Direct observation of the target cell for leaf-movement factor using novel fluorescence-labeled probe compounds: Fluorescence studies of nyctinasty in legumes. Part 1

Article abstract of DOI:10.1016/S0040-4039(01)00589-5

We synthesized fluorescence-labeled probe compounds bearing AMCA (1), NBD (2), and 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.

Full text of DOI:10.1016/S0040-4039(01)00589-5

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 3869–3872
Direct observation of the target cell for leaf-movement factor
using novel fluorescence-labeled probe compounds:
fluorescence studies of nyctinasty in legumes. Part 1
Minoru Ueda,* Yoko Wada and Shosuke Yamamura*
Laboratory of Natural Products Chemistry, Department of Chemistry, Faculty of Science and Technology, Keio University,
Hiyoshi, Yokohama 223-8522, Japan
Received 14 March 2001; revised 5 April 2001; accepted 6 April 2001
Abstract—We synthesized fluorescence-labeled probe compounds bearing AMCA (1), NBD (2), and 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. © 2001 Elsevier Science Ltd. All rights reserved.
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 identified several bioactive substances
that regulate this leaf movement,3 and chemical study
using these bioactive substances gave significant infor-
mation on the mechanism for the control of nycti-
nasty.3 The next issue is to determine how these
compounds induce leaf movement. Because of the high
hydrophobicity of the leaf-movement factors, it is
assumed that some receptors would exist on a plasma
membrane of the plant motor cell, which is essential for
nyctinastic leaf movement. The bioactive substances
concerning leaf movement can be used as probe com-
pounds that are highly useful for the identification of
their receptors in the plant body, which leads to bioor-
ganic studies of nyctinasty. Investigation of the site
where bioactive substances are perceived at the cellular
level is the first step towards the bioorganic study of
their receptor molecule. Here, we report chemical syn-
thesis of novel fluorescence-labeled probe compounds
bearing AMCA (1), NBD (2), and dansyl (3) groups as
the fluorescent functionality, and a fluorescence study
on the nyctinastic movement using these probe
compounds.
O
OH
OH
O
COOH
S
H
N
OH
N
H
O
R1
N
H
HO
O
O
O
O
O
3
R2
HO
HO
OH
OH
COOK
COOMe
4
Potassium galactoisolespedezate
(5, R1=OH, R2=H)
Potassium isolespedezate
(6, R =H, R =OH)
Keywords: nyctinasty; leaf-opening substance; fluorescence; probe compound; motor cell.
* Corresponding authors. Fax: 81 45 563 5967; e-mail: ueda@chem.keio.ac.jp; yamamura@chem.keio.ac.jp
0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(01)00589-5

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M. Ueda et al. / Tetrahedron Letters 42 (2001) 3869–3872
Recently, we synthesized FITC-labeled potassium
galactoisolespedezate (4), based on the artificial leaf-
opening substance 5, which showed leaf-opening activ-
ity against the leaf of Cassia mimosoides L.4–6 However,
the bioactivity of 4 was one-fiftieth as strong as that of
the natural product (6); thus, a fluorescence labeled
probe compound of much stronger bioactivity was
required for the bioorganic study of nyctinasty. The
decrease of bioactivity in 4 would be attributed to the
largeness of the FITC functionality. Based on this
result, we chose AMCA [6-((7-amino-4-methylcou-
marin-3-acetyl)amino)hexanoyl], NBD [6-N-(7-nitro-
benz - 2 - oxa - 1,3 - diazol - 4 - yl)aminohexanoyl], and
dansyl [6-(4-((5-dimethylaminonaphthalene-1-sulfonyl)-
amino))hexanoyl] groups as the fluorescent func-
tionalities.7 All these groups are much smaller than the
FITC group. We synthesized probe compounds bearing
those fluorescent functionalities according to the proce-
dure previously reported in Ref. 6. As shown in Scheme
1, compound 7, which was prepared according to the
previously established procedure,6 was coupled with
C. mimosoides. The bioactivities of all the probe com-
pounds are shown in Table 1. Especially, the NBD-
labeled probe compound (2) showed the strongest
bioactivity, and was effective at 5×10−6 M, which was
one-fifth as strong as that of the natural product (6).
We used probe 1 for fluorescence study of the interac-
tion between the leaf-opening substance and the plant
motor cell, which gains volume in the process of leaf-
opening and loses volume in the process of leaf-closing
and plays a central role in the plant leaf movement.11
A
leaf of C. mimosoides was cut by a microslicer
(Dousaka EM Co., Ltd.) to a thickness of thirty
micrometers. Then the section containing a motor cell
was incubated overnight in an aqueous solution con-
taining 5×10−5 M of 1. After staining, the stained
section was incubated for 30 minutes with washing
buffer to remove excess fluorescent probes. Then, the
stained section was monitored by using a fluorescence
microscope with an appropriate filter. The use of an
antifadant reagent (Slow FadeTM Antifade Kits, Molec-
ular Probes Inc.) was essential to prevent photobleach-
ing (fading of fluorescence). Fig. 1 shows photographs
succinimide-type
activated
esters
(8–10).
An
aminosugar and a hexanoic acid linker in the fluores-
cent functionalities were connected through an amide
bond, which is assumed to be stable against the hydroly-
sis by esterase in the plant body. After alkaline hydroly-
sis in the final step from ester (11–13) to potassium salt
(1–3), pH of reaction mixtures were adjusted to weakly
basic (pH 7–8) with monitoring pH by a pH meter to
circumvent the decomposition of fluorescent group in
the course of work-ups.
Table 1. The leaf-opening activity of each fluorescence-
labeled probe (status of leaf at 9:00 p.m.)
1×10−4
M
1×10−5
M
5×10−6
M
1×10−6
M
1
2
3
++
++
++
++
+
+
–
–
–
–
++
+–
The resulting fluorescence-labeled probe compounds
(1–3)8–10 showed leaf-opening activity against the leaf of
++: Completely open; +: weakly open; +–: random; –: closed.
OH
R
1) H2/Pd-CaCO3,
MeOH
OH
OH
R
O
CH2
KOH aq.
5
O
CH2
5
NH
MeOH - H2O
(pH 7-8)
HO
HO
2) 8-10, MeOH
O
N3
NH
O
O
HO
HO
HO
HO
O
O
O
OH
COOK
OH
OH
COOMe
COOMe
7
(11-13)
(1-3)
NMe2
NO2
H2N
O
O
O
N
N
O
R =
N
H
CH3
SO2
HN
N
H
(1 or 11)
(2 or 12)
(3 or 13)
NMe2
NO2
N
H2N
O
O
8
O
O
O
O
O
O
CH2
5
O
N
N
N
O
N
H
SO2
CH2
O
5
CH3
O
HN
N
O
N
H
CH
2 5
O
O
O
9
10
Scheme 1. The preparation of fluorescence labeled probe compounds (1–3).

M. Ueda et al. / Tetrahedron Letters 42 (2001) 3869–3872
3871
Figure 1. Fluorescence study of pant pulvini containing motor cells using a probe compound (1); left: control, right: a section
treated with 1.
of plant pulvini, which contains motor cell, under a
fluorescence microscope. The staining pattern for the
fluorescence of probe compound (1) was observed on
the surface of motor cell (Fig. 1). No stain was
observed in the control section, which was treated with
a solution containing no 1 (Fig. 1). Probe 2, the fluores-
cence of which is similar to the background fluores-
cence (autofluorescence) of the plant tissue under a
fluorescence microscope, was inappropriate for the
fluorescence study. In conclusion, we have succeeded in
visualization of the binding site of the leaf-opening
substance in the plant body. These results suggest that
the specific binding site for 1 (or 5) should exist on the
plasma membrane of the motor cell.
Univ. Press: London, 1973.
3. Ueda, M.; Yamamura, S. Angew. Chem., Int. Ed. 2000,
39, 1400–1414.
4. Shigemori, H.; Sakai, N.; Miyoshi, E.; Shizuri, Y.; Yama-
mura, S. Tetrahedron Lett. 1989, 30, 3991–3994.
5. Shigemori, H.; Sakai, N.; Miyoshi, E.; Shizuri, Y.; Yama-
mura, S. Tetrahedron 1990, 46, 383–394.
6. Ueda, M.; Sawai, Y. M.; Yamamura, S. Tetrahedron
Lett. 2000, 41, 3433–3436.
7. Haugland, R. P. Handbook of Fluorescent Probes and
Research Chemicals; Molecular Probes, 1996.
8. Potassium
(Z)-2-[6%-(6-((7-amino-4-methylcoumarin-3-
-galactopyranosyloxy]-3-p-
acetyl)amino)hexanamide)-b-
D
hydroxyphenylacrylate (1): 1H NMR (270 MHz, CD3OD,
rt): 7.80 (2H, d, J=8.7 Hz), 7.51 (1H, d, J=8.7 Hz), 6.98
(1H, s), 6.76 (2H, d, J=8.7 Hz), 6.67 (1H, dd, J=8.7, 2.1
Hz), 6.52 (1H, d, J=2.1 Hz), 3.82 (1H, t, J=8.9 Hz),
3.73 (1H, d, J=2.3 Hz), 3.65–3.23 (4H, m), 3.17 (2H, t,
J=6.9 Hz), 2.40 (3H, s), 2.03 (2H, t, J=7.1 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): [M-K]−. Found m/z 668.2471, C33H38N3O12
requires m/z 668.2455; IR (film) w: 3341, 1677, 1605,
1557, 1515 cm−1; [h]2D2 +60.0°(c 0.14, MeOH).
Acknowledgements
We are indebted to the Ministry of Education, Science,
Sports and Culture (Japan) for Grants-in-Aid for Scien-
tific Research (Nos. 12045259 and 12680598), Pioneer-
ing Research Project in Biotechnology given by the
Ministry of Agriculture, Forestry and Fisheries, Kihara
Foundation, Saneyoshi Foundation, Sumitomo Foun-
dation, Inamori Foundation, and Kanagawa Academy
of Science and Technology Research Grant for finan-
cial support. Also we deeply thank Professor Masao
Tasaka (NAIST) for helpful suggestions regarding the
fluorescence study of the plant and Mr. Yuichi
Ishikawa (Keio University) for technical support.
9. Potassium (Z)-2-[6%-(6-N-(7-nitrobenz-2-oxa-1,3-diazol-4-
yl)-amino)hexanamide)-b-D -galactopyranosyloxy]-3-p-
hydroxyphenylacrylate (2): 1H NMR (270 MHz, CD3OD,
rt): 8.54 (1H, d, J=8.9 Hz), 7.80 (2H, d, J=8.7 Hz), 7.04
(1H, s), 6.76 (2H, d, J=8.7 Hz), 6.35 (1H, d, J=8.9 Hz),
3.82 (1H, t, J=8.9 Hz), 3.74 (1H, d, J=3.1 Hz), 3.65–
3.44 (4H, m), 2.09 (2H, t, J=7.3 Hz), 1.76 (2H, quintet,
J=7.3 Hz), 1.59 (2H, quintet, J=7.2 Hz), 1.41 (2H,
quintet, J=7.1 Hz) ppm; 13C NMR (100 MHz, CD3OD,
30°C): 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,
References
1. Darwin, C. The Power of Movement in Plants. Third
Thousand; John Murray: London, 1882.
2. Bu¨nning, E. The Physiological Clock, 3rd ed; English
.

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M. Ueda et al. / Tetrahedron Letters 42 (2001) 3869–3872
41.1, 36.8, 30.8, 28.9, 27.5, 26.4, 23.7 ppm. HR FAB MS
3.70 (1H, d, J=3.1 Hz), 3.58–3.40 (3H, m), 3.28–3.23
(1H, m), 2.88 (6H, s), 2.82 (2H, t, J=6.9 Hz), 1.84 (2H,
t, J=7.3 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): [M-K]−. Found m/z 686.2334,
C33H40N3O11S requires m/z 686.2384; IR (film) w: 3307,
1651, 1607, 1575, 1514 cm−1; [h]2D0 +54.9° (c 0.11, MeOH).
11. Cote, G. G. Plant Physiol. 1995, 109, 729–734.
(negative): [M-K]−. Found m/z 616.1937, C27H30N5O12
requires m/z 616.1891; IR (film) w: 3341, 1584, 1510 cm−1
[h]1D9 +62.5° (c 0.26, MeOH).
;
10. Potassium (Z)-2-[6%-(6-(4-((5-dimethylaminonaphthalene-
- galactopyranosy-
1 - sulfonyl)amino))hexanamide) - b -
D
loxy]-3-p-hydroxyphenylacrylate (3): 1H NMR (270
MHz, CD3OD, rt): 8.57 (1H, d, J=8.6 Hz), 8.37 (1H, d,
J=8.7 Hz), 8.20 (1H, d, J=7.4 Hz), 7.73 (2H, d, J=8.7
Hz), 7.63–7.55 (2H, m), 7.28 (1H, d, J=6.8 Hz), 6.95
(1H, s), 6.73 (2H, d, J=8.7 Hz), 3.81 (1H, t, J=9.7 Hz),
.