Direct observation of a target cell of the leaf-closing factor by using novel fluorescence-labeled phyllanthurinolactone

Article abstract of DOI:10.1016/j.tetlet.2005.06.044

We report the synthesis of the fluorescence-labeled probe 2 based on phyllanthurinolactone 1, which is a leaf-closing substance of Phyllanthus urinaria L. Bioorganic studies using probe 2 showed leaf-closing activity at 1 × 10^-5 M, which was on

Full text of DOI:10.1016/j.tetlet.2005.06.044

Tetrahedron
Letters
Tetrahedron Letters 46 (2005) 5537–5541
Direct observation of a target cell of the leaf-closing factor by
using novel fluorescence-labeled phyllanthurinolactone
Hirotaka Sato, Masayoshi Inada, Takanori Sugimoto, Nobuki Kato and Minoru Ueda*
Tohoku University, Department of Chemistry, Aoba-ku, Sendai 980-8578, Japan
Received 5 May 2005; revised 7 June 2005; accepted 9 June 2005
Available online 1 July 2005
Abstract—We report the synthesis of the fluorescence-labeled probe 2 based on phyllanthurinolactone 1, which is a leaf-closing sub-
stance of Phyllanthus urinaria L. Bioorganic studies using probe 2 showed leaf-closing activity at 1 · 10ꢀ5 M, which was one-hun-
dredth of that of the natural product 1. The fluorescence study using 2 revealed that the target cell for 1 is a motor cell and suggested
that some receptors for 1 exist on the plasma membrane of the motor cell as with leaf-opening substances.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Most leguminous plants close their leaves in the evening,
as if to sleep, and open them early in the morning
according to the circadian rhythm controlled by a bio-
logical clock.1 Nyctinastic leaf movement is induced by
the swelling and shrinking of motor cells in the pulvini,
a small organ located in the joint of the leaf to the stem.
Motor cells play a key role in plant leaf-movement. Flux
of potassium ions across the plasma membranes of the
Figure 1.
motor cells is followed by massive water flux, which re-
sults in swelling and shrinking of these cells.2 We have
revealed that nyctinasty is controlled by a pair of leaf-
movement factors: leaf-opening and leaf-closing sub-
large enough for the structure modification that is essen-
stances.3 Recently, we revealed that the target cell of
tial for a synthetic probe. The synthesis of 1 was com-
the leaf-opening substance is a motor cell,4 which plays
pleted by Audran and Mori in 1998.6 They also
a key role in nyctinastic leaf-movement.2 On the other
showed that the stereochemistry of natural 1 is essential
hand, no attempt has been carried out to clarify the tar-
for its bioactivity. We also carried out structure–activity
get cell of the leaf-closing substances because the struc-
relationship studies by using synthetic analogs of 1 with
ture of most leaf-closing substances is so simple3 that
sugars other than D-glucose, and it was revealed that
structural modification toward a molecular probe, such
L-glucose-type and D-galactose-type analogs were as
as a fluorescence-labeled one, seems to be difficult. This
effective as natural 1.7 This result showed that we can
is because large fluorescence dye or a photoaffinity unit
develop a biologically active molecular probe based on
would cause serious decrease in the bioactivity of the
1 when a large functional unit such as a fluorescence
synthetic probe.
dye or a photoaffinity labeling unit is introduced into
the sugar moiety. In this letter, we report a synthesis
of fluorescence-labeled 1 (2) and the direct observation
of its target cell in the plant body by using 2.
Phyllanthurinolactone (1), a leaf-closing substance of
Phyllanthus urinaria L., is a glycoside-type leaf-closing
substance (Fig. 1).5 The structure of 1 is comparatively
We already reported the enantioselective synthesis of 1
and its sugar analogs.7 However, total yield to 1 was
too low to prepare an adequate quantity of the probe.
Keywords: Nyctinasty; Leaf-closing substance; Fluorescence; Probe
compound; Motor cell; Mitsunobu reaction; Glycosidation.
*
Thus, we tried to improve yields in the synthesis of
1; especially low yields in the synthesis of the aglycon
Corresponding author. Tel./fax: +81 22 795 6553; e-mail:
ueda@mail.tains.tohoku.ac.jp
0040-4039/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2005.06.044

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H. Sato et al. / Tetrahedron Letters 46 (2005) 5537–5541
Scheme 1. Synthesis of aglycon 12.
moiety from D-glucose and glycosidation reaction
incurred serious problems.
Mitsunobu product was obtained as a mixture of stereo-
isomers (R:S = 10:1) in moderate yield by using excess
amount of the Mitsunobu reagents. When stoichio-
metric amount of Mitsunobu reagent was used, the
Mitsunobu product was hardly obtained. However, the
desired 10 was obtained as a major product. Addition-
ally, despite our best efforts, the epimerization could
not be suppressed. Intramolecular Hornor–Emmons
reaction using t-BuOK as the base afforded the cyclized
product 11. Finally, deprotection of the PMB group by
DDQ gave the aglycon 12 as a 10:1 diastereomixture.
Total yield of 12 from D-glucose was improved from
0.25% to 2.0% compared with our previous enantioselec-
tive synthesis.7
First, we improved the synthesis of aglycon by using the
Mitsunobu reaction.8 The improved synthesis of agly-
con is shown in Scheme 1. Intermediate 3 that was pre-
pared according to the previous letter,7 was protected by
the TBS group, and then subjected to elimination of io-
dide to afford the enol ether 4. A catalytic amount of
Hg(OCOCF3)2-mediated FerrierÕs carbocyclization9 of
4 and followed by treatment of the alcohol 5 with mesyl-
chloride and triethylamine, that is a one-pot reaction
involving mesylation and b-elimination, afforded the
cyclohexanone derivative 6 in 70% overall yield in
two-steps. Deprotection of the TBS group and the ob-
tained alcohol 7 was coupled with diethyl phospho-
noacetate by the Mitsunobu reaction.8 In general, the
Mitsunobu reaction is known to be difficult to proceed
in a cyclohexanol system.10 However, in this case, the
Next, we synthesized 1-fluorosugar. The synthesis of 1-
fluorosugar 16 is shown in Scheme 2. The acetate 13,
prepared from D-galactose according to the procedure
in Ref. 11, was treated with hydrazine monohydrate
Scheme 2. Syntheses of 1-fluorosugar 16.

H. Sato et al. / Tetrahedron Letters 46 (2005) 5537–5541
5539
and fluorination of the resulting hydroxyl group affor-
ded the fluoride 14. Removal of acetates with sodium
methoxide, reduction of azide, and followed by protec-
tion of amine with Fmoc group, gave a triol 15. Finally,
acetylation of the triol yielded the 1-fluorosugars 16.
stants between H4 and H5ax, H4 and H5eq, H5ax and
H6 and H5eq and H6.
With the fluorescence-labeled probe 2 in hand, we inves-
tigated the bioactivity of 2. The young leaves detached
from the stem of the plant P. urinaria with a sharp razor
blade were used for bioassay. One leaf was placed in H2O
(ca. 1.0mL) using a 20mL glass tube in the greenhouse
kept at 25–35 ꢁC and allowed to stand overnight. The
leaves, which closed again in the evening were used for
the bioassay. The test solution was carefully poured into
test tubes with a microsyringe around 10:00 a.m. The
bioactive fraction was judged by the leaf-closing
after the leaf-opening of the plant leaf in the blank
solution. The probe 2 was effective for the leaf-closing
of P. urinaria at 1 · 10ꢀ5 M that is one-fiftieth as effective
as natural product 1. And the epimer of 2 was biologi-
cally inactive.
Synthesis of fluorescence-labeled probe 2 was then
examined (Scheme 3). Glycosidation of 1-fluorosugar
16 via the Suzuki method12 gave the glycoside 17 in
moderate yield. Acetyl groups of the glycoside 17 were
deprotected with KCN to give the triol 18 as a 4:1 mix-
ture of stereoisomers. At this stage, further epimeriza-
tion occurred. Because the C4 proton in 18 would be
easily abstracted by KCN and the resulting stable fur-
an-type intermediate gave a mixture of 1and its epimer
by protonation. Deprotection of the Fmoc group and
introduction of AMCA-X, which is a fluorescence
group gave the fluorescence-labeled probe 2 as a 4:1
diastereomixture. Diastereomerically pure 213 and its
epimer epi-214 were obtained after purification by
HPLC (COSMOSIL 5C18-AR, 20% CH3CN aq,
260nm). The desired epimer 2 was confirmed by cou-
pling constants in the 1H NMR spectra (Scheme 3).
The relation of H4 and H6 was determined to be syn
on the basis of the observation of the coupling con-
We used probe 2 to seek the target cell for 1. For this
purpose, the binding experiment using a plant section
was carried out. The leaf of P. urinaria opening in the
daytime was cut in an appropriate size and fixed in agar.
The agar was sliced perpendicular to the petiole by a
microslicer (Dousaka EM CO., Ltd.) to a thickness of
Scheme 3. Syntheses of fluorescence-labeled probe 2.

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H. Sato et al. / Tetrahedron Letters 46 (2005) 5537–5541
Figure 2. Fluorescence study using Phyllanthus pulvini containing motor cell with a probe 2; left: Nomarskii image of plant section, center:
fluorescence image of control section, right: fluorescence image of a section treated with 2.
thirty micrometers and the sections containing the pul-
vini were floated on distilled water. The sections were
immersed in a solution containing 7 · 10ꢀ6 M of 2 and
allowed to stand overnight under shielded condition at
room temperature for staining. After staining, the
stained section was incubated for 30min with equilib-
rium buffer (Amersham CO., Ltd.) to remove excess
fluorescence probes. Then, the stained section was
placed on a slide glass and covered by a cover glass after
adding a drop of antifadant reagent (Slow FadeTM Anti-
fade Kits, Molecular Probes Inc.). The observation of
these sections was carried out by a fluorescence micro-
scope (ECLIPSE E800, Nicon CO., Ltd.) with an appro-
priate filter (B-2A, Nicon CO., Ltd; excitation
wavelength 450–490 nm). At this time, the use of an
antifadant reagent was essential to prevent photobleach-
ing (fading of fluorescence). Figure 2 shows photo-
graphs of plant pulvini, which contains motor cell,
under a fluorescence microscope. The staining pattern
for the fluorescence of probe 2 was observed on the sur-
face of the motor cell (Fig. 2). No stain was observed in
the control section, which was treated with an aqueous
solution containing no 2 (Fig. 2). These results revealed
that the target cell for 1 is a motor cell, which plays a
central role in the plant leaf movement,15 as with leaf-
opening substances.16
for the 21st Century COE program of Tohoku Univer-
sity from the Ministry of Education, Culture, Sports,
Science, and Technology, Japan.
References and notes
1. Schildknecht, H. Angew. Chem., Int. Ed. Engl. 1983, 22,
695–710.
2. Satter, R. L.; Gorton, H. L.; Vogelmann, T. C. The
Pulvinus: Motor Organ for Leaf Movement, American
Society of Plant Physiologists, 1990.
3. Ueda, M.; Yamamura, S. Angew. Chem., Int. Ed. 2000, 39,
1400–1414.
4. Sugimoto, T.; Wada, Y.; Yamamura, S.; Ueda, M.
Tetrahedron 2001, 57, 9817–9825.
5. Ueda, M.; Shigemori-Suzuki, T.; Yamamura, S. Tetra-
hedron Lett. 1995, 36, 6267–6270.
6. Audran, G.; Mori, K. Eur. J. Org. Chem. 1998, 57–
62.
7. Urakawa, Y.; Sugimoto, T.; Sato, H.; Ueda, M. Tetra-
hedron Lett. 2004, 45, 5885–5888.
8. (a) Itzstein, M. V.; Jenkins, M. J.; Mocerino, M. Carbo-
hydr. Res. 1990, 208, 287–288; (b) Mitsunobu, O. Synthesis
1981, 1–28.
9. Amano, S.; Takemura, N.; Ohtsuka, M.; Ogawa, S.;
Chida, N. Tetrahedron 1999, 55, 3855–3870.
10. Martin, S. F.; Dodge, J. A. Tetrahedron Lett. 1991, 32,
3017–3020.
The binding of probe can be strongly correlated with
leaf-closing activity. Biologically inactive epi-2 did not
bind with motor cell at all. This result strongly suggested
that some receptor for 1, which is located in the motor
cell and specifically recognizes the stereochemistry of
probe 2, would be involved in the leaf-closing movement
of P. urinaria. In conclusion, we have succeeded in visu-
alization of the target cell of the leaf-closing substance in
the plant body by using novel probe 2. Some receptor
for 1 would be involved in the leaf-closing movement
in P. urinaria. To reveal a receptor protein of 1, the syn-
theses of photoaffinity labeling probes based on 1 are
now in progress.
11. Szarek, W. A.; Jones, J. K. N. Can. J. Chem. 1965, 43,
2345–2356.
12. Suzuki, K.; Maeta, H.; Suzuki, T.; Matsumoto, T.
Tetrahedron Lett. 1989, 30, 6879–6882.
13. Compound 2: 1H NMR (400 MHz, D2O, rt): 6.75 (1H, dd,
J = 10.0, 2.4 Hz), 6.47 (1H, br d, J = 10.0 Hz), 5.95 (1H, br
s), 5.15 (1H, ddd, J = 13.6, 5.2, 2.0Hz), 4.85 (1H, dddd,
J = 10.4, 5.2, 2.4, 2.0 Hz), 4.69 (1H, d, J = 8.0Hz), 3.91
(1H, dd, J = 12.4, 2.4 Hz), 3.72 (1H, dd, J = 12.4, 5.6 Hz),
3.50(1H, t, J = 8.8 Hz), 3.48 (1H, ddd, J = 8.8, 5.6,
2.4 Hz), 3.39 (1H, t, J = 8.8 Hz), 3.27 (1H, dd, J = 8.8,
8.0Hz), 3.03 (1H, dt, J = 10.4, 5.2, Hz), 1.78 (1H, dt,
J = 13.6, 10.4 Hz); 13C NMR (100 MHz, D2O, rt) 178.3,
167.1, 142.1, 122.1, 112.1, 103.5, 80.9, 77.5, 77.1, 75.7,
74.9, 71.1, 62.6, 39.5 ppm; HR ESI-MS (positive):
[M+H]+. Found: m/z 315.1054, C14H19O8 requires m/z
315.1080; IR (film) m: 3388, 1735, 1637, 1585, 1387 cmꢀ1
;
25
21
Acknowledgments
½aꢁD ꢀ30.0 (c 0.11, H2O); ½aꢁD ꢀ6.0( c 0.20, H2O) in
naturally occurring 1.
14. epi-2: 1H NMR (400 MHz, D2O, rt): 6.75 (1H, dd,
J = 10.0, 2.4 Hz), 6.47 (1H, br d, J = 10.0 Hz), 5.95 (1H,
br s), 5.15 (1H, ddd, J = 13.6, 5.2, 2.0Hz), 4.85 (1H, dddd,
This work was supported by the Ministry of Education,
Science, Sports and Culture (Japan) Grant-in-Aid for
Scientific Research (No. 15681013) and Grant-in-Aid

H. Sato et al. / Tetrahedron Letters 46 (2005) 5537–5541
5541
J = 10.4, 5.2, 2.4, 2.0 Hz), 4.69 (1H, d, J = 8.0Hz), 3.91
(1H, dd, J = 12.4, 2.4 Hz), 3.72 (1H, dd, J = 12.4, 5.6 Hz),
3.50(1H, t, J = 8.8 Hz), 3.48 (1H, ddd, J = 8.8, 5.6,
2.4 Hz), 3.39 (1H, t, J = 8.8 Hz), 3.27 (1H, dd, J = 8.8,
8.0Hz), 3.03 (1H, dt, J = 10.4, 5.2, Hz), 1.78 (1H, dt,
J = 13.6, 10.4 Hz); HR ESI-MS (positive): [M+H]+.
Found: m/z 315.1054, C14H19O8 requires m/z 315.1080.
15. Cote, G. G. Plant Physiol. 1995, 109, 729–734.
16. Ueda, M.; Wada, Y.; Yamamura, S. Tetrahedron Lett.
2001, 42, 3869–3872.