A study of substrate specificity of toluene dioxygenase in processing aromatic compounds containing benzylic and/or remote chiral centers

Article abstract of DOI:10.1039/b006545p

A series of substituted arenes containing remote chiral centers were screened as substrates for toluene dioxygenase (TDO). The absolute stereochemistry of the new metabolites was determined by chemical and spectroscopic correlation with synthetic standards. There was no evidence for kinetic resolution; enantiomers were indiscriminately processed by the enzyme to diastereomeric pairs, which were separable upon derivatization. Some of these new metabolites are useful as synthons for morphine synthesis. Full experimental details are reported for those new compounds stable to isolation and for derivatives of those that are unstable.

Full text of DOI:10.1039/b006545p

View Article Online / Journal Homepage / Table of Contents for this issue
A study of substrate speciÐcity of toluene dioxygenase in processing
aromatic compounds containing benzylic and/or remote chiral centers
Vu P. Bui,a Trond Vidar Hansen,¤a Yngve StenstrÊm,¤a Tomas Hudlicky*a and
Douglas W. Ribbonsb
a Department of Chemistry, University of Florida, Gainesville, FL 32611-7200, USA.
E-mail: hudlicky=chem.uÑ.edu
b SFB Biocatalysis at the Institute of Organic Chemistry, T echnical University of Graz,
Stremayrgasse 16, A-8010 Graz, Austria. E-mail: sekretariat=orgc.tu-graz.ac.at
Received (in Cambridge, UK) 4th August 2000, Accepted 17th October 2000
First published as an Advance Article on the web 19th December 2000
A series of substituted arenes containing remote chiral centers were screened as substrates for toluene dioxygenase
(
TDO). The absolute stereochemistry of the new metabolites was determined by chemical and spectroscopic
correlation with synthetic standards. There was no evidence for kinetic resolution; enantiomers were
indiscriminately processed by the enzyme to diastereomeric pairs, which were separable upon derivatization. Some
of these new metabolites are useful as synthons for morphine synthesis. Full experimental details are reported for
those new compounds stable to isolation and for derivatives of those that are unstable.
putida 39D was used. No preference was detected in the oxida-
Introduction
tions mediated by TDO expressed in the recombinant
organism Escherichia coli JM109 (pDTG601).23 Here, we
report the experimental details and absolute stereochemistry
correlations of the new metabolites.
In 1987, nearly 20 years after the report by Gibson et al. of
enzymatic dihydroxylation of p-chlorotoluene,1 Ley et al.
described the Ðrst application of cis-cyclohexadienediols in
synthesis.2 Since then the number of syntheses taking full
advantage of enzymatically introduced asymmetry has bur-
geoned, as has the number of reports of isolation and identiÐ-
cation of new metabolites derived from biooxidation of
aromatic compounds. The isolation of diol metabolites and
their synthetic applications have been reviewed recently.3h15
The two latest reviews list structures of most of the metabo-
lites produced to date,3,4 grouped by the corresponding class
of arenes from which they originate. Fig. 1 summarizes the
structural diversity available through this transformation,
which still does not have an asymmetric chemical equiva-
lent.16 Of the several hundred metabolites identiÐed to date,
only 21 have been employed in synthesis, most of which (ca.
90%) are the diols derived from benzene, chlorobenzene or
bromobenzene.3
There are fewer than ten metabolites reported from the bio-
oxidation of aromatic compounds bearing chiral substituents;
Gibson et al. described the Ðrst such oxidation, of 1-phenyl-1-
ethanol, in 1973.17 Other reports of oxidations of 1-phenyl-1-
ethanol and related substrates catalyzed by toluene
dioxygenase (TDO) have generally been connected to studies
of mono vs. dihydroxylation.18,19 A recent paper by Boyd et
al.20 discusses the issue of hydroxylation and the ability of
TDO to recognize enantiomers of 1-phenyl-1-ethanol.
We have published a preliminary report on the oxidation of
several substrates having either a chiral center at a benzylic
position or at other chiral centers located a or b to the
benzene ring.21 We found no evidence of discrimination, with
the exception of a slight preference for the oxidation of S-1-
phenyl-1-ethanol when the blocked mutant22 Pseudomonas
¤
On leave from: Department of Chemistry and Biotechnology, the
Agricultural University of Norway, N-1432 As, Norway. E-mail:
yngve.stenstrom=ikb.nlh.no.
Ž
Fig. 1 Structural diversity of substrates processed by toluene
dioxygenase (TDO) and naphthalene dioxygenase (NDO).
116
New J. Chem., 2001, 25, 116È124
DOI: 10.1039/b006545p
This journal is ( The Royal Society of Chemistry and the Centre National de la Recherche ScientiÐque 2001

View Article Online
Results and discussion
Our interest was focused primarily on phenyl-substituted
cyclohexanols and cyclohexanones (Table 1) because of the
relevance of these compounds to our synthetic approaches to
morphine (23). With these cases, the issue of kinetic resolution
of enantiomers is less important than the enzymeÏs acceptance
of both enantiomers of the substrates, because it is ultimately
our intention to re-aromatize the biooxidation productsÈby
means of either E. coli JM109 (pDTG601), followed by chemi-
cal oxidation, or E. coli JM109 (pDTG602), an organism that
expresses both TDO and catechol dehydrogenase
(
DHCD)24Èto produce advanced morphine intermediates
Fig. 3 Substrates not oxidized by toluene dioxygenase (TDO) or
with the catechol unit already attached to ring A, Fig. 2.25
If TDO actually discriminates enantiomers in more highly
substituted cases, it would be useful for approaches to the syn-
theses of both enantiomers of morphine by kinetic resolution.
However, biooxidation of enantiomers of the substrates does
not appear to discriminate between enantiomers; it produces
a mixture of diastereomers which, on separation, would also
be useful in an enantiodivergent approach to the target.
Nevertheless, for the time being, it was more important for us
to probe the limits of substitution on the substrates and how
the presence of chiral centers would be tolerated by the
enzyme. That both substrates 9 and 11 were processed by
TDO, albeit without enantiomeric preference, bodes well for
attempting to generate the required catechol units from com-
pounds such as 13, 15, or 25, as well as the relatively complex
advanced intermediate 24.26
naphthalene dioxygenase (NDO).
substrates by the two enzymes.21 Because none were oxidized,
we also looked at a series of phenethyl alcohols related to 1
having ortho, meta and para methyl groups. Of these, only the
para isomer was recognized by TDO, and this may explain
why 28 and 30 were not processed by the enzyme. Di†erences
in recognition rates for ortho, meta and para substitution have
been studied previously on several compounds, for example
halostyrenes and ring-halogenated ethylbenzenes,27h30 with
the conclusion that ortho or meta substitution leads to lower
yields of metabolites.
Trial fermentations were carried out in shake Ñasks and
monitored by UV for the presence of the desired metabolites.
On detection of a cyclohexadienediol, the process was scaled
up in either a 10 or 15 L fermentor. Isolated compounds were
subjected to rigorous structure proof, including absolute
stereochemistry, as described in the Experimental. Pure enan-
tiomers as well as racemic mixtures of substrates 1, 9 and 11
were subjected to oxidation by TDO.
Structure and absolute stereochemistry of metabolites
Although the diols derived from 1-phenyl-1-ethanols (1a, 1b)
are known,17h19 we carried out a potassium azodicarboxylate
(
PAD) reduction of racemic 2a,b to 31 (Scheme 1) and con-
Ðrmed a 1 : 1 ratio of diastereomers by NMR. Diols 4, 6 and 8
were detected by UV (j \ 262, 270 and 266 nm,
max
respectively) only in shake-Ñask experiments and were not iso-
In only one case was any enantiomeric preference
detectedÈin the oxidation of (^)-1 with the blocked
mutant22 P. putida 39D, in agreement with an earlier obser-
vation by Ribbons and Ahmed.19 The use of recombinant
clones did not reveal any preference for enantiomers, and we
rationalized this by slight di†erences in the topology of the
enzymes from the two organisms as a result of post-
translational folding and structural modiÐcation. The sub-
strates shown in Fig. 3 were not recognized by TDO in either
of the organisms, nor were they recognized by naphthalene
dioxygenase (NDO).21
lated.
Substrates 5 and 7 were screened in order to probe the tol-
erance of the enzyme for increased size and functionality of
the aromatic substituent. We expected that the relatively
bulky nature of these substrates would lead to preferential
oxidation of one enantiomer; however, (^)-9 was oxidized to
a 1 : 1 mixture of 10a and 10b, and (^)-11 to a 2 : 1 : 1
mixture of 12a, 12b and 12c. These observations conÐrmed
that the enzyme did not discriminate enantiomers. Diols 10a
and 10b were also reduced with PAD to 32a and 32b, respec-
tively, which were subsequently converted to the diastereo-
meric bromides and reduced to 33, a compound that was
independently synthesized (via cuprate coupling with cyclo-
hexyl triÑate) from the vinyl bromide 34, whose absolute con-
Ðguration is known.31 The two materials were identical in all
respects, thus providing a proof of the absolute stereoche-
mistry of 10a and 10b. In addition, 18 was reduced to 35, pro-
tected as acetonide 33, and found to be identical to the
material derived from 34 or 10a and 10b. The absolute con-
Ðguration of 16 was similarly proven by conversion of 16 to
diene 36, which was matched with the compound synthesized
from 34 via its trimethylstannane derivative and a palladium-
catalyzed coupling with an enol triÑate derived from cyclo-
hexanone (Scheme 1). Diol 14 (which we considered for its
potential use in our approach to morphine, Fig. 2) was
Initially, we subjected compounds 28, 29 and 30 (Fig. 3) to
both TDO and NDO because of the overlap in recognition of
detected by UV (j \ 266 nm) but was not isolated because
max
of difficulties in scale-up.
To prove its absolute stereochemistry, diol 20 was subjected
to PAD reduction with the expectation that both of the
unhindered double bonds would be reduced to provide 35, a
compound of known absolute stereochemistry; however, the
reduction provided a mixture of inseparable diols 37 and 38.
These two compounds were protected as acetonides and sub-
Fig. 2 Introduction of the catechol unit to morphine synthons.
New J. Chem., 2001, 25, 116È124
117

View Article Online
Table 1 Biotransformations of aromatics containing remote chiral centers
Substrate
Product
Substrate
Product
1a
2a [1.0]a
1b
2b [1.0]a
b
(^)-1
2a : 2b \ 1 : 1 (JM109)
2a : 2b \ 2 : 3 (Pp39D) [5.0]a
3
4
b
b
5
6
7
8
(
])-9
^)-9
10a [1.0]a
([)-9b
10b [1.0]a
(
9a : 9b \ 1 : 1 (JM109) [1.2]a
(])-11a
12a
([)-11b
12b
12c
b
(^)-11
12a : 12b : 12c \ 2 : 1 : 1 (JM109) [1.0]
13
14
1
5
9
16 [3.0]a
17
18 [1.8]a
1
20 [0.8]a
21
22 [1.1]a
a Numbers in brackets denote the isolated yield of metabolite in g L~1; yields are not optimized. b Detected by UV in shake-Ñask experiments
only.
118
New J. Chem., 2001, 25, 116È124

View Article Online
Scheme 1 Reagents: i, PAD, AcOH, MeOH; ii, DMP, p-TsOH; iii, CBr , PPh ; iv, Bu SnH, AIBN; v, Mg, Et O, Li CuCl ; vi, cyclohexanyl
4
3
3
2
2
4
triÑate; vii, sec-BuLi, Me SnCl; viii, (CH CN) PdCl , cyclohexenyl triÑate, DMF; ix, THF, H O, TFA; x, sec-BuLi, BnCl; xi, NaBH , EtOH, rt;
3
3
2
2
2
4
xii, MeOH, (PPh ) RhCl, H , 30 psi.
3
3
2
jected to catalytic hydrogenation with WilkinsonÏs catalyst to
furnish compound 33, of known absolute stereochemistry,
thus conÐrming the absolute stereochemistry of 20 (Scheme 1).
To establish the absolute stereochemistry of 22, the com-
pound was reduced with PAD to diol 39, which was also
chemically synthesized by coupling vinyl bromide 34 with
benzyl chloride using sec-BuLi as the metalÈhalogen exchange
reagent (Scheme 1). The biooxidation of 21 to diol 22 served
as a background experiment for the attempted kinetic
resolution study of 29, shown in Fig. 3.
Pure enantiomers of 11a and 11b, by pyridinium chloroch-
romate (PCC) oxidation of commercially available alcohols 9a
and 9b, were each subjected to fermentation. The products
were isolated and characterized, as shown in (Scheme 2).
It is clear that cyclohexyl-substituted arenes are good sub-
strates for TDO, with both enantiomers being processed by
the enzyme to a†ord the corresponding diasteromeric diols.
Because some of the compounds contain structural elements
of morphine, it will be worthwhile to investigate greater limits
of substitution.
New J. Chem., 2001, 25, 116È124
119

View Article Online
or DMSO-d . Coupling constants are measured in Hz. Multi-
6
plicities in 13C NMR are based on APT or DEPT. IR spectra
were recorded on a Perkin-Elmer FT-IR (KBr) and are given
as wavenumbers in cm~1. For the mass spectra, relative abun-
dance is reported in parentheses. High resolution mass spectra
and elemental analyses were performed at the University of
Florida and at Atlantic Microlab, Inc, respectively. Optical
rotations were recorded on a Perkin-Elmer 241 digital polari-
meter (10~1 deg cm2 g~1). Melting points were obtained on a
Thomas-Hoover capillary melting point apparatus and are
uncorrected. Gas chromatographic analysis of enantiomeric
mixtures was performed on a cyclodextrin column (Chirasil
DexCB, 25 m ] 0.32 mm) with helium as the carrier gas.
Reversed-phase HPLC analyses were performed on a C18
column (Phenomenex 250 ] 10 mm) using mixtures of water
and acetonitrile as eluents. For chromatographic puriÐcation,
silica gel (230È400 mesh) was used.
General procedure for the large-scale (15 L) fermentation with
Escherichia coli
A mineral salts broth (MSB, 600 mL) containing K HPO
2
4
(
9.6 g), KH PO (8.4 g), (NH ) SO (3 g), yeast extract (9 g),
4 2
glucose (18 g), and MgSO É 7H O (1.2 g) was divided into two
2
4
4
4 2
2.8 L Fernbach Ñasks and sterilized. After the broth had
Scheme 2 Reagents: i, PCC, CH Cl ; ii, E. coli JM109 (pDTG601).
cooled to ambient temperature, ampicillin (100 mg L~1) was
added, and each Ñask inoculated with a single colony of E.
coli JM109 (pDTG 601) from a fully grown plate. The inocu-
lum was grown overnight on an orbital shaker (35 ¡C, 150
rpm) and added to sterilized production medium (8 L) in a 15
L fermentor. The production medium consists of an aqueous
mixture of KH PO (60 g), citric acid (16 g), MgSO É 7H O
2
2
Conclusions
Several new diol metabolites were isolated and identiÐed from
arenes having chiral substituents. We found that TDO did not
discriminate between enantiomers. Now, the more important
issue with respect to morphine synthesis is the introduction of
the catechol unit directly onto an aromatic ring, a process dif-
Ðcult to accomplish by chemical means when there are sensi-
tive functionalities elsewhere. We have performed preliminary
experiments with JM109 (pDTG602) on several functionalized
aromatics and found that they can be cleanly converted to
catechols.32 Of direct relevance to morphine synthesis was the
successful oxidation of phenylcyclohexane, which led to the
corresponding catechol33 (Scheme 3).
2
4
4
2
(
40 g), trace metal solution [16 mL: Na SO (1 g L~1),
2
4
MnSO (2 g L~1), ZnCl (2 g L~1), CoCl É 6H O (2 g L~1),
4
4
2
2
2
CuSO É 5H O (0.3 g L~1), FeSO É 7H O (10 g L~1), pH 1.0],
2
4
4
2
conc. H SO (9.6 mL) and ferric ammonium citrate (9.6 mL,
2
2
70 g L~1). The mixture was neutralized with conc. ammonia
and supplemented with ampicillin (800 mg) and thiamine
hydrochloride (2.69 g). The culture was grown for 24 h with
concentrated glucose solution (720 g L~1) as the carbon
source, then induced with 80 mg isopropyl b-D-thiogalactopy-
ranoside (IPTG) after the optical density (OD) had reached at
least 15%. Once the OD was 45% or higher, the cells were
used for screening new substrates or large-scale fermentations.
General procedure for screening of new substrates
1
L of the high OD culture was centrifuged (3000 rpm, 15
Scheme 3
min). The cells were suspended in 300 mL of 0.20 M KPO4
bu†er (pH 7.0) supplemented with 2% glucose. The substrate
was added in small portions with the pH maintained at 7.0
with 10 M NaOH. The progress of the biotransformation was
monitored by UV absorbance of the broth in the region 260È
270 nm and by TLC. The substrates metabolized under these
conditions were then subjected to large-scale biooxidation in a
15 L fermentor. (Compounds 4, 6, 8 and 14 were not scaled
up.)
These endeavors were intended to identify substrates in
which enantiomeric discrimination (by TDO), processing of
both enantiomers to the corresponding diols (TDO), or direct
introduction of a catechol unit (TDO and DHCD) could be
achieved. The second topic will be expanded to other sub-
strates useful in morphine synthesis, as diastereomeric cyclo-
hexadienediols can be utilized separately in an
enantiodivergent approach to the alkaloid. Further develop-
ment in these areas will be reported in due course. The last
project was successful and bodes well for direct biocatalytic
synthesis of functionalized catechols. It will be investigated
further for its potential in the environmentally benign synthe-
sis of functionalized catechols.32
General method for the PAD reduction of diene-diols
The crude fermentation product (10 mmol) was dissolved in
cold methanol (5 mL), and the mixture cooled in an ice bath.
Potassium azodicaboxylate (PAD) (4.9 g, 25 mmol) was added
with stirring. A solution of acetic acid (4.6 mL, 80 mmol) in
methanol (20 mL) was added dropwise to the reaction vessel
at such a rate that 1È2 bubbles were produced per second.
Acetic acid addition took 3È4 h. The progress of the reaction
was monitored by UV or by 1H NMR for the disappearance
of oleÐnic protons. The mixture was allowed to warm to
ambient temperature, with the color changing from yellow to
Experimental
General
1
H and 13C NMR spectra were recorded at 300 and 75 MHz,
respectively, on a Varian 300 spectrometer, unless otherwise
stated. Chemical shifts are reported relative to TMS, CDCl3
120
New J. Chem., 2001, 25, 116È124

View Article Online
milky white. The mixture was stirred for 3 h, then saturated
according to the method described for that of (^)-1-phenyl-1-
ethanol. The crude material was immediately subjected to
PAD reduction. PuriÐcation by Ñash chromatography (1 : 4
hexaneÈEtOAc) a†orded 100 mg (49%) of 32a.
NaHCO (aq) added until e†ervescence ceased and the meth-
3
anol removed under reduced pressure. The residue was diluted
with brine (10 mL), and the aqueous solution extracted with
EtOAc (4 ] 20 mL). The combined organic phases were dried
(
MgSO ) and concentrated to yield the reduced product.
(1S,2R,1ºR,2ºS)-3-(2º-Hydroxycyclohexanyl)-3-cyclohexene-
4
1,2-diol (32a)
Biooxidation of (»)-1-phenyl-1-ethanol (1) with Pseudomonas
putida 39D
R \ 0.68 (EtOAc); mp \ 89È91 ¡C; [a]28 [82.3 (c 0.6,
f
D
CH Cl ); IR (Ðlm): 3329, 1447 cm~1; 1H NMR (300 MHz,
2
2
The culture was prepared according to the procedure of Hud-
licky et al.34 The culture was induced by adding 150È200 mg
of toluene and shaken for 2È3 h, after which time the cells
were centrifuged (3000 rpm, 10 min) and resuspended in the
same nutrient broth (250 mL). Racemic 1 (300 mg) was added
to the culture. The conversion was monitored by the increase
of the UV absorption of the formed diene at 266 nm and by
TLC (EtOAc). The biooxidation was allowed to proceed for
another 58 h. The cells were removed by centrifugation, and
the supernatant was extracted with EtOAc (5 ] 250 mL). The
organic layers were combined, dried over Na SO and con-
DMSO-d ): d 5.50 (m, 1H), 4.83 (d, J \ 4.5, 1H), 4.73 (d,
6
J \ 3.4, 1H), 4.30 (d, J \ 6.1, 1H), 3.73 (t, J \ 3.9 Hz, 1H),
3.46È3.38 (m, 1H), 3.26È3.15 (m, 1H), 2.1È1.0 (m, 13H); 13C
NMR (75 MHz, CDCl ): d 139.9, 125.5, 75.0, 69.9, 69.5, 49.9,
3
35.3, 32.3, 25.9, 25.1, 24.8, 24.2; MS (CI ] ): m/z 213 (22), 195
(100), 177 (87), 159 (29), 147 (59), 105 (24); HRMS: calc. for
C
C
H O , 213.1490. Found 213.1456. Anal. calc. for
1
2 21 3
H
O : C, 67.89; H, 9.50%. Found: C, 67.60; H, 9.55%.
1
2 20 3
Biooxidation of (1R,2S)-(Ô)-trans-2-phenyl-1-cyclohexanol
(9b)
2
4
centrated to give 138 mg of residue, which was analyzed by
GLC on an optically active cyclodextrin column, showing it
to be a mixture of dienediols and starting material. Compari-
son to standard samples showed the residual 1-phenylethanol
to be a 60 : 40 mixture of the R- and the S-enantiomers,
respectively.
The biooxidation of 9b with E. coli JM109 (pDTG601) was
carried out as described for the small-scale transformation for
racemic 1-phenyl-1-ethanol (1). The progress of the biotrans-
formations was monitored by UV absorbance (j \ 270 nm)
and TLC (EtOAc). The crude metabolite 10b was reduced
with PAD and puriÐed by Ñash chromatography (1 : 4
hexaneÈEtOAc) a†orded 102 mg (50%) of 32b.
Biooxidation of (R)-1-phenyl-1-ethanol (1a) and
(
S)-1-phenyl-1-ethanol (1b)
(
1S, 2R, 1ºS, 2ºR)-3-(2-hydroxycyclohexanyl)-3-cyclohexene-
Biooxidation of each enantiomer was carried out as described
for racemic 1-phenyl-1-ethanol with P. putida 39D. The con-
1,2-diol (32b)
R \ 0.68 (EtOAc); mp \ 103È106 ¡C; [a]31 [148.0 (c 1,
version was monitored by UV spectroscopy (j \ 270 nm).
f
D
max
CH Cl ); IR (Ðlm): 3364, 1448 cm~1; 1H NMR (300 MHz,
(
R)-1-Phenyl-1-ethanol (1a) yielded 3-[1(R)-hydroxyethyl]
2
2
CDCl ): d 5.70 (dd, J \ 2.6, 4.5, 1H), 4.16 (d, J \ 3.3 Hz, 1H),
.70È3.60 (m, 2H), 3.10 (bs, 1H), 2.85 (bs, 1H), 2.30È2.00 (m,
H), 1.90È1.62 (m, 4H), 1.58È1.50 (m, 1H), 1.41È1.20 (m, 4H);
cyclohexa-3,5-diene-1(S),2(R)-diol (2a) and (S)-1-phenyl-1-
ethanol (1b) yielded 3-[1(S)-hydroxyethyl]cyclohexa-3,5-diene-
3
3
5
1
(S),2(R)-diol (2b) after respective work-up. In each case, the
1
3C NMR (75 MHz, CDCl ): d 138.9, 129.5, 72.1, 70.7, 65.8,
yield was 1 g L~1.
3
5
4.1, 34.5, 32.2, 25.9, 25.3, 25.2, 24.7; MS (CI ] ): m/z 213 (29),
Biooxidation of (»)-1-phenyl-1-ethanol (1) with Escherichia
coli JM109 (pDTG601)
195 (78), 177 (100), 159 (28), 147 (16), 105 (14); HRMS: calc.
for C
H
H O , 213.1490. Found: 213.1511. Anal. calc. for
C12 20 3
1
2 21 3
O : C, 67.89; H, 9.50%. Found: C, 67.78; H, 9.62%.
The cultures were prepared according to the procedure of
Hudlicky et al.,30,34 and 1.0 g of substrate was added to the
cell culture. The progress was monitored by UV absorbance
Biooxidation of (»)-trans-2-phenyl-1-cyclohexanol (9)
Into a 12 L fermentor with fully grown cells (OD
\ 45),
(j \ 266 nm). GLC analysis revealed a 1 : 1 mixture of dia-
640 nm
(^)-trans-2-phenyl-1-cyclohexanol (10.29 g) was added (2 g
stereomers 2a and 2b. The 1H NMR spectrum was in agree-
ment with that published in the literature.17 The biooxidation
of racemic 1-phenyl-1-ethanol was also carried out on a large
scale to a†ord a crude yield of 40 g (5 g L~1) of a 1 : 1 mixture
of as reddish oil.
every 30 min). The progress of the biotransformation was
monitored by UV absorbance (j \ 270 nm). Three hours after
the Ðrst addition, the absorbance at 270 nm ceased to
increase. The pH of the medium was adjusted to 7.8 with
conc. NH OH. Cells were removed by centrifugation, and the
4
Biooxidation of (»)-1-(4-methylphenyl)-1-ethanol (3),
supernatant was extracted with base-washed ethyl acetate to
(»)-2-phenyl-1-propanol (5) and (»)-1-phenyl-2-propyn-1-ol
(7)
a†ord 10.25 g (1.3 g L~1) of a gray solid as a 1 : 1 mixture of
inseparable diastereomers of 3-(2-hydroxycyclohexanyl)-3,5-
cyclohexadien-1,2-diol (10a and 10b).
The biooxidations of 3, 5 and 7 with E. coli JM109
pDTG601) were carried out as described for the small scale
(
(
R)- and (S)-2-Phenylcyclohexanone (11a and 11b)
transformation for racemic 1-phenyl-1-ethanol (1). The
progress of the biotransformations was monitored by UV
11a and 11b were made by oxidizing the corresponding opti-
cally active trans-2-phenylcyclohexanol with pyridinium chloro-
chromate (PCC) in CH Cl at 5 ¡C with the usual work-up.
absorbance (j \ 262È272 nm). The metabolites 4, 6 and 8
max
were detected by UV only, with j
of 262, 270 and 266 nm,
max
2 2
respectively, and were not isolated.
(
R)-2-Phenylcyclohexanone (11a)
Biooxidation of (1S,2R)-(+ )-trans-2-phenyl-1-cyclohexanol
R \ 0.30 (hexaneÈEt O 4 : 1); mp 48È51 ¡C; [a]27 ]108.7 (c
(9a)
f
2
D
0
.20, benzene), lit.35 [a]26 ]114.7 (c 0.45, benzene); IR (KBr):
D
The biooxidation of 9a with E. coli JM109 (pDTG601) was
carried out as described for the small-scale transformation of
racemic 1-phenyl-1-ethanol (1). The progress of the biotrans-
formations was monitored by UV absorbance (j \ 270È272
nm) and TLC (EtOAc). The metabolite 10a was isolated
3090, 1699 cm~1; 1H NMR (300 MHz, CDCl ): d 7.34È7.16
3
(m, 5H), 3.62 (dd, J \ 12.0, 5.5 Hz, 2H), 2.55È2.38 (m, 2H),
2.27È2.22 (m, 1H), 2.14È2.08 (m, 1H) 2.02È1.95 (m, 2H), 1.85È
1.77 (m, 2H); 13C NMR (300 MHz, CDCl ): d 210.2, 138.8,
3
128.8, 128.5, 126.7, 57.7, 42.0, 35.3, 27.6, 25.0.
New J. Chem., 2001, 25, 116È124
121

View Article Online
(
S)-2-Phenylcyclohexanone (11b)
1-phenylcyclohexene (15.7 g, 99.2 mmol) a†orded a crude yield
of 18.4 g (3 g L~1) of 16 as an o†-white solid.
[
a]27 [109.8 (c 0.30, benzene), lit.35 [a]24 [113.5 (c 0.60,
D
D
benzene); IR (KBr): 3092, 1695 cm~1; 1H NMR (300 MHz,
(
1S,2R)-3-(1-Cyclohexenyl)-3,5-cyclohexadien-1,2-diol (16)
CDCl ): d 7.36È7.15 (m, 5H), 3.62 (dd, J \ 12.0, 5.5 Hz, 2H),
3
j
\ 302 nm; mp 90È93 ¡C; [a]28 ]47.5 (c 0.5, MeOH); IR
2
1
.56È2.39 (m, 2H), 2.28È2.24 (m, 1H), 2.13È2.08 (m, 1H) 2.02È
max
D
(
KBr): 3226, 3050, 1435 cm~1; 1H NMR (300 MHz, CDCl ):
.95 (m, 2H), 1.85È1.78 (m, 2H); 13C NMR (75 MHz, CDCl ):
3
3
d 6.16 (bt, J \ 4.2, 1H), 6.01È5.89 (m, 2H), 5.75 (bd, J \ 9.5,
1
d 210.2, 138.8, 128.8, 128.5, 126.7, 57.7, 42.0, 35.3, 27.6, 25.0.
H), 4.49È4.43 (m, 1H), 4.34 (bd, J \ 5.5 Hz, 1H), 2.70 (bs,
Biooxidation of (»)-2-phenylcyclohexanone (11a and 11b)
2H), 2.27È2.14 (m, 4H), 1.79È1.55 (m, 4H); 13C NMR (75
MHz, CDCl ): d 138.8, 133.8, 130.7, 125.7, 124.0, 117.9, 71.4,
Biooxidation of 11a and 11b with E. coli JM109 (pDTG601)
was carried out as described for the small-scale transform-
ation of racemic 1-phenyl-1-ethanol (1). This a†orded the
acetals 12a and 12c along with the ketone 12b in a 2 : 1 : 1
mixture, respectively.
The proof of structures and the correlation of absolute
stereochemistry was provided by transforming the two enan-
tiomers 11a and 11b separately, as for racemic 11.
3
6
6.7, 26.0, 25.3, 22.6, 22.0; MS (CI): m/z 193 (1), 191 (27), 175
(
(
100), 174 (79), 173 (22); HRMS (CI): m/z calc. for C
M ] H): 193.1228. Found: 193.1213.
H O
12 17 2
Biooxidation of phenylcyclohexane (17)36
The biooxidation of 17 was performed according to the
general procedure for the small-scale fermentation. Addition
of phenylcyclohexane (26.5 g, 165 mmol) a†orded 14.0 g (1.8 g
L~1) of 18 as an o†-white solid.
(4S,4aR,9aR)-4,6,7,8,9,9a-Hexahydro-4aH-dibenzofuran-
4
,5a-diol (12a)
(
1S,2R)-3-(1-Cyclohexyl)-3,5-cyclohexadien-1,2-diol (18)
j
\ 266 nm; R \ 0.58 (1 : 1 hexaneÈEtOAc); [a]28 ]130.3
max
f
D
R \ 0.32 (hexaneÈEtOAc 1 : 1); mp \ 63È65 ¡C; j \ 268
(
c 0.7, MeOH); IR (Ðlm): 3378, 1449 cm~1; 1H NMR (300
f
max
nm; [a]30 [73.8 (c 1.0, CH Cl ); IR (KBr): 3312, 3048, 1646,
MHz, CDCl ): d 6.09 (dd, J \ 4.8, 9.4, 1H), 5.98 (dd, J \ 4.3,
9
4
2
D
2 2
3
1
588, 1447 cm~1; 1H NMR (DMSO-d ): d 5.80 (ddd, J \ 2.3,
.4, 1H), 5.84È5.78 (m, 1H), 4.76È4.70 (m, 1H), 4.25 (bs, 1H),
.12 (bs, 1H), 3.97 (t, J \ 5.5 Hz, 1H), 2.47È2.38 (m, 1H), 2.18È
.06 (m, 1H), 1.83È1.74 (m, 1H), 1.62È1.56 (m, 3H), 1.48È1.08
6
5
.4, 9.5, 1H), 5.62È5.56 (m, 2H), 4.67 (d, J \ 6.4, OH), 4.30 (d,
J \ 6.3, OH), 4.08È4.00 (m, 1H), 3.75 (bt, J \ 5.4 Hz, 1 H),
2
.10È1.98 (m, 1 H), 1.86È1.60 (m, 5 H), 1.34È0.96 (m, 5 H); 13C
(
1
m, 3H); 13C NMR (75 MHz, CDCl ): d 144.3, 127.8, 124.2,
3
NMR (DMSO-d ): d 146.9, 129.5, 123.0, 116.7, 69.7, 67.0, 41.4,
14.9, 106.9, 79.7, 61.6, 49.1, 33.9, 30.2, 24.0, 22.8; MS (CI):
6
3
2.8, 31.2, 26.3, 26.2, 25.9.
m/z 192 (12), 191 (40), 132 (32), 128 (35), 119 (32), 107 (31), 105
(
29), 91 (100), 83 (21), 81 (37); HRMS (CI) m/z calc. for
Biooxidation of 3-phenylcyclohexene (19)37
C12 13 2
(
(
H O (M [ OH): 191.1072. Found: 191.1090.
The biooxidation of 19 was performed according to the
general procedure for the small-scale fermentation. Addition
of 3-phenylcyclohexene (500 mg) a†orded a crude yield of 120
mg of 20 as a reddish oil.
1S,5ºS,6ºR)-5º,6º-Dihydroxybicyclohexyl-1º,3º-dien-2-one
12b)
R \ 0.42 (hexaneÈEtOAc 1 : 1); [a]28 ]58.1 (c 0.6, MeOH);
f
D
IR (Ðlm): 3352, 1702, 1444 cm~1; 1H NMR (300 MHz,
(
1S,2R)-3-(2-Cyclohexenyl)-3,5-cyclohexadien-1,2-diol (20)
CDCl ): d 6.06 (dd, J \ 4.7, 9.5, 1H), 5.95 (dd, J \ 5.6, 9.5,
3
1
H), 5.81È5.77 (m, 1H), 4.74È4.69 (m, 1H), 4.20 (bs, 2H), 3.95 (t,
R \ 0.53 (hexaneÈEtOAc 2 : 3); j \ 262 nm; 1H NMR
f
max
J \ 5.6 Hz, 1H), 2.45È2.38 (m, 1H), 2.14È2.04 (m, 1H), 1.80È
1
(DMSO-d ): d (5.86È5.75 (m, 4H), 5.66È5.51 (m, 6H), 4.64 (d,
6
.72 (m, 1H), 1.68È1.58 (m, 3H), 1.46È1.12 (m, 3H); 13C NMR
J \ 5.97, 2H), 4.41 (d, J \ 6.36, 2H), 4.05 (bs, 2H), 3.78 (t,
J \ 5.97 Hz, 2H), 2.95 (bs, 2H), 1.98È1.91 (m, 4H), 1.86È1.70
(m, 2H), 1.60È1.40 (m, 6H).
(
6
75 MHz, CDCl ): d 213.2, 139.2, 129.2, 124.0, 114.6, 68.7,
3
8.5, 42.1, 30.1, 27.3, 25.9, 22.8; HRMS (CI): m/z calc. for
C12 17 3
(
4
H O (M ] H): 209.2675. Found: 209.2669.
Biooxidation of diphenylmethane (21)
4S,4aR,9aS)-4,6,7,8,9,9a-Hexahydro-4aH-dibenzofuran-
,5a-diol (12c)
The biooxidation of 21 was carried out according to the
general procedure for large-scale production. Diphenyl-
methane (17 g, 101 mmol) was slowly added to a fully grown
(OD \ 65) culture (8 L). Progress was monitored by UV
j
\ 266 nm; R \ 0.49 (hexaneÈEtOAc; 1 : 1); [a]28
max
f
D
[
126.3 (c 0.5, MeOH); IR (Ðlm): 3371, 1454 cm~1; 1H NMR
(
300 MHz, CDCl ): d (6.05 (dd, J \ 4.8, 9.4, 1H), 5.92 (dd,
(j \ 266 nm). The biooxidation a†orded 22 (8.8 g, 1.1 g
3
max
J \ 4.3, 9.4, 1H), 5.80È5.75 (m, 1H), 4.72È4.68 (m, 1H), 4.20
(
L~1) as an o†-white solid.
bs, 1H), 4.10 (bs, 1H), 3.98 (t, J \ 5.5 Hz, 1H), 2.48È2.40 (m,
H), 2.17È2.07 (m, 1H), 1.85È1.77 (m, 1H), 1.60È1.54 (m, 3H),
1
1
1
(1S,2R)-3-Benzyl-3,5-cyclohexadien-1,2-diol (22)
.45È1.10 (m, 3H); 13C NMR (75 MHz, CDCl ): d 144.8,
3
R \ 0.47 (hexaneÈEtOAc 2 : 3); mp \ 69È71 ¡C; [a]33 ]117
27.8, 124.4, 114.4, 105.7, 79.7, 61.7, 49.0, 33.8, 30.1, 24.2, 22.7;
f
D
(
c 1.0, CH Cl ); IR (Ðlm): 3440, 3054, 2987, 1643, 1422, 1265,
HRMS (CI): m/z calc. for C
Found: 191.1081.
H
O
(M [ OH): 191.1072.
2 2
1
2 13 2
896, 745, 705 cm~1; 1H NMR (300 MHz, DMSO-d ): d 7.34È
.26 (m, 2H), 7.23È7.16 (m, 3H), 5.85È5.75 (m, 1H), 5.68È5.60
6
7
(
m, 1H), 5.54 (d, J \ 4.43, 1H), 4.64 (d, J \ 6.36, 1H), 4.52 (d,
Biooxidation of 2-phenyl-2-cyclohexenone (13)36
J \ 6.17, 1H), 3.70 (t, J \ 6.16 Hz, 1H), 3.46 (s, 2H); 13C
The biooxidation of 13 with E. coli JM109 (pDTG601) was
carried out as described for the small-scale transformation of
racemic 1-phenyl-1-ethanol (1). The progress of the reaction
was monitored by UV (j \ 266 nm). The metabolite (14) was
detected by UV only and was not isolated.
NMR (75 MHz, CDCl ): d 141.3, 139.1, 129.2, 128.7, 128.2,
3
126.6, 124.9, 121.1, 69.9, 69.3, 40.3; MS (FAB): m/z 202 (12),
185 (56), 184 (14), 107 (14), 93 (31), 91(100); HRMS (CI): m/z
calc. for C13 14 2
PAD reduction of racemic 2 to 31
H O : 202.0994. Found: 202.0997.
Biooxidation of 1-phenylcyclohexene (15)
The diasteromeric mixture of 2 was reduced with PAD
The biooxidation of 15 was performed according to the
general procedure for the large-scale fermentation. Addition of
according to the general procedure to a†ord 31 as a colorless
oil.
122
New J. Chem., 2001, 25, 116È124

View Article Online
3
-(Hydroxyethyl)cyclohexa-3-ene-1(S),2(R)-diol (31)
to a†ord crude 33 (589 mg, 72%). PuriÐcation by chromatog-
raphy (EtOAcÈhexane 1 : 9) yielded an analytical sample
whose spectroscopic and physical data were identical to those
of synthetic 33 M[a]28 ]50.1 (c 0.6, CHCl )N.
1
H NMR (300 MHz, CDCl ): d 6.00 (bt, J \ 3.5, 1H), 5.89 (bt,
3
J \ 3.7 Hz, 1H), 4.47È4.42 (m, 1H), 4.40È4.26 (m, 4H), 4.17È
3
.90 (m, 5H), 3.76È3.52 (m, 2H), 2.30È2.12 (m, 2H), 2.11È1.90
D
3
(
m, 2H), 1.85È1.56 (m, 4H), 1.36È1.20 (m, 6H); 13C NMR (75
Correlation of absolute stereochemistry of (18)
MHz, CDCl ): d 140.3, 139.2, 126.5, 124.9, 71.0, 69.6, 69.2,
6
3
The cis-diene diol 18 was reduced with PAD according to the
general procedure. PuriÐcation by Ñash chromatography
(hexaneÈEtOAc 1 : 4) a†orded 35 as a crystalline compound
9.1, 67.6, 65.8, 24.9, 24.7, 23.9, 23.4, 21.8, 21.6.
Correlation of absolute stereochemistry of 10a and 10b
(
0.98 g, 86%).
1S,2R)-3-(1-Cyclohexyl)-3-cyclohexene-1,2-diol (35)
R \ 0.35 (hexaneÈEtOAc 1 : 1); mp \ 68È69 ¡C; [a]28 [75.2
The crude mixture from the biooxidation of (^)-9 was
reduced with PAD to a†ord 32a and 32b. Protection of the
mixture of the vic-cis-diols 32a and 32b (390 mg, 2.0 mmol)
was carried out in neat dimethoxypropane with a catalytic
amount of p-toluenesulfonic acid to yield the corresponding
ketals. The crude mixture (378 mg, 1.5 mmol) was dissolved in
dry CH Cl (10 mL) at 0 ¡C and CBr (746 mg, 2.25 mmol)
was added followed by PPh (393 mg, 2.25 mmol) portionwise
over 10È15 min. The mixture was left for another two hours at
ambient temperature. Chromatography (2.5% EtOAc in
hexane) yielded the two diastereomeric bromides (441 mg,
(
f
D
(
c 1.1, CH Cl ); IR (KBr): 3330, 1448 cm~1; 1H NMR (300
2
2
MHz, DMSO-d ): d 5.34 (t, J \ 3.5, 1H), 4.31 (d, J \ 5.9, 1H,
6
OH), 4.27 (d, J \ 5.6, 1H, OH), 3.80 (t, J \ 4.5 Hz, 1H), 3.45È
2
2
4
3
.33 (m, 1H), 2.12È1.85 (m, 3H), 1.82È1.52 (m, 6H), 1.49È1.39
3
(
m, 1H), 1.32È0.90 (m, 5H); 13C NMR (75 MHz, DMSO-d ):
6
d 143.8, 120.9, 69.4, 67.0, 41.5, 38.0, 31.6, 26.6, 26.4, 26.0, 24.9,
2
HRMS (CI): m/z calc. for C
Found: 179.1436. Anal. calc. for C
4.2; MS (CI): m/z 195 (3, M [ 1), 179 (100), 161 (18), 93 (33);
H
O (M [ OH): 179.1436.
90%) which were reduced in the general manner using
1
2 19
H
O : C, 73.43; H,
12 20 2
0.27%. Found: C, 73.46; H, 10.38%.
Bu SnH and AIBN in dry, reÑuxing THF, a†ording crude 33
3
1
(
377 mg, 80%). PuriÐcation by chromatography (EtOAcÈ
The spectroscopic and physical data of the acetonide of 35
were identical to those of synthetic 33.
hexane 1 : 9) yielded an analytical sample with all spectro-
scopic and physical data in accord with 33 obtained from the
synthetic approach described below. [a]28 ]49.6 (c 0.5,
D
Correlation of absolute stereochemistry of 20
CHCl ).
3
The crude metabolites (7.5 g, 39 mmol) from a large-scale bio-
oxidation of 19 were subjected to PAD reduction according to
the general procedure to a†ord a mixture of inseparable diols
Synthesis of (2,2-dimethyl-3a,7a-dihydrobenzo[1,3]dioxol-
4-yl)cyclohexane (33)
3
7 and 38. The crude mixture of products were protected as
The procedure for coupling was adapted from a report by
their acetonides, then subjected to catalytic hydrogenation (30
psi, MeOH, WilkinsonÏs catalyst). The product was puriÐed
by Ñash chromatography to a†ord a colorless oil (2.1 g) whose
spectroscopic and physical data M[a]28 ]52.8 (c 0.6, CHCl )N
Tamura and Kochi.38 Magnesium (98 mg, 4.0 mmol) was
covered with 5 mL dry Et O in a 100 mL 3-necked round-
2
bottomed Ñask. The mixture was degassed with ultrasound
under argon for 30 min. 7-Bromo-2,2-dimethyl-3a,4,5,7a-
D
3
were in agreement with those of synthetic 33.
tetrahydrobenzo[1,3]dioxole (34) (0.70 g, 3.0 mmol) was added
together with dry Et O (5 mL) and left in the ultrasound bath
2
Correlation of absolute stereochemistry of
for 1 h. The reaction mixture was then cooled to [78 ¡C, and
(
1S,2R)-3-benzyl-3,5-cyclohexadien-1,2-diol (22)
with rigorous stirring Li CuCl in Et O (30 mL, 0.1 M) was
2
4
2
added over 15 min. Stirring was continued for another 30 min,
In a Ñame-dried 25 mL round-bottomed Ñask equipped with a
3-way stopper, vinyl bromide 34 (172 mg, 0.746 mmol) was
dissolved in freshly distilled THF (10 mL). The reaction vessel
was cooled to [78 ¡C, then sec-BuLi (0.86 mL, 1.1 mmol) was
added dropwise via syringe. The mixture was stirred at
[78 ¡C for 45 min, then benzyl bromide (0.19 g, 1.1 mmol)
was added. The reaction was allowed to proceed for 1 h at
[78 ¡C, then the mixture was allowed to warm slowly to
room temperature. The reaction was then quenched with 10%
aq NH Cl (5 mL) and ethyl acetate (10 ml) added. The layers
were separated and the organic layer was washed with water
(2 ] 10 mL), then dried with MgSO . The solvent was
removed under vacuum to a†ord the crude product (123 mg,
68%) as a light yellow oil. The crude product was dissolved in
6
stir at room temperature for 1 h. The mixture was concen-
trated under vacuum and the residue diluted with ethyl
acetate (10 mL), then washed with 5% NaHCO (aq) (2 ] 5
mL). The organic layers were combined, dried over Na SO ,
then cyclohexyl triÑate (390 mg, 2.0 mmol) in dry Et O (10
2
mL) was added. The reaction mixture was stirred for 3 h.
Work-up in the usual manner followed by chromatography
(
5% EtOAc in hexane) a†orded 18 mg (38%) of the product.
[
5
2
a]27 ]49.2 (c 0.9, CHCl ); 1H NMR (300 MHz, CDCl ): d
D
3
3
.52 (t, J \ 3.0, 1H), 4.39 (d, J \ 5.6, 1H), 4.22È4.17 (m, 1H),
.18È2.04 (m, 1H), 2.02È1.93 (bt, J \ 11.3 Hz, 1H), 1.87È1.54
(
m, 7H), 1.35 (s, 6H), 1.34È0.93 (m, 6H); 13C NMR (75 MHz,
CDCl ): d 141.2, 122.9, 107.9, 73.6, 72.9, 40.8, 33.3, 31.5, 28.0,
3
4
2
6.8, 26.6, 26.5, 26.4, 25.6, 20.7; MS (FAB): m/z 236 (0.6), 207
(
1
40), 191 (21), 177 (12), 161 (18), 153 (42), 147 (100), 133 (42),
05 (21), 95 (35), 91 (63), 83 (70); HRMS (FAB): m/z calc. for
4
C15 24 2
H
O : 236.1776. Found: 236.1774.
mL of a 4 : 1 : 1 mixture of THFÈH OÈTFA and allowed to
2
Correlation of absolute stereochemistry of 12a and 12b
The crude mixture from the biooxidation of (^)-11 was
treated with NaBH in MeOH to a†ord a mixture of dia-
3
4
stereomeric alcohols 10, then the vicinal cis diol was protected
2
4
and concentrated. The product was puriÐed by Ñash chroma-
tography to a†ord a white solid (84 mg, 82%). Physical and
spectroscopic data for the compound obtained from this route
matched those of the PAD-reduced diol 39 obtained from the
biooxidation.
as its acetonide with dimethoxypropane and cat. p-
toluenesulfonic acid. This mixture was reduced with PAD
according to the general procedure to yield 32. The crude
product (800 mg, 3 mmol) was dissolved in dry CH Cl (25
2
2
mL) at 0 ¡C and CBr (1.5 g, 4.5 mmol) was added, followed
4
by portionwise addition of PPh (790 mg, 4.5 mmol) over 15
min. The mixture was stirred for 2 h at ambient temperature.
3
Synthesis of (1S,2R)-3-(1-cyclohexenyl)-3-cyclohexene-1,2-
diol (36)
Chromatography (2.5% EtOAc in hexane) yielded 700 mg
(
85%) of the two diastereomeric bromides, which were subse-
Into a Ñame-dried 50 mL round-bottomed Ñask equipped
with a 3-way stopper, (CH CN) PdCl
quently reduced with Bu SnH and AIBN in dry THF at reÑux
2 (5 mg, 0.019 mmol)
3
3
2
New J. Chem., 2001, 25, 116È124
123

View Article Online
7
8
H. A. J. Carless, T etrahedron: Asymmetry, 1992, 3, 795.
and anhydrous DMF (5 mL) were added. A solution of cyclo-
hexanone enol triÑate39 (216 mg, 0.94 mmol) in dry DMF (5
mL) was added to the reaction Ñask via syringe, under argon
atmosphere. The trimethyltin derivative of the vinyl bromide
G. N. Sheldrake, in Chirality and Industry, ed. A. N. Collins,
G. N. Sheldrake and J. Crosby, John Wiley and Sons, Chichester,
UK, 1992, p. 127.
T. Hudlicky and J. W. Reed, in Advances in Asymmetric Synthesis,
ed. A. Hassner, JAI Press, Greenwich, CT, 1995, p. 271.
A. D. Grund, SIM News, 1995, 45, 59.
9
3
4 (300 mg, 0.94 mmol), prepared according to the procedure
of Stille,40 was dissolved in dry DMF (5 mL), and the solution
added dropwise to the reaction Ñask via syringe. The starting
material was consumed after 4 h of stirring at room tem-
perature, according to TLC. The reaction mixture was washed
10
11
12
T. Hudlicky and A. J. Thorpe, Chem. Commun., 1996, 1993.
T. Hudlicky, Chem. Rev., 1996, 96, 3.
13 T. Hudlicky, D. A. Entwistle, K. K. Pitzer and A. J. Thorpe,
with 5% NaHCO (aq) (2 ] 10 mL), and the aqueous layer
Chem. Rev., 1996, 96, 1195.
3
1
1
4
5
T. Hudlicky, ACS Symp. Ser., 1996, 626, 180.
was extracted several times with pentane. The organic layers
T. Hudlicky, in Green Chemistry: Frontiers in Benign Chemical
Syntheses and Processes, ed. P. T. Anastas and T. C. Williamson,
Oxford University Press, Oxford, UK, 1998, ch. 10, p. 166.
A racemic bis-hydroxylation of benzene with OsO under pro-
were combined, dried with Na SO , and the solvent was
2
4
removed under vacuum to a†ord a red oil. The residue was
dissolved in a solvent mixture of THFÈH OÈTFA (4 : 1 : 1; 10
1
6
7
2
4
mL) and stirred for 1 h. The mixture was concentrated under
tolytic conditions has been reported: W. B. Motherwell and A. S.
vacuum and the residue washed with 5% NaHCO (2 ] 5
Williams, Angew. Chem., Int. Ed. Engl., 1995, 34, 2031.
D. T. Gibson, B. Gschwendt, W. K. Yeh and V. M. Kobal, Bio-
chemistry, 1973, 12, 1520.
3
1
mL) and water (2 ] 5 mL). The organic layer was dried over
Na SO and the product puriÐed by Ñash chromatography
2
4
18 R. E. Cripps, P. W. Trudgill and G. Whately, Eur. J. Biochem.,
(
1
hexaneÈEtOAc 1 : 4) to a†ord 36 as an o†-white solid (43 mg,
1
978, 86, 175.
8%). R \ 0.54; mp \ 143È144 ¡C; [a]28 [88.8 (c 1.0,
1
9
S. Ahmed, Ph.D. Thesis, Imperial College of Science, Technology
and Medicine, London, 1991. D. W. Ribbons, thesis director.
f
D
CHCl ); IR (KBr): 3222, 3060, 1451 cm~1; 1H NMR (300
3
MHz, DMSO-d ): d 5.99 (m, 1H), 5.64 (m, 1H), 4.41 (d,
20 D. R. Boyd, N. D. Sharma, N. I. Bowers, J. Du†y, J. S. Harrison
6
and H. Dalton, J. Chem. Soc., Perkin T rans. 1, 2000, 1345.
V. Bui, T. V. Hansen, Y. StenstrÔm, D. W. Ribbons and T. Hud-
licky, J. Chem. Soc., Perkin T rans. 1, 2000, 1669.
J \ 6.1, 1H), 4.24 (d, J \ 5.2 Hz, 1H), 4.12 (m, 1H), 3.44È3.33
2
1
(
m, 1H), 2.19È1.98 (m, 6H), 1.75È1.38 (m, 6H); 13C NMR (75
MHz, CDCl ): d 137.6, 134.3, 124.3, 123.5, 70.0, 65.9, 25.84,
3
22 A blocked mutant is a bacterial strain that lacks a particular
enzyme in a metabolic pathway, resulting in accumulation of the
metabolic intermediate produced immediately before the step
blocked. In this case (Pp 39D), the enzyme that dehydrogenates
cis-dihydrodiols to catechols is missing.
2
5.77, 24.94, 24.85, 22.87, 22.21; MS (FAB): m/z 194 (5), 177
(
1
22), 93 (100); HRMS (CI): m/z calc. for C H O (M`):
94.1307. Found: 194.1253.
1
2 18 2
2
3
All organisms used in this study were provided by David T.
Gibson. (a) Pseudomonas putida 39D, see: D. T. Gibson, J. R.
Koch and R. E. Kallio, J. Biol. Chem., 1968, 7, 2653; (b) Escheri-
chia coli JM109 (pDTG 601) expressing TDO, see: G. J. Zylstra
and D. T. Gibson, J. Biol. Chem., 1989, 264, 14940; (c) E. coli
JM109 (pDTG 141) expressing NDO, see: M. J. Simon, T. D.
Osslund, R. Saunders, B. D. Ensley, S. Suggs, A. Harcourt, W. C.
Suen, D. L. Cruden and D. T. Gibson, Gene, 1993, 127, 31.
(
1S,2R)-3-Benzyl-cyclohex-3-ene-1,2-diol (39)
An aliquot of the crude extract (2 g, 10 mmol) from bio-
oxidation of substrate 21 was subjected to PAD (5.8 g, 30
mmol) reduction according to the general procedure. The
product was puriÐed by chromatography (hexaneÈEtOAc
1
: 1) to a†ord a white solid (0.98 g, 49%): mp \ 73.5È75 ¡C;
[
1
a]26 [160.4 (c 1.0, MeOH); IR (KBr): 3281, 3024, 1602,
24 For E. coli JM109 (pDTG 602), see ref. 23b.
D
2
5
It is difficult to introduce the catechol unit into highly functional-
ized compounds because of the harsh oxidizing conditions
usually required. On the other hand, the presence of a catechol
functionality in the starting materials alters the reactivity of the
arene and may complicate the subsequent chemistry. Thus, the
enzymatic introduction of this functionality into advanced inter-
mediates has the potential to greatly simplify synthetic routes to
catechol-containing targets.
494, 1453, 1260, 1078 cm~1; 1H NMR (300 MHz, DMSO-
d ): d 7.32È7.23 (m, 2H), 7.21È7.12 (m, 3H), 5.36 (s, 1H), 4.47
6
(
d, J \ 5.7, 1H), 4.31 (dd, J \ 5.7, 1H), 3.62 (t, J \ 4.5 Hz, 1H),
3
1
1
.44È3.31 (m, 3H), 2.11È2.08 (m, 2H), 1.70È1.52 (m, 1H), 1.50È
.38 (m, 1H); 13C NMR (75 MHz, CDCl ): d 139.7, 137.4,
3
29.2, 128.6, 127.2, 126.4, 69.8, 68.2, 41.0, 25.5, 24.1; MS
(
FAB): m/z: 188 (12), 187 (100), 185 (26), 169 (19), 93 (31),
2
6
D. Gonzales, V. Schapiro, G. Seoane, T. Hudlicky and K.
Abboud, J. Org. Chem., 1997, 62, 1194; T. Hudlicky, K. Oppong,
C. Duan, C. Stanton, M. G. Natchus and M. J. Laufersweiler,
Bioorg. Med. Chem. L ett., 2001, in press.
9
1
7
1(38); HRMS (CI): m/z calc. for C H O : (M [ OH):
1
3 16 2
87.1123. Found: 187.1125. Anal. calc. for C
6.44; H, 7.90%. Found: C, 76.20; H, 7.93%.
H
O : C,
1
3 16 2
2
2
7
8
T. Hudlicky, E. E. Boros and C. H. Boros, T etrahedron: Asym-
metry, 1993, 4, 1365.
Acknowledgements
We are grateful to Professor David Gibson (University of
Iowa) for providing us with a sample of E. coli JM 109
K. Ko
Ž
nigsberger and T. Hudlicky, T etrahedron: Asymmetry,
1
993, 4, 2469.
2
3
9
0
T. Hudlicky, E. E. Boros and C. H. Boros, Synlett., 1992, 391.
M. R. Stabile, T. Hudlicky, M. L. Meisels, G. Butora, A. G.
Gunn, S. P. Fearnley, A. J. Thorpe and M. R. Ellis, Chirality,
1995, 7, 556.
(
pDTG602). Financial support from NSF (CHE-9615112 and
CHE-9910412), EPA (R826113) and TDC Research, Inc., as
well as from the Agricultural University of Norway (T. V. H.
and Y. S.) and the Norwegian Research Council (T. V. H.) is
gratefully acknowledged.
31 L. A. Paquette, J. H. Kuo and J. Doyon, T etrahedron, 1996, 52,
1625.
3
3
3
1
2
3
4
V. P. Bui, T. V. Hansen, Y. StenstrÔm and T. Hudlicky, Green
Chem., 2000, 2, 263.
Phenylcyclohexane was converted to the corresponding catechol
with JM109 (pDTG602), see Scheme 3.
References and notes
T. Hudlicky, M. R. Stabile, D. T. Gibson and G. M. Whited, Org.
Synth., 1999, 76, 77.
1
2
3
D. T. Gibson, J. R. Koch, C. L. Schmid and R. E. Kallio, Bio-
chemistry, 1968, 7, 3795.
35 G. Berti, B. Macchia, F. Macchia and L. Monti, J. Chem. Soc. C,
1971, 3371.
36 W. E. Bachmann and L. B. Wick, J. Am. Chem. Soc., 1950, 72,
V. Ley, F. Sternfeld and S. Taylor, T etrahedron L ett., 1987, 28,
2
25.
T. Hudlicky, D. Gonzalez and D. T. Gibson, Aldrichim. Acta,
1
3388.
999, 32, 35.
37 R. W. Murray, M. Singh, B. L. Williams and H. M. Moncrie†, J.
Org. Chem., 1996, 61, 1830.
4
5
D. R. Boyd and G. N. Sheldrake, Nat. Prod. Rep., 1998, 309.
S. M. Brown and T. Hudlicky, in Organic Synthesis: T heory and
Applications, ed. T. Hudlicky, JAI Press, Greenwich, CT, 1993,
vol. 2, p. 113.
38 M. Tamura and J. Kochi, Synthesis, 1971, 303.
39 A. Garcia Martinez, A. Herrera, R. Martinez, E. Teso, A. Garcia,
J. Osio, L. Pargada, R. Unanue, L. R. Subramian and M.
Hanack, J. Heterocycl. Chem., 1988, 25, 1237.
6
D. A. Widdowson, D. W. Ribbons and S. D. Thomas, Janssen
Chim. Acta, 1990, 8, 3.
40 J. K. Stille and B. L. Groh, J. Am. Chem. Soc., 1987, 109, 813.
124
New J. Chem., 2001, 25, 116È124