Whole-cell biotransformation of m-ethyltoluene into 1S,6R-5-ethyl-1,6-dihydroxycyclohexa-2,4-diene-1-carboxylic acid as an approach to the C-ring of the binary indole-indoline alkaloid vinblastine

Article abstract of DOI:10.1071/CH04185

The title compound 3, a potential building block for the construction of analogues of the clinically important anticancer agent vinblastine (1), has been prepared in an efficient manner through a whole-cell biotransformation of m-ethyltoluene (4) using th

Full text of DOI:10.1071/CH04185

Rapid Communication
CSIRO PUBLISHING
Aust. J. Chem. 2005, 58, 14–17
www.publish.csiro.au/journals/ajc
Whole-Cell Biotransformation of m-Ethyltoluene into
1S,6R-5-Ethyl-1,6-dihydroxycyclohexa-2,4-diene-1-carboxylic Acid
as an Approach to the C-Ring of the Binary Indole–Indoline
Alkaloid Vinblastine
Martin G. Banwell,A,C Alison J. Edwards,A David W. Lupton,A and Gregg WhitedB
A Research School of Chemistry, Institute of Advanced Studies, Australian National University,
Canberra ACT 0200, Australia.
B Genencor International Inc., 925 Page Mill Road, Palo Alto, CA 94304, USA.
C Corresponding author. Email: mgb@rsc.anu.edu.au
The title compound 3, a potential building block for the construction of analogues of the clinically important anti-
cancer agent vinblastine (1), has been prepared in an efficient manner through a whole-cell biotransformation of
m-ethyltoluene (4) using the microorganism Pseudomonas putida BGXM1 which expresses the enzyme toluate
dioxygenase (TADO). Metabolite 3 was readily converted into derivatives 5–8 using conventional chemical tech-
niques and the structure, including absolute stereochemistry, of the last of these was established by single-crystal
X-ray analysis.
Manuscript received: 11 August 2004.
Final version: 11 October 2004.
Thebinaryindole–indolinealkaloidvinblastine(1, Scheme1)
and several derivatives are currently in clinical use for the
treatment of various human cancers including Hodgkin’s and
non-Hodgkin’s lymphoma, testicular cancer, bladder cancer,
and lung cancer as well as acute childhood leukaemia.[1]
As such, compounds of this class have been the target of
many synthetic studies over more than three decades.[1]
Notwithstanding important contributions from the laborato-
ries of Kuehne, Kutney, Langlois/Potier, and Magnus,[2] the
daunting structural complexity of vinblastine meant that it
eluded de novo total synthesis until 2002 when Fukuyama
and coworkers first reported their landmark work.[3] For
the same reasons, there has been an attendant lack of
medicinal-chemistry-type studies so that the minimum struc-
ture responsible for the therapeutically useful anti-mitotic
properties of compound 1 remains ill-defined.[1,4] We have
recently established, using Pd[0]-catalyzed Ullmann cross-
coupling chemistries, new methods for the construction of
the indole and indoline ring systems,[5,6] each of which is
incorporated within the vinblastine framework. Accordingly,
we have begun a program directed towards the synthesis of
‘stripped down’ vinblastine analogues that might retain clin-
ically useful therapeutic properties. During the course of
analyzing structure 1 we recognized that enzymatic dihy-
droxylation of m-ethylbenzoic acid (2) using the toluate
dioxygenase (TADO) enzyme system[7] might be expected[8]
to give metabolite 3 embodying key structural features
associated with the C-ring of vinblastine. While this con-
version has been described previously by Whited et al.,[9]
these researchers were unable to assign the absolute stereo-
chemistry of the metabolite and actually displayed (with an
appropriate caveat) ent-3 in their paper. As a consequence,
this structure (namely ent-3) has been propagated, without
qualification, in a review[10] even though it is at variance with
the well-defined actions of this same enzyme system on the
parent benzoic acid which delivers the des-ethyl derivative
of compound 3.[8] In order to resolve this seeming stereo-
chemical contradiction and to establish serviceable routes to
HO
N
H
N
OAc
C
N
OH
CO2Me
MeO
N
H
H
CO2Me
Me
1
OH
OH
CO2H
CO2H
2
3
4
Scheme 1.
© CSIRO 2005
10.1071/CH04185
0004-9425/05/010014

An Approach to the C-Ring of Vinblastine
15
6
O19
OH
O21
1
OH
O
O
O22
Br
O18
O
O
O20
O
O
O
O
CO2R
O
5 R ꢀ Me
6 R ꢀ H
7
8
Br1
Br
Fig. 1. ADEP derived from a single-crystal X-ray analysis of
compound 8.
Scheme 2.
6 was now contaminated with significant quantities of aro-
matic products, making this a less efficient process than the
three-step protocol just described.
compound 3 we have investigated a closely related biotrans-
formation and now detail the outcomes of our studies in this
area.
The microorganism used by Whited et al.[9] in carrying
out the biotransformation 2 → ent-3/3 was the Pseudomonas
putida (Pp) strain BGXM1 which is an NTG mutant of the
wild-type strain PpBG1 containing a TOL plasmid encod-
ing the pathway for the catabolism of toluene. The mutation
in strain BGXM1 inactivates the gene encoding the enzyme
cis-toluate diol dehydrogenase which oxidizes cis-toluate
diols to the corresponding catechols. As such, the mutant
accumulates the cis-diols metabolically formed from the
corresponding acids. Since the P. putida strain BGXM1 also
expresses enzymes capable of oxidizing toluenes to benzoic
acids, commercially available m-ethyltoluene (4) was exam-
ined as a substrate for this microorganism in the expectation
this would be oxidized to acid 2 in situ. If this approach were
successful it would avoid the need to prepare m-ethylbenzoic
acid (2), which is not readily accessible.
Subjection of the β,γ-unsaturated acid 6 to standard
trans-selective bromolactonization conditions[11] afforded
the expected brominated β-lactone 7 in 92% yield. While
it is anticipated that this highly functionalized compound
will serve as a useful intermediate in the construction of vin-
blastine analogues, it was, unfortunately, only ever obtained
as an oil. Following work reported by Widdowson et al.,[8a]
we eventually achieved access to a solid derivative of com-
pound 3 through its reaction with commercially available
α,p-dibromoacetophenone in the presence of triethylamine.
By such means the ester 8 was obtained in 65% yield and
this crystalline material proved amenable to single-crystal
X-ray analysis, details of which are provided in Fig. 1 and
the Experimental section. This analysis clearly reveals that
compound 3 possesses the illustrated absolute configuration,
a result in keeping with reports[8] on related biotransforma-
tions of benzoic acid and as required for exploitation of the
title metabolite in the preparation of vinblastine analogues
incorporating C-ring surrogates.The crystal structure reveals
that the diene moiety within compound 8 assumes P helicity
with a torsion angle of +13.6 and that the C1 and C6 OH
groups (see structure 8, Scheme 2) are pseudo-axially and
pseudo-equatorially orientated, respectively.
In the event, presentation of compound 4 to the above-
mentioned mutant resulted in the smooth formation (>55%
yield) of the targeted metabolite 3 which could be extracted
from the fermentation broth by conventional methods. This
rather thermally sensitive material was immediately sub-
jected to the derivatization protocols outlined below and
detailed in the Experimental section.
The work detailed herein serves to further highlight the
extraordinary utility of dioxygenases in the large-scale prepa-
ration of monochiral and highly functionalized building
blocks likely to be suitable for use in the synthesis of biolog-
ically active natural products and their analogues.[12] Work
along such lines and directed towards the synthesis of ana-
logues of vinblastine (1) from metabolite 3 is now underway
in these laboratories. Results will be reported in due course.
Derivatization of compound 3 was undertaken with a view
to obtaining a crystalline product, ideally one incorporating
a heavy atom since such features should allow for a single-
crystal X-ray analysis, and, thereby, determination of the
absolute stereochemistry of this metabolite. To such ends the
acid was treated with diazomethane in a 10 : 1 v/v mixture
of dichloromethane/ethanol and the resulting methyl ester
immediately treated with 2,2-dimethoxypropane in the pres-
ence of p-toluenesulfonic acid ( p-TsOH). In this manner the
acetonide ester 5 (Scheme 2) was obtained as a clear, colour-
lessoilandin49%overallyieldfromthestartinghydrocarbon
used in the biotransformation. Saponification of this mate-
rial using potassium carbonate in methanol then gave, after
careful acidic workup, the free acid 6 (100%), which was
also obtained as a clear, colourless oil. A more direct route
to this same acid involved treatment of metabolite 3 with
2,2-dimethoxypropaneinthepresenceofp-TsOHbutproduct
Experimental
Melting points were measured on a Reichert hot-stage microscope appa-
ratus and are uncorrected. Proton (1H) and carbon (13C) NMR spectra
were recorded on either a Varian Inova 500 spectrometer, operating at
500 MHz (1H) and 126 MHz (13C), or a Gemini 300 NMR spectro-
meter, operating at 300 MHz (1H) and 75 MHz (13C). Unless otherwise
specified, spectra were acquired at 20◦C in CDCl3 that had been fil-
tered through basic alumina immediately before use. Chemical shifts
are recorded as δ values in parts per million (ppm). Infrared spectra

16
M. G. Banwell et al.
(νmax) were recorded on a Perkin–Elmer 1800 FTIR spectrometer and
samples were analyzed as KBr disks (for solids) or plates (for oils). Low-
resolutionmassspectrawererecordedonaMicromass-WatersLC-ZMD
single quadrupole liquid chromatograph-MS or a VG Quattro II triple
quadrupole MS instrument using electron-impact techniques. High-
resolution mass spectra were recorded on an AUTOSPEC spectrometer.
Optical rotations were measured with a Perkin–Elmer 241 polarimeter
122.5 (CH), 116.3 (CH), 75.0 (C), 72.7 (CH), 25.0 (CH2), 12.0 (CH3).
This material was used in all the derivatization reactions detailed below.
Methyl (3aS,7aR)-7-ethyl-2,2-dimethyl-1,3-benzodioxole-
3a(7aH)-carboxylate 5
A solution of compound 3 (300 mg of crude material obtained by
ethyl acetate extraction of the processed fermentation broth—see above)
in dichloromethane/ethanol (4 mL of a 10 : 1 v/v mixture) was treated
with diazomethane (8 mL of a 0.24 M solution in ether) and the result-
ing mixture was left to stand at 18◦C for 0.5 h. Excess diazomethane
was then blown off by passing a stream of nitrogen gas through the
solution. After 10 min the residual solvent was removed under reduced
pressure to yield a pale orange residue, that was then dissolved in
2,2-dimethoxypropane (2 mL) and treated with p-toluenesulfonic acid
(5 mg). The ensuing mixture was stirred at 18◦C for 16 h then concen-
trated under reduced pressure. The resulting light yellow oil was subject
to column chromatography (silica; 5 : 95 v/v ethyl acetate/hexane elu-
tion) and concentration of the relevant fractions (RF 0.3) afforded the
title compound 5 (190 mg, 49% from compound 4) as a c+lear, colour-
less oil, [α]D −226 (c 0.9 in CHCl3) [Found: (M − H•) 237.1133.
C13H18O4 requires 237.1127]. δH (300 MHz) 6.07 (1H, dd, J 5.7 and
9.3), 5.80 (1H, dm, J 5.7), 5.69 (1H, dm, J 9.3), 4.81 (1H, s), 3.77
(3H, s), 2.31 (2H, m), 1.43 (3H, s), 1.36 (3H, s), 1.11 (3H, app. t, J
7.2). δC (75 MHz) 172.4 (C), 139.7 (C), 125.0 (CH), 122.0 (CH), 117.3
(CH), 107.3 (C), 80.5 (C), 76.0 (CH), 52.8 (CH3), 26.9 (CH3), 26.7
(CH2), 25.5 (CH3), 11.4 (CH3). νmax (neat)/cm−1 2968, 1736, 1454,
1435, 1378, 1371, 1256, 1176, 1031, 892, 798, 720. m/z (EI, 70 eV) 237
[(M − H•)+, 2%], 207 (17), 191 (70), 173 (57), 148 (52), 146 (100), 145
(81), 120 (57), 77 (36).
at the sodium-d line (589 nm) and at the concentrations (c; g 100 mL−1
)
indicated using spectroscopic-grade CHCl3 unless otherwise specified.
The measurements were carried out between 19 and 21◦C in a cell with
a path length (l) of 1 dm. Specific rotations [α]D were calculated using
the equation [α]D = 100α/(cl) and are given in 10−1 deg cm2 g−1. Ele-
mental analyses were performed by theAustralian National University’s
Microanalytical Services Unit based at the Research School of Chem-
istry. Dichloromethane was distilled from calcium hydride, and THF
was distilled, under nitrogen, from sodium benzophenone ketyl. Where
necessary, reactions were performed under a nitrogen atmosphere.
Biotransformation and Chemical Derivatization Studies
Whole-Cell Biotransformation of m-Ethyltoluene into 1S,6R-5-
Ethyl-1,6-dihydroxycyclohexa-2,4-diene-1-carboxylic Acid 3
Psuedomonas putida strain BGXM1[9] was used to produce the title
compound 3 through biotransformation of m-ethyltoluene (4) in a 14 L
fed batch culture. Frozen cells that had been maintained at −70◦C in
Lauria broth containing 25% glycerol were used for the biotransfor-
mation. An isolated colony of strain BGXM1 grown on Lauria agar at
30◦C was used as inoculum for a 500 mL preculture. The preculture
was grown in MSB (2) medium supplemented with 0.4% glycerol and
0.05% yeast extract at 30◦C on a rotary shaker overnight. A 14 L fer-
mentor was charged with 8.5 L of MSB medium supplemented with
0.4% glycerol and 0.05% yeast extract. The fermentor was inoculated
with the preculture and allowed to grow at 30◦C until the initial glycerol
was depleted. The growth conditions were maintained at 25% dissolved
oxygen by automatic adjustment of stirring, the pH was controlled at
7.2 with a 25% solution of ammonium hydroxide, and the air flow was
constant at 8 L min−1.After the initial depletion of glycerol (approx. 7 h)
the culture was fed at a constant rate of 0.25 g min−1 with a 25% solu-
(3aS,7aR)-7-Ethyl-2,2-dimethyl-1,3-benzodioxole-
3a(7aH)-carboxylic Acid 6
Method A: A magnetically stirred solution of ester 5 (40 mg,
0.168 mmol) in methanol (3 mL) was treated with K2CO3 (1.5 mL of a
1 M aqueous solution) and stirred at 18◦C for 4 h after which time TLC
analysis indicated the complete consumption of starting material. As a
consequence, the reaction mixture was acidified to a pH of 6 with HCl
(1 M aqueous solution) and extracted with diethyl ether (3 × 15 mL).
The combined organic fractions were then dried (MgSO4), filtered, and
concentrated under reduced pressure to afford the title acid 6 (38 mg,
100%) as a pale yellow oil, [α]D −246 (c 0.6 in CHCl3) (Found: M+•
224.1049. C12H16O4 requires 224.1049). δH (300 MHz) 6.11 (1H, dd, J
6.0 and 9.6), 5.90 (1H, br s), 5.82 (1H, dm, J 6.0), 5.70 (1H, dm, J 9.6),
4.82 (1H, s), 2.29 (2H, m), 1.47 (3H, s), 1.14 (3H, s), 1.11 (3H, t, J 6.9),
signal due to CO2H not observed. δC (75 MHz) 175.9, 139.6, 125.5,
121.3, 117.3, 107.9, 80.3, 76.1, 26.8, 26.6, 25.5, 11.4. νmax (neat)/cm−1
3184, 2988, 2938, 1732, 1458, 1435, 1382, 1372, 1247, 1215, 1163,
1074, 1054, 1035, 892, 741. m/z (EI, 70 eV) 224 [M+•, 2%], 166 (40),
149 (36), 148 (39), 121 (100), 107 (51), 77 (43), 43 (47).
Method B:A magnetically stirred solution of diol 3 (300 mg of crude
material obtained by ethyl acetate extraction of the processed fermen-
tation broth—see above) in 2,2-dimethoxypropane (3 mL) and acetone
(0.5 mL) was treated with p-TsOH (5 mg) and the resulting mixture
allowed to stir at 18◦C for 16 h. Concentration of the reaction mixture
afforded the desired acid 6 (as judged by 1H NMR spectroscopic com-
parisons with the material obtained by method A) albeit contaminated
with significant quantities of aromatized material.
tion of glycerol for 10 h after which the rate was increased to 1 g min−1
.
The culture reached an optical density of approx. 18 at 600 nm about
19 h after inoculation. At this time a slow drip of m-ethyltoluene (4, ex.
Aldrich) was delivered directly to the fermentation broth using an HPLC
pump. Metabolite 3 began to accumulate immediately as determined by
measuring the spectrum of a sample of the clarified broth and quantify-
ing the absorbance peak at 268 nm. The diol absorbance peak continued
to increase proportionally to the addition of hydrocarbon until about
20 g of substrate was delivered.
After all of the hydrocarbon substrate had been delivered the pH of
the fermentation medium (now approx. 10 L in volume) was adjusted to
8.0 with KOH (1 M aqueous solution) and the cells were then removed
by centrifugation. The 8.5 L of clarified supernatant thus obtained was
concentratedto1.6 Lunderreducedpressureusingawaterbathtempera-
ture of approximately 55◦C. This concentrated material was centrifuged
again, to remove residual cells and precipitated salts, then shipped from
San Francisco to Canberra. The yield of diol in this concentrate was
estimated, by UV-vis spectroscopy and using the molar extinction coef-
ficient of 3000 (at 268 nm) reported[9] for the corresponding cis-diol
derived from p-toluic acid, to be approximately 16.9 g (about 55%
yield). Appropriate portions of this material were cooled to 0◦C and
treated with HCl (10 M aqueous solution) until the pH was between 2
and 3. The resulting orange-brown solution was extracted three times
with ethyl acetate and the combined extracts were then dried (MgSO4),
filtered, and concentrated under reduced pressure on a rotary evapo-
rator (with the associated bath temperature maintained below 37◦C)
to afford samples of compound 3[9] contaminated with ethyl acetate.
δH (300 MHz) 6.25 (2H, br s), 6.10 (1H, dd, J 5.5 and 9.3), 5.69 (2H,
m), 4.82 (1H, s), 2.24 (2H, q, J 7.5), 1.05 (3H, t, J 7.5), signal due to
CO2H not observed. δC (75 MHz) 171.5 (C), 144.6 (C), 127.7 (CH),
(3aR,6S,6aS,8aR)-6-Bromo-4-ethyl-6,6a-dihydro-2,2-dimethyl-
8-oxo-3aH,8H-oxeto[3,2-d]-1,3-benzodioxole 7
Following protocols developed by Ganem and coworkers,[11] a mag-
netically stirred solution of acid 6 (50 mg, 0.22 mmol) in NaHCO3
(1.5 mL of a saturated aqueous solution) was cooled to 0◦C then treated,
dropwise, with a solution of molecular bromine (36 mg, 0.22 mmol) in
dichloromethane (1 mL). After 20 min the organic layer was separated
and the aqueous phase extracted with dichloromethane (2 × 10 mL).The
combined organic fractions were washed with brine (1 × 20 mL) then
dried (MgSO4), filtered, and concentrated under reduced pressure to

An Approach to the C-Ring of Vinblastine
17
yield an orange oil. Subjection of this material to flash chromatography
(silica; 1 : 9 v/v ethyl acetate/hexane elution) gave, after concentration of
the appropriate fractions (RF 0.3), the title bromolactone 7 (58 mg, 92%)
as a light yellow oil, [α]D −27 (c 1.0 in CHCl3) [Found: (M − H3C•)+
286.9922. C12H1579BrO4 requires 286.9919]. δH (300 MHz) 5.85 (1H,
m), 4.92 (1H, dd, J 5.1 and 6.0), 4.58 (1H, d, J 5.1), 4.55 (1H, m), 2.34
(2H, m), 1.71 (3H, s), 1.58 (3H, s), 1.15 (3H, t, J 7.5). δC (75 MHz)
170.6, 147.1, 119.1, 114.7, 81.0, 76.3, 74.0, 45.6, 27.0, 25.7, 25.3, 11.1.
νmax (neat)/cm−1 2986, 2938, 1805, 1384, 1335, 1302, 1247, 1217,
1157, 1109, 1075, 1055, 939, 874, 787. m/z (EI, 70 eV) 289 and 287
[(M − H3C•)+, both 2%], 179 (35), 121 (100), 109 (27), 93 (32), 69 (40).
Acknowledgments
We thank the Institute ofAdvanced Studies as well as theAus-
tralian Research Council Discovery Program for generous
financial support.
References
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ANIE4217>3.0.CO;2-U
2-(4-Bromophenyl)-2-oxoethyl (1S,6R)-5-Ethyl-1,6-
dihydroxycyclohexa-2,4-diene-1-carboxylate 8
A magnetically stirred solution of diol 3 (300 mg of crude mate-
rial obtained by ethyl acetate extraction of the processed fermentation
broth—see above) in acetone (5 mL) maintained at 18◦C was treated
with triethylamine (184 mg, 1.83 mmol) then α,p-dibromoacetophenone
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124–130◦C, [α]D +11 (c 0.9 in CHCl3) (Found: C 53.4, H 4.3, Br 21.1;
M+• 380.0266. C17H1781BrO5 requires C 53.6, H 4.5, Br 21.0%; M+•
380.0259). δH (300 MHz) 7.76 (2H, dm, J 8.7), 7.66 (2H, dm, J 8.7),
6.17 (1H, dd, J 5.7 and 9.0), 5.87 (1H, d, J 9.0), 5.71 (1H, m), 5.51 (1H,
d, J 18), 5.43 (1H, d, J 18), 4.95 (1H, m), 3.28 (2H, br s), 2.32 (2H,
m), 1.10 (3H, t, J 7.2). δC (75 MHz) 191.4, 174.7, 144.6, 132.4, 132.1,
129.8, 129.3, 128.0, 122.1, 116.2, 75.5, 73.3, 67.0, 25.1, 12.1. νmax
(neat)/cm−1 3493, 3413, 2963, 1739, 1698, 1587, 1428, 1399, 1284,
1224, 1121, 1069, 995, 961, 815, 747, 563. m/z (EI, 70 eV) 382 and 380
[M+•, both 12%], 364 and 362 [(M − H2O)+•, both 22%], 200 and 198
(both 26), 185 and 183 (both 100), 149 (57), 148 (97), 138 (54), 120
(57), 95 (25), 77 (35).
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Crystal Data for Ester 8
C17H17BrO5, M 381.227,T 200 K, orthorhombic, space group P212121,
Z 4, a 5.3466(1), b 15.8093(3), c 18.8706(4) Å, V 1595.06(5) Å3, Dx
1.588 Mg m−3, 3646 unique data (2θmax 54.96◦), 2546 with I > 3σ(I);
R 0.022, wR 0.026, S 1.072.
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Images were measured on a Nonius Kappa CCD diffractometer
(MoKα, graphite monochromator, λ 0.71073 Å) and the data extracted
using the DENZO package.[13] Structure solution was by direct methods
(SIR97)[14] and refinement was by full-matrix least-squares on F using
the CRYSTALS program package.[15] The final Flack parameter of
0.012(8) demonstrates that the compound is of the absolute stereo-
chemistry reported here. CCDC 246605 contains the supplementary
crystallographic data for this paper. These data can be obtained free
of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing
data_request@ccdc.cam.ac.uk.