J1,2 6.27, J1,5 2.8, J1,6 2.4, H-1), 4.95 (1 H, d, J2,1
6.3, H-2), 6.07 (1 H, dd, J6,1 2.4, J6,5 7.2, H-6), 6.27 (1 H, ddd, J5,1
2.8, J5,4 5.9, J5,6 7.2, H-5), 6.63 (1 H, d, J4,5 5.9, H-4), 6.68 (1 H,
dd, J3¢,2¢ 1.9, J3¢,4¢ 2.5, H-3¢), 7.89 (1 H, d, J2¢,3¢ 1.9, H-2¢), 8.29 (1 H,
d, J4¢,3¢ 2.5, H-4¢); dC (125 MHz, CD3OD) 70.20, 73.49, 109.53,
114.38, 124.38, 131.16, 132.72, 142.36, 143.97; m/z (EI) 178 (M+,
22%), 160 (36), 93 (11); CD: De 1.04 (316 nm), De -3.37 (226 nm);
ee >98% (MEBBA).
Substrate 1c, S. yanoikuyae B8/36: light yellow oil (0.718 g, 49%);
Rf 0.16 (50% ethyl acetate–hexane); [a]D +249 (c 0.83, THF)
(Found: M+, 189.0793. C11H11NO2 requires 189.0789); nmax (neat)
3367 cm-1 (O–H); dH (500 MHz, CDCl3) 4.50 (1 H, d, J2,1 5.9,
H-2), 4.62 (1 H, dd, J1,2 5.9, J1,6 3.1, H-1), 5.99 (1 H, dd, J6,1 3.1,
J6,5 9.6, H-6), 6.13 (1 H, m, J5,4 5.5, J5,6 9.6, H-5), 6.40 (1 H, d,
J
4,5 5.5, H-4), 7.30 (1 H, dd, J5¢,4¢ 7.9, J5¢,6¢ 4.8, H-5¢), 7.87 (1 H, dd,
J4¢,2¢ 2.0, J4¢,6¢ 7.9, H-4¢), 8.48 (1 H, d, J6¢,4¢ 4.8, H-6¢), 8.76 (1 H, d,
2¢,4¢ 2.0, H-2¢); dC (125 MHz, CDCl3) 67.13, 69.20, 122.32, 122.50,
J
122.65, 131.61, 132.31, 134.03, 134.11, 145.53, 146.84; m/z: (EI)
189 (M+, 30%), 171 (91), 160 (100), 93 (9); CD: De 6.0 (312 nm),
De -10.3 (229 nm); ee >98% (MEBBA).
(+)-(9S,10R)-9,10-Dihydrobenzo[h]quinoline-9,10-diol 5b
Substrate 4b, S. yanoikuyae B8/36: light yellow crystalline solid
(3.0 g, 50%); mp 128–129 ◦C (from CHCl3); Rf 0.25 (7%
MeOH/CHCl3); [a]D +167 (c 0.3, MeOH) (Found: M+, 213.0781.
C13H11NO2 requires 213.0789); nmax (KBr) 3408 cm-1 (O–H); dH
(500 MHz, CDCl3) 4.46 (1 H, dd, J9,10 5.0 J9,8 5.4, 9-H), 5.57 (1 H,
d, J10,9 5.0, 10-H), 6.38 (1 H, dd, J8,7 9.5, J8,9 5.4, 8-H), 6.72 (1 H,
d, J7,8 9.6, 7-H), 7.26–7.42 (2 H, m, 5-H, 3-H), 7.73 (1 H, d, J6,5
8.2, 6-H), 8.16 (1 H, dd, J4,3 8.3, J4,2 1.8, 4-H), 8.81 (1H, dd, J2,3
8.2, J2,4 1.8, 2-H); dC (125 MHz, CDCl3) 65.25, 71.72, 120.63,
125.89, 126.78, 127.52, 128.26, 129.40, 129.89, 133.10, 137.23,
147.39, 148.31; m/z (EI) 213 (M+, 19%), 194 (61), 86 (100), 93
(9); CD: De 2.78 (344 nm), De 3.44 (330 nm), De -0.92 (280 nm);
De 5.29 (262 nm); De 6.86 (253 nm); De 1.64 (225 nm); ee >98%
(MEBBA).
(+)-(1S,2R)-1,2-Dihydroxy-3-(4¢-pyridyl)cyclohexa-3,5-diene 2d
Substrate 1d, S. yanoikuyae B8/36: light yellow oil (0.381 g,
31%); Rf 0.11 (50% EtOAc–hexane); [a]D +181 (c 0.85, THF)
(Found: M+, 189.0793. C11H11NO2 requires 189.0789); nmax (neat)
3370 cm-1 (O–H); dH (500 MHz, CDCl3) 4.40 (1 H, dd, J2,1 5.9,
J2,4 1.3, H-2), 4.54 (1 H, dd, J1,2 5.9, J1,6 2.3, H-1), 5.94 (1 H, dd,
J6,1 2.3, J6,5 9.5, H-6), 6.06 (1 H, dd, J5,4 5.6, J5,6 9.5, H-5), 6.47
(1 H, d, J4,5 5.6, H-4), 7.34 (2 H, d, J3¢,2¢ 5.9, J5¢,4¢ 5.9, H-3¢, H-5¢),
8.49 (2 H, d, J2¢,3¢ 5.9, J6¢,5¢ 5.9, H-2¢, H-6¢); dC (125 MHz, CDCl3)
68.21, 70.50, 119.75 (2 ¥ C), 123.64, 124.82, 133.53, 135.74, 146.07,
150.13 (2 ¥ C); m/z (EI) 189 (M+, 25%), 171 (61), 143 (100), 93 (9);
CD: De 3.61 (317 nm), De -6.13 (220 nm); ee >98% (MEBBA).
Substrate 4b, P. putida 9816/11: (0.01 g, 16%) [a]D +167 (c 0.3,
MeOH); ee >98% (MEBBA).
(+)-(1S,2R)-1,2-Dihydroxy-3-(1¢-pyrrolyl)cyclohexa-3,5-diene 2e
Substrate 1e, P. putida UV4: crystalline solid (0.08 g, 12%); mp
102–104 ◦C (EtOAc–hexane); [a]D +144 (c 0.23, THF) (Found:
C, 67.4; H, 6.2, C10H11NO2 requires C, 67.8; H, 6.3%); nmax (KBr)
3347 cm-1 (O–H); dH (500 MHz, CDCl3) 4.46 (1 H, dd, J1,2 6.2,
(+)-(7R,8S)-7,8-Dihydrobenzo[h]quinoline-7,8-diol 6b
Substrate 4b, S. yanoikuyae B8/36: white crystalline solid (0.76 g,
13%); mp 125–126 ◦C (from CHCl3); Rf 0.32 (5% MeOH/CHCl3);
[a]D +46 (c 0.97, MeOH) (Found: M+, 213.0799. C13H11NO2
requires 213.0789); nmax (KBr) 3319 cm-1 (O–H); dH (500 MHz,
CDCl3) 4.48 (1 H, dd, J8,7 = J8,9 4.8, 8-H), 4.89 (1 H, d, J7,8
4.9, 7-H), 6.35 (1 H, dd, J9,10 9.9, J9,8 4.6, 9-H), 7.42 (1 H, dd,
J3,4 8.2, J3,2 4.2, 3-H), 7.79 (1 H, d, J5,6 8.3, 5-H), 7.83 (1 H,
d, J6,5 8.3, 6-H), 7.93 (1 H, d, J10,9 9.9, 10-H), 8.14 (1 H, dd,
J4,3 8.3, J4,2 1.8, 4-H), 8.93 (1 H, dd, J2,3 4.2, J2,4 1.8, 2-H); dC
(125 MHz, CDCl3) 67.71, 71.45, 121.59, 125.21, 125.98, 128.19,
128.40, 128.99, 129.06, 136.57, 137.10, 144.30, 150.55; m/z (EI)
213 (M+, 58%), 194 (60), 167 (100); CD: De +0.17 (273 nm), De
-1.05 (247 nm), De +1.59 (224 nm); ee >98% (MEBBA).
Substrate 4b, P. putida 9816/11: (0.006 g, 10%); [a]D +43 (c 0.3,
MeOH); ee >98% (MEBBA).
J
1,6 5.9, H-1), 4.64 (1 H, dd, J2,1 6.2, J2,4 2.4, H-2), 5.74 (1 H, d, J5,4
2.6, H-5), 5.93 (1 H, d, J6,1 5.9, H-6), 6.00 (1 H, d, J4,2 2.4, J4,5 2.5,
H-4), 6.28 (2 H, m, J2¢,3¢ 4.4, H-2¢, H-5¢), 7.01 (2 H, J3¢,2¢ 4.4, m,
H-3¢, H-4¢); dC (125 MHz, CDCl3) 68.73, 71.11, 110.04, 110.60 ¥
2, 118.95 ¥ 2, 123.20, 128.35, 138.52; m/z (EI) 177 (M+, 24%), 159
(4), 83 (100); CD: De 4.65 (320 nm), De -9.99 (229 nm); ee >98%
(MEBBA).
Crystal data for 2e. C10H11NO2, M = 177.2, monoclinic,
˚
a = 13.076(4), b = 4.980(2), c = 21.362(11) A, b = 103.18(3),
3
˚
U = 1354.5(10) A , T = 293(2) K, Cu-Ka radiation, l =
˚
1.5418 A, space group P21 (no. 4), Z = 6, F(000) = 564, Dx =
1.303 g cm-3, m = 0.75 mm-1, Siemens P3 diffractometer, w
scans, scan range 2◦, 4.0◦ < 2q < 110.1◦, measured/independent
reflections: 3971/3307, Rint = 0.11, direct methods solution, full-
matrix least squares refinement on Fo2, anisotropic displacement
parameters for non-hydrogen atoms; all hydrogen atoms located in
a difference Fourier synthesis but included at positions calculated
from the geometry of the molecules using the riding model, with
(-)-(7R,8S)-7,8-Dihydrobenzo[h]quinoline-7,8-diol 6b
Substrate 4b, E. coliF352V: (0.004 g, 11%); [a]D -46 (c 0.4, MeOH);
CD: De -0.09 (273 nm), De +0.66 (247 nm), De -1.043 (224 nm);
ee >98%. (MEBBA).
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(+)-(9S,10R)-9,10-Dihydrobenzo[f ]quinoline-9,10-diol 5c
6.40 (1 H, dd, J8,7 9.9, J8,9 2.4, 8-H), 7.14 (1 H, dd, J7,8 9.9, J7,9 2.0,
7-H), 7.74 (2 H, m, 3-H, 2-H), 8.34 (2 H, m, 1-H, 4-H); m/z: 214
(M+, 46%), 195 (29), 87 (100); CD: De -0.26 (287 nm), De -0.75
(263 nm); De +1.26 (257 nm); ee >98% (MEBBA).
Substrate 4e, P. putida 9816/11: (0.02 g, 16%); [a]D -280 (c 0.3,
MeOH); ee >98% (MEBBA).
Substrate 4c, S. yanoikuyae B8/36: light yellow crystalline solid
(0.08 g, 67%); Rf 0.25 (7% MeOH–CHCl3); mp 126–128 ◦C
(from CHCl3); [a]D +153 (c 0.3, MeOH) (Found: M+, 213.0791.
C13H11NO2 requires 213.0789); nmax (KBr) 3408 cm-1 (O–H); dH
(500 MHz, CDCl3) 4.74 (1 H, ddd, J9,10 5.5, J9,8 2.1, J9,7 2.5, 9-H),
5.26 (1 H, d, J10,9 5.5, 10-H), 6.07 (1 H, dd, J8,7 9.7, J8,9 2.1, 8-H),
6.57 (1 H, dd, J7,8 9.7, J7,9 2.5, 7-H), 7.45 (2 H, m, 5-H, 2-H), 8.01
(1 H, d, J6,5 8.5, 6-H), 8.51 (1 H, dd, J1,2 8.5, J1,3 1.4, 1-H), 8.82
(1H, dd, J3,2 5.6, J3,1 1.4, 3-H); m/z (EI) 213 (M+, 65%), 194 (33),
86 (100); CD: De -1.74 (311 nm), De +1.13 (257 nm); De -4.82
(223 nm); ee >98% (MEBBA).
Stereochemical correlation sequence of
(+)-(9S,10R)-9,10-dihydrobenzo[h]quinoline-9,10-diol 5b and
(+)-(9S,10R)-9,10-dihydrophenanthridine-9,10-diol 5d with
(-)-(2S,3S)-dimethyl (2,3-diacetoxy)adipate 10
(-)-(9S,10R)-7,8,9,10-Tetrahydrobenzo[h]quinoline-9,10-diol 7.
To a solution of enantiopure cis-dihydrodiol metabolite 5b or
5d (0.2 g, 1.12 mmol) in MeOH (15 cm3) was added 10% Pd/C
(0.010 g), and the mixture stirred (4 h) under an atmosphere of
hydrogen at 1 atm pressure. The catalyst was removed by filtration
and the filtrate concentrated under reduced pressure to give cis-
tetrahydrodiol 7 or 11.
Colourless oil (0.192 g, 80%); [a]D -43 (c 0.5, MeOH) (Found:
C, 72.3; H, 6.0; N, 6.3. C13H13NO2 requires C, 72.5; H, 6.1; N,
6.5%); nmax (neat) 3340 cm-1 (O–H); dH (500 MHz, CDCl3) 1.93–
2.01 (1 H, m, 8-H), 2.25–2.35 (1 H, m, 8¢-H), 2.77–2.86 (1 H, m,
7-H), 2.26–2.38 (1 H, m, 7¢-H), 4.39 (1 H, m, 9-H), 5.48 (1 H, d,
J10,9 3.5, 10-H), 7.34 (1 H, d, J6,5 8.4, 6-H), 7.40 (1 H, dd, J3,4 8.2,
J3,2 6.1, 3-H), 7.67 (1 H, d, J5,6 8.4, 5-H), 8.16 (1 H, dd, J4,3 8.3,
J4,2 1.8, 4-H), 8.83 (1 H, dd, J2,3 6.1, J2,4 1.8, 2-H); dC (125 MHz,
CDCl3) 25.40, 27.04, 65.18, 70.21, 123.67, 126.49, 127.77, 130.18,
131.66, 133.53, 138.35, 147.37, 148.29; m/z (EI) 215 (M+, 21%),
196 (14), 186 (69), 171 (43), 143 (100); CD: De +4.84 (235 nm), De
-1.58 (218 nm), De -2.22 (201 nm).
Substrate 4c, P. putida 9816/11: (0.02 g, 17%); [a]D +153 (c 0.3,
MeOH); ee >98% (MEBBA).
(+)-(9S,10R)-9,10-Dihydrophenanthridine-9,10-diol 5d
Substrate 4d, S. yanoikuyae B8/36: crystalline solid (0.21 g, 72%);
mp 190–192 ◦C (from EtOAc); Rf 0.15 (5% MeOH–CHCl3); [a]D
+82 (c 0.5, MeOH) (Found: C, 72.7; H, 4.7; N, 6.3. C13H11NO2
requires C, 73.2; H, 5.2; N, 6.6%); nmax (KBr) 3390 cm-1 (O–H); dH
(500 MHz, CDCl3) 4.75 (1 H, ddd, J9,10 5.3, J9,8 2.0, J9,7 2.6, 9-H),
5.34 (1 H, d, J10,9 5.4, 10-H), 6.13 (1 H, dd, J8,7 9.8, J8,9 2.0, 8-H),
6.60 (1 H, dd, J7,8 9.8, J7,9 2.6, 7-H), 7.57–7.70 (2 H, m, 2-H, 3-H),
8.05 (1 H, d, J4,3 7.5, 4-H), 8.18 (1H, d, J1,2 8.3, 1-H), 8.67 (1 H, s,
6-H); dC (125 MHz, CDCl3) 65.30, 69.73, 120.51, 125.12, 127.78,
127.95, 128.96, 129.40, 129.81, 133.10, 139.07, 146.93, 152.96; m/z
(EI) 213 (M+, 55%), 194 (34), 141 (100); CD: De -0.48 (305 nm),
De +1.99 (255 nm), De -0.79 (227 nm); ee >98%. (MEBBA).
Substrate 4d, P. putida 9816/11: (0.004 g, 7%); [a]D +83 (c 0.2,
MeOH); ee >98% (MEBBA).
(-)-(9S,10R)-7,8,9,10-Tetrahydrophenanthridine-9,10-diol 11.
Colourless oil (0.180 g, 75%); [a]D -72 (c 0.5, MeOH) (Found: C,
72.3; H, 5.9; N, 6.4 C13H13NO2 requires C, 72.5; H, 6.1; N 6.5%);
nmax (KBr) 3325 cm-1 (O–H); dH (500 MHz, CDCl3) 2.03–2.07
(1 H, m, 8-H), 2.09–2.15 (1 H, m, 8¢-H), 2.95–2.97 (1 H, m, 7-H),
3.07–3.11 (1 H, m, 7¢-H), 4.07 (1 H, m, 9-H), 5.36 (1 H, d, J10,9
4.0, 10-H), 7.51–7.68 (2 H, m, 2-H,3-H), 8.08 (1 H, d, J4,3 8.5,
4-H), 8.23 (1 H, d, J1,2 8.3, 1-H), 8.68 (1 H, s, 6-H); dC (125 MHz,
CDCl3) 25.42, 26.24, 65.13, 69.72, 123.36, 125.90, 127.33, 128.66,
129.81, 131.27, 139.17, 146.90, 151.74; m/z (EI) 215 (M+, 44%),
197 (29), 171 (74), 143 (100); CD: De -0.63 (267 nm), De -0.16
(241 nm), De -5.60 (212 nm).
Substrate 4d, E. coliF352V: (2.98 g, 10%); [a]D +42 (c 0.4, MeOH);
ee 51% (MEBBA).
(-)-(3R,4S)-3,4-Dihydrophenanthridine-3,4-diol 6d
Substrate 4d, E. coliF352V: (3.6 g, 12%); mp 168–170 ◦C
(from EtOAc); Rf 0.20 (5% MeOH–CHCl3); [a]D -26 (c 0.49,
MeOH) (Found: M+, 213.0799. C13H11NO2 requires 213.0790); dH
(500 MHz, CD3OD) 4.62 (1 H, ddd, J3,4 5.2, J3,2 3.2, J3.1 2.0, 3-H),
4.76 (1 H, dd, J4,3 5.2, J4,2 0.7, 4-H), 6.28 (1 H, ddd, J2,1 10.0, J2,3
3.2, J2,4 0.7, 2-H), 7.29 (1 H, dd, J1,2 10.0, J1,3 2.0, 1-H), 7.67 (1 H,
ddd, J8,7 8.0, J8,9 7.0, J8,10 1.0, 8-H), 7.82 (1 H, ddd, J9,10 8.4, J9,8
7.0, J9,7 1.4, 9-H), 8.07 (1 H, d, J7,8 8.3, 7-H), 8.23 (1H, d, J10,9 8.6,
10-H), 9.10 (1 H, s, 6-H); dC (125 MHz, CD3OD) 68.69, 71.37,
120.53, 121.77, 121.82, 127.10, 127.93, 128.45, 130.88, 132.09,
132.54, 147.66, 150.21; m/z (EI) 213 (M+, 27%), 196 (9), 195 (29),
184 (48), 166 (29), 156 (15), 149 (22), 112 (38), 105 (64), 97 (45),
83 (50), 77 (33), 71 (55), 57 (100); CD: De -0.29 (269 nm), De
-2.14 (236 nm), De -0.24 (220 nm), De -0.48 (212 nm); ee 84%
(MEBBA).
Degradation of cis-tetrahydrodiols 7 and 11. cis-Tetrahydrodiol
7 (or 11, 0.1 g, 0.47 mmol) was converted into diacetate 8 (or 11)
by treatment with Ac2O–pyridine. After identification by infra-
red and 1H NMR spectroscopy, the crude diacetate 8 (or 11)
(0.12 g) was dissolved in a mixture of CCl4 (2 cm3), MeCN
(2 cm3) and water (3 cm3). Sodium periodate (3.26 g, 15 mmol)
and ruthenium(II) oxide hydrate (0.005 g) were then added to the
solution. The reaction mixture was stirred at room temperature
for 4 days, a solution of HCl (20 cm3, 1.5 M) added, and the
mixture saturated with NaCl. From the mixture, the product
was extracted with EtOAc (3 ¥ 20 cm3), dried (Na2SO4) and the
solvent evaporated. The residue was dissolved in MeOH (1.5 cm3)
and treated with an excess of diazomethane solution in Et2O
(4 h, 0 ◦C). The solvents and excess diazomethane were removed
under a stream of nitrogen. Purification of the residue by flash
(-)-(9S,10R)-9,10-Dihydrobenzo[c]cinnoline-9,10-diol 5e
Substrate 4e, S. yanoikuyae B8/36: (0.08 g, 62%); Rf 0.35 (7%
MeOH–CHCl3); mp 132–133 ◦C (from MeOH); [a]D -280 (c 1.3,
pyridine) (Found: M+, 214.0760. C12H10N2O2 requires 214.0784);
nmax (KBr) 3408 cm-1 (O–H); dH (500 MHz, CDCl3) 4.73 (1 H,
ddd, J9,10 5.3, J9,8 2.4, J9,7 2.0, 9-H), 5.38 (1 H, d, J10,9 5.3, 10-H),
3964 | Org. Biomol. Chem., 2008, 6, 3957–3966
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-3
chromatography on silica gel (hexane : Et2O, 90 : 10 → 50 : 50)
gave (-)-(2S,3S)-dimethyl (2,3-diacetoxy)adipate 10.
˚
parameter = -0.07(17), Drmin,max = -0.12/0.11 e A . CCDC
reference number 691142.
(ii) (1S,2S)-1,2-Dihydroxy-3-(3¢-pyridyl)cyclohex-3-ene 13. To a
stirring solution of (1S,2R)-1,2-dihydroxy-3-(3¢-pyridyl)cyclohex-
(-)-(9S,10R)-9,10-Diacetoxy-7,8,9,10-tetrahydrobenzo[h]quino-
line 8. Colourless oil; nmax (neat) 1744 cm-1 (C O); dH (500 MHz,
=
3-ene 7 (1.0 g, 5.24 mmol, [a]D -50, THF) in anhydrous benzene
CDCl3) 2.04 (3 H, s, Ac), 2.11 (3 H, s, Ac), 2.23–2.46 (2 H, m, 8-H,
8¢-H), 3.08–3.19 (2 H, m, 7-H, 7¢-H), 5.23 (1 H, m, 9-H), 7.23–7.39
(3 H, m, 3-H, 5-H, 10-H), 7.75 (1 H, d, J6,5 8.2, 6-H), 8.08 (1 H, d,
J4,3 8.3, 4-H), 8.89 (1 H, d, J2,3 5.9, 2-H).
3
˚
(20 cm ) were added dry 3 A molecular sieves (1 g), triphenyl
phosphine (1.51 g, 5.76 mmol) and diethyl azodicarboxylate
(1.0 g, 5.76 mmol). The mixture was stirred for 30 min, p-
nitrobenzoic acid (0.875 g, 5.24 mmol) was then added and the
stirring continued for another 30 min at room temperature. The
mixture was refluxed at 90 ◦C until diol 7 had reacted completely
(monitored by TLC). The reaction mixture was filtered and
the filtrate evaporated under reduced pressure. The residue was
dissolved in MeOH (15 cm3), a 10% aq solution of potassium
carbonate (10 cm3) added to it and the mixture stirred (4 h) at
room temperature. On completion of hydrolysis (3 h, monitored
by TLC), the solvents were removed in vacuo and the crude product
taken up in ethyl acetate (40 cm3). The extract was washed with
saturated brine solution (10 cm3), dried (Na2SO4), and the solvent
removed under reduced pressure. Purification of the residue by
PLC (Rf 0.19, 50% EtOAc–hexane) yielded trans-diol 13 as a
yellow crystalline solid (0.62 g, 62%), mp 183 ◦C (CHCl3–hexane);
[a]D +26 (c 1.25, D2O) (Found: M+, 191.0942. C11H13NO2 requires
191.0946); nmax (KBr) 3355 cm-1 (O–H);dH (500 MHz, CD3OD):
1.68 (1 H, m, 6-H), 1.88 (1 H, m, 6¢-H), 2.18 (1 H, m, J5A,4 4.0, 5-H),
2.29 (1 H, m, 5¢-H), 3.83 (1 H, m, J1,2 4.7, 1-H), 4.28 (1 H, d, J2,1
4.7, 2-H), 6.14 (1 H, t, J4,5A 4.0, 4-H), 7.28 (1 H, dd, J5¢,4¢ 4.1, J5¢,6¢
4.9, 5¢-H), 7.83 (1 H, dd, J4¢,2¢ 2.1, J4¢,5¢ 4.1, 4¢-H), 8.28 (1 H, d, J6¢,5¢
4.9, 6¢-H), 8.53 (1 H, d, J2¢,4¢ 2.1, 2¢-H); dC (125 MHz, CD3OD):
21.49, 24.33, 69.20, 70.99, 122.77, 129.27, 133.59, 133.90, 136.44,
145.99, 146.41; m/z (EI) 191 (M+, 19%), 173 (25), 147 (100).
(-)-(9S,10R)-9,10-Diacetoxy-7,8,9,10-tetrahydrophenanthri-
dine-9,10-diol 12. Colourless oil; nmax (neat) 1740 cm-1 (C O); dH
=
(500 MHz, CDCl3) 2.10 (3 H, s, Ac), 2.12 (3 H, s, Ac), 2.25–2.38
(2 H, m, 8-H, 8¢-H), 3.08–3.29 (2 H, m, 7-H, 7¢-H), 5.25 (1 H, m,
9-H), 6.88 (1 H, d, J10,9 2.9, 10-H), 7.58–7.69 (2 H, m, 2-H, 3-H),
7.84 (1 H, d, J4,3 8.0, 4-H), 8.15 (1 H, d, J1,2 8.3, 1-H), 8.79 (1 H, s,
6-H).
(-)-(2S,3S)-Dimethyl (2,3-diacetoxy)adipate 10. Colourless
oil (0.015 g, 13%), [a]D -14.0 (c 1.0, CHCl3) (lit.35 [a]D -14.1,
CHCl3); nmax (neat) 1736 cm-1 (C O); dH (500 MHz, CDCl3) 1.97
=
(1 H, m, CHH), 2.07 (3 H, s, OAc), 2.06–2.15 (1 H, m, CHH),
2.18 (3 H, s, OAc), 2.37 (2 H, m, CH2), 3.68 (3 H, s, CO2Me), 3.79
(3 H, s, CO2Me), 5.30 (2 H, m, 2-H, 3-H).
Synthesis of trans-(1S,2S)-1,2-Dihydroxy-3-(3¢-pyridyl)-
cyclohexa-3,5-diene 17
(i) (1S,2R)-1,2-Dihydroxy-3-(3¢-pyridyl)cyclohex-3-ene
2g.
cis-Dihydrodiol 2c (0.1 g, 0.53 mmol, [a]D +249, THF, ee >98%)
in methanol solution (20 cm3) containing quinoline (50 ml)
was catalytically hydrogenated (3% Pd/C) at room temperature
and 1 atm pressure. Hydrogen absorption was complete after
4.5 h. The catalyst was removed by filtration and the filtrate
evaporated. The hydrogenated product was purified by PLC to
give yellow coloured crystalline solid (0.089 g, 88%); mp 86–88 ◦C
(CHCl3–hexane); Rf 0.16 (50% EtOAc–hexane); [a]D -50 (c 0.56,
THF) (Found: C, 68.9; H, 7.0; N, 7.1. C11H13NO2 requires C,
69.1; H, 6.9; N, 7.3%); nmax (KBr) 3344 cm-1 (O–H);dH (500 MHz,
CDCl3) 1.77 (1 H, m, 6-H), 1.86 (1 H, m, 6¢-H), 2.24 (1 H, m,
5-H), 2.35 (1 H, m, 5-H¢), 3.82 (1 H, m, J1,2 3.7, 1-H), 4.48 (1 H,
d, J2,1 3.7, 2-H), 6.18 (1 H, m, 4-H), 7.18 (1 H, dd, J5¢,4¢ 3.8, J5¢,6¢
4.9, 5¢-H), 7.78 (1 H, dd, J4¢,2¢ 2.0, J4¢,5¢ 3.8, 4¢-H), 8.33 (1 H, d,
(iii) trans-(1S,2S)-1,2-Diacetyloxy-3-(3¢-pyridyl)cyclohex-3-ene
14. trans-(1S,2S)-1,2-Dihydroxy-3-(3-pyridyl)cyclohex-3-ene
(13) (0.62 g, 3.25 mmol, [a]D +26) was acetylated using Ac2O–
pyridine. PLC yielded diacetate 14 as a light yellow oil (0.867 g,
97%); Rf 0.01 (30% Et2O–hexane); [a]D +83 (c 1.32, CH3OH)
(Found: M+, 275.3011. C15H17NO4 requires 275.3020); nmax (neat)
1759 cm-1 (C O); dH (500 MHz, CDCl3) 1.91 (3 H, s, Ac), 1.99
=
(2 H, m, 6-H, H-6¢-H), 2.01 (3 H, s, Ac), 2.39 (2 H, m, 5-H, 5-H¢),
5.15 (1 H, m, J1,2 4.8, J1,6 6.5, 1-H), 5.91 (1 H, dd, J2,1 4.8, J2,4 0.5,
2-H), 6.31 (1 H, dd, J4,2 0.5, J4,5 2.4, 4-H), 7.24 (1 H, dd, J5¢,4¢ 7.9,
J
6¢,5¢ 4.9, 6¢-H), 8.61 (1 H, d, J2¢,4¢ 2.0, 2¢-H); dC (125 MHz, CDCl3)
24.96, 25.50, 67.64, 69.94, 123.74, 130.60, 134.10, 135.06, 136.49,
147.28, 147.77; m/z (EI) 191 (M+, 13%), 173 (56), 147 (100).
J
J
5¢,6¢ 5.1, 5¢-H), 7.59 (1 H, dd, J4¢,2¢ 1.6, J4¢,5¢ 4.8, 4¢-H), 8.51 (1 H, d,
6¢,5¢ 5.1, 6¢-H), 8.59 (1 H, d, J2¢,4¢ 1.6, 2¢-H); dC (125 MHz, CDCl3)
20.74, 21.04, 22.55, 23.53, 68.67, 70.92, 123.02, 131.86, 133.12,
134.47, 147.57, 148.56, 170.13; m/z (EI) 276 (M + 1, 2%), 275 (6),
215 (25), 155 (100).
Crystal data for 2g. C11H13NO2, M = 191.2, monoclinic, a =
˚
15.561(6), b = 6.152(2), c = 11.984(6) A, b = 122.08(3), U =
3
˚
˚
972.0(7) A , T = 293(2) K, Cu-Ka radiation, l = 1.5418 A, space
group C2 (no. 5), Z = 4, F(000) = 408, Dx = 1.31 g cm-3, m =
0.73 mm-1, Siemens P3 diffractometer, w scans, scan range 2◦,
4.0◦ < 2q < 110.1◦, measured/independent reflections: 2214/997,
Rint = 0.037, direct methods solution, full-matrix least squares
refinement on Fo2, anisotropic displacement parameters for non-
hydrogen atoms; all hydrogen atoms located in a difference
Fourier synthesis but included at positions calculated from the
geometry of the molecules using the riding model, with isotropic
vibration parameters. R1 = 0.032 for 987 data with Fo > 4s(Fo),
130 parameters, wR2 = 0.085 (all data), GoF = 1.14, Flack ¥
(iv) trans-(1S,2S)-1,2-Diacetyloxy-3-(3¢-pyridyl)-5-bromocyclo-
hex-3-ene 15. Freshly crystallised N-bromosuccinimide (0.591 g,
3.32mmol) and a,a-azoisobisbutyronitrile (0.01 g) were added to a
solution of trans-(1S,2S)-1,2-diacetyloxy-3-(3¢-pyridyl)cyclohex-
3-ene 14 (0.83 g, 3.02 mmol, [a]D +83, MeOH) in CCl4 (10 cm3).
The mixture was gently refluxed (~90 ◦C) using a heat lamp. When
the reaction had gone to completion (1 h, 1H-NMR analysis), the
mixture was cooled to room temperature, succinimide filtered off,
and the solvent removed in vacuo. The crude product, obtained as
a yellow oil (0.997 g, 93%), was identified as the title compound by
1H-NMR spectroscopy. dH (500 MHz, CDCl3) 1.90 (3 H, s, Ac),
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2.02 (3 H, s, Ac), 2.50 (1 H, m, 6-H), 2.62 (1 H, m, J6B,1 4.9, 6¢-H),
4.93 (1 H, m, 5-H), 5.45 (1 H, m, J1,6B 4.9, J1,2 3.6, 1-H), 6.06 (1 H,
d, J2,1 3.6, 2-H), 6.37 (1 H, d, 4-H), 7.27 (1 H, dd, J5¢,4¢ 4.9, J5¢,6¢ 5.2,
5¢-H), 7.61 (1 H, dd, J4¢,2¢ 1.6, J4¢,5¢ 4.9, 4¢-H), 8.55 (1 H, d, J6¢,5¢ 5.2,
H-6¢), 8.60 (1H, s, 2¢-H). Due to its unstable nature, it was used
without further purification for the next step of the synthesis.
strain. We gratefully acknowledge valuable assistance provided
by Eric Becker and Friedrich Schmitt (Mannheim University of
Applied Sciences) during preliminary biotransformation studies.
References
1 D. A. Widdowson and D. W. Ribbons, Janssen Chim. Acta, 1990, 8, 3.
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4 S. M. Brown and T. Hudlicky, in Organic Synthesis: Theory and
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(v) trans-(1S,2S)-1,2-Diacetyloxy-3-(3¢-pyridyl)cyclohexa-3,5-
diene 16.
A mixture of trans-(1S,2S)-1,2-diacetyloxy-3-(3¢-
pyridyl)-5-bromocyclohex-3-ene 15 (0.97 g, 2.73 mmol), anhy-
drous lithium chloride (0.325 g, 7.64 mmol) and anhydrous
lithium carbonate (0.505 g, 6.83 mmol) in freshly distilled hex-
amethylphosphoramide (3 cm3) was heated, with stirring at 95 ◦C,
under nitrogen for 2 h. The ice cooled reaction mixture was diluted
with diethyl ether (15 cm3) and then treated dropwise with stirring
with HCl (13.7 cm3, 1 M solution). The ether layer was separated
and the aqueous layer extracted with diethyl ether (2 ¥ 15 cm3). The
combined ether extract was washed with 2.5% NaHCO3 solution
(15 cm3), dried (Na2SO4) and the solvent evaporated. Purification
of the residue by PLC (Rf 0.27, Et2O) yielded trans-diacetate 16 as
a white crystalline solid (0.708 g, 95%); mp 84 ◦C (Et2O–hexane);
[a]D +570 (c 1.03, CHCl3) (Found: M+, 273.2867. C15H15NO4
6 D. R. Boyd and G. N. Sheldrake, Nat. Prod. Rep., 1998, 15, 309.
7 T. Hudlicky, D. Gonzalez and D. T. Gibson, Aldrichimica Acta, 1999,
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8 D. T. Gibson and R. E. Parales, Curr. Opin. Biotechnol., 2000, 11, 236.
9 D. R. Boyd, N. D. Sharma and C. C. R. Allen, Curr. Opin. Biotechnol.,
2001, 12, 564.
10 R. A. Johnson, Org. React., 2004, 63, 117.
11 D. R. Boyd and T. Bugg, Org. Biomol. Chem., 2006, 4, 181.
12 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.
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13 D. R. Boyd, N. D. Sharma, L. V. Modyanova, J. G. Carroll, J. F. Malone,
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requires 273.2862); nmax (KBr) 1727 cm-1 (C O); dH (500 MHz,
=
CDCl3) 2.01 (3 H, s, Ac), 2.07 (3 H, s, Ac), 5.43 (1 H, dd, 1-H),
6.06–6.10 (2 H, m, 2-H, 6-H), 6.36 (1 H, dd, J5,4 5.9, 5-H), 6.60
(1 H, d, J4,5 5.9, 4-H), 7.28 (1 H, dd, J5¢,4¢ 4.2, J5¢,6¢ 4.8, 5¢-H),
7.68 (1 H, dd, J4¢,5¢ 4.2, J4¢,2¢ 2.3, 4¢-H), 8.53 (1 H, d, J6¢,5¢ 4.8, 6¢-
H), 8.70 (1 H, d, J2¢,4¢ 2.3, 2¢-H); dC (125 MHz, CDCl3) 21.37,
21.46, 68.99, 69.56, 123.76, 124.25, 124.95, 127.45, 131.87, 133.13,
147.60, 149.49, 170.5; m/z (EI) 273 (10%), 213 (18%), 171 (100).
14 D. R. Boyd, N. D. Sharma, J. G. Carroll, C. C. R. Allen, D. A. Clarke
and D. T. Gibson, Chem. Commun., 1999, 1201.
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Malone, S. M. Resnick and D. T. Gibson, J. Org. Chem., 1999, 64,
4005.
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and D. T. Gibson, Science, 1983, 222, 167.
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Okazaki, S. Harayama and K. Furukawa, Tetrahedron, 2002, 58, 9605.
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28 D. M. Jerina, H. Selander, H. Yagi, M. C. Wells, J. F. Davey, V.
Mahadevan and D. T. Gibson, J. Am. Chem. Soc., 1976, 98, 5988.
29 M. Koreeda, M. N. Akhtar, D. R. Boyd, J. D. Neill, D. T. Gibson and
D. M. Jerina, J. Org. Chem., 1978, 43, 1023.
30 D. Kim, Y.-S Kim, S.-K. Kim, G. J. Zylstra, Y. M. Kim and E. Kim,
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(vi) trans-(1S,2S)-1,2-Dihydroxy-3-(3¢-pyridyl)cyclohexa-3,5-
diene 17. To a solution of trans-(1S,2S)-1,2-diacetyloxy-3-(3¢-
pyridyl)cyclohexa-3,5-diene 16 (0.75 g, 3.97 mmol, [a]D +570)
in MeOH (10 cm3) were added water and K2CO3 (1.24 g,
9 mmol). The mixture was stirred at room temperature. When
the deacetylation was complete (3 h, by TLC), the inorganic salts
were filtered off and the filtrate concentrated in vacuo. The crude
product was extracted into EtOAc (25 cm3) and subsequently
purified by PLC to yield the trans-dihydrodiol 17 as a light
yellow crystalline solid (0.720 g, 96%); mp 157 ◦C (decomp.)
(CHCl3–hexane); Rf 0.2 (50% EtOAc–hexane); [a]D +189 (c 0.51,
CHCl3) (Found: M+, 189.0791. C11H11NO2 requires 189.0790);
n
max (KBr) 3816 cm-1 (O–H); dH (500 MHz, CDCl3) 4.46 (1 H, dd,
J1,2 6.2, J1,6 5.3, 1-H), 4.73 (1 H, d, J2,1 6.2, 2-H), 6.12 (1 H, dd,
J6,1 5.3, J6,5 4.4, 6-H), 6.22 (1 H, dd, J5,4 5.4, J5,6 4.4, 5-H), 6.36
(1 H, d, J4,5 5.4, 4-H), 7.29 (1 H, dd, J5¢,4¢ 4.3, J5¢,6¢ 5.5, 5¢-H), 7.85
(1 H, d, J4¢,5¢ 4.3, 4¢-H), 8.50 (1 H, d, J6¢,5¢ 5.5, 6¢-H), 8.75 (1 H, s,
2¢-H); dC (125 MHz, CDCl3) 71.08, 71.77, 122.04, 123.62, 124.73,
128.02, 129.05, 133.07, 133.91, 146.98, 148.25; m/z (EI) 189 (M+,
11%), 171 (36), 43 (100); CD: De 4.45 (300 nm), De 3.49 (247 nm),
De 3.66 (227 nm), De -1.641(203 nm).
Acknowledgements
We thank Science Foundation Ireland for funding (Grant no.
04/IN3/B581, NDS), the Queen’s University of Belfast for
studentships (GPC, FH, VL), and Professors David Gibson and
Rebecca Parales (University of Iowa) for providing the E. coli F352V
3966 | Org. Biomol. Chem., 2008, 6, 3957–3966
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