Studies on taxol biosynthesis. Preparation of taxa-4(20),11(12)-dien-5 alpha-acetoxy-10 beta-ol by deoxygenation of a taxadiene tetraacetate obtained from Japanese yew.

Article abstract of DOI:10.1021/jo025679z

Taxa-4(20),11(12)-dien-5 alpha-acetoxy-10 beta-ol 6 has been identified as an early stage intermediate involved in the biosynthesis of taxol (Paclitaxel). This compound has been efficiently prepared by Barton deoxygenation of the C-2- and C-14-hydroxyl groups on a derivative semisynthetically prepared from taxa-4(20),11(12)-dien-2 alpha,5 alpha,10 beta-triacetoxy-14 beta-(2-methyl)butyrate (7), a major taxoid metabolite isolated from Japanese yew heart wood. The synthetic methodology is amenable for the preparation of isotopically labeled congeners that will be useful to probe further intermediate steps in the biosynthesis of taxol.

Full text of DOI:10.1021/jo025679z

Stu d ies on Ta xol Biosyn th esis. P r ep a r a tion of
Ta xa -4(20),11(12)-d ien -5r-a cetoxy-10â-ol by Deoxygen a tion of a
Ta xa d ien e Tetr a a ceta te Obta in ed fr om J a p a n ese Yew
Tohru Horiguchi,† Christopher D. Rithner,† Rodney Croteau,‡ and Robert M. Williams*,†
Department of Chemistry, Colorado State University, Fort Collins, Colorado 80523 and
Institute of Biological Chemistry, Washington State University, Pullman, Washinton 99164
rmw@chem.colostate.edu
Received March 4, 2002
Taxa-4(20),11(12)-dien-5R-acetoxy-10â-ol 6 has been identified as an early stage intermediate
involved in the biosynthesis of taxol (Paclitaxel). This compound has been efficiently prepared by
Barton deoxygenation of the C-2- and C-14-hydroxyl groups on a derivative semisynthetically
prepared from taxa-4(20),11(12)-dien-2R,5R,10â-triacetoxy-14â-(2-methyl)butyrate (7), a major taxoid
metabolite isolated from J apanese yew heart wood. The synthetic methodology is amenable for the
preparation of isotopically labeled congeners that will be useful to probe further intermediate steps
in the biosynthesis of taxol.
SCHEME 1. Ea r ly Step s in Ta xol Biosyn th esis
In connection with ongoing studies on the biosynthesis
of taxol (Paclitaxel, 1),1,2 our laboratory needed to prepare
several lightly oxygenated taxoids with the aim of
elucidating intermediate hydroxylation steps in the bio-
synthetic pathway. To fully gain command of taxol
biosynthesis in genetically engineered Taxus sp. cell
culture systems, a detailed understanding of the steps
of taxol biosynthesis and the identification of the associ-
ated genes is essential.
A combination of in vivo feeding studies and investiga-
tions with cell-free enzyme systems, using Yew stem
tissue or suspension cultured Taxus sp. cells as biocon-
version vectors, have recently revealed that the early
steps of the taxol biosynthetic pathway proceed in
sequence from the initial conversion of geranylgeranyl
pyrophosphate (2) to the parent diene 3 catalyzed by
taxadiene synthase (Scheme 1).3,4 Following this first
committed step in taxol biosynthesis, taxadiene hydroxy-
lase, a cytochrome P-450-dependent enzyme, regioselec-
* Corresponding author.
†Colorado State University.
‡ Washington State University.
(1) (a) Suffness, M. In Taxane anticancer agents: Basic science and
current status; Georg, G. I.; Chen, T. T.; Ojima, I.; Vyas, D. M., Eds.;
American Chemical Society: Washington, DC, 1995; pp 1-17. (b)
Suffness, M.; Wall, M. E. In Taxol: Science and applications; Suffness,
M., Ed.; CRC Press: Boca Raton, FL, 1995; pp 3-25. (c) Holmes, F.
A.; Kudelka, A. P.; Kavanagh, J . J .; Huber, M. H.; Ajani, J . A.; Valero,
V., In Taxane anticancer agents: Basic science and current status;
Georg, G. I.; Chen, T. T.; Ojima, I.; Vyas, D. M.; Eds.; American
Chemical Society: Washington, DC, 1995; pp 31-57. (d) Golspiel, B.
R. Pharmacotherapy 1997, 17, 110S-125S.
tively hydroxylates 3 with allylic rearrangement, to
taxa-4(20),11(12)-dien-5R-ol (4).5 Taxadienol-O-acetyl-
transferase subsequently acylates 4 to provide the acetate
5, which has proven to be a superior substrate for
downstream hydroxylation reactions.6
(2) Paclitaxel is the generic name for taxol, a registered trademark
of Bristol-Myers Squibb; because of its greater familiarity, the term
“taxol” is used throughout.
Initial observations from our laboratories reveal that
after the formation of 5, downstream hydroxylations
(3) (a) Hezari, M.; Croteau, R. Planta Med. 1997, 63, 291-295. (b)
Walker, K.; Croteau, R. Phytochemistry 2001, 58, 1-7.
(4) (a) Koepp, A. E.; Hezari, M.; Zajicek, J .; Vogel, B. S.; LaFever,
R. E.; Lewis, N. G.; Croteau, R. J . Biol. Chem. 1995, 270, 8686-8690.
(b) Hezari, M.; Lewis, N. G.; Croteau, R. Arch. Biochem. Biophys. 1995,
322, 437-444. (c) Lin, X.; Hezari, M.; Koepp, A. E.; Floss, H. G.;
Croteau, R. Biochemistry 1996, 35, 2968-2977.
(5) Hefner, J .; Rubenstein, S. M.; Ketchum, R. E. B.; Gibson, D. M.;
Williams, R. M.; Croteau, R. Chem. Biol. 1996, 3, 479-489.
(6) (a) Walker, K.; Ketchum, R. E. B.; Hezari, M.; Gatfield, D.;
Goleniowski, M.; Barthol, A.; Croteau, R. Arch. Biochem. Biophys.
1999, 364, 273-279. (b) Walker, K.; Schoendorf, A.; Croteau, R. Arch.
Biochem. Biophys. 2000, 374, 371-380.
10.1021/jo025679z CCC: $22.00 © 2002 American Chemical Society
Published on Web 06/05/2002
J . Org. Chem. 2002, 67, 4901-4903
4901

SCHEME 2
diol monoactetate 5 appears to enter an oxygenation
“matrix” and is converted into a host of polyols, we have
focused on the downstream oxygenation reactions of 6, a
verified taxol pathway metabolite. However, production
of 6 by bioconversion of 5 gives at best, sub-milligram
quantities of this substance, which is insufficient for the
substrate requirements of our planned bioconversion
experiments. We have thus devoted considerable effort
to devising totally synthetic and semisynthetic methods
to prepare this compound. A further restriction we have
placed on our synthetic planning requires that the
convenient installation of radioisotopic tags be easily
attainable.
Herein, we report a method to semisynthetically
prepare 6 from taxa-4(20),11(12)-dien-2R,5R,10â-triac-
etoxy-14â-(2-methyl)butyrate (7), a component of J apa-
nese Yew heart wood.11 This method appears easily
applicable for the preparation of multi-milligram quanti-
ties of tritium-labeled 6.
Taxa-4(20),11(12)-dien-2R,5R,10â-triacetoxy-14â-(2-me-
thyl)butyrate 7 was prepared by extraction from J apa-
nese yew heart wood. Although the yield of 7 is less than
that of taxusin,12 the main taxoid component in Yew
heart wood, compound 7 does not possess the C-13 allylic
hydroxyl group. We have devoted considerable efforts to
deoxygenate taxusin at C-13, but all attempts to reduc-
tively remove this hydroxyl function were attended by
allylic transposition of the bridgehead olefin to the 12-
(13) position.13 Compound 7, therefore, appeared to be
an excellent precursor for the synthesis of lightly oxygen-
ated C-13-deoxy taxoids.
As shown in Scheme 2, all of the acyl groups of 7 were
removed by treatment with LAH to give taxa-4(20),11-
(12)-dien-2R,5R,10â,14â-tetraol 814 in good yield. Next,
the C-10-hydroxyl group was selectively protected as the
corresponding tert-butyldimethylsilyl ether (TBS) 9a or
as the corresponding diethylisopropylsilyl ether (DEIPS)
9b by treatment with the corresponding silyl chlorides
at low temperature. Deoxygenation via hydride attack
on C-2- and/or C-14- mesyl or tosyl groups was consid-
ered, but appeared to be difficult due to steric hindrance
on the congested and convex R-face at C-14 as well as to
steric hindrance imposed by the C-17 methyl group on
the â-face at C2.
enter a very complex matrix and the elucidation of a
single linear path to taxol has proven extremely chal-
lenging. This appears to be due to the softening of
substrate specificity by several of the remaining cyto-
chrome P-450 enzymes that can accept 5 as a suitable
substrate in vitro. Two major approaches are being
concurrently investigated to identify the genes and
associated intermediates from 5 to taxol. In the first
approach, we have obtained a set of related full-length
cytochrome P-450 clones by the method of differential
display of mRNA-reverse transcription-PCR, followed by
traditional library screening. Clones were selected based
on homology to other plant cytochrome P-450s and used
to individually transform Saccharomyces cerevisiae and
the transformed yeast clones were screened for oxygen-
ation activity with several taxoids as substrates. One
such clone produced taxa-4(20),11(12)-dien-5R-acetoxy-
10â-ol (6) using taxa-4(20),11(12)-dien-5R-acetate (5) as
a substrate.7 This material has been characterized by 1H
NMR and mass spectroscopy and has been shown to
incorporate into taxol in vivo.
In a second approach, microsomal bioconversion of 5
with Taxus sp. microsomes to more polar products,
however, yields several diol monoacetates including
taxa-4(20),11(12)-dien-5R-acetoxy-2R-ol8 and taxa-4(20),-
11(12)-dien-5R-acetoxy-13R-ol along with other, as yet
unidentified oxygenation products.9
Thus, Barton deoxygenation strategies were exam-
ined.15 Xanthate esters were formed from the sterically
more accessible C-2 hydroxyl group of triols 9a and 9b
by treatment with base followed by O-alkylation with
carbon disulfide and methyl iodide. This protocol fur-
nished the requisite substrates 10a and 10b in 55% and
44% yields, respectively (Scheme 3). Resubjecting 10a
and 10b to the xanthate ester acylation conditions yielded
Due to the very low yield of the intermediate metabo-
lites that may be obtained from natural sources, we have
relied heavily on synthetic, tritium-labeled taxadienes
3,10 4,5 and 55 as substrates from which in vivo and in
vitro bioconversion strategies have been utilized to
identify lightly oxenated taxoids downstream of these
substances. Using tritium-labeled 5 as a substrate, we
have identified several diol, triol, and higher polyols from
Taxus sp. suspension cell cultures by GC-MS, and, in a
few instances where more than 20 µg was produced and
purified, 1H NMR techniques were applied to identify the
structures of three diol monoacteates (vida supra). Since
(10) (a) Rubenstein, S. M.; Williams, R. M. J . Org. Chem. 1995, 60,
7215-7223. (b) Rubenstein, S. M.; Vazquez, A.; Williams, R. M. J .
Labelled Cmpd. Radiopharm. 2000, 43, 481-491.
(11) Sugiyama, T.; Oritani, T.; Oritani, Takashi. Biosci. Biotech.
Biochem. 1994, 54, 1923-1924.
(12) Ho, T.-L.; Lee, G.-H.; Peng. S, -M.; Yeh, H.-M.; Chen, F. C.;
Yang, W. L. Acta Crystallogr. 1987, C43, 1378.
(13) Nicolaou, K. C.; Nantermet, P. G.; Ueno, H.; Guy, R. K. J . Chem.
Soc., Chem. Commun. 1994, 295-296.
(14) Keki, C.; Weiming, C.; Weihuua, Z. Qicheng, F.; XiaoTian, L.;
J iyu, G. PCT Int. Appl. 1994, 31. J apanese Patent Appl. No. Wo
9406740.
(7) Schoendorf, A.; Rithner, C. D.; Williams R. M.; Croteau, R. Proc.
Natl. Acad. Sci. U.S.A. 2001, 98, 1501-1506.
(8) Vazquez, A.; Williams, R. M. J . Org. Chem. 2000, 65, 7865-
7869.
(9) (a) Wheeler, A. L.; Long, R. M.; Ketchum, R. E. B.; Rithner, C.
D.; Williams R. M.; Croteau, R. Arch. Biochem. Biophys. 2001, 390,
265-278. (b) J ennewein, S.; Rithner, C. D.; Williams, R. M.; Croteau,
R. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 13595-13600.
(15) Barton, D. H. R.; McCombie, S. W. J . Chem. Soc., Perkin Trans.
1 1975, 1574-1585.
4902 J . Org. Chem., Vol. 67, No. 14, 2002

Studies on Taxol Biosynthesis
SCHEME 3
the O-TBS group from 13a required treatment with
TBAF in THF for 20 days, affording 6 in 40% yield. The
DEIPS group was more easily removed requiring treat-
ment with TBAF for 1 day to give 6 in 80% isolated yield.
The semisynthetic 6 prepared by either route proved to
1
be identical by H NMR and 13C NMR to that of natural
taxa-4(20),11(12)-dien-5R-acetoxy-10â-ol (6).7 The sub-
stitution of n-Bu3Sn3H for n-Bu3SnH in the Barton
deoxygenation step (11 f 12) is reasonably expected to
3
yield the requisite H-labeled 6.
In summary, taxa-4(20),11(12)-dien-5R-acetoxy-10â-ol
(6) has been efficiently prepared from the readily avail-
able taxa-4(20),11-dien-2R,5R,10â-triacetoxy-14â-(2-me-
thyl)butyrate 7, one of the main taxoid components in
J apanese Yew heart wood. The synthetic methodology
described here is readily amenable for the preparation
of isotopically labeled congeners that are required for
biosynthetic studies. In addition, the synthesis recorded
here provides further corroboration of the structure
assigned to 6 that was based on extensive 2D NMR
methods. Utilization of this procedure is presently being
explored to probe further steps in the biosynthesis of
taxol and will be reported on in due course.
Ack n ow led gm en t. We thank the National Insti-
tutes of Health (Grant CA 70375 to R.M.W., and Grant
CA 55254 to R.C) for financial support. We thank Prof.
Takayuki Oritani (Laboratory of Applied Bioorganic
Chemistry, Division of Life Science, Graduate School of
Agricultural Science, Tohoku University, J apan) and
Hida Ichii Ittoubori Kyoudou Kumiai of The Engraving
Craftsman Association, Hida, J apan, for the generous
gift of the J apanese Yew heart wood. We thank the
Yamada Science Foundation for fellowship support (to
T.H.). We thank Dr. J ohn Greaves (Department of
Chemistry, University of California, Irvine) for exact
mass measurement of compound 6.
the bis-xanthates 11a and 11b in 79% and 59% yields,
respectively.
Treatment of 11a and 11b with tri-n-butyltin hydride
in the presence of AIBN in toluene at 100-120 °C gave
the desired deoxygenation products 12a and 12b in 73%
and 46% yields, respectively. Acetylation of these sub-
stances with acetic anhydride in pyridine containing
DMAP provided the corresponding acetates 13a and 13b
in 39% and 96% isolated yields, respectively. Removal of
Su p p or tin g In for m a tion Ava ila ble: Full experimental
procedures and 1H NMR spectra of all new compounds
reported in this study. This material is available free of charge
via the Internet at http://pubs.acs.org.
J O025679Z
J . Org. Chem, Vol. 67, No. 14, 2002 4903