Enantiodivergent, biocatalytic routes to both taxol side chain antipodes

Article abstract of DOI:10.1021/jo0516077

Two enantiocomplementary bakers' yeast enzymes reduced an α-chloro-β-keto ester to yield precursors for both enantiomers of the N-benzoyl phenylisoserine Taxol side chain. After base-mediated ring closure of the chlorohydrin enantiomers, the epoxides were

Full text of DOI:10.1021/jo0516077

SCHEME 1
Enantiodivergent, Biocatalytic Routes to
Both Taxol Side Chain Antipodes
Brent D. Feske, Iwona A. Kaluzna,† and
Jon D. Stewart*
Department of Chemistry, University of Florida, 127
Chemistry Research Building, Gainesville, Florida 32611
jds2@chem.ufl.edu
Received August 1, 2005
plored a biocatalytic approach to a â-lactam derivative
of the Taxol side chain in which whole bakers’ yeast cells
were used to kinetically resolve a ketone intermediate
by asymmetric reduction.18 In addition to a maximal 50%
yield, this approach suffered from limited stereoselectiv-
ity in the crucial biocatalytic reduction, even when
engineered cells were employed.19 Since that time, we
have systematically examined reductases encoded by the
bakers’ yeast genome.20 In nearly all cases, individual
yeast reductases afforded much higher stereoselectivities
than whole cells of the native organism.21 Our recent
discovery that some yeast reductases show high diaste-
reo- and enantioselectivity toward a variety of R-chloro-
â-keto esters22 suggested that the alcohols resulting from
such dynamic kinetic resolutions might be useful glycidic
ester precursors that could be converted to the Taxol side
chain. Moreover, given the diversity of yeast reductases,
we were intrigued by the possibility that both target
enantiomers could be synthesized from a common pre-
cursor. This indeed proved to be the case.
Two enantiocomplementary bakers’ yeast enzymes reduced
an R-chloro-â-keto ester to yield precursors for both enan-
tiomers of the N-benzoyl phenylisoserine Taxol side chain.
After base-mediated ring closure of the chlorohydrin enan-
tiomers, the epoxides were converted directly to the oxazo-
line form of the target molecules using a Ritter reaction with
benzonitrile. These were hydrolyzed to the ethyl ester form
of the Taxol side chain enantiomers under acidic conditions.
This brief and atom-efficient route to both target enanti-
omers demonstrates both the synthetic utility of individual
yeast reductases and the power of genomic strategies in
making these catalysts available.
We selected oxazoline 2 as a key intermediate for our
synthesis since both the oxygen and nitrogen moieties
are protected in this derivative, allowing its coupling to
Taxol 1 has emerged as the drug of choice for treating
certain types of ovarian and breast cancers because it
blocks microtubule disassembly (Scheme 1).1,2 Because
the quantities supplied by isolation from the natural
source proved insufficient to meet commercial demand,
routes for its semi-synthesis from a terpene core such as
10-deacetylbaccatin III 3 have been developed.3,4 Most
biological activity resides in the natural (2R,3S)-diaste-
reomer of the N-benzoyl-phenyisoserine side chain, and
its small size, dense functionality, and medical impor-
tance have made this molecule an important test-bed for
methods in asymmetric synthesis.5-17 Earlier, we ex-
(9) Lee, D.; Kim, M.-J. Tetrahedron Lett. 1998, 2163-2166.
(10) Lee, K.-Y.; Kim, Y.-H.; Park, M.-S.; Ham, W.-H. Tetrahedron
Lett. 1998, 8129-8132.
(11) Wuts, P. G. M. Curr. Opin. Drug Discovery Dev. 1998, 1, 329-
337.
(12) Hamamoto, H.; Mamedov, V. A.; Kitamoto, M.; Hayashi, N.;
Tsuboi, S. Tetrahedron: Asymmetry 2000, 11, 4485-4497.
(13) Mandai, T.; Oshitari, T.; Susowake, M. Synlett 2002, 1665-
1668.
(14) Voronkov, M. V.; Gontcharov, A. V.; Wang, Z.-M. Tetrahedron
Lett. 2003, 407-409.
(15) Zhou, Z. Q.; Mei, X. G. Synth. Commun. 2003, 33, 723-728.
(16) Borah, J. C.; Gogoi, S.; Boruwa, J.; Kalita, B.; Barua, N. C.
Tetrahedron Lett. 2004, 3689-3691.
† Present address: BioCatalytics, Inc., 129 North Hill Avenue, Suite
103, Pasadena, CA 91106.
(1) Wall, M. E.; Wani, M. C. In Taxane Anticancer Agents: Basic
Science and Current Status; Georg, G. I., Ed.; ACS Symposium Series
583; American Chemical Society: Washington, DC, 1995; pp 18-30.
(2) Morrissey, S. R. Chem. Eng. News 2003, 37, 17-20.
(3) Denis, J.-N.; Greene, A. E.; Serra, A. A.; Luche, M.-J. J. Org.
Chem. 1986, 51, 46-50.
(17) Castagnolo, D.; Armaroli, S.; Corelli, F.; Botta, M. Tetrahe-
dron: Asymmetry 2004, 15, 941-949.
(18) Kayser, M. M.; Mihovilovic, M. D.; Kearns, J.; Feicht, A.;
Stewart, J. D. J. Org. Chem. 1999, 64, 6603-6608.
(19) Yang, Y.; Kayser, M. M.; Rochon, F. D.; Rodriguez, S.; Stewart,
J. D. J. Mol. Catal. B: Enzym. 2005, 32, 167-174.
(20) Stewart, J. D.; Rodriguez, S.; Kayser, M. M. In Enzyme
Technologies for Pharmaceutical and Biotechnological Applications;
Kirst, H. A., Yeh, W.-K., Zmijewski, M. J., Eds.; Marcel Dekker: New
York, 2001; pp 175-208.
(4) Holton, R. A. U.S. Patent 5,015,744, 1989.
(5) Deng, L.; Jacobsen, E. N. J. Org. Chem. 1992, 57, 4320-4323.
(6) Srivastava, R. P.; Zjawiony, J. K.; Peterson, J. R.; McChesney,
J. D. Tetrahedron: Asymmetry 1994, 5, 1683-1688.
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59, 5104-5105.
(21) Kaluzna, I. A.; Matsuda, T.; Sewell, A. K.; Stewart, J. D. J.
Am. Chem. Soc. 2004, 126, 12827-12832.
(22) Kaluzna, I. A.; Feske, B. D.; Wittayanan, W.; Ghiviriga, I.;
Stewart, J. D. J. Org. Chem. 2005, 70, 342-345.
(8) Cardillo, G.; Gentilucci, L.; Tolomelli, A.; Tomasini, C. J. Org.
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10.1021/jo0516077 CCC: $30.25 © 2005 American Chemical Society
Published on Web 10/13/2005
9654
J. Org. Chem. 2005, 70, 9654-9657

SCHEME 2
the terpene core prior to acid-catalyzed hydrolysis to yield
the final target 1 (Scheme 1).23 We planned to form
glycidic esters 7 and ent-7 by ring closure of the corre-
sponding syn-chlorohydrin enantiomers, the products of
enantiodivergent enzymatic reductions (Scheme 2). While
facile enolization allows reductions of 5 to proceed by
dynamic kinetic resolution, previous efforts to use chiral
hydrogenation catalysts24 or baker’s yeast cells25,26 were
met with disappointingly low stereoselectivities. We
hoped that individual yeast reductases might be more
suitable. We also hoped that a Ritter reaction might allow
one-step conversion of 7 and its enantiomer directly to
the corresponding trans-oxazolines that are direct pre-
cursors of the final targets.27,28 This would eliminate the
need for lengthier sequences from epoxide 7 to 8, some
of which involve azide intermediates.3,5,6,9,15
R-Chloro-â-keto ester 5 was prepared by treating
commercially available 4 with sulfuryl chloride.29 We
examined its NADPH-mediated reduction by 19 isolated
Saccharomyces cerevisiae reductases expressed as fusion
proteins with glutathione S-transferase.22 Two reductions
were successful: aldose reductase YDL124w produced
syn-(2S,3R)-6 as the only detectable product ([R]D -3.0°,
c 0.70, CHCl3; lit.25 [R]D -3.0°, c 1.7, CHCl3), whereas
short chain dehydrogenase YGL039w afforded a 9:1
mixture of syn-(2R,3S)- and anti-(2S,3S)-alcohols. By
comparison, reduction of 5 with whole baker’s yeast cells
yielded a mixture of all four stereosiomeric chlorohydrins
in which the desired syn-(2S,3R)-product made up only
half of the total yield.25,26 Clearly, the use of individual
yeast reductases allowed for a much cleaner conversion.
Moreover, the ability to prepare both 6 and ent-6 opened
the door to synthesizing both the natural Taxol side chain
as well as its enantiomer.
The use of an NADPH-regenerating system was con-
venient for small-scale reductions of 5; however, larger-
scale bioconversions were carried out with whole Escher-
ichia coli cells that overexpressed one of the two key S.
cerevisiae reductases (as GST fusion proteins). Glucose
fed batch conditions with nondividing cells were em-
ployed30 with the pH maintained at 5.6 to avoid sponta-
neous substrate decomposition under neutral or basic
conditions. The ketone substrate was toxic to the cells
at levels above 1 mM. We therefore added a nonpolar
resin to the reaction mixture (XAD-4) to act as a reservoir
for both substrate and product and used a slow-feeding
strategy.31 We observed reductive dechlorination of 5
during the early phases of the bioconversions. A similar
side reaction has been observed by others,32,33 and the
extent of this dechlorination varied widely from batch to
batch. Under optimized conditions, <5% of the total
added substrate was lost to spontaneous decomposition
and reductive dechlorination, which allowed isolation of
g1.33 g/L of optically pure 6 from 1 L of fermentations.
We did not attempt to improve the volumetric productiv-
ity of this process further since it was sufficient for
laboratory-scale synthesis. Unreacted starting material
was easily removed by column chromatography, and
reported yields were corrected for any recovered 5.
Conversion of 6 to cis-epoxide 7 proceeded smoothly
according to the method of Azerad.26 The cis stereochem-
istry and absolute configuration of 7 were confirmed by
the J2,3 value of 4.6 Hz and the optical rotation value ([R]D
+24°, c 1.5, CHCl3; lit.26 [R]D +25°, c 1.1, CHCl3),
respectively. A Ritter reaction employing benzonitrile and
a stoichiometric amount of BF3‚OEt2 converted 7 directly
to a 5:1 mixture of trans- and cis-oxazolines. Systematic
variation in reaction conditions (temperature, solvent,
(23) Kingston, D. G. I. Chem. Commun. 2001, 867-880.
(24) Geneˆt, J.-P.; de Andrade, M. C. C.; Ratovelomanana-Vidal, V.
Tetrahedron Lett. 1995, 2063-2066.
(25) Cabon, O.; Larcheveˆque, M.; Buisson, D.; Azerad, R. Tetra-
hedron Lett. 1992, 7337-7340.
(26) Cabon, O.; Buisson, D.; Larcheveˆque, M.; Azerad, R. Tetra-
hedron: Asymmetry 1995, 6, 2211-2218.
(30) Walton, A. Z.; Stewart, J. D. Biotechnol. Prog. 2004, 20, 403-
411.
(27) Fotadar, U.; Becu, C.; Borremans, F. A. M.; Anteunis, M. J. O.
Tetrahedron 1978, 34, 3537-3544.
(31) Anderson, B. A.; Hansen, M. M.; Harkness, A. R.; Henry, C.
L.; Vicenzi, J. T.; Zmijewski, M. J. J. Am. Chem. Soc. 1995, 117, 12358-
12359.
(28) Legters, J.; van Dienst, E.; Thijs, L.; Zwanenburg, B. Recl. Trav.
Chim. Pays-Bas 1992, 111, 69-74.
(29) Matsuki, K.; Sobukawa, M.; Kawai, A.; Inoue, H.; Takeda, M.
Chem. Pharm. Bull. 1993, 41, 643-648.
(32) Jo¨rg, G.; Bertau, M. Anal. Biochem. 2004, 328, 22-28.
(33) Jo¨rg, G.; Bertau, M. ChemBioChem 2004, 4, 87-92.
J. Org. Chem, Vol. 70, No. 23, 2005 9655

to 28 °C over 15 min, then GST fusion protein expression was
induced by adding isopropylthio-â-D-galactoside to a final con-
centration of 0.1 mM. After being stirred for an additional 6 h
under these conditions, the cells were collected by centrifugation
(6000g for 10 min at 4 °C). These could be stored at 4 °C for
later use, if desired. Approximately 25 g (wet weight) of cells
was resuspended in 1 L of 10 mM KPi (pH ) 5.6) containing 4
g/L glucose in a Braun Biostat B fermenter vessel (2 L total size)
containing 0.5 g of XAD-4 resin. The temperature, pH, and
dissolved oxygen level were maintained at 30 °C, 5.6 (3 M NaOH
titrant), and 75% saturation (fixed air flow of 0.25 vvm and
variable stirring rate), respectively. Aliquots of neat 5 (0.2 mL)
were added approximately every hour over a total of 12 h to a
final concentration of 6 mM. Glucose was added portionwise after
3 and 6 h to maintain a glucose concentration of approximately
4 g/L. Consumption of both 5 and glucose slowed significantly
after 8 h. After 24 h, the entire reaction mixture was gently
extracted with CH2Cl2 (2 × 300 mL) to avoid an emulsion, using
a procedure described in detail elsewhere.22 The organic layer
was dried with MgSO4, concentrated in vacuo, and purified by
flash chromatography (cyclohexane/Et2O 85:15) to afford 1.3 g
of 6 as a colorless oil (91% yield). [R]D -3.0°, c 0.68, CHCl3; lit.33
[R]D -3.0°, c 1.7, CHCl3. Anal. Calcd for C11H13O3Cl: C, 57.78;
H, 5.73. Found: C, 57.92; H, 5.86. Spectral data matched those
reported in the literature.35
Epoxides 7 and ent-7. To a solution of chlorohydrin 6 (1.3
g, 5.7 mmol) in DMF (28 mL) was added K2CO3 (2.2 g, 17 mmol,
3 equiv) and water (525 µL). After being stirred for 5 h at room
temperature, the mixture was diluted with water (75 mL) and
extracted with ether (3 × 75 mL). The combined organics were
washed with water to remove residual DMF (6 × 5 mL), dried
with MgSO4, and concentrated in vacuo to yield 1.1 g of 7 as a
colorless oil (99% yield). No further purification was necessary.
[R]D +24°, c 1.5, CHCl3; lit.26 [R]D +25°, c 1.1, CHCl3. Anal. Calcd
for C11H12O3: C, 68.74; H, 6.29. Found: C, 68.34; H, 6.57.
Spectral data matched those reported in the literature.26 Crude
ent-7 was prepared in the same way and subjected to silica gel
chromatography to remove a small amount of trans-epoxide,
affording 0.86 g of ent-7 as a colorless oil (66% overall yield from
5). [R]D -29°, c 2.9, CHCl3.
Oxazolines 2 and ent-2. A solution of glycidic ester 7 (0.50
g, 2.6 mmol) in benzonitrile (4 mL) was cooled to 0 °C under
argon atmosphere, then BF3‚OEt2 (330 µL, 2.6 mmol, 1.0 equiv)
was added over 10 min. The ice bath was removed, and the
reaction was stirred for 3 h before aqueous saturated NaHCO3
(4 mL) was added. After being stirred for an additional 2 h, the
reaction mixture was diluted with water (20 mL) and extracted
with CH2Cl2 (3 × 20 mL). The combined organics were dried
with MgSO4, concentrated in vacuo, and purified by silica gel
chromatography (cyclohexane/ether 85:15) to afford 0.42 g of 2
as a colorless oil (55% yield). 2: [R]D +11°, c 1.1, CHCl3. ent-2:
[R]D -12°, c 1.7, CHCl3. Spectral data for both 2 and ent-2
matched those reported in the literature for the racemic com-
pound.36,37
Taxol Side Chain Ethyl Esters 8 and ent-8. A solution of
oxazoline 2 (0.32 g, 1.1 mmol) in 0.5 M HCl (2.5 mL) and EtOH
(7 mL) was heated at reflux for 6 h. The solvent was removed
under reduced pressure, and then the residue was dissolved in
CH2Cl2 (10 mL), washed with water (2 × 7.5 mL), dried with
MgSO4, and concentrated under reduced pressure. The residue
was purified by silica gel chromatography (hexanes/Et2O 85:15)
to afford 0.30 g of 8 (85% yield) as a white solid, mp 162-163
°C, lit.38 mp 164-165 °C. [R]D -12 °, c 2.0, CHCl3; 1H NMR
(CDCl3) δ 1.30 (t, 3H, J ) 7.1), 3.30 (d, 1H, J ) 3.9), 4.30 (m,
Lewis acid) failed to improve this ratio. Fortunately, pure
trans-oxazoline 2 could be isolated from the crude mix-
ture by column chromatography in 55% yield. This was
hydrolyzed under mildly acidic conditions to yield the
natural enantiomer of the Taxol side chain ethyl ester
1
8. Its syn-configuration was evident from the H NMR
spectrum, particularly by the chemical shift (5.76 ppm)
and J2,3 value (1.8 Hz) of the C3 proton (in the case of
the trans-analogue, this signal lies 0.16 ppm upfield and
the corresponding J2,3 value is 3.5 Hz).34 The optical
purity of 8 was assessed by 1H NMR after derivatization
with (S)-R-methoxy-R-phenylacetic acid.
A complementary route was used to prepare the
antipode of the Taxol side chain. In this case, an E. coli
strain overexpressing the yeast YGL039w short chain
dehydrogenase was used for the bioconversion. Because
the C2-epimeric chlorohydrins resulting from this reaction
were inseparable by column chromatography, the mix-
ture was subjected to mild base-mediated ring closure.
The desired cis-(2S,3S)-epoxide ent-7 was isolated from
the crude product mixture in 66% overall yield from 5.
Its stereochemistry and absolute configuration were
confirmed by proton coupling and optical rotation values.
This material was carried on to ent-2 and ent-8 in the
same manner described above.
The ability to produce directly either enantiomer of the
chlorohydrin cis-epoxide precursor was a direct conse-
quence of the highly syn-selective biocatalysts used to
reduce 5 and our success in identifying individual yeast
reductases with the required stereoselectivities. These
results further underscore the power of genome-wide
expression libraries for solving synthetic problems. In
addition, the observation that two enzymes within the
same organism possessed complementary enantioselec-
tivities re-emphasizes the importance of screening indi-
vidual enzymes, rather than whole cells where such
enzymes would compete with one another.21
Using the Ritter reaction to install the nitrogen has
significant safety advantages over earlier methods that
require azide opening of epoxide 7. Our approach also
eliminated the need for a separate conversion to the
oxazoline, thereby enhancing the brevity and atom
economy of our synthesis. In fact, only the chlorine atom
was lost in the conversion of 5 to the target compounds.
It should be noted that the Taxol side chain is only
one member of a large family of R-hydroxy-â-amino acids
that are key substructures in pharmaceutically active
compounds, and our chemoenzymatic strategy may also
be useful for other members of this family. Our success
in the present case bodes well for such future applica-
tions.
Experimental Section
(2S,3R)-Ethyl 2-Chloro-3-hydroxy-3-phenylpropionate 6.
A 45-mL portion of LB medium supplemented with 30 µg/mL
kanamycin was inoculated with a single colony of E. coli BL21-
(DE3)(pIK8). After being shaken overnight at 37 °C, 40 mL of
this preculture was added to 4 L of the same medium containing
4 g/L glucose in a New Brunswick M19 fermenter. The culture
was grown for 2 h at 37 °C with a stir rate of 800 rpm and an
air flow of 0.5 vessel volumes per min (vvm) until it reached an
optical density at 600 nm of 0.6. The cell suspension was cooled
(35) Cabon, O.; Buisson, D.; Larcheveˆque, M.; Azerad, R. Tetra-
hedron: Asymmetry 1995, 6, 2199-2210.
(36) Yokoyama, M.; Menjo, Y.; Watanabe, M.; Togo, H. Synthesis
1994, 1467-1470.
(37) Repeated attempts to obtain consistent elemental analysis data
for 2 and ent-2 were unsuccessful, probably because of spontaneous
hydrolysis.
(38) Patel, R. N.; Banerjee, A.; Howell, J. M.; McNamee, C. B.;
Brozozowski, D.; Mirfakhrae, D.; Nanduri, V.; Thottathil, J. K.; Szarka,
L. J. Tetrahedron: Asymmetry 1993, 4, 2069-2084.
(34) Mader, M. M.; Edel, J. C. J. Org. Chem. 1998, 63, 2761-2764.
9656 J. Org. Chem., Vol. 70, No. 23, 2005

2H), 4.62 (dd, 1H, J ) 3.9, 1.9), 5.76 (dd, 1H, J ) 9.0, 1.9), 6.99
(br d, 1H, J ) 9.0), 7.30-7.55 (m, 8H), 7.77 (m, 2H); 13C NMR
(CDCl3) δ 14.3/14.4, 55.0, 60.6/62.9, 73.5, 127.1, 127.3, 128.1,
Acknowledgment. We thank the National Science
Foundation for financial support of this research (CHE-
0130315) and Dr. Ion Ghiviriga for carrying out NMR
experiments to assess the optical purity of 8 and ent-8.
A Ruegamer Fellowship provided to I.A.K. is also
gratefully acknowledged.
128.8, 128.9, 132.0, 134.4, 139.0, 167.1, 173.1;39 IR (KBr):
ν
(cm-1) 3416, 1718, 1638, 1529; HRMS (FAB) m/z calcd for C18H19-
NO4‚Na+ 336.1206, found 336.1209. ent-8: [R]D +12°, c 1.0,
CHCl3. The optical purities of 8 and ent-8 were determined by
1H NMR following derivatization with (S)-R-methoxy-R-phenyl
acetic acid (carried out in the NMR tube with 1.0 equiv of (S)-
MPA and 1.5 equiv of DCC and 0.5 equiv of DMAP in CDCl3).
The sample of ent-8 showed only a single enantiomer within the
limits of instrumental detection; that of 8 showed ca. 5%
contamination by its enantiomer.
Supporting Information Available: General experimen-
tal information, details for the preparation of ent-6, spectral
data for intemediates, and NMR optical purity determination
for 8 and ent-8. This material is available free of charge via
the Internet at http://pubs.acs.org.
(39) Both the 1H and 13C NMR spectra showed evidence for two ethyl
group conformers.
JO0516077
J. Org. Chem, Vol. 70, No. 23, 2005 9657