Visualization of the precise structure recognition of leaf-opening substance by using biologically inactive probe compounds: Fluorescence studies of nyctinasty in legumes 2

Article abstract of DOI:10.1246/cl.2002.1118

Intimate correlation between bioactivity and binding activity was shown using biologically active and inactive fluorescence-labeled probe compounds for nyctinasty. This results suggested the existence of a receptor which recognizes the aglycon of a leaf-o

Full text of DOI:10.1246/cl.2002.1118

1118
Chemistry Letters 2002
Visualization of the Precise Structure Recognition of Leaf-opening Substance by Using
Biologically Inactive Probe Compounds:Fluorescence Studies of Nyctinasty in Legumes 2
Takanori Sugimoto, Shosuke Yamamura, and Minoru Uedaꢀ
Laboratory of Natural Products Chemistry, Department of Chemistry, Faculty of Science and Technology,
Keio University, Hiyoshi, Yokohama 223-8522
(Received January 21, 2002; CL-020068)
Intimate correlation between bioactivity and binding activity
was used in the next step. Compound 7 was derivatised to an
aminosugar and then coupled with 8. After alkaline hydrolysis, 49
was obtained. The absolute stereochemistry of 4 was determined
by the PGME method10 to be (R) by using the pentaacetate of final
product 4.
was shown using biologically active and inactive fluorescence-
labeled probe compounds for nyctinasty. This results suggested
the existence of a receptor which recognizes the aglycon of a leaf-
opening substance on the motor cell.
For the synthesis of a probe compound with methyl ether (5),
95 was treated with diazomethane to give 10, which was coupled
with 8 and hydrolyzed to give 5.11 The geometry of the double
bond in 5 changed to (E)-form. However, it was already shown
that the geometry of the double bond in 1 easily isomerized in
aqueous solution to give a mixture of both geometrical isomers
which are equally effective against plant leaf.6 The resulting 4 and
5 showed no leaf-opening activity against C. mimosoides even at
1 ꢁ 10ꢂ3 M. The bioactivities of 4 and 5 were no more than one-
thousandth of that of probe 3.
Most legumes close their leaves in the evening, as if to sleep,
and open them in the morning (nyctinasty).1;2 We have isolated
bioactive substances that regulate nyctinasty from several
nyctinastic plants.3 Recently, we reported that potassium
lespedezate (1),4 a leaf-opening substance, directly binds with a
motor cell existing in the pulvini of Cassia mimosoides L . by
using a biologically active fluorescence-labeled probe compound
(3).5 However, to prove the existence of a receptor molecule, it is
also essential to demonstrate that biologically inactive probe
compounds cannot bind to the motor cell. In this paper, we report
the design and syntheses of the biologically inactive fluorescence-
labeled probe compounds (4 and 5) and binding studies using
them.
OH
OTBS
OH
R
HO
HO
e, f, g, h, i
NH
O
HO
a, b, c, d
AcO
AcO
OH
OTs
O
O
O
O
O
HO
OH
COOK
OH
OAc
7
COOMe
OH
COOMe
OMe
4 (bioactivity : > 1 × 10-3 M )
6
OH H N
O
O
OH
2
O
R
O
N
H
2
HO
NH
l, m
j, k
HO
HO
HO
HO
NH2
N3
R2
OH
NH
O
O
CH
3
HO
HO
O
O
O
O
O
O
HO
O
O
OH
KOOC
R
OH
OH
1
COOMe
H2N
OMe
5 (bioactivity : > 1 × 10-3 M )
COOMe
HO
OH
OH
COOK
COOK
10
9
1 (R1=OH, R2=H)
2 (R1=H, R2=OH)
O
O
3
O
O
H2N
R in 4, 5 =
O
O
O
O
8 :
O N
N
5
N
5
O
H
CH3
O
H
CH3
Scheme 1. The preparation of fluorescence labeled probe compounds
(4 and 5): reagents: (a) TsCl, pyr; (b) Ac2O, pyr; (c) TBAF, THF; (d) H2/
Pd-C, EtOAc; (e) NaN3, 15-Crown-5, DMF; (f) MeONa, MeOH; (g)
H2/Pd-C, MeOH; (h) 8, DMF; (i) KOH, MeOH-H2O; (j) CH2N2,
MeOH; (k) H2/Pd-CaCO3, MeOH; (l) 8, DMF; (m) KOH, DMF-H2O.
Molecular designs 4 and 5 were based on the structure-
activity relationship of 1. The structure modification in the
glucose moiety of 1 did not affect its bioactivity,6 on the other
hand, modification in the aglycon of 1, such as the reduction of the
double bond, protection of the carboxylate or phenolic hydroxyl
group, greatly diminished the bioactivity.7 Thus, the biologically
inactive probe compounds were designed on inactive analogs of 1
with modification in its aglycon moiety. The sugar part of the
probe was changed togalactose, which prevents decomposition of
the probe compounds by ꢀ-glucosidase in plant body.5 The
introduction of a fluorescence dye was carried out at the 6-
position of the galactose moiety through the amide bond.5 We
chose 6-[(7-amino-4-methylcoumarin-3-acetyl) amino]-hexanoy
(AMCA) groups8 as a fluorescent dye which is the same as that
contained in 3.
We carried out fluorescence studies of the interaction
between biologically inactive probe (4 and 5) and the plant motor
cell using plant sections. A leaf of C. mimosoides was cut by a
microslicer to a thickness of thirty micrometers. Then the section
containing a motor cell was incubated overnight in an aqueous
solution containing 5 ꢁ 10ꢂ5 M of 4 and 5. After staining, the
stained section was incubated with washing buffer to remove
excess probe compounds. The same experiment was carried out
by using 3 as a control experiment.
We synthesized two biologically inactive probe compounds
(4 and 5). For the synthesis of a probe compound with a reduced
double bond (4), 65 was protected and reduced with catalytic
hydrogenation with Pd-C to give 7. Resulting two epimers on the
ꢁ-carbon of carboxylate (3 : 2) were separated and major product
Figure 1 shows photographs of sections of plant pulvini,
which contains a motor cell, under a fluorescence microscope. A
staining pattern for the fluorescence of probe compound (3) was
observed on the motor cell (Figure 1). On the other hand, no stain
was observed in the section treated with (R)-4 and 5 (Figure 1).
Copyright Ó 2002 The Chemical Society of Japan

Chemistry Letters 2002
1119
Figure 1. Fluorescence staining of pulvini containing motor cell; sections treated with; left: 3, center: 4, right: 5.
(2001).
Thus, it was proved that biologically inactive probe compounds
cannot bind to the plant motor cell. And also, binding of probe 3
was inhibited by the coexistence of 1000-fold concentration of
non-labeled 2. When the section was treated by 5 ꢁ 10ꢂ5 M of 3
together with 5 ꢁ 10ꢂ2 M of 2, no staining was observed in the
plant section. Results from the structure activity relationship were
consistent with the results from binding experiments. Also, it was
clearly shown that the binding of biologically active AMCA-
labeled probe (3) with a motor cell is due to the specific binding of
the aglycon moiety which is the active site of this molecule, and is
not a nonspecific binding due to the hydrophobic AMCA group.
These results strongly suggested that some receptor for the 1
exists on the motor cell. Along with the previous result,5 some
properties were revealed on a receptor molecule of 1, that is, this
receptor recognizes the precise structure of aglycon moiety; on
the other hand, it does not recognize the sugar moiety precisely.
6
7
8
9
H. Shigemori, N. Sakai, E. Miyoshi, Y. Shizuri, and S. Yamamura,
Tetrahedron, 46, 383 (1990).
M. Ueda, Y. Sawai, and S. Yamamura, Tetrahedron Lett., 41, 3433
(2000).
R. P. Haugland, ‘‘Handbook of Fluorescent Probes and Research
Chemicals,’’ Molecular Probes, Inc., Leiden (1996).
Properties of 4: 1H NMR (400 MHz, CD3OD, RT): ꢂ 7.48 (1H, d,
J ¼ 8:8 Hz), 7.11 (2H, d, J ¼ 8:6 Hz), 6.66 (2H, d, 8.6 Hz), 6.64
(1H, dd, J ¼ 8:8, 2.2 Hz), 6.50 (1H, d, J ¼ 2:2 Hz), 4.21 (1H, dd,
J ¼ 5:4, 6.4 Hz), 4.18 (1H, d, J ¼ 7:6 Hz), 3.66 (1H, d,
J ¼ 3:4 Hz), 3.48–3.57 (4H, m), 3.43 (1H, dd, J ¼ 8:6, 4.6), 3.34
(1H, dd, J ¼ 3:4, 10.0 Hz), 3.20 (1H, dd, J ¼ 8:6, 13.7 Hz), 3.17
(2H, t, J ¼ 6:8 Hz), 2.97 (1H, dd, J ¼ 13:9, 5.4 Hz), 2.94 (1H, dd,
J ¼ 13:9, 6.4 Hz), 2.36 (3H, s), 2.20 (2H, t, J ¼ 7:3 Hz), 1.45–1.63
(4H, m), 1.32 (2H, m); 13C NMR (100 MHz, D2O, 30 ꢃC): ꢂ 181.3,
179.5, 175.1, 156.6, 156.0, 155.8, 153.7, 133.1, 131.6, 129.0, 117.6,
115.6, 115.5, 113.9, 104.9, 103.1, 83.2, 75.5, 75.0, 73.1, 71.7, 42.3,
41.8, 40.0, 38.1, 36.4, 30.6, 28.0, 27.5, 17.1; HRMS-FAB
(negative); [M-K]ꢂ found m/z 670.2597, calcd for C33H40O12N3,
670.2612; IR (film) ꢃ: 3344, 1635, 1603, 1557 cmꢂ1; [ꢁ]D21 þ20:0ꢃ
(c 0.14, H2O).
This work was supported by a Grant-in-Aid for Scientific
Research from the Ministry of Education, Culture, Sports,
Science and Technology (No. 12045259 and No. 12680598),
Pioneering Research Project in Biotechnology given by the
Ministry of Agriculture, Forestry and Fisheries, Inamori Founda-
tion, Mitsubishi Chemical Corporation Fund, Naito Foundation
for financial support.
10 T. Yabuuchi and T. Kusumi, J. Org. Chem., 65, 397 (2000).
1
11 Properties of 5: H NMR (400 MHz, D2O, 30 ꢃC): ꢂ 7.76 (2H, d,
J ¼ 9:0 Hz), 7.57 (1H, d, J ¼ 8:5 Hz), 6.89 (2H, d, J ¼ 9:0 Hz),
6.82 (1H, s), 6.80 (1H, dd, J ¼ 2:2, 8.5 Hz), 6.64 (1H, d,
J ¼ 2:2 Hz), 4.88 (1H, d, J ¼ 7:8 Hz), 3.87 (1H, d, J ¼ 3:4 Hz),
3.82 (1H, dd, J ¼ 7:8, 9.8 Hz), 3.78 (3 H, s), 3.71 (1H, dd, J ¼ 13:9,
3.4 Hz), 3.68 (1H, dd, J ¼ 9:8, 3.7 Hz), 3.55 (2H, s), 3.45 (1H, dd,
J ¼ 13:9, 3.7 Hz), 3.28 (1H, dd, J ¼ 13:9, 9.8 Hz), 3.15 (2H, dt,
J ¼ 2:9, 5.6 Hz), 2.40 (3H, s), 2.04 (2H, t, J ¼ 7:3 Hz), 1.10–1.44 (6
H, m); 13C NMR (100 MHz, D2O, 30 ꢃC): ꢂ 174.5, 173.2, 166.1,
160.6, 155.3, 155.2, 153.1, 147.2, 133.4, 132.5, 128.4, 128.2, 122.8,
115.4, 115.0, 114.7, 113.3, 104.6, 102.5, 75.0, 74.9, 73.0, 70.9, 57.0,
41.7, 41.0, 37.2, 35.7, 29.6, 27.2, 26.5, 16.5; HRMS-FAB
(negative); [M-K]ꢂ found m/z 682.2628, calcd for C33H40O12N3,
682.2612; IR (film) ꢃ: 3351, 1601, 1556, 1516 cmꢂ1; [ꢁ]D22 þ50:5ꢃ
(c 0.20, H2O).
References and Notes
1
2
3
4
5
C. Darwin, ‘‘The Power of Movement in Plants. Third Thousand,’’
John Murray, London (1882).
E. Bunning, ‘‘The Physiological Clock,’’ 3rd ed., English
¨
University Press, London (1973).
M. Ueda and S. Yamamura, Angew. Chem., Int. Ed. Engl., 39, 1400
(2000).
H. Shigemori, N. Sakai, E. Miyoshi, Y. Shizuri, and S. Yamamura,
Tetrahedron Lett., 30, 3991 (1989).
M. Ueda, Y. Wada, and S. Yamamura, Tetrahedron Lett., 42, 3869