Processing of cyclopropylarenes by toluene dioxygenase: Isolation and absolute configuration of metabolites

Article abstract of DOI:10.1016/j.tetasy.2005.09.020

Several arenes possessing a cyclopropyl substituent were subjected to enzymatic oxidation with toluene dioxygenase. The absolute configuration of metabolites was established by chemical means.

Full text of DOI:10.1016/j.tetasy.2005.09.020

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 16 (2005) 3606–3613
Processing of cyclopropylarenes by toluene dioxygenase:
isolation and absolute configuration of metabolites
Kevin J. Finn, Lena Rochon and Tomas Hudlicky*
Department of Chemistry, Brock University, St. Catharines, Ontario, Canada L2S 3A1
Received 15 August 2005; accepted 7 September 2005
Available online 10 November 2005
Abstract—Several arenes possessing a cyclopropyl substituent were subjected to enzymatic oxidation with toluene dioxygenase. The
absolute configuration of metabolites was established by chemical means.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
ideally suited for many further reactivity options and
cascade processes, as illustrated in Figure 1.
Enzymatic oxidation of aromatics with recombinant
organisms expressing toluene dioxygenase (TDO)
provides valuable synthons for asymmetric synthesis.
Several hundreds of cyclohexadiene cis-diols are already
known1 and many have found application in total
synthesis of natural products.1c,2 Reliable procedures
have been published for the whole-cell fermentation of
arenes using either a recombinant organism3 (Escherichia
coli JM 109 pDTG601) or a blocked mutant4 (Pseuda-
monas putida 39D), both of which express toluene
dioxygenase. To extend the functional content of such
metabolites, we wished to examine a series of cyclo-
propylarenes that would provide diene diols of type 1,
2. Results and discussion
Cyclopropylbenzene, previously investigated in connec-
tion with the mechanism of the enzymatic oxidation,5
has proven to be an excellent substrate for the enzyme.
We prepared the cyclopropylarenes6 listed in Table 1
and subjected them to the whole-cell fermentation
protocol.
The products of fermentation were isolated by extrac-
tion of the fermentation broth with base-washed ethyl
acetate, then characterized and subjected to the proof
of absolute configuration, as outlined in Schemes 1–3.
In all cases, the absolute configuration was proven by
comparison with synthetic material derived from diol
15, whose absolute stereochemistry has already been
firmly established,7 with the exception of metabolite 8,
whose match was made with the known diol 23.8
R
[5+2]
cycloadditions
{
OH
[4+2]
electrophilic
cycloadditions
radical cyclizations
tether attachment
OH
Oxidation of racemic trans-cyclopropylstilbene gave
diene diols 12a and 12b, which was reduced with potas-
sium azodicarboxylate/acetic acid and the diol protected
as an acetonide,5 to provide 20a and 20b (Scheme 1).
1H and 13C NMR spectroscopic data for compounds
20a and 20b revealed an approximate 1:1 ratio of diaste-
reoisomers. The absolute configuration was proven by
conversion of phenylacetylene 17 to the corresponding
cyclopropylboronic acid,9 which was subjected to a
Suzuki coupling protocol with vinyl bromide 21,
prepared from homochiral bromodiene diol 15.10
epoxidation
nucleophilic tether
attachment
1
Figure 1. Reactivity options of homochiral dienylcyclopropanes
obtained from enzymatic oxidation of the corresponding cyclo-
propylarenes.
*
Corresponding author. Tel.: +1 905 688 5550x4956; fax: +1 905 984
4841; e-mail: thudlicky@brocku.ca
0957-4166/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2005.09.020

K. J. Finn et al. / Tetrahedron: Asymmetry 16 (2005) 3606–3613
3607
Table 1. Results of enzymatic oxidation of cyclopropylarenes by E. coli JM 109 (pDTG601)
Entry
Substrate
Product
Yield (mg/L)
OH
1a
2500
OH
2
4
3
5
OH
OH
2
3
56
OH
OH
32
Br
Br
7
6
OH
OH
OH
OH
4
90
( )-8
9a
(1:1) 9b
5b
—
—
(
)-10
6
140
OH
OH
OH
OH
(
)- 11
12a
12b
(1:1)
7b
—
—
13
8c
—
—
14
a Biotransformation of substrate 2 was performed in a 15-L fermentor.
b 1H NMR spectra of the crude residue from biooxidation of cis-configured cyclopropanes revealed the presence of only trace amounts of the
corresponding diene diols.
c Only starting material was recovered.
Spectroscopic data and the specific rotations of 20a and
20b, synthesized from compound 15, provided the proof
of the absolute configuration of the trans-isomer 12a
and 12b.
to the literature methods.11 When this approach failed,
the methyl derivative was prepared from a known com-
pound, triene 23,8 as shown in Scheme 2. Fermentation
of trans-b-methylstyrene 22, initially reported by Boyd
et al., gave the corresponding chiral triene diol 23,
which was reduced with diimide to the more stable
diene. The diol functionality was protected as its
acetonide and the less-substituted olefin converted to
The proof of the absolute configuration for diols 9a and
9b was initially attempted by oxidative cleavage and
reduction of phenylcyclopropyl metabolite 12 according

3608
K. J. Finn et al. / Tetrahedron: Asymmetry 16 (2005) 3606–3613
Et2Zn/
1) diimide
TFA/CH2I2
TDO
+
OH
OH
OH
OH
2) DMP/TsOH
( )-11
12a
16
12b
O
HO
B
O
B
OH
1) borane
2) 2,3-butanediol
1) Pd(OAc)2/CH2N2
2) NaOH
"Pd(0)"
21
O
O
( )-19
17
18
20a
+
Br
Br
O
O
OH
OH
ref 10
O
15
21
O
20b
1:1 diastereomers
by NMR
Scheme 1.
its corresponding cyclopropane6 25a, formed as a single
diastereoisomer whose specific rotation sign matched
that of compounds 25a and 25b prepared by fermenta-
tion of cyclopropyl benzene 8.12 The enantiomeric
excesses of the inseparable diastereomeric mixtures of
20a and 20b and 25a and 25b cannot be determined
accurately. We base the estimate on the fact over 200
diols derived from mono-substituted arenes are pro-
duced in >95% ee.
metric synthesis. It is interesting to note that the
enzyme did not differentiate between the enantiomers
of racemic substrates 8 and 11, producing a 1:1 mixture
of diastereomers. This observation is consistent with
previous results obtained from the fermentation of var-
ious substrates containing stereogenic centers proximal
to the aromatic ring.10b It appears that the substituents
containing the remote stereogenic centers lie outside the
active site and therefore no enantiomeric discrimination
takes place during the oxidation; this rationale is consis-
tent with the model of enzymatic oxidations proposed
by Boyd.1a
Proof of the absolute configuration of metabolite 7 was
accomplished by conversion to vinylcyclopropane 26 by
hydrogenation with AdamsÕ catalyst in triethylamine.
The configuration of diol 26 was correlated to diol 3, as
shown in Scheme 3. The absolute configuration of diol 3
had previously been matched to diol 15.5 It has been
shown that oxidation of cyclopropylbenzene proceeds
to give essentially enantiomerically pure diene diol,
whereas enzymatic oxidation of p-bromocyclopropylben-
zene provides the product with only 27% enantiomeric ex-
cess13 in the absolute configuration shown in Scheme 3.
It should also be noted that the cis-isomers 10 and 13
proved not to be substrates at all. This is surprising, espe-
cially in case of 13, whose molecular volume resembles
phenanthrene, an excellent substrate for both toluene
dioxygenase and the closely related enzyme naphthalene
dioxygenase.14 Equally surprising is the fact that p-dicy-
clopropylbenzene was not oxidized by toluene dioxygen-
ase, especially since p-bromocyclopropylbenzene is a
substrate. Further studies on the enzymatic oxidation
of substrates containing remote stereogenic centers will
be conducted. The currently available cyclopropylarene
metabolites will be employed in various cycloaddition
and cascade processes as outlined in Figure 1. The results
of these endeavors will be reported in due course.
3. Conclusion
In summary, new metabolites derived from cyclopro-
pylarenes have been identified for future use in asym-

K. J. Finn et al. / Tetrahedron: Asymmetry 16 (2005) 3606–3613
3609
1) Potassium
OH
azodicarboxylate, AcOH
O
O
TDO
ref 8
2) 2,2-Dimethoxypropane
OH
pTsOH
22
23
24
Et2Zn/ trifluoroacetic acid
CH2I2 producing only 25a
1) Potassium
OH
OH
azodicarboxylate, AcOH
O
O
TDO
2) 2,2-Dimethoxypropane
pTsOH
25a
25b
( )-8
9a
+
O
O
OH
OH
9b
Scheme 2.
PtO2/ NEt3
3 atm H2
toluene
dioxygenase
toluene
OH
OH
OH
OH
OH
diimide
dioxygenase
OH
Br
Br
6
7
2
26
3
Scheme 3.
4. Experimental
4.1. General biotransformation procedure
ampicillin (50mg L ꢀ1). A sterile plastic culture tube con-
taining 3 mL LB medium was inoculated with a single
colony of E. coli JM 109 (pDTG601) and the preculture
grown at 35 °C on an orbital shaker (180rpm) for 6 h.
4.1.1. Shake-flask fermentation using E. coli JM 109
(pDTG601).
4.1.1.3. Fernbach flask preparation. LB liquid med-
ium consisted of bactotryptone (10g L ꢀ1), yeast extract
(5 g Lꢀ1), NaCl (5 g Lꢀ1), glucose (5 g Lꢀ1), and ampicil-
lin (50mg L ꢀ1). A 2-L sterile fernbach flask containing
500 mL of LB medium was inoculated with 1 mL of
E. coli JM 109 (pDTG601) preculture and the resulting
culture grown at 35 °C on an orbital shaker (180rpm)
for 5 h. A chemical inducer of protein synthesis, b-iso-
propylthiogalactopyranoside (IPTG) (10mg L ꢀ1), was
added via a sterile filter, and the cells grown for an addi-
tional 7 h at 35 °C on an orbital shaker (180rpm).
4.1.1.1. Growth of cꢀo1lonies. Agar plates consisted of
bactotryptone (10g L ), yeast extract (5 g Lꢀ1), NaCl
(5 g Lꢀ1), agar (30g L ꢀ1), and ampicillin (50mg L ꢀ1).
E. coli JM 109 pDTG601 cells were streaked onto a
plate and incubated at 35 °C for 18 h. Only single bacte-
rial colonies were selected for preculture preparations.
4.1.1.2. Preparation of preculture. Luria Bertani
(LB) liquid medium consisted of bactotryptone
(10g L ꢀ1), yeast extract (5 g Lꢀ1), NaCl (5 g Lꢀ1), and

3610
K. J. Finn et al. / Tetrahedron: Asymmetry 16 (2005) 3606–3613
4.1.1.4. Substrate addition. The supernatant was
tone-d6) d: 144.2, 126.6, 124.2, 115.6, 72.6, 70.3, 13.2,
6.4, 6.0.
separated from the cells by centrifugation at 7000 rpm
for 15 min. The supernatant was decanted and the cell
pellet resuspended 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 (250
mg Lꢀ1) was added as a solution in isopropyl alcohol
(for solid substrates) or as a neat liquid (for oils). Prod-
uct formation was monitored by thin-layer chromato-
graphy (silica gel, hexane–ethyl acetate, 1:1).
4.1.4. (1S,2R)-3-(2-Methyl-1-cyclopropyl)-cyclohexa-3,5-
diene-1,2-diols 9a and 9b 1:1 mixture of diastereoiso-
mers. The crude mixture of diols was isolated from fer-
mentation with E. coli JM 109 (pDTG601) according to
the general procedure for shake-flask fermentation. The
product was purified by flash column chromatography
on 10% deactivated silica gel (hexanes–ethyl acetate,
1:1) to provide the crystalline diols 9a and 9b as an
4.1.1.5. 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
obtained by centrifugation at 7000 rpm and 4 °C for
20min. The supernatant liquid was extracted with
acid-free ethyl acetate prepared by stirring with a satu-
rated solution of Na2CO3. The extract was dried over
MgSO4, filtered, and the solvent removed under reduced
pressure. The crude material was purified by crystalliza-
tion or flash column chromatography (silica gel deacti-
vated with 10% distilled water) immediately after
concentration of the solvent in order to minimize
decomposition of the unstable diene diols.
inseparable mixture of diastereoisomers (90mg/L). Mp
22
42–46 °C; ½aŠ ¼ þ71:3 (c 0.5, CHCl3); Rf 0.3 (hex-
D
anes–ethyl acetate, 1:1); IR (film) m 3350, 2951, 1585,
1
1384, 1074 cmꢀ1; H NMR (300 MHz, CDCl3) d: 5.83
(m, 1H), 5.62 (d, J = 9.8 Hz, 1H), 5.55 (m, 1H), 4.25
(s, 1H), 3.78 (s, 1H), 2.30(br s, 1H), 1.87 (br s, 1H),
1.20(m, 1H), 1.05 (d, J = 2.8 Hz, 3H), 0.97 (m, 1H),
0.65 (m, 1H), 0.45 (m, 1H); 13C NMR (100 MHz,
CDCl3) d: 143.5, 128.0, 127.9, 124.9, 124.8, 120.7,
117.4, 117.3, 70.3, 70.2, 69.6, 69.5, 23.4, 23.3, 19.2,
16.4, 16.0, 15.2, 14.6, 14.5; HRMS (EI) calcd for
C10H14O2 (M+), 166.0993; found, 166.0994.
4.1.5. (1S,2R)-3-(2-Phenyl-cyclopropyl)-cyclohexa-3,5-
diene-1,2-diols 12a and 12b 1:1 mixture of diastereoiso-
mers. The crude mixture of diols was isolated from fer-
mentation with E. coli JM 109 (pDTG601) according to
the general procedure for shake-flask fermentation. The
product was purified by flash column chromatography
on 10% deactivated silica gel (hexanes–ethyl acetate,
1:1) to provide the title compound as a clear oil, which
4.1.1.6. Large-scale fermentations. These were
carried out in a 15-L (8 L working volume) B. Braun
Fermentor according to a published procedure.3
4.1.2. (1S,2R)-3-(1-Methyl-cyclopropyl)-cyclohexa-3,5-
diene-1,2-diol 5. The diol was isolated from a crude
mixture from the biotransformation of the correspond-
ing cyclopropylbenzene by E. coli JM 109 (pDTG601)
according to the general procedure for shake-flask
fermentation. After purification by flash silica gel chro-
matography (2:1 Hex/EA, 10% deactivated silica), the
cis-diene diol was recrystallized from ethyl acetate/
was an inseparable mixture of diastereoisomers
22
(140mg/L): ½aŠ ¼ þ38:6 (c 0.89, CHCl3); Rf 0.28 (hex-
D
anes–ethyl acetate, 1:1); IR (film) m 3346, 2922, 1603,
1498, 1403 cmꢀ1
;
1H NMR (300 MHz, CDCl3) d:
7.24–7.19 (m, 2H), 7.13–7.01 (m, 3H), 5.86 (m, 1H),
5.67–5.60(m, 2H), 4.27 (s, 1H), 3.87 (d, J = 5.8 Hz,
1H), 2.58–2.41 (br s, 1H), 2.07 (m, 1H), 1.81–1.71 (m,
1H), 1.33 (m, 1H), 1.17 (m, 2H); 13C NMR (100 MHz,
CDCl3) d: 142.4, 142.3, 142.1, 142.0, 128.9, 128.8,
128.6, 128.5, 128.2, 126.4, 126.3, 126.2, 126.2, 124.8,
124.7, 120.9, 118.5, 118.3, 70.3, 70.1, 69.4, 27.1, 26.9,
26.5, 25.5, 25.3, 16.9, 16.2, 15.7; HRMS (EI) calcd for
C15H16O2 (M+): 228.1150; found, 228.1139.
pentane to afford the cis-dihydroarene diol as a white
26
D
crystalline solid, 56 mg/L; mp 81–81.5 °C; ½aŠ
¼
þ65:5 (c 1.0, MeOH); Rf 0.30 (hexanes–ethyl acetate,
1:1); IR (film) m 3290, 1639, 1582, 1453 cmꢀ1 1H
;
NMR (300 MHz, CDCl3) d: 5.90(d, J = 5.6 Hz, 1H),
5.85 (d, J = 6.0Hz, 1H), 5.63 (d, J = 9.0Hz, 1H), 4.32
(s, 1H), 4.10(dd, J = 14, 10Hz, 1H), 3.72 (d, J =
5.0 Hz, 1H), 1.23 (s, 3H), 0.92 (m, 2H), 0.56 (m, 2H);
HRMS (EI) calcd for C10H14O2, 166.0994; found,
166.0995.
4.1.6. (3aR,7aS)-2,2-Dimethyl-4-(2-phenylcyclopropyl)-
3a,4,5,7a-tetrahydrobenzo[1,3]dioxole 20 1:1 mixture of
diastereoisomers. Diene diols 12a and 12b (140mg,
0.61 mmol, 1 equiv) were dissolved in 5 mL methanol
and the solution then transferred to a 25-mL round-bot-
tomed flask equipped with magnetic stirring bar and
addition funnel. The flask was cooled externally to
0 °C at which time a PAD reagent (potassium azodi-
carboxylate, 331 mg, 1.71 mmol, 2.8 equiv) was added
to the reaction flask. The yellow slurry was stirred for
10min, then the addition funnel charged with acetic acid
(236 lL, 3.99 mmol, 6.5 equiv) dissolved in 5 mL meth-
anol. The acetic acid solution was added dropwise over
a 1 h period and the yellow slurry allowed to warm to rt
overnight. A saturated solution of sodium bicarbonate
was used to adjust the pH to approximately 8.5 and
the reaction mixture diluted with 20mL ethyl acetate.
4.1.3.
(1R,2R)-3-Bromo-6-cyclopropyl-cyclohexa-3,5-
diene-1,2-diol 7. The diol was isolated from a crude
mixture from the biotransformation of the correspond-
ing cyclopropylbenzene by E. coli JM 109 (pDTG601)
according to the general procedure for shake-flask fer-
mentation. Purification by flash silica gel chromatogra-
phy (3:2 Hex/EA, 10% deactivated silica) furnished the
cis-dihydroarene diol as a white crystalline solid,
26
32 mg/L; ½aŠ ¼ ꢀ17:5 (c 0.65, CHCl3); Rf 0.76 (hex-
D
anes–ethyl acetate, 1:1); IR (film) m 3290, 1631,
1
1550cm ꢀ1; H NMR (300 MHz, acetone-d6) d: 6.27 (d,
J = 6.3 Hz, 1H), 5.47 (d, J = 6.3 Hz, 1H), 5.63 (d,
J = 9 Hz, 1H), 4.23 (d, J = 2.4 Hz, 2H), 4.18 (m, 1H),
3.92 (d, J = 7.5 Hz, 1H), 2.88 (s, 1H), 1.65 (m, 1H),
0.76 (m, 2H), 0.60 (m, 2H); 13C NMR (100 MHz, ace-

K. J. Finn et al. / Tetrahedron: Asymmetry 16 (2005) 3606–3613
3611
The layers were separated and the aqueous layer
extracted with portions of ethyl acetate. The combined
organic layers were washed with brine, dried (MgSO4),
and the drying agent removed by filtration. The solvent
was removed under reduced pressure to provide 125 mg
crude product, which was used directly in the subse-
quent reaction without further purification.
4.1.8. (3aS,7aR)-2,2-Dimethyl-4-propenyl-3a,4,5,7a-tetra-
hydro-benzo[1,3]dioxole 24. The diol derived from
diimide reduction of diene diol 23 (828 mg, 5.37 mmol,
1 equiv) was transferred to a 50-mL round-bottomed
flask and dissolved in 5 mL acetone. 2,2-Dimethoxy pro-
pane (1.243 mL, 10.1 mmol, 1.88 equiv) was added fol-
lowed by 1 spatula tip of p-toluenesulfonic acid. The
solution was stirred at rt for 2.5 h before the reaction
was quenched with 10mL of saturated sodium carbon-
ate solution and the acetone removed under reduced
pressure. The cloudy mixture was diluted with 15 mL
ethyl acetate and 2 mL of distilled water. The layers
were separated and the aqueous layer back-extracted
with fresh ethyl acetate. The combined organic layers
were washed with brine and dried over anhydrous mag-
nesium sulfate. The solvent was removed under vacuum
to provide a tan oil, which was purified by flash column
chromatography (98:2 hexanes–ethyl acetate) to afford
The crude product from diimide reduction was dissolved
in the minimum amount of acetone and transferred to a
25 mL round-bottomed flask equipped with magnetic
stirring bar. 2,2-Dimethoxypropane (471 lL, 3.9 mmol,
6.4 equiv) was added as a neat solution followed by sev-
eral crystals of p-toluenesulfonic acid. The reaction mix-
ture was stirred for 1.5 h at which time DMP was
removed under vacuum. The residue was purified by
flash column chromatography (hexanes–ethyl acetate,
3:1), affording the title compound, which was an insep-
arable mixture of diastereoisomers, as a clear and color-
the title compound as a clear and colorless oil, 900 mg
24
less oil (66 mg, 40% yield over two-step sequence);
(86%): ½aŠ ¼ þ68:1 (c 0.72, CHCl3); Rf 0.56 (10% ethyl
D
22
½aŠ ¼ þ30:2 (c 1.0, CHCl3); Rf 0.55 (hexanes–ethyl ace-
acetate in hexanes); IR (neat) m 2984, 2932, 1451,
tatDe, 5:1); IR (film) m 2984, 2931, 1604, 1498, 1455, 1367,
1367 cmꢀ1 1H NMR (300 MHz, CDCl3) d: 5.96 (d,
;
1241 cmꢀ1
;
1H NMR (300 MHz, CDCl3) d: 7.16 (m,
J = 15.9 Hz, 1H), 5.83 (m, 1H), 5.73 (t, J = 4.2 Hz,
1H), 4.58 (d, J = 6.0Hz, 1H), 4.22 (m, 1H), 2.15 (m,
1H), 1.96 (m, 1H), 1.71 (d, J = 6.3 Hz, 3H), 1.69 (m,
2H), 1.42 (s, 6H); 13C NMR (100 MHz, CDCl3) d:
134.3, 131.9, 129.3, 124.5, 108.3, 73.6, 71.4, 28.1, 26.3,
26.1, 21.6, 18.4; HRMS (EI) Calcd for C12H18O2,
194.1306; found, 194.1304.
2H), 7.05 (m, 3H), 5.72/5.50 (t, J = 3.8 Hz, 1H), 4.35
(m, 1H), 4.25 (m, 1H), 2.20–2.05 (m, 1H), 2.05 (m,
1H), 1.90–1.75 (m, 2H), 1.70–1.60 (m, 1H), 1.35 (d,
J = 3.5 Hz, 3H), 1.30(d, J = 3.2 Hz, 3H), 1.22 (m,
1H), 1.05 (m, 2H); 13C NMR (100 MHz, CDCl3) d:
142.0, 141.9, 135.3, 135.1, 127.5, 127.2, 124.9, 124.7,
124.4, 124.4, 123.0, 122.0, 119.4, 107.3, 73.3, 72.9,
72.4, 27.0, 26.9, 25.7, 25.7, 25.5, 24.5, 24.1, 22.5, 19.6,
19.5, 14.2, 14.0; HRMS (EI) calcd for C18H22O2 (M+):
270.1619; found, 270.1603; Anal. Calcd for C18H22O2:
C, 79.56; H, 8.20. Found, C, 79.23; H, 8.15.
4.1.9. (3aR,7aS)-2,2-Dimethyl-4-(2-methylcyclopropyl)-
3a,4,5,7a-tetrahydro-benzo[1,3]dioxole 25. To a solu-
tion of diethyl zinc (16.5 mL of 1.0M solution in
hexanes, 16.5 mmol, 4 equiv) in 16 mL anhydrous
methylene chloride at 0 °C was added freshly distilled tri-
fluoroacetic acid (0.65 mL, 8.25 mmol, 2 equiv) in 8 mL
methylene chloride very slowly (ca. 20min). The thick,
white slurry was stirred at 0 °C for 20min at which time
diiodomethane (0.66 mL, 8.25 mmol, 2 equiv) in 8 mL
was introduced to the reaction flask by cannulation.
The resulting gray slurry was stirred for 20min before
addition of propenyl protected diol 24 (800 mg,
4.06 mmol, 1 equiv) dissolved in 8 mL methylene chlo-
ride. The reaction flask was removed from the ice bath
and the slurry allowed to warm to rt over 30min. Pro-
gress of the reaction was monitored by TLC (10% ethyl
acetate in hexanes, KMnO4 stain). When deemed com-
plete, the reaction was quenched by the addition of
20mL saturated solution of NH 4Cl and the layers sepa-
rated. The aqueous layer was extracted with two portions
of methylene chloride and the combined organic layers
dried over anhydrous MgSO4. Evaporation of the solvent
under reduced pressure provided an oil, which was puri-
fied by flash column chromatography (10% ethyl acetate
4.1.7. (1S,2R)-3-Propenyl-cyclohex-3-ene-1,2-diol. To a
solution of diene diol 23 (1.35 g, 8.8 mmol, 1.0equiv) in
20mL MeOH at 0 °C was added portionwise potassium
azodicarboxylate (5.16 g, 26.6 mmol, 3 equiv) over
10min. The yellow slurry was stirred for 10min before
the addition of AcOH (3.55 mL, 61.6 mmol, 7 equiv)
in 20mL MeOH over a 1 h period. The reaction mixture
was stirred overnight, warming slowly to rt over 12 h.
The reaction mixture was quenched by addition of
12 mL saturated solution of sodium carbonate. Metha-
nol was removed under vacuum and replaced with ethyl
acetate. The layers were separated and the aqueous por-
tion extracted three times with 15 mL ethyl acetate. The
combined organic layers were washed with brine and
dried over anhydrous magnesium sulfate. Filtration of
the drying agent and removal of the solvent under
reduced pressure provided a solid, which was recrystal-
lized to give 828 mg of the title compound as a tan solid
(61%). Mp 107–108 °C (from methylene chloride-
24
pentane); ½aŠ ¼ ꢀ133 (c 0.5, CHCl3); Rf 0.21 (hex-
in hexanes) to afford the corresponding cyclopropane as
D
24
anes–ethyl acetate, 1:1); IR (film) m 3260, 2945, 2871,
a clear and colorless oil (567 mg, 67%). ½aŠ ¼ þ64:3 (c
D
1633 cmꢀ1
;
1H NMR (300 MHz, CDCl3) d: 5.93 (d,
0.4, CHCl3); Rf 0.44 (hexanes–ethyl acetate, 9:1); 1H
NMR (300 MHz, CDCl3) d: 5.32 (m, 1H), 4.17 (m,
1H), 4.15 (m, 1H), 1.98 (s, 1H), 1.73 (m, 2H), 1.27
(m, 6H), 0.94 (m, 4H), 0.85 (2H), 0.42 (m, 1H), 0.21
(m, 1H); 13C NMR (100 MHz, CDCl3) d: 137.1, 122.4,
108.2, 74.2, 73.6, 28.0, 26.5, 25.7, 23.1, 20.7, 18.9, 14.2,
13.4 ppm (single diastereomer).
J = 16 Hz, 1H), 5.84 (m, 1H), 5.63 (t, J = 3.9 Hz, 1H),
4.30(d, J = 3.6 Hz, 1H), 3.63 (m, 1H), 2.23 (br s, 2H),
2.15 (m, 2H), 1.72 (d, J = 5.9 Hz, 3H), 1.66 (m, 2H);
13C NMR (100 MHz, CDCl3) d: 135.9, 131.5, 128.9,
123.7, 69.7, 65.9, 25.3, 25.1, 18.3; HRMS (EI) calcd
for C9H14O2 (M+): 154.0993; found, 154.0994.

3612
K. J. Finn et al. / Tetrahedron: Asymmetry 16 (2005) 3606–3613
4.1.10. (3aR,7aS)-2,2-Dimethyl-7-(2-methyl-cyclopropyl)-
3a,4,5,7a-tetrahydrobenzo[1,3]dioxole 25 1:1 mixture of
diastereoisomers. Diols 9a and 9b (40mg, 0.24 mmol,
1 equiv) were transferred to a 10-mL round-bottomed
flask and dissolved in 2 mL acetone. 2,2-Dimethoxy pro-
pane (250 lL, 2.4 mmol, 10equiv) was added followed
by 1 crystal p-toluenesulfonic acid. The solution was
stirred at rt for 24 h before the reaction was quenched
with 1 mL of saturated sodium bicarbonate solution.
The cloudy mixture was diluted with 15 mL ethyl acetate
and 2 mL of distilled water. The layers were separated
and the aqueous layer back-extracted with fresh ethyl
acetate. The combined organic layers were washed with
brine and dried over anhydrous magnesium sulfate. The
solvent was removed under vacuum to provide a tan oil
(24 mg), which was purified by flash column chromatog-
raphy (10% ethyl acetate in hexanes) to afford the title
by the American Chemical Society (PRF-38075-AC);
TDC Research Inc.; and TDC Research Foundation.
We also thank Brock University for providing a gradu-
ate fellowship for K.J.F. We are indebted to Dr. Gregg
Whited (Genencor International, Inc.), Professor Rebec-
ca Parales (University of California-Davis), and Dipl.
Ing. Hannes Leisch (Brock University) for their gener-
ous help and discussions regarding whole-cell fermenta-
tions. We appreciate the helpful suggestions provided by
Professor Yian Shi (Colorado State University) regard-
ing the cyclopropanation reactions described in the gen-
eral procedure.
References
1. (a) Boyd, D. R.; Sheldrake, G. N. Nat. Prod. Rep. 1998,
15, 309; (b) Johnson, R. A. Org. React. 2004, 63, 117; (c)
Hudlicky, T.; Gonzalez, D.; Gibson, D. T. Aldrichim. Acta
1999, 32, 35.
2. (a) Brown, S. M.; Hudlicky, T. In Organic Synthesis:
Theory and Applications; Hudlicky, T., Ed.; JAI Press:
Greewich, CT, 1993; Vol. 2, p 113; (b) Hudlicky, T.; Reed,
J. W. In Advances in Asymmetric Synthesis; Hassner, A.,
Ed.; JAI Press: Greenwich, CT, 1995; p 271.
3. Biotransformations of all substrates were carried out in
either a 2-L shake-flask or 15-L fermentor. A detailed
protocol for shake-flask fermentation is described in the
Experimental. The procedure for medium-scale fermenta-
tion using a 15-L fermentor has been previously published:
Endoma, M. A.; Bui, V. P.; Hansen, J.; Hudlicky, T. Org.
Process Res. Dev. 2002, 6, 525.
compound as a clear oil and inseparable mixture of dia-
22
stereoisomers, 16 mg (23%): ½aŠ ¼ þ47:5 (c 0.4,
D
CHCl3); Rf 0.44 (10% ethyl acetate in hexanes); IR (film)
;
m 3353, 2984, 2930, 1430, 1366 cmꢀ1 1H NMR
(300 MHz, CDCl3) d: 5.43/5.37 (t, J = 3.8 Hz, 1H),
4.47 (m, 1H), 4.17 (m, 1H), 2.15–2.00 (m, 1H), 1.85–
1.70(m, 2H), 1.65–1.50(m, 2H), 1.34 (d,
J = 6 Hz,
1H), 1.03 (d, J = 5.8 Hz, 2H), 0.9–0.75 (m, 1H), 0.68
(m, 1H), 0.51 (dd, J = 8.9, 4.8 Hz, 1H), 0.30 (m, 1H);
13C NMR (100 MHz, CDCl3) d: 137.5, 122.8, 122.5,
108.5, 74.8, 74.6, 73.9, 28.3, 26.9, 26.1, 23.5, 23.4, 21.1,
21.0, 19.6, 19.2, 15.2, 14.5, 13.8, 13.6; HRMS (EI) calcd
for C13H20O2, 208.1463; found, 208.1468.
4.1.11. (1S,2R)-3-Cyclopropyl-cyclohexa-3-ene-1,2-diol
26. From (1R,2R)-3-bromo-6-cyclopropyl-cyclohexa-
3,5-diene-1,2-diol: Diol 7 (100 mg, 0.43 mmol, 1 equiv)
was dissolved in 4 mL of MeOH and transferred to a
thick-walled hydrogenation flask. Distilled triethylamine
(62 lL, 0.43 mmol, 1 equiv) was added followed by PtO2
(Adams catalyst, 19 mg, 0.087 mmol, 20 mol %). The
flask was placed on a Parr hydrogenation apparatus,
evacuated, and the headspace replaced with hydrogen
gas (3 atm). The flask was allowed to shake at rt for a
period of 1.5 h. The reaction mixture was filtered
through Celite and washed with several portions of
methanol, concentrated, and purified by flash column
chromatography (hexanes–ethyl acetate, 3:2) and the
solvent removed under reduced pressure to provide the
title compound as an oily solid (27 mg, 41%). Spectral
data are consistent with that of the same compound
prepared by enzymatic oxidation from cyclopropyl
4. 1-Chloro-(2S,3S)-dihydroxycyclo-hexa-4,6-diene
Hud-
licky, T.; Stabile, M.; Gibson, D. T.; Whited, G. M.
Org. Synth. 1999, 76, 77.
5. Bui, V. P.; Nguyen, M.; Hansen, J.; Baker, J.; Hudlicky,
T. Can. J. Chem. 2002, 80, 708.
6. Cyclopropanation protocols were adopted from Yang,
Z.; Lorenz, J. C.; Shi, Y. Tetrahedron Lett. 1998, 39,
8621; Generalprocedure for Simmons–Smith cycolpropa-
nation. The preparation of trans-1-methyl-2-phenyl-cyclo-
propane is representative: To a solution of diethyl zinc
(85 mL of 1.0M solution in hexanes, 85 mmol, 2 equiv)
in 75 mL anhydrous methylene chloride at 0 °C was
added freshly distilled trifluoroacetic acid (7.8 mL,
85 mmol, 2 equiv) in 35 mL methylene chloride very
slowly (ca. 30min). The gray solution was stirred at 0 °C
for 20min at which time diiodomethane (8.4 mL,
85 mmol, 2 equiv) in 30mL was introduced to the
reaction flask by cannulation. The resulting slurry was
stirred for 20min before the addition of b-methyl styrene
(5.0g, 43 mmol, 1 equiv) dissolved in 30mL methylene
chloride. The reaction flask was removed from the ice
bath and the slurry allowed to warm to rt over 30min.
Progress of the reaction was monitored by TLC (100%
hexanes, KMnO4 stain). When deemed complete, the
reaction was quenched by the addition of 50mL
saturated solution of NH4Cl and the layers separated.
The aqueous layer was extracted with two portions of
hexanes and the combined organic layers dried over
anhydrous MgSO4. Evaporation of the solvent under
reduced pressure provided an oil, which was purified by
flash column chromatography (100% hexanes) to afford
the corresponding cyclopropane as a clear and colorless
oil (4.19 g, 75%).
benzene and subsequent diimide reduction.5 Rf 0.27
26
(hexanes–ethyl acetate, 1:1); ½aŠ ¼ ꢀ33 (c 1.0, MeOH),
D
23
lit. ½aŠ ¼ ꢀ126 (c 1.0, MeOH). 26% enantiomeric
D
excess.
Acknowledgments
The authors express their gratitude to the following
agencies for generous financial 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 Petroleum Research Fund, administered
7. Tian, X.; Hudlicky, T.; Ko¨nigsberger, K. J. Am. Chem.
Soc. 1995, 117, 3643.

K. J. Finn et al. / Tetrahedron: Asymmetry 16 (2005) 3606–3613
3613
8. Boyd, D. R.; Sharma, N. D.; Bowers, N. I.; Brannigan, I.
N.; Groocock, M. R.; Malone, J. F.; McConville, G.;
Allen, C. C. R. Adv. Synth. Catal. 2005, 347, 1081.
9. Rossi, R.; Carpita, A.; Ribecai, A.; Mannina, L. Tetra-
hedron 2001, 57, 2847.
12. Specific rotation of 25 prepared by fermentation of
22
cyclopropylbenzene 8: ½aŠ ¼ þ47:5 (c 0.4, CHCl3);
D
specific rotation of 25 prepared by Simmons–Smith
24
D
cyclopropanation of propenyl diol 24: ½aŠ ¼ þ64:3 (c
0.4, CHCl3).
10. (a) Ylidirim, S.; Zezula, J.; Hudlicky, T.; Witholt, B.;
Schmid, A. Adv. Synth. Catal. 2004, 346, 933; (b) Bui, V.;
Hansen, T. V.; Stenstrøm, Y.; Ribbons, D. W.; Hudlicky,
T. J. Chem. Soc., Perkin Trans. 1 2000, 1669.
13. Specific rotation of 22 obtained from homochiral diol 2:
24
½aŠ ¼ ꢀ126 (c 1.0, MeOH), specific rotation of 22
D
24
D
prepared from compound 7: ½aŠ ¼ ꢀ33 (c 1.0, MeOH).
14. Resnick, S. M.; Gibson, D. T. Appl. Environ. Microbiol.
1996, 62, 3355.
11. Crigee, R.; Ho¨ver, H. J. Prakt. Chem. 1960, 2521.