Fluorescence study on the nyctinasty of Cassia mimosoides L. using novel fluorescence-labeled probe compounds

Article abstract of DOI:10.1016/S0040-4020(01)00999-1

We synthesized fluorescence-labeled probe compounds bearing 6-((7-amino-4-methylcoumarin-3-acetyl)amino)-hexanoyl (AMCA, 1), 6-N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)-aminohexanoyl (NBD, 2), and 6-(4-((5-dimethylaminonaphthalene-1-sulfonyl)-amino))-hexanoyl (dansyl, 3) groups as the fluorescent functionality. In these probe compounds, NBD-type probe, 2, showed leaf-opening activity at 5×10^-6 M. The bioactivity of 2 is one-fifth as strong as that of the natural product, potassium isolespedezate (6). We carried out the binding experiment using 1 in a plant body. Then, it was suggested that fluorescence-labeled probe compound directly bound to a motor cell in pulvina of Cassia mimosoides. And this binding was specific to C. mimosoides. Probe compounds cannot bind plant sections of other nyctinastic plants.

Full text of DOI:10.1016/S0040-4020(01)00999-1

TETRAHEDRON
Pergamon
Tetrahedron 57 (2001) 9817±9825
Fluorescence study on the nyctinasty of Cassia mimosoides L.
using novel ¯uorescence-labeled probe compounds
Takanori Sugimoto, Yoko Wada, Shosuke Yamamurap and Minoru Uedap
Laboratory of Natural Products Chemistry, Department of Chemistry, Faculty of Science and Technology, Keio University, Hiyoshi,
Yokohama 223-8522, Japan
Received 21 September 2001; accepted 15 October 2001
AbstractÐWe synthesized ¯uorescence-labeled probe compounds bearing 6-((7-amino-4-methylcoumarin-3-acetyl)amino)-hexanoyl
(AMCA, 1), 6-N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)-aminohexanoyl (NBD, 2), and 6-(4-((5-dimethylaminonaphthalene-1-sulfonyl)-
amino))-hexanoyl (dansyl, 3) groups as the ¯uorescent functionality. In these probe compounds, NBD-type probe, 2, showed leaf-opening
activity at 5£1026 M. The bioactivity of 2 is one-®fth as strong as that of the natural product, potassium isolespedezate (6). We carried out the
binding experiment using 1 in a plant body. Then, it was suggested that ¯uorescence-labeled probe compound directly bound to a motor cell
in pulvina of Cassia mimosoides. And this binding was speci®c to C. mimosoides. Probe compounds cannot bind plant sections of other
nyctinastic plants. q 2001 Elsevier Science Ltd. All rights reserved.
1. Introduction
the plant body, which leads to bioorganic studies of
nyctinasty. Investigation of the target cells where bioactive
substances are perceived is the ®rst step towards the bio-
organic study of their receptor molecule. Here, we
report chemical synthesis of novel ¯uorescence-labeled
probe compounds bearing 6-((7-amino-4-methylcoumarin-
3-acetyl)amino)-hexanoyl (AMCA, 1), 6-N-(7-nitrobenz-2-
oxa-1, 3-diazol-4-yl)-aminohexanoyl (NBD, 2), and 6-(4-
((5-dimethylaminonaphthalene-1-sulfonyl)-amino))-hexa-
noyl (dansyl, 3) groups as the ¯uorescent functionality, and
the direct observation of the target cell for the leaf-
movement factor using these probe compounds.
Most legumes close their leaves in the evening, as if to
sleep, and open them in the morning.1 This is called
nyctinasty, and such a circadian rhythmic movement has
been known to be controlled by their biological clocks.2
We have identi®ed several bioactive substances that
regulate this leaf-movement,3±16 and our recent studies
revealed the mechanism for the control of nyctinasty by
the biological clock.12±14 The next issue is to determine
how these compounds induce leaf-movement. For this
reason, our current study is focused on the mode of action
of the leaf-movement factor. However, different from the
studies using some cell-lines, our bioassay is carried out by
using whole plant-leaf. Thus, we should start from deter-
mining a cell to which leaf-movement factor binds. Our
leaf-movement factor is supposed to induce the swelling
and shrinking of motor cells by direct or indirect interaction
with motor cells, which exists in the pulvini of the plant and
is most important for the leaf-movement. The cell exert their
function by swelling and shrinking in the process of leaf-
opening and -closing and play a central role in the plant leaf-
movement.17 The ion ¯uxes followed by massive water
¯uxes across the plasma membrane of these cells produce
the swelling and shrinking behavior of the motor cells. To
determine the role of leaf-movement factor in plant leaf-
movement, we developed molecular probes based on the
leaf-movement factor for identi®cation of its target cell in
2. Results and discussion
2.1. Synthesis of ¯uorescence-labeled probe compounds
with small ¯uorescence dye
Recently, we synthesized FITC-labeled potassium galacto-
isolespedezate (4),18,19 based on the arti®cial leaf-opening
substance 5,20,21 which showed leaf-opening activity against
the leaf of Cassia mimosoides L. However, the bioactivity of
4 was one-®ftieth as strong as that of the natural product (6);
thus, a ¯uorescence labeled probe compound of much
stronger bioactivity was required for the bioorganic study
of nyctinasty. We examined the length of linker moiety in
FITC-labeled probe compounds, to improve their bio-
activities. However, according to this methodology, bio-
activity of 4, for example, was at most improved to one-
®ftieth of 6, which is insuf®cient for bioorganic study. These
results suggest that the weak bioactivity of 4 would be
attributed to the large size of the FITC group. Based on
Keywords: nyctinasty; leaf-opening substance; ¯uorescence; probe
compound; motor cell.
Corresponding authors. Tel.: 181-45-563-1141; fax: 181-45-563-5967;
e-mail: yamamura@chem.keio.ac.jp; ueda@chem.keio.ac.jp
p
0040±4020/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(01)00999-1

9818
T. Sugimoto et al. / Tetrahedron 57 -2001) 9817±9825
Scheme 1. Synthetic route of ¯uorescence probe compounds (1±3).
this idea, we examine AMCA, NBD, and dansyl groups as
the ¯uorescence dyes.22 All these ¯uorescence dyes are
much smaller than the FITC. We synthesized probe
compounds bearing those ¯uorescent dyes according to
the procedure previously reported in Ref. 19. As shown in
Scheme 1, compound 7, which was prepared according to
the previously established procedure,19 was coupled with
succinimide-type ¯uorescence reagents (8±10). An amino-
sugar and a hexanoic acid linker in the ¯uorescent reagents
were connected through an amide bond, which is assumed to
be stable against the hydrolysis by an esterase in the plant
body. However, the reactivity of the amino group in the
aminosugar was so low that secondary hydroxyl groups in
the sugar moiety or phenolic hydroxyl group reacted pre-
dominantly to give by-products which have plural ¯uores-
cent functionalities in a molecule. Addition of some bases,
such as DMAP or TEA, did not improve the reaction yield.
Moreover, puri®cation of the resulting coupling product
(11±13) in this step caused a serious loss of the yield
because of the aggregative properties of 11±13 to form an
insoluble precipitate; accordingly, the product was
subjected to the following hydrolysis reaction without puri-
®cation to improve the yield. After alkaline hydrolysis in the
®nal step from an ester (11±13) to a potassium salt (1±3),
pH of the reaction mixtures must be adjusted to weakly
basic (pH 7±8) with monitoring pH by a pH meter to
circumvent the decomposition of a ¯uorescent group in
the course of work-ups. Probe compounds (1±3) were
mainly obtained as the (Z)-form along with a trace amount
of the (E)-form. The double bond in 1±3 isomerized in the
puri®cation process to give a trace amount of corresponding
geometrical isomer. On these probe compounds, different

T. Sugimoto et al. / Tetrahedron 57 -2001) 9817±9825
9819
Table 1. The leaf-opening activity of each ¯uorescence-labeled probe
(status of leaf at 9:00 p.m.) (11: completely open, 1: weakly open,
12: random, 2: closed)
as a linker part to improve their bioactivity. Thus, we
synthesized AMCA-labeled compound with a triglycine
linker (Scheme 2), and compared the bioactivity with that
of 1. The azide was reduced with Lindler's catalyst and was
coupled with Boc-triglycine using the DCC±HOBt method.
The product (14) was deprotected and coupled with an
activated-ester type ¯uorescence reagent (15) to give 16.
After hydrolysis with KOH, AMCA-labeled probe
compound with the triglycine linker (17) was obtained as
a potassium salt. Compound 17 was mainly obtained as the
(Z)-form along with a trace amount of the (E)-form. The
stereochemistry of 17 was determined from the chemical
shifts of the ole®nic protons compared with that of 4 and
its geometrical isomer.19
1£1024 M
1£1025
M
5£1026
M
1£1026
M
1
2
3
17
11
11
11
11
11
11
1
2
2
2
2
2
11
12
2
11
chemical shifts were observed on the ole®nic protons of two
geometrical isomers. The stereochemistry of the probe
compound was determined from the chemical shifts of the
ole®nic protons compared with that of 4 and its geometrical
isomer.19
However, probe compound (17) showed leaf-opening
activity against the leaf of C. mimosoides at 1£1025 M,
which is as strong as 1 (Table 1). From these results, it
was concluded that the size of the ¯uorescence functionality
is more important than the structure of the linker moiety for
the bioactivity of probe compounds. Thus, ¯uorescence-
labeled probe compounds with a ¯exible chain, such as 1,
2, and 3, would be suf®cient for the ¯uorescence study of
nyctinasty.
The resulting ¯uorescence-labeled probe compounds (1±3)
showed leaf-opening activity against the leaf of C.
mimosoides.22 The bioactivities of all the probe compounds
are shown in Table 1. Especially, the NBD-labeled probe
compound (2) showed the strongest bioactivity, and was
effective at 5£1026 M, which was one-®fth as strong as
that of the natural product (6). And all these probe
compounds retain their leaf-opening activity for a week.
This result suggested that probe compounds (1±3) were not
hydrolyzed in the plant body by internal b-glucosidase, and
can be used in the ¯uorescence study using a plant section.
2.2. Direct observation of the target cell for the leaf-
movement factor
According to our recent report,18 an improvement of bio-
activity for the probe compound was expected by the intro-
duction of a triglycine linker. That is because the
hydrophilic property of oligoglycine chain would be more
effective than carboxylic acids with long-chain alkyl group
We used probe 1 for the detection of the target cell for the
leaf-movement factor. On the other hand, probe 2 and 3, the
¯uorescence of which are similar to the background one
(auto¯uorescence) of the plant tissue under a ¯uorescence
microscope, was inappropriate for the ¯uorescence study.
Scheme 2. Synthetic route of ¯uorescence probe compound with triglycine linker (17).

9820
T. Sugimoto et al. / Tetrahedron 57 -2001) 9817±9825
Figure 1. Fluorescence study using probe compound (1, 17); upper left: DIG image of plant section, upper right: blank ¯uorescence image of plant section,
lower left: ¯uorescence image of plant section treated with 1, lower right: ¯uorescence image of plant section treated with 17.
And FITC labeled probe (4) did not give a sharp and bright
¯uorescence image compared with 1 because of its weak
binding ability.
moiety. These results suggest that the binding site for 1
(and thus, 5) should exist on the surface of the motor cell.
2.3. Speci®c binding ability of ¯uorescent probe
compound to C. mimosoides
A leaf of C. mimosoides was cut by a microslicer perpen-
dicular to the petiole to a thickness of 30 mm. Then the
section was incubated overnight in an aqueous solution
containing 5£1025 M of 1 under dark condition to avoid
photobleaching (fading of ¯uorescence) of ¯uorescent dye.
When the incubation period was too short, such as 30 min,
no staining was observed in the plant section. After staining,
the stained section was incubated for 15 min with a washing
buffer to remove excess ¯uorescent probes. Careful exami-
nation on washing time revealed that very long washing
time decreased the reproducibility of staining. Then, the
stained section was monitored by using a ¯uorescence
microscope with an appropriate ®lter. The use of an anti-
fadant reagent was essential to prevent photobleaching. Fig.
1 shows photographs of the plant sections under a ¯uores-
cence microscope. Strong auto¯uorescence was observed in
the plant section. However, when the section was treated
with probe compound (1), the staining pattern for 1 was
observed only in the motor cells contained in pulvini (Fig.
1).23 No other part of the plant section showed the ¯uores-
cent stain for 1. Also, no stain was observed in the control
section, which was treated with a solution without 1 (Fig. 1).
And probe compound 17 gave the same result (Fig. 1).
Moreover, no stain was observed in the binding experiment
using AMCA reagent (8) itself, which is easily hydrolyzed
to give corresponding potassium carboxylate under the
incubation condition. Thus, the binding of 1 and motor cell
would be speci®c to the structure of leaf-opening substance
moiety in 1, and not non-speci®c resulting from AMCA
From our previous studies, it was revealed that each
nyctinastic plant has different leaf-movement factor whose
bioactivity is speci®c to the original plant.3±16 Thus,
synthetic probe compounds is expected to show speci®c
leaf-opening activity against the leaves of C. mimosoides,
and not to be effective for the leaf of other plants even at
higher concentrations. We examined the speci®city of
bioactivity on the probe compounds 1 and 17. Probe
compound 1 and 17 did not show leaf-opening activity
against the leaves of Leucaena leucocephala, Albizzia
julibrissin Durazz., and Aeschynomene indica L. at
5£1025 M. On the other hand, 1 and 17 showed leaf-
opening activity against the leaves of C. mimosoides even
at 1£1025 M. These results showed that the bioactivities of
1 and 17 were speci®c to the leaves of C. mimosoides. From
this result, the binding of probe compound is expected to be
speci®c against the section of C. mimosoides 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, L. leucocephala, A. julibrissin,
and A. indica. The binding experiments were carried out in
absolutely the same manner described in Section 2.2. An
aqueous solution containing 5£1025 M of probe 1 was used
for the staining of each plant section. After the treatment
with 1, section of each plant was monitored by ¯uorescence

T. Sugimoto et al. / Tetrahedron 57 -2001) 9817±9825
9821
Figure 2. Fluorescence images of plant sections treated with 1 (upper: sections before treatment, lower: sections after treatment).
microscope. Thus, it was revealed that only the section of C.
mimosoides gave the ¯uorescence image resulting from 1
(Fig. 1) and no other sections did not (Fig. 2). Moderately
strong red ¯uorescence observed on each plant section is
due to the auto¯uorescence from chlorophylls contained in
the plant section, which was also observed on the blank
sample (Fig. 2). This result showed that the binding of
probe compound with a motor cell is also speci®c to the
plant species and suggested that the speci®c receptor
molecule would be involved in the binding of 1 with a
motor cell. And, it was estimated that each plant would
have different receptor molecule on a motor cell which is
speci®c to each leaf-movement factor.
be carried out on other nyctinastic plants, such as L.
leucocephala, A. julibrissin and A. indica, etc. to con®rm
this hypothesis by using ¯uorescence probe compounds
designed on the structure of the leaf-movement factor of
each plant. The systematic studies on the identi®cation of
the receptor protein for leaf-movement factor and investi-
gations on the difference of receptor molecule in each
nyctinastic plants will give a clue for the question: why
different leaf-movement factor operates in each nyctinastic
plants?
In our previous works,12,14 we have shown two examples of
nyctinastic plants in which the concentration of the leaf-
movement factor in a plant body changes through a day
according to a circadian rhythm, and this rhythmic change
in concentration agrees with the nyctinastic leaf-movement.
And now, we have shown that our leaf-movement factor
binds to the motor cell. So far, the behavior of our leaf-
movement factor can fully account for this physiological
phenomenon in the molecular level. The next issue of our
research is focused on investigation of the role of leaf-
movement factors in this biological event, especially, the
interaction between leaf-movement factor and its
receptor molecule which invokes the signal transduction
in the motor cell which leads to the swelling and shrinking
of motor cell.
3. Conclusion
Because of its high hydrophilicity, leaf-movement factors
would not be able to be transmitted into the plasma
membrane. Thus, it was assumed that some receptors
would exist on a plasma membrane of the target cell for
the leaf-movement factor.24 Now, we have succeeded in
the visualization of binding between the leaf-movement
factor and the motor cell, and demonstrated that the target
cell for the leaf-movement factor is a motor cell.
Also, it was revealed that the binding of ¯uorescent probe
compound (17) is speci®c to C. mimosoides. Fluorescent
probe compound (17) cannot bind with the section of
other nyctinastic plants. This result strongly suggest that
each nyctinastic plant has different receptor molecule on
the motor cell to perceive external chemical signal for
introduction of swelling and shrinking of motor cell.
However, more examples of ¯uorescence study should
4. Experimental
4.1. General Procedures
1H NMR (270 MHz) was recorded on a Jeol EX270 spectro-
meter, and 13C NMR spectra (100 MHz) were recorded on a

9822
T. Sugimoto et al. / Tetrahedron 57 -2001) 9817±9825
Jeol ALPHA 400 spectrometer equipped with microprobe
NTH3-FG (NAROLAC) using TMS in CDCl3 or t-BuOH
(1H; 1.23 ppm, 13C; 32.1 ppm) in D2Oas internal standards
at various temperatures. The FAB-MS and HR FAB-MS
spectra were measured on a Jeol JMS-700 spectrometer,
using glycerol or m-nitrobenzylalcohol as a matrix. The
IR spectra were measured by JASCOFT/IR-410. The
speci®c rotations were measured by JASCODIP-360 polari-
meter. The HPLC puri®cation was carried out with a
Shimadzu LC-6A pump equipped with an SPD-6A detector
using Cosmosil 5C18AR column (B 20£250 mm2) (Nakalai
Tesque). The solvents used for HPLC were available from
Kanto Chemical and were ®ltered through a Toyo Roshi
membrane ®lter (cellulose acetate of 0.45 mm pore size,
47 mm. dia.) before use. Silica gel column chromatography
was performed on Silica Gel 60 K070 (Katayama Chemical)
or BW-300 (Fuji Silicia). Reversed-phase open-column
chromatography was performed on Cosmosil 75C18-OPN
(Nakalai Tesque). TLC was performed on Silica gel F254
(0.25 or 0.5 mm, MERCK) or RP-18F254S (0.25 mm,
MERCK). Fluorescence reagents were purchased from
Funakoshi.
(30 mL), and TEA (0.68 mL, 4.8 mmol) was added to this
solution. A solution of AMCA 8 (0.8 mg, 1.9 mmol) in DMF
(30 mL) was added to this solution, and the reaction mixture
was allowed to stand overnight. After the addition of TEA
(0.68 mL, 4.9 mmol), the reaction mixture was allowed to
stand for one more day. After evaporation, the reaction
mixture was dissolved in MeOH±H2Oˆ1:1 (200 mL), and
0.1 M KOHaq. (9.6 mL, 9.6 mmol) was added to this
mixture at 08C. After 3.5 h of stirring, additional 0.1 M
KOHaq. (9.6 mL, 9.6 mmol) was added to this solution
and the reaction mixture was stirred for a day. Then,
Amberlite IR-120B (H1) was added to this solution to adjust
pH to 6.5±6.8. Then the solution was ®ltered, neutralized
with 0.1 M K2CO3, and evaporated. Separation of the reac-
tion mixture with ODS±pTLC (CH3CN±H2Oˆ1:1) gave 1
(0.7 mg, 24% in 3 steps). Repeated coupling and hydrolysis
reactions gave 3.7 mg of 1 in total amount.
Compound 1: 1H NMR (270 MHz, CD3OD, rt): 7.80 (2H, d,
Jˆ8.7 Hz), 7.51 (1H, d, Jˆ8.7 Hz), 6.98 (1H, s), 6.76 (2H,
d, Jˆ8.9 Hz), 6.67 (1H, dd, Jˆ8.6, 2.1 Hz), 6.52 (1H, d,
Jˆ2.1 Hz), 3.82 (1H, t, Jˆ8.7 Hz), 3.73 (1H, d,
Jˆ2.3 Hz), 3.65±3.23 (4H, m), 3.17 (2H, t, Jˆ6.9 Hz),
2.40 (3H, s), 2.03 (2H, t, Jˆ7.1 Hz), 1.50±1.45 (4H, m),
1.31±1.27 (4H, m) ppm; 13C NMR (100 MHz, CD3OD, rt):
177.3, 160.1, 156.7, 154.8, 153.7, 134.3, 128.2, 127.4,
126.9, 125.3, 115.8, 115.3, 114.1, 112.6, 105.7, 101.3,
76.0, 75.6, 73.7, 71.4, 41.8, 41.2, 37.6, 35.9, 31.6, 30.8,
28.2, 27.2, 16.2 ppm; HR FAB-MS (negative): [M2K]2.
Found m/z 668.2471, C33H38O12N3 requires m/z 668.2455;
4.1.1. Synthesis of methyl 0Z)-2-[60-06-007-amino-4-
methylcoumarin-3-acetyl)amino)-hexanamide)-b-d-
galactopyranosyloxy]-3-p-hydroxyphenyl-acrylate 011).
Compound 7 (57.0 mg, 150 mmol) was dissolved in
MeOH (1.0 mL). After addition of Pd±CaCO3 (15 mg),
the reaction mixture was stirred for 2 h under a hydrogen
atmosphere. After ®ltration with celite and then evapora-
tion, crude amine (69.1 mg) was obtained. The resulting
crude amine (10.0 mg, 25.6 mmol) was dissolved in
MeOH (300 mL), and TEA (4.2 mL, 29 mmol) was added
to this solution. A solution of AMCA 8 (5.0 mg, 11 mmol) in
MeOH (1.2 mL) was added to this solution, and the reaction
mixture was allowed to stand for 4 h. After evaporation, the
residue was separated by pTLC repeatedly using MeOH±
H2Oˆ3:1 to give 11 (4.7 mg, 32% in 2 steps).
IR (®lm) n: 3341, 1677, 1605, 1557, 1515 cm21
[a]D ˆ160.08 (c 0.14, MeOH).
;
22
4.1.3. Synthesis of methyl 0Z)-2-[60-06-N-07-nitrobenz-2-
oxa-1,3-diazol-4-yl)-amino)hexanamide)-b-d-galacto-
pyranosyloxy]-3-p-hydroxyphenyl-acrylate 012). Com-
pound 7 (57.0 mg, 150 mmol) was dissolved in MeOH
(1.0 mL). After addition of Pd±CaCO3 (15 mg), the reaction
mixture was stirred for 2 h under a hydrogen atmosphere.
After ®ltration with celite and then evaporation, crude
amine (69.1 mg) was obtained. The resulting crude amine
(10.0 mg, 25.6 mmol) was dissolved in MeOH (500 mL),
and TEA (3.6 mL, 26 mmol) was added to this solution. A
solution of NBD 9 (3.0 mg, 7.7 mmol) in DMF (500 mL)
was added to this solution, and this reaction mixture was
stirred overnight. After evaporation, the residue was sepa-
rated by pTLC using MeOH±H2Oˆ2:1 to give 12 (2.3 mg,
17% in 2 steps).
Compound 11: 1H NMR (270 MHz, CD3OD, rt): 7.80 (2H,
d, Jˆ6.8 Hz), 7.50 (1H, d, Jˆ8.9 Hz), 7.03 (1H, s), 6.78
(2H, d, Jˆ7.1 Hz), 6.67 (1H, dd, Jˆ7.8, 2.1 Hz), 6.52
(1H, d, Jˆ2.3 Hz), 3.82 (4H, s), 3.74 (1H, d, Jˆ2.8 Hz),
3.55 (4H, s), 3.52±3.14 (2H, m), 2.39 (3H, s), 2.07 (2H, t,
Jˆ7.3 Hz), 1.55±1.47 (4H, m), 1.30 (4H, m) ppm; 13C
NMR (100 MHz, CD3OD, 358C): 176.5, 172.9, 167.0,
164.9, 160.0, 155.9, 153.9, 152.9, 140.1, 134.0, 127.5,
127.4, 125.9, 116.2, 114.6, 113.3, 111.9, 104.1, 100.6,
74.8, 74.6, 72.8, 70.5, 52.7, 41.0, 40.4, 36.9, 35.2, 30.7,
30.0, 27.5, 26.4, 15.4 ppm; HR FAB-MS (positive):
[M1H]1. Found m/z 684.2816, C34H42O12N3 requires m/z
684.2768; IR (®lm) n: 3350, 3239, 1683, 1633, 1604, 1556,
Compound 12: 1H NMR (270 MHz, CD3OD, rt): 8.53 (1H,
d, Jˆ8.9 Hz), 7.79 (2H, d, Jˆ7.0 Hz), 7.00 (1H, s), 6.77
(2H, d, Jˆ7.0 Hz), 6.34 (1H, d, Jˆ8.9 Hz), 4.94 (1H, d,
Jˆ7.8 Hz), 3.82 (3H, s), 3.85±3.51 (6H, m), 3.43 (1H, dd,
Jˆ13.7, 5.4 Hz), 3.24 (1H, dd, Jˆ13.7, 5.4 Hz), 2.12 (2H, t,
Jˆ7.3 Hz), 1.78 (2H, quintet, Jˆ7.3 Hz), 1.61 (2H, quintet,
Jˆ7.3 Hz), 1.43 (2H, quintet, Jˆ7.3 Hz) ppm; 13C NMR
(100 MHz, CD3OD, 358C): 176.3, 167.0, 160.1, 140.1,
138.5, 134.0, 127.3, 125.8, 116.2, 104.1, 74.8, 74.6, 72.8,
70.6, 52.7, 41.1, 36.8, 30.7, 29.0, 27.5, 26.4 ppm; HR FAB
MS (positive): [M1H]1. Found m/z 632.2173, C28H34O12N5
requires m/z 632.2204; IR (®lm) n: 3322, 1699, 1618,
1514 cm21; [a]D ˆ184.48 (c 0.60, MeOH).
22
4.1.2. Synthesis of potassium 0Z)-2-[60-06-007-amino-4-
methylcoumarin-3-acetyl)amino)-hexanamide)-b-d-
galactopyranosyloxy]-3-p-hydroxyphenyl-acrylate 01).
Compound 7 (57.0 mg, 150 mmol) was dissolved in
MeOH (1.0 mL). After addition of Pd±CaCO3 (15 mg),
the reaction mixture was stirred for 2 h under a hydrogen
atmosphere. After ®ltration with celite and then evapora-
tion, crude amine (69.1 mg) was obtained. The resulting
crude amine (1.9 mg, 4.8 mmol) was dissolved in DMF
1605, 1584, 1528, 1509 cm21; [a]D ˆ176.08 (c 0.45,
24
MeOH).

T. Sugimoto et al. / Tetrahedron 57 -2001) 9817±9825
9823
4.1.4. Synthesis of potassium 0Z)-2-[60-06-N-07-nitrobenz-
2-oxa-1,3-diazol-4-yl)-amino) hexanamide)-b-d-galacto-
pyranosyloxy]-3-p-hydroxyphenyl-acrylate 02). Com-
pound 7 (57.0 mg, 150 mmol) was dissolved in MeOH
(1.0 mL). After addition of Pd±CaCO3 (15 mg), the reaction
mixture was stirred for 2 h under a hydrogen atmosphere.
After ®ltration with celite and then evaporation, crude
amine (69.1 mg) was obtained. The resulting crude amine
(9.5 mg, 24.3 mmol) was dissolved in DMF (500 mL), and
TEA (3.4 mL, 24.3 mmol) was added to this solution. A
solution of NBD 9 (3.5 mg, 8.7 mmol) in DMF (500 mL)
was added to this solution, and this reaction mixture was
stirred overnight. After evaporation, the residue was
dissolved in MeOH±H2Oˆ1:1 (1.0 mL), then, 1.0 M
KOHaq. (48.6 mL, 48.6 mmol) was added at 08C. After
2 h of stirring, 1.0 M KOHaq. (48.6 mL, 48.6 mmol) was
added. After 3 h of stirring, Amberlite IR-120B (H1) was
added to the reaction mixture to adjust pH among pH 6.5±
6.8. Then the solution was ®ltered, neutralized with 0.1 M
K2CO3, and evaporated. Twice separation of the reaction
mixture with ODS±pTLC (MeOH±H2Oˆ1:1) gave 2
(3.1 mg, 23% in 3 steps). Repeated coupling and hydrolysis
reactions gave 6.7 mg of 2 in total amount.
130.2, 129.1, 127.4, 125.8, 124.3, 120.6, 116.5, 116.3,
104.1, 74.8, 74.6, 72.8, 70.5, 52.8, 45.8, 43.7, 40.9, 36.8,
30.3, 27.1, 26.2 ppm; HR FAB-MS (positive): [M1H]1.
Found m/z 702.2678, C34H44O11N3S requires m/z
702.2697; IR (®lm) n: 3348, 1698, 1637, 1604, 1586,
1513 cm21; [a]D ˆ168.38 (c 0.60, MeOH).
23
4.1.6. Synthesis of potassium 0Z)-2-[60-06-04-005-dimethyl-
aminonaphthalene-1-sulfonyl)-amino))-hexanamide)-b-
d-galactopyranosyloxy]-3-p-hydroxyphenyl-acrylate 03).
Compound 7 (57.0 mg, 150 mmol) was dissolved in MeOH
(1.0 mL). After addition of Pd±CaCO3 (15 mg), the reaction
mixture was stirred for 2 h under a hydrogen atmosphere.
After ®ltration with celite and then evaporation, crude
amine (69.1 mg) was obtained. The resulting crude amine
(1.9 mg, 4.8 mmol) was dissolved in DMF (30 mL), and
TEA (0.68 mL, 4.8 mmol) was added to this solution.
Dansyl reagent (10, 2.2 mg, 3.3 mmol) in DMF (70 mL)
was added to this solution, and the reaction mixture was
allowed to stand overnight. After the second addition of
TEA (0.68 mL, 4.9 mmol), the reaction mixture was allowed
to stand one more day. After the third addition of TEA
(0.68 mL, 4.9 mmol), the reaction mixture was allowed to
stand one more day. After evaporation, the reaction mixture
was dissolved in MeOH±H2Oˆ1:1 (200 mL), and 0.1 M
KOHaq. (9.6 mL, 9.6 mmol) was added to this mixture at
08C. After 3 h of stirring, another 0.1 M KOHaq. (9.6 mL,
9.6 mmol) was added to this solution and the reaction
mixture was stirred for a day. Then, Amberlite IR-120B
(H1) was added to this solution to adjust pH among 6.5±
6.8. Then the solution was ®ltered, neutralized with 0.1 M
K2CO3, and evaporated. Separation of the reaction mixture
with ODS±pTLC (CH3CN±H2Oˆ1:1) gave 3 (1.0 mg, 34%
in 3 steps). Repeated coupling and hydrolysis reactions gave
3.9 mg of 3 in total amount.
Compound 2: 1H NMR (270 MHz, CD3OD, rt): 8.54 (1H, d,
Jˆ8.9 Hz), 7.80 (2H, d, Jˆ8.7 Hz), 7.04 (1H, s), 6.76 (2H,
d, Jˆ8.7 Hz), 6.35 (1H, d, Jˆ8.9 Hz), 3.82 (1H, t,
Jˆ8.9 Hz), 3.74 (1H, d, Jˆ3.1 Hz), 3.65±3.44 (4H, m),
2.09 (2H, t, Jˆ7.3 Hz), 1.76 (2H, quintet, Jˆ7.3 Hz), 1.59
(2H, quintet, Jˆ7.2 Hz), 1.41 (2H, quintet, Jˆ7.1 Hz) ppm;
13C NMR (100 MHz, CD3OD, 308C): 179.3, 178.1, 176.3,
163.9, 159.8, 138.6, 133.8, 126.5, 126.4, 126.1, 116.2,
104.7, 75.0, 74.8, 72.9, 70.6, 41.1, 36.8, 30.8, 28.9, 27.5,
26.4, 23.7 ppm; HR FAB-MS (negative): [M2K]2. Found
m/z 616.1937, C27H30O12N5 requires m/z 616.1891; IR (®lm)
n: 3341, 1584, 1510 cm21; [a]D ˆ162.58 (c 0.26, MeOH).
19
Compound 3: 1H NMR (270 MHz, CD3OD, rt): 8.57 (1H, d,
Jˆ8.6 Hz), 8.37 (1H, d, Jˆ8.7 Hz), 8.20 (1H, d, Jˆ7.4 Hz),
7.73 (2H, d, Jˆ8.9 Hz), 7.63±7.55 (2H, m), 7.28 (1H, d,
Jˆ6.8 Hz), 6.95 (1H, s), 6.73 (2H, d, Jˆ8.7 Hz), 3.81 (1H, t,
Jˆ9.7 Hz), 3.70 (1H, d, Jˆ3.1 Hz), 3.58±3.40 (3H, m),
3,28±3.23 (1H, m), 2.88 (6H, s), 2.82 (2H, t, Jˆ6.9 Hz),
1.84 (2H, t, Jˆ7.3 Hz), 1.31±1.05 (6H, m) ppm; 13C NMR
(100 MHz, CD3OD, rt): 176.3,170.4, 159.2, 153.2, 144.2,
137.2, 133.5, 131.2, 131.1, 131.0, 130.2, 129.1, 126.6,
124.3, 120.6, 116.4, 116.0, 104.9, 75.2, 74.8, 72.9, 70.6,
45.8, 43.6, 41.0, 36.7, 30.8, 30.2, 27.0, 26.1 ppm; HR
FAB-MS (negative): [M2K]2. Found m/z 686.2334,
C33H40O11N3S requires m/z 686.2384; IR (®lm) n: 3307,
4.1.5. Synthesis of methyl 0Z)-2-[60-06-04-005-dimethyl-
aminonaphthalene-1-sulfonyl)-amino))-hexanamide)-b-
d-galactopyranosyloxy]-3-p-hydroxyphenyl-acrylate
013). Compound 7 (57.0 mg, 150 mmol) was dissolved in
MeOH (1.0 mL). After addition of Pd±CaCO3 (15 mg),
the reaction mixture was stirred for 2 h under a hydrogen
atmosphere. After ®ltration with celite and then evapora-
tion, crude amine (69.1 mg) was obtained. The resulting
crude amine (12.2 mg, 31.2 mmol) was dissolved in
MeOH (500 mL), and TEA (4.3 mL, 32 mmol) was added
to this solution. Dansyl reagent (10, 9.8 mg, 25.0 mmol) in
MeOH (500 mL) was added to this solution, and the reaction
mixture was allowed to stand overnight. After evaporation,
the residue was puri®ed by HPLC with Cosmosil 5C18Ar
column using 80% MeOHaq. to give 13 (6.0 mg, 32% in 2
steps).
1651, 1607, 1575, 1514 cm21; [a]D ˆ154.98 (c 0.11,
20
MeOH).
4.1.7. Synthesis of methyl 0Z)-2-[60-0N-t-butoxycarbonyl-
glycylglycylglycylamino)-b-d-galactopyranosyloxy]-3-p-
1
Compound 13: H NMR (400 MHz, CD3OD, 358C): 8.49
hydroxyphenyl-acrylate 014). Azide
7
(68.5 mg,
(1H, d, Jˆ8.8 Hz), 8.30 (1H, d, Jˆ8.8 Hz), 8.13 (1H, d,
Jˆ7.3 Hz), 7.70 (2H, d, Jˆ8.3 Hz), 7.55±7.49 (2H, m),
7.20 (1H, d, Jˆ7.3 Hz), 6.93 (1H, s), 6.70 (2H, d,
Jˆ8.8 Hz), 4.86 (1H, d, Jˆ7.3 Hz), 3.73 (3H, s), 3,78±
3.12 (6H, m), 2.81 (6H, s), 2.78 (2H, t, Jˆ6.8 Hz), 1.83
(2H, t, Jˆ7.3 Hz), 1.24 (4H, m), 1.06 (2H, m) ppm; 13C
NMR (100 MHz, CD3OD, rt): 176.3, 167.0, 160.1, 153.2,
140.1, 137.2, 134.0, 133.7, 131.4, 131.3, 131.1, 131.0,
180 mmol) was dissolved in MeOH (1.0 mL), and to this
solution was added Pd±CaCO3 (15 mg). After stirring for
2 h under a hydrogen atmosphere, the reaction mixture was
®ltered with celite and the ®ltrate was dried up to give crude
amine (59.9 mg). The amine (22.6 mg) was dissolved in
DMF (0.5 mL) without puri®cation, and to this solution,
Boc-GlyGlyGly (22.1 mg, 76.5 mmol), 1-hydroxybenzo-
triazole monohydrate (12.9 mg, 95.6 mmol), and DCC

9824
T. Sugimoto et al. / Tetrahedron 57 -2001) 9817±9825
(19.7 mg, 95.6 mmol) was added at 08C. The reaction
mixture was stirred under an argon atmosphere at room
temperature overnight. The reaction mixture was evapo-
rated to dryness. The resulting residue was dissolved in
MeOH, and ®ltered with hy¯o-supercell to remove
DCUrea. The ®ltrate was evaporated and puri®ed with
ODS±TLC (MeOH±H2Oˆ1:1) to give 14 (15.6 mg, 40%
in 2 steps).
neutralized with 10% K2CO3. After evaporation to dryness,
17 (1.7 mg, 65%) was obtained.
1
Compound 17: H NMR (400 MHz, D2O, rt): 7.65 (2H, d,
Jˆ8.8 Hz), 7.44 (1H, d, Jˆ8.3 Hz), 6.77 (2H, d, Jˆ8.8 Hz),
6.72 (1H, s), 6.71 (1H, m), 6.52 (1H, d, Jˆ2.4 Hz), 4.89
(1H, d, Jˆ7.8 Hz), 3.97 (1H, d, Jˆ17.6 Hz), 3.95 (1H, d,
Jˆ17.6 Hz), 3.94 (2H, s), 3.84 (1H, d, Jˆ3.4 Hz), 3.75±
3.81 (3H, m), 3.66 (1H, dd, Jˆ10.3, 3.4 Hz), 3.60 (1H,
m), 3.59 (2H, s), 3.43 (1H, dd, Jˆ14.2, 3.9 Hz), 3.29 (1H,
dd, Jˆ14.2, 8.3 Hz), 2.29 (3H, s) ppm; 13C NMR (100 MHz,
CD3OD, 308C): 174.9, 173.2, 172.7, 172.0, 165.4, 156.9,
156.6, 154.6, 154.4, 152.2, 146.1, 132.6, 127.5, 126.7,
121.1, 116.0, 114.1, 113.6, 112.3, 102.9, 101.6, 73.9, 73.8,
72.1, 69.8, 65.4, 43.7, 43.4, 43.2, 40.5, 34.6 ppm; HR FAB-
MS (negative): [M2K]2. Found m/z 726.2283, C33H36O14N5
requires m/z 726.2259; IR (®lm) n: 3352, 1652, 1605,
Compound 14: 1H NMR (400 MHz, CD3OD, rt): 7.76 (2H,
d, Jˆ8.8 Hz), 7.00 (1H, s), 6.76 (2H, d, Jˆ8.8 Hz), 4.93
(1H, d, Jˆ7.8 Hz), 3.90 (1H, m), 3.88 (2H, s), 3.81 (3H,
s), 3.77 (2H, s), 3.74 (1H, m), 3.73 (2H, s), 3.50±3.55 (2H,
m), 3.40 (1H, dd, Jˆ13.7, 6.8 Hz), 3.26 (1H, m), 1.43 (9H,
s) ppm; 13C NMR (100 MHz, CD3OD, rt): 173.5, 172.0,
171.7, 166.7, 159.7, 139.8, 133.8, 127.1, 125.7, 116.1,
103.6, 81.0, 74.6, 74.3, 72.7, 69.9, 52.7, 49.8, 44.8, 43.8,
43.4, 40.6, 28.7 ppm; HR FAB-MS (positive): [M1H]1.
Found m/z 627.2516, C27H39O13N4 requires m/z 627.2514;
24
1557 cm21; [a]D ˆ135.08 (c 1.0, H2O).
IR (®lm) n: 3327, 1663, 1605, 1514 cm21; [a]D ˆ176.68
22
4.1.10. Bioassay. The young leaves detached from the stem
of the plant C. mimosoides, which was grown in the green-
house of Keio University, with a sharp razor blade were
used for the bioassay. One leaf was placed in H2O(ca.
1.0 mL) using a 20-mL glass tube in the greenhouse kept
at 25±358C and allowed to stand overnight. The leaves
which opened again the next morning (around 10:00 a.m.)
were used for the bioassay. Each test solution was carefully
poured into test tubes with a microsyringe around 10:00
a.m. The bioactive fraction was judged by the leaf-opening
after the leaf-closing of the plant leaf in the blank
solution containing no sample. Other nyctinastic plants, L.
leucocephalam, A. julibrissin and A. indica, used in bio-
assay were also grown in the greenhouse of Keio University.
(c 0.2, MeOH).
4.1.8. Synthesis of methyl 0Z)-2-[60-007-amino-4-methyl-
coumarin-3-acetyl)amino)glycylglycylglycyl)-b-d-galac-
topyranosyloxy]-3-p-hydroxyphenyl-acrylate 016). Com-
pound 14 (15.6 mg, 24.9 mmol) was dissolved in 180 mL of
THF±H2O(30:1). To this solution, TFA (180 mL) was
added at 08C. After being stirred for 1 h, the reaction
mixture was evaporated to dryness with H2Oto remove
TFA as an azeotrope to give TFA salt of crude amine
(15). Crude 15 was dissolved in DMF (0.5 mL) without
puri®cation, and to this solution, AMCA±NHS (3.3 mg,
10 mmol) was added at 08C. After being stirred at room
temperature for 2 h, the reaction mixture was evaporated
and puri®ed with ODS±TLC (MeOH±H2Oˆ1:2) to give
16 (4.2 mg, 56% in 2 steps).
4.1.11. Fluorescence study using a ¯uorescence micro-
scope. The leaf of C. mimosoides opening in the daytime
was cut in an appropriate size and ®xed in agar. The agar
was sliced perpendicular to the petiole by a microslicer
(DSK-1000, DOUSAKA EM) by 20±30 mm, and the
sections containing the pulvini were ¯oated on distilled
water. The sections were immersed in a solution containing
the various concentration of ¯uorescent-labeled probe
compound, and allowed to stand overnight under shielded
condition at room temperature for staining. After staining,
the sections were washed by being incubated with equilibra-
tion buffer (SlowFadee Light Antifadant Kit, Molecular
Probes) for 15 min. This section was placed on a slide
glass and covered by a coverglass after adding a drop of
antifade reagent (SlowFadee Light Antifadant Kit,
Molecular Probes). The observation of these sections was
carried out by using an ECLIPSE E-800 microscope
(Nikon) equipped with VFM ¯uorescence instrument.
Appropriate ®lter was used against each ¯uorescent dye:
UV-2A (Nikon) ®lter for NBD, B-2A (Nikon) ®lter for
FITC and NBD. The plant sections of other nyctinastic
plants, L. leucocephalam, A. julibrissin and A. indica,
were prepared and treated with ¯uorescent-labeled probe
compound in the same procedure.
1
Compound 16: H NMR (400 MHz, CD3OD, 308C): 7.75
(2H, d, Jˆ8.8 Hz), 7.48 (1H, d, Jˆ8.8 Hz), 6.98 (1H, s),
6.74 (2H, d, Jˆ8.8 Hz), 6.64 (1H, dd, Jˆ2.2, 8.8 Hz),
6.49 (1H, d, Jˆ2.2 Hz), 4.95 (1H, d, Jˆ7.8 Hz), 3.89 (1H,
d, Jˆ16.6 Hz), 3.87 (1H, d, Jˆ16.6 Hz), 3.86 (2H, s), 3.72±
3.81 (4H, m), 3.79 (3H, s), 3.61 (2H, s), 3.49±3.54 (2H, m),
3.38 (1H, dd, Jˆ13.4, 6.8 Hz), 3.28 (1H, m), 2.38 (3H,
m) ppm; 13C NMR (100 MHz, DMSO-d6, rt): 169.9,
169.5, 169.1, 169.0, 164.2, 161.7, 158.2, 154.1, 152.3,
150.1, 138.7, 132.2, 126.2, 124.1, 122.9, 115.1, 113.2,
111.3, 109.3, 101.2, 98.3, 72.9, 72.8, 72.2, 70.9, 60.2,
51.9, 42.2, 42.0, 41.9, 33.7, 33.6 ppm. HR FAB-MS (posi-
tive): [M1H]1. Found m/z 742.2582, C34H40O14N5 requires
m/z 742.2572; IR (®lm) n: 3342, 1659, 1603, 1552 cm21
[a]D ˆ176.98 (c 0.77, DMF).
;
24
4.1.9. Synthesis of potassium 0Z)-2-[60-007-amino-4-
methylcoumarin-3-acetyl)amino)glycylglycylglycyl)-b-
d-galactopyranosyloxy]-3-p-hydroxyphenyl-acrylate
017). Compound 16 (2.6 mg, 3.5 mmol) was dissolved in
0.3 mL of MeOH±H2O(1:1), and to this solution, 1.0 M
KOHaq. (14 mL, 14 mmol) was added at 08C. After stirring
overnight, the reaction mixture was acidi®ed by the addition
of Amberlite IR-120B (H1), ®ltered, and evaporated to
dryness. The residue was puri®ed with ODS±TLC (aceto-
nitrile±H2Oˆ1:2), dissolved in H2O(5 mL), and
Acknowledgements
We are indebted to the Ministry of Education, Science,

T. Sugimoto et al. / Tetrahedron 57 -2001) 9817±9825
9825
10. Ueda, M.; Ohnuki, T.; Yamamura, S. Phytochemistry 1998,
49, 633±635.
Sports and Culture (Japan) for Grant-in-Aid for Scienti®c
Research (No. 13024267 and No. 12680598), Pioneering
Research Project in Biotechnology given by the Ministry
of Agriculture, Forestry and Fisheries, Sumitomo Founda-
tion, Inamori Foundation, and Mishima-Kaiun Foundation
for ®nancial support. And we deeply thank Professor Masao
Tasaka (NAIST) for helpful suggestions regarding the
¯uorescence study of the plant and Mr Yuichi Ishikawa
(Keio University) for technical support.
11. Ueda, M.; Asano, M.; Yamamura, S. Tetrahedron Lett. 1998,
49, 9731±9734.
12. Ueda, M.; Asano, M.; Sawai, Y.; Yamamura, S. Tetrahedron
1999, 55, 5781±5792.
13. Ueda, M.; Ohnuki, T.; Yamamura, S. Chem. Lett. 1998, 179±
180.
14. Ohnuki, T.; Ueda, M.; Yamamura, S. Tetrahedron 1998, 54,
12173±12184.
15. Ueda, M.; Yamamura, S. Tetrahedron Lett. 1999, 40, 353±
356.
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