Enzyme-catalysed synthesis and absolute configuration assignments of cis-dihydrodiol metabolites from 1,4-disubstituted benzenes

Article abstract of DOI:10.1002/chem.200601852

A series of ten cis-dihydrodiol metabolites has been obtained by bacterial biotransformation of the corresponding 1,4-disubstituted benzene substrates using Pseudomonas putida UV4, a source of toluene dioxygenase (TDO). Their enantiomeric excess (ee) values have been established using chiral stationary phase HPLC and ¹H NMR spectroscopy. Absolute con_figurations of the majority of cis-dihydrodiols have been established using stereochemical correlation and X-ray crystallography and the remainder have been tentatively assigned using NMR spectroscopic methods but finally confirmed by circular dichroism (CD) spectroscopy. These configurational assignments support and extend the validity of an empirical model, previously used to predict the preferred stereochemistry of TDO-catalysed cis-dihydroxylation of ten 1,4-disubstituted benzene substrates, to more than twenty-five examples.

Full text of DOI:10.1002/chem.200601852

DOI: 10.1002/chem.200601852
Enzyme-Catalysed Synthesis and Absolute Configuration Assignments of
cis-Dihydrodiol Metabolites from 1,4-Disubstituted Benzenes
Derek R. Boyd,*[a] Narain D. Sharma,[a] Gerard P. Coen,[a] Peter J. Gray,[a]
John F. Malone,[a] and Jacek Gawronski[b]
Abstract: A series of ten cis-dihydro-
diol metabolites has been obtained by
bacterial biotransformation of the cor-
responding 1,4-disubstituted benzene
substrates using Pseudomonas putida
UV4, a source of toluene dioxygenase
(TDO). Their enantiomeric excess (ee)
values have been established using
chiral stationary phase HPLC and
1H NMR spectroscopy. Absolute con-
figurations of the majority of cis-dihy-
drodiols have been established using
stereochemical correlation and X-ray
crystallography and the remainder
have been tentatively assigned using
NMR spectroscopic methods but final-
ly confirmed by circular dichroism
(CD) spectroscopy. These configura-
tional assignments support and extend
the validity of an empirical model, pre-
viously used to predict the preferred
stereochemistry of TDO-catalysed cis-
dihydroxylation of ten 1,4-disubstituted
benzene substrates, to more than
twenty-five examples.
Keywords: absolute configuration ·
conformation analysis
· diols ·
enantiopurity · enzymes
Introduction
The initial step in a major
pathway for the bacterial bio-
degradation of aromatic rings
A involves dioxygenase-cata-
lysed cis-dihydroxylation to
Scheme 1. Bacterial mineralisation of 1,2-disubstituted benzenes.
yield the corresponding cis-di-
hydrodiols B. The wild-type
bacterial strains present in the environment contain cis-diol
dehydrogenases, which can in turn convert the cis-dihydro-
diols to catechols C prior to ring opening and further bio-
degradation to yield carbon dioxide and water, that is, min-
eralisation (Scheme 1).
Toluene dioxygenase is the most widely used dioxygenase
enzyme for bacterial biotransformations and has been found
to catalyse the cis-dihydroxylation of benzene[1] and a large
number of mono- and disubstituted benzene substrates in a
regio- and stereoselective manner.[2–12] The TDO enzyme
present in whole cells of the UV4 constitutive mutant strain
of Pseudomonas putida, favours the cis-dihydroxylation of
unsubstituted arene bonds and since the corresponding de-
hydrogenase enzyme (toluene cis-diol dehydrogenase) is
blocked, accumulation of the initial cis-dihydrodiol metabo-
lites occurs.
Based on preliminary biotransformation results obtained
using P. putida UV 4 and range of substituted benzene sub-
strates, it was possible to predict the preferred regio-and ste-
reochemistry of the cis-dihydrodiol bioproducts using a
simple model controlled by the relative steric requirements
of substituents X and Y in substrate A.[13] Initially the model
was developed for the TDO-catalysed cis-dihydroxylation of
a relatively small number of mono- and 1,4-disubstituted
benzene substrates bearing spherically symmetrical substitu-
ents, for example, F, Cl, Br, I, Me, and CF3.[13,14] According
to this predictive model, the cis-dihydroxylation step will
[a] Prof. D. R. Boyd, Dr. N. D. Sharma, Dr. G. P. Coen, P. J. Gray,
Dr. J. F. Malone
School of Chemistry and Chemical Engineering
CenTACat and QUESTOR Centre
The Queenꢀs University of Belfast
Belfast BT9 5AG (UK)
Fax : (+44)909-74-687
E-mail: dr.boyd@qub.ac.uk
[b] Prof. J. Gawronski
Department of Chemistry, A. MickiewiczUniversity, Grunwaldzka 6,
60-780 Poznan (Poland)
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occur on the less substituted bond in the benzene ring A
and the direction of facial attack will favour the absolute
configuration shown by enantiomer B when the steric re-
quirements of substituent X, are significantly larger than
those of Y, for example, using the Charton steric parameter
(u).[15] This model has recently been updated to also allow
predictions for substrates bearing substituents which are
non-spherically symmetrical and whose size will be confor-
mationally dependent but which have a dominant stereodi-
recting effect, for example, SMe,[16] CH=CH2,[17] Et,[18] Pr.[18]
With increasing use now being made of disubstituted ben-
zene cis-dihydrodiols as chiral precursors in synthesis,[19–27]
the development of more convenient and rigorous methods
for stereochemical assignment is very important. The sim-
plest literature method used for the direct determination of
enantiopurity of benzene cis-dihydrodiols is chiral stationary
phase HPLC (CSPHPLC) at ambient temperature.[13,16]
Chiral stationary phase GC (CSPGC) has also been used
but this required the formation of n-butyl boronate deriva-
tives and elevated temperatures.[28,29] Comparison of optical
rotations, and in some cases X-ray crystallographic analy-
sis,[30,31] of cis-dihydrodiols and derivatives, has also been
used to confirm their enantiopurity. Other methods that
have been developed to confirm ee values include NMR
analysis after stabilization of the cis-dihydrodiol metabolites
of monosubstituted benzene substrates by formation of a
single diastereoisomeric cycloadduct or regioselective hydro-
genation of the unsubstituted alkene bond. This is followed
by formation of diastereoisomeric di-MTPA esters using
both R and S forms of the a-methoxy-a-(trifluoromethyl)
phenylacetic acid (MTPA).[31,32] The synthesis of diastereo-
isomeric boronate dervatives using (R)- and (S)-2-(1-me-
thoxyethyl)phenyl boronic acids followed by 1H NMR analy-
sis has also been used.[15,33]
site of the TDO enzyme,[13] and in particular the stereodi-
recting effect of the non-spherically symmetrical but rigid
nitrile group and ii) the general applicability of confronta-
tion of experimental and calculated CD spectra as a method
for determining both the preferred conformations and abso-
lute configurations.[35]
Results and Discussion
Biotransformation of the 1,4-disubstituted benzene sub-
strates A using P. putida UV4 yielded the corresponding cis-
dihydrodiols B (1a–j) and in some cases a minor proportion
of the corresponding enantiomers B’. The yields were varia-
ble for different substrates (5–95% isolated yield) but were
generally lower than those obtained from the corresponding
monosubstituted benzene substrates (Table 1). The ee values
(20–98%) were determined using a combination of methods
(methods i–v, Schemes 2–6) and are given in Table 1. To
maintain uniformity, the numbering system used in Table 1
has also been adopted in the Experimental Section.
Table 1. Isolated yields, optical rotations ([a]D), ee values and absolute
configurations (ACs) of the major cis-dihydrodiol enantiomers 1a–j (B).
[a]
cis-Diol
X
Y
F
Yield [%] [a]D
Enantiopurity AC
[% ee]
1a
1b
1c
1d
1e
1 f
1g
1h
1i
Br
CF3 Br
95
81
5
+52
ꢀ67
ꢀ12
ꢀ5
76[b–d]
B (1R,2S)
>98[b,d,e]
>98[b,d,e]
20[b–e]
B (1R,2R)
B (1R,2R)
B (1S,2S)
B (1R,2R)
B (1R,2R)
B (1R,2R)
B (1S,2R)
B (1S,2R)
B (1S,2R)
CN
Br
CF3
CN
Me
Br
Me 30
F
F
F
Several of the above methods used for ee determination
have also been employed for the assignment of absolute
configurations of benzene cis-dihydrodiols. These include
stereochemical correlation,[15] X-ray crystallography,[30,31] and
1H NMR analysis of di-MTPA esters[31,32] or chiral boro-
nates.[16,33] Unfortunately the chromatographic methods
(CSPHPLC and CSPGC) did not give information on the
absolute configuration and the NMR methods (di-MTPA
esters and chiral boronates), and early CD studies[34] provid-
ed only tentative assignments.
75
50
40
ꢀ41
+68
+143
ꢀ32
ꢀ118
+92
>98[b,d,f]
>98[b,d,e]
93[b–d]
CF3 CN 80
CF3 Me 75
CN
>98[b,d,e]
>98[b–d]
>98[b,d,e]
1j
Me 74
[a] MeOH. [b] Method i. [c] Method iv. [d] Method v. [e] Method ii.
[f] Method iii.
The present study is focussed on selected cis-dihydrodiol
metabolites 1a–j, obtained by biotransformation (P. putida
UV4) of the corresponding 1,4-disubstituted benzene sub-
strates, containing combinations of the substituents, that is,
F, Br, CF3, CN, Me. Several of the previously reported meth-
ods (CSPHPLC, di-MTPA ester and chiral boronate forma-
tion, stereochemical correlation and X-ray crystallography),
allied to experimental and calculated CD spectrocopy,[35]
have been used to determine their ee values and absolute
configurations. This reliable stereochemical information is,
in turn, used to verify i) the validity of the preliminary ste-
reochemical model used for prediction of absolute stereo-
chemisty of cis-dihydrodiol metabolites formed at the active
Enantiopurity determination methods for cis-dihydrodiols
1a–j: The enantiopurity values for the cis-dihydrodiols were
measured using CSPHPLC with Chiralcel OJ (1a, c, d, f and
g) or Chiralcel AD (1j) columns (method i). Only metabo-
lites 1a, d and g appeared to contain both enantiomers after
biotransfomation of the parent 1,4-disubstituted arene sub-
strates but three other cis-diols (1c, f and h) were obtained
as separable enantiomeric mixtures after chemoenzymatic
synthesis (Scheme 4). As the other cis-diols (1b, e, i and j)
showed only single peaks on CSPHPLC analysis (OJ
column), this could have resulted from poor peak resolution
of enantiomers rather than an ee value of >98%. Addition-
al methods for ee determination were therefore adopted.
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D. R. Boyd et al.
Alternative approaches involved stereochemical correla-
tion (method ii, Schemes 2–4) and comparison of enantio-
mer ratios from the resulting derivatives by CSPHPLC
(method i), optical rotation measurements or X-ray crystal-
lography (method iii) These methods were used to confirm
that the cis-diols 1b, c, e, f, h, i and j (Table 1) were enantio-
pure (>98% ee).
discussed earlier for the measurement of ee values including
stereochemical correlation with compounds of established
absolute configurations, X-ray crystallography using the
1
anomalous dispersion method and H and 19F NMR analyses
of diastereoisomeric di-MTPA ester derivatives of cycload-
ducts.
1H and 19F NMR analysis of the diastereoisomeric (R)-
and (S)-di-MTPA esters of the corresponding cycloadducts,
for example, 4gR/4gS and 4iR/4iS (formed using cis-dihydro-
diols as dienes and 4-phenyl-1,2,4-triazoline-3,5-dione as di-
enophile) provided ee values (1a, d, g, and i, Table 1,
method iv, Scheme 2). Unfortunately this method was not
generally applicable since formation of the di-MTPA esters
of some cis-dihydrodiols from 1,4-disubstituted benzenes
failed due to reactant unreactivity or product instability.
Stereochemical correlation: The correlation method, used
for the determination of absolute configurations in the
study, was based on that reported earlier[16] for the synthesis
of a series of cis-dihydrodiol metabolites of 4-substituted
methylphenyl sulfides (1k–m) where Stille coupling, with or-
ganostannanes as the organometallic component, was em-
ployed. This earlier stereochemical correlation sequence in-
volved substitution of an iodine atom by an SMe group in
the cis-dihydrodiols of 4-substituted iodobenzenes (1n–p)
using [Pd(PPh3)4]-catalysed organotin chemistry to give sul-
G
fides (1k–m) and hydrogenolysis of compounds 1n–p to
yield monosubstituted benzene cis-dihydrodiols (1q–s) of
known absolute configurations (Scheme 3).
The absolute configurations of seven cis-dihydrodiol me-
tabolites (1a–d, f, h and j) have been determined by rigor-
ous stereochemical correlation methods, during the current
programme, as shown in Schemes 4–6. The procedure in-
volved direct substitution of a halogen atom by a vinyl
group (1a!1v, and 1d!1w, Scheme 5) or a nitrile group
(1n!1 f, 1o!1c, 1p!1j, 1t!1h, Scheme 6) using
Bu3SnSMe/Bu3SnCN and [Pd(PPh3)4] as catalyst.
U
Removal of iodine or bromine atoms by catalytic hydro-
genolysis (1n!1q, 1o!1r, 1p!1s, 1t!1u, Scheme 4) or
hydrogenolysis/hydrogenation (1b!2b, Scheme 5) was then
used to correlate their absolute configurations to cis-diols of
known configurations (1q, r, s, u, and 2b). Concomitant cat-
alytic hydrogenolysis and hydrogenation of the substituted
bromobenzene cis-dihydrodiol 1b was achieved using a Pd/
C catalyst (Scheme 5). The residual alkene bond in com-
pound 2b proved to be very resistant to further hydrogena-
tion and was of identical 1S,2R absolute configuration to
that obtained from the partial hydrogenation of the cis-dihy-
drodiol metabolite of 1,1,1-trifluoromethylbenzene (1u)
based on a comparison of optical rotations and CD spectra.
Treatment of the cis-dihydrodiol metabolites 1a (and 1n)
Scheme 2.
Formation of the corresponding diastereoisomeric boro-
nate esters (1a(R)-MEBBA and 1a(S)-MEBBA) using (R)- and (S)-2-
(1-methoxyethyl) benzeneboronic acid (MEBBA), followed
by 1H NMR analysis of the diastereoisomeric composition
(method v, Scheme 3), proved to be successful for all the
cis-dihydrodiols 1a–j.[32] The ee values shown in Table 1 are
an average of those obtained using methods i–v.
or 1d (and 1p) with Bu3SnCH=CH2 and [Pd(PPh3)4] catalyst
G
yielded the vinyl cis-dihydrodiols 1v or 1w, respectively
(Scheme 5). cis-Diols 1n and 1p were stereochemically cor-
related with the corresponding (1R,2R)-1q and (1R,2S)-1s
cis-diol enantiomers (Scheme 4). The absolute configura-
Scheme 3.
Absolute configuration determination methods for cis-dihy-
drodiols 1a–j: The absolute configurations of cis-dihydro-
diols 1a–j were determined using several of the methods
Scheme 4.
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Enzyme-Catalysed Synthesis
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Scheme 5.
Figure 1. X-ray structure of one of the two independent molecules of
tions of the metabolites 1a and 1d were thus unequivocally
established as (1R,2S)and (1S,2S), respectively (Table 1).
The 1,4-disubstituted iodobenzene cis-dihydrodiol metab-
olites 1n–p were all available from an earlier study[16] while
compound 1t was obtained during the current study by a
similar TDO-catalysed cis-dihydroxylation of the 4-trifluoro-
methyl substituted iodobenzene substrate under similar con-
ditions. Replacement of the iodine atom in cis-dihydrodiols
1n–p and t by a cyano group using Bu3SnCN in the presence
(1R,2R)-1e.
tentatively assigned as (1R,2R) by the Bijvoet method, at
about the 90% confidence level. In common with other cis-
dihydrodiols derived from monosubstituted benzene sub-
strates.[25] compound 1e was also found to adopt an M heli-
cal diene conformation.
An X-ray crystal structure analysis of cis-dihydrodiol 1j
allowed the absolute configuration to be unequivocally as-
signed as (1S,2R), from the anomalous dispersion of the
oxygen atoms. This configuration assignment had earlier
been determined by the stereochemical correlation method
(Scheme 4) and is consistent with the CN group having the
dominant stereodirecting effect at the active site of the
enzyme. However, the conformation appears to be affected
predominantly by the Me group, possibly due to its greater
steric bulk (u 0.52). The OH group proximate to the Me was
found to be pseudoaxial while the other OH group, closer to
the smaller CN group (u 0.40), was pseudoequatorial. In
contrast to the earlier X-ray crystal studies of monosubsti-
tuted benzene cis-dihydrodiols, compound 1e was found to
have a P helical conformation (Figure 2).
of [Pd(PPh3)4] yielded compounds 1 f, c, j and h, respectively
A
(Scheme 6). Hydrogenolysis of cis-diols 1n–p and (H2, Pd/
C) yielded the ent-forms of the corresponding cis-diol me-
tabolites of fluorobenzene (1R,2R)-1q, bromobenzene
(1R,2R)-1r and toluene (1R,2S)-1s (Scheme 4). Conversely
the cis-dihydrodiol 1u derived from metabolism of 1,1,1-tri-
fluoromethyl benzene and also from hydrogenolysis of cis-
diol 1t both had the natural (1S,2R) absolute configuration.
Scheme 6.
X-ray crystallography: The cis-dihydrodiol 1e could not be
readily assigned an absolute configuration using the un-
equivocal stereochemical correlation methods shown in
Schemes 5 and 6 since neither the hydrogenolysis, hydroge-
nation nor Stille coupling procedures were applicable and
thus X-ray crystallography was used (Figure 1). The hydrox-
yl group at C1 was pseudoequatorial while that on C2 was
pseudoaxial in the solid state. This would also be expected
to be the preferred conformation in the solution phase as
the steric requirements of the CF3 group (u 0.90) are much
greater than those of a F atom (u 0.27). Thus the OH group
proximate to the bulky CF3 group would prefer to adopt the
pseudoaxial conformation. The absolute configuration was
Figure 2. X-ray structure of (1S,2R)-1j.
A crystal structure analysis of cis-dihydrodiol 1 f gave the
relative configuration and preferred conformations of the
OH groups (Figure 3). The absolute configuration was not
determined crystallographically but had been unambiguous-
ly assigned as (1R,2R) by stereochemical correlation
(Scheme 6). This configuration is consistent with the CN
group (u 0.40) having the dominant stereodirecting effect at
the active site, as compared with an F atom (u 0.27), but the
conformation does not appear to be determined by steric ef-
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D. R. Boyd et al.
used to make the tentative assignments (1R,2R)-1g and
(1S,2R)-1i respectively (Table 2).
Table 2. Characteristic 1H NMR d values for the cycloadduct di-MTPA
diastereoisomers 4gR, 4gS, 4iR and 4iS.
cis-Diol
Cycloadduct
di-MTPA
dOMe
dOMe
cis-Diol Config.
B (1R,2R)
1g
3g[a]
4gR
4gS
4iR
4iS
3.07
3.19
3.41
3.34
3.77
3.47
3.55
3.52
1i
3i
B (1S,2R)
Figure 3. X-ray structure of (1R,2R)-1 f.
[a] NMR data from the major enantiomer of cis-dihydrodiol 1g.
fects. The OH group proximate to the F was found to be
pseudoaxial while the other OH group, closer to the CN
group, was pseudoequatorial. Diene 1 f was similar to diene
1j in again having P helicity in the solid state.
In this and earlier studies,[16] ee measurements and un-
equivocal absolute configuration assignments have been
made for the 1,4-disubstituted benzene cis-dihydrodiols 1a–j
(Table 1 and Scheme 2) and also for a further eighteen ex-
amples, based on independent experimental and calculated
CD spectroscopy.[35] From this list of 28 cis-dihydrodiol de-
rivatives only five (including compounds 1d and 1g) have
been reported from other laboratories[28,34] but their absolute
configurations had not been rigorously established. The ste-
reochemical assignment methods adopted herein and in the
other paper,[35] now allow us to confirm and extend the val-
idity of the predictive model for the preferred absolute con-
figuration of bioproducts formed during TDO-catalysed cis-
dihydroxylation.[7,13,14]
The model shown in Scheme 7 assumes a stereopreference
for configuration B over the enantiomeric configuration B’,
based on the relative differences in size between large (X)
and small (Y) spherically symmetrical atoms or substituents,
using the Charton steric parameter as an indicator or sub-
stituent size.[13,14] This has been demonstrated for cis-dihy-
drodiols 1a, b, d, e, g and i. Thus, the dominant stereodirect-
ing effect of the larger (X) atom or group was found to
follow the sequence CF3 (u 0.90) > I (u 0.78) > Br (u 0.65)
> Cl > (u 0.53) > Me (u 0.52) > F (u 0.27) > H (u 0.00)
and decreased as the size difference between atoms or
groups X and Y became smaller.
Spectroscopic methods: The absolute configurations of most
of the cis-dihydrodiols in the series 1a–j were determined
by stereochemical correlation or X-ray crystallographic
methods with the exception of two compounds (1g and i). A
spectroscopic method used successfully for the determina-
tion of enantiopurity values of the cis-dihydrodiol metabo-
lites of monosubstituted benzenes, for example, 1q–s and u,
involved the initial formation of cycloadducts with 4-phenyl-
1,2,4-triazoline-3,5-dione exclusively syn to the diol moiety.
This was followed by the reaction with the (S)- and (R)-
enantiomers of MTPA chloride in pyridine to yield the cor-
responding di-MTPA diastereoisomers which were readily
distinguishable by 1H and 19F NMR spectrosopy.[31,32]
This approach also provided an empirical assignment of
absolute configurations to a range of monosubstituted ben-
zene cis-dihydrodiols, based on
a consistent trend in
1H NMR signals for MeO groups.[31,32] Thus, for cis-dihydro-
diols B (X=F, Cl, Br, I, Me, CF3, CN, Y=H) having a (1S)
configuration, the more downfield 1H NMR signal for the
two MeO groups had a larger dH value using (S)-MTPA
chloride (derived from (R)-MTPA). The validity of this
method for absolute configuration assignment was con-
firmed by X-ray crystallography of the di-MTPA esters and
other methods. The di-MTPA ester method was not as rigor-
ous as methods used earlier. However, in our experience, it
was found to be applicable for all cis-dihydrodiols B, formed
from the corresponding monosubstituted benzene rings A
(Y=H), we had tested.
In the current study, it was anticipated that a similar trend
1
in the H NMR spectra would be found for cis-dihydrodiols
Scheme 7.
derived from 1,4-disubstituted benzene substrates providing
that there was again a significant differential between the
group sizes of X and Y (Scheme 5). It was assumed that for
cis-dihydrodiols 1g (X=Me, Y=F) and 1i (X=CF3, Y=
Me) a sufficient difference in the sizes of substituents X and
Y (CF3 > Me > F) existed, for a similar empirical ap-
proach to be valid. Thus, cis-dihydrodiols 1g and 1i gave the
corresponding cycloaddducts (3g and 3i) and di-MTPA
esters (4gR/4gS and 4iR/4iS). The relevant chemical shifts for
the MeO signals (dOMe) from the di-MTPA esters were then
To accommodate substituents of conformationally depen-
dent size, the predictive model has recently been refined.
Thus, SMe (u 0.60)[16] CH=CH2 (u 0.60),[16] Et (u 0.56)[18] and
nPr (u 0.68)[18] groups having smaller u values were found to
have a stronger stereodirecting effect than the substituents
having larger u values (e.g. CF3, I) studied earlier. The re-
sults shown in Table 1 indicate that the conformationally in-
dependent size of the CN group, present in the 1,4-disubsti-
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Enzyme-Catalysed Synthesis
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An alternative method (method iv) involved formation of cycloadducts
by reaction with Cooksenꢀs reagent (4-phenyl-1,2,4-triazoline-3,5-dione)
followed by reaction with the acid chloride derivatives of (R)- and (S)-a-
methoxy-a-(trifluoromethyl)phenyl acetic acid (MTPA) to yield the cor-
responding bis-MTPA esters which were analysed by 1H NMR.[31, 32] The
formation of the corresponding diastereoisomeric boronate esters using
(R)- and (S)-2-(1-methoxyethyl)benzeneboronic acid followed by
1H NMR analysis of the diastereoisomeric composition provided a fur-
ther approach (method v).[16, 32]
tuted benzene substrates 1c, f, h and j, exerts a stronger ste-
reodirecting effect than the Br and Me substituents despite
having a relatively small u value (0.40). CD spectroscopic
analysis of the cis-dihydrodiol metabolite, derived from 4-
chlorobenzonitrile, has also confirmed that the CN group is
dominant over the Cl atom during TDO-catalysed cis-dihy-
droxylation.[35] On the basis of the current study, the domi-
nant stereodirecting effect of the X atom or group was
found to follow the sequence CF3 > CN > Br > Cl ꢁMe
> F > H. This is again consistent with the view that non-
spherically symmetrical substituents such as CN, SMe, CH=
CH2, Et and Pr have a dominant stereodirecting effect at
the active site during the TDO-catalysed cis-dihydroxylation
of arenes. It also implies that in addition to substituent size
(u), the length of the relatively rigid CN group (or of a
planar conformation the SMe, CH=CH2, Et and Pr groups)
could also be an important factor during binding and cataly-
sis at the active site of the TDO enzyme.
The absolute configurations were determined by empirical methods in-
cluding formation of chiral boronate derivatives, bis-MTPA ester deriva-
tives of Cooksen adducts[31, 33] and 1H NMR analysis. Unequivocal meth-
ods used include X-ray crystallography and stereochemical correlation.
Shake flask (0.5 g) biotransformations were carried out using P. putida
[31]
UV4 under reported conditions.
The cis-dihydrodiols (1a–j) obtained
after bioconversion of the corresponding arene substrates, were separated
and purified by PLC (silica gel, 50% EtOAc in hexane).
Physical properties and spectroscopic data for metabolites 1a–j and t
(1R,2S)-1a (X=Br, Y=F): 0.567 g, 95%; m.p. 90–938C (CH2Cl2/
hexane); [a]D = +52 (c = 0.92, MeOH); 76% ee; 1H NMR (500 MHz,
CDCl3): d = 2.75 (brs, 1H, OH), 2.66 (brs, 1H, OH), 4.49 (m, 2H, 1-H,
2-H), 5.52 (dd, 1H, J5,4 = 6.6, J5,F = 10.4 Hz, 5-H), 6.26 ppm (dd, 1H,
J4,5 = 6.6, J4,F = 5.2 Hz, 4-H); EI MS: m/z (%): 210 (33) [M]+, 190 (2),
83 (100); elemental analysis calcd (%) for C6H6O2BrF: C 34.5, H 2.9;
found: C 34.7, H 2.8.
Conclusion
A
A series of cis-dihydrodiol metabolites 1a–j has been ob-
tained, using the corresponding 1,4-disubstituted benzenes
as substrates and TDO from P. putida UV4 as biocatalyst.
The enantiopurity values and absolute configurations have
been determined employing a range of chemical methods in-
volving halogen substitution, hydrogenolysis, hydrogenation,
cycloaddition and esterification reactions. X-ray crystallog-
raphy, chiral stationary phase HPLC and NMR spectroscop-
ic methods have also been used for stereochemical analysis.
Taken in conjunction with the accompanying comprehensive
calculated and experimental CD studies on compounds 1a–j
(and 18 other cis-dihydrodiol metabolites from 1,4-disubsti-
tuted benzene substrates), the absolute configurations can
now be assigned in an unequivocal manner.[35] This study
has allowed the validity of a stereochemical model, and the
dominant stereodirecting effect of the relatively small but
inflexible CN group, during TDO-catalysed cis-dihydroxyla-
tion of substituted benzene substrates, to be further estab-
lished.
1
hexane); [a]D = ꢀ67 (c = 0.82, MeOH); >98% ee; H NMR (500 MHz,
CDCl3): d = 4.54 (m, 2H, 1-H, 2-H), 6.42 (m, 1H, 4-H), 6.48 ppm (d,
1H, J4,5 = 6.3 Hz, 5-H); EI MS: m/z (%): 260 (39) [M]+, 258 (40), 242
(5), 240(4), 159 (100); elemental analysis calcd (%) for C7H6O2F3Br: C
32.5, H 2.3; found: C 32.4, H 2.4.
A
hexane); [a]D = ꢀ12 (c = 1.4, MeOH); >98% ee; 1H NMR (500 MHz,
CDCl3): d = 4.42 (d, 1H, J1,2 = 6.2 Hz, 1-H), 4.55 (dd, 1H, J2,1 = 6.2,
J
2,4 = 1.3 Hz, 2-H), 6.52 (d, 1H, J5,4 = 6.3 Hz, 5-H), 6.56 ppm (dd, 1H,
J4,5 = 6.3, J4,2 = 1.3 Hz, 4-H); EI M: m/z (%): 217 (29) [M]+, 215 (31),
199(6), 197 (5), 136 (100), 118 (23); HR MS (EI): calcd for C7H6O2N79Br:
214.9582; found: 214.9577 [M]+.
A
= ꢀ5 (c = 0.53, MeOH); 20% ee (lit.:[13] ꢀ9 and 37% ee; lit.:[34] 0%
ee); 1H NMR (300 MHz, CDCl3): d = 1.91 (s, 3H, Me), 4.30 (m, 2H, 1-
H, 2-H), 5.61 (m, 1H, 5-H), 6.29 ppm (d, 1H, J5,4 = 6.0 Hz, 5-H); EI
MS: m/z (%): 206 (31) [M]+, 204 (29), 79 (100); HR MS (EI): calcd for
C7H9O279Br: 203.9786; found: 203.9791 [M]+.
A
1
hexane); [a]D = ꢀ41 (c = 0.86, MeOH); >98% ee; H NMR (500 MHz,
CDCl3): d = 4.51 (m, 1H, J1,2 = 6.1 Hz, 1-H), 4.57 (d, 1H, J2,1 = 6.2 Hz,
2-H), 5.64 (dd, 1H, J5,4 = 6.6, J5,F = 9.9 Hz, 5-H), 6.54 ppm (m, 1H, 4-
H); EI MS: m/z (%): 198 (90) [M]+, 180 (16), 152 (79), 101 (100); ele-
mental analysis calcd (%) for C7H6O2F4: C 42.4, H 3.1; found: C 42.1, H
3.0.
Experimental Section
Crystal data for 1e: C7H6F4O2, M=198.1, orthorhombic, a=10.195(5),
b=19.487(8), c=8.391(4) Š, V=1667.0(13) Š3, T=293(2) K, CuKa radia-
General information: 1H NMR spectra were recorded at 300 MHz
(Bruker Avance DPX-500) and at 500 MHz(Bruker Avance DRX-500)
in CDCl3 solvent unless stated otherwise. Chemical shifts (d) are report-
ed in ppm relative to SiMe4 and coupling constants (J) are given in Hz.
Mass spectra were recorded at 70 eV on a VG Autospec Mass Spectrom-
eter, using a heated inlet system. Accurate molecular weights were deter-
mined by the peak matching method with perfluorokerosene as standard.
Elemental microanalyses were obtained on a Perkin–Elmer 2400 CHN
microanalyser. CSPHPLC was carried out using a Shimadzu LC-6 A
liquid chromatograph connected to Hewlett Packard diode array detec-
tor. The 1,4-disubstituted benzene substrates and reagents used in the for-
mation of cis-dihydrodiol derivatives were obtained commercially.
tion, l=1.54178 Š, space group P21212 (no. 18), Z=8, F(000)=800,
R
1calcd =1.579 gcmꢀ3, m=1.552 mmꢀ1, Siemens P3 diffractometer, w scans,
10.6 < 2q < 1108, measured/independent reflections: 7892/1993, direct
methods solution, full-matrix least squares refinement on Fo2, anisotropic
displacement parameters for non-hydrogen atoms; all hydrogen atoms lo-
cated in a difference Fourier synthesis but included at positions calculat-
ed from the geometry of the molecules using the riding model, with iso-
tropic vibration parameters. R1 =0.064 for 1839 data with Fo > 4s(Fo),
243 parameters, wR2 =0.148 (all data), GoF=1.16, Flack parameter x=
0.2(4), D1kl;min,max =ꢀ0.21/0.21 eŠꢀ3. CCDC-631365 contains the supple-
mentary crystallographic data for this paper. These data can be obtained
free of charge from The Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data_request/cif.
The enantiomeric excess values of the cis-dihydrodiols (1a–j) were deter-
mined by CSPHPLC using a Daicel OJ or AD column (method i).[13,14]
Chem. Eur. J. 2007, 13, 5804 – 5811
ꢁ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
5809

D. R. Boyd et al.
A
Halogen-substitution reactions of cis-dihydrodiols 1a, d, n, o, p and t
with tributyltin reagents and [Pd(PPh3)4]
A
=
=
(500 MHz, CDCl3): d = 4.38 (dd, 1H, J1,2 = 6.5 Hz, 2-H), 4.63 (m, 1H,
1-H), 5.77 (dd, 1H, J5,4 = 6.5, J5,F = 9.6 Hz, 5-H), 6.69 ppm (m, 1H, 4-
H); EI MS : m/z (%): 155 (60) [M]+, 138 (21), 126 (100); HR MS (EI):
calcd for C7H6O2NF: 155.0383; found: 155.0386.
Substitution of a bromine atom by a vinyl group in cis-dihydrodiols 1a
and 1d toyield cis-dihydrodiols 1v and 1w: To a solution of cis-diol (1a
and 1d, 2 mmol), palladium(II) acetate (10 mol%) and triphenylphos-
phine (20 mol%) in dry THF (20 mL) was added vinyltributyltin
(2.3 mmol). The mixture was stirred at room temperature for 16 h under
nitrogen. THF was removed under reduced pressure and the resulting
residue purified by flash chromatography followed by PLC (5% MeOH
in CHCl3) to give the corresponding vinyl cis-dihydrodiols 1v and 1w
(35–45% yields). Samples of cis-dihydrodiols 1v and 1w of identical ab-
solute configuration were obtained using the same procedure with the
iodo-substituted analogues 1n and 1p of known configuration.
Crystal data for 1 f: C7H6FNO2, M=155.1, monoclinic, a=7.450(2), b=
6.244(1), c=7.712(2) Š, b=108.06(2)8, V=341.1(1) Š3, T=298(2) K,
MoKa radiation, l=0.71073 Š, space group P21 (no. 4), Z=2, F(000)=
T
160, 1calcd =1.510 gcmꢀ3, m=0.13 mmꢀ1, Marresearch image plate diffrac-
tometer, w scans, 5.6 < 2q < 40.88, measured/independent reflections:
1335/649, direct methods solution, full-matrix least squares refinement on
Fo2, anisotropic displacement parameters for non-hydrogen atoms; hydro-
gen atoms included at positions calculated from the geometry of the mol-
ecules using the riding model, with isotropic vibration parameters. R1 =
A
= +85 (c = 0.59, MeOH); 76% ee; 1H NMR (300 MHz, CDCl3): d =
4.54 (m, 1H, 1-H), 4.62 (m, 1H, 2-H), 5.19 (d, 1H, Jcis = 10.9 Hz, CH=
CH2), 5.43 (d, 1H, Jtrans = 17.6 Hz, CH=CH2), 5.59 (m, 1H, 5-H), 5.90
(m, 1H, 4-H), 6.38 ppm (dd, 1H, Jcis = 10.9, Jtrans = 17.6 Hz, CH=CH2);
EI MS: m/z (%): 156 (13) [M]+, 138 (90), 109 (100), 83 (21); HR MS
(EI): calcd for C8H9O2F: 156.0586: found: 156.0669 [M]+.
0.069 for 637 data with Fo > 4s(Fo), 103 parameters, wR2 =0.131 (all
ꢀ3
data), GoF=1.48, D1min,max =ꢀ0.55/0.58 eŠ
. CCDC-631366 contains
the supplementary crystallographic data for this paper. These data can be
obtained free of charge from The Cambridge Crystallographic Data
Centre via www.ccdc.cam.ac.uk/data_request/cif.
A
(1R,2S)-1g (X=Me, Y=F): 0.262 g, 40%; m.p. 120–1218C (CHCl3);
[a]D = +16 (c = 1.13, MeOH); 30% ee; 1H NMR (300 MHz, CDCl3): d
= 1.94 (s, 3H, Me), 4.31 (m, 1H, 1-H), 4.42 (m, 1H, 2-H), 5.14 (d, 1H,
Jcis = 10.9 Hz, CH=CH2), 5.44 (d, 1H, Jtrans = 17.6 Hz, CH=CH2), 5.77
[a]D = +143 (c = 1.01, MeOH); 93% ee (lit.[13] 90 and 49% ee); 1H
NMR (300 MHz, CDCl3): d = 1.92 (s, 3H, Me), 4.32 (m, 2H, 1-H, 2-H),
5.54 ppm (m, 2H, 4-H, 5-H); EI MS: m/z (%): 144 (86) [M]+, 126 (72),
97 (100); HR MS (EI): calcd for C7H9O2F: 144.0587; found: 144.0587
[M]+.
(m, 1H, 5-H), 5.91 (m, 1H, 4-H), 6.39 ppm (dd, 1H, Jcis = 10.9, Jtrans
17.6 Hz, CH=CH2); EI MS: m/z (%): 152 (57) [M]+, 134 (45), 106 (42),
91 (100), 79 (62); HR MS (EI): m/z: calcd for C9H12O2: 152.0837; found:
152.0836 [M]+.
A
1
hexane); [a]D = ꢀ32 (c = 0.96, MeOH); >98% ee; H NMR (500 MHz,
CDCl3): d = 4.44 (d, 1H, J1,2 = 6.0 Hz, 1-H), 4.58 (m, 1H, 2-H), 6.66 (d,
1H, J4,5 = 6 Hz, 4-H), 6.78 ppm (m, 1H, J5,4 = 6 Hz, 5-H); EI MS: m/z:
205 (100) [M]+, 187 (14), 126 (100); elemental analysis calcd (%) for
C8H6NO2F3: C 46.8, H 2.9, N 6.8; found: C 46.3, H 2.7, N 6.5.
Substitution of an iodine atom in cis-dihydrodiols 1n, o, p and t by a ni-
trile group to yield cis-dihydrodiols 1 f, c, j and h: To a solution of cis-
diol (1n, o, p and t, 2 mmol), containing [Pd(PPh3)4] (3 mol%) in dry
THF (15 mL) was added tributyltin cyanide (4 mmol). The mixture was
stirred under nitrogen at 508C for 3 h. The cooled reaction mixture was
filtered, the THF removed under reduced pressure and the resulting resi-
due purified by PLC (5% MeOH in CHCl3) to give the corresponding
cyano cis-dihydrodiols 1 f, c, j and h. The nitrile substituted cis-dihydro-
diols 1 f, c, j and h, obtained by this method, had identical spectroscopic
characteristics and absolute configurations to the same compounds pro-
duced as metabolites of the parent 1,4-disubstituted benzene substrates.
ACHTREUNG
=
ꢀ118 (c
=
(500 MHz, CDCl3): d = 1.99 (s, 3H, Me), 4.35 (m, 2H, 1-H, 2-H), 5.82
(d, 1H, J5,4 = 5.0 Hz, 5-H), 6.51 ppm (dd, 1H, J5,4 = 5.0, J4,2 = 1.8 Hz, 4-
H); EI MS: m/z: 194 (14) [M]+, 176 (61), 79 (100); elemental analysis
calcd (%) for C8H9O2F3: C 49.5, H 4.6; found: C 49.3, H 4.4.
ACHTREUNG
Catalytic reduction of cis-(1R,2R)-1b:
A solution of cis-diol 1b
=
=
(2.0 mmol) in MeOH (15 mL) containing Et3N (ꢁ50 mL) was stirred at
room temperature under hydrogen (1 atm) in the presence of 10% Pd/C
(0.06 g). When all the starting material had been consumed (5 h, TLC)
the resulting residue was purified by flash chromatography (5% MeOH
in CHCl3) followed by PLC using the same solvent system.
(500 MHz, CDCl3): d = 2.04 (s, 3H, Me), 4.17 (d, 1H, J1,2 = 6.1 Hz, 1-
H), 4.39 (d, 1H, J2,1 = 6.1 Hz, 2-H), 5.86 (d, 1H, J5,4 = 5.4 Hz, 5-H),
6.68 ppm (d, 1H, J4,5 = 5.4 Hz, 4-H); EI MS: m/z: 151 (16) [M]+, 133
(57), 86 (100); elemental analysis calcd (%) for C8H9NO2: C 63.5, H 6.0,
N 9.3; found: C 62.9, H 5.9, N 9.1.
A
(CHCl3/hexane), (lit.[36] m.p. 105–107); [a]D = ꢀ129 (c = 0.35, MeOH),
Crystal data for 1j: C8H9NO2, M=151.2, orthorhombic, a=8.044(2), b=
8.151(2), c=11.400(2) Š, V=747.5(3) Š3, T=293(2) K, CuKa radiation,
l=1.54178 Š, space group P212121 (no. 19), Z=4, F(000)=320, 1calcd =
(lit.[36] [a]D = ꢀ129).
N
Cycloadducts 3g and 3i from cis-dihydrodiols 1g and 1i: A solution of
sublimed 4-phenyl-1,2,4-triazoline-3,5-dione (2 mmol) in CH2Cl2 (10 mL)
was added, dropwise at room temperature, to a stirring solution of the
cis-dihydrodiol 1g or 1i (1.5 mmol) in CH2Cl2 (10 mL), until the pink
colour of the reagent persisted. Removal of solvent, after stirring the
mixture for 1 h, and purification of the residue by PLC (75% EtOAc in
hexane) yielded the respective cycloadduct 3g or 3i.
1.343 gcmꢀ3, m=0.81 mmꢀ1, Siemens P3 diffractometer, w scans, 13.4 <
2q < 114.18, measured/independent reflections: 1230/994, direct methods
solution, full-matrix least squares refinement on Fo2, anisotropic displace-
ment 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 vi-
bration parameters. R1 =0.041 for 954 data with Fo > 4s(Fo), 104 param-
eters, wR2 =0.113 (all data), GoF=1.03, Flack parameter x=ꢀ0.5(4),
D1min,max =ꢀ0.16/0.12 eŠꢀ3. CCDC-631367 contains the supplementary
crystallographic data for this paper. These data can be obtained free of
charge from The Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data_request/cif.
A
[a]D = +8 (c = 0.92, MeOH); 1H NMR (300 MHz, CD3 COCD3): d =
1.96 (s, 3H, Me), 3.80 (dd, 1H, J8,F = 1.5, J8,9 = 8.8 Hz, 8-H), 4.19 (dd,
1H, J9,F = 5.8, J9,8 = 8.8 Hz, 9-H), 6.40 (dd, 1H, J11,F = 4.6, J11,10
=
8.9 Hz, 11-H), 6.64 (dd, 1H, J10,F = 9.6, J10,11 = 8.9 Hz, 10-H), 7.36–
7.51 ppm (m, 5H, Ar-H); EI MS: m/z (%): 319 (23) [M]+, 119 (100); HR
MS (EI): m/z: calcd for C15H14FN3O4: 319.0968; found: 319.0973 [M]+.
(1R,2R)-1t (X=CF3, Y=I): 0.300 g, 53%; m.p. 117–1188C (CHCl3);
1
[a]D = ꢀ56 (c = 0.94, MeOH); >98% ee; H NMR (300 MHz, CDCl3):
d = 4.43 (m, 2H, 1-H, 2-H), 6.19 (d, 1H, J4,5 = 6.3 Hz, 4-H), 6.77 ppm
(d, 1H, J5,4 = 5.7 Hz, 5-H); EI MS: m/z: 306 (100) [M]+, 179 (30); ele-
mental analysis calcd (%) for: C7H6O2F3I: C 27.5, H 1.9; found: C 27.8,
H 1.6.
AHCTREUNG
=
=
CD3COCD3): d = 1.98 (s, 3H, Me), 3.83 (d, 1H, J8,9 = 8.5 Hz, 8-H), 4.27
(d, 1H, J9,8 = 8.5 Hz, 9-H), 6.58 (m, 2H, 10-H, 11-H), 7.45–7.47 ppm (m,
5H, Ar-H); EI MS: m/z (%): 369 (7) [M]+, 119 (100); elemental analysis
5810
www.chemeurj.org
ꢁ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2007, 13, 5804 – 5811

Enzyme-Catalysed Synthesis
FULL PAPER
calcd (%) for C16H14O4N3F3: C 52.0, H 3.8, N 11.4; found: C 51.6, H 3.5,
N 11.1.
[8] T. Hudlicky, D. Gonzalez, D. T. Gibson, Aldrichimica Acta 1999, 32,
35–62.
[9] D. T. Gibson, R. E. Parales, Curr. Opin. Biotechnol. 2000, 11, 236–
243.
[10] D. R. Boyd, N. D Sharma, C. C. R. Allen, Curr. Opin. Biotechnol.
2001, 12, 564–573.
[11] R. A. Johnson, Org. React. 2004, 63, 117–264.
[12] D. R. Boyd, T. Bugg, Org. Biomol. Chem. 2006, 4, 181–192.
[13] D. R. Boyd, N. D. Sharma, M. V. Hand, M R. Groocock, N. A.
Kerley, H. Dalton, J. Chima, G. N. Sheldrake, C. C. R. Allen, J.
Chem. Soc. Chem. Commun. 1993, 974–976.
Bis-(R)-(+)- and bis-(S)-(ꢀ)-MTPA esters 4gR/4gS and 4isR/4iS: To a so-
lution of the cycloadduct 3g or 3i (0.1 mmol) in dry pyridine, containing
4-dimethylaminopyridine (5 mg), was added (S)-(+)- or (R)-(ꢀ)-2-me-
thoxy-2-phenyl-2-trifluoromethylacetyl chloride (0.25 mmol). The mix-
ture was stirred at 608C until the esterification reaction had gone to com-
pletion (48 h, by 1H NMR analysis). Pyridine was removed under reduced
pressure and the residue purified by PLC (2% MeOH in CHCl3) to yield
the respective bis-(R)- or bis-(S)-MTPA esters (ca. 90% yield).
(8R,9R)-4gR: Light yellow semi-solid: 68 mg, 90%; 1H NMR (500 MHz,
[14] D. R. Boyd, N. D. Sharma, S. A. Barr, H. Dalton, J. Chima, G. M.
Whited, R. Seemayer, J. Am. Chem. Soc. 1994, 116, 1147–1148.
[15] M. B. Smith, J. March in Advanced Organic Chemistry: Reactions,
mechanism and structure, 5th ed., Wiley, New York, 2001, pp. 374.
[16] D. R. Boyd, N. D. Sharma, A. King, B. Byrne, S. A. Haughey, M. A.
Kennedy, C. C. R. Allen, Org. Biomol. Chem. 2004, 2, 2530–2537.
[17] D. R. Boyd, N. D. Sharma, I. N. Brannigan, M. R. Groocock, J. F.
Malone, G. McConville, C. C. R. Allen, Adv. Synth. Catal. 2005, 347,
1081–1089.
[18] D. R. Boyd, N. D. Sharma, N. I. Bowers, H. Dalton, M. D. Garrett,
J. S. Harrison, G. N. Sheldrake, Org. Biomol. Chem. 2006, 4, 3343–
3349.
[19] D. Gonzalez, T. Martinot, T. Hudlicky, Tetrahedron Lett. 1999, 40,
3077–3080.
[20] T. Hudlicky, H. Akgun, Tetrahedron Lett. 1999, 40, 3081–3084.
[21] M.A. Endoma, V. P. Bui, J. Hansen, T. Hudlicky, Org. Process Res.
Dev. 2002, 6, 525–532.
[22] T. Hudlicky, U. Rinner, D. Gonzalez, H. Akgun, S. Schilling, P. Sien-
galewicz, T. A. Martinot, G. R. Petit, J. Org. Chem. 2002, 67, 8726–
8743.
CDCl3): d = 1.54 (s, 3H, Me), 3.07 (s, 3H, OMe), 3.77 (s, 3H, OMe),
4.70 (dd, 1H, J8,F = 1.7, J8,9 = 9.2 Hz, 8-H), 5.90 (dd, 1H, J9,F = 3.9, J9,8
= 9.2 Hz, 9-H), 6.32 (m, 1H, 11-H), 6.73 (dd, 1H, J10,F = 8.3, J10,11
=
8.6 Hz, 10-H), 7.29–7.47 ppm (m, 15H, Ar-H); EI MS: m/z (%): 751 (9)
[M]+, 189 (100); HR MS (EI): m/z: calcd for C35H28O8F7N3: 751.1765;
found: 751.1782.
(8R,9R)-4gS: Light yellow semi-solid: 65 mg, 86%; 1H NMR (500 MHz,
CDCl3): d = 1.95 (s, 3H, Me), 3.19 (s, 3H, OMe), 3.47 (s, 3H, OMe),
5.07 (dd, 1H, J8,F = 1.4, J8,9 = 9.2 Hz, 8-H), 5.86 (dd, 1H, J9,F = 4.7, J9,8
= 9.2 Hz, 9-H), 6.38 (dd, 1H, J11,F = 5.3, J11,10 = 8.9 Hz, 11-H), 6.73 (t,
1H, J10,F
= 8.5, J10,11 = 8.9 Hz, 10-H), 7.31–7.51 (m, 13H, Ar-H),
7.62 ppm (d, 2H, J = 7.4 Hz, Ar-H); EI MS: m/z (%): 751 (6) [M]+, 189
(100); HR MS (EI): m/z: calcd for C35H28O8F7N3: 751.1765; found:
751.1782.
A
CHCl3); [a]D = +16 (c = 1.28, MeOH); 1H NMR (500 MHz, CDCl3): d
= 1.98 (s, 3H, Me), 3.41 (s, 3H, OMe), 3.55 (s, 3H, OMe), 5.40 (d, 1H,
J8,9 = 8.9 Hz, 8-H), 5.47 (d, 1H, J9,8 = 8.9 Hz, 9-H), 6.54 (d, 1H, J11,10
=
8.1 Hz, 11-H), 6.58 (d, 1H, J10,11
= 8.1 Hz, 10-H), 7.27–7.54 ppm (m,
15H, Ar-H); EI MS: m/z (%): 801 (10) [M]+, 568 (9), 189 (100); HR MS
(EI): m/z: calcd for C36H28O8F9N3: 801.1733; found: 801.1745 [M]+.
[23] L. M. Newman, H. Garcia, T. Hudlicky, S. A. Selifonov, Tetrahedron
2004, 60, 729–734.
[24] K. J. Finn, P. Cankar, T. R. B. Jones, T. Hudlicky, Tetrahedron:
Asymmetry 2004, 15, 2833–2836.
[25] M. Banwell, A. J. Edwards, D. W. Lupton, G. Whited, Aust. J. Chem.
2005, 58, 14–17.
A
CHCl3); [a]D = +66 (c = 1.4, CHCl3); 1H NMR (500 MHz, CDCl3): d
= 1.53 (s, 3H, Me), 3.34 (s, 3H, OMe), 3.52 (s, 3H, OMe), 5.03 (d, 1H,
J8,9 = 8.8 Hz, 8-H), 5.76 (d, 1H, J9,8 = 8.8 Hz, 9-H), 6.48 (d, 1H, J10,11
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Received: December 21, 2006
Published online: April 12, 2007
Chem. Eur. J. 2007, 13, 5804 – 5811
ꢁ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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5811