Synthetic probe compounds for bioorganic studies of nyctinasty, based on the leaf-opening substance of Lespedeza cuneata G. Don

Article abstract of DOI:10.1016/S0040-4020(00)00421-X

In a previous paper, the syntheses of potassium galactolespedezate (1) and potassium galactoisolespedezate (2), artificial leaf-opening substances useful for bioorganic studies of nyctinasty were reported. The fluorescent probe compounds, fluorescence-labeled galactoisolespedezate (3) and iodo-one (4), designed on the basis of 1 and 2 were prepared. Especially, compound 3 was bioactive at 5x10^-5 M, one-fiftieth as strong as the natural leaf-opening substance. This fluorescent probe would be useful for bioorganic studies of nyctinasty. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4020(00)00421-X

TETRAHEDRON
Pergamon
Tetrahedron 56 (2000) 5123±5130
Synthetic Probe Compounds for Bioorganic Studies of Nyctinasty,
Based on the Leaf-opening Substance of Lespedeza cuneata G. Don
*
Minoru Ueda, Yoshiyuki Sawai, Yoko Wada and Shosuke Yamamura
*
Department of Chemistry, Faculty of Science and Technology, Keio University, Hiyoshi, Yokohama 223-8522, Japan
Received 23 March 2000; accepted 17 May 2000
AbstractÐIn a previous paper, the syntheses of potassium galactolespedezate (1) and potassium galactoisolespedezate (2), arti®cial leaf-
opening substances useful for bioorganic studies of nyctinasty were reported. The ¯uorescent probe compounds, ¯uorescence-labeled
galactoisolespedezate (3) and iodo-one (4), designed on the basis of 1 and 2 were prepared. Especially, compound 3 was bioactive at
5£1025 M, one-®ftieth as strong as the natural leaf-opening substance. This ¯uorescent probe would be useful for bioorganic studies of
nyctinasty. q 2000 Elsevier Science Ltd. All rights reserved.
Most Leguminosae plants 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
Recently, we have identi®ed several bioactive substances
that regulate this leaf-movement.3±16 These bioactive
substances can be used for probe compounds which would
be highly useful for the puri®cation of their receptors to
enable bioorganic studies of nyctinasty.
The most important problem involved in the design of the
leaf-movement factor-based probe compounds is the
unstability of 5 and 6 in the plant body, which are easily
hydrolyzed by b-glucosidase. Thus, we designed some
probe compounds based on the structures of the arti®cial
leaf-opening substances, potassium galactolespedezate (1)
and potassium galactoisolespedezate (2), which could not be
hydrolyzed in a plant body.23,24
The introduction of large functional groups, such as a
¯uorescent FITC (¯uorescein-5-isothiocyanate) group or
an iodine group, to arti®cial leaf-opening substances
requires careful consideration of the structure±activity
relationship in 5 and 6. Previous studies showed that the
substituents at the phenolic hydroxyl group of 5 and 6 as
well as the carboxyl group led to decrease of activity to 1023
or 1024 M.5 However, considering the bioactivity of 1 and 2,
the substitution of the sugar moiety in 5 and 6, from glucose
to galactose, did not affect the bioactivity at all. From these
results, it is expected that molecular recognition of 5 and 6
by its receptor molecule is attributable to the phenol moiety
of these compounds. Thus, the introduction of a large func-
tional group in the hydroxyl group at the 60 position of the
galactose moiety would not weaken the bioactivity of 1 and
2 to any extent.
Because of the high hydrophobicity of the leaf-movement
factors, it is unlikely that these compounds pass through the
plasma membrane of the plant cell and interact with target
molecule in the cell. Investigation of the site where bio-
active substances are perceived at the cellular level is a
®rst step towards the bioorganic study of their receptor
molecule. Various bioactive probe compounds are used
for the study of their binding-site, e.g. ¯uorescence-labeled
compounds which are widely used for the identi®cation of
receptor molecules,17±19 and also, 125I-labeled compounds
which are often used for the localization of the binding
site of bioactive substances because of their extremely
high sensitivity for the detection of the radioisotope.20±22
Recently, we synthesized arti®cial leaf-opening substance
(1 and 2) useful for the bioorganic studies on nyctinasty.23,24
Based on this result, we report now the syntheses of a
¯uorescence-labeled leaf-movement factor (3) and an
iodo-one (4) designed on the leaf-opening substance of
Lespedeza cuneata G. Don, potassium lespedezate (5) and
potassium isolespedezate (6).4,5
Moreover, because of the resistance to the esterase in a plant
body, an amide bond would be better than an ester bond to
connect the FITC group with 1 and 2. Thus, to introduce an
FITC group at the 60 position of 1 (or 2), we should convert
the hydroxyl group on the 60 position of 1 (or 2) into an
amino group.
Compound 723,24 was deprotected with sodium methoxide,
and the primary alcohol in the product was tosylated. After
acetylation 8 was obtained. By the deprotection of the silyl
Keywords: plants; natural products; biologically active compounds; ¯uor-
escent probe.
* Corresponding authors. Fax: 181-45-563-5967;
e-mail: yamamura@chem.keio.ac.jp
0040±4020/00/$ - see front matter q 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(00)00421-X

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M. Ueda et al. / Tetrahedron 56 (2000) 5123±5130
protective group using tetra-n-butyl ammonium ¯uoride, 8
was converted to tosylate 9 (Scheme 1). The resulting 9 was
treated with sodium azide to give azide 10, which was
treated with sodium methoxide to give 11. The (Z)-con-
®guration of the enol double bond in 11 was determined
reduced by catalytic hydrogenation, and the resulting
amine was coupled with FITC without puri®cation.
After deprotection of 12, ¯uorescence-labeled potassium
galactoisolespezate (13) was obtained together with a
trace amount of ¯uorescence-labeled potassium galactole-
spedezate (14).25 However, 13 showed leaf-opening activity
0
by NOE between H2 and H2. Compound 11 was then
Scheme 1. Chemical synthesis of ¯uorescent-labeled probe compound (13).

M. Ueda et al. / Tetrahedron 56 (2000) 5123±5130
5125
Scheme 2. Chemical synthesis of ¯uorescent-labeled probe compound (18).
Table 1. Bioactivities of ¯uorescence-labeled probe compounds (3, 13, 18)
Scheme 2 shows the synthetic route of ¯uorescence-labeled
potassium galactoisolespedezate (18). Azide 11 was
reduced by catalytic hydrogenation, and then coupled with
Z-Gly using WSC in DMF to give 16. Compound 16 was
deprotected with Pearlman's catalyst. When Pd±C was used
as a catalyst instead of Pearlman's catalyst, overreduction of
the enol double bond occurred. The resulting amine was
coupled with the FITC reagent in situ to give 17, which
was further treated with KOH to give 18.25
Compound
Bioactivity (M)
6
1£1026
5£1024
1£1024
5£1025
13
18
3
at 5£1024 M, which is one-®ve hundredth as strong as 1 and
2. The decrease of bioactivity could be due to the steric
hindrance of the large ¯uorescent functional group with
p-coumaroyl group; thus, the insertion of some linker
between the FITC group and the active site of the arti®cial
leaf-opening substance would be effective for the improve-
ment of bioactivity. We used glycine and triglycine as a
linker for the straightness of the peptide chain instead of
long-chain carboxylic acid which is usually used as a
linker.26
The bioactivity of 18 was improved up to ®ve times the
strength of 13, thus providing the effectiveness of the spacer
(Table 1).
Finally, we synthesized the third ¯uorescent probe with a
linker of glycylglycylglycine (Scheme 3). According to the
same procedures as those of 18, using Z-Gly-Gly-Gly
instead of Z-Gly, 3 could not be obtained because of the
Scheme 3. Chemical synthesis of ¯uorescent-labeled probe compound (3).

5126
M. Ueda et al. / Tetrahedron 56 (2000) 5123±5130
Scheme 4. Chemical synthesis of iodo-probe compound (4).
reduction of the enol double bond in catalytic hydrogenation
for removal of the protective group. Thus, we changed the
protective group of amine from a benzyloxycarbonyl to a
tert-butoxycarbonyl group. Glycylglycylglycine was treated
with (Boc)2O and the resulting protected tripeptide was
coupled with the amine derived in situ from 11 using the
DCC-HOBt method to give 19. Compound 19 was then
coupled with FITC to give 20, which was further depro-
tected to give 3.25 The leaf-opening activity of ¯uorescent
probe compound 3 was improved up to 5£1025 M. The
bioactivity of 3 was one-®ftieth as strong as that of the
natural product; thus, 3 would be useful as a molecular
probe for the bioorganic studies of nyctinasty.
Material and Methods
General procedures
1H NMR (400 MHz) and 13C NMR spectra (100 MHz) were
recorded on a Jeol GX400 spectrometer, using TMS in
CDCl3 or t-BuOH (1H; 1.23 ppm, 13C; 32.1 ppm) in D2O
as internal standards at various temperatures. ROESY
experiment was carried out in CD3OD (12) or CD3OD/
D2Oˆ8:2 (3, 17) at 408C. The FAB-MS and HR FAB-MS
spectra were measured on a Jeol JMS-700 spectrometer,
using glycerol as a matrix. The IR spectra were measured
by JASCO FT/IR-410. The HPLC puri®cation was carried
out with a Shimadzu LC-6A pump equipped with a SPD-6A
detector using Cosmosil 5C18AR column (f 20£250 mm)
(Nakalai Tesque Co. Ltd). The solvents used for HPLC were
available from Kanto Chemical Co. 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 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 or
0.5 mm, MERCK) or RP-18F254S (0.25 mm, MERCK).
The bioactivity of probe compounds (13, 18, 3) became
higher with the increase of the length of the linker moiety
(Table 1). In the ROESY experiments using 13, 18, and 3,
the correlation between the ¯uorescein moiety (H7; d 7.05)
and the phenol moiety (H2; d 7.70) was observed only in 13
(Scheme 1); on the other hand, no correlation between the
two parts was observed in 18 and 3. Thus, the ¯uorescein
group exists near the phenol moiety in 13. This result
suggests that the large ¯uorescein moiety in 13 blocks the
binding of the phenol moiety to the receptor molecule to
weaken the bioactivity.
Synthesis of methyl (Z)-2-(60-O-p-toluenesulfonyl-
20,30,40,60-tetra-O-acetyl-b-d-galactopyranosyloxy)-3-p-
t-butyldimethylsilyloxyphenyl-acrylate (8). Compound 7
(1.75 g, 2.74 mmol) was dissolved in methanol (30 mL),
and then sodium methoxide (583 mg, 11.0 mmol) was
added to the solution at 2508C. The reaction mixture was
stirred for 3 h, neutralized by Amberlite IR-120B (H1), and
then ®ltered. After evaporation, the resulting residue was
puri®ed by silica gel column chromatography
(CHCl3:MeOHˆ5:1) to give the corresponding alcohol
(1.11 g, 86%).
Moreover, we have synthesized an iodo-probe compound
(Scheme 4). To use the 125I-labeled compound, we synthe-
sized the probe compound containing a cold iodine atom at
the 60 position for the study of its bioactivity and stability
under the conditions of the bioassay. Compound 9 was
treated with sodium iodide to give 21. Compound 21 was
deprotected by 3.0 M KOHaq., and then the product was
puri®ed by ODS TLC to give 4. The (Z)-con®guration of
the enol double bond in 4 was determined by NOE between
0
H2 and H2. The bioactivity of the resulting 4 and 22 was as
strong as that of the original leaf-opening substance 5 and 6.
Also, its stability in the aqueous solution under the con-
ditions of the bioassay is suf®cient for usage as a molecular
probe. Iodo-galactoisolespedezate (4) suffered no
decomposition after one week. Thus, the radioactive
125I-derivative of 4 would be used as a highly sensitive
probe compound for the bioorganic study of receptors
with respect to nyctinasty.
The resulting alcohol (600 mg, 1.28 mmol) was dissolved in
pyridine (5 mL), and then p-toluene sulfonyl chloride
(293 mg, 1.54 mmol) was added to this solution at 08C.
The reaction mixture was allowed to stand at rt for 5 h,
with stirring under argon atmosphere, and was evaporated
with the toluene azeotrope. The residue was chromato-
graphed on silica gel (CHCl3:MeOHˆ9:1) to give depro-
tected 7 (221 mg, 28%), along with the recovered alcohol
(401 mg, 67%). Deprotected 7 (566 mg, 0.882 mmol) was
dissolved in pyridine (5 mL), and acetic anhydride (1 mL,
1.06 mmol) was added to this solution with stirring
Here, we have succeeded in the chemical synthesis of
several probe compounds for the bioorganic study of
nyctinasty. Bioorganic studies using these probe compounds
are now in progress.

M. Ueda et al. / Tetrahedron 56 (2000) 5123±5130
5127
overnight at rt. The reaction mixture was evaporated as the
toluene azeotrope, and the residue was chromatographed on
silica gel (n-hexane:EtOAcˆ2:1) to give 8 (627 mg, quant.).
164.0, 156.6, 137.7, 132.8, 130.8, 126.7, 125.5, 115.1,
99.3, 73.1, 70.1, 69.2, 67.8, 52.2, 50.4, 20.9, 20.8,
20.7 ppm; HR FAB MS (positive): [M1H]1 Found m/z
508.1615, C22H26O11N3 requires m/z 508.1567; IR (®lm)
1H NMR (400 MHz, CD3OD, rt): 7.68 (2H, d, Jˆ8.8 Hz),
7.67 (2H, d, Jˆ8.8 Hz), 7.28 (2H, d, Jˆ8.8 Hz), 7.00 (1H,
s), 6.81 (2H, d, Jˆ8.8 Hz), 5.41 (1H, dd, Jˆ10.3, 8.3 Hz),
5.36 (1H, d, Jˆ3.4 Hz), 5.31 (1H, d, Jˆ8.3 Hz), 5.05 (1H,
dd, Jˆ10.3, 3.4 Hz), 3.89 (3H, m), 3.84 (3H, s), 2.42 (3H, s),
2.06 (3H, s), 2.04 (3H, s), 1.98 (3H, s), 099 (9H, s), 0.22
(6H, s) ppm; 13C NMR (100 MHz, CDCl3, rt): 169.7, 169.5,
163.9, 145.0, 137.9, 132.4, 132.0, 129.8, 127.9, 126.1,
125.9, 119.9, 99.2, 70.8, 70.6, 68.9, 66.7, 65.6, 52.3, 25.8,
25.7, 25.5, 21.7, 20.9, 20.6, 19.3, 24.21 ppm; HR FAB MS
(positive): [M1H]1 Found m/z 751.2418, C35H47O14SiS
requires m/z 751.2436; IR (®lm) n: 1755, 1716, 1639,
1601, 1508 cm21; [a]D19ˆ12.868(cˆ0.93, CHCl3).
Synthesis of methyl (Z)-2-(60-O-p-toluenesulfonyl-
20,30,40,60-tetra-O-acetyl-b-d-galactopyranosyloxy)-3-p-
hydroxyphenyl-acrylate (9). The resulting tosylate
(569 mg, 0.759 mmol) was dissolved in THF (7 mL), and
tetra-n-butyl ammonium ¯uoride (1.0 M in THF, 1.0 mL,
1.0 mmol) was added to this solution at 08C with stirring
for 5 min. Water (10 mL) was added to this solution for the
extraction with EtOAc. The resulting organic layer was
washed with brine, dried over anhydrous Na2SO4, and
then evaporated. The residue was chromatographed on silica
gel (CHCl3:EtOAcˆ2:1) to give 9 (520 mg, quant.).
1H NMR (400 MHz, CD3OD, rt): 7.69 (2H, d, Jˆ8.3 Hz),
7.68 (2H, d, Jˆ8.3 Hz), 7.29 (2H, d, Jˆ8.3 Hz), 7.01 (1H,
s), 6.81 (2H, d, Jˆ8.3 Hz), 5.52 (1H, s), 5.40 (1H, dd,
Jˆ10.3, 7.8 Hz), 5.36 (1H, d, Jˆ3.4 Hz), 5.29 (1H, d,
Jˆd 7.8 Hz), 5.04 (1H, dd, Jˆ10.3, 3.4 Hz), 3.90 (3H,
m), 3.84 (3H, s), 2.42 (3H, s), 2.06 (3H, s), 2.05 (3H, s),
1.99 (3H, s) ppm; 13C NMR (100 MHz, CDCl3, rt.): 169.8,
163.9, 156.6, 145.1, 137.9, 132.7, 131.9, 129.8, 127.8,
126.1, 125.3, 115.2, 99.3, 70.8, 70.6, 69.0, 66.7, 65.6,
52.3, 21.7, 20.9, 20.7 ppm; HR FAB MS (positive):
[M1H]1 Found m/z 637.1558, C29H33O14S requires m/z
637.1591; IR (®lm) n: 3437, 1754, 1715, 1638, 1606,
1585, 1514 cm21; [a]D19ˆ12.138 (c 0.44, CHCl3).
Synthesis of methyl (Z)-2-(60-azido-20,30,40,60-tetra-O-
acetyl-b-d-galactopyranosyloxy)-3-p-hydroxyphenyl-
acrylate (10). Compound 9 (520 mg, 0.818 mmol) was
dissolved in DMF (4 mL). After the addition of 15-
crown-5 (1.6 mL, 8.18 mmol) and NaN3 (531 mg,
8.18 mmol), the reaction mixture was stirred at 708C for
2 days, mixed with water (20 mL), and then extracted
with EtOAc. The organic layer was washed with brine and
dried over anhydrous Na2SO4. After evaporation, the
residue was chromatographed on silica gel (n-hexane:E-
tOAcˆ1:1) to give 10 (260 mg, 63%).
n: 3422, 2103, 1751, 1720, 1638, 1606, 1585, 1514 cm21
;
[a]1D9ˆ114.38 (cˆ0.50, CHCl3).
Synthesis of methyl (Z)-2-(60-azido-b-d-galactopyrano-
syloxy)-3-p-hydroxyphenyl-acrylate (11). Compound 10
(260 mg, 0.513 mmol) was dissolved in MeOH, and sodium
methoxide (83.1 mg, 0.513 mmol) was added to this solu-
tion at 08C. After stirring for 30 min, Amberlite IR-120B
(H1) was added to the reaction mixture for neutralization.
The reaction mixture was then ®ltered and evaporated, and
the residue was chromatographed on silica gel
(CHCl3:MeOHˆ5:1) to give 11 (165 mg, 84%).
1H NMR (400 MHz, CD3OD, rt): 7.75 (2H, d, Jˆ8.8 Hz),
7.02 (1H, s), 6.75 (2H, d, Jˆ8.8 Hz), 5.07 (1H, d,
Jˆ7.8 Hz), 3.83 (1H, dd, Jˆ9.8, 7.8 Hz), 3.80 (3H, s),
3.72 (1H, d, Jˆ3.4 Hz), 3.60 (1H, m), 3.54 (1H, dd,
Jˆ9.8, 3.4 Hz), 3.48 (1H, dd, Jˆ13.2, 8.5 Hz), 3.17 (1H,
dd, Jˆ13.2, 3.9 Hz) ppm; 13C NMR (100 MHz, CD3OD,
rt.): 166.5, 159.7, 139.5, 133.7, 127.3, 125.7, 116.0, 103.1,
76.1, 74.6, 72.7, 70.7, 52.6, 52.4 ppm; IR (®lm) n: 3362,
2103, 1703, 1639 1605, 1586, 1514 cm21; HR FAB MS
(positive): [M1Na]1 Found m/z 404.1040, C16H19O8N3Na
requires m/z 404.1070; [a]1D9ˆ184.28 (cˆ0.77, MeOH).
Synthesis of methyl (Z)-2-[60-(5-thioureidyl¯uorescein)-
b-d-galactopyranosyloxy]-3-p-hydroxyphenyl-acrylate
(12). Compound 11 (3.4 mg, 8.9 mmol) was dissolved in
MeOH (0.5 mL), and then Pd±CaCO3 (5 mg) was added
to this solution. After stirring for 4 h under hydrogen
atmosphere, the reaction mixture was ®ltered and evapo-
rated to yield amine. Fluorescein isothiocyanate (isomer I,
4.1 mg, 10 mmol) was added to the acetone (1.0 mL) solu-
tion of the resulting crude amine, and then the solution was
stirred at rt for 2 h. After evaporation, the residue was chro-
matographed on silica gel TLC to give coupling product 12
(2.7 mg, 41%).
1H NMR (400 MHz, CD3OD, 408C): 8.08 (1H, br. s), 7.67
(1H, d, Jˆ7.8 Hz), 7.61 (2H, d, Jˆ7.8 Hz), 7.04 (1H, d,
Jˆ8.3 Hz), 6.84±6.92 (5H, m), 6.70 (2H, d, Jˆ8.8 Hz),
6.61 (2H, d, Jˆ8.3 Hz), 4.96 (1H, d, Jˆ7.8 Hz), 3.76±
3.71 (4H, m), 3.69 (3H, s), 3.58 (1H, m), 3.48 (1H, dd,
Jˆ8.8, 3.4 Hz) ppm; IR (®lm) n: 3323, 1703, 1605, 1542,
1509 cm21; 13C NMR (100 MHz, CD3OD, 308C): 183.0,
174.7, 166.7, 159.9, 154.3, 140.0, 133.8, 130.4, 127.1,
126.9, 125.9, 122.8, 116.3, 114.0, 111.7, 103.6, 74.8, 72.9,
71.0, 52.7, 46.5 ppm; HR FAB MS (positive): [M2H]2
Found m/z 743.1573, C37H31O13N2S requires m/z
743.1547; [a]1D9ˆ193.18 (cˆ1.0, MeOH).
Synthesis of potassium (Z)-2-[60-(5-thioureidyl¯uores-
cein)-b-d-galactopyranosyloxy]-3-p-hydroxyphenyl-
acrylate (13). Compound 12 (2.7 mg, 3.6 mmol) was
dissolved in MeOH:H2Oˆ3:1 (0.5 mL), and 3.0 M
KOHaq. (12 mL, 36 mmol) was added to this solution at
08C. After stirring for 3 h, Amberlite IR-120B (H1) was
added to the solution. The solution was ®ltered and
evaporated. The residue was dissolved in water (0.2 mL),
1H NMR (400 MHz, CDCl3, rt): 7.73 (2H, d, Jˆ8.8 Hz),
7.05 (1H, s), 6.81 (2H, d, Jˆ8.8 Hz), 5.47 (1H, dd,
Jˆ10.3, 7.8 Hz), 5.38 (1H, d, Jˆ7.8 Hz), 5.34 (1H, d,
Jˆ3.4 Hz), 5.08 (1H, dd, Jˆ10.3, 3.4 Hz), 3.84 (3H, s),
3.77 (1H, m), 3.34 (1H, dd, Jˆ13.2, 7.8 Hz), 3.02 (1H,
dd, Jˆ13.2, 4.4 Hz), 2.19 (3H, s), 2.08 (3H, s), 2.01 (3H,
s) ppm; 13C NMR (100 MHz, CDCl3, rt.): 170.0, 169.9,

5128
M. Ueda et al. / Tetrahedron 56 (2000) 5123±5130
neutralized with 10% K2CO3aq., and then evaporated.
Finally, the residue was chromatographed on an ODS
TLC to give 13 (2.3 mg, 79%).
(3H, m) ppm; 13C NMR (100 MHz, CD3OD, 308C): 190.6,
183.5, 171.8, 170.9, 166.7, 159.7, 154.1, 142.1, 139.9,
133.8, 131.7, 130.2, 127.1, 125.7, 120.0, 116.1, 113.7,
111.5, 103.7, 103.5, 74.7, 74.6, 72.8, 70.4, 52.8,
40.9 ppm; HR FAB MS (positive): [M1H]1 Found m/z
802.1940, C39H36O14N3S requires m/z 802.1918; IR (®lm)
n: 3272, 1699, 1638, 1604, 1511 cm21; [a]D20ˆ180.68
(cˆ0.94, MeOH).
Synthesis of potassium (Z)-2-[60-(glycyl 5-thioureidyl-
¯uorescein)-b-d-galactopyranosyloxy]-3-p-hydroxy-
phenyl-acrylate (18). Compound 17 (3.9 mg, 4.9 mmol)
was dissolved in MeOH:H2Oˆ3:1 (0.5 mL), and 1.0 M
KOHaq. (23.5 mL, 23.5 mmol) was added to this solution
at 08C. After stirring for 6 h, the solution was acidi®ed with
Amberlite IR-120B (H1), ®ltered, and then evaporated. The
residue was dissolved in MeOH:H2Oˆ1:1 (0.2 mL),
neutralized with 10% K2CO3aq., and then evaporated.
Finally, the residue was chromatographed on ODS TLC to
give 18 (3.6 mg, 85%).
1H NMR (400 MHz, CD3OD, 308C): 8.09 (1H, d,
Jˆ2.0 Hz), 7.70 (2H, d, Jˆ8.8 Hz), 7.69 (1H, dd, Jˆ8.3,
2.0 Hz), 7.05 (1H, d, Jˆ8.3 Hz), 6.85 (1H, s), 6.78 (2H, d,
Jˆ8.8 Hz), 6.64±6.69 (4H, m), 6.51±6.55 (2H, m), 5.02
(1H, d, Jˆ7.8 Hz), 4.06 (1H, m), 3.77±3.84 (3H, m), 3.63
(1H, dd, Jˆ14.2, 8.4 Hz), 3.58 (1H, dd, Jˆ9.7, 3.4 Hz) ppm;
13C NMR (100 MHz, CD3OD, 308C): 182.8, 171.0, 161.4,
158.9, 154.0, 142.2, 133.1, 131.5, 130.2, 128.9, 126.5,
125.5, 123.7, 116.0, 113.6, 111.5, 103.9, 103.4, 75.1, 74.9,
72.9, 70.1, 46.6 ppm; HR FAB MS (negative): [M2H]2
Found m/z 805.0540, C36H27O13N2SK2 requires m/z
805.0508; IR (®lm) n: 3290, 1634, 1575, 1510 cm21
;
[a]2D0ˆ198.48 (cˆ1.0, MeOH).
Synthesis of methyl (Z)-2-[60-(N-benzyloxycarbonyl-
glycylamino)-b-d-galactopyranosyloxy]-3-p-hydroxy-
phenyl-acrylate (16). Compound 11 (9.2 mg, 24 mmol)
was dissolved in MeOH (0.5 mL), and the solution was
stirred for 4 h under hydrogen atmosphere after the addition
of Pd±CaCO3 (8 mg). The reaction mixture was ®ltered
with celite, and evaporated to yield amine. The resulting
crude amine was dissolved in DMF (0.5 mL). To this solu-
tion was added Z-Gly (7.5 mg, 35 mmol), and then WSC
(5.5 mg, 29 mmol) at 08C. This reaction mixture was
allowed to stand at rt overnight under argon atmosphere,
and evaporated. The residue was chromatographed on silica
gel to give 16 (10.3 mg, 79%).
1H NMR (400 MHz, D2O, 308C): 7.80 (1H, d, Jˆ2.0 Hz),
7.78 (2H, d, Jˆ8.8 Hz), 7.62 (1H, dd, Jˆ8.3, 2.0 Hz), 7.32
(2H, dd, Jˆ8.3, 2.9 Hz), 7.23 (1H, d, Jˆ8.3 Hz), 6.78±6.82
(6H, m), 6.72 (1H, s), 4.95 (1H, d, Jˆ7.8 Hz), 4.25 (1H, d,
Jˆ17.1 Hz), 4.13 (1H, d, Jˆ17.1 Hz), 3.94 (1H, d,
Jˆ3.4 Hz), 3.80 (1H, m), 3.60±3.73 (2H, d, m), 3.56 (1H,
d, Jˆ14.2, 3.9 Hz), 3.42 (1H, d, Jˆ14.2, 9.3 Hz) ppm; 13C
NMR (100 MHz, CD3OD:D2Oˆ4:1, 308C): 183.0, 172.3,
172.1, 157.2, 156.5, 146.4, 140.9, 132.6, 131.6, 129.4,
129.1, 128.7, 126.9, 123.6, 121.1, 118.3, 115.9, 114.0,
103.7, 103.4, 74.5, 74.4, 72.5, 70.2, 49.8, 40.8 ppm; HR
FAB MS (negative): [M2K]2 Found m/z 824.1158,
C38H31O14N3SK requires m/z 824.1163; IR (®lm) n: 3290,
1638, 1587, 1510 cm21; [a]D22ˆ143.08 (cˆ0.48, H2O).
Synthesis of methyl (Z)-2-[60-(N-t-butoxycarbonylglycyl-
glycylglycylamino)-b-d-galactopyranosyloxy]-3-p-
hydroxyphenyl-acrylate (19). Compound 11 (57.0 mg,
150 mmol) was dissolved in MeOH (1.0 mL). After the
addition of Pd±CaCO3 (15 mg), the reaction mixture was
stirred for 2 h under hydrogen atmosphere. After ®ltration
with celite and then evaporation, amine was obtained. The
resulting crude amine was dissolved in DMF (0.5 mL), and
then Boc-GlyGlyGly (52.0 mg, 180 mmol) and 1-hydroxy-
benzotriazole (26.8 mg, 225 mmol), DCC(46.4 mg,
225 mmol) were added to this solution at 08C. The reaction
mixture was stirred overnight under argon atmosphere, and
then evaporated. The residue was suspended in
MeOH:H2Oˆ3:2, and precipitated DCUrea was ®ltered
out. After evaporation, the resulting residue was chromato-
graphed on Cosmosil 75C18-OPN (MeOH:H2Oˆ1:2) to
yield 19 (78.6 mg, 84%).
1H NMR (400 MHz, CD3OD, 308C): 7.75 (2H, d, Jˆ
8.8 Hz), 7.3 (5H, m), 6.99 (1H, s), 6.76 (2H, d,
Jˆ8.8 Hz), 5.08 (2H, s), 4.92 (1H, d, Jˆ7.8 Hz), 3.80
(3H, s), 3.64±3.77 (4H, m), 3.44±3.51 (3H, m) ppm; 13C
NMR (100 MHz, CD3OD, 308C): 172.3, 166.7, 159.7,
139.9, 133.7, 131.5, 129.3, 128.9, 128.7, 127.0, 125.7,
116.1, 115.9, 103.7, 74.6, 74.5, 72.7, 70.2, 67.9, 52.7,
44.9, 40.8 ppm; HR FAB MS (positive): [M1Na]1 Found
m/z 569.1747, C26H30O11N2Na requires m/z 569.1747; IR
(®lm) n: 3364, 1704, 1660, 1606, 1585, 1540, 1514 cm21
[a]2D0ˆ183.98(cˆ1.0, MeOH).
Synthesis of methyl (Z)-2-[60-(glycyl 5-thioureidyl¯uor-
escein)-b-d-galactopyranosyloxy]-3-p-hydroxyphenyl-
acrylate (17). Compound 16 (10.3 mg, 18.9 mmol) was
dissolved in MeOH (0.5 mL). After the addition of Pearl-
man's catalyst (8 mg), the solution was stirred for 2 h under
argon atmosphere. The reaction mixture was then ®ltered
with celite and evaporated to yield amine. FITC (isomer I,
8.8 mg, 23 mmol) was added to the crude amine dissolved in
MeOH (0.5 mL), and then allowed to stand for 1 h. The
reaction mixture was then evaporated, and the residue was
chromatographed on ODS to give 17 (3.9 mg, 26%).
1H NMR (400 MHz, CD3OD, 30): 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, CDCl3, 308C): 173.5, 171.9, 171.7,
166.7, 159.7, 139.9, 133.7, 127.1, 125.7, 116.1, 103.7,
81.0, 74.6, 74.3, 72.8, 69.9, 52.7, 49.8, 44.9, 43.8, 43.5,
40.6, 28.7 ppm; HR FAB MS (positive): [M1H]1 Found
1H NMR (400 MHz, CD3OD, 308C): 8.18 (1H, d,
Jˆ2.0 Hz), 7.80 (1H, dd, Jˆ8.3, 2.0 Hz), 7.75 (2H, d,
Jˆ8.8 Hz), 7.15 (1H, d, Jˆ8.3 Hz), 6.98 (1H, s), 6.75
(2H, d, Jˆ8.8 Hz), 6.69 (2H, d, Jˆ8.8 Hz), 6.67 (2H, d,
Jˆ2.4 Hz), 6.54 (2H, dd, Jˆ8.8, 2.4 Hz), 4.95 (1H, d,
Jˆ7.8 Hz), 4.22 (1H, d, Jˆ16.6 Hz), 4.14 (1H, d,
Jˆ16.6 Hz), 3.78±3.82 (2H, m), 3.80 (3H, s), 3.49±3.58

M. Ueda et al. / Tetrahedron 56 (2000) 5123±5130
5129
m/z 627.2517, C27H39O13N4 requires m/z 627.2514; IR (®lm)
n: 3323, 1691, 1681, 1667, 1605, 1583, 1538, 1514 cm21
Synthesis of methyl (Z)-2-(60-iodo-20,30,40,60-tetra-O-ace-
tyl-b-d-galactopyranosyloxy)-3-p-hydroxyphenyl-acry-
late (21). Compound 9 (100 mg, 0.157 mmol) was dissolved
in DMF (1.0 mL). After the addition of NaI (236 mg,
1.573 mmol), the solution was stirred at 808C for 2 days.
After the addition of water (5 mL), the reaction mixture
was extracted with EtOAc. The resulting organic layer
was washed with brine, dried over anhydrous Na2SO4, and
then evaporated. The residue was chromatographed on silica
gel (benzene:acetoneˆ4:1) to give 21 (49.9 mg, 54%).
1H NMR (400 MHz, CD3OD, rt): 7.72 (2H, d, Jˆ8.8 Hz),
7.04 (1H, s), 6.82 (2H, d, Jˆ8.8 Hz), 5.55 (1H, d, Jˆ
3.4 Hz), 5.43 (1H, dd, Jˆ10.3, 8.3 Hz), 5.40 (1H, s), 5.34
(1H, d, Jˆ8.3 Hz), 5.09 (1H, dd, Jˆ10.3, 3.4 Hz), 3.84 (3H,
s), 3.80 (1H, t, Jˆ7.3 Hz), 3.03 (2H, d, Jˆ7.3 Hz), 2.18 (3H,
s), 2.07 (3H, s), 2.01 (3H, s) ppm; 13C NMR (100 MHz,
CDCl3, rt.): 170.0, 169.8, 156.5, 138.0, 132.8, 130.8,
126.1, 125.5, 115.1, 114.9, 99.0, 74.1, 70.8, 68.9, 68.2,
60.4, 52.3, 21.7, 20.9, 20.8, 20.7, 14.3 ppm; HR FAB MS
(positive): [M1Na]2 Found m/z 615.0371, C22H25O11INa
requires m/z 615.0339; IR (®lm) n: 3421, 1750, 1716,
1606, 1585, 1514 cm21; [a]D22ˆ121.88 (cˆ0.36, CHCl3).
Synthesis of potassium (Z)-2-(60-iodo-b-d-galactopyra-
nosyloxy)-3-p-hydroxyphenyl-acrylate (4). Compound
21 (38.1 mg, 69.4 mmol) was dissolved in MeOH:H2Oˆ3:1
(0.50 mL). To this solution was added 3.0 M KOHaq.
(171 mL, 0.513 mmol) at 08C. After stirring for 2 h, the
reaction mixture was neutralized with Amberlite IR-
120B(H1). After ®ltration, the ®ltrate was evaporated, and
the residue was chromatographed on ODS TLC
(MeOH:H2Oˆ4:3) to give 4 (23.1 mg, 73%) and its geome-
trical isomer (0.3 mg).
;
[a]1D8ˆ178.88 (cˆ1.0, MeOH).
Synthesis of methyl (Z)-2-[60-(glycylglycylglycyl 5-thio-
ureidyl¯uorescein)-b-d-galactopyranosyloxy]-3-p-hydroxy-
phenyl-acrylate (20). Compound 19 (24.0 mg, 38.3 mmol)
was dissolved in THF-H2Oˆ30:1 (0.3 mL). To this solution
was added TFA (0.3 mL) at 08C. After stirring for 1 h, the
reaction mixture was evaporated to dryness as the water
azeotrope to yield amine as TFA salt. The resulting crude
amine was dissolved in MeOH (1 mL), and then trimethy-
amine (5.3 mL, 38.3 mmol) and FITC (isomer I, 17.9 mg,
46.0 mmol) were added to this solution at 08C. After stirring
for 2 h, the reaction mixture was evaporated and the residue
was puri®ed by ODS TLC (MeOH:H2Oˆ2:1) to give 20
(14.1 mg, 40%).
1H NMR (400 MHz, CD3OD, 308C): 8.25 (1H, d,
Jˆ2.0 Hz), 7.81 (1H, dd, Jˆ8.3, 2.0 Hz), 7.73 (2H, d,
Jˆ8.8 Hz), 7.11 (1H, d, Jˆ8.3 Hz), 6.97 (1H, s), 6.73
(2H, d, Jˆ8.8 Hz), 6.67 (4H, m), 6.54 (2H, dd, Jˆ9.3,
2.2 Hz), 4.93 (1H, d, Jˆ7.8 Hz), 4.35 (1H, d, Jˆ16.6 Hz),
4.28(1H, d, Jˆ16.6 Hz), 3.90 (2H, s), 3.77±3.93 (7H, m),
3.51±3.55 (2H, m), 3.37 (1H, dd, Jˆ13.2, 6.9 Hz), 3.26 (1H,
m) ppm; 13C NMR (100 MHz, CDCl3, 308C): 202.4, 189.4,
183.8, 173.0, 172.1, 171.7, 166.8, 159.7, 154.2, 142.0,
139.8, 133.7, 131.6, 130.3, 127.2, 125.9, 125.7, 118.3,
116.1, 114.0, 111.6, 103.7, 103.5, 74.6, 74.3, 73.7, 72.8,
69.8, 47.2, 44.1, 43.4, 40.4 ppm; HR FAB MS (negative):
[M2H]2 Found m/z 914.2238, C43H40O16N5S requires m/z
914.2191; IR (®lm) n: 3357, 1654, 1648, 1641, 1606, 1590,
1510 cm21; [a]1D9ˆ139.48 (cˆ1.0, MeOH).
Synthesis of potassium (Z)-2-[60-(glycylgylcylglycyl 5-
thioureidyl¯uorescein)-b-d-galactopyranosyloxy]-3-p-
hydroxyphenyl-acrylate (3). Compound 20 (11.5 mg,
12.5 mmol) was dissolved in MeOH:H2Oˆ3:1 (0.5 mL),
and then 1.0 M KOH aq. (0.1 mL, 0.1 mmol) was added
to this solution at 08C. After stirring for 2 h, the reaction
mixture was acidi®ed with Amberlite IR-120B (H1),
®ltered, and then evaporated. The residue was dissolved in
MeOH:H2Oˆ1:1 (0.2 mL), and then neutralized with 10%
K2CO3aq. After evaporation, the residue was puri®ed by
ODS TLC (MeOH:H2Oˆ3:2) to give 3 (6.0 mg, 49%).
1H NMR (400 MHz, D2O, rt): 7.72 (2H, d, Jˆ8.8 Hz), 6.85
(2H, d, Jˆ8.8 Hz), 6.79 (1H, s), 4.96 (1H, d, Jˆ7.8 Hz),
4.06 (1H, d, Jˆ3.4 Hz), 3.76 (1H, dd, Jˆ10.3, 7.8 Hz),
3.73 (1H, t, Jˆ6.8 Hz), 3.66 (1H, dd, Jˆ10.3, 3.4 Hz),
3.27 (1H, dd, Jˆ10.3, 6.8 Hz), 3.23 (1H, dd, Jˆ10.3,
6.8 Hz) ppm; 13C NMR (100 MHz, D2O, 308C): 158.9,
147.0, 143.1, 134.8, 127.4, 117.8, 116.9, 104.1, 102.9,
78.0, 75.1, 73.3, 71.9, 51.3 ppm; HR FAB MS (negative):
[M2K]2. Found m/z 450.9898, C15H16O8I requires m/z
450.9890; IR (®lm) n: 3348, 1606, 1577, 1512 cm21
[a]1D9ˆ159.98 (cˆ0.35, H2O).
:
1H NMR (400 MHz, CD3OD, 308C): 8.15 (1H, d,
Jˆ2.0 Hz), 7.75 (1H, dd, Jˆ8.3, 2.0 Hz), 7.63 (2H, d,
Jˆ8.8 Hz), 7.02 (1H, d, Jˆ8.3 Hz), 6.81 (1H, s), 6.70±
6.77 (6H, m), 6.61 (2H, dt, Jˆ8.7, 2.0 Hz), 4.85 (1H, d,
Jˆ8.8 Hz), 4.85 (1H, d, Jˆ17.1 Hz), 3.86 (1H, d,
Jˆ17.1 Hz), 4.30 (1H, d, Jˆ17.1 Hz), 3.92 (1H, d,
Jˆ17.1 Hz), 3.74±3.79 (4H, m), 3.55±3.59 (2H, m), 3.40
(1H, d, Jˆ13.7, 5.4 Hz), 3.24 (1H, dd, Jˆ13.7, 8.3 Hz) ppm;
13C NMR (100 MHz, CD3OD, 308C): 184.9, 173.9, 173.2,
172.8, 172.1, 162.6, 159.6, 155.1, 150.2, 143.2, 133.9,
132.8, 131.2, 130.0, 127.7, 126.5, 123.4, 121.2, 116.8,
114.7, 112.4, 104.9, 104.4, 75.9, 75.5, 73.8, 71.2, 48.7,
44.7, 44.2, 41.7 ppm; HR FAB MS (negative):
[M22K1H]2 Found m/z 900.2019, C42H38O16N5S
requires m/z 900.2034; IR (®lm) n: 3294, 1736, 1661,
Bioassay. The young leaves detached from the stem of the
plant Cassia mimosoides L. 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 am.)
were used for the bioassay. Each test solution was carefully
poured into test tubes with a microsyringe around 10:00 am.
The bioactive fraction was judged by the leaf-opening after
the leaf-closing of the plant leaf in the blank solution
containing no sample.
Acknowledgements
1639, 1606, 1586, 1546, 1513 cm21
(cˆ0.38, MeOH).
;
[a]2D0ˆ158.08
We are indebted to the Ministry of Education, Science,

5130
M. Ueda et al. / Tetrahedron 56 (2000) 5123±5130
12. Ueda, M.; Asano, M.; Sawai, Y.; Yamamura, S. Tetrahedron
1999, 55, 5781±5792.
Sports and Culture (Japan) for a Grant-in-Aid for Scienti®c
Research on Special Promotion Research (No. 12045259
and 12680598) and Pioneering Research Project in Bio-
technology given by the Ministry of Agriculture, Forestry
and Fisheries for ®nancial support.
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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