Synthesis and in vitro evaluation of taxol oxetane ring D precursors

Article abstract of DOI:10.1016/j.tetlet.2010.02.033

A series of potential taxoid substrates was prepared in radiolabeled form to probe in vitro for the oxirane formation step and subsequent ring expansion step to the oxetane (ring D) presumably involved in the biosynthesis of the anticancer agent Taxol. None of the taxoid test substrates underwent transformation in cell-free systems from Taxus suggesting that these surrogates bore substitution patterns inappropriate for recognition or catalysis by the target enzymes, or that taxoid oxiranes and oxetanes arise by independent biosynthetic pathways.

Full text of DOI:10.1016/j.tetlet.2010.02.033

Tetrahedron Letters 51 (2010) 2017–2019
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Tetrahedron Letters
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Synthesis and in vitro evaluation of taxol oxetane ring D precursors
a,*
b
c
a
a
Rüdiger Kaspera , Jonathan L. Cape , Juan A. Faraldos , Raymond E. B. Ketchum , Rodney B. Croteau
a
Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340, United States
Department of Chemistry, Washington State University, Pullman, WA 99164-4630, United States
Department of Chemistry, University of Illinois, 600 South Mathews Avenue, Urbana, IL 61801, United States
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of potential taxoid substrates was prepared in radiolabeled form to probe in vitro for the oxirane
formation step and subsequent ring expansion step to the oxetane (ring D) presumably involved in the
biosynthesis of the anticancer agent Taxol. None of the taxoid test substrates underwent transformation
in cell-free systems from Taxus suggesting that these surrogates bore substitution patterns inappropriate
for recognition or catalysis by the target enzymes, or that taxoid oxiranes and oxetanes arise by indepen-
dent biosynthetic pathways.
Received 28 January 2010
Revised 3 February 2010
Accepted 5 February 2010
Available online 11 February 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Taxol is an established antineoplastic agent that represents a
platform for the development of new, second generation drugs
for the treatment of cancers and other diseases. The supply of
This mechanism is thought to proceed via a reactive 1,3-dioxo-
lan-2-ylium cation formed by acetate-assisted opening of the pro-
1,2
15
tonated b4,20-oxirane ring, but it could also be formulated as a
7
Taxol and its immediate precursors, by isolation from Taxus tis-
concerted reaction (Scheme 1, II).
3
4
sues or cell cultures, is expected to be replaced by synthetic biol-
From a biochemical perspective, the intermediate 4b,20-func-
tion (e.g., 4, Scheme 2) could be formed from the corresponding
double bond by a cytochrome P450 oxygenase or a flavin-depen-
dent monooxygenase, since double bond epoxidations involving
5
,6
ogy approaches
necessitating knowledge of the underlying
biosynthetic pathway. Whereas many of the biosynthetic steps
7
en route to Taxol have been characterized, the biochemistry of for-
1
6,17
mation of the oxetane D-ring is still uncertain.
these enzyme types have been observed previously.
The subse-
The complexity of the Taxol biosynthetic pathway has impeded
the determination of the timing of oxetane ring formation. Within
the proposed 19 distinct enzymatic steps leading to Taxol, oxetane
ring formation is anticipated to occur in mid-pathway, presumably
following the formation of an acetylated taxadien-2,5,7,9,10,13-
hexaol but before the formation of baccatin III, to which the C13-
side chain is appended in three steps to complete the pathway to
quent ring expansion from epoxide to oxetane with acetate migra-
tion could involve a transferase-type enzyme or a mutase of
unknown type; this transformation might also be mediated by a
cytochrome P450 oxygenase, as somewhat related rearrangements
to cyclic ethers catalyzed by P450-enzymes have been recently
1
8,19
reported.
To explore the enzymology of the presumed epoxidation and
oxetane formation reactions in vitro, a series of accessible sub-
strate surrogates was synthesized in radiolabeled form (Scheme
2). Although the precise nature (oxidation and acylation state) of
the taxoid substrates involved in these reactions are yet to be
7
,8
Taxol.
Theoretically feasible reaction mechanisms to account for the
formation of the Taxol oxetane ring D have been proposed by sev-
9
–14
eral groups.
Based on an evaluation of known structures of nat-
simple and plausible
urally occurring taxoid derivatives,
a
mechanism leading to the oxetane D-ring of Taxol was first pro-
posed by Potier and co-workers.12 The co-occurrence of epoxy
and oxetanyl ester taxoids in Taxus species led the authors to pro-
pose an enzyme-mediated acid-catalyzed epoxyester/oxetaneester
rearrangement mechanism involving protonation of a proposed
b4,20-epoxide intermediate, backside attack of a C5-acetate moi-
ety onto C4, and rearrangement to the expanded oxetane ring with
the formal migration of the secondary 5a-acetoxy group to the ter-
tiary C4 position of the taxane core (Scheme 1, I).
a
*
Corresponding author.
E-mail addresses: rkaspera@gmail.com, kaspera@u.washington.edu (R. Kaspera).
Scheme 1. Proposed pathway to taxol oxetane ring D.
0
040-4039/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2010.02.033

2
018
R. Kaspera et al. / Tetrahedron Letters 51 (2010) 2017–2019
O.29 After structural
2
AcO
AcO
OAc
followed by benzoylation of the latter with Bz
confirmation of the unlabeled compounds, the syntheses of 2 and 7
were carried out with tritiated Ac O and Bz O, respectively.
2
2
5
4
1
3
2
Surrogates (1, 2, 6, and 7) were evaluated as potential sub-
strates for the proposed oxirane and oxetane ring formation reac-
tions in crude, soluble, and membranous enzyme preparations
OAc
AcO
H
O
HO OAc
4
1
7,28
from Taxus cells under a broad range of redox-type
ferase-type
and trans-
reaction conditions. To rule out any possible arte-
facts from non-enzymatic reactions, incubations of compounds 2,
, and 7 were initially conducted without or with boiled Taxus cell
2
9,30
a) cat K CO
b) MeLi, THF
2
3
MeOH, RT, 10 min
º
-
60 C, 2h
6
AcO AcO
2
AcO AcO OAc
preparations; both experiments revealed that these epoxy ester
surrogates were stable under incubation conditions. Since the oxe-
tane (ring D) formation step en route to Taxol is thought to occur at
5
4
13
OAc
7,8
AcO
H
OAc
mid biosynthetic pathway, the Taxus cell-free preparations were
R O
H
O
1
H
30
31
1
20
HO OR
2
tested for taxoid hydroxylase and taxoid acetyl transferase
5
a R = Ac, R = H (43%)
activities, confirming their catalytic competency for these early
and intermediate pathway transformations. Radio-HPLC or HPLC–
MS analysis of the reaction mixtures using authentic standards
1
2
5
b R = H, R = Ac (90%)
1
2
CH OOOH
3
CH Cl , RT, o/n
c) Ac O, cat DMAP d) Bz O, cat DMAP
CH Cl , RT, o/n
2
2
2
2
(
e.g., baccatin IV and VI, 8) showed that none of the surrogates em-
CH Cl , RT, o/n
2
2
2
2
ployed in the present study was converted to the expected epoxide
AcO
AcO
or oxetane derivatives, hence demonstrating that compounds 1, 2,
AcO
AcO
OAc
6, and 7 did not act as functional substrates under the conditions
used herein.
In contrast to previous Taxol biosynthetic studies, in which sur-
rogate substrates were of value in defining the target reaction,
OAc
OAc
32,33
AcO
H
O
R O
1
H
O
H
HO OR
3
2
the present case did not yield useful information regarding precur-
sors of taxoid oxiranyl or oxetanyl esters. These negative results
suggest that the presumed epoxidation (1?2) and oxetane forma-
tion (2?3 and 6/7?8) reactions may require different conditions,
or substrates with different substitution patterns to permit recog-
nition and catalysis by the relevant enzymes. Alternatively, these
results may indicate that 4b,20-epoxy ester and oxetanyl esters
represent two distinct types of advanced taxoids formed by sepa-
rate biosynthetic routes.
c 6 R = [2'- H]-Ac, R = Ac (90%)
d 7 R = Ac, R = [4'- H]-Bz (5%)
1
2
2
4β,20-epoxy 5%
3
2
a 4α,20-epoxy 30%
1
2
oxetane ring formation
AcO
AcO AcO
AcO
OAc
OAc
O
O
AcO
H
R1O
H
OAc
HO OR2
H
Acknowledgments
3
8
Scheme 2. Synthesis of radiolabeled surrogates.
We thank Gregory Helms (NMR measurements), Mark B. Lange
(
HRMS) for assistance. This investigation was supported by Grant
CA-55254 from the National Institutes of Health and by the McIn-
tire-Stennis Project 0967 from the Washington State University
Agricultural Research Center.
discovered, we reasoned that allylic ester 1 could serve as a probe
to study the initial oxirane ring formation, and the possibility that
the resulting epoxide 2 might be processed further to oxetanyl tax-
oid 3 by subsequent ester-assisted ring expansion. In addition, the
epoxy ester surrogates 6 and 7, displaying a more advance oxida-
tion–acylation state, were thought to be good candidates to test
the ester-assisted ring expansion reaction outlined in Scheme 1,
since oxetanyl taxoids with similar functionalities are known in
Supplementary data
Supplementary data (additional NMR data and methods for en-
zyme preparation) associated with this article can be found, in the
online version, at doi:10.1016/j.tetlet.2010.02.033.
2
0
nature. Exhaustive literature searches to evaluate the relative
abundances of the several hundred defined taxoids from Taxus spe-
References and notes
2
0–23
cies,
ies,
in combination with a wide range of biochemical stud-
have shown that none of the known 4b,20-epoxy taxoids
7
,9,11
1.
2.
3.
4.
5.
Brown, D. T. In Taxus—The Genus Taxus; Itokawa, H., Lee, K. H., Eds.; Taylor &
Francis: London, UK, 2003; pp 387–435.
Wang, X. In Taxus—The Genus Taxus; Itokawa, H., Lee, K. H., Eds.; Taylor &
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Taylor & Francis: London, UK, 2003; pp 151–178.
Takeya, K. Taxus—The Genus Taxus; Taylor & Francis: London, UK, 2003. pp 134–
bears a benzoate ester at the C2 position (C2-acetates are com-
mon), whereas over three quarters of the characterized oxetane
derivatives do bear a benzoate group at the C2 position.2
0,21
Based
on these structural observations, it is tempting to suggest that
b,20-epoxy taxoids -benzoylated at C2 (such as synthesized 7)
4
a
150.
might be transient intermediates of oxetane ring formation.
Engels, B.; Dahm, P.; Jennewein, S. Metab. Eng. 2008, 10, 201–206.
Labeled taxusin 1 was prepared by regioselective deacetylation
6. Roberts, S. C. Nat. Chem. Biol. 2007, 3, 387–395.
7. Croteau, R.; Ketchum, R. E. B.; Long, R. M.; Kaspera, R.; Wildung, M. R.
Phytochem. Rev. 2006, 5, 433–444.
2
4
at C13 of 1 using MeLi, followed by re-acetylation with tritiated
2
5
2
Ac O. Taxusin-b4,20-epoxide 2 was obtained in low yield by per-
8
.
Ketchum, R. E. B.; Croteau, R. In Plant Metabolomics, Biotechnology in Agriculture
and Forestry; Saito, K., Dixon, R., Eds.; Springer: Heidelberg, 2006; pp 291–309.
Floss, H. G.; Mocek, U. In Taxol—Science and Application; Suffness, M., Ed.; CRC:
Boca Raton, USA, 1995; pp 191–208.
2
6
acetic acid epoxidation. Commercially available 1-hydroxy bacc-
9.
atin I 4 was prepared, via diol 5b, in radiolabeled form (6)
following the C13 deacetylation24/reacetylation25 procedure de-
10. Swindell, C. S.; Britcher, S. F. J. Org. Chem. 1986, 51, 793–797.
scribed for allylic alcohol 1. Benzoate 7 was synthesized by regio-
11. Walker, K.; Ketchum, R. E.; Hezari, M.; Gatfield, D.; Goleniowski, M.; Barthol,
28
selective hydrolysis (K
2
CO
3
/MeOH)27 at C2 of 4 affording diol 5a,
A.; Croteau, R. Arch. Biochem. Biophys. 1999, 364, 273–279.

R. Kaspera et al. / Tetrahedron Letters 51 (2010) 2017–2019
2019
12. Gueritte-Voegelein, F.; Guenard, D.; Potier, P. J. Nat. Prod. 1987, 50, 9–18.
13. Giner, J. L.; Li, X.; Mullins, J. J. J. Org. Chem. 2003, 68, 10079–10086.
14. Willenbrink, D.; Tantillo, D. J. Russ. J. Gen. Chem. 2008, 78, 723–731.
15. Giner, J. L.; Faraldos, J. A. Helv. Chim. Acta 2003, 86, 3613–3622.
16. Abe, I.; Prestwich, G. D. In Comprehensive Natural Product Chemistry; Cane, D. E.,
Ed.; Elsevier: Amsterdam, NL, 1999; pp 267–298.
mixture was1 extracted with AcOEt to give diol 5a (4 mg, 43%) after LC
purification. H NMR (CDCl , 600 MHz), d 6.18 (1H, d, J = 11 Hz, H-10), 6.08 (1H,
br m, not resolved, H-13), 5.92 (1H, d, J = 11.1 Hz, H-9), 5.49 (1H, dd, J = 4.5 and
12.1 Hz, H-7), 4.27 (1H, t, J = 3.3 Hz, H-5), 4.20 (1H, d, J = 3.5 Hz, H-2), 3.79 (1H,
d, J = 4.8 Hz, Ha-20), 3.68, (1H, s, OH at C1), 3.21 (1H, s, OH at C2), 3.19 (1H, d,
J = 3.5 Hz, H-3), 2.56 (1H, dd, J = 9.9, 14.9 Hz, H-14), 2.41 (1H, d, J = 4.8 Hz, Hb-
3
1
7. Hallahan, D. L.; Lau, S. M.; Harder, P. A.; Smiley, D. W.; Dawson, G. W.; Pickett, J.
A.; Christoffersen, R. E.; O’Keefe, D. P. Biochim. Biophys. Acta 1994, 1201, 94–100.
8. Mizutani, M.; Todoroki, Y. Phytochem. Rev. 2006, 5, 385–404.
9. Rontein, D.; Onillon, S.; Herbette, G.; Lesot, A.; Werck-Reichhart, D.; Sallaud, C.;
Tissier, A. J. Biol. Chem. 2008, 283, 6067–6075.
0. Baloglu, E.; Kingston, D. G. J. Nat. Prod. 1999, 62, 1448–1472.
1. Itokawa, H. In Taxus—The Genus Taxus; Itokawa, H. L., Lee, K. H., Eds.; Taylor &
Francis: London, UK, 2003; pp 35–78.
20), 2.19 (3H s, C(O)CH
(3H, s, C(O)CH at C7 or 13), 2.08 (3H, s, C(O)CH
C(O)CH at C9), 1.97 (3H, s, C(O)CH at C5), 1.83 (1H, dd, J = 6.2, 15.0 Hz, H-14),
1.78 (1H, br m, H-6), 1.56 (3H, s, CH ), 1.28 (3H, CH -19), 1.23 (3H, s, CH ).
HRMS m/z found 633.25194 [M +Na], C30 13Na requires 633.2523.
29. Compound 7: Bz O was prepared as follows: A 200 L portion of dry benzene
containing dry pyridine (16 L) was supplemented with benzoyl chloride
(7.2 L) and stirred for 10 min. 7.5 mg benzoic acid (8.0 mg, 62.5 mol) or 4-
[ H]-benzoic acid [450 Ci] for radiolabeling) in benzene was added in
portions over 5 min period. After 10 min, the reaction mixture was diluted
with CH Cl (1 mL) and filtered through a SiO -column to elute pure benzoic
anhydride. A stoichiometric amount of Bz O in CH Cl (0.5 mL) and DMAP
(1 mg) in CH Cl (0.5 mL) were added to 5a (2.0 mg, 3.3
solution was stirred at room temperature over night. H
3
at C10), 2.17 (3H, s, CH
3
-18), 2.15 (1H, m, H-6), 2.09
3
3
at C7 or 13), 2.04 (3H, s,
1
1
3
3
3
3
3
+
42
H O
2
2
2
l
l
l
l
3
2
2
2
2
2
2. Ketchum, R. E.; Horiguchi, T.; Qiu, D.; Williams, R. M.; Croteau, R. B.
l
Phytochemistry 2007, 68, 335–341.
3. Ketchum, R. E.; Rithner, C. D.; Qiu, D.; Kim, Y. S.; Williams, R. M.; Croteau, R. B.
Phytochemistry 2003, 62, 901–909.
4. Ketchum, R. E. B.; Tandon, M.; Gibson, D. M.; Begley, T.; Schuler, M. L. J. Nat.
Prod. 1999, 62, 1395–1398.
5. Samaranyake, G.; Neidigh, K. A.; Kingston, D. G. I. J. Nat. Prod. 1993, 56, 884–
2
2
2
2
2
2
2
2
lmol). The resulting
2
O was added, and the
mixture was extracted with EtOAc. Purification by LC gives benzoate 7 in 5%.
1
8
98.
6. Compound 2: Peracetic acid (10
5 mg, 10 mol) dissolved in dry CH
3
H NMR (CDCl , 600 MHz), d 7.95 (2H, d, J = 8.5 Hz, benzoyl ortho-H), 7.58 (1H
l
L, 50
l
2
mol) was added to radiolabeled 1
t, J = 7.34 Hz, benzoyl para-H), 7.46 (2H, t, J = 7.8 Hz, benzoyl meta-H), 6.26 (1H,
d, J = 10.9 Hz, H-10), 6.14 (1H, d, J=11.3 Hz, H-9), 6.13 (1H, m, H-13,
overlapping with C9), 5.79 (1H, d, J=3.4 Hz, H-2), 5.53 (1H, dd, J = 4.4,
12.0 Hz, H-7), 4.20 (1H, t, J = 3.1 Hz, H-5), 3.59 (1H, d, J = 4.7 Hz, H-20a), 3.32
(1H, d, J = 3.6 Hz, H-3), 2.65 (1H, dd, J = 9.8, 14.6 Hz, Hb-14), 2.30 (1H, d,
(
l
2
Cl
(1 mL). The reaction mixture was
stirred over night at 6–10 °C. Purification by LC gave epoxide 2 (250
NMR (CDCl 600 MHz) 6.02 (1H, d, J = 10.69 Hz, H-10), 5.83 (1H, d,
J = 10.69 Hz, H-9), 5.81 (1H, m, overlapping H-13), 4.38 (1H, t, J = 2.57 Hz, H-
l H
g, 5%). 1
3
,
d
5
2
2
), 2.79 (1H, dd, J = 4.92 Hz, 2.13 Hz, H-3), 2.72 (1H, d, J = 3.86 Hz, H-20a), 2.77–
.68 (1H, m, H-14b), 2.42 (1H, d, J = 3.86 Hz, H-20b), 2.16 (3H, s, C(O)CH at C5),
.09 (3H, d, J = 0.95 Hz, CH -18), 2.08 (3H, s, C(O)CH at C13), 2.06 (3H, s,
at C9), 2.00 (3H, s, C(O)CH at C10), 1.95 (1H, m, H-6b), 1.84 (1H, m, H-
b), 1.76 (1H, m, H-1), 1.74 (1H, m, H-6 ), 1.71 (1H, m, H-7 ), 1.58 (3H, s, CH
7), 1.52 (1H, m, H-2b), 1.07 (3H, s, CH -16, overlapping H-14 , through
-19), 0.88 (1H, m, H-2 ); HRMS m/z found 543.25572
Na requires 543.25699.
7. Lin, H.-X.; Jiang, Y.; Chen, J.-M.; Chen, J.-K.; Chen, M.-Q. J. Mol. Struct. 2005, 738,
9–65.
8. Compound 5a: To a solution of 4 (10 mg, 15.4
a catalytic amount of K CO . After 2 h, aq satd NH
J = 5.0 Hz, H-20b), 2.26 (3H, s, CH
3
-18), 2.22 (3H, s, C(O)CH
3
at C-10), 2.14 (3H,
1
3
s, C(O)CH
s, C(O)CH
2.02 (3H, s, C(O)CH
16), 1.36 (3H, s, CH
[M+Na], C37
3
at C-13), 2.14 (Hb-6, through HMQC, covered in H NMR), 2.10 (3H,
at C7), 2.08 (3H, s, C(O)CH at C9), 2.07 (m, H -14, through HMQC),
at C5), (1H, ddd, J = 3.1, 3.9, 14.8, H -6), 1.716 (3H, s, CH
-19), 1.26 (3H, s, CH -17); HRMS m/z found 737.27884
14Na requires 737.27853.
3
3
3
3
a
C(O)CH
7
1
3
3
3
a
3
-
a
a
3
-
3
3
3
a
46
H O
HMQC), 0.97 (3H, s, CH
3
a
30. Wheeler, A. L.; Long, R. M.; Ketchum, R. E.; Rithner, C. D.; Williams, R. M.;
Croteau, R. Arch. Biochem. Biophys. 2001, 390, 265–278.
31. Walker, K.; Croteau, R. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 583–587.
32. Chau, M.; Croteau, R. Arch. Biochem. Biophys. 2004, 427, 48–57.
33. Walker, K.; Croteau, R. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 13591–13596.
+
[
40 9
M +Na], C28H O
2
2
5
l
mol) in MeOH (1 mL) was added
Cl (1 mL) was added. The
2
3
4