Toluene dioxygenase-mediated oxidation of dibromobenzenes. Absolute stereochemistry of new metabolites and synthesis of (-)-conduritol E

Article abstract of DOI:10.1016/j.tet.2006.05.012

Dibromobenzenes (o-, m-, and p-isomers) were converted to the corresponding cis-cyclohexadiene diols by whole-cell fermentation with Escherichia coli JM 109 (pDTG601A), an organism over-expressing the enzyme toluene dioxygenase (TDO). Absolute stereochemi

Full text of DOI:10.1016/j.tet.2006.05.012

Tetrahedron 62 (2006) 7471–7476
Toluene dioxygenase-mediated oxidation of dibromobenzenes.
Absolute stereochemistry of new metabolites
and synthesis of (L)-conduritol E
*
Kevin J. Finn, Jonathan Collins and Tomas Hudlicky
Department of Chemistry and Centre for Biotechnology, Brock University, 500 Glenridge Avenue, St. Catharines, ON L2S 3A1, Canada
Received 20 March 2006; revised 28 April 2006; accepted 4 May 2006
Available online 2 June 2006
Abstract—Dibromobenzenes (o-, m-, and p-isomers) were converted to the corresponding cis-cyclohexadiene diols by whole-cell fermen-
tation with Escherichia coli JM 109 (pDTG601A), an organism over-expressing the enzyme toluene dioxygenase (TDO). Absolute stereo-
chemistry of new metabolites was determined, and (ꢀ)-conduritol was synthesized.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
all three regioisomers: ortho, meta, and para. The metabo-
lites of these are shown in Table 1. A large number of diene
diols is known for at least two of the three possible isomers,
and their metabolites have been listed in several recent com-
pilations.1,2 Many diol metabolites have been observed in
variously substituted biphenyls but were not rigorously char-
acterized. A total of nearly 100 such compounds has been
noted.1 This manuscript reports the results of oxidation of a
series of dibromobenzenes as well as the determination of
absolute stereochemistry for a new diol derived from o-di-
bromobenzene by chemical conversion to (ꢀ)-conduritol E.
Oxidation of aromatic compounds to the corresponding cis-
cyclohexadiene diols with toluene dioxygenase represents
a reaction that has, as yet, no equivalent in synthetic method-
ology. To date over 400 metabolites have been isolated1 and
many served as optically pure material in total synthesis of
natural products.2,3 The whole-cell fermentation of aromatic
compounds with the recombinant organism Escherichia coli
JM 109 (pDTG601)4 produces moderate to excellent yields
of the corresponding diols, which are easily extracted from
the fermentation broth using base-washed ethyl acetate.3a
The mechanism of the enzymatic oxidation remains un-
known although some predictive models have been proposed
regarding the expected regio- and stereochemistry of oxida-
tion in single-ring, disubstituted aromatic compounds.5 For
several such compounds the fate of oxidation is known for
2. Results and discussion
Of the three isomers of dibromobenzene, only one, m-di-
bromobenzene (28), had previously been subjected to
ortho-
meta-
para-
Br
Br
Br
Br
Br
Br
OH
OH
Br
OH
OH
OH
OH
Br
Br
Br
26
27
Br
28
29
Br
30
31
1 regioisomer
1 regioisomer
2 enantiomers
1 regioisomer
2 enantiomers
1 meso compound
(1 meso compound)
Figure 1. Possible modes of oxidation for the series of dibromobenzenes.
* Corresponding author. Tel.: +1 905 688 5550 x4596; e-mail: thudlicky@brocku.ca
0040–4020/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2006.05.012

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K. J. Finn et al. / Tetrahedron 62 (2006) 7471–7476
Table 1. Oxidation of isomeric series of disubstituted arenes
diol with only 20% ee and may be enriched by either subse-
quent lipase resolution of acetyl derivatives or by resubmis-
sion of the scalemic diol to further fermentation with a wild
Pseudomonas strain in which one of the enantiomer is con-
sumed. Such procedures have been applied in the prepara-
tion of ent-diene diols22 and ent-7-deoxypancratistatin.8
The symmetrical nature of the molecule may be exploited
through the use of double radical or Heck-type cyclizations
from the vinyl bromides to appropriately tethered function-
alities.
Substrate
Products (references in superscript)
I
I
I
I
OH
OH
F
OH
OH
OH
OH
F
F
F
I
1
26
36
46,7
I
I
I
OH
OH
Br
OH
OH
The absolute configuration of 27 was established by its con-
version to conduritol E,23 as shown in Scheme 1, and its
enantiomeric excess was conveniently determined by 19F
NMR evaluation of the Mosher ester derived from mono-
protected diol 38, (Scheme 2). The Mosher ester 39 was pre-
viously prepared from homochiral and racemic alcohols 38
in connection with a study on the oxidation of a series of
methylsulfanyl bromobenzenes.15,16 Analysis of the crude
19F NMR spectrum indicated a single peak corresponding
to an enantioselectivity of greater than 95% in the enzymatic
oxidation of o-dibromobenzene.
OH
Br
OH
Br
Br
Cl
5
66
76
86,8
OH
OH
Cl
OH
OH
OH
OH
Cl
F
Cl
9
109,10
119
129
CO2H
F
CO2H
OH
CO2H
OH
CO2H
OH
OH
F
OH
a1611,12,14
OH
a1411,12,13
F
Enantiomerically pure diol 33 can easily be converted into
D-mannaric acid by ozonolysis according to established
procedures for the conversion of vinyl bromides of this type
into hexoses.24 The provision of isomeric sugars D-glucaric
acid and D-altaric acid is also possible, as all four diastereo-
isomers at C-4 and C-5 are accessible by directed hydroxyl-
ation or epoxidation procedures. Diol 27 would appear to be
suitable for synthesis of such acids (Fig. 2).
13
a1514
SMe
SMe
OH
SMe
SMe
Br
OH
OH
OH
OH
OH
Br
Br
Br
17
Cl
1815,16
1915,16
2015,16
Cl
Cl
CO2H
H
H
OH
OH
Br
HO
HO
H
Cl
OH
O
O
Br
OH
OH
OH
HO
H
OH
2217,18
OH
OH
2418
CO2H
21
2317
34
33
D-mannaric acid
a
Absolute stereochemistry not determined.
Figure 2.
toluene dioxygenase-mediated oxidation. A single isomer,
29, was produced in a yield of 4 g Lꢀ1 3a and served as a con-
venient starting material for a short synthesis of the amaryl-
lidaceae constituent narciclasine.19
3. Experimental
3.1. General
All non-hydrolytic reactions were carried out under an argon
atmosphere. Glassware used for moisture-sensitive reactions
was flame-dried under vacuum and subsequently purged
with argon. THF was distilled from potassium/benzo-
phenone. Methylene chloride was distilled from calcium
hydride. Flash column chromatography was performed
using Kieselgel 60 (230–400 mesh). Analytical thin-layer
chromatography was performed using silica gel 60-F254
plates. Melting points are reported uncorrected. IR spectra
were recorded as a film, unless otherwise specified. 1H
and 13C NMR spectra were obtained on a Bruker instru-
ment at 300 and 75 MHz, respectively. Specific rotation
measurements are given in deg cm3 gꢀ1 dmꢀ1. Ultraviolet
spectroscopy was performed using a diode array spectro-
photometer. Large-scale fermentation was performed in a
15-L B. Braun Biostat C-15 Fermentor. All biological media
was purchased through Fisher Canada. Combustion analyses
Because of the symmetry of dibromobenzenes, only one re-
gioisomer of a diol is possible, as shown in Figure 1. In prin-
ciple, o-dibromobenzene could also form a meso compound
if the oxidation took place with a 3,4-regiochemistry, how-
ever, TDO oxidations of disubstituted arenes yield exclu-
sively 1,2-regioisomers with respect to the directing group.
Whole-cell oxidation of m-dibromobenzene with E. coli
JM 109 (pDTG601) produced a single enantiomer (>99%
ee). Similarly, the o-isomer provided diol 27 in a yield of
4.1 g Lꢀ1, and the p-isomer furnished the meso-diol 31 in
55 mg Lꢀ1 20
Though this particular metabolite is meso, it
.
may find application in asymmetric synthesis through fur-
ther desymmetrization.21 Procedures such as lipase resolu-
tion have been used to enrich enantiomeric excesses of
those diols that are produced as scalemic mixtures. For
example, p-bromoiodobenzene provides the corresponding

K. J. Finn et al. / Tetrahedron 62 (2006) 7471–7476
7473
Br
Br
Br
OsO4, NMO,
acetone-H2O
2,2-DMP,
Br
O
O
Br
Br
OH
OH
O
O
pTsOH
95%
71%
HO
OH
27
33
32
nBu3SnH,
AIBN, THF
64%
OH
OH
3% conc HCl,
MeOH
O
O
HO
81%
HO
OH
OH
(-)-conduritol E
34
35
21 -285 (c 1.0, H2O)
[α]D
lit: [α]D20 -294 (c 1.0, H2O)ref 23b
Scheme 1. Correlation of absolute configuration by conversion to (ꢀ)-conduritol E.
Br
Br
potassium
azodicarboxylate,
AcOH
Br
Br
OH
OH
Br
OH
Br
OH
OH
THSCl, imidazole
86%
95%
OTHS
36
37
27
H2,
PtO2, NEt3
52%
O
Ph
CF3
Mosher's acid
chloride, NEt3
O
MeO
OH
OTHS
OTHS
39
38
Scheme 2. Synthesis of Mosher’s ester 39.
were performed by Atlantic Microlabs, Norcross, Georgia,
USA.
(100 mg Lꢀ1). LB medium (500 mL) was inoculated with
1 mL of E. coli JM 109 (pDTG601) of the preculture
medium. This inoculum was grown at 35 ꢁC on an orbital
shaker (180 rpm) for 5 h. A chemical inducer, isopropyl-1-
thio-b-D-galactopyranoside (IPTG) (10 mg Lꢀ1), was added
via sterile filter and the cells were grown for an additional 7 h
at 35 ꢁC on an orbital shaker (200 rpm).
3.2. General biotransformation procedure
3.2.1. Small-scale fermentation with E. coli JM 109
(pDTG601).
3.2.1.1. Growth of colonies. Agar plates consisted of
bactotryptone (10 g Lꢀ1), yeast extract (5 g Lꢀ1), NaCl
(5 g Lꢀ1), agar (30 g Lꢀ1), and ampicillin (100 mg Lꢀ1).
E. coli JM 109 (pDTG601) cells were streaked onto a plate
and were incubated at 35 ꢁC for 12–24 h. A single bacterial
colony was selected for preculture preparations as described
in the following section.
3.2.1.4. Substrate addition. The supernatant was sepa-
rated from the cells by centrifugation at 7000 rpm for
15 min. The cell pellet was re-suspended in 500 mL of
0.1 M phosphate buffer consisting of KH2PO4 (6.8 g Lꢀ1),
K2HPO4 (8.7 g Lꢀ1), and glucose (2 g Lꢀ1). The aromatic
substrate (400 mg Lꢀ1) was added as a solution in isopropyl
alcohol. Product formation was monitored by thin-layer
chromatography (silica gel, hexane/ethyl acetate, 1:1).
3.2.1.2. Preparation of preculture. Luria Bertani (LB)
liquid medium consisted of bactotryptone (10 g Lꢀ1), yeast
extract (5 g Lꢀ1), NaCl (5 g Lꢀ1), and ampicillin
(100 mg Lꢀ1). The preculture medium (3 mL) was inocu-
lated with a single colony of E. coli JM 109 (pDTG601)
and the resulting inoculum was grown at 35 ꢁC on an orbital
shaker (200 rpm) for 6 h.
3.2.1.5. Product isolation. After 5 h of incubation with
substrate the pH of the culture medium was adjusted with
6 M NaOH to 8.5, and a cell pellet was obtained by centrifu-
gation at 7000 rpm and 4 ꢁC for 20 min. The supernatant
liquid was extracted with acid-free ethyl acetate, prepared
by stirring the organic solvent with a saturated solution of
Na2CO3 and separation of the organic layer from the aque-
ous layer. The extract was dried over anhydrous MgSO4, fil-
tered, and the solvent was removed under reduced pressure.
3.2.1.3. Fernbach flask preparation. LB liquid me-
dium consisted of bactotryptone (10 g Lꢀ1), yeast extract
(5 g Lꢀ1), NaCl (5 g Lꢀ1), glucose (5 g Lꢀ1), and ampicillin

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K. J. Finn et al. / Tetrahedron 62 (2006) 7471–7476
The crude material was purified by crystallization or flash
column chromatography (silica gel deactivated with 10%
distilled water) immediately after concentration of the sol-
vent in order to minimize decomposition of the unstable
dienediols.
An analytical sample was obtained after chromatography
over 10% deactivated silica gel to afford the title compound
as a clear oil. [a]2D2 +101 (c 0.75, CHCl3); Rf 0.50 (50% ethyl
acetate in hexanes); IR (film) n 2988, 2933, 2896,
1
1634 cmꢀ1; H NMR (300 MHz, CDCl3) d 6.14 (d, J¼
9.9 Hz, 1H), 5.93 (dd, J¼9.9, 3.6 Hz, 1H), 4.82–4.72
(m, 2H), 1.45 (s, 6H); 13C NMR (75 MHz, CDCl3)
d 128.7, 125.6, 123.8, 120.3, 106.9, 77.3, 71.3, 26.6, 25.0;
HRMS (EI) Calcd for C9H10Br2O2, 307.9047; Found,
307.9037.
3.2.2. Large-scale fermentations. Large-scale fermenta-
tions were carried out in a 15-L (8-L working volume)
B. Braun Fermentor according to a published procedure.3
3.2.3. Extraction of products. Dienediols obtained from
large-scale (8-L fermentation) were extracted from the aque-
ous fermentation broth into ethyl acetate either by standard
manual extraction or by continuous extraction. Large-scale
manual extraction requires up to 20 L of ethyl acetate,
whereas the use of a rotary-evaporator-driven continuous ex-
tractor facilitates the extraction of up to 9 L of aqueous broth
using as little as 3 L of ethyl acetate. The diene diols derived
from small-scale fermentations (1-L) were extracted manu-
ally. Progress of either manual or continuous extraction was
monitored by thin-layer chromatographic analysis of the
aqueous layer.
3.2.3.3. (3aS,4R,5S,7aS)-2,2-Dimethyl-6,7-dibromo-
4,5-dihydroxybenzo[1,3]dioxole (33). The protected diene-
diol 32 (3.1 g, 10.0 mmol, 1 equiv) was suspended in 60 mL
of 8:1 (by volume) mixture of acetone/water. N-Methyl-
morpholine-N-oxide (2.34 g, 20.0 mmol, 2 equiv) was added
followed by the addition of four crystals of osmium tetraox-
ide. The reaction mixture darkened slightly and was stirred
for 18 h until consumption of starting material was com-
plete. The reaction mixture was quenched by addition of
5 mL satd aq sodium bisulfite and 2 g solid sodium bisulfite,
and the pH of the mixture was adjusted by addition of concd
HCl. The mixture was stirred for 15 min, and the acetone
was removed under reduced pressure. The aqueous portion
was extracted repeatedly with ethyl acetate, and the com-
bined organic extracts were washed with 1 N HCl, 20% aq
solution of KOH, and brine before being dried over anhy-
drous magnesium sulfate. The extracts were filtered through
a short column of silica gel and the solvent evaporated to fur-
nish 2.4 g of a white crystalline solid, 71% yield, which re-
quired no further purification for the subsequent reaction. An
analytically pure sample was obtained by recrystallization
from ethyl acetate/pentane. Mp 155–156 ꢁC; [a]D22 +5.47
(c 0.75, MeOH); Rf 0.3 (50% ethyl acetate in hexanes); IR
3.2.3.1. (1S,2S)-3,4-Dibromo-cyclohexa-3,5-diene-1,2-
diol (27). The biooxidation of o-dibromobenzene was per-
formed according to the general procedure for large-scale
fermentation.3 o-Dibromobenzene 26 (60 g) was added
dropwise over 45 min to a 15-L fermentor containing a grow-
ing culture of E. coli JM 109 (pDTG601). After stirring the
media for an additional 1 h, the cell broth was separated
from the cells by centrifugation. The broth was extracted
using a rotary-evaporator-driven continuous extractor over
a three-days period with 3 L of ethyl acetate. The combined
organic layers were washed twice with approximately
10% w/v sodium carbonate solution to remove any phenolic
residue. The organic extracts of the fermentation broth were
concentrated in vacuo and the dienediol precipitated by addi-
tion of pentane. Recrystallization from ethyl acetate/pentane
provided the title compound as a white solid (32.8 g,
4.1 g Lꢀ1). Mp 144–146 ꢁC (from ethyl acetate/pentane);
[a]2D2 +104 (c 0.15, diethyl ether); Rf 0.36 (hexanes/ethyl
(KBr) n 3435, 3360, 2994, 2905, 1606 cmꢀ1 1H NMR
;
(300 MHz, CDCl3) d 4.77 (dd, J¼1.2, 5.4 Hz, 1H), 4.50–
4.45 (m, 2H), 4.34 (m, 1H), 2.68 (br s, 2H), 1.43 (s, 6H);
13C NMR (75 MHz, CDCl3) d 127.6, 125.6, 77.6, 75.3,
70.9, 69.1, 27.6, 26.1; HRMS (EI) Calcd for C8H9O4Br,
326.8867; Found, 326.8863; Anal. Calcd for C9H12Br2O4:
C, 31.42; H, 3.52. Found: C, 31.59; H, 3.53.
1
acetate, 1:1); IR (film) n 3175, 1621 cmꢀ1; H NMR (300
MHz, acetone-d6) d 6.03 (dd, J¼9.9, 2.1 Hz, 1H), 6.00 (dd,
J¼6.6, 2.4 Hz, 1H), 4.62 (d, J¼6.9 Hz, 1H), 4.55 (m, 1H),
4.28 (m, 2H); 13C NMR (75 MHz, acetone-d6) d 133.4,
126.5, 126.2, 120.6, 74.2, 69.0; HRMS (EI) Calcd for
C6H6Br2O2 (M+), 267.8731; Found, 267.8733; Anal. Calcd
for C6H6Br2O2: C, 26.70; H, 2.24. Found: C, 27.04; H, 2.32.
3.2.3.4. (L)-Conduritol E (35). Dibromide 33 (0.50 g,
1.4 mmol, 1 equiv) was dissolved in 50 mL distilled THF
and transferred to a flame-dried 100-mL round-bottomed
flask equipped with a reflux condenser. The solution was de-
gassed in an ultrasound bath and under positive argon pres-
sure for 10 min. Azoisobutyronitrile (23 mg, 0.14 mmol,
0.1 equiv) was added, and the solution was heated to steady
reflux (83 ꢁC external temp). At this time, tributyl tin hy-
dride (1.0 mL, 3.36 mmol, 2 equiv) was added in portion.
Reflux was maintained for 1.5 h until complete consumption
of starting material was witnessed by TLC analysis. The re-
action mixture was cooled, and potassium fluoride (2 g) was
added. The resulting precipitate was filtered, and the filtrate
concentrated under reduced pressure. The residue was puri-
fied by flash column chromatography using 4:1 hexanes/
ethyl acetate to 100% ethyl acetate to provide 170 mg
(64%) of the de-brominated material. The solid was dis-
solved in 5 mL of methanol and to this solution was added
2 mL of a 3% (by volume) solution of concd HCl in metha-
nol and the resulting solution was stirred for 40 h after which
time the solvent was removed under reduced pressure to
3.2.3.2.
(3aS,7aS)-2,2-Dimethyl-4,5-dibromo-4,6-
benzo[1,3]dioxole (32). Diene diol 27 (3.0 g, 11.1 mmol,
1 equiv) was transferred to a 100-mL round-bottomed flask
and suspended in 5 mL acetone and 15 mL of 2,2-dimeth-
oxypropane. A few crystals of p-toluenesulfonic acid were
added, and the reaction mixture was stirred at rt for 3 h.
The reaction was quenched with 12 mL of 10% aq sodium
hydroxide solution, and the acetone was removed under re-
duced pressure. The residue was diluted with ethyl acetate,
and the layers were separated. The aqueous layer was then
extracted with several portions of ethyl acetate. The com-
bined organic layers were washed with brine and dried
over anhydrous magnesium sulfate. The solvent was re-
moved under vacuum to provide a light oil (3.21 g, 95%).

K. J. Finn et al. / Tetrahedron 62 (2006) 7471–7476
7475
provide a crude white solid. The solid was purified by flash
column chromatography (4:1 chloroform/methanol) to give
conduritol E as a white crystalline solid (78 mg, 81%). Mp
194–195 ꢁC (lit.23b mp 193 ꢁC); [a]D20 ꢀ285 (c 1.0, H2O),
lit.23b: [a]D20 ꢀ294 (c 1.0, H2O); Rf 0.18 (chloroform/
3.2.3.7.
(1R,2S)-2-[(Hexyldimethylsilyl)oxy]cyclo-
hexan-1-ol (38). A flask containing a magnetic stirring bar
was charged with dibromide 37 (0.219 g, 0.53 mmol,
1 equiv), triethylamine (0.5 mL, 3.56 mmol, 7 equiv), plati-
num oxide (Adam’s catalyst, 24 mg, 0.11 mmol, 0.2 equiv),
and 0.5 mL MeOH. The reaction flask was evacuated,
flushed with hydrogen via a balloon (1 atm), and stirred until
total consumption of starting material as was observed by
TLC control (6 h). The crude mixture was filtered through
a short plug of Celite, and the solvent was removed under
reduced pressure. The crude residue was purified by flash
column chromatography (pentane/Et2O, 10:1) to give the
title compound as a clear and colorless oil (71 mg, 52%)
with spectral data matching that of previously reported
compound 38. [a]D23 +3.4 (c 1.0, CHCl3), lit.16: [a]D23 +3.3
(c 1.0, CHCl3).
methanol, 4:1); IR (film) n 3434, 1634 cmꢀ1 1H NMR
;
(300 MHz, MeOD) d 5.79 (d, J¼2.1 Hz, 2H), 4.27 (s, 2H),
3.93 (d, J¼0.9 Hz, 2H); 13C NMR (75 MHz, MeOD)
d 130.7, 70.9, 67.6; HRMS (EI) Calcd for C6H8O3
(M+ꢀH2O), 128.0473; Found, 128.0455.
3.2.3.5. (1S,2S)-3,4-Dibromo-cyclohexa-3-ene-1,2-diol
(36). Diol 27 (0.38 g, 1.47 mmol, 1 equiv) was dissolved
in 6 mL MeOH, and the round-bottomed flask containing the
solution was subsequently placed into an ice/NaCl bath.
Potassium azodicarboxylate (0.93 g, 4.27 mmol, 3 equiv)
was added in two portions to the methanolic solution. Acetic
acid (0.85 mL, 12.78 mmol, 9 equiv) in 2 mL MeOH was
added dropwise over 40 min. The reaction flask was allowed
to warm to room temperature overnight (15 h). The reaction
was quenched by adding 2 mL saturated Na2CO3 solution
and stirring for 20 min. Methanol was removed under
reduced pressure and the residue diluted with 10 mL EtOAc.
The layers were separated and the aqueous phase was ex-
tracted with 3ꢂ10 mL EtOAc. The combined organic layers
were washed with brine, dried over MgSO4, and treated with
activated charcoal. Filtration and concentration of the filtrate
under reduced pressure afforded 36 as a white crystalline
solid (0.367 g, 95%). Mp 175–176 ꢁC; [a]D21 ꢀ50.4
(c 0.75, MeOH); Rf 0.23 (Hex/EtOAc, 1:1); IR (KBr pellet)
n 3246, 1626 cmꢀ1; 1H NMR (300 MHz, acetone-d6) d 4.61
(d, J¼6 Hz, 1H), 4.26 (s, 1H), 3.96–3.84 (m, 2H), 2.79–2.51
(m, 2H), 2.05–1.92 (m, 1H), 1.85–1.73 (m, 1H); 13C NMR
(75 MHz, acetone-d6) d 127.1, 124.9, 73.5, 68.25, 35.2,
26.9; HRMS (EI) Calcd for C6H8Br2O2, 271.8872; Found,
271.8871; Anal. Calcd for C6H8Br2O2: C, 26.50; H, 2.97.
Found: C, 27.34; H, 3.16.
3.3. General procedure for the formation of Mosher
ester derivative 39
Alcohol 38 (20 mg, 0.076 mmol, 1 equiv) was transferred
to a flame-dried round-bottomed flask containing magnetic
stirring bar under an argon atmosphere. Anhydrous triethyl-
amine (17 mL) was added followed by 4-dimethylaminopyr-
idine (4.8 mg, 0.038 mmol, 0.5 equiv). (R)-(ꢀ)-a-Methoxy-
a-trifluoromethyl-phenylacetic acid chloride (23 mL,
0.11 mmol, 1.5 equiv) was added dropwise. Within minutes
a white precipitate was observed. The reaction was stirred
overnight. The reaction mixture was then diluted with
5 mL methylene chloride, transferred to a separatory funnel,
and washed with 5 mL saturated solution of sodium bicar-
bonate. The layers were separated, the organic layer dried
(MgSO4), and the solvent evaporated to provide the ester as
a crude oil. The compound was purified by flash column
chromatography (pentane/Et2O, 10:1) to afford the ester as
a clear and colorless oil (20 mg, 57%). Physical and spectral
data matched that for the compound previously reported.16
3.2.3.6. (1S,2S)-1-[(Hexyldimethylsilyl)oxy]-3,4-dibro-
mocyclohexa-3-ene-2-ol (37). A 5-mL round-bottom flask
was charged with diol 36 (200 mg, 0.74 mmol, 1 equiv),
imidazole (65 mg, 0.96 mmol, 1.3 equiv), and 1 mL anhy-
drous dimethylformamide. The flask was cooled externally
to ꢀ30 ꢁC, then hexyldimethylsilyl chloride (0.15 mL,
0.78 mmol, 1.05 equiv) was added. The mixture was stirred
at ꢀ30 ꢁC for 1 h and then the reaction flask was placed in
a freezer (ꢀ18 ꢁC) for 21 h. The mixture was allowed to
warm to room temperature and diluted with 50 mL ether,
washed with 10ꢂ1 mL distilled H2O, brine, and dried over
MgSO4. After filtration, the filtrate was concentrated under
reduced pressure. The crude silyl ether was purified by flash
column chromatography (pentane/Et2O, 10:1) to give 37 as
a clear and colorless oil (0.26 g, 86%). [a]2D1 ꢀ41.1
(c 0.75, MeOH); Rf 0.23 (pentane/Et2O, 10:1); IR (film)
Rf 0.46 (pentane/Et2O, 10:1); IR (film) n 2948, 2867, 1745,
1
1463, 1450, 1379, 1263, 1169 cmꢀ1; H NMR (300 MHz,
CDCl3) d 7.60 (m, 2H), 7.40 (m, 3H), 5.12 (m, 1H), 3.80
(m, 1H), 3.55 (m, 3H), 1.75–1.55 (m, 7H), 1.5–1.2 (m,
2H), 0.88 (dd, J¼6.8, 5.2 Hz, 6H), 0.81 (d, J¼4.1 Hz, 6H),
0 (s, 3H), ꢀ0.10 (s, 3H); 19F NMR (188 MHz, CDCl3)
d ꢀ72.4 ppm. Lit.15: 19F NMR (188 MHz, CDCl3) d
ꢀ72.8 ppm.
Acknowledgements
We are indebted to the following agencies for generous
support of this work: National Science and Engineering
Research Council (NSERC); the Canadian Foundation for
Innovation; the Ontario Innovation Trust; Brock University;
the donors of the Peteroleum Research Fund, administered
by the American Chemical Society (PRF-38075-AC);
TDC Research Inc.; and TDC Research Foundation. We
thank Brock University for providing a graduate fellowship
for K.J.F. We are also grateful to Dr. Gregg Whited
(Genencor International, Inc.) and Dipl. Ing. Hannes Leisch
(Brock University) for their generous help and discussions
regarding whole-cell fermentations.
n 3547, 2958, 2868, 1628 cmꢀ1 1H NMR (300 MHz,
;
CDCl3) d 4.26–4.17 (m, 1H), 4.03–3.93 (m, 1H), 2.84 (d,
J¼4 Hz, 1H), 2.77–2.64 (m, 1H), 2.63–2.48 (m, 1H),
2.10–1.9 (m, 1H), 1.77–1.57 (m, 2H), 0.95–0.84 (m, 13H),
0.17 (s, 6H); 13C NMR (75 MHz, CDCl3) d 130.8,
127.8, 122.9, 74.1, 73.6, 69.6, 35.1, 34.1, 27.2, 24.8,
20.2, 20.0, 18.5, 18.4; HRMS (EI) Calcd for C14H26Br2O2Si,
328.9032; Found, 328.9026; Anal. Calcd for
C14H26Br2O2Si: C, 40.59; H, 6.33. Found: C, 40.96; H, 6.36.

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K. J. Finn et al. / Tetrahedron 62 (2006) 7471–7476
ˇ
15. Finn, K. J.; Cankar, P.; Jones, R. T. B.; Hudlicky, T.
Tetrahedron: Asymmetry 2004, 15, 2833.
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