Synthetic inhibitor of leaf-closure that reveals the biological importance of leaf-movement for the survival of leguminous plants

Article abstract of DOI:10.1016/S0040-4020(03)00906-2

Nyctinasty has been known since pre-Christian era, whereas the question 'Why do leguminous plants sleep?' has always puzzled scientists. This paper gives a clue to the historical mystery: by using synthetic inhibitors for nyctinasty, we found that legumes cannot survive without nyctinasty.

Full text of DOI:10.1016/S0040-4020(03)00906-2

Tetrahedron 59 (2003) 5909–5917
Synthetic inhibitor of leaf-closure that reveals the biological
importance of leaf-movement for the survival of
leguminous plants
*
Eisuke Kato, Hideharu Nagano, Shosuke Yamamura and Minoru Ueda
Laboratory of Natural Products Chemistry, Faculty of Science and Technology, Keio University, Hiyoshi 3-14-1, Yokohama 2238522, Japan
Received 9 May 2003; revised 6 June 2003; accepted 6 June 2003
Abstract—Nyctinasty has been known since pre-Christian era, whereas the question ‘Why do leguminous plants sleep?’ has always puzzled
scientists. This paper gives a clue to the historical mystery: by using synthetic inhibitors for nyctinasty, we found that legumes cannot survive
without nyctinasty.
q 2003 Elsevier Ltd. All rights reserved.
1. Introduction
keeping system from moonlight, because moonlight falling
on leaves during the night might prevent accurate
measurement of night length.5 Leaves that were folded
together intercepted much less light. However, no experi-
mental evidence to date has been reported that explains the
biological significance of nyctinasty. Research has been
hindered because the leaf movement could not be inhibited.
Also, no mutant without nyctinasty has been reported so far.
This result strongly suggests that the mutant that lacks leaf-
movement is lethal and cannot survive. Thus, a genetic
approach to this issue will be difficult. Now we have
succeeded in inhibiting leaf movement using a synthetic
inhibitor of leaf closure based on the substance that
naturally induces leaf-opening.6 Our result provides the
first experimental evidence to answer the question ’Why do
leguminous plants sleep?’
Plants are unable to move from one place to another.
However, the folding and opening movements of the leaves
according to circadian rhythm have been widely observed in
leguminous plants (Fig. 1). This periodic leaf-movement is
called nyctinasty and has been known since the age of
Alexander the Great.1
On the other hand, the question ’Why do leguminous plants
sleep?’ has always puzzled many scientists studying
nyctinasty. Darwin concluded that nyctinasty might protect
plants from chilling or from frost.2 However, later studies
revealed that nyctinasty failed to provide soybeans with
significant protection from freezing. This result suggested
that Darwin’s results were probably artifactual.3,4 Bu¨nning
proposed that nyctinasty protected the photoperiodic time-
Schildknecht’s turgorin had been widely believed to be an
endogeneous factor controlling leaf movement,1 but we
revealed that it did not show any bioactivity under
physiological conditions.6,7 Nyctinasty is controlled by
two endogenous factors of contrasting bioactivities: leaf-
closing substance which makes the leaf close and leaf-
opening substance which makes the leaf open.6 The
bioactivity of these factors is extremely specific to the
original plant from which they were isolated. In the plant
body, the rhythm of nyctinasty is generated by change in
balance of concentrations between two leaf-movement
factors according to circadian rhythm.6 This change in
balance can be attributed to hydrolysis of the glucoside-type
leaf-movement factor into the corresponding aglycon by
b-glucosidase, whose activity is controlled by a biological
clock (Fig. 2).6
Figure 1. Nyctinastic leaf-movement of Cassia mimosoides L. (left: at
10:00 a.m., right: at 9:00 p.m.).
Keywords: leguminous plants; synthetic inhibitors; nyctinasty.
*
Corresponding author. Tel.: þ81-45-566-1702; fax: þ81-45-566-1697;
According to the mechanism shown in Figure 2, it was
e-mail: ueda@chem.keio.ac.jp
0040–4020/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0040-4020(03)00906-2

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E. Kato et al. / Tetrahedron 59 (2003) 5909–5917
cleaved by b-glucosidase. We synthesized thioglucoside
(9a and 9b) by using SN2 reaction with 1-bromo-2,3,4,6-
tetra-O-acetyl-b-D-glucopyranoside and thioenol (14)
which was prepared according to Cline’s method
(Scheme 1).11 The separation of the resulting two
geometrical isomers was achieved by HPLC equipped
with a recycle unit using a high-resolution column packed
with 3 mm particles (Cadenza CD-C18). The assignment of
geometry on the0 olefin was determined by NOE. However,
NOE between 2 -H in the glycon and 2-H in the aromatic
ring which was used for the assignment of geometry in 1 and
its analogs,7–9 was not observed because of the larger
atomic radius of the sulfur atom compared to that of oxygen.
Thus, we prepared methyl ester of 9a and 9b (16a and 16b)
by TMS-diazomethane, and the geometry was determined
from NOE (0.3%) between the methyl proton in the
methoxy group (3.75 ppm) and aromatic H-2 proton
(7.14 ppm) for 16a and NOE (0.7%) between methyl proton
in the methoxy group (3.83 ppm) and the olefinic
proton (7.864 ppm) for 16b (Scheme 2).
Figure 2. The control of internal concentration of 1 by a biological clock
that causes the nyctinastic leaf-movement.
expected that a structurally modified leaf-opening substance
that cannot be hydrolyzed by b-glucosidase would keep the
leaf open constantly, and, thus, inhibit the leaf-closure
(causing ‘insomnia’).
2. Results and discussion
Interestingly, the thioglucoside-type analogs of 1 (9a and
9b) did not show leaf-opening activity at all. There would be
two possibilities to explain this phenomenon. The first
hypothesis is that the oxygen atom in the glycosidic linkage
is important for the leaf-opening activity, and the second
one is that thioglucoside-type analogs (9a and 9b) would be
susceptible to the hydrolysis by b-glucosidase compared to
the glucoside-type ones. However, the latter was ruled out
by the hydrolysis of 9a and 9b by commercially available
b-glucosidase which is described later. These results
showed the importance of the oxygen atom in the glucoside
linkage of 1 for the development of leaf-opening activity.
We showed that some receptor of 1 would be concerned
with the recognition of 1 by plant motor cell.11 Our result
suggests that this receptor would recognize the oxygen atom
in the glycosidic linkage of 1. The results of structure-
activity relationship study are summarized in Table 1. From
these results, the potentially most effective leaf-movement
inhibitor that has both leaf-opening activity and resistance
against hydrolysis by b-glucosidase would be potassium
L-lespedezate (2a) and potassium L-isolespedezate (2b)
(Fig. 2).6,9,10 Thus, we synthesized 2a and 2b (Scheme 3).
Potassium lespedezate (1) was isolated as a glucoside-type
leaf-opening substance that is effective for the leaf of a
leguminous plant, Cassia mimosoides L.6 As shown in
Figure 2, the molecular design of a leaf-closure inhibitor
requires both retention of leaf-opening activity and
resistance to hydrolysis by b-glucosidase. Structure–
activity relationship studies were carried out using some
analogs of 1, such as those with various glycons (2, 4, and
5),8,9 one with a reduced double bond (6a and 6b),10 one
with a phenolic methyl ether (7),10 and one with a methyl
ester (8).8,9 Bioassays using them showed that structural
modification on the sugar moiety of 1 (2, 4, and 5) caused no
decrease in bioactivity (Table 1).8–10 In contrast, bioactivity
was greatly diminished by modification of the aglycon
moiety in 6a,6b, 7 and 8 (Table 1). These results strongly
suggest that the aglycon part of 1 would be recognized by
the receptor of 1, and the recognition of the glycon part in 1
would be very weak.
Also, we examined the thioglucoside-type analogs of 1 (9a
and 9b) that are expected to be best as an inhibitor because
those glucoside linkages containing heteroatom could not be
Coupling of L-acetobromogalactose (17) and 18 was carried
˚
out with AgOTf-Molecular sieves 4 A to give mainly
Table 1. Bioactivity of 1 and its analogs
b-glycoside (19). A racemic mixture of 18 was used in the
glycosidation reaction, and the following reactions were
carried out using both diastereomers without separation. A
trace amount of an a-anomer was also observed in the
reaction mixture; however, it was separated from 19 by
silica gel column chromatography. In the glycosidation
Compound
Bioactivity (mol/l)
Compound
Bioactivity (mol/l)
1a (or 1b)
2a (or 2b)
4
5
1£1026
1£1026
1£1026
1£1026
6a and 6b
7
8
9a and 9b
.1£1023
.1£1023
.1£1023
.1£1023

E. Kato et al. / Tetrahedron 59 (2003) 5909–5917
5911
Scheme 1. Synthesis of thioglucoside (9).
corresponding (Z)-23. Finally, potassium L-lespedezate (2a)
and L-isolespedezate (2b) were obtained from 23 on
treatment with KOH in MeOH at 08C. The separation of
2a and 2b was achieved by HPLC under acidic condition.
The reaction mixture was acidified by ion exchange resin
and separated with HPLC under acidic condition. The
isolated acids were then neutralized with potassium
carbonate to give 2a and 2b, respectively. The synthetic
route is summarized in Scheme 3. The geometry of the
double bond of 2a and 2b was confirmed by the NOE
0
experiments: NOE (1.7%) was observed between H2
(3.61 ppm) and H2 (7.76 ppm) in 2b. L-Glucose type
analogs (2a and 2b) were as effective as 1 in the bioassay.
Scheme 2. NOE experiment using 16a and 16b.
It was already known that 1a and 1b afforded a mixture of
these two compounds on standing at room temperature as an
aqueous solution.8 And no difference could be observed in
the result of bioassay using 1 and related analogs as a single
geometrical isomer or a mixture of two isomers.8,9 Thus, 2a
and 2b that are optical isomers of 1a and 1b should give the
same result in the bioassay. Thus, we used a mixture of 2a
and 2b in the following experiments.
reaction, both diastereomers at the a-position of the
carbonyl group were equally formed and the following
reactions were carried out using a mixture of both
diastereomers. After deprotection by catalytic hydrogen-
ation, the phenolic hydroxyl group of 20 was protected with
TBDMSCl, and following DDQ oxidation of 21 using
4 equiv. of DDQ for 42 h gave mainly one isomer, 22, which
was deprotected with nBu4NF in THF at 08C to give the
Scheme 3. Syntheses of biologically inactive analogs of 2.

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E. Kato et al. / Tetrahedron 59 (2003) 5909–5917
experiment using thioglucoside-type analogs (9a and 9b).
These results strongly suggested that 2a and 2b were not
hydrolyzed by b-glucosidase in the plant body during
bioassay and the leaves were kept open.
However, the death of leaf could be also attributable to the
potential toxic feature of 2. However, we have two
experimental proofs that 2 operates as a leaf-movement
inhibitor, and not as toxins in the plant body: Strong
correlation was observed between the leaf-opening activity
and the death of plant leaf.
Figure 3. The effect of a leaf-movement inhibitor (2a and 2b) on the leaf of
C. mimosoides (From the left, ‘insomnia’ at second day, fourth day, ninth
day, and control on the ninth day).
We synthesized two biologically inactive analogs of 2 from
21 and 23 (Scheme 3) such as one with a reduced double
bond (24a and 24b) and one with phenolic methyl ether (26a
and 26b). The geometry of the double bond was confirmed
by the NOE experiments: NOE (1.9%) was observed
Table 2. Time course change of the leaf-opening activities in 1, 2a and 2b
0
between H2 (3.39 ppm) and H2 (7.59 ppm) in 26b.
Compounds 24, 26a and 26b did not show leaf-opening
activity even at 1£1023 mol/l. When the leaves were treated
with (a) a diastereomeric mixture of 24, (b) 26a, and (c) 26b,
the leaves suffered no damage and did not wither and die
even after 2 weeks in all cases.
Status of the leaves at 21:00
First
day
Second
day
Third
day
Fourth
day
Fifth
day
Sixth
day
1
2a
2b
þþ
þþ
þþ
þþ
þþ
þþ
22
þþ
þþ
22
þþ
þþ
22
þþ
þþ
22
þþ
þþ
Moreover, 2 showed extremely specific bioactivity to the
leaf of C. mimosoides, that is a special feature also observed
in 1.6 And 2 showed no leaf-opening activity with leaves of
other plants, such as Mimosa pudica L., Albizzia julibrissin
Durazz, Aeschynomene indica L., and Phyllanthus urinaria
L., even at 3£1025 mol/l. Also, after 2 weeks, no damage to
the leaf was observed in these plants. This specific
bioactivity cannot be interpreted if 2 operated as some
toxin in the plant body of C. mimosoides. These results
strongly suggested that 2 operated as a leaf-movement
inhibitor in the plant body and caused withering and death
of C. mimosoides by inhibiting leaf closure.
Movement of the leaf was represented by the following marks: þþ
completely open; þ nearly open; 2 nearly closed; 22 completely closed.
Leaf-movement inhibitor (2a and 2b) showed novel
bioactivities in bioassay; the leaves detached from the
stem of C. mimosoides and placed in H2O continued the
circadian rhythmic leaf movement (Fig. 3). Both of leaf-
opening substance (1) and leaf-movement inhibitor (2a and
2b) can keep the leaves open even at night at 1£1026 mol/l.
When the leaves were treated with 3£1026 mol/l of 1, its
leaf-opening activity lasted for only 2 days (Table 2). After
that, the leaves closed at night again. This is because 1 is
gradually hydrolyzed into 3 within a few days in the plant
body (Fig. 2). On the other hand, leaf-opening activity of 2a
and 2b lasted even after 6 days (Table 2). The leaves treated
with 3£1026 mol/l of 2a and 2b remained open until it
completely withered and died after 9 days (Fig. 3). These
results clearly showed that leaf closure is essential for the
survival of this plant. This is because no mutant without
nyctinasty has been reported so far.
Our result gives an important clue for to the historic
mystery, ‘Why do leguminous plants sleep?’ We showed
that nyctinastic leaf-movement is essential for the survival
of leguminous plants by using a synthetic inhibitor of leaf-
closure that was designed on the chemical mechanism of
nyctinasty. This result clearly showed the effectiveness of a
chemical approach using a synthetic inhibitor of some
biological phenomenon that would be appropriate for
solving biological problems to which a genetic knockout
approach cannot be applied.
We also examined the hydrolysis of 2a and 2b by
commercially available b-glucosidase. We treated 2a and
2b with b-glucosidase to examine the reactivity against this
enzyme. The potential product of this reaction, 3, existed
mainly as an enol form in aqueous solutions: about 95% of 3
isomerized to an enol form within an hour under the reaction
conditions used for the enzymatic reaction. Therefore, we
observed the content of an enol form of 3 by the HPLC
analyses of the reaction mixture. After 30 min of incubation
in 0.1 M citrate buffer (pH 5.0) at 378C with commercially
available b-glucosidase, 0% of 2a and 2b was hydrolyzed
into its aglycon; on the other hand, 78% of 1 was easily
hydrolyzed into its aglycon under the same conditions.
Therefore, remarkable difference was observed in the rate of
enzymatic hydrolysis of 1 and 2a (or 2b) by using
b-glucosidase. Also, no hydrolysis was observed in the
3. Experimental
3.1. General
NMR (400 MHz) and 13C NMR (100 MHz) spectra were
recorded on a Jeol GX400 spectrometer or a Jeol ALPHA
400 spectrometer equipped with microprobe NTH3-FG
(NAROLAC Co., Ltd) using TMS in CDCl3 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 spectrometer,
using glycerol or m-nitrobenzylalcohol as a matrix. The IR
spectra were measured by JASCO FT/IR-410. The specific
rotations were measured by JASCO DIP-360 polarimeter.

E. Kato et al. / Tetrahedron 59 (2003) 5909–5917
5913
The HPLC purification was carried out with a JASCO LC
2000 system equipped with RV-2080-02 recycle unit using
COSMOCIL 5C18-AR column (f 20£250 mm) (Nakalai
Tesque Co. Ltd) or Cadenza CD-C18 column (f
10£250 mm) (Intact 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 mm pore size, 47 mm dia.) before use. Silica
gel column chromatography was performed on Silica Gel
60N (Kanto Chemical 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).
pyranosyl-thio)-3-p-benzyloxyphenyl acrylate (15).
Compound 14 (238.8 mg, 1.14 mmol) was dissolved in
DMF (11 ml) under argon atmosphere, and then, potassium
carbonate (314.6 mg, 2.28 mmol) was added to this
solution. After stirring for 10 min, 2,3,4,6-tetra-O-acetyl-
a-D-glucopyranosyl bromide (514.0 mg, 1.25 mmol) was
added to this solution. Then the solution was stirred for 4 h.
Then, 80 ml of water was added to this solution and this
mixture was extracted three times with 40 ml each of ethyl
acetate. The resulting organic layer was washed by brine
and dried over absolute sodium sulfate. After evaporation,
the residue was purified by silica gel column chromato-
graphy with CHCl3/MeOH¼20/1 to give 15 (374.7 mg,
61%).
3.1.1.
5-(4-Hydroxybenzylidene)-rhodanine
(12).
1H NMR (400 MHz, CDCl3, rt): 8.05 (1H, s), 7.85 (2H, d,
J¼8.8 Hz), 6.85 (2H, d, J¼8.8 Hz), 6.56 (1H, s, -OH), 5.20
(1H, dd, J¼9.6, 9.6 Hz), 5.09 (1H, dd, J¼9.6, 10.2 Hz), 5.08
(1H, dd, J¼9.6, 9.9 Hz), 4.84 (1H, d, J¼10.2 Hz), 4.18 (1H,
dd, J¼5.2, 12.4 Hz), 4.02 (1H, dd, J¼2.0, 12.4 Hz), 3.87
(3H, s), 3.63 (1H, ddd, J¼2.0, 5.2, 9.9 Hz), 2.02 (3H, s),
2.01 (3H, s), 2.00 (3H, s), 1.96 (3H, s) ppm; 13C NMR
(100 MHz, CDCl3, rt): 170.8, 170.2, 169.6, 169.4, 166.9,
158.0, 148.2, 133.7 (2C), 126.3, 118.2, 115.2 (2C), 84.8,
75.7, 73.9, 70.9, 68.2, 62.1, 52.9, 20.68, 20.66, 20.63
(2C) ppm; HR FAB MS (positive): [MþNa]þ found m/z
563.1199, C24H28O12SNa requires m/z 563.1199; IR (film)
4-Hydroxybenzaldehyde (10, 506.6 mg, 4.15 mmol),
rhodanine (11, 555.9 mg, 4.18 mmol) were dissolved in
acetic acid (20 ml). To this solution, potassium acetate
(2.44 g, 24.9 mmol) was added and refluxed for 18 h. After
adding 80 ml of water, the solution was cooled over ice bath
and resulting orange crystal was collected and dried to give
5-(4-hydroxybenzylidene)-rhodanine (12) (938.1 mg, 95%).
1H NMR (400 MHz, acetone-d6, rt): 7.56 (1H, s), 7.50 (2H,
d, J¼8.4 Hz), 7.02 (2H, d, J¼8.4 Hz) ppm; 13C NMR
(100 MHz, acetone-d6, rt): 195.7, 169.5, 160.9, 133.7 (2C),
133.0, 125.6, 122.4, 117.2 (2C) ppm; HR FAB MS
(positive): [MþH]þ found m/z 238.0012, C10H8O2NS2
requires m/z 237.9996; IR (film) n: 3427, 1695, 1564,
n: 3396, 1753, 1716, 1606, 1589, 1512 cm21
[a]2D6¼þ106.08 (c 1.00, CHCl3).
;
1516 cm21
.
3.1.5. Potassium (Z)-2-b-D-glucopyranosythio-3-p-
benzyloxy-phenyl-acrylate (9a and 9b). A mixture of
15a and 15b (98.3 mg, 0.128 mmol) was dissolved in 2 ml
of methanol, and then 1 ml of 4 M NaOH aq. was added to
this solution. After stirring for 12 h at rt, the reaction
mixture was acidified by Amberlite-IR120B (Hþ). After
filtration, the filtrate was dried up to give a mixture of
isomers of 9a and 9b (66.0 mg). This mixture (51.9 mg) was
purified by HPLC [COSMOCIL 5C18-AR, 1% AcOH/40%
MeOHaq.], and each fraction was neutralized by potassium
carbonate. Another purification by recycle-HPLC [Cadenza
CD-C18, 5% CH3CNaq.] gave 9a (3.7 mg, 9%) and 9b
(25.4 mg, 66%), respectively.
3.1.2. 3-p-Hydroxyphenyl-2-mercaptoacrylic acid (13).
Compound 12 (430.1 mg, 1.81 mmol) was dissolved in
18 ml of 15% NaOH aq. After refluxed for 10 min, the
solution was cooled to room temperature and neutralized
with 6N HCl aq. The resulting precipitate was filtered and
dried to give 13 (299.1 mg, 84%).
1H NMR (400 MHz, CD3OD, rt): 7.73 (1H, s), 7.54 (2H, d,
J¼8.6 Hz), 6.84 (2H, d, J¼8.6 Hz) ppm; 13C NMR
(100 MHz, CD3OD, rt): 168.5, 159.4, 135.9, 132.9 (2C),
127.9, 120.7, 116.3 (2C) ppm; HR FAB MS (negative):
[M2H]2 found m/z 195.0090, C9H7O3S requires m/z
195.0116; IR (film) n: 3313, 1682, 1604, 1570, 1508 cm21
.
Compound 9a. H NMR (400 MHz, D2O, rt): 7.21 (2H, d,
1
J¼8.8 Hz), 6.72 (2H, d, J¼8.8 Hz), 6.61 (1H, s), 4.55 (1H,
d, J¼10.0 Hz), 3.75 (1H, dd, J¼1.6, 12.8 Hz), 3.56 (1H, dd,
J¼5.2, 12.8 Hz), 3.39 (1H, t, J¼8.6, 8.6 Hz), 3.35–3.21
(3H, m) ppm; 13C NMR (100 MHz, D2O, rt): 176.8, 157.1,
133.2, 131.1 (2C), 129.4, 128.9, 116.9 (2C), 87.4, 81.6,
78.7, 73.4, 70.9, 62.5 ppm; HR FAB MS (negative):
[M2K]2 found m/z 357.0648, C15H17O8S requires m/z
3.1.3. Methyl 3-p-hydroxyphenyl-2-mercaptoacrylate
(14). Compound 13 (322.5 mg, 1.65 mmol) was dissolved
in methanol (5 ml). To this solution, acetyl chloride (1 ml)
in methanol (10 ml) was added, and then the solution was
stirred for 34 h. After evaporation, the residue was purified
with silica gel column chromatography with CHCl3/
MeOH¼10/1 to give 14 (249.7 mg, 72%).
357.0644; IR (film) n: 3298, 1568, 1510 cm21
[a]2D6¼þ13.18 (c 0.62, H2O).
;
1H NMR (400 MHz, CD3OD, rt): 7.69 (1H, s), 7.54 (2H, d,
J¼8.8 Hz), 6.84 (2H, d, J¼8.8 Hz), 3.84 (3H, s) ppm; 13C
NMR (100 MHz, CD3OD, rt): 167.5, 159.6, 136.4, 133.1
(2C), 127.7, 119.8, 116.4 (2C), 53.8 ppm; HR FAB MS
(positive): [MþH]þ found m/z 211.0408, C10H11O3S
requires m/z 211.0429; IR (film) n: 3392, 1672, 1603,
1
Compound 9b. H NMR (400 MHz, D2O, rt): 7.52 (2H, d,
J¼8.2 Hz), 7.36 (1H, s), 6.79 (2H, d, J¼8.2 Hz), 4.61 (1H,
d, J¼9.6 Hz), 3.66 (1H, broad d, J¼12.4 Hz), 3.49 (1H, dd,
J¼5.0, 12.4 Hz), 3.35 (1H, t, J¼8.8, 8.8 Hz), 3.26–3.17
(3H, m) ppm; 13C NMR (100 MHz, D2O, rt): 175.1, 157.6,
139.2, 133.1 (2C), 128.9, 128.3, 116.2 (2C), 86.3, 81.1,
78.2, 74.0, 70.3, 61.8 ppm; HR FAB MS (negative):
[M2K]2 found m/z 357.0648, C15H17O8S requires m/z
1577, 1508 cm21
.
3.1.4. Methyl (Z)-2-(20,30,40,60-tetra-O-acetyl-b-D-gluco-

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E. Kato et al. / Tetrahedron 59 (2003) 5909–5917
357.0644; IR (film) n: 3294, 1599, 1568, 1510 cm21
[a]2D6¼þ20.38 (c 1.00, H2O).
;
Compound 19a. 1H NMR (400 MHz, CDCl3, rt): 7.43–7.30
(5H, m), 7.09 (2H, d, J¼8.8 Hz), 6.86 (2H, d, J¼8.8 Hz),
5.20 (1H, dd, J¼9.5, 9.6 Hz), 5.07 (1H, dd, J¼9.6, 9.8 Hz),
5.06 (1H, dd, J¼8.0, 9.5 Hz), 5.03 (2H, s), 4.57 (1H, d,
J¼8.0 Hz), 4.55 (1H, t, J¼6.0, 6.0 Hz), 4.19 (1H, dd, J¼4.8,
12.2 Hz), 4.09 (1H, dd, J¼2.4, 12.2 Hz), 3.65–3.61 (1H,
m), 3.64 (3H, s), 3.06 (1H, dd, J¼6.0, 13.4 Hz), 3.00 (1H,
dd, J¼6.0, 13.4 Hz), 2.08 (3H, s), 2.02 (3H, s), 2.01 (3H, s),
1.98 (3H, s) ppm; 13C NMR (100 MHz, CDCl3, rt): 171.0,
170.6, 170.2, 169.6, 169.4, 157.6, 137.0, 130.6 (2C), 128.5
(2C), 128.0, 127.9, 127.4 (2C), 114.4 (2C), 99.0, 76.5, 72.4,
71.8, 70.8, 69.9, 68.4, 61.8, 51.8, 37.9, 20.7, 20.63, 20.62,
20.59 ppm; HR FAB MS (positive): [MþNa]þ found m/z
639.2084, C31H36O13Na requires m/z 639.2054; IR (film) n:
1753, 1512 cm21; [a]D26¼þ30.88 (c 1.00, CHCl3).
3.1.6. Methyl (E)-2-b-D-glucopyranosythio-3-p-benzyl-
oxy-phenylacrylate (16a). (E)-Thioglucoside (9a)
(12.0 mg, 0.034 mmol) was dissolved in MeOH and to
trimethylsilyldiazomethane (2 M solution in hexane, 50 ml)
was added to this solution under argon atmosphere at 08C.
After stirring for 1 h, additional trimethylsilyldiazomethane
(2 M solution in hexane, 40 ml) was added. After 1 h
stirring, reaction mixture was evaporated in vacuo and
purified by prep.TLC with 50% MeOHaq. to give methyl
ester (16a) (7.0 mg, 55%).
Compound 16a. 1H NMR (400 MHz, CD3OD, rt): 7.17 (1H,
s), 7.14 (2H, d, J¼8.8 Hz), 6.73 (2H, d, J¼8.8 Hz), 4.46
(1H, d, J¼9.2 Hz), 3.81 (1H, dd, J¼2.2, 12.0 Hz), 3.75 (3H,
s), 3.61 (1H, dd, J¼5.2, 12.0 Hz), 3.36–3.25 (4H, m); 13C
NMR (100 MHz, CD3OD, rt): 170.9, 159.7, 142.6, 131.1
(2C), 127.6, 121.3, 116.3 (2C), 88.1, 82.2, 79.4, 73.6, 71.2,
62.9, 53.0 ppm; HR FAB MS (negative): [M2H]2 found
m/z 371.0796, C16H19O8S requires m/z 371.0801; IR (film)
n: 3379, 1703, 1608, 1512 cm21; [a]D21¼þ42.58 (c 0.70,
MeOH).
Compound 19b. 1H NMR (400 MHz, CDCl3, rt): 7.43–7.30
(5H, m), 7.09 (2H, d, J¼8.8 Hz), 6.88 (2H, d, J¼8.8 Hz),
5.10 (1H, dd, J¼9.4, 9.4 Hz), 5.06–5.00 (2H, m), 5.04 (2H,
s), 4.44 (1H, d, J¼7.6 Hz), 4.16 (1H, dd, J¼5.3, 12.2 Hz),
4.11 (1H, dd, J¼4.4, 9.1 Hz), 4.07 (1H, dd, J¼2.5, 12.2 Hz),
3.72 (3H, s), 3.62 (1H, ddd, J¼2.4, 5.3, 9.6 Hz), 2.97 (1H,
dd, J¼9.1, 14.5 Hz), 2.90 (1H, dd, J¼4.4, 14.5 Hz), 2.10
(3H, s), 2.01 (3H, s), 1.98 (3H, s), 1.78 (3H, s) ppm; 13C
NMR (100 MHz, CDCl3, rt): 171.8, 170.6, 170.2, 169.3,
169.0, 157.6, 136.9, 130.3 (2C), 128.6 (3C), 128.0, 127.4
(2C), 114.7 (2C), 101.3, 81.5, 72.7, 71.9, 70.9, 69.9, 68.2,
61.8, 52.1, 37.8, 20.7, 20.6 (2C), 20.5 ppm; HR FAB MS
(positive): [MþNa]þ found m/z 639.2078, C31H36O13Na
3.1.7. Methyl (Z)-2-b-D-glucopyranosythio-3-p-benzyl-
oxy-phenylacrylate (16b). (Z)-Thioglucoside (9b)
(6.6 mg, 0.018 mmol) was dissolved in MeOH and to
trimethylsilyldiazomethane (2 M solution in hexane, 23 ml)
was added to this solution under argon atmosphere at 08C.
After stirring for 12 h, reaction mixture was evaporated in
vacuo and purified by prep.TLC with 50% MeOHaq. to give
methyl ester (16b) (2.4 mg, 35%) with recovered 9b
(3.2 mg, 48%).
requires m/z 639.2054; IR (film) n: 1755, 1512 cm21
[a]2D6¼20.78 (c 1.00, CHCl3).
;
3.1.9. Methyl 2-(20,30,40,60-tetra-O-acetyl-b-L-glucopyra-
nosy-loxy)-3-p-hydroxyphenylpropionate (20a and 20b).
To a solution of 19 (353.5 mg, 0.574 mmol) in EtOAc/
MeOH¼1/1 (6 ml), 10% Pd–C was added and stirred at rt
under hydrogen atomosphere for 9 h. The reaction mixture
was filtered on celite and the filtrate was concentrated in
vacuo. The residue was purified with silica gel column
chromatography (n-hexane/EtOAc¼2/3) to give a mixture
of 20a and 20b (293.4 mg£97%). From 25.9 mg of this
mixture, each diastereomer was separated with preparative
TLC (benzene/acetone¼5/1) to give 20a (15.7 mg, 59%)
and 20b (8.8 mg, 33%), respectively.
1
Compound 16b. H NMR (400 MHz, CD3OD, rt): 7.864
(1H, s), 7.862 (2H, d, J¼8.6 Hz), 6.80 (2H, d, J¼8.6 Hz),
4.72 (1H, d, J¼9.2 Hz), 3.83 (3H, s), 3.72 (1H, dd, J¼2.4,
12.4 Hz), 3.58 (1H, dd, J¼5.4, 12.4 Hz), 3.34–3.24 (3H,
m), 3.15 (1H, ddd, J¼2.4, 5.4, 9.2 Hz); 13C NMR
(100 MHz, CD3OD, rt): 169.2, 160.6, 146.4, 134.6 (2C),
127.1, 120.5, 116.1 (2C), 87.2, 82.1, 79.4, 75.3, 71.2, 62.7,
53.3 ppm; HR FAB MS (positive): [MþH]þ found m/z
373.0928, C16H21O8S requires m/z 373.0957; IR (film) n:
3335, 1668, 1606, 1568, 1508 cm21; [a]D20¼þ59.78 (c 1.00,
MeOH).
Compound 20a. 1H NMR (400 MHz, CDCl3, rt): 7.03 (2H,
d, J¼8.6 Hz), 6.72 (2H, d, J¼8.6 Hz), 5.41 (1H, s, –OH),
5.20 (1H, dd, J¼9.3, 9.5 Hz), 5.07 (1H, dd, J¼9.5, 10.0 Hz),
5.05 (1H, dd, J¼8.1, 9.3 Hz), 4.56 (1H, d, J¼8.1 Hz), 4.52
(1H, dd, J¼5.4, 6.1 Hz), 4.17 (1H, dd, J¼4.5, 12.5 Hz), 4.08
(1H, dd, J¼2.3, 12.5 Hz), 3.66 (3H, s), 3.63 (1H, ddd,
J¼2.3, 4.5, 10.0 Hz), 3.04 (1H, dd, J¼5.4, 14.3 Hz), 2.98
(1H, dd, J¼6.1, 14.3 Hz), 2.09 (3H, s), 2.02 (3H, s), 2.013
(3H, s), 2.005 (3H, s) ppm; 13C NMR (100 MHz, CDCl3, rt):
171.0, 170.7, 170.1, 169.6, 169.4, 154.5, 130.7 (2C), 127.7,
114.9 (2C), 99.1, 76.7, 72.4, 71.7, 70.9, 68.4, 61.9, 51.9,
38.0, 20.8, 20.72, 20.69, 20.66 ppm; HR FAB MS
(positive): [MþNa]þ found m/z 549.1591, C24H30O13Na
3.1.8. Methyl 2-(20,30,40,60-tetra-O-acetyl-b-L-glucopyra-
nosyl-oxy)-3-p-benzyloxyphenylpropionate (19). To a
suspension of 2,3,4,6-tetra-O-acetyl-b-L-glucopyranosyl-
bromide (17) (789.3 mg, 1.92 mmol), methyl 3-p-benzyl-
oxyphenyl-2-hydroxypropionate
(18)
(377.2 mg,
˚
1.32 mmol), dry powdered molecular sieves 4 A (2.0 g) in
CH2Cl2 (19 ml), and silver triflate (0.61 g£2.37 mmol) were
added at 08C under argon atmosphere. The reaction mixture
was stirred for 18 h at room temperature, and then, filtered
on celite and the filtrate was concentrated in vacuo. The
residue was separated with silica gel column chromato-
graphy (n-hexane/EtOAc¼11/9) to give a mixture of
diastereomers of 19 (306.8 mg£38%). From 36.9 mg of
19, each diastereomer was separated with preparative TLC
(benzene/acetone¼10/1) to give 19a (16.1 mg, 17%), 19b
(14.0 mg, 14%), respectively.
requires m/z 549.1584; IR (film) n: 3438, 1753, 1518 cm21
[a]2D5¼þ23.48 (c 1.00, CHCl3).
;
Compound 20b. 1H NMR (400 MHz, CDCl3, rt): 7.04 (2H,
d, J¼8.6 Hz), 6.74 (2H, d, J¼8.6 Hz), 5.25 (1H, s, –OH),

E. Kato et al. / Tetrahedron 59 (2003) 5909–5917
5915
5.11 (1H, dd, J¼8.8, 9.1 Hz), 5.04 (1H, dd, J¼8.3, 8.8 Hz),
5.03 (1H, dd, J¼9.1, 9.3 Hz), 4.46 (1H, d, J¼8.3 Hz), 4.15
(1H, dd, J¼5.1, 12.3 Hz), 4.11 (1H, dd, J¼4.6, 9.3 Hz), 4.08
(1H, dd, J¼2.4, 12.3 Hz), 3.72 (3H, s), 3.62 (1H, ddd,
J¼2.4, 5.1, 9.3 Hz), 2.96 (1H, dd, J¼9.3, 14.1 Hz), 2.89
(1H, dd, J¼4.6, 14.1 Hz), 2.10 (3H, s), 2.01 (3H, s), 1.98
(3H, s), 1.83 (3H, s) ppm; 13C NMR (100 MHz, CDCl3, rt):
171.8, 170.6, 170.2, 169.3, 169.0, 154.5, 130.4 (2C), 128.2,
115.2 (2C), 101.2, 81.5, 72.7, 71.9, 71.0, 68.2, 61.9, 52.1,
37.9, 20.8, 20.64 (2C), 20.57 ppm; HR FAB MS (positive):
[MþNa]þ found m/z 549.1599, C24H30O13Na requires m/z
549.1584; IR (film) n: 3419, 1753, 1518 cm21; [a]D25¼þ2.78
(c 0.81, CHCl3).
3.1.11. Methyl (Z)-2-(20,30,40,60-tetra-O-acetyl-b-L-gluco-
pyranosyloxy)-3-t-butyldimethylsiloxyphenyl-2-acrylate
(22). To a solution of 21 (332.6 mg, 0.519 mmol) in 1,4-
dioxane (5 ml) was added 2,3-dichloro-5,6-dicyano-1,4-
benzoquinone (DDQ, 482.3 mg, 2.12 mmol) and refluxed
for 42 h under argon atmosphere. The reaction mixture was
mixed with sat. NaHCO3aq. and then extracted with diethyl
ether. The organic layer was washed with saturated NaClaq.
and dried over anhydrous Na2SO4. After evaporation, the
residue was purified by silica gel column chromatography to
give 22 (295.6 mg, 89%).
1H NMR (400 MHz, CDCl3, rt): 7.67 (2H, d, J¼8.8 Hz),
7.05 (1H, s), 6.81 (2H, d, J¼8.8 Hz), 5.30–2.25 (3H, m),
5.18–5.14 (1H, m), 4.13 (1H, dd, J¼4.4, 12.4 Hz), 4.01
(1H, dd, J¼2.6, 12.4 Hz), 3.83 (3H, s), 3.67 (1H, ddd,
J¼2.6, 4.4, 10.0 Hz), 2.03 (6H, s), 2.02 (3H, s), 1.98 (3H, s),
0.99 (9H, s), 0.22 (6H, s) ppm; 13C NMR (100 MHz, CDCl3,
rt): 170.5, 170.2, 169.6, 169.5, 164.2, 157.0, 138.1 (2C),
132.5, 126.9, 125.8, 119.9 (2C), 99.2, 72.8, 71.8, 71.6, 68.3,
61.5, 52.2, 25.6 (3C), 20.65, 20.62, 20.59, 20.52, 18.2,
24.38, 24.42 ppm; HR FAB MS (positive): [MþNa]þ
found m/z 661.2282, C30H42O13SiNa requires m/z 661.2292;
IR (film) n: 1757, 1718, 1601, 1508 cm21; [a]D25¼þ1.98 (c
1.00, CHCl3).
3.1.10. Methyl 2-(20,30,40,60-tetra-O-acetyl-b-L-gluco-
pyranosyl-oxy)-3-p-t-butyldimethylsiloxyphenylpropio-
nate (21a and 21b). To a solution of 20 (293.4
mg£0.558 mmol) in DMF (5 ml), TBDMSCl (179.8 mg,
1.19 mmol), imidazole (153.1 mg£2.25 mmol), and DMAP
(7.3 mg, 0.060 mmol) were added at 08C under argon
atmosphere and then the reaction mixture was stirred for
2 h. After the addition of water (35 ml), the reaction mixture
was partitioned with EtOAc (20 ml) three times. The
organic layer was washed with saturated NaClaq. and
dried over absolute Na2SO4 and concentrated in vacuo. The
residue was purified with silica gel column chromatography
(n-hexane/EtOAc¼3/2) to give 21 (336.6 mg, 94%). From
29.4 mg of 21, each diastereomer was separated with
preparative TLC (benzene/acetone¼10/1) to give 21a
(14.9 mg, 48%) and 21b (8.5 mg, 27%), respectively.
3.1.12. Methyl (Z)-2-(20,30,40,60-tetra-O-acetyl-b-L-gluco-
pyranosyloxy)-3hydroxyphenyl-2-acrylate (23). To a
solution of 22 (290.0 mg, 0.454 mmol) in dry tetrahydro-
furan (THF, 4 ml) was added tetra-n-butyl ammonium
fluoride (1.0 M in THF, 0.60 ml, 0.60 mmol) at 08C for
10 min. After adding water, the reaction mixture was
partitioned with EtOAc, and washed with NaHCO3aq. The
organic layer was evaporated in vacuo and the residue was
purified by preparative TLC (CHCl3/acetone¼10/1) to
afford 23 (243.2 mg, quant.).
Compound 21a. 1H NMR (400 MHz, CDCl3, rt): 7.02 (2H,
d, J¼8.6 Hz), 6.72 (2H, d, J¼8.6 Hz), 5.21 (1H, dd, J¼9.5,
9.6 Hz), 5.08 (1H, dd, J¼9.6, 10.0 Hz), 5.07 (1H, dd, J¼8.0,
9.5 Hz), 4.562 (1H, d, J¼8.0 Hz), 4.556 (1H, t, J¼5.8 Hz),
4.21 (1H, dd, J¼8.8, 12.0 Hz), 4.10 (1H, dd, J¼2.4,
12.0 Hz), 3.63 (1H, ddd, J¼2.4, 8.8, 10.0 Hz), 3.62 (3H,
s), 3.04 (1H, dd, J¼5.8, 14.2 Hz), 3.00 (1H, dd, J¼5.8,
14.2 Hz), 2.09 (3H, s), 2.019 (3H, s), 2.015 (3H, s), 2.00
(3H, s), 0.97 (9H, s), 0.17 (6H, s) ppm; 13C NMR (100 MHz,
CDCl3, rt): 170.9, 170.5, 170.1, 169.4, 169.3, 154.4, 130.5
(2C), 128.3, 119.7 (2C), 99.0, 76.5, 72.4, 71.8, 70.8, 68.4,
61.8, 51.8, 38.1, 25.7 (3C), 20.8, 20.72, 20.70, 20.66, 18.25,
24.4 (2C) ppm; HR FAB MS (positive): [MþNa]þ found
m/z 663.2458, C30H44O13SiNa requires m/z 663.2449; IR
(film) n: 1755, 1510 cm21; [a]D27¼þ27.08 (c 1.00, CHCl3).
Compound 21b. 1H NMR (400 MHz, CDCl3, rt): 7.03 (2H,
d, J¼8.6 Hz), 6.73 (2H, d, J¼8.6 Hz), 5.11 (1H, dd, J¼9.4,
9.5 Hz), 5.05 (1H, dd, J¼7.8, 9.5 Hz), 5.03 (1H, dd, J¼9.4,
9.5 Hz), 4.45 (1H, d, J¼7.8 Hz), 4.16 (1H, dd, J¼5.3,
12.4 Hz), 4.11 (1H, dd, J¼4.6, 9.0 Hz), 4.07 (1H, dd, J¼2.5,
12.4 Hz), 3.71 (3H, s), 3.62 (1H, ddd, J¼2.5, 5.3, 9.5 Hz),
2.95 (1H, dd, J¼9.0, 14.6 Hz), 2.89 (1H, dd, J¼4.6,
14.6 Hz), 2.10 (3H, s), 2.01 (3H, s), 1.98 (3H, s), 1.85
(3H, s), 0.97 (9H, s), 0.176 (3H, s), 0.173 (3H, s) ppm; 13C
NMR (100 MHz, CDCl3, rt): 171.7, 170.5, 170.1, 169.2,
168.9, 154.4, 130.2 (2C), 128.8, 119.9 (2C), 101.2, 81.4,
72.7, 71.9, 71.0, 68.2, 61.8, 52.1, 38.0 25.8 (3C), 20.8, 20.65
(2C), 20.60, 18.2, 24.4 (2C) ppm; HR FAB MS (positive):
[MþNa]þ found m/z 663.2458, C30H44O13SiNa requires
m/z 663.2449; IR (film) n: 1757, 1512 cm21; [a]D27¼þ2.98
(c 0.85, CHCl3).
1H NMR (400 MHz, CDCl3, rt): 7.67 (2H, d, J¼8.8 Hz),
7.06 (1H, s), 6.80 (2H, d, J¼8.8 Hz), 5.68 (1H, s, –OH),
5.29–5.26 (3H, m), 5.18–5.12 (1H, m), 4.11 (1H, dd,
J¼12.2, 4.9 Hz), 4.01 (1H, dd, J¼12.2, 2.4 Hz), 3.84 (3H,
s), 3.66 (1H, ddd, J¼9.8, 4.4, 2.4 Hz), 2.04 (3H, s), 2.03
(6H, s), 2.02 (3H, s) ppm; 13C NMR (100 MHz, CDCl3, rt):
170.5, 170.0, 169.7, 169.4, 164.1, 156.8, 137.9, 132.7,
130.8, 126.9, 125.2, 115.2, 99.3, 72.8, 71.8, 71.6, 68.4, 61.6,
20.8, 20.7, 20.6 ppm; HR FAB MS (positive): [MþNa]þ
found m/z 547.1444, C24H28O13Na requires m/z 547.1428;
IR (film) n: 3419, 1756, 1717, 1639, 1606, 1585,
1514 cm21; [a]1D9¼þ4.38 (c 0.39, CHCl3).
3.1.13. Potassium L-isolespedezate (2a) and potassium L-
lespedezate (2b). Compound 23 (72.0 mg, 0.137 mmol)
was dissolved in metahol/H2O¼3/1 (6 ml). To this solution
was added 3.0 M KOHaq. (0.5 ml, 0.15 mmol) at 08C, and
then the reaction mixture was stirred for 40 min. The
reaction mixture was concentrated and then acidified by
Amberlite IR-120B(Hþ) column. The eluate was concen-
trated and purified by using HPLC[COSMOSIL 5C18-
AR(f 20£250 mm), 1% AcOH/30%MeOHaq.] to afford
L-lespedezic acid (5.7 mg) and L-isolespedezic acid
(34.9 mg), respectively. Each acid was neutralized by
K2CO3aq. to afford potassium L-lespedezate (2a) (6.3 mg,
12%) and potassium L-isolespedezate (2b) (38.8 mg, 75%).

5916
E. Kato et al. / Tetrahedron 59 (2003) 5909–5917
Compound 2a. 1H NMR (400 MHz, D2O, 308C): 7.23 (2H,
d, J¼8.3 Hz), 6.83 (2H, d, J¼8.3 Hz), 6.19 (1H, s), 4.94
(1H, d, J¼7.8 Hz), 3.96 (1H, dd, J¼12.2, 2.0 Hz), 3.77 (1H,
dd, J¼12.2, 5.9 Hz), 3.61 (1H, t, J¼9.0 Hz), 3.57 (1H, m),
3.53 (1H, dd, J¼9.3, 7.8 Hz), 3.48 (1H, t, J¼9.0 Hz) ppm;
13C NMR (100 MHz, D2O, 308C): 172.5, 156.6, 149.7,
130.2, 126.7, 116.4, 109.6, 101.0, 77.0, 76.3, 73.7, 70.3,
61.5 ppm; HR FAB MS (negative): [M2K]2 found m/z
341.0914, C15H17O9 requires m/z 341.0872; IR (film) n:
3247, 1634, 1593, 1513 cm21; [a]D21¼þ40.98 (c 0.57, H2O).
0.130 mmol) was dissolved in DMF (1.3 ml) and K2CO3
(34.3 mg, 0.249 mmol) was added to this solution. After
adding iodomethane (50 ml, 0.942 mmol) at 08C under
argon atmosphere, the solution was stirred for 2 h. To this
solution was added water, and then this mixture was
extracted by EtOAc. The organic layer was washed by
saturated NaClaq., dried over Na2SO4, and evaporated to
dryness in vacuo. The residue was separated by preparative
TLC (n-hexane/EtOAc¼1/1) to give methyl ether (25
65.8 mg, 94%).
Compound 2b. 1H NMR (400 MHz, D2O, 308C): 7.76 (2H,
d, J¼8.3 Hz), 6.93 (2H, d, J¼8.3 Hz), 6.76 (1H, s), 5.07
(1H, d, J¼7.4 Hz), 3.82 (1H, dd, J¼12.7, 2.0 Hz), 3.70 (1H,
J¼12.7, 5.0 Hz), 3.61 (1H, dd, J¼9.0, 7.4 Hz), 3.56 (1H, t,
J¼9.0 Hz), 3.47 (1H, t, J¼9.0 Hz), 3.39 (1H, ddd, J¼9.0,
5.0, 2.0 Hz) ppm; 13C NMR (100 MHz, D2O, 308C): 172.1,
156.5, 145.9, 132.4, 126.7, 120.8, 116.1, 101.9, 77.2, 76.7,
74.6, 70.2, 61.3 ppm; HR FAB MS (negative): [M2K]2
found m/z 341.0914, C15H17O9 requires m/z 341.0872; IR
(film) n: 3290, 1642, 1574, 1511 cm21; [a]D20¼250.78 (c
1.0, H2O).
1H NMR (400 MHz, CDCl3, rt): 7.73 (2H, d, J¼8.8 Hz),
7.07 (1H, s), 6.88 (2H, d, J¼8.8 Hz), 5.30–5.26 (3H, m),
5.17–5.12 (1H, m), 4.11 (1H, dd, J¼4.8, 12.4 Hz), 4.01
(1H, dd, J¼2.4, 12,4 Hz), 3.84 (3H, s), 3.83 (3H, s), 3.67
(1H, ddd, J¼2.4, 4.8, 9.8 Hz), 2.04 (3H, s), 2.03 (3H, s),
2.02 (3H, s), 1.96 (3H, s) ppm; 13C NMR (100 MHz, CDCl3,
rt): 170.3, 170.0, 169.5, 169.3, 164.0, 160.4, 137.9, 132.5
(2C), 126.9, 125.2, 113.6 (2C), 99.2, 72.7, 71.8, 71.6, 68.3,
61.6, 55.3, 52.2, 20.7, 20.64 (2C), 20.57 ppm; HR FAB MS
(positive): [MþNa]þ found m/z561.1573, C25H30O13Na
requires m/z 561.1584; IR (film) n: 1755, 1716, 1604,
1512 cm21; [a]2D6¼þ3.38 (c 1.0, CHCl3).
3.1.14. Potassium 2-b-L-glucopyranosyloxy-3-p-
hydroxyphenyl-propionate (24a and 24b). A mixture of
21a and 21b (101.0 mg, 0.192 mmol) was dissolved in
MeOH/H2O¼3/1 (1 ml). To this solution was added 4 M
KOHaq. (0.5 ml, 2.0 mmol) at 08C. After stirring for 19 h,
the solution was acidified by Amberlite IR-120, filtered, and
then concentrated in vacuo. The resulting residue was
neutralized by K2CO3, purified by HPLC (Cosmocil 5C18-
AR, 1% AcOH–30% MeOH aq.), and neutralized by
K2CO3 again to give a mixture of 24a (16.5 mg, 25%) and
24b (37.1 mg, 56%).
3.1.16. Potassium-2-b-L-glucopyranosyloxy-3methoxy-
phenyl-2-acrylate (26a and 26b). To a solution of 25
(54.4 mg, 0.101 mmol) in MeOH/H2O¼3/1 (1 ml) 4 M
KOHaq. (0.3 ml, 0.12 mmol) was added at 08C. The
reaction mixture was stirred at rt for 19 h and then acidified
with Amberlite IR-120 B (Hþ) column chromatography.
The eluate was concentrated in vacuo, purified by HPLC
using Cosmosil 5C18-AR column (f 20£mm) with 30%
MeOHaq. containing 1% AcOH, and neutralized with
K2CO3, to give 26a (1.9 mg, 5%) and 26b (18.4 mg,
51%), respectively.
Compound 24a. 1H NMR (400 MHz, D2O, rt): 7.19 (2H, d,
J¼8.0 Hz), 6.82 (2H, d, J¼8.0 Hz), 4.30 (1H, d, J¼7.8 Hz),
4.17 (1H, dd, J¼5.2, 7.4 Hz), 3.80 (1H, dd, J¼2.4, 12.4 Hz),
3.67 (1H, J¼5.0, 12.4 Hz), 3.39 (1H, dd, J¼8.0, 8.8 Hz),
3.37 (1H, dd, J¼8.0, 9.6 Hz), 3.29 (1H, dd, J¼7.8, 8.8 Hz),
3.27 (1H, ddd, J¼2.4, 5.0, 9.6 Hz), 2.97 (1H, dd, J¼5.2,
14.4 Hz), 2.91 (1H, dd, J¼7.4, 14.4 Hz) ppm; 13C NMR
(100 MHz, D2O, rt): 181.3, 155.4, 132.3 (2C), 130.9, 116.7
(2C), 103.6, 83.4, 77.3, 77.1, 74.6, 70.9, 62.0, 39.1 ppm; HR
FAB MS (negative): [M2K]2 found m/z 343.1042,
C15H19O9 requires m/z 343.1029; IR (film) n: 3307, 1597,
1516 cm21; [a]2D6¼þ22.18 (c 1.0, H2O).
Compound 26a. 1H NMR (400 MHz, D2O, rt): 7.06 (2H, d,
J¼8.6 Hz), 6.73 (2H, d, J¼8.6 Hz), 5.97 (1H, s), 4.72 (1H,
d, J¼7.8 Hz), 3.72 (1H, dd, J¼2.0, 12.8 Hz), 3.61 (3H, s),
3.42 (1H, dd, J¼5.8, 12.8 Hz), 3.37 (1H, dd, J¼9.4, 9.4 Hz),
3.34 (1H, m), 3.30 (1H, dd, J¼7.8, 9.4 Hz), 3.24 (1H, dd,
J¼9.4, 9.4 Hz) ppm; 13C NMR (100 MHz, D2O, rt): 173.2,
159.2, 151.0, 130.8 (2C), 129.0, 115.4 (2C), 109.8, 101.6,
77.7, 77.0, 74.4, 71.0, 62.2, 56.9 ppm; HR FAB MS
(negative): [M2K]2 found m/z355.1012, C16H19O9
requires m/z355.1029; IR (film) n: 3269, 1595,
1512 cm21; [a]2D6¼þ41.08 (c 0.63, H2O).
Compound 24b. 1H NMR (400 MHz, D2O, rt): 7.17 (2H, d,
J¼9.0 Hz), 6.80 (2H, d, J¼9.0 Hz), 4.43 (1H, d, J¼7.8 Hz),
4.41 (1H, dd, J¼6.1, 7.2 Hz), 3.85 (1H, dd, J¼2.0, 12.2 Hz),
3.66 (1H, J¼5.2, 12.2 Hz), 3.46 (1H, dd, J¼9.0, 9.0 Hz),
3.38 (1H, ddd, J¼2.0, 5.2, 9.2 Hz), 3.36 (1H, dd, J¼9.0,
9.2 Hz), 3.30 (1H, dd, J¼7.8, 9.0 Hz), 3.00 (1H, dd, J¼6.1,
13.2 Hz), 2.95 (1H, dd, J¼7.2, 13.2 Hz) ppm; 13C NMR
(100 MHz, D2O, rt): 180.5, 156.2, 132.3 (2C), 130.3, 116.9
(2C), 103.2, 82.7, 77.5, 77.2, 74.8, 71.0, 62.2, 39.6 ppm; HR
FAB MS (negative): [M2K]2 found m/z 343.1008,
C15H19O9 requires m/z 343.1029; IR (film) n: 3222, 1597,
1512 cm21; [a]2D6¼þ14.78 (c 1.0, H2O).
Compound 26b. 1H NMR (400 MHz, D2O, rt): 7.59 (2H, d,
J¼8.8 Hz), 6.80 (2H, d, J¼8.8 Hz), 6.55 (1H, s), 4.85 (1H,
d, J¼7.6 Hz), 3.65 (3H, s), 3.59 (1H, dd, J¼2.2, 12.6 Hz),
3.47 (1H, dd, J¼5.2, 12.6 Hz), 3.39 (1H, dd, J¼7.6, 9.2 Hz),
3.34 (1H, dd, J¼9.0, 9.2 Hz), 3.24 (1H, dd, J¼9.0, 9.4 Hz),
3.16 (1H, ddd, J¼2.2, 5.2, 9.4 Hz) ppm; 13C NMR
(100 MHz, D2O, rt): 172.9, 160.5, 147.2, 133.1 (2C),
128.3, 121.3, 115.6 (2C), 102.7, 77.9, 77.5, 75.3, 70.9,
62.1, 57.0 ppm; HR FAB MS (negative): [M2K]2 found
m/z355.1012, C16H19O9 requires m/z355.1029; IR (film) n:
3303, 1604, 1577, 1510 cm21; [a]D20¼254.98 (c 1.0, H2O).
3.2. Bioassay
3.1.15. Methyl (Z)-2-b-L-glucopyranosyloxy-3methoxy-
phenyl-2-acrylate (25). Compound 23 (68.3 mg,
C. mimosoides, which was used for the bioassay, was grown

E. Kato et al. / Tetrahedron 59 (2003) 5909–5917
5917
in the greenhouse of Keio University. The young leaves
Acknowledgements
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
25–308C 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 around 10:00 a.m. 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. Other nyctinastic plants, Mimosa pudica L.,
Albizzia julibrissin Durazz, Aeschynomene indica L. and
Phyllanthus urinaria L.,used in the bioassay were also
grown in the greenhouse of Keio University.
This work was supported by the Ministry of Education,
Science, Sports and Culture (Japan) Grant-in-Aid for
Scientific Research (No. 1302467 and 12680598), Pioneer-
ing Research Project in Biotechnology given by the
Ministry of Agriculture, Forestry and Fisheries, Kato
Memorial Bioscience Foundation, Naito Foundation, Kurata
Foundation, Moritani Foundation. 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.
3.3. Bioassay using leaf-movement inhibitors
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