Enzymatic oxidation of cyclopropylbenzene: Structures of new metabolites and possible mechanistic implications

Article abstract of DOI:10.1139/v02-098

Cyclopropylbenzene was subjected to whole-cell fermentation with either Escherichia coli JM109 (pDTG601) or E. coli JM109 (pDTG602), expressing toluene dioxygenase and toluene dioxygenase - dihydrodiol dehydrogenase enzymes, respectively. The corresponding metabolites, 3-cyclopropylcyclohexa-3,5-diene-1,2-diol (3) and 3-cyclopropylbenzene-1,2-diol (5) have been isolated in yields of 2.5 and 1 g L^-1, respectively. The absolute stereochemistry correlation for 3 is provided, along with a preliminary discussion of its potential in asymmetric synthesis. Possible mechanistic implications are indicated for the enzymatic oxygenation through the use of calculations. Experimental data are provided for all new compounds.

Full text of DOI:10.1139/v02-098

708
Enzymatic oxidation of cyclopropylbenzene:
structures of new metabolites and possible
mechanistic implications
Vu P. Bui, Minh Nguyen, Jeff Hansen, John Baker, and Tomas Hudlicky
Abstract: Cyclopropylbenzene was subjected to whole-cell fermentation with either Escherichia coli JM109
(pDTG601) or E. coli JM109 (pDTG602), expressing toluene dioxygenase and toluene dioxygenase – dihydrodiol
dehydrogenase enzymes, respectively. The corresponding metabolites, 3-cyclopropylcyclohexa-3,5-diene-1,2-diol (3) and
3-cyclopropylbenzene-1,2-diol (5) have been isolated in yields of 2.5 and 1 g L–1, respectively. The absolute
stereochemistry correlation for 3 is provided, along with a preliminary discussion of its potential in asymmetric synthe-
sis. Possible mechanistic implications are indicated for the enzymatic oxygenation through the use of calculations. Ex-
perimental data are provided for all new compounds.
Key words: cyclopropylbenzene, bio-oxidation, cis-diene diol, catechol, JM109 (pDTG601), JM109 (pDTG602),
dioxygenase.
Résumé : On a soumis le cyclopropylbenzène à une fermentation à l’aide de cellules entières d’Escherichia coli
JM109 (pDTG601) ou d’E. coli JM109 (pDTG602) qui laissent écouler respectivement les enzymes dioxygénase du to-
luène et dioxygénase du toluène/déshydrogénase du dihydrodiol. On a isolé les métabolites correspondants, le 3-
cyclopropylcyclohexa-3,5-diène-1,2-diol (3) et le 3-cyclopropylbenzène-1,2-diol (5) avec des rendements respectifs de
2,5 g/L et 1 g/L. On présente une corrélation absolue de la stéréochimie du produit 3 ainsi qu’une discussion prélimi-
naire de son potentiel éventuel en synthèses asymétriques. En se basant sur des calculs théoriques, on indique les im-
plications mécanistiques possibles pour l’oxygénation enzymatique. On rapporte les données expérimentales relatives à
tous les nouveaux composés.
Mots clés : cyclopropylbenzène, biooxydation, cis-diènediol, catéchol, JM109 (pDTG601), JM109 (pDTG602), dioxygénase.
[Traduit par la Rédaction] Bui et al. 713
Introduction
Fig. 1. Natural degradation pathway of aromatics by eukaryotic
The historically significant disclosure of isolation of the
first homochiral cis-cyclohexadiene diol by Gibson and co-
workers (1) in 1968 was soon followed by detailed studies of
the degradation pathways employed by soil organisms that
grow on aromatic compounds. Gibson provided the first
blocked mutants of Pseudomonas putida, whose use led to
isolation of homochiral diols of type 1 (2). Eventually, the
elucidation of the degradation pathway (Fig. 1) allowed the
protein synthesis of the first two enzymes employed in the
pathway, namely, toluene dioxygenase (TDO) and dihy-
drodiol dehydrogenase (DHDH), to be expressed in recom-
binant clones of Escherichia coli JM109 (3). Similar
organisms have also been constructed to express the topolog-
ically similar enzymes, naphthalene (4) and biphenyl
organisms, and mutation points for specific enzyme expression.
dioxygenase (5). The X-ray crystal structure has been solved
for naphthalene dioxygenase (6).
Metabolites of type 1 have been extensively used in asym-
metric synthesis (7) since 1987, when the first such applica-
tion was disclosed by Ley et al. (8). Recently, we reported
the synthetic applications of E. coli JM109 (pDTG602) to
the practical preparation of substituted catechols (2), com-
pounds which are difficult to prepare efficiently by tradi-
tional means (9). Although cyclopropyl catechol has been
previously reported (10), the corresponding diene diol was
unknown.
Received 15 March 2002. Published on the NRC Research
Press Web site at http://canjchem.nrc.ca on 3 June 2002.
This paper is dedicated to Professor Bryan Jones in
recognition and appreciation of his many contributions
to the field of biocatalysis.
V.P. Bui, M. Nguyen, J. Hansen, J. Baker, and T. Hudlicky.1
Department of Chemistry, University of Florida, Gainesville,
FL 32611–7200, U.S.A.
In this paper we report the results of the oxidation of
cyclopropylbenzene with the organisms that express TDO
1Corresponding author (e-mail: hudlicky@chem.ufl.edu).
Can. J. Chem. 80: 708–713 (2002)
DOI: 10.1139/V02-098
© 2002 NRC Canada

Bui et al.
709
Scheme 1. Reagents: (i) JM109 (pDTG601); (ii) JM109
Scheme 2. Reagents and conditions: (i) PAD, AcOH, MeOH;
(pDTG602).
(ii) DMP, p-TsOH; (iii) Mg, I2, Et2O; (iv) Pd(PPh3)4, THF, re-
flux; (v) THF–H2O–TFA (4:1:1); (vi) Ac2O, pyridine, DMAP,
CH2Cl2; (vii) JM109 (pDTG601).
and TDO–DHDH. The isolation of the metabolites may pro-
vide clues to the mechanism by which oxygen is introduced
into the aromatic nucleus. In addition, the vinylcyclopro-
panes generated by such means may serve as versatile inter-
mediates for asymmetric syntheses.
Results and discussion
Fig. 2. Exploration of reactivity of oxygenated cyclopropylhex-
enyl systems.
Whole-cell fermentation of either E. coli JM109
(pDTG601), expressing toluene dioxygenase (TDO) or E.
coli JM109 (pDTG602) expressing both TDO and
dihydrodiol dehydrogenase (DHDH), was performed in either
a 10-L or 15-L fermentor, as previously described (9b, 11).
When the cells were grown to optimum optical density (OD
= 50–60 at 640 nm) (9b, 11), the substrate was introduced at
a rate of 5 g h–1 and the biotransformation allowed to pro-
ceed for 5–6 h. After the separation of cells from the broth,
diol 3 was isolated in a yield of 2.5 g L–1. Diol 3 is essen-
tially pure after extraction from the broth, but is somewhat
unstable and soon thereafter begins to dehydrate to phenol 4
(Scheme 1).
Since this dehydration is greatly accelerated by the pres-
ence of acid, it is essential to use base-washed solvents
when isolating 3. The amount of 4 produced varies depend-
ing on the time and the amount of acid present. The dehy-
dration process is self-catalytic, insofar as 4 is sufficiently
acidic to catalyze further dehydration. Diol 3 can be stored
for several weeks at –78°C.
afforded diol 9, whose [ꢀ]D (–126°) matched that of the
material obtained via fermentation of cyclopropylbenzene.
Diol 3 offers new opportunities for asymmetric synthesis
through further transformations of the reactive vinylcyclo-
propane moiety. Two obvious applications are the tethered
[5+2] annulation to 7-membered ring systems, discovered
and developed by Wender et al. (14), simple thermolysis to
cyclopentenes (15), or cascade radical cyclizations with con-
comitant unraveling of the cyclopropylcarbinyl system
(Fig. 2). The electrochemical behavior of the vinylcyclo-
propane moiety will also be investigated (16).2
Exposure of cyclopropylbenzene to E. coli JM109 (pDTG602)
led to the formation of cyclopropyl catechol 5 in an isolated
yield of 1 g L–1. The identity of the metabolites was estab-
lished by spectroscopic techniques. The absolute stereo-
chemistry of diol 3 was confirmed by an independent
synthesis of vinylcyclopropane 9 from diol 6 whose absolute
stereochemistry is known (Scheme 2). Compound 9 was pre-
pared by the selective reduction of diol 3 with potassium
azadicarboxylate (PAD) in 73% yield. Diol 6 was also re-
duced with PAD (86%) and then protected to provide
acetonide 7 (78%) (12). Grignard reagent 11, derived from
cyclopropyl bromide 10, was coupled with 7 via a modified
Corey–House synthesis (13) to give 8 in a low yield (16%).
Deprotection of this material with THF–H2O–TFA (4:1:1)
The development of such new technology also depends on
our ability to efficiently couple cyclopropyl units to the vinyl
bromide in 6 or 7 via a Suzuki-type process. The research on
the synthesis of cyclopropyl boronates is ongoing and will
be reported in due course.
Mechanistic implications
The mechanism of aromatic dioxygenation remains un-
known, except for initial proposals by Gibson (17), who sug-
gested dioxetane 14 as an intermediate. This proposal would
account for the cis-configuration of diols 1 found in metabo-
lites from biotransformation by prokaryotic organisms
(Fig. 3) (18) and was supported by incorporation of 18O
from labeled dioxygen into both hydroxyl groups of the diol
2 The vinylcyclopropane to cyclopentene rearrangement has been shown to be greatly accelerated by catalytic amounts of triarylaminium ions
via a one-electron oxidation (16). We are investigating the initiation of the rearrangement by a one-electron electrochemical oxidation. Cy-
clic voltammetry of vinylcyclopropane 9 indicates an irreversible oxidation at E1/2 = 1.2 V (0.15 M LiClO4 in MeOH). Initial attempts at
oxidative rearrangement consumed 9 and produced products that do not contain a cyclopropane ring and may contain a cyclopentene ring.
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Can. J. Chem. Vol. 80, 2002
Fig. 4. Mechanistic possibilities for cis-dihydroxylation.
Fig. 3. Oxygenation of aromatics by prokaryotic and eukaryotic
systems.
(19). On the other hand, eukaryotic oxidations are much
better understood and known to proceed via arene oxides 15
(incorporation of only one oxygen from O2), which are hy-
drolyzed to trans diols 16 (20).
The suggestion of a dioxetane as an intermediate would
require the participation of a singlet oxygen-type species.
Alternatively, radical species such as iron-bound peroxy rad-
icals or anionic species (peroxides) of either type 18 or 19
might lead to a stepwise formation of C–O bonds for the for-
mer cases or synchronous formation of cycloadducts of type
20 in the latter, as indicated in a very speculative way in
Fig. 4 (21).
Since the full structure of naphthalene dioxygenase
(NDO) has been determined (6), Gibson and co-workers (21a)
provided further details on an alternative mechanism of
dioxygenation by an iron-peroxide species either of a radical
nature or as a partially reduced metal-bound peroxide. Inter-
mediates analogous to 18 or 19 have been observed in the
oxidation of indole by NDO in crystals grown in a rich me-
dium containing indole.
faster than that of radical 22 (k cralcd = 1.1 s–1). Radical
stabilizing substituents at the carbinyl position are known to
retard the rates of ring opening (30). Even if a radical is
formed at C3, ring opening may not occur if the radical has
a short lifetime.
Conclusions
The calculation described here for species such as 19 does
not eliminate the possibility of intermediates of this type in
the oxidation of cyclopropyl benzene even though products
derived from the ring opening of the cyclopropane have not
been observed in the reaction mixtures. While the nature of
the cis-dihydroxylation remains a mechanistic enigma, re-
cent evidence by Gibson points to a possible stepwise mech-
anism for this unique reaction. Whether the mechanism
involves radical or anionic iron-bound species is at the mo-
ment unknown.
From a synthetic viewpoint, the cyclopropyl system fur-
ther offers various synthetic manipulations and can be
viewed as a chiral vinylcyclopropane synthon for incorpora-
tion into future synthetic schemes. Such endeavors will be
reported in due course.
Cyclopropylcarbinyl-type rearrangements have been ob-
served during the investigation of the mechanism in the iso-
penicillin N-synthase reaction (22). Iron(IV)–sulfur-bound
species have been proposed to account for the results, where
trapping of the butenyl radical by the sulfur atom has been
observed.
We therefore wished to compare the reactive tendency of
a conjugated cyclopropylcarbinyl radical such as 19 to the
well-known cyclopropylcarbinyl rearrangement (23). Since
unrestricted Becke’s hybrid three-parameter functional the-
ory (UB3LYP) (24) provides a reliable level of theory for
examining the cyclopropylcarbinyl radical system (25), the
density functional theory methodology was used to examine
the kinetics of the ring opening of the cyclopropylcarbinyl
radical system (22) to the conjugated butenyl system (23)
(Fig. 4). Calculations were performed using the Gaussian 98
program package (26). Structures were optimized and vibra-
tional frequency calculations were performed using UB3LYP
level of theory and the 6–31G(d) basis set (27). The transi-
tion structure was characterized by a single imaginary fre-
quency, and thermochemical information was obtained using
unscaled frequencies. Single-point energies were calculated
using UB3LYP level of theory using the 6–311+G(2df,2p)
basis set (28). At 35°C the ring opening of the cyclopro-
pylcarbinyl radical (17) (kr = 1.4 × 108 s–1) (29) is 108 times
Experimental
3-Cyclopropylcyclohexa-3,5-diene-1,2-diol (3)
3-Cyclopropylcyclohexa-3,5-diene-1,2-diol was isolated
from the crude mixture collected from the biotransformation
of cyclopropyl benzene. This was accomplished by
centrifugation of the cells and extraction of the broth with
base-washed ethyl acetate. This process provides essentially
pure diene diol, which can be further purified by flash col-
umn chromatography (hexanes–EtOAc, 4:1) to afford a white
solid; mp 45–46°C (dec); [ꢀꢁ 2D8 +11.39 (c 1.75, CD3OD); Rf =
0.4 (hexanes–EtOAc, 1:1). IR (film) (cm–1): 3274, 3008,
1
2911, 1453, 1090, 992, 804. H NMR (300 MHz, acetone-
d6) ꢂ: 5.75–5.85 (m, 1H), 5.54–5.66 (m, 2H), 4.17–4.26 (m,
1H), 3.63–3.81 (m, 2H), 3.40–3.51 (d, J = 7 Hz, 1H),
1.48–1.62 (m, 1H), 0.40–0.79 (m, 4H). 13C NMR (75 MHz,
© 2002 NRC Canada

Bui et al.
711
acetone-d6) ꢂ: 130.2, 127.4, 124.7, 118.7, 71.2, 69.0, 15.8,
7.6, 6.6.
dropwise to the reaction mixture at such a rate that 1–2
bubbles were produced per second. The mixture was al-
lowed to warm to ambient temperature, with the color
changing from yellow to milky white. The reaction mixture
was stirred overnight and then quenched with saturated
NaHCO3 until effervescence ceased. Methanol was re-
moved under reduced pressure. The residue was diluted
with brine (20 mL) and extracted with ethyl acetate (4 ×
40 mL). The combined organic phases were dried (MgSO4)
and concentrated to yield a reddish oil. The crude product
was purified by flash silica gel chromatography (hexane–
EtOAc, 3:2) to afford 9 (12.411 g, 73%); as a white solid;
mp 96–98°C; [ꢀ]2D9 –126.0 (c 1.0, CH3OH); Rf = 0.20 (hex-
ane–EtOAc, 3:2). IR (film) (cm–1): 3272, 3012, 1461, 989.
1H NMR (300 MHz, DMSO-d6) ꢂ: 5.32 (s, 1H), 4.32–4.36
(m, 1H), 3.65 (t, J = 4 Hz, 1H), 3.35–3.42 (m, 2H), 1.92–
1.99 (m, 2H), 1.52–1.63 (m, 1H), 1.38–1.46 (m, 2H), 0.33–
0.56 (m, 4H). 13C NMR (75 MHz, CDCl3) ꢂ: 138.6, 123.4,
70.0, 68.9, 25.3, 24.2, 14.9, 5.6. FAB-MS m/z (%): 137 (8),
93 (100). FAB-HRMS calcd. for C9H13O ([MH – H2O]+):
137.0966; found: 137.0967. Anal. calcd. for C9H14O2:
C 70.1, H 9.15; found: C, 70.17, H 9.18.
2-Cyclopropylphenol (4)
2-Cyclopropylphenol was isolated from the crude mixture
collected from the biotransformation of cyclopropyl ben-
zene. The phenol was purified by distillation to afford a
clear oil; Rf = 0.42 (hexane–EtOAc, 8:1). IR (film) (cm–1):
1
3536, 1609, 1580, 1491, 752. H NMR (300 MHz, DMSO-
d6) ꢂ: 9.26 (s, 1H), 6.86–6.92 (m, 1H), 6.63–6.76 (m, 3H),
2.02–2.12 (m, 1H), 0.80–0.87 (m, 2H), 0.55–0.61 (m, 2H).
13C NMR (75 MHz, CDCl3) ꢂ: 155.8, 129.4, 125.9, 124.5,
119.0, 114.6, 9.3, 7.7. EI-MS m/z: 136 (23), 134 (100), 133
(24), 115 (27), 107 (39), 106 (21), 105 (28), 91 (34), 89
(41), 78 (24), 77 (96), 61 (43). EI-HRMS calcd. for C9H10O
(M+): 134.0732; found: 134.0732.
3-Cyclopropylbenzene-1,2-diol (5)
Metabolite 5 was prepared by biotransformation of the
corresponding arene with E. coli JM109 (pDTG602); mp
41–42°C; Rf = 0.52 (hexane–EtOAc, 3:2). IR (film) (cm–1):
1
3418, 1622, 1592, 1477, 775. H NMR (300 MHz, DMSO-
d6) ꢂ: 9.11 (s, 1H), 8.11 (s, 1H), 6.50–6.58 (m, 1H), 6.49 (t,
J = 7.8 Hz, 1H), 6.16–6.21 (m, 1H), 2.01–2.12 (m, 1H),
0.79–0.86 (m, 2H), 0.50–0.57 (m, 2H). 13C NMR (75 MHz,
CDCl3) ꢂ: 143.4, 142.9, 128.8, 120.6, 119.7, 113.5, 9.4, 6.1.
EI-MS m/z (%): 150 (100), 149 (6), 107 (26), 77 (44). EI-
HRMS calcd. for C9H10O2 (M+): 150.0681; found:
150.0675. Anal. calcd. for C9H10O2: C 71.98, H 6.71; found:
C 72.07, H 6.69.
Method B: From vinyl bromide 7
A 50-mL round-bottom flask equipped with a condenser
and an addition funnel under argon atmosphere was charged
with magnesium metal (0.76 g, 31.2 mmol) and a crystal of
iodine. The addition funnel was charged with a solution of
cyclopropyl bromide (0.76 g, 6.24 mmol) in anhydrous di-
ethyl ether (10 mL) and this solution was added dropwise
into the reaction flask. Once the reaction proceeded on its
own, the addition was continued to maintain a constant re-
flux. After the addition was completed the reaction flask was
heated to reflux for an additional 45 min. Grignard reagent
11 was then transferred into another reaction flask contain-
ing bromoacetonide 7 (1.29 g, 5.61 mmol) and tetra-
kis(triphenylphosphine) palladium (0.194 g, 0.17 mmol) in
THF (25 mL). The reaction was heated at reflux for 5 h and
then the reaction mixture was filtered through a thin layer of
silica. The filtrate was concentrated under reduced pressure.
Flash chromatography provided the coupled product 8
(0.174 g, 16%). Acetonide 8 was deprotected using a THF–
H2O–TFA (4:1:1, 10 mL) solvent mixture to afford diol 9
(0.104 g, 75%) whose physical data matched the compound
obtained via the biotransformation of cyclopropylbenzene
followed by PAD reduction.
7-Cyclopropyl-2,2-dimethyl-3a,4,5,7a-tetrahydroben-
zo[1,3]dioxole (8)
To a well-stirred solution of diol 9 (0.150 g, 1.0 mmol) in
2,2-dimethoxypropane (DMP) (20 mL), a catalytic amount
of p-toluenesulfonic acid was added. The reaction mixture
was stirred for 30 min and then quenched with 5% NaHCO3
(2 mL). Solvent was removed under reduced pressure. The
residue was diluted with brine (20 mL) and extracted with
CH2Cl2 (3 × 30 mL). The organic layers were combined,
dried (MgSO4), and concentrated under reduced pressure.
The crude residue was purified by flash silica gel chroma-
tography (hexane–EtOAc, 8:1) to afford 8 (0.151 g, 80%);
[ꢀ]2D0 +58.99 (c 1.0, CH3OH); Rf = 0.57 (hexane–EtOAc,
8:1). IR (film) (cm–1): 3082, 2985, 1456, 1367, 870. 1H
NMR (300 MHz, CDCl3) ꢂ: 5.51 (t, J = 3.9 Hz, 1H), 4.37
(d, J = 4 Hz, 1H), 4.25–4.31 (m, 1H), 2.09–2.22 (m, 1H),
1.75–1.96 (m, 2H), 1.60–1.72 (m, 2H), 1.42 (d, J = 5 Hz,
6H), 0.32–0.71 (m, 4H). 13C NMR (75 MHz, CDCl3) ꢂ:
137.3, 123.4, 108.5, 74.5, 73.8, 28.2, 26.8, 25.9, 20.9, 14.6,
5.9, 5.1. EI-MS m/z (%): 179 (57), 137 (33), 136 (66), 121
(29), 108 (25), 107 (28), 95 (21), 91 (100), 79 (45), 77 (23),
55 (26). EI-HRMS calcd. for C12H18O2 (M+): 194.1307;
found: 194.1317.
Acetic acid 6-acetoxy-2-cyclopropylcyclohex-2-enyl ester
(12)
To a well-stirred solution of diol 9 (0.105 g, 0.68 mmol)
in CH2Cl2 (20mL), pyridine (0.16 g, 2.0 mmol) was added.
Addition of acetic anhydride (0.21 g, 2.0 mmol) was fol-
lowed by dimethylaminopyridine catalyst (0.2 mg, 0.20
mol%). The reaction mixture was allowed to stir for 20 h
and quenched with saturated citric acid (2 mL). The reaction
mixture was diluted with brine (20 mL) and extracted with
CH2Cl2 (3 × 30 mL). The organic layers were combined,
dried (Na2SO4), and concentrated under reduced pressure.
The crude residue was purified by flash silica gel chroma-
tography (hexane–EtOAc, 3:2) to afford 12 (0.156 g, 96%)
as a white solid; mp 80.5–81.5°C; [ꢀ]2D9 –196.9 (c 1.0,
3-Cyclopropylcyclohex-3-ene-1,2-diol (9)
Method A: From diol 3
To a well-stirred and ice-cooled solution of diol 3 (17 g,
0.11 mol) in methanol (50 mL), potassium azadicar-
boxylate (PAD) (76 g, 0.35 mol) was added. A solution of
acetic acid (64 mL, 1 mol) in methanol (50 mL) was added
© 2002 NRC Canada

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Can. J. Chem. Vol. 80, 2002
CH3OH); Rf = 0.79 (hexane–EtOAc, 3:2). IR (film) (cm–1):
1739, 1460, 1248. H NMR (300 MHz, CDCl3) ꢂ: 5.82
press (2002); (c) V.P. Bui, T. Hudlicky, T.V. Hansen, and Y.
Stenstrom. Tetrahedron Lett. 43, 2839 (2002).
1
(t, J = 3.2 Hz, 1H), 5.52 (d, J = 3.2 Hz, 1H), 4.92–4.99
(m, 1H), 2.14–2.22 (m, 2H), 2.10 (s, 3H), 2.01 (s, 3H),
1.84–1.91 (m, 1H), 1.70–1.74 (m, 1H), 1.22–1.28 (m, 1H),
0.50–0.64 (m, 2H), 0.30–0.47 (m, 2H). 13C NMR (75 MHz,
CDCl3) ꢂ: 170.9, 170.6, 135.6, 125.5, 70.8, 68.4, 24.3, 22.6,
21.3, 21.2, 14.8, 5.6, 5.0. EI-MS m/z (%): 179 (100), 119 (92),
91 (24). EI-HRMS calcd. for C13H19O4 (MH+): 239.1283;
found: 239.1289. Anal. calcd. for C13H18O4: C 65.63,
H 7.61; found: C, 65.43, H 7.60.
10. (a) J.B. Johnston and V. Renganathan. Enzyme Microb.
Technol. 9, 706 (1987); (b) J.B. Johnston and V. Renganathan.
Applied Microb. Biotechnol. 31, 419 (1989).
11. (a) V.P. Bui, T.V. Hansen, Y. Stenstrom, T. Hudlicky, and
D.W. Ribbons. New J. Chem. 25, 116 (2001); (b) V.P. Bui,
T.V. Hansen, Y. Stenstrom, D.W. Ribbons, and T. Hudlicky. J.
Chem. Soc. Perkin Trans 1, 1669 (2000); (c) T. Hudlicky,
M.R. Stabile, D.T. Gibson, and G.M. Whited. Org. Synth. 76,
77 (1999).
12. D.R. Boyd, M.R. Hand, N.D. Sharma, J. Chima, H. Dalton,
and G.N. Sheldrake. J. Chem. Soc. Chem. Commun. 1630
(1991).
Acknowledgments
13. M. Yamamura, I. Moritani, and S.I. Murahashi. J. Organomet.
Chem. 91, C39 (1975).
The generous research support from the National Science
Foundation (CHE-9910412), TDC Research, Inc., and TDC
Research Foundation is acknowledged. We thank Professor
William Dolbier (UF) and his group for assistance with the
calculations performed by John M. Baker.
14. (a) P.A. Wender, H. Takahashi, and B. Witulski. J. Am. Chem.
Soc. 117, 4720 (1995); (b) P.A. Wender, J.M. Nuss, D.B. Smith,
A. Suarez-Sobrino, J. Vagberg, D. Decosta, and J. Bordner. J.
Org. Chem. 62, 4908 (1997); (c) P.A. Wender and A.J. Dyckman.
Org. Lett. 1, 2089 (1999); (d) P.A. Wender, M. Fuji, C.O.
Husfeld, and J.A. Love. Org. Lett. 1, 137 (1999); (e) P.A. Wender,
A.J. Dyckman, C.O. Husfeld, and M.J. Scanio. Org. Lett. 2, 1609
(2000); ( f ) P.A. Wender, F.C. Bi, M.A. Brodney, and F. Gosselin.
Org. Lett. 3, 2105 (2001); (g) P.A. Wender, C.M. Barzilay, and
A.J. Dyckman. J. Am. Chem. Soc. 123, 179 (2001).
15. T. Hudlicky. J.W. Reed. In Comprehensive organic synthesis:
Selectivity, stategy and efficiency in modern organic chemis-
try. Vol. 5. Edited by B.M. Trost. Pergamon Press, Oxford.
1991. p. 899.
16. (a) J.P. Dinnocenzo and D.A. Conlon. J. Am. Chem. Soc. 110,
2324 (1988); (b) J.P. Dinnocenzo and D.A. Conlon. Tetrahe-
dron Lett. 36, 7415 (1995).
References
1. D.T. Gibson, J.R. Koch, C.L. Schuld, and R.E Kallio. Bio-
chemistry, 7, 3795 (1968).
2. D.T. Gibson, M. Hensley, H. Yoshioka, and T.J. Mabry. Bio-
chemistry, 9, 1626 (1970).
3. G.J. Zylstra and D.T. Gibson. J. Biol. Chem. 264, 14940 (1989).
4. J.J. Simon, T.D. Osslund, R. Saunders, B.D. Ensley, S. Suggs,
A. Harcourt, W.C. Suen, D.L. Cruden, D.T. Gibson, and
G.J. Zylstra. Gene, 127, 31 (1993).
5. F.J. Mondello. J. Bacteriol. 171, 1725 (1989).
6. B. Kauppi, K. Lee, E. Carredano, R.E. Parales, D.T. Gibson,
H. Eklund, and S. Ramaswamy. Structure, 6, 571 (1998).
7. (a) T. Hudlicky, D. Gonzalez, and D.T. Gibson. Aldrich. Acta,
32, 35 (1999); (b) D.R. Boyd and G.N. Sheldrake. Nat. Prod.
Rep. 15, 309 (1998); (c) T. Hudlicky. In Green chemistry:
Frontiers in benign chemical syntheses and process. Edited by
P.T. Anastas and T.C. Williamson. Oxford University Press,
Oxford. 1998. Chap. 10. p. 166; (d) T. Hudlicky. In Green
chemistry: Designing chemistry for the environment. Vol. 626.
Edited by P.T. Anastas and T.C Williamson. ACS Symposium
Series, American Chemical Society, Washington, DC. 1996.
p. 180; (e) T. Hudlicky, D.A. Entwistle, K.K. Pitzer, and
A.J. Thorpe. Chem. Rev. 96, 1195 (1996); ( f ) T. Hudlicky.
Chem. Rev. 96, 3 (1996); (g) T. Hudlicky and A.J. Thorpe.
Chem. Commun. 1993 (1996); (h) T. Hudlicky and J.W. Reed.
In Advances in asymmetric synthesis. Edited by A. Hassner.
JAI Press, Greenwich, CT. 1995. p. 271; (i) A.D. Grund. SIM
News, 45, 59 (1995); (j) S.M. Brown and T. Hudlicky. In
Organic synthesis: Theory and applications. Vol. 2. Edited
by T. Hudlicky. JAI Press, Greenwich, CT. 1993. p. 113;
(k) H.A.J. Carless. Tetrahedron: Asymmetry, 3, 795 (1992);
(l) G.N. Sheldrake. In Chirality and industry. Edited by
A.N. Collins, G.N. Sheldrake, and J. Crosby. John Wiley and
Sons, Chichester, U.K. 1992. p. 127; (m) D.A. Widdowson,
D.W. Ribbons, and S.D. Thomas. Janssen Chimica Acta, 8, 3
(1990).
17. D.T. Gibson. Zentralbl. Bakt. P. 157 (1976).
18. D.T Gibson. In Microbial degradation of organic compounds.
Vol 13. Edited by D.T. Gibson. Marcell Dekker, Inc., New
York, NY. 1984. Chap. 7. p. 181.
19. D.T. Gibson, G.E. Cardini, F.C. Maseles, and R.E. Kallio. Bio-
chemistry, 9, 1631, (1970).
20. D.M. Jerina and J.W. Daly. In Natl. Inst. Arthritis Metab. Dis.
Edited by T. E. King. Natl. Inst. Health, Bethesda, MD, U.S.A.
Oxidases Relat. Redox Syst., Proc. Int. Symp. 2nd (1973),
Meeting Date 1971, 1, 143.
21. (a) Gibson has recently reported evidence suggesting a step-
wise mechanism. E. Carredano, A. Karlsson, B. Kauppi,
D. Choudhury, R.E. Parales, J.V. Parales, K. Lee, D.T. Gibson,
H. Eklund, and S. Ramaswamy. J. Mol. Biol. 296, 701 (2000);
For a review of dioxygenase-type enzymes see: (b) K. Chen
and L. Que, Jr. Angew. Chem. Int. Ed. 38, 2227 (1999);
(c) L. Que, Jr. and R.Y.N. Ho. Chem. Rev. 96, 2607 (1996).
22. J.E. Baldwin, R.M. Adlington, B.P. Domayne-Hayman, G. Knight,
and H.H. Ting. J. Chem. Soc. Chem. Commun. 1661 (1987)
23. L. Birladeanu, T. Hanafusa, B. Johnson, and S. Winstein. J.
Am. Chem. Soc. 88, 2316 (1966); (b) C.D. Poulter and S.
Winstein. J. Am. Chem. Soc. 91, 3649 (1969); (c) C.D. Poulter
and S. Winstein. J. Am. Chem. Soc. 91, 3650 (1969).
24. A.D. Becke. J. Chem. Phys. 98, 5648 (1993).
25. D.M. Smith, A. Nicolaides, B.T. Golding, and L. Radom. J.
Am. Chem. Soc. 120, 10223 (1998).
8. S.V. Ley, F. Sternfeld, and S. Taylor. Tetrahedron Lett. 28, 225
(1987).
9. (a) V.P. Bui, T.V Hansen, Y. Stenstrom, and T. Hudlicky. Green
Chem. 2, 263 (2000); (b) for an up-to-date description of the
medium-scale fermentation procedure see: M.A. Endoma,
V.P. Bui, J. Hansen, and T. Hudlicky. Org. Process Res. Dev. In
26. M.J.T. Frisch, G.W. Trucks; H.B. Schlegel, G.E. Scuseria,
M.A. Robb, J.R. Cheeseman, V.G. Zakrzewski, J.A. Montgom-
ery, Jr., R.E. Stratmann, J.C. Burant, S. Dapprich, J.M. Millam,
A.D. Daniels, K.N. Kudin, M.C. Strain, O. Farkas, J. Tomasi,
V. Barone, M. Cossi, R. Cammi, B. Mennucci, C. Pomelli,
© 2002 NRC Canada

Bui et al.
C. Adamo, S. Clifford, J. Ochterski, G.A. Petersson, P.Y. Ayala,
713
28. K. Raghavachari and G.W. Trucks. J. Chem. Phys. 91, 1062
(1989).
Q. Cui, K. Morokuma, D.K. Malick, A.D. Rabuck, K. Raghavachari,
J.B. Foresman, J. Cioslowski, J.V. Ortiz, A.G. Baboul,
B.B. Stefanov, G. Liu, A. Liashenko, P. Piskorz, I. Komaromi,
R. Gomperts, R.L. Martin, D.J. Fox, T. Keith, M.A. Al-Laham,
C.Y. Peng, A. Nanayakkara, C. Gonzalez, M. Challacombe,
P.M.W. Gill, B. Johnson, W. Chen, M.W. Wong, J.L. Andres,
C. Gonzalez, M. Head-Gordon, E.S. Replogle, and J.A. Pople.
Gaussian 98. Revision A.7. Gaussian, Inc., Pittsburgh PA. 1998.
27. P.C. Hariharan and J.A. Pople. Theor. Chem. Acta, 28, 213
(1973).
29. (a) M. Newcomb and A.G. Glenn. J. Am. Chem. Soc. 111, 275
(1989); (b) J.M. Tanko and R.E. Drumright. J. Am. Chem.
Soc. 112, 5362 (1990); (c) J.M. Tanko, R.E. Drumright, N.K.
Suleman, and L.E. Brammer, Jr. J. Am. Chem. Soc. 116, 1785
(1994); (d) J.M. Tanko and J.P. Phillips. J. Am. Chem. Soc.
121, 6078 (1999).
30. A.L.J. Beckwith and V.W. Bowry. J. Am. Chem. Soc. 116,
2710 (1994).
© 2002 NRC Canada