Novel fluoro- and hydroxyl-containing jasmonate derivatives as highly efficient elicitors in suspension cultures of Taxus chinensis

Article abstract of DOI:10.1016/j.bmcl.2004.06.082

Some of jasmonate derivatives with fluoro- or hydroxyl groups on their ester parts are found to be novel and effective elicitors, which enhanced the production of taxuyunnanine C (Tc) up to 60% more than that by methyl jasmonate in Taxus chinensis cell cultures. To develop more effective abiotic elicitors for cell suspension cultures of T. chinensis to meet the needs for paclitaxel as anti-tumor drug, some fluoro- or hydroxyl-containing groups are introduced to the ester moiety of jasmonic acid by the esterification or acylation with bis(trichloromethyl) carbonate and corresponding alcohol. Some of them are found to be novel and effective elicitors, which can enhance the production of taxuyunnanine C (Tc) up to 60% more than that by methyl jasmonate (MJA) in T. chinensis cell cultures.

Full text of DOI:10.1016/j.bmcl.2004.06.082

Bioorganic & Medicinal Chemistry Letters 14 (2004) 4755–4758
Novel fluoro- and hydroxyl-containing jasmonate derivatives
as highly efficient elicitors in suspension cultures of Taxus chinensis
a
Zhenjiang Zhao, Yufang Xu, Zhigang Qian, Wenhong Tian,
a
c
a
b,
Xuhong Qian and Jian-Jiang Zhong
c,
*
*
a
Shanghai Key Lab. of Chemical Biology, Institute of Pesticides and Pharmaceuticals, East China University of Science
and Technology, 130 Meilong Road, Shanghai 200237, China
State Key Laboratory of Fine Chemicals, Dalian University of Technology, 158 Zhongshan Road, Dalian 116012, China
b
c
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China
Received 25 February 2004; revised 20 May 2004; accepted 22 June 2004
Available online 23 July 2004
Abstract—To develop more effective abiotic elicitors for cell suspension cultures of T. chinensis to meet the needs for paclitaxel as
anti-tumor drug, some fluoro- or hydroxyl-containing groups are introduced to the ester moiety of jasmonic acid by the esterifica-
tion or acylation with bis(trichloromethyl) carbonate and corresponding alcohol. Some of them are found to be novel and effective
elicitors, which can enhance the production of taxuyunnanine C (Tc) up to 60% more than that by methyl jasmonate (MJA) in
T. chinensis cell cultures.
Ó 2004 Elsevier Ltd. All rights reserved.
In Taxus plant cell culture, methyl jasmonate (MJA) is
recognized as the most effective abiotic elicitor to obtain
the valuable anti-tumor drug paclitaxel (commercial
trade name: taxol) and a novel physiologically active
on C-1 and C-2, some of them had stronger bioactivities
9
than MJA. For example, a compound with octanoic
acid on C-1 rather than acetic acid of MJA mimicked
hydrogenated 12-oxophytodienoic acid (OPDA), a
10
lipid-derived signal molecule in higher plants.
1
–5
substance taxuyunnanine C (Tc). Tc was reported to
have a neuron growth factor (NGF)-like activity and
6
facilitate the treatment of AlzheimerÕs disease.
However, most of modifications on MJA are not suc-
cessful and impractical for application to biological sys-
tems. In comparison with MJA, Miersch et al.
investigated jasmonic acid (JA) and 66 structurally re-
lated compounds in gene expression of barley leaves,
and found that all structural modifications on C-1,
Structure–bioactivity relationship of MJA suggests that
structural moieties for high bioactivity are mainly acetyl
side chain at C-1, pentenyl chain at C-2 and keto at C-3.
Kiyota et al. synthesized two compounds 1,2- and 4,5-
didehydrojasmonates and found methyl 1,2-didehydro-
jasmonate inhibited the germination of lettuce seed more
strongly than that by MJA, and methyl 4,5-didehydro-
jasmonate was comparable to that by MJA in rice seed-
1
1
C-2, and C-3 moiety would decrease their bioactivities.
Koda et al. found that the introduction of locking
methyl group at C-2 position considerably lowered
1
2
tuber-inducing activities compared with MJA.
7
lings assays. Another jasmonate derivative was methyl
0
0
0
5
,5 ,5 -trifluorojasmonate synthesized also by Kiyota
et al., which induced sessile microtubers 20% more effec-
In cell suspension cultures of T. media, Yukimune et al.
investigated two elicitors derived from MJA, that is,
methyl cucurbate, which was obtained by reducing the
keto into hydroxyl at C-3 position of MJA, and cis-jas-
mone, which does not have the carboxyl group at C-1
position, and both of them had a weak elicitation activ-
8
tively than that by MJA. Blechert et al. synthesized a
series of MJA derivatives with modification of chains
Keywords: Taxus chinensis; Taxanes; Methyl jasmonate derivatives;
Plant cell culture; Abiotic elicitors.
1
ity or almost no activity. Dong et al. tried HMJA
*
Corresponding authors. Tel.: +86-21-64252091; fax: +86-21-
4253904 (J.-J.Z.); tel.: +86-4113673466; fax: +86-4113673488
X.Q.); e-mail addresses: xhqian@dlut.edu.cn; jjzhong@ecust.edu.cn
whose 2-pentenyl was substituted by 2-pentyl and
found that dihydro-methyl jasmonate (HMJA) had less
inducing effect on the taxane accumulation than that of
6
(
0
960-894X/$ - see front matter Ó 2004Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.06.082

4756
Z. Zhao et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4755–4758
1
3
1
structures of compounds were identified by H NMR
and HR-MS.
MJA. In addition, stereo-configuration of MJA also
affected production of paclitaxel and cell growth.
1
9
(
tion, while (1R,2R)-MJA had the highest activity in pro-
1R,2S)-MJA showed the strongest cell growth inhibi-
Suspension cultures of T. chinensis cells were performed
in a 250-mL Erlenmeyer flask containing 50-mL med-
1
4
moting paclitaxel biosynthesis. This means that the
configuration of C-1 and C-2 is important for its bioac-
1
3,16,17
ium as described elsewhere.
The elicitation activ-
7
,12
tivity.
Commercially available MJA is a mixture of
ity of MJA and synthesized elicitors on Tc was dose
dependent, and each elicitor at 100lM was found to
be optimal concentration for Tc production as a typical
example shown in Table 1. An elicitor was added to the
cell cultures in 1lL of ethanol per 1mL of culture med-
ium on day 7 after inoculation, and an equal volume of
ethanol was also added to control cultures. All experi-
ments were performed in triplicate, and the data are ex-
pressed as the mean of three independent samples with
standard deviations (i.e., mean± S.D.). Tc content was
measured by a reverse phase HPLC (Hewlett–Packard
series 1100 HPLC system, Agilent Palo Alto, CA). The
maximum Tc content and production obtained in 21
days are summarized in Table 2.
about 5% each of (1R,2S)- and (1S,2R)-cis isomers,
and 45% each of (1R,2R)- and (1S,2S)-trans isomers.
MJA in our experiments was bought from Tokyo Kasei
Kogyo Co., and it was also a mixture with its ratio of
cis:trans at 6:44 as determined by GC analysis.
Traditionally, the modifications on the ester group of
MJA do not affect bioactivity too much, and the
replacement of methyl by other groups usually lowered
1
1
their bioactivities. Until now, little work on the ester
modification has been reported. Our previous work indi-
cated that MJA could strongly stimulate taxanes biosyn-
thesis by enhancing activities of key enzymes such as
1
3
taxadiene synthase. Besides the special structure and
stereo configuration of MJA, the introduction of H-
bond on it should affect its interaction with these cata-
lyzing enzymes. We suppose that H-bond acceptor
fluorine, H-bond donor and acceptor OH may change
the above interaction and the lipophilicity/hydrophilici-
ty for permeation of plant cell membranes, which might
be beneficial to their bioactivity. Therefore, a series of
jasmonate derivatives with fluoro- or hydroxyl-contain-
ing groups were synthesized in this paper, and their elic-
iting activities on cell suspension cultures of T. chinensis
were studied.
MJA has four stereoisomers and the isomeric ratios of
novel jasmonate derivatives prepared in this paper were
the same as those of MJA, which was determined by
GC. Therefore, it is reasonable to compare the eliciting
activity of new jasmonate derivatives with that of
1
1,14
MJA.
In cell culture experiments, novel jasmonate derivatives
all displayed a certain degree (around 5–10%) of cell
growth inhibition at their optimal concentration of
100lM for Tc biosynthesis as a typical example shown
in Table 1. As the dynamic profiles shown in Figure 1,
Fluoro-containing compounds 1b, 1c, and 2c (Scheme 1)
were prepared through the esterification of jasmonate
chloride, which resulted from JA and bis-(trichloro-
methyl) carbonate, with fluoro-containing alcohol or
D-(+)-glucose in the presence of triethylamine. JA was
obtained from alkaline hydrolysis of MJA. The com-
pound 2a and the hydroxyl-containing compounds 2b,
1
5
Table 1. Effect of elicitor concentrations on the maximum cell
concentration (12days), Tc content and Tc production (21days) in
cell cultures of T. chinensis treated by newly synthesized MJA
derivatives on day 7 by taking the compound 2b as a typical example
Concentration Cell concentration Tc content
(g DW/L)
Tc production
2
e, and 2f were prepared through the esterification of
a
(mg/g DW) (mg/L)
of 2b (lM)
JA and corresponding alcohol. All compounds were sep-
arated and purified by gel silica chromatography. The
b
0
1
18.3± 0.2
9.4± 0.7
11.6± 1.3
27.0± 1.8
43.2± 2.1
49.8± 0.8
128± 5
160± 8
358± 19
544± 12
522± 7
18.5± 0.3
17.3± 0.2
16.6± 0.2
14.9± 0.6
1
1
0
00
O
O
500
1'
4'
a
2' 3'
mg/g DW: Tc amount (mg) per gram dry cells.
All data are expressed as an average± S.D.
3
4
5'
ROH
H SO
b
2
5
COOH
CO2R
1
2
4
2
a,2b,2e,2f
(
CCl3O)2CO
COCl
(R=OH-containing alkyl)
Table 2. Eliciting activities of novel MJA derivatives
O
O
No.
R (in Scheme 1)
Tc content
(
Tc production
(mg/L)
mg/g DW)
ROH
MJA
CH
CH
CH
CH
CH
3
2
2
2
2
29.2± 0.6
386± 10
440.3± 5.9
480.5± 0.6
357± 7
N(C H )
2
5 3
CO2R
1b
1c
2a
2b
2c
2e
2f
CF
CF
3
34.94± 0.1
38.22± 0.9
27.9± 1.6
47.2± 0.5
40.5± 0.0
38.1± 0.9
31.4± 1.5
2
CF
CH
CHOHCH
3
1
b,1c,2c
CH
2
3
(
R=F-containing alkyl and
2
OH
550± 13
492± 6
D-(+)-glucosyl for 2c)
C
6
H
11
O
5
2
2
(2-)
CH
CH
2
CH
CH
OH
464± 20
417± 2
Scheme 1. Preparation of MJA derivatives. The details of R groups
are shown in Table 2.
2
2 2
OCH CH OH

Z. Zhao et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4755–4758
4757
5
4
3
2
1
0
0
0
0
0
0
The introduction of hydroxyl groups gave us excellent
results. Novel elicitors 2b, 2c, and 2e had much improve-
ment in the Tc accumulation. Compared with MJA, the
compound 2b could even increase Tc content and pro-
duction up to 62% and 43% more, respectively. For
2a, 2f, 2e, 2b, and 2c, their number of hydroxyl groups
may contribute remarkably to their bioactivities. For
example, the obvious difference in structure between
2a and 2b is that the latter has two hydroxyl groups,
0
5
10
15
20
25
and the Tc eliciting activity for 2b was about 1.7-fold
that of 2a. However, although 2c has four hydroxyl
groups, its eliciting activity was lower than that of 2b.
The reason might be that the lipophilicity of 2c is too
low for it to transport cell membrane effectively
Cultivation time (d)
Figure 1. Time course of Tc content as elicited by fluorine-containing
jasmonate derivatives. The bar in the figure shows standard deviations
calculated from three independent samples. Symbols: ctrl-n; MJA-m;
(
1
A log P98 of 2b, 2c, 2e, and 2f is 1.17, 0.06, 1.68 and
.55, respectively as calculated by Cerius ). Thus, the
1b-j; 1c-h.
2
optimal lipophilicity and the number of hydroxyl groups
on jasmonate derivatives are two key factors for high
eliciting activity.
the Tc content induced on day 7 by the novel fluorine-
containing compounds was higher than that by MJA,
especially around the harvest time of cell cultivation
(
i.e., day 21). Also, all chemically synthesized hydroxyl-
This work demonstrates that the introduction of fluoro-
and hydroxyl-containing groups on jasmonate esters
is a successful strategy to promote Tc production by
T. chinensis cell cultures. Pentafluoropropyl jasmonate
(1c) and 2,3-dihydroxypropyl jasmonate (2b) are the
most effective jasmonate derivatives. It was found that
the number of fluorine, hydroxyl and optimal lipophilic-
ity are beneficial to their eliciting activities. The poten-
tial applications of these novel and effective elicitors in
Taxus plant cell cultures may be helpful for obtaining
a novel physiologically active substance Tc and the val-
uable anti-tumor drug paclitaxel.
containing compounds had strong Tc stimulation
activities than that by MJA (Fig. 2).
It was reported that a fluoro-containing compound
0
0
0
ꢀ7
methyl 5 ,5 ,5 -trifluorojasmonate at 10 M induced
sessile microtubers tuberization rate 20% more effec-
8
tively than that by MJA. Compared with MJA, tri-
fluoroethyl jasmonate 1b increased Tc content and
production by about 20% and 15% higher, and penta-
fluoropropyl jasmonate 1c increased Tc content and
production by about 30% and 25% higher, respectively.
The effect of fluorine introduction could be observed by
comparing 2a and 1c, and about 35% increase in Tc con-
tent and production was observed by introducing five
fluorine atoms (Fig. 1 and Table 2). With the calculation
of A log P98 by Cerious 4.8 (Accelrys Inc., San Diego,
CA, USA), fluorine introduction markedly increased
lipophilicity as represented by A log P98 (the A log P98
values of MJA, 1b and 1c are estimated to be 2.22,
Acknowledgements
2
This study was under the auspices of National Natural
Science Foundation of China (NSFC project nos.
1
8
20376023, 20225619, and 20236040), and the National
Key Project for Basic Research, the Ministry of Science
and Technology of China (2003CB114400).
3
.22, and 3.72, respectively). Interestingly, the bioactivi-
ties elicited by MJA, 1b and 1c seem to be quantitatively
correlated with the lipophilicity properties of these com-
pounds, because the bioactivity was enhanced (Fig. 1
and Table 2) with an increase of the fluoro-groups intro-
duced into MJA (an increase in their lipophilicity).
References and notes
1
2
3
4
5
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1
793–1796.
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0
6
7
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. Cheng, K. D.; Chen, W. M.; Zhu, W. H.; Fang, Q. C.;
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Gundlach, H., et al. J. Chem. Soc., Perkin Trans. 1 1997,
3549–3559.
0
0
5
10
15
20
25
Cultivation time (d)
Figure 2. Time profiles of Tc content as elicited by hydroxyl-
containing jasmonate derivatives. The bar in the figure shows standard
deviations calculated from three independent samples. Symbols: ctrl-
n; MJA-m; 2b-j; 2c-h; 2e-d; 2f-s.

4758
Z. Zhao et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4755–4758
0
0 0
=10.80Hz, 3 -H), 5.38 (1H, dt,
3 ,2
=10.81Hz, 2 -H). 2b: colorless oil. H
1
1
1
1
1
0. Sch u¨ ler, G.; Mith o¨ fer, A.; Baldwin, I. T.; Berger, S.; Ebel,
J.; Santos, J. G., et al. FEBS Lett. 2004, 563, 17–22.
1. Miersch, O.; Kramell, R.; Parthier, B.; Wasternack, C.
Phytochemistry 1999, 50, 353–361.
dt, J
=7.28Hz, J
NMR (500MHz, DMSO-d6) d=0.90 (3H, t,
3
0
,4
0
=7.50Hz, J
0
1
J
2
0
,1
0
0 0
2 ,3
0
5 ,4
J =7.55Hz, 5 -H), 1.39–1.49 (1H, m), 1.93–2.09 (5H,
0 0
m), 2.16–2.25 (4H, m), 2.30–2.35 (1H, m), 2.63–2.67 (1H,
m, 2-H), 3.30–3.38 (2H, m), 3.61–3.64(1H, m), 3.90–3.94
(1H, m), 4.04–4.07 (1H, m), 4.63 (1H, s, OH), 4.86 (1H, s,
2. Koda, Y.; Ward, J. L.; Beale, M. H. Phytochemistry 1995,
3
8, 821–823.
3. Dong, H. D.; Zhong, J. J. Biochem. Eng. J. 2000, 8,
45–150.
0
0 0
=10.64Hz, 3 -H),
3 ,2
=10.63Hz, 2 -H). HR-
1
OH), 5.22 (1H, dt, J
5.38 (1H, dt, J =7.28Hz, J
MS (EI, m/e), Found M =284.1618; C H O requires
3
0
,4
0
=7.56Hz, J
0
4. Yukimune, Y.; Hara, Y.; Nomura, E.; Seto, H.; Yoshida,
S. Phytochemistry 2000, 54, 13–17.
5. Gottos M. W. WO 9317019A1, 1993.
2
0
,1
0
0 0
2 ,3
+
1
5
24
5
1
1
1
284.1624. 2c: colorless oil. H NMR (500MHz, DMSO-
0
6. Pan, Z. W.; Wang, H. Q.; Zhong, J. J. Enzyme. Microb.
Technol. 2000, 27, 714–723.
7. Wang, H. Q.; Zhong, J. J.; Yu, J. T. Biotechnol. Lett. 1997,
d ) d=0.89 (3H, t, J =7.50Hz, 5 -H), 1.42–1.52 (1H,
6 5 ,4
0 0
m), 1.97–2.10 (5H, m), 2.20–2.26 (4H, m), 2.43–2.46 (1H,
m), 2.78–2.81 (1H, m, 2-H), 3.09–3.44 (4H, m), 3.57–3.76
(2H, m), 4.45–4.54 (2H, m), 4.80–4.88 (3H, m, OH), 5.16
1
19, 353–355.
0
1
1
8. Smart, B. E. J. Fluorine Chem. 2001, 109, 3–11.
(1H, dt, J
0
3
0
,4
0
=7.68Hz, J
0
3
0
,2
0
=10.56Hz, 3 -H), 5.38 (1H,
0
1
9. Compound 1b: colorless oil. H NMR (500MHz, DMSO-
dt, J =7.34Hz, J
0
0
=10.54Hz, 2 -H). MS (EI, m/e),
2
,1
2
,3
0
+
d6) d=0.89 (3H, t, J
0
0
=7.50Hz, 5 -H), 1.42–1.52 (1H,
m), 1.97–2.10 (5H, m), 2.20–2.26 (4H, m), 2.43–2.46 (1H,
Found M =372; C H O requires 372.42. 2e: colorless
18 28 8
5 ,4
1
oil. H NMR (500MHz, DMSO-d
6
) d=0.89 (3H, t,
0 0
J =7.50Hz, 5 -H), 1.39–1.48 (1H, m), 1.95–2.07 (5H,
5 ,4
0
m), 2.78–2.81 (1H, m, 2-H), 4.75 (2H, q, J=9.11Hz,
0
OCH CF ), 5.16 (1H, dt, J
2
0
=7.68Hz, J =10.56Hz,
0 0
m), 2.20–2.25 (4H, m), 2.30–2.35 (1H, m), 2.63–2.67 (1H,
m, 2-H), 3.55 (2H, t, J=5.06Hz, OCH ), 4.03 (2H, t,
3
3 ,4
3 ,2
0
0
3
-H), 5.38 (1H, dt, J2
HRMS (EI, m/e), Found M =292.1273; C14
0
0
=7.34Hz, J
0
0
=10.55Hz, 2 -H).
,1
2 ,3
2
+
H
19
O
3
F
3
J=5.06Hz, OCH
2
), 5.22 (1H, dt,
=10.69Hz, 3 -H), 5.38 (1H, dt,
0
J
J
3
0
,4
,1
0
=7.60Hz,
=7.30Hz,
1
requires 292.1286. 1c: colorless oil. H NMR (500MHz,
0
J
J
3
0
,2
0
2
0
0
0
+
DMSO-d ) d=0.89 (3H, t, J
0
0
=7.47Hz, 5 -H), 1.42–1.49
1H, m), 1.96–2.09 (5H, m), 2.17–2.27 (4H, m), 2.43–2.46
0
0
=10.68Hz, 2 -H). HR-MS (EI, m/e), Found M =
6
5 ,4
2 ,3
(
254.1517; C14
H
22
O
4
requires 254.1518. 2f: colorless oil.
NMR (500MHz, DMSO-d d=0.90 (3H, t,
=7.53Hz, 5 -H), 1.42–1.46 (1H, m), 1.92–2.08 (5H,
m), 2.15–2.25 (4H, m), 2.31–2.36 (1H, m), 2.64–2.68 (1H,
m, 2-H), 3.41 (2H, t, J=5.00Hz, OCH ), 3.47 (2H, t,
J=4.69Hz, OCH ), 3.59 (2H, t, J=4.99Hz, OCH ), 4.14
1
(
OCH
1H, m), 2.78–2.82 (1H, m, 2-H), 4.83 (2H, t, J=13.89Hz,
H
6
)
0
CF
=10.67Hz, 3 -H), 5.38 (1H, dt,
2
CF
3
),
5.20
(1H,
dt,
J
J
3
0
0
,4
0
0
=7.56Hz,
=7.31Hz,
J
5 ,4
0 0
2
0
J3
0
0
,2
2 ,1
0
J
2
0
,3
0
=10.65Hz, 2 -H). HRMS (EI, m/e), Found
requires 342.1254. 2a: color-
2
+
M =342.1218; C15
H
19
O
3
F
3
2
2
1
less oil. H NMR (500MHz, DMSO-d ) d=0.86–0.91 (6H,
(2H, t, J=4.70Hz, OCH ), 5.21 (1H, dt, J
2
0
0
=7.51Hz,
=7.28Hz,
6
3 ,4
0
m, 2CH ), 1.41–1.48 (1H, m), 1.53–1.61 (2H, m), 1.91–2.08
J
J
0
0
=10.77Hz, 3 -H), 5.37 (1H, dt,
J
0
0
3
3 ,2
,3
2 ,1
0
+
=10.78Hz, 2 -H). HR-MS (EI, m/e), Found M =
(
(
5H, m), 2.16–2.25 (4H, m), 2.29–2.34 (1H, m), 2.62–2.66
1H, m, 2-H), 3.98 (2H, t, J=6.58Hz, OCH ), 5.21 (1H,
2
0
0
2
26 5
298.1794; C16H O requires 298.1790.