Fluorescence studies on nyctinasty which suggest the existence of genus-specific receptors for leaf-movement factor.

Article abstract of DOI:10.1039/b308764f

Periodic leaf-movement of legumes is called nyctinasty and has been known since the age of Alexander the Great. We found that nyctinasty is controlled by a periodic change of the internal concentration of leaf-opening and leaf-closing substances in the plant body. Now, we have developed novel fluorescent probes (1) based on the structure of cis-p-coumaroylagmatine (3), which was isolated as a leaf-opening substance of Albizzia juribrissin Durazz. Binding experiments using probe 1 showed that Albizza plants have receptors for a leaf-opening substance in their motor cells. By using probes 1 we then found that genus-specific receptors are involved in nyctinasty.

Full text of DOI:10.1039/b308764f

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Fluorescence studies on nyctinasty which suggest the existence
of genus-specific receptors for leaf-movement factor
Hideharu Nagano, Eisuke Kato, Shosuke Yamamura and Minoru Ueda*
Laboratory of Natural Products Chemistry, Department of Chemistry,
Faculty of Science and Technology, Keio University, Hiyoshi, Yokohama 223-8522, Japan.
E-mail: ueda@chem.keio.ac.jp; Fax: ϩ81-45-566-1697; Tel: ϩ81-45-566-1702
Received 28th July 2003, Accepted 5th August 2003
First published as an Advance Article on the web 18th August 2003
Periodic leaf-movement of legumes is called nyctinasty and has been known since the age of Alexander the Great. We
found that nyctinasty is controlled by a periodic change of the internal concentration of leaf-opening and leaf-closing
substances in the plant body. Now, we have developed novel fluorescent probes (1) based on the structure of
cis-p-coumaroylagmatine (3), which was isolated as a leaf-opening substance of Albizzia juribrissin Durazz. Binding
experiments using probe 1 showed that Albizza plants have receptors for a leaf-opening substance in their motor cells.
By using probes 1 we then found that genus-specific receptors are involved in nyctinasty.
receptors for this leaf-movement factor are located on motor
Introduction
cells.3–5
Plants are unable to move from one place to another. However,
the folding and opening movement of the leaves according to
the circadian rhythm have been widely observed in leguminous
cis-p-Coumaroylagmatine (3) was isolated as a leaf-opening
substance of A. juribrissin.6 We revealed that 3 operates as a
leaf-opening substance among the Albizzia genus based on
plants (Fig. 1). This periodic leaf-movement is called nyctinasty
analyses of other Albizzia plants such as A. lebbeck and A.
and has been known since the age of Alexander the Great.1 It is
saman.7 In contrast, 3 was not effective for plants belonging
widely known that Charles Darwin carried out the pioneering
to any other genus, such as Cassia mimosoides, Phyllnathus
work in this field, and Since Darwin, many scientists have
urinaria, Mimosa pudica. These data suggest that a common
worked on research in this field. It was revealed that nyctinastic
receptor for 3 would operate in the Albizzia genus, whereas
leaf movement is induced by the swelling and shrinking of
receptors operating in plants belonging to any other genus
motor cells in the pulvini, an organ located in the joint of the
would not bind to 3. Bioactivity of the leaf-movement factor is
leaf.1 Motor cells play a key role in plant leaf-movement. A flux
known to be highly genus-specific.2 Thus, it will be important
of potassium ions across the plasma membrane of the motor
to clarify whether the genus-specific bioactivity of the leaf-
cells is followed by a massive water flux, which results in swell-
movement factor could be due to the difference in the specificity
ing and shrinking of these cells. Extensive studies on nyctinastic
of the receptor on the motor cell. We carried out fluorescence
studies using a fluorescent probe designed on 3 to address this
issue.
In this paper, we report the development of a novel fluor-
escent probe (1) based on the structure of cis-p-coumaroyl-
agmatine (3), a leaf-opening substance of Albizzia julibrissin
plants led to the isolation of a variety of leaf-closing and
leaf-opening substances.2 We found that the biological clock
regulates the balance of concentration between leaf-opening
and -closing substances in the plant body during the day.2
Recently, we studied the mechanisms by which leaf-movement
factors induce leaf movement and developed molecular probes
Durazz. We found that genus-specific receptors are involved in
consisting of modified leaf-movement factors in order to
nyctinasty by using probe 1.
identify the target cells of these factors. For example, we
synthesized fluorescence-labeled potassium lespedezate (2), a
leaf-opening substance of Cassia plants, and showed that some
Results and discussion
The molecular design of a fluorescent probe required structure–
activity relationship studies of 3. Thus, we synthesized some
analogs of 3, such as one with a trans-double bond (4),8 one
with a reduced double bond (5), one with a protected hydroxy
group (6), and one which contains -arginine instead of
agmatine (7) (Schemes 1 and 2). Commercially available trans-
p-coumaric acid was reduced by hydrogen gas using Pd–C and
resulting 13 was coupled with agmatine to give 5 (Scheme 1).
An analog 6 was synthesized from 16 which was prepared from
trans-p-coumaric acid with methyl iodide (Scheme 2). Analog 7
was synthesized from N-hydroxysuccinimide ester of cis-p-
coumaric acid which was prepared according to the method in
reference 8.
Analogs with structure modification in the cis-p-coumaroyl
moiety, such as 4, 5, and 6, showed no leaf-opening activity
against the leaves of A. juribrissin, whereas 7 was as effective as
3 that is effective at 5 × 10Ϫ5 M. These data suggested that the
cis-p-coumaroyl moiety in 3 is indispensable for its bioactivity,
and the agmatine moiety in 3 can be structurally modified
with almost no decrease in bioactivity. Thus, in the molecular
Fig. 1 Nyctinasty of Albizzia julibrissin Durazz. (left: leaf in the
daytime, right: leaf at night).
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 1 8 6 – 3 1 9 2
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 3
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Scheme 1 Synthetic route of analog of cis-p-coumaroylagmatine (5).
at 5 × 10Ϫ5 M. Both of the probes were of moderate bioactivity
and can be used in binding experiments using plant sections
containing motor cells.
We used probe 1 for a fluorescence study of the interaction
between the leaf-opening substance and the plant motor cell. A
leaf of A. julibrissins was cut by a microslicer. Then the section
containing a motor cell was incubated for 4.5 hours at 25 ЊC in a
0.1 M citrate–phosphate buffer (pH 7.0) containing 1 × 10Ϫ4 M
of 1. After staining, the stained section was incubated for
10 minutes with washing buffer to remove excess fluorescent
probes. Then, the stained section was monitored by using a
fluorescence microscope. The use of an antifadant reagent was
essential to prevent photobleaching (fading of fluorescence).
Fig. 2 shows photographs of plant pulvini (Fig. 2, left), which
contains motor cells. The staining pattern for the fluorescence
of 1 was observed on both the plasma membrane and nuclei
(Fig. 2, center and right) of the motor cell. No staining was
observed in the cells contained in other parts of the plant
section treated with 1, and the control section which was treated
with a solution containing no 1. Clear staining could be
observed up to a concentration of 5 × 10Ϫ5 M of 1. Also, bind-
ing of probe 1 was inhibited by the coexistence of a 1000-fold
concentration of non-labeled 3. When the section was treated
design of a fluorescent probe, a large fluorescence dye should be
introduced in the agmatine moiety in 3.
We used -arginine, instead of agmatine, to introduce a
fluorescence dye in the agmatine moiety. Use of the -form
of arginine circumvented the hydrolysis of the amide bond by
peptidase in the plant body. Also, a fluorescence dye was
connected to the carboxylate group via an amide bond to
circumvent the hydrolysis by esterase in the plant body. The
fluorescent probe was synthesized according to Scheme 3.
-Arginine was coupled with a diamine-ether type linker by
using BOP (benzotriazole-1-yl-oxy-tris(dimethylamino)phos-
phonium hexafluorophosphate) reagent. After deprotection
it was coupled with cis-p-coumaric acid by using DMTMM
(dimethoxythiazolyl methylmorphorine),9 and then with
AMCA–NHS [AMCA
: 6-((7-amino-4-methylcoumarin-3-
acetyl) amino)hexanoyl] or FITC [fluorescein-5-isothiocyanate]
to give FITC-labeled probe (1) or AMCA labeled probe (24).
The bioassays using these two probes were carried out with
the leaves of A. julibrissin. AMCA-labeled probe (24) was
effective at 1 × 10Ϫ5 M, which was half as strong as that of the
native factor (3), and FITC-labeled probe (1) was also effective
Fig. 2 Binding experiment of fluorescent probe 1 with a plant section
of A. juribrissin containing a motor cell (left: Nomarskii image of the
plant section containing motor cell, right: fluorescence image of plant
section after treatment with 1 × 10Ϫ4 M of probe 1; [Excitation: 450–490
nm]. Right: expansion of the fluorescence image of motor cell).
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Scheme 2 Synthetic route of analogs of cis-p-coumaroylagmatine (6 and 7).
with 5 × 10Ϫ5 M of 1 together with 5 × 10Ϫ2 M of 3, no staining
genuses contained some analogs of cis-p-coumaric acid in the
was observed in the plant section on both membrane and
nuclei. These results suggest that the specific binding site for
1 (or 3) should exist in the plasma membrane and nuclei of the
motor cell.
molecule.2 The structural variation of the leaf-opening sub-
stances is due to the variety of the other parts. Thus, it would
be estimated that 25 would be a genuine bioactive substance for
leaf-opening, and an agmatine moiety would be important for
transport in the plant body and recognition by a membrane
receptor. After recognition by a receptor molecule on the
membrane, 1 would be transferred into the cytosol and trans-
ported to the nuclei to develop the leaf-opening activity.
From the result of the fluorescence studies mentioned above,
the specific recognition of 1 by the membrane receptor could be
due to the whole structure of the molecule containing a cis-p-
coumaroyl moiety and agmatine part. A. juribrissin contains
two types of receptor: one is the membrane receptor that
recognizes the whole structure of the molecule, and the other is
the nucleus receptor that binds to the cis-p-coumaric acid. This
result suggests that the leaf-opening substance 3 is perceived by
the membrane receptor, and then the cis-p-coumaric acid is
released, transported into the cytosol, and bound to a nucleus
receptor to induce leaf-opening.
Interestingly, we found that the binding of 1 with nuclei was
inhibited in the presence of cis-p-coumaric acid (25). We carried
out the binding examination of probe 1 at 1 × 10Ϫ4 M in the
presence of 1000-fold molar excess of 25, that was not effective
for the leaf-opening of A. juribrissin even at 1 × 10Ϫ4 M. Binding
of 1 to the nuclei was strongly inhibited by the coexistence of
25, whereas binding to the plasma membrane was not (Fig. 3).
Together with the result using 3, the competitive inhibition of
the binding of 1 to the plasma membrane was strongly corre-
lated to the leaf-opening activity of the coexisting molecule.
These results strongly suggest that the binding of 1 to the nuclei
is attributed to cis-p-coumaric acid which has no leaf-opening
activity on the bioassay using the whole leaf. Thus, the genuine
receptor of leaf-opening substance 3 would be located in the
plasma membrane of the motor cell.
On the other hand, this result showed that some receptors for
25 could be located in the nuclei of the motor cell as well as leaf-
opening substances that have so far been isolated from several
This model will provide a molecular basis for discussion of
the following issue: Why does each nyctinastic plant have a
different leaf-movement factor whose bioactivity is specific to
the plant genus? The genus specificity on the recognition of the
leaf-movement factor can be attributed to the ligand specificity
of the membrane receptor, and the nucleus receptor which
binds to cis-p-coumaric acid would be common among legum-
inous plants because almost all leaf-opening substances contain
the cis-p-coumaroyl moiety in the molecule.
From our previous studies, it was revealed that each nyctin-
astic plant has a different leaf-movement factor whose bio-
activity is specific to the plant genus.2,7 We revealed that 3 is
commonly contained in three Albizzia plants, such as A.
julibrissin, A. lebbeck, and A. saman.7 Compound 3 was effect-
ive for these Albizzia plants, whereas it was not effective for the
plants belonging to other plant genera. This result strongly
suggested the existence of some specific receptor for 3 that
is common among genus Albizzia. First, we examined the
Fig. 3 Expansion of the fluorescence image of an Albizzia section
treated with 1 (left), and of the section treated with 1 in the presence of
1000 molar exess of cis-p-coumaric acid (25) (right).
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 1 8 6 – 3 1 9 2
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Scheme 3 Synthetic route of fluorescence labeled cis-p-coumaroylagmatines (1 and 24).
specificity of the bioactivity on the probe compound 1. Probe
specific receptor would be involved in the binding between the
compound 1 did not show leaf-opening activity against the
leaves of Cassia mimosoides L., and Aeschynomene indica L. at 1
× 10Ϫ4 M, whereas it was effective at the same concentration for
the leaf-opening of Albizzia saman and Albizzia lebbeck, which
belong to the same Albizzia genus as A. julibrissin. From these
results, the binding of 1 is expected to be specific to the section
of plants belonging to genus Albizzia, and no binding would be
observed in the experiment using the section of other plants.
Then, we used probe 1 for the binding experiment with the
sections of C. mimosoides, Aeschynomene indica together with
that of A. saman and A. lebbeck. The binding experiments were
carried out according to the same method used in the case of A.
juribrissin. Thus, it was revealed that the sections of A. saman
and A. lebbeck gave a fluorescence image resulting from 1
and no other sections gave the image (Fig. 4). Red stains seen in
the fluorescence images are due to the porphyrin in the plant
tissue. These results showed that the binding of a probe 1 with a
motor cell is specific to genus Albizzia and suggested that
a genus-specific receptor molecule for the genus-specific leaf-
movement factor on a motor cell would be involved in
nyctinasty.
leaf-movement factor and a motor cell. It was estimated that
each plant genus would have different receptor molecules on
the motor cells which are specific to each genus-specific leaf-
movement factor. Genus specific recognition of the ligand by
a receptor of a leaf-movement factor strongly suggests that
membrane receptors concerning nyctinasty would be differ-
entiated in the comparatively latter process of evolution in the
plant kingdom.
Experimental
General
NMR spectra were recorded on a Jeol JNM-A400 spectrometer
[1H (400 MHz) and 13C (100 MHz)] or a Jeol JNM-EX 270
spectrometer [1H (270 MHz) and 13C (67.5 MHz)] using TMS
in CDCl3, CD2HOD in CD3OH (1H; 3.33 ppm, 13C; 49.8 ppm),
or t-BuOH (1H; 1.23 ppm, 13C; 32.1 ppm) in D2O as internal
standards at various temperatures. The FAB-MS and HR
FAB-MS spectra were recorded on a Jeol JMS-700 spectro-
meter, using glycerol or m-nitrobenzylalcohol as a matrix. The
IR spectra were recorded on a JASCO FT/IR-410. The specific
rotations were measured by JASCO DIP-360 polarimeter and
are given in units of 10Ϫ1 deg cm2 gϪ1. The HPLC purification
was carried out with a Shimadzu LC-6A pump equipped with
From these results, we have shown that Albizza plants have
receptors for leaf-opening substances in their motor cells. The
specificity of the receptors for the ligands is common among
the plant genus and this result strongly suggests that the genus
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 1 8 6 – 3 1 9 2
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Fig. 4 Photographs of plant sections in the binding experiments which show specific binding of probe 1 with the motor cells of Albizzia plants
(upper: Nomarskii image of the plant section, lower: fluorescent image of the plant section after treatment with 1 × 10Ϫ4 M of probe 1 [excitation:
450–490 nm]).
a SPD-6A detector using a COSMOCIL 5C18-AR column
(ꢀ 20 × 250 mm) (Nakalai Tesque Co. Ltd.). The solvents used
for HPLC were available from Kanto Chemical Co. and were
filtered through a Toyo Roshi membrane filter (cellulose acetate
of 0.45 µm pore size, 47 mm diameter) before use. Silica gel
column chromatography was performed on silica gel 60 K070
(Katayama Chemical Co. Ltd.) or silica gel 60N (Kanto Chem-
ical Co. Ltd.). Reversed-phase open-column chromatography
was performed on Cosmosil 75C18-OPN (Nakalai Tesque Co.
Ltd.). TLC was performed on Silica gel F254 (0.25 mm or
0.5 mm, MERCK) or RP-18F254S (0.25 mm, MERCK). Photo-
chemical reactions were performed with a high-pressure
Hg-lump system (UM-452 equipped with UM-453B-A, Ushio
Electronics Co. Ltd.)
3-(4-Hydroxyphenyl) propionyl agmatine (5)
To a solution of 13 (30.6 mg, 0.184 mmol) in THF (0.5 mL),
HO–NSu (36.2 mg, 0.315 mmol) and then DCC (58.2 mg, 0.282
mmol) was added at 0 ЊC. After stirring for 2 hours under an
argon atmosphere, the solution was filtered through Celite, and
was diluted with ethyl acetate. The filtrate was washed with
saturated NaHCO3 aq., dried over abs. Na2SO4, and evaporated
to dryness. The residue was purified by silica gel column
chromatography and TLC (CHCl3 : MeOH = 9 : 1) to give
activated ester 14 (39.2 mg, 81%). Compound 14 (32.1 mg) was
dissolved in acetone (1 mL) at 0 ЊC and was then mixed with
agamatine sulfate (26.3 mg, 0.115 mmol) in 1 mL of H2O, which
was adjusted to pH 8 by saturated NaHCO3aq. After stirring
for 19 hours, the reaction mixture was adjusted to pH 3 by
acetic acid and was washed with ethyl acetate. The aqueous
layer was evaporated to dryness, and the residue was dissolved
in 30% MeOH aq. The residue was purified by HPLC Cosmosil
5C18AR (30% MeOH aq.) to give 5 (29.2 mg, 91%).
cis-p-Coumaroyl-D-arginine methyl ester (7)
To a solution of 10 (16.2 mg, 98.7 µmol) in DMF (0.2 mL) was
added HO–NSu (17.3 mg, 150 µmol) and DCC (23.2 mg,
112 µmol) at 0 ЊC and then the solution was stirred for 24 hours.
The reaction mixture was concentrated in vacuo, and the
residue was mixed with ethyl acetate, and then filtered with
Hyflo super-cell (Wako Pure Chemical Industries). The filtrate
was washed with saturated cold NaHCO3aq. and then cold
brine. The organic layer was concentrated in vacuo, and the
residue was separated by silica gel column chromatography
(CHCl3–MeOH = 10 : 1) to give 11. To the solution of 11 in
acetone (0.7 mL), -arginine methyl ester sulfate (11.1 mg,
49.4 µmol) in H2O (1.0 mL), which was adjusted to pH 8.0 with
saturated NaHCO3 aq., was added at 0 ЊC. This reaction
mixture was stirred for 24 hours, adjusted to pH 3 with acetic
acid, and then evaporated to remove acetone. This solution was
extracted by ethyl acetate, and the aqueous layer was concen-
trated to dryness. The residue was purified by HPLC Cosmosil
5C18AR (30% MeOH aq. containing 1% AcOH) to give 7 (5.6
mg, 25%).
5:1H-NMR (270 MHz, CD3OD, 18 ЊC): 6.93 (2H, d, J = 8.3
Hz), 6.63 (2H, d, J = 8.3 Hz), 2.66 (2H, t, J = 6.9 Hz), 2.32 (2H,
t, J = 6.9 Hz), 1.13–1.05 (2H, m), 1.01–0.95 (2H, m) ppm;
13C-NMR (100 MHz, CD3OD, 20 ЊC): 175.6, 156.9, 132.8,
130.4, 116.2, 42.1, 39.5, 39.3, 32.2, 27.7, 27.0 ppm; HR FAB
MS (positive): [M ϩ H]ϩ Found m/z 279.1796, C14H23O2N4
requires m/z 279.1821; IR (film) ν: 3172, 1641, 1552, 1514, 1406,
1248 cmϪ1
.
4-O-Methyl-cis-p-coumaryl succinimide ester (18)
To a solution of 17 (25.1 mg, 0.14 mmol) in DMF (0.3 mL) was
added N-hydroxysuccinimide (HO–NSu, 32.9 mg, 0.29 mmol)
and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydro-
chloride (EDCI, 54.0 mg, 0.28 mmol) at 0 ЊC under an argon
atmosphere. After stirring for 4 hours, additional EDCI
(39.2 mg, 0.20 mmol) was added to this solution. After stirring
overnight, the reaction mixture was concentrated in vacuo, and
the residue was purified with silica gel column chromatography
and preparative TLC (CHCl3–MeOH = 10 : 1) to give 18 (37.7
mg, 97.2%).
7: 1H-NMR (270 MHz, CD3OD, 19 ЊC): 7.46 (2H, d, J = 8.9
Hz), 6.71 (2H, d, J = 8.9 Hz), 6.69 (1H, d, J = 12.5 Hz), 5.86
(1H, d, J = 12.5 Hz), 4.49 (1H, dd, J = 8.6, 4.9 Hz), 3.73 (3H, s),
3.18 (2H, dd, J = 12.9, 6.3 Hz), 1.92–1.87 (1H, m), 1.75–1.57
(3H, m) ppm; 13C-NMR (100 MHz, CD3OD, 20 ЊC): 176.1,
173.4, 159.3, 158.4, 139.5, 132.5, 127.8, 120.2, 115.8, 53.2, 52.8,
41.9, 29.6, 26.4 ppm.; HR FAB MS (positive): [M ϩ H]ϩ Found
m/z 335.1749, C16H23O4N4 requires m/z 335.1719; IR (film)
ν: 3197, 1736, 1653, 1608, 1514, 1219, 1173 cmϪ1; [α]2D0 48.9
(c 0.17, MeOH).
18:1H-NMR (270 MHz, CDCl3, 18 ЊC): 7.77 (2H, d, J = 8.9
Hz), 7.18 (1H, d, J = 12.5 Hz), 6.89 (2H, d, J = 8.9 Hz), 6.00
(1H, d, J = 12.5 Hz), 3.83 (3H, s), 2.84 (4H, s) ppm; 13C-NMR
(100 MHz, CDCl3, 19 ЊC): 169.3, 161.3, 160.8, 150.3, 133.0,
126.1, 113.5, 108.8, 55.3, 25.6 ppm; HR FAB MS (positive):
[M ϩ Na]ϩ Found m/z 298.0680, C14H13O5N1Na1 requires m/z
298.0691; IR (film) ν: 1767, 1736, 1597, 1512, 1208, 1071 cmϪ1
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cis-p-Methoxycinnamoylagmatine (6)
(68.7 mg, 0.25 mmol) was added. After 3 hours, the reaction
mixture was evaporated, and the residue was purified with silica
gel column chromatography and preparative TLC (CHCl3–
MeOH–H2O = 24 : 8 : 1) to give 23 (22.6 mg, 27%).
To a solution of Agmatin sulfate (26.8 mg, 0.12 mmol) in H2O
(0.8 mL) was added saturated NaHCO3 aq. in order to adjust
the pH to 8. To this solution was added compound 18 (36.1 mg,
0.13 mmol) in acetone (1 mL) at 0 ЊC. After stirring for 3 days,
the reaction mixture was evaporated to remove acetone,
extracted with EtOAc, and the resulting aqueous layer was
evaporated to dryness. The residue was purified with HPLC
using a Cosmosil 5C18AR column (1% AcOH–30% MeOH
aq.) to give 6 (31.0 mg, 81%).
23: 1H-NMR (270 MHz, CD3OD, 18 ЊC): 7.45 (2H, d, J = 8.6
Hz), 6.73 (2H, d, J = 8.6 Hz), 6.69 (1H, d, J = 12.5 Hz), 5.89
(1H, d, J = 12.5 Hz), 4.40 (1H, m), 3.53–3.44 (4H, m), 3.39–3.34
(2H, m), 3.19 (4H, t, J = 5.6 Hz), 1.87–1.79 (1H, m), 1.71–1.52
(3H, m), 1.43 (9H, s) ppm; 13C-NMR (67.5 MHz, CD3OD, 19
ЊC): 181.7, 172.4, 169.8, 167.8, 159.2, 139.4, 132.5, 127.8, 120.6,
115.8, 82.2, 70.9, 70.2, 54.2, 42.0, 41.3, 40.4, 30.2, 28.8, 26.4
ppm; HR FAB MS (positive): [M ϩ H]ϩ Found m/z 507.2932,
C24H39O6N6 requires m/z 507.2931; IR (film) ν: 3273, 1655,
1514, 1252, 1173, 843 cmϪ1; [α]2D0 ϩ31.7 (c 0.5, MeOH).
6:1H-NMR (270 MHz, CD3OD, 20 ЊC): 7.49 (2H, d, J = 8.9
Hz), 6.86 (2H, d, J = 8.9 Hz), 6.66 (1H, d, J = 12.5 Hz), 5.90
(1H, d, J = 12.5 Hz), 3.78 (3H, s), 3.24 (2H, br s), 3.15 (2H, br
s), 1.57 (4H, br s) ppm; 13C-NMR (100 MHz, CD3OD, 20 ЊC):
175.5, 161.4, 158.6, 138.0, 132.2, 129.2, 122.3, 114.5, 55.7, 42.0,
39.7, 27.4, 27.2 ppm; HR FAB MS (positive): [M ϩ H]ϩFound
m/z 291.1823, C15H23O2N4 requires m/z 291.1821; IR (film)
N-{2-[(Fluorescein-4-isothiocyanato)amino]ethoxyethyl} Nꢀ-cis-
p-coumaroyl-D-arginine amide (1)
ν: 3163, 1643, 1512, 1255, 1176, 1093, 613 cmϪ1
.
Compound 23 (22.6 mg, 44.7 µmol) was dissolved in THF–H2O
(1 : 1) (0.6 mL) and TFA (1.5 mL) was added to this solution at
0 ЊC. After stirring for 2 hours, this solution was evaporated to
dryness to give a TFA salt of the resulting amine. The TFA salt
of the resulting amine (10.7 mg) was dissolved in methanol
(1 mL). To this solution, triethylamine (4.8 µL, 34.5 µmol) and
then fluorescein isothiocyanate (FITC) (isomer I, 6.6 mg, 17.0
µmol) were added at 0 ЊC. After stirring overnight at rt, the
reaction mixture was evaporated to dryness and the residue was
purified with preparative TLC (CHCl3–MeOH–H2O = 24 : 8 : 1)
to give 1 (21.0 mg, quant.).
N-(2-(tert-Butoxycarbonylamino)ethoxyethyl) Nꢀ-benzyloxy-
carbonyl-D-arginine amide (21)
Compound 19 (60.7 mg, 0.18 mmol, Fluka Co., Ltd.) was
dissolved in pyridine (1.3 mL), and to this solution was added
20 (27.0 mg, 0.13 mmol) followed by benzotriazole-1-yloxy-
tris(dimethylamino)phosphonium hexafluorophosphate (BOP)
(83.2 mg, 0.19 mmol) at 0 ЊC under an argon atmosphere. After
stirring for 15 hours, the reaction mixture was evaporated with
the addition of toluene to remove pyridine. The resulting resi-
due was purified with silica gel column chromatography and
preparative TLC (CHCl3–MeOH–AcOH = 10 : 2 : 1) to give 21
(54.2 mg, 85%).
1:1H-NMR (270 MHz, CD3OD, 18 ЊC): 8.03 (1H, d, J = 2.0
Hz), 7.66 (1H, br s), 7.38 (2H, d, J = 8.6 Hz), 7.20 (1H, d, J = 8.4
Hz), 7.13 (2H, d, J = 8.6 Hz), 6.70–6.58 (7H, m), 5.89 (1H, d,
J = 12.7), 4.30 (1H, m,), 3.94–3.37 (8H, m), 3.01 (2H, t, J = 7.0
Hz), 1.86–1.41 (4H, m) ppm; 13C-NMR (100 MHz, DMSO-d6,
18 ЊC): 180.2, 171.8, 169.6, 166.1, 165.5, 159.1, 158.2, 156.9,
155.2, 140.2, 137.7, 132.3, 130.0, 129.3, 127.8, 126.1, 125.2,
120.8, 120.2, 118.4, 115.8, 114.8, 111.0, 102.6, 69.0, 68.2, 52.3,
45.7, 43.9, 29.1, 25.0, 11.5 ppm; HR FAB MS (positive): [M ϩ
H]ϩ Found m/z 796.2778, C40H42O9N7S requires m/z 796.2765;
IR (film) ν: 3273, 1589, 1466, 1387, 1265, 1209, 1111 cmϪ1; [α]2D0
ϩ40.2 (c 1.0, DMSO).
21: 1H-NMR (270 MHz, CD3OD, 17 ЊC): 7.37–7.29 (5H, m),
5.11 (1H, d, J = 12.8 Hz), 5.07 (1H, d, J = 12.8 Hz), 4.10 (1H,
m), 3.49 (2H, t, J = 5.2 Hz), 3.45 (2H, t, J = 5.2 Hz), 3.36 (2H,
m), 3.20–3.16 (4H, m), 1.84–1.58 (4H, m), 1.42 (9H, s) ppm;
13C-NMR (67.5 MHz, CD3OD, 19 ЊC): 174.3, 158.3, 158.2,
137.9, 129.3, 128.9, 128.7, 80.1, 70.8, 70.2, 67.7, 56.0, 41.9, 41.3,
40.3, 30.4, 28.8, 26.3 ppm.; [M ϩ H]ϩ Found m/z 495.2912,
C23H39O6N6 requires m/z 495.2931; IR (film) ν: 3411, 1668,
1523, 1252, 1169, 845 cmϪ1; [α]2D0 Ϫ5.7 (c 0.5, MeOH).
N-{[2-(7-Amino-4-methylcoumarin-3-acetyl)amino]ethoxyethyl}
Nꢀ-cis-p-coumaroyl-D-arginine amide (24)
N-(2-(tert-Butoxycarbonylamino)ethoxyethyl) D-arginine amide
Compound 23 (31.1 mg, 61 µmol) was dissolved in THF–H2O
(1 : 1) (0.6 mL) and TFA (1.0 mL) was added to this solution
at 0 ЊC. After stirring for one hour, the reaction mixture
was evaporated to give a TFA salt of the resulting amine. The
TFA salt of the resulting amine was dissolved in DMF (0.5 mL)
and triethylamine (17.7 µL, 127.8 µmol) and then 7-amino-
4-methylcoumarin-3-acetic acid–NHS (AMCA–NHS) (8.4 mg,
25.5 µmol) in DMF (0.5 mL) was added to this solution
at 0 ЊC. After stirred overnight, the reaction mixture was
adjusted to pH 5 with acetic acid and evaporated to dryness.
The residue was then purified by HPLC using a Cosmosil
5C18AR column (1% AcOH–30% MeOH aq.) to give 24 (14.8
mg, 79%).
(22)
Compound 21 (146.6 mg, 0.27 mmol) was dissolved in
methanol (3 mL) and 10% Pd–C powder was added to this
solution. This solution was stirred for one hour under a
hydrogen atmosphere, filtered with Celite, and evaporated to
dryness to give 22 (100.8 mg, 91%).
22: 1H-NMR (270 MHz, CD3OD, 25 ЊC): 3.61 (1H, t, J = 6.3
Hz), 3.54 (2H, t, J = 4.9 Hz), 3.47 (2H, t, J = 5.6 Hz), 3.42 (2H,
m), 3.21 (4H, t, J = 5.9 Hz), 1.94 (3H, s), 1.81–1.72 (2H, m),
1.67–1.62 (2H, m), 1.43 (9H, s) ppm; 13C-NMR (67.5 MHz,
CD3OD, 19 ЊC): 181.8, 170.2, 158.8, 157.3, 81.7, 70.0, 69.2,
53.7, 41.2, 40.5, 39.9, 28.9, 28.5, 24.5, 24.1 ppm; HR FAB MS
(positive): [M ϩ H]ϩ Found 361.2550 m/z, C15H33O4N6 requires
361.2563 m/z; IR (film) ν: 3263, 1676, 1547, 1406, 1173, 1124
cmϪ1; [α]2D0 Ϫ4.4 (c 1.0, MeOH)
24:1H-NMR (400 MHz, CD3OD, 25 ЊC): 7.49 (1H, d, J = 9
Hz), 7.42 (2H, d, J = 9 Hz), 6.69 (2H, d, J = 9 Hz), 6.67 (1H, dd,
J = 2, 8 Hz), 6.64 (1H, d, J = 12 Hz), 6.51 (1H, d, J = 2 Hz), 5.86
(1H, d, J = 13 Hz), 4.42 (1H, dd, J = 6, 7 Hz), 3.54 (2H, s), 3.51
(5H, m), 3.37 (3H, m), 3.16 (2H, t, J = 7 Hz), 2.35 (3H, s), 1.82
(1H, m), 1.72–1.51 (3H, m) ppm; 13C-NMR (100 MHz,
CD3OD, 18 ЊC): 175.2, 173.9, 173.3, 170.0, 164.9, 159.4, 158.6,
155.9, 153.6, 153.0, 139.5, 132.6, 127.9, 127.5, 120.7, 116.0,
114.6, 113.5, 112.0, 100.7, 70.3, 70.2, 54.3, 42.0, 40.6, 40.5, 35.2,
30.2, 26.3, 20.7, 15.4 ppm; HR FAB MS (positive): [M ϩ H]ϩ
Found 622.2983 m/z, C31H40O7N7 requires 622.2989 m/z;
IR (film) ν: 3344, 1668, 1554, 1516, 1201, 1136, 721 cmϪ1; [α]2D0
ϩ14.1 (c 0.5, MeOH).
N-(2-(tert-Butoxycarbonylamino)ethoxyethyl) Nꢀ-cis-p-
coumaroyl-D-arginine amide (23)
Compound 22 (78.9 mg, 0.164 mmol) was dissolved in DMF
(1.5 mL) with a trace amount of water. To this solution was
added 10 (40.4 mg, 0.246 mmol) and then 4-(4,6-dimethoxy-
1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM)
(75.2 mg, 0.272 mmol) at 0 ЊC. This solution was stirred under
an argon atmosphere. After 12 hours, additional DMTMM
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 1 8 6 – 3 1 9 2
3191

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Bioassay
of other nyctinastic plants, C. mimosoides, L. leucocephalam, A.
saman, A. lebbeck and Aeschnomene. indica, were prepared and
treated with a fluorescent-labelled probe compound in the same
procedure.
Albizzia julibrissin Durazz, Albizzia saman, Albizzia lebbeck,
Cassia mimosoides, Aeschynomene indica L, and Leucaena
leucocephala, which were used for bioassay, were grown in the
greenhouse of Keio University. The young leaves detached from
the stem of the plant with a sharp razor blade were used for the
bioassay. One leaf was placed in H2O (ca. 1.0 mL) using a 5 mL
glass tube in the greenhouse kept at 25–30 ЊC and was allowed
to stand overnight. The leaves which opened again the next
morning (around 10:00 am) were used for the bioassay. Each
test solution was carefully poured into test tubes around 10:00
am. The bioactive fraction was judged by the leaf–opening
at 21:00 after the leaf-closing of the plant leaf in the blank
solution containing no sample.
Acknowledgements
This work was supported by the Ministry of Education,
Science, Sports and Culture (Japan) for Grant-in-Aid for
Scientific Research (No.15681013), Pioneering Research
Project in Biotechnology given by the Ministry of Agriculture.
This work was also supported by Grant-in-Aid for the 21st
Century COE program “KEIO Life Conjugate Chemistry”
from the Ministry of Education, Culture, Sports, Science, and
Technology, Japan.
Fluorescence study using a fluorescence microscope
The leaf of A. julibrissin opening in the daytime was cut into
an appropriate size and fixed in agar. The agar was sliced
perpendicular to the petiole by a microslicer (DSK-1000,
DOUSAKA EM Co. Ltd.) by 20–30 micrometers, and the
sections containing the pulvini were floated on a 0.1 M citrate–
0.2 M phosphate buffer (pH 7.0). The sections were immersed
in a solution containing various concentrations of fluorescent-
labelled probe compounds, and were allowed to stand for
4.5 hours under shielded conditions at room temperature for
staining. After staining, the sections were washed and
incubated with equilibration buffer (SlowFade Light Anti-
fadant Kit, Molecular Probes Inc.) for 10 min. This section was
placed on a slide glass and was covered by a cover glass after
adding a drop of antifade reagent (SlowFade Light Anti-
fadant Kit, Molecular Probes Inc.). The observation of these
sections was carried out by using an ECLIPSE E-800 micro-
scope (Nikon Co. Ltd.) equipped with VFM fluorescence
instrument. B-2A (Nikon Co. Ltd.) filter (λem: 450–490 nm) was
used on the observation with microscope. The plant sections
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