Rational design and efficient synthesis of a fluorescent-labeled jasmonate

Article abstract of DOI:10.1016/j.tetlet.2012.06.006

A fluorescent-labeled jasmonate was rationally designed based on examination of the model of interaction between the jasmonate and its receptor. An efficient synthetic route has been developed for this molecule. The biological activity of this fluorescent probe was retained which was similar to that of the methyl jasmonate as examined by root growth inhibition bioassay. This fluorescent probe will greatly facilitate biological studies of jasmonates through fluorescent imaging.

Full text of DOI:10.1016/j.tetlet.2012.06.006

Tetrahedron Letters 53 (2012) 4235–4239
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Rational design and efficient synthesis of a fluorescent-labeled jasmonate
Songbai Liu , Wu-Hong Wang b, , Ya-Li Dang , Yuanqing Fu , Ruocheng Sang
a,
⇑
c
a
a
a
Department of Food Science and Nutrition, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058, China
Institute of Vegetable, Zhejiang Academy of Agricultural Sciences, 198 Shiqiao Road, Hangzhou 310021, China
Institute of Health Food, Zhejiang Academy of Medical Science, 182 Tianmushan Road, Hangzhou 310013, China
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
A fluorescent-labeled jasmonate was rationally designed based on examination of the model of interac-
tion between the jasmonate and its receptor. An efficient synthetic route has been developed for this mol-
ecule. The biological activity of this fluorescent probe was retained which was similar to that of the
methyl jasmonate as examined by root growth inhibition bioassay. This fluorescent probe will greatly
facilitate biological studies of jasmonates through fluorescent imaging.
Received 18 April 2012
Revised 25 May 2012
Accepted 1 June 2012
Available online 12 June 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Jasmonate
Fluorescent probe
Synthesis
Design
Biological activity
Jasmonic acid (JA) and its derivatives, collectively referred to as
jasmonates, occur widespread in plants and some lower eukary-
otes. Jasmonates originate from oxidation of linolenic acid and
Herein we described a rational design and efficient synthesis of a
fluorescent-labeled jasmonate.
1
To design a fluorescent-labeled biomolecule a few requirements
should be fulfilled. First the fluorescent group does not interfere
with the biological activity. Second the fluorescent group can be
share notable structural and functional similarities with prosta-
2
glandins in animals. They are of general biological importance,
8
not only regulating plant growth and development but also medi-
ating environmental stress responses of plants through reprogram-
ing of gene expression.3 Recently several studies indicated that
jasmonates were promising in cancer treatment which induced
apoptosis in various cancer cell lines including breast, prostate,
melanoma, and leukemia.4 Significant anti-inflammatory activity
was also found for jasmonate analogues that exhibited enhanced
easily introduced. Coumarin 343 was carefully chosen as fluores-
cent carrier due to its biocompatibility, high quantum yield, ease of
handling, and stability. Next we needed to decide where and how
to install the fluorescent group to maintain integrity of the biolog-
ical activity. According to known studies, the aliphatic side chain of
JA is important to keep its biological activities and derivatization of
the carboxyl group does not severely interfere with the activities.7b
5
activity than natural anti-inflammatory prostaglandins. These re-
The endogenous amino acid conjugate of JA, jasmonoyl-L-isoleu-
9
sults have provoked much research interest in jasmonates as a
class of versatile bioactive molecules either in plant or animal
kingdom.
cine (JA-Ile) represents an active jasmonate derivative. Scrutiniz-
ing the model of interaction between JA-Ile and its receptor
7
b
COl1 revealed the carboxyl group of isoleucine moiety is far away
from the active site. Therefore modification of the carboxyl group
of JA-Ile has more chance to realize an active form of fluorescent
probe of JA. To best mimic JA-Ile and keep a reasonable space for
To elucidate biological mechanisms of jasmonates on molecular
basis a way to visualize the molecules is highly desirable. Fluores-
cent probes have been established as the most powerful way to
monitor the events in proteomics, functional genomics, and cell
the fluorescent group from the jasmonate moiety, L-lysine was se-
6
biology studies. Although a few labeled jasmonates have been de-
lected as the link chain with an additional advantage of synthetic
7
10
signed, a fluorescent-labeled jasmonate is still not available. One
simplicity. Taken all of the factors together, a possibly bioactive
fluorescent probe at hand will undoubtedly facilitate the biological
studies. Inspired by this notion, we initiated the development of
fluorescent-labeled jasmonates retaining biological activities.
fluorescent probe of jasmonates was designed (Fig. 1).
The synthetic study was commenced with the synthesis of the
fluorescent coumarin 343 (6) (Scheme 1). Although the preparation
1
1
of coumarin 343 is known in the literature, it is far from straight-
forward always requiring multiple protection and deprotection
operations. To eliminate the employment of protecting groups,
we started from commercially available 3-aminophenol. Following
⇑
Corresponding author. Tel./fax: +86 571 88982186.
E-mail address: songbailiu@zju.edu.cn (S. Liu).
The author contributed equally as first author to this work.
0
040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2012.06.006

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S. Liu et al. / Tetrahedron Letters 53 (2012) 4235–4239
Figure 1. Designed bioactive fluorescent-labeled jasmonate.
Scheme 1. Synthesis of coumarin 343 (6).
Scheme 2. Synthesis of JA-Ile.
Scheme 3. Attempt to synthesis of the fluorescent-labeled jasmonate.

S. Liu et al. / Tetrahedron Letters 53 (2012) 4235–4239
4237
Scheme 4. Synthesis of the fluorescent-labeled jasmonate.
the published procedure12 8-hydroxyjulolidine derived from
alkylation with 1-bromo-3-chloropropane was obtained in very
low yield (<5%). Optimization of reaction conditions by intensive
investigations including solvents, temperatures, bases, and stoichi-
ometry gave an acceptable yield (35%) for further synthesis. The
major side reaction, double substitution of 1-bromo-3-chloropro-
pane by 3-aminophenol was greatly diminished by applying exces-
sive 1-bromo-3-chloropropane. Treatment of 8-hydroxyjulolidine
The synthesis of the jasmonate fragment was commenced with
mild hydrolysis of commercially available methyl jasmonate 7 (a
racemic mixture of stereoisomers) by LiOH in a mixture of THF
and water (1:1) (Scheme 2). After the acid was activated by
N-hydroxysuccinimide with treatment of DCC, the coupling with
3
L-isoleucine in aqueous NaHCO and THF went smoothly affording
JA-Ile in good yield (54%, three steps).
With the two fragments in hand it is the time to connect them
together. Initially Fmoc-Lys-OHÁHCl was coupled with the couma-
rin succinimidyl ester 12 prepared from Steglich esterification13 by
DCC (Scheme 3). The carboxyl group of the lysine conjugated
coumarin was protected with 2-(trimethylsilyl) ethanol through
Steglich esterification. Then Fmoc group was deprotected by the
action of morpholine in DMF. The coupling of the resultant lysine
conjugated coumarin employing 2-chloro-4,6-dimethoxy-1,3,5-tri-
3
with POCl in DMF provided the aldehyde product in high yield
(
84%). The strongly fluorescent coumarin methyl ester was readily
generated by the action of piperidine and dimethyl malonate in a
refluxing mixture of toluene and acetonitrile (2:1) for 6 h. Hydroly-
sis of the methyl ester in concentrated HCl for 24 h afforded the
acid in good yield (61%, two steps). Through this newly developed
route the fluorescent coumarin can be easily prepared on gram
scale.
1
4
azine (CDMT) and N-methyl morpholine (NMM) with JA-Ile 11
Figure 2. Fluorescent properties of the fluorescent probe 1. (a) Fluorescent spectra of 1. (b) Fluorescent image of coumarin 343 and 1.

4
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S. Liu et al. / Tetrahedron Letters 53 (2012) 4235–4239
Figure 3. Biological activity of the fluorescent-labeled jasmonate tested by root growth inhibition of cabbage seedlings. Seedlings of Chinese cabbage hybrid ‘Zhebai 6’ were
grown on MS medium without additive (MS) or with coumarin 343, 1, MeJA at 10
Error bars indicating SE (n >30).
lM for 9 days. (a) Picture of the seedlings. (b) Primary root lengths of 9-day-old seedlings.
underwent smoothly. Unfortunately the last step of deprotection of
-(trimethylsilyl) ethanol was unsuccessful under various condi-
tions with TBAF.
Hence an alternative strategy for protection of the carboxyl
group via copper complexation15 was used (Scheme 4). The cou-
marin succinimidyl ester 12 was coupled with the copper pro-
tected lysine produced from the complexation of lysine with
(negative control) did not inhibit root growth. It confirmed that
the fluorescent-labeled jasmonate 1 is a bioactive jasmonate.
In conclusion, a bioactive fluorescent-labeled jasmonate was
rationally designed and efficiently synthesized. The fluorescent
probe will greatly facilitate biological studies of jasmonates
through fluorescent imaging. Studies on this fluorescent probe in
plants by fluorescent imaging are underway.
2
4 3
CuSO in aqueous NaHCO and THF. After decomplexation with
EDTA, the resultant lysine conjugated coumarin 17 was coupled
with succinimidyl ester of JA-Ile successfully to provide the target
molecule 1 in good yield (49%, three steps).
Upon completion of synthesis of the fluorescent-labeled jasmo-
nate, fluorescent properties of the molecule were studied. The fluo-
Acknowledgment
This work was supported by ‘The Fundamental Research Funds
for the Central Universities’.
rescent excitation and emission spectra of aqueous NaHCO
solution of the compound 1 exhibited its absorption maxima at
56 nm and emission maxima at 494 nm (Fig. 2a). The significant
Stokes shift (38 nm) would render this molecule very useful as a
fluorescent probe. The fluorescent image of the compound in aque-
3
Supplementary data
4
Supplementary data (experimental details and spectroscopic
data) associated with this article can be found, in the online ver-
sion, at http://dx.doi.org/10.1016/j.tetlet.2012.06.006.
3
ous NaHCO solution was collected (Fig. 2b). As compared with
coumarin 343, molecule 1 showed strong comparable fluorescent
signal.
References and notes
Finally the biological activity of the fluorescent probe was
examined in cabbage using root growth inhibition bioassay which
is a well established model based on the fact that jasmonates sup-
plemented to the growth medium cause a significant inhibition of
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