Detection of 210 kDa receptor protein for a leaf-movement factor by using novel photoaffinity probes

Article abstract of DOI:10.1016/j.tet.2005.06.022

Circadian rhythmic plant leaf-movement, called nyctinasty, is controlled by a time-course change in the internal concentration of the leaf-movement factor in the plant body. We revealed that specific binding proteins (210 and 180 kDa) for the leaf-movement factor, potassium lespedezate (1), are contained in the plasma membrane of the plant motor cell by using novel synthetic photoaffinity probes. These proteins are localized on the motor cell in the plant body, and would be potential receptors for the leaf-movement factor to control the leaf-movement. Our study is a rare successful result of the detection of membrane receptors by using a synthetic photoaffinity probe designed on a biologically active natural product. And these results also advance a guideline for probe design towards successful photoaffinity labeling.

Full text of DOI:10.1016/j.tet.2005.06.022

Tetrahedron 61 (2005) 7874–7893
Detection of 210 kDa receptor protein for a leaf-movement factor
by using novel photoaffinity probes
*
Tomohiko Fujii, Yoshiyuki Manabe, Takanori Sugimoto and Minoru Ueda
Department of Chemistry, Tohoku University, Aoba-ku, Sendai 980-8578, Japan
Received 11 May 2005; revised 7 June 2005; accepted 8 June 2005
Available online 5 July 2005
Abstract—Circadian rhythmic plant leaf-movement, called nyctinasty, is controlled by a time-course change in the internal concentration of
the leaf-movement factor in the plant body. We revealed that specific binding proteins (210 and 180 kDa) for the leaf-movement factor,
potassium lespedezate (1), are contained in the plasma membrane of the plant motor cell by using novel synthetic photoaffinity probes. These
proteins are localized on the motor cell in the plant body, and would be potential receptors for the leaf-movement factor to control the leaf-
movement. Our study is a rare successful result of the detection of membrane receptors by using a synthetic photoaffinity probe designed on a
biologically active natural product. And these results also advance a guideline for probe design towards successful photoaffinity labeling.
q 2005 Elsevier Ltd. All rights reserved.
1. Introduction
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 the biological clock
(Fig. 1).
In recent years, the importance of post-genomic studies is
highly emphasized. The existence of many receptors are
predicted and found in the sequence of genomic DNA.
However, many of them are orphan receptors whose ligand
is unknown. They have some types of trans-membrane
domain and are thought to be potential receptors. Then,
detection of the ligand of the orphan receptor is one of the
most important issues of the post-genomic era, and the
detection of a receptor protein for a biologically active
natural product is a central issue in bioorganic chemistry or
chemical genetics.1
Charles Darwin, well-known for his theory of evolution,
was the first to establish the basis of this field in the 19th
century.2 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 motor cells is
followed by massive water flux, which results in swelling
and shrinking of these cells.3 This also suggests that
mechanism of nyctinasty is concerned with the water
control in the plant body of legumes, which is an important
food resource of human race. An issue of great interest is the
regulation of the opening and closing of the potassium
channels involved in nyctinastic leaf movement. We have
revealed that nyctinasty is controlled by a pair of leaf-
movement factors: leaf-opening and leaf-closing sub-
stances.4 Recently, we revealed that the target cell of the
leaf-opening substance is the motor cell,5 which plays a key
role in nyctinastic leaf-movement.3 Thus, the next important
issue is the detection and isolation of a receptor molecule for
the leaf-movement factor, that will be located on the plasma
membrane of the motor cell.
Our study focuses on the bioorganic chemistry of
biologically intriguing phenomena, such as nyctinasty:
Figure 1. Cassia mimosoides L: in the daytime (left), and at night (right).
Keywords: Photoaffinity labelling; Receptor; Potassium lespedezate.
Photoaffinity labeling is a powerful method for the detection
of receptors from the viewpoint of bioactive substances.6
However, in general, it is difficult to get a successful result
in photoaffinity labeling experiments. One reason for this
difficulty in photoaffinity labeling would be attributable to
*
Corresponding author. Tel./fax: C81 22 795 6553;
e-mail: ueda@mail.tains.tohoku.ac.jp
0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2005.06.022

T. Fujii et al. / Tetrahedron 61 (2005) 7874–7893
7875
the unsuitable molecular design of the probe. Molecular
design of the probe molecule incurs many difficulties: the
most important one is that the molecular design of the probe
requires the cost of either high binding affinity or high
biological activity because these two factors are never
compatible. The arrangement of a large photoaffinity group
against the binding site of a bioactive substance presents a
serious dilemma. The shorter the distance between a large
photoaffinity group and the binding site of the molecule is,
the higher the ability to capture its receptor becomes, but the
weaker its bioactivity becomes, and vice versa. The location
of the photoaffinity labeling group in the probe should be
discussed systematically for solving this issue. However, no
guidelines have been reported for the discussion of the
location of photoaffinity probes because of the difficulty of
synthesizing systematic structural variation of the probes
without important loss of the bioactivity. In this paper, we
report the detection of a receptor molecule for the leaf-
movement factor using novel synthetic probes, and advance
a guideline for the probe design toward successful
photoaffinity labeling.
photoaffinity group and the binding site of the molecule is,
the higher the ability to capture its receptor becomes, but the
weaker its bioactivity becomes, and vice versa.
We assumed that the 60-and 20-positions of the glycone
moiety are suitable for the photoaffinity group. A 20-modi-
fied probe is expected to give higher labeling yield than a
60-modified one because of the short distance between the
photoaffinity group and the potential0 binding site to the
receptor. On the other hand, the 6 -modified0 probe is
expected to show higher bioactivity than the 2 -modified
one because of the long distance between the photoaffinity
group 0and a potential binding site. Thus, we synthesized
both 2 - and 60-modified probes to compare the efficiency of
the photoaffinity labeling.
We compared the results of labeling experiments using
these probes designed based on different concepts.
We synthesized biologically active photoaffinity probes
(2, 3 and 4) based on the structure of 1. These probes are
efficient tools for the biotinylation of the receptor proteins
for 1. Novel probes 2 and 3 have trifluoromethydiazirine10
as photoaffinity groups, and 4 has a benzophenone group.11
Probe 2, which bears a photoaffinity group on the 60-position
of the sugar moiety, was designed for higher bioactivity than
3 and 4 by reducing the steric hindrance caused by a large
photolabeling group. On the other hand, probes 3 and 4 bear
a photolabeling group on the 20-position of the sugar moiety,
which is adjacent to the p-hydroxyphenylpyruvate group.
These probes were designed for high labeling yield with the
receptor molecule. We compared the results of labeling
experiments using these probes that were designed on
different concepts.
2. Results
2.1. Molecular design of photoaffinity probes
We developed novel synthetic probes for photoaffinity
labeling to look for the native factor receptor based on the
structure of potassium isolespedezate (1),7 a bioactive
substance effective for leaf-opening of a leguminous plant,
Cassia mimosoides L (Fig. 1). Structure–activity relation-
ship studies on 1 revealed that the p-hydroxyphenylpyruvate
moiety of 1 is indispensable for the leaf-opening activity of
1, and is expected to be a binding site to its receptor.8 On the
other hand, structural modification in the glycon moiety of 1
did not cause any drop in bioactivity. For example, b-D-
galactopyranoside, a-D-mannopyranoside, and b-L-gluco-
pyranoside analogs of 1 were as effective as 1 in the
bioassay using Cassia leaves.9 Thus, we planned to
introduce a large photoaffinity group to the glycon moiety
of 1. The photoaffinity group should be connected through
an amide bond, which is expected to be stable against the
hydrolysis by esterase in the plant body.
However, molecular design of the probe molecule incurs
many difficulties: the most important one is that the
molecular design of the probe requires the cost of either
high photoaffinity labeling yield or high biological activity
because these two factors are never compatible. The
arrangement of a large photoaffinity group against the
binding site of a bioactive substance presents a serious
dilemma (Fig. 2). The shorter the distance between a large
2.2. Synthesis of probe 2
Synthesis of photoaffinity probe 2 was carried out according
to the synthetic route in Scheme 1.12 A photoaffinity group
was introduced using commercially available Affilight-
CHOe (19), which has a trifluoromethydiazirine and biotin
group.13 Introduction of 19 requires an aldehyde or a ketone
group. Thus, we prepared the 60-aldehyde analog of 1. For
Figure 2. Molecular design of probe compound based on 1.

7876
T. Fujii et al. / Tetrahedron 61 (2005) 7874–7893
oxime moiety of photoaffinity probe 2 (34% yield of (E)-2
and 25% yield of (Z)-2, respectively).
2.3. Synthesis of probe 3
Probe 3 was synthesized according to Scheme 2. We used
galactosamine as a starting material to introduce the
photolabeling unit on an amino group of the 20-position.
The photolabeling unit was connected with the aglycon
moiety via an amide linkage. This molecular design would
be effective for the improvement of labeling yield This
change in molecular design requires revision of the
synthetic route that was used in 2.
Scheme 1. Synthesis of photoaffinity probe 2.
the sake of later oxidation, the primary hydroxyl group of
D-galactose should be protected by a different protective
group from secondary hydroxyl groups. Thus, 10, which has
different protective groups on primary and secondary
hydroxyl groups was synthesized via 6-O-benzyl diaceto-
negalactose according to the method by Xia et al.14
Selective acetonide formation between secondary hydroxyl
groups using copper sulfate with catalytic sulfuric acid and
following protection of the residual primary hydroxyl group
with a benzyl group gave 10.
Scheme 2. Synthesis of photoaffinity probe 3.
Compound 9 was synthesized as shown in Scheme 1.
Coupling of 9 and 10 gave 11, and then the product was
subjected to deacetylation, and the resulting secondary
hydroxyl groups were protected with TBSOTf to give 13.
After conversion of the protective group on the 60-position
of the galactose moiety in 13 from the benzyl to an acetyl
group, resulting 15 was treated with excess amount of DDQ
(7 equiv) for 2 days to give 16. This oxidation gave an 8:1
mixture of (Z)- and (E)-16. The stereochemistry was
assigned by NOE correlation observed in (Z)-16 (Scheme
1). These stereoisomers were separable by column
chromatography. The selective deacetylation of 16 was
achieved by carrying out the reaction at K20 8C. When the
reaction was carried out at higher temperature, TBS on the
phenolic hydroxyl group was deprotected simultaneously.
Then, the resulting primary hydroxyl group was oxidized to
give aldehyde (18). Coupling reaction of 18 with 20, which
was prepared from 19 was carried out in aq methanol, and
the following deprotection and purification by HPLC using
Develosil C8 UG-5 column gave the two isomers on the
Aglycon 22 was synthesized from 5, which was prepared
according to Scheme 1. On the other hand, we synthesized
3, 4, 6-tri-O-acetyl-2-deoxy-2-phtalimido- a, b-D-galacto-
pyranosyl bromide (23) from D-galactosamine hydro-
chloride. We modified previously reported synthetic
conditions of 2415 from D-galactosamine hydrochloride
because of the low yield (17%). In Ref. 15, we synthesized
23 from 32 prepared by Lee’s method.16 However, Lee’s
method gave 32 in a very low yield (32% from
D-galactosamine hydrochloride) because of the difficulty
in a protection of amine by phtalimide. We modified Lee’s
method and a one-pot protection of D-galactosamine by
phtalimide and then acetic anhydride was carried out using
DMF as a solvent to give 32 in a quantitative yield (Scheme
3). Compound 23 prepared from 32 was coupled with 22 by
¨
modified Konigs-Knorr’s method to give 24 (Scheme 3).
When we used normal Konigs-Knorr’s condition, only 56%
¨
of coupling product 24 was obtained together with

T. Fujii et al. / Tetrahedron 61 (2005) 7874–7893
7877
Scheme 3. Synthesis of 24.
carboxylate without tert-butyl group. However, addition of
diisopropylethylamine (DIPEA) prevented the deprotection
of tert-butyl group to give 24 in 86% yield. Thus, total yield
of 24 from galactosamine hydrochloride was improved from
17%15 to 67%. After conversion of the benzyl group in 24 to
TBS, DDQ oxidation was carried out to give 27.
Deprotection of 27 with TBAF and then sodium methoxide
gave 29. Resulting 29 was further deprotected with
hydrazine monohydrate, and then coupled with the
photolabeling unit (30) bearing trifluoromethydiazirine
and biotin, which is separately prepared according to the
method by Hatanaka.13 Low yield in coupling reaction was
due to the low solubility of biotin unit 30 in any solvent.
Free amine, which was obtained in the deprotection of 31,
was used in the coupling reaction without purification.
t-Butyl ester in 29 was essential for the synthesis of 31.
When we used the methyl ester of 29 (34)9 instead of t-butyl
Scheme 5. Synthesis of photoaffinity probe 4.
0
ester (29), lactam (35) [dH-2 3.63 ppm] was obtained
quantitatively in the following deprotection of the amino
group on the 20-position (Scheme 4). The t-butyl group
would prevent the formation of lactam by its steric
hindrance.
the glycon moiety, and then 4-bromomethylbenzophenone
was introduced on its 20 position.
Synthetic intermediate 27 was prepared according to the
same method as Scheme 2. Similar to the case of probe 3,
t-butyl ester in 27 was essential for avoiding the lactam
formation in the following deprotection of the amino group
on the 20-position. Compound 27 was deprotected with
sodium methoxide to give 36. Compound 36 was then
treated with p-toluenesulfonyl chloride to give 37 with 47%
of recovered 36. After deprotection with TBAF, the
resulting 38 was treated with sodium azide to give 39.
After catalytic hydrogenation with Pd–CaCO3, the resulting
crude amine was coupled without purification with biotinyl
glycylglycylglycine (40), which is prepared separately
(Scheme 6). Low yield in coupling reaction was due to
the low solubility of biotin unit 40 in any solvent. Coupling
product 41 was deprotected with hydrazine monohydrate,
and the resulting crude amine was used for coupling
reaction with 4-bromomethyl benzophenone (42) without
purification in the presence of TEA. Coupling product 43
was then deprotected by neat TFA to give probe 4.
Deprotection was not achieved by 30 s treatment with
TFA at rt, which is the condition used in the synthesis of
probe 3. Deprotection of 43 required much longer reaction
time (25 min at rt) than in case of probe 3.
Scheme 4. Lacton formation from 34.
However, deprotection of the t-butyl ester in 31 raises a
serious problem. We examined the reaction conditions on
the deprotection of 31 thoroughly. But the use of several
weak acids, such as formic acid, acetic acid, and benzoic
acid gave a complex mixture of decomposed products or
ended in no reaction. The resulting complex mixture mainly
contained the fragments obtained by the dissociation of the
glycosidic bond or the amide bond. After many trials, we
found that the treatment of 31 by neat TFA within 30 s at rt
provided good results. Under this condition, deprotection
proceeded in 82% yield. Following neutralization and
purification gave probe 3. In probe 3, the photolabeling
group on the 20-position is extruded to the direction of
aglycon, which is expected to be a binding site with the
receptor.
2.4. Synthesis of probe 4
Synthesis of novel probe 4 was carried out according to the
route in Scheme 5.17 In this probe, a large benzophenone
group and a large biotin unit were separately introduced on
the 20- and 60-position of the sugar moiety, respectively. For
this purpose, we synthesized 20, 60-diamino galactose
skeleton from D-galactosamine. We synthesized properly
protected diamine as intermediate, and then, biotin coupled
with a triglycine linker was introduced on the 60 position of
Scheme 6. Synthesis of biotin unit (40).

7878
T. Fujii et al. / Tetrahedron 61 (2005) 7874–7893
2.5. Bioactivity of the probes
ultracentrifugation (1st; 3000!g, 15 min, 4 8C, 2nd; 100,
000!g, 60 min, 4 8C) gave a pellet of the crude membrane
fraction. The content of protein in that fraction was
determined to be 134 mg by the Bradford method with
BSA as a reference. The membrane ATPase activity, which
is a reference for the purity of the plasma membrane was
determined to be 0.67 mmol/mg protein min by Sandstom’s
method.18 The crude membrane fractions were suspended
and incubated with 3 mM aq solutions of probe 2 or 3 for
60 min at rt, respectively. After cross-linking, by irradiation
of UV-light (365 nm) for 10 min, the suspended membrane
fraction was solubilized by the addition of an electro-
phoresis buffer containing SDS. The membrane fraction was
analyzed by SDS-PAGE (7.5% T). After western blotting,
detection of the bands of the potential receptor for leaf-
opening substance was carried out by chemiluminescence
detection with ECL Advance Western Blotting Detection
Kit (Amersham Bioscience Co. Ltd), which is a method for
the detection of bands of biotinylated proteins. Photolabel-
ing experiment with probe 3 gave two bands corresponding
to binding proteins for 1. One is due to a protein of 210 kDa
molecular weight, and the other to a protein of 180 kDa
(lane 2 in Fig. 3). The molecular weight was estimated from
comparison with a biotinylated molecular weight marker
(Amersham Bioscience Co. Ltd).
The resulting probes were tested in a bioassay using the
leaves of C. mimosoides. When the leaves of C. mimosoides
were separated from the stem, they continued leaf move-
ment according to the circadian rhythm: open in the daytime
and closed at night. The leaf-opening activity of the probe
was judged by its bioactivity based on the leaves opening
after 7:00 PM.
Photoaffinity probe 2, which bears a photoaffinity group and
a biotin group on the 60-position of the galactose unit,
showed leaf-opening activity at 5!10K5 M for C.
mimosoides. This is one-fiftieth as active as the natural
product. On the other hand, the bioactivity of probe 3 (8!
10K5 M) was one-eightieth that of the natural product.
Similarly, probe 4 was effective at 1!10K4 M, which is
one-hundredth as effective as the natural product. Thus, all
three synthetic probes were bioactive in the bioassay using
the leaves of C. mimosoides. Their bioactivity changed
according to the distance between the aglycone moiety and
the large photoaffinity group. The molecular size of the
photoaffinity group also affected bioactivity. These results
showed that the nearer the large photoaffinity group was
arranged to the aglycone unit, the weaker the bioactivity of
the probe became.
We then compared the results of photolabeling exami-
nations using these synthetic probes.
2.6. Photoaffinity labeling with synthetic probes17
Photoaffinity probes 3 and 4, which bear a photolabeling
group close to the potential binding site, are expected to
label their receptor more effectively than probe 2. On the
other hand, probe 2, which bears a photolabeling group far
from the potential binding site showed stronger bioactivity
than 3 and 4 in the bioassay, and is estimated to be easier to
bind with its receptor molecule than probe 3 and 4. Which
type of probe gives a better result in the photolabeling
examination? This is a very important problem because the
result would give a general guideline for the molecular
design of photoaffinity probes. Both 2 and 3 have a
trifluoromethyldiaziridine group as a photoaffinity group.
We then compared the results of photolabeling examin-
ations using these probes 2 and 3.
Figure 3. SDS PAGE analyses (10–20% T gradient gel) of potential
membrane receptor using probe 2 and 3 by the chemiluminescence
detection (Lane 1: molecular weight marker, lane 2: membrane protein of
motor cell incubated with 3, lane 3: membrane protein of motor cell
incubated with 3 in the presence of 5000-fold molar excess of 1, lane 4:
membrane protein of leaf cell incubated with 2, and lane 5: membrane
protein of motor cell incubated with 2 in the presence of 5000-fold molar
excess of 1).
Specific bindings of the probe 3 were confirmed by the
disappearance of the corresponding bands in the photo-
labeling examination in the presence of 5000-fold molar
excess of non-labeled leaf-opening substance 1 (lane 3 in
Fig. 3). Binding of probe 3 with binding proteins of 210 and
180 kDa would be inhibited competitively under this
condition.
From previous experiments using fluorescence-labeled 1,
the target cell of 1 is revealed to be a motor cell, which is
contained in the pulvini of C. mimosoides. Thus, photo-
labeling examination needed a large amount of the plasma
membrane fraction of the motor cell, which is expected to
contain a receptor of the leaf-opening substance 1. We
collected a large amount of plant motor cells by cutting off a
large number of sections of plant pulvini (size; ca.
0.5 mm!0.5 mm) containing the motor cell one by one
from plant leaves under a stereoscopic microscope. One
cross-linking experiment needed about nine hundred plant
sections.
The bands for proteins smaller than 100 kDa were also
observed in both lane 2 and lane 3 in Figure 3. However,
these bands were concluded to be non-specific bands due to
the biotin unit, because they were also detected in the cross-
linking examination using photolableing unit 20 without the
structure of 1. The reproducibility was checked by labeling
experiments repeated 10 times.
On the other hand, no specific band was detected in the
labeling experiments with probe 2 under the same
conditions (lane 4 and 5 in Fig. 3). And no band was
Successive homogenization in extraction buffer (0.25 M
sucrose, 3 mM EDTA, 2.5 mM DTT, 25 mM Tris–MES, pH
7.8) at 4 8C, filtration with nylon mesh, and twice

T. Fujii et al. / Tetrahedron 61 (2005) 7874–7893
7879
detected even in the experiment using 1.8-fold concen-
tration of probe 2 (5 mM). These results suggested that the
close arrangement of the photolabeling group and the
binding site in a photoaffinity probe is most important for a
successful result of a photolabeling examination. Molecular
design of a photoaffinity probe should be carried out by
evaluating the close arrangement of photoaffinity group to
the binding site even at the cost of strong bioactivity due to
its steric hindrance.
Next, we examined the difference between probe 3 and 4 in
the efficiency of photoaffinity labeling for these potential
receptor proteins. Photoaffinity labeling experiment using
probe 4 was carried out under the same conditions as in the
case of probe 3. As shown in Figure 4, probe 4 with
benzophenone as a photolabeling group seems to give
clearer band corresponding to 210 and 180 kDa (lane 2 in
Fig. 4) than probe 3 with trifluoromethyldiazirine (lane 2 in
Fig. 3). Specific bindings of the probe 4 were also confirmed
by the disappearance of the corresponding bands in the
photolabeling examination in the presence of 5000-fold
molar excess of non-labeled leaf-opening substance 1 (lane
3 in Fig. 4).
Figure 5. The localization of potential receptor protein for 1 in the plant.
specific binding activity for 1 are located in the plasma
membrane of the motor cell (Fig. 5). And no other part of
the plant contain these potential receptors. This result was
consistent with the result from fluorescence study.
2.8. Photoaffinity labeling using biologically inactive
probes based on analogs of 1
Next, we carried out a photoaffinity labeling examination
using probes based on a biologically inactive analog of 1. If
this potential receptor concerns leaf-opening of this plant,
biologically inactive probe should not give the band
corresponding to the potential receptor protein. Based on
the result of structure–activity relationship study of 1,8,9 we
synthesized a biologically inactive probe 51 and 58
according to a synthetic route in Schemes 7 and 8,
respectively. Compound 38, which is a synthetic inter-
mediate of probe 4, was methylated by TMSCHN2 to give
47. Biologically inactive probe 51 was synthesized from 47
according to the same procedure as in the case of probe 4
(Scheme 7). And 24 was used for the synthesis of probe 58
as shown in Scheme 8.
Figure 4. SDS PAGE analyses of potential membrane receptor for 1 by the
chemiluminescence detection (Lane 1: molecular weight marker, lane 2:
membrane protein of motor cell incubated with 4, lane 3: membrane protein
of motor cell incubated with 4 in the presence of 5000-fold molar excess of
1, and lane 4: membrane protein of leaf cell incubated with 4).
Resulting 51 whose phenolic hydroxyl group was protected
as a methyl ether and 58 whose olefin was reduced to
single bond, did not show leaf-opening activity even at 1!
10K4 M. Probe 51 gave a band at 200 kDa, a different band
From these results, probe 4 which bears a benzophenone
group near the binding site gave the best result in
photoaffinity labeling examination. And it must be
emphasized that probe with strongest bioactivity is not
always the best one in photoaffinity labelling experiments.
This result suggest the important information for the design
of photoaffinity probe for the successful photoaffinity
labeling.
2.7. Localization of the potential receptor protein
We also examined the localization of these binding proteins.
In the fluorescence study with a fluorescent probe,5 it was
revealed that leaf-opening substance 1 exclusively binds to
the motor cell, and not with other parts of the plant body at
all. If these binding proteins were the genuine receptor for
the leaf-movement factor, they would be localized in the
motor cell. Photolabeling experiment with a crude
membrane fraction prepared from the section of plant
leaves, which contain no motor cell gave no specific band on
the chemiluminescence detection for biotinylated proteins
(lane 4 in Fig. 4). From these results, we proposed that
potential receptor proteins for 4 (210 and 180 kDa) that have
Scheme 7. Synthesis of biologically inactive probe (51).

7880
T. Fujii et al. / Tetrahedron 61 (2005) 7874–7893
concerning the control of potassium channels. Trials for the
cloning of this receptor are now in progress.
Photoaffinity labeling is known to provide important
information on the binding site of some bioactive substance
to the known receptor proteins. However, photoaffinity
labeling has provided very few successful results in the
search for unknown receptors of some bioactive substances,
especially, in the case of membrane receptor.20 Our study
showed that careful molecular design is necessary for
success in each unknown receptor by photoaffinity labeling.
Our study also showed that discovery of a new biologically
active substance led to the discovery of an unknown
membrane receptor.
The chemical approach could be compatible with the
molecular biological approach in the study of cell biology,
and it will become more important in this field.
Scheme 8. Synthesis of biologically inactive probe (58).
from the 210 and 180 kDa ones, which were detected using
a biologically active probe (4) (Fig. 6). And probe 58 gave
no band in SDS-PAGE analysis (Fig. 6). These results
showed that the bands of 210 and 180 kDa were strongly
correlated with the biological activity of the probe, which
strongly suggests that these proteins are the genuine
receptors of the leaf-opening substance.
4. Experimental
4.1. General experimental
NMR spectra were recorded on a Jeol JNM-A600
spectrometer [1H (600 MHz) and 13C (150 MHz)], Jeol
JNM-A400 [1H (400 MHz) and 13C (100 MHz)], JNM-
AL300 [1H (300 MHz) and 13C (75 MHz)], a Jeol JNM-EX
270 spectrometer [1H (270 MHz) and 13C (67.5 MHz)]
using TMS in CDCl3, CD2HOD in CD3OH (1H; 3.33 ppm,
13C; 49.8 ppm), 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 or JMS-SX102 spectrometer, using glycerol or
m-nitrobenzylalcohol as a matrix. The HR ESI-MS spectra
were recorded on a Bruker APEX-III. The IR spectra were
recorded on a JASCO FT/IR-410. The specific rotations
were measured by JASCO DIP-360 polarimeter. The HPLC
purification was carried out with a Shimadzu LC-6A pump
equipped with SPD-6A detector using COSMOCIL 5C18-
AR column (f20!250 mm) (Nakalai Tesque 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 60 K070 (Katayama Chemical
Co. Ltd) or silica gel 60N (Kanto Chemical Co. Ltd).
Reversed-phase open-column chromatography was per-
formed 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).
Figure 6. SDS PAGE analyses using biologically inactive probe 51
(detected by the chemiluminescence detection, Lane 1: molecular weight
marker, lane 2: membrane protein of motor cell incubated with 51, lane 3:
membrane protein of motor cell incubated with 58, lane 4: molecular
weight marker, lane 5: membrane protein of motor cell incubated with 4).
3. Discussion
These results are an important advance in the bioorganic
studies of nyctinasty, and would be an important clue for the
molecular mechanism of nyctinasty that has been a
historical mystery since the era of Darwin.
Potential receptor proteins reported in this paper, are key
molecules connecting the bioorganic study using low-
molecular weight natural products with molecular biology
within the cell. Because nyctinastic leaf-movement is
induced by opening and closing of potassium channels, it
is generally considered that potential receptor proteins
would be a subunit of the potassium channels or HC-
ATPase, which is known to be involved in the regulation of
channel movements. However, no subunit of potassium
channels and no HC-ATPase of a plant have been reported
to have such large molecular weight as 200 kDa.19 Thus,
these potential receptors might be a new type of protein
4.1.1. Methyl 3-p-benzyloxyphenyl-2-t-butyldimethyl-
silyloxy propionate (6). Compound 5 (1.00 g, 3.50 mmol)
was dissolved in DMF (35 mL). To this solution, TBSCl
(845 mg, 5.25 mmol), imidazole (714 mg, 10.5 mmol), and
a catalytic amount of DMAP (10 mg, 82.0 mmol) were
added, and then this reaction mixture was stirred overnight
under argon atmosphere. This reaction mixture was then
mixed with water, and extracted by n-hexane/ethyl acetate
1:1, and the organic layer was washed with brine, dried over
abs Na2SO4, concentrated in vacuo, and purified by silica

T. Fujii et al. / Tetrahedron 61 (2005) 7874–7893
7881
gel column chromatography (n-hexane/ethyl acetate 3:1) to
give 6 (1.53 g, quant.).
column chromatography (n-hexane/ethyl acetate 1:1) to
give 9 (55.2 mg, 98%).
1
Compound 6. H NMR (400 MHz, CDCl3, rt): 7.26–7.43
Compound 9. 1H NMR (400 MHz, CDCl3, rt): 7.21 (2H, d,
JZ8.3 Hz), 7.00 (2H, d, JZ8.3 Hz), 4.43 (1H, dd, JZ4.4,
6.8 Hz), 3.76 (3H, s), 3.10 (1H, dd, JZ4.4, 14.2 Hz), 2.94
(1H, dd, JZ6.8, 14.2 Hz), 2.27 (3H, s) ppm; 13C NMR
(67.8 MHz, CDCl3, rt): 174.2, 169.4, 149.3, 133.9, 130.3,
121.3, 71.1, 52.4, 39.7, 21.1 ppm; HR FAB MS (positive):
[MCNa]C Found m/z 261.0716, C12H14O5Na requires m/z
(5H, m), 7.11 (2H, d, JZ8.3 Hz), 6.87 (2H, d, JZ8.3 Hz),
5.03 (2H, s) 4.27 (1H, dd, JZ3.9, 8.8 Hz), 3.70 (3H, s), 2.99
(1H, dd, JZ3.9, 13.7 Hz), 2.81 (1H, dd, JZ8.8, 13.7 Hz),
0.77 (9H, s), K0.15 (3H, s), K0.22 (3H, s) ppm; 13C NMR
(100 MHz, CDCl3, rt): 173.5, 157.4, 136.9, 130.6, 129.6,
128.4, 127.7, 127.3, 114.6, 73.9, 69.9, 51.9, 40.8, 25.7, 18.3,
K5.4, K5.5 ppm; HR FAB MS (positive): [MCNa]C
Found m/z 423.1986, C23H32O4NaSi requires m/z 423.1968;
261.0739; IR (film) n: 3482, 1743, 1508 cmK1
.
IR (film) n: 1756, 1612, 1512 cmK1
.
4.1.5. Methyl 2-(20,30,40-tri-O-acetyl-60-benzyl-b-D-galac-
topyranosyl-oxy)-3-p-acetoxyphenyl propionate (11).
Compound 9 (25.0 mg, 0.105 mmol) and 10 (48.3 mg,
0.105 mmol) were dissolved in dry CH2Cl2 (1.0 mL). To
this solution, MS4A (50 mg), and then AgOTf (30 mg,
0.117 mmol) were added at K30 8C. The reaction mixture
was stirred for 2 h warming slowly to rt. The reaction
mixture was filtered with Celite, and washed with chloro-
form. Then, the organic layer was washed with satd
NaHCO3 and then brine, dried over abs Na2SO4, and
evaporated in vacuo. The residue was then purified by silica
gel column chromatography (toluene/acetone 4:1) to give
11a (18.3 mg, 28%) and 11b (13.6 mg, 21%) with recovered
10 (9.2 mg, 37%).
4.1.2. Methyl 3-p-hydroxyphenyl-2-t-butyldimethylsilyl-
oxy propionate (7). Compound 6 (1.53 g, 3.82 mmol) was
dissolved in ethyl acetate (1.5 mL). To this solution, a
catalytic amount of Pd–C (200 mg) was added. The reaction
mixture was stirred overnight under argon atmosphere,
filtered with Celite, concentrated in vacuo, and purified by
silica gel TLC (n-hexane/ethyl acetate 1:1) to give 7 (1.03 g,
87%).
Compound 7. 1H NMR (400 MHz, CDCl3, rt): 7.06 (2H, d,
JZ8.3 Hz), 6.73 (2H, d, JZ8.3 Hz), 4.27 (1H, dd, JZ3.9,
9.3 Hz), 3.70 (3H, s), 2.97 (1H, dd, JZ3.9, 13.7 Hz), 2.80
(1H, dd, JZ9.3, 13.7 Hz), 0.79 (9H, s), K0.14 (3H, s),
K0.21 (3H, s) ppm; 13C NMR (67.8 MHz, CDCl3, rt):
173.7, 154.3, 130.8, 129.2, 115.0, 73.9, 51.9, 40.8, 25.7,
18.3, K5.37, K5.43 ppm; HR FAB MS (positive): [MC
H]C Found m/z 311.1653, C16H27O4Si requires m/z
Compound 11a. 1H NMR (400 MHz, CDCl3, rt): 7.25–7.35
(5H, m), 7.16 (2H, d, JZ8.8 Hz), 6.94 (2H, d, JZ8.8 Hz),
5.42 (1H, d, JZ3.4 Hz), 5.21 (1H, dd, JZ7.8, 10.3 Hz),
4.98 (1H, dd, JZ3.4, 10.3 Hz), 4.59 (1H, t, JZ5.9 Hz), 4.50
(1H, d, JZ12.2 Hz), 4.48 (1H, d, JZ7.8 Hz), 4.38 (1H, d,
JZ12.2 Hz), 3.77 (1H, t, JZ6.3 Hz), 3.60 (3H, s), 3.49 (1H,
dd, JZ6.3, 9.8 Hz), 3.44 (1H, dd, JZ6.3, 9.8 Hz), 3.10 (1H,
dd, JZ5.9, 14.2 Hz), 3.04 (1H, dd, JZ5.9, 14.2 Hz), 2.26
(3H, s), 2.06 (3H, s), 1.96 (3H, s), 1.95 (3H, s) ppm;13C
NMR (100 MHz, CDCl3, rt): 170.7, 170.1, 169.9, 169.7,
169.3, 149.4, 137.4, 133.3, 130.7, 128.4, 127.81, 127.78,
121.1, 99.6, 75.9, 73.5, 72.2, 70.7, 68.6, 67.43, 67.41, 51.8,
38.2, 21.2, 20.8, 20.74, 20.66 ppm; HR FAB MS (positive):
[MCH]C Found m/z 617.2250, C31H37O13 requires m/z
617.2234 IR (film) n: 1749, 1508 cmK1; [a]D25 K38.18 (c
1.0, CHCl3).
311.1679; IR (film) n: 3419, 1736, 1614, 1597, 1516 cmK1
.
4.1.3. Methyl 3-p-acetoxyphenyl-2-t-butyldimethylsilyl-
oxy propionate (8). Compound 7 (1.03 g, 3.32 mmol) was
dissolved in pyridine (20 mL). To this solution acetic
anhydride (0.94 mL, 9.95 mmol) was added at 0 8C. The
reaction mixture was then stirred overnight under argon
atmosphere, and evaporated with toluene to remove
pyridine as an azeotropic mixture. The residue was then
purified by silica gel column chromatography (n-hexane/
ethyl acetate 4:1) to give 8 (1.03 g, 88%).
Compound 8. 1H NMR (400 MHz, CDCl3, rt): 7.20 (2H, d,
JZ8.8 Hz), 6.98 (2H, d, JZ8.8 Hz), 4.29 (1H, dd, JZ3.9,
9.3 Hz), 3.70 (3H, s), 3.04 (1H, dd, JZ3.9, 13.7 Hz), 2.85
(1H, dd, JZ9.3, 13.7 Hz), 2.26 (3H, s), 0.76 (9H, s), K0.14
(3H, s), K0.23 (3H, s) ppm; 13C NMR (67.8 MHz, CDCl3,
rt): 173.2, 169.3, 149.3, 134.9, 130.6, 121.2, 73.6, 51.9,
40.9, 25.6, 21.2, 18.2, K5.4, K5.6 ppm; HR FAB MS
(positive): [MCNa]C Found m/z 375.1608, C18H28O5 NaSi
Compound 11b. 1H NMR (400 MHz, CDCl3, rt): 7.18–7.30
(5H, m), 7.13 (2H, d, JZ8.8 Hz), 6.92 (2H, d, JZ8.8 Hz),
5.34 (1H, d, JZ3.4 Hz), 5.16 (1H, dd, JZ7.8, 10.3 Hz),
4.84 (1H, dd, JZ3.4, 10.3 Hz), 4.44 (1H, d, JZ11.7 Hz),
4.41 (1H, d, JZ7.8 Hz), 4.36 (1H, d, JZ11.7 Hz), 4.16 (1H,
dd, JZ3.9, 9.3 Hz), 3.76 (1H, t, JZ6.4 Hz), 3.64 (3H, s),
3.45 (1H, dd, JZ6.4, 9.3 Hz), 3.40 (1H, dd, JZ6.4, 9.3 Hz),
3.02 (1H, dd, JZ9.3, 14.1 Hz), 2.92 (1H, dd, JZ3.9,
14.1 Hz), 2.21 (3H, s), 1.98 (3H, s), 1.88 (3H, s), 1.71 (3H,
s) ppm; 13C NMR (100 MHz, CDCl3, rt): 171.6, 170.2,
170.0, 169.4, 169.2, 149.5, 137.3, 133.9, 130.3, 128.4,
127.80, 127.79, 121.5, 101.8, 80.9, 73.4, 72.1, 71.0, 68.5,
67.3, 67.2, 52.1, 38.0, 21.1, 20.6, 20.5 ppm; HR FAB MS
(positive): [MCH]C Found m/z 617.2250, C31H37O13
requires m/z 617.2234 IR (film) n: 1751, 1510 cmK1; [a]D25
K14.38 (c 1.0, CHCl3).
requires m/z 375.1604; IR (film) n: 1760, 1508 cmK1
.
4.1.4. Methyl 3-p-acetoxyphenyl-2-hydroxy propionate
(9). Compound 9 (83.1 mg, 0.238 mmol) was dissolved in
THF (1.2 mL). To this solution, THF solution (1.2 mL) of
TBAF (1 M THF solution, 0.47 mL, 0.470 mmol) and acetic
acid (27 mL, 0.473 mmol) was added. The rection mixture
was stirred overnight under argon atmosphere, mixed with
water, and extracted with ethyl acetate. The organic layer
was then washed with brine, dried over abs Na2SO4, and
concentrated in vacuo. The residue was purified by silica gel
4.1.6. Methyl 2-(60-benzyl-b-D-galactopyranosyloxy)-3-
p-hydroxy-phenyl propionate (12). Compound 11a

7882
T. Fujii et al. / Tetrahedron 61 (2005) 7874–7893
1
Compound 13a. H NMR (400 MHz, acetone-d6, 40 8C):
(71.0 mg, 0.115 mmol) was dissolved in MeOH (1.5 mL),
and sodium methoxide (6.0 mg, 0.111 mmol) was added to
this solution. After 7 h stirring at rt, Amberlite IR-120B
(HC) was added to this solution for neutralization. After
filtration, the filtrate was concentrated in vacuo and purified
by silica gel TLC (CHCl3/MeOH 10:1) to give 12a
(47.6 mg, 92%).
7.19–7.36 (5H, m), 7.03 (2H, d, JZ8.3 Hz), 6.66 (2H, d, JZ
8.3 Hz), 4.70 (1H, br s), 4.53 (3H, m), 4.17 (1H, br s), 3.82–
3.94 (2H, m), 3.75 (1H, br s), 3.66 (2H, m), 3.50 (3H, s),
3.01 (1H, dd, JZ5.9, 13.7 Hz), 2.93 (1H, m), 0.94 (9H, s),
0.93 (9H, s), 0.90 (9H, s), 0.89 (9H, s), 0.15 (6H, s), 0.14
(3H, s), 0.12 (3H, s), 0.09 (3H, s), 0.08 (3H, s), 0.06 (3H, s),
0.01 (3H, s) ppm; 13C NMR (100 MHz, acetone-d6, 40 8C):
171.9, 155.0, 139.7, 131.1, 130.3, 128.9, 128.2, 128.0,
120.4, 101.7, 79.1, 77.0, 75.6, 73.7, 73.6, 73.4, 70.7, 51.5,
39.0, 27.1, 26.9, 26.6, 26.1, 19.1, 18.8, 18.7, K3.8, K3.9,
K4.2, K4.4 ppm; HR FAB MS (positive): [MCNa]C
Found m/z 927.5061, C47H84O9Si4Na requires m/z 927.5090
IR (film) n: 1751, 1510 cmK1; [a]2D5 K15.68 c¸(c 1.0,
CHCl3).
Similarly, 11b (68.6 mg, 0.111 mmol) was dissolved in
MeOH (1.5 mL), and sodium methoxide (6.0 mg,
0.111 mmol) was added to this solution. After 7 h stirring
at rt, Amberlite IR-120B (HC) was added to this solution for
neutralization. After filtration, the filtrate was concentrated
in vacuo and purified by silica gel TLC (CHCl3/MeOH
10:1) to give 12b (41.0 mg, 82%).
1
Compound 13b. H NMR (400 MHz, acetone-d6, 40 8C):
1
Compound 12a. H NMR (400 MHz, CD3OD, rt): 7.22–
7.17–7.33 (5H, m), 7.03 (2H, d, JZ8.3 Hz), 6.72 (2H, d, JZ
8.3 Hz), 4.26–4.55 (4H, m), 4.11 (1H, s), 3.88 (1H, dd, JZ
5.4, 7.4 Hz), 3.54–3.77 (4H, m), 3.44 (3H, s), 3.06 (1H, dd,
JZ5.4, 13.2 Hz), 2.92 (1H, dd, JZ7.4, 13.2 Hz), 0.75–0.95
(36H, m), 0.00–0.16 (24H, m) ppm; 13C NMR (100 MHz,
acetone-d6, rt): 171.9, 155.2, 131.2, 129.02, 128.97, 128.3,
128.1, 120.7, 120.5, 103.8, 80.2, 76.8, 74.4, 73.4, 72.9, 69.5,
51.6, 51.5, 38.2, 27.1, 26.8, 26.5, 26.0, 19.4, 19.3, 19.1,
18.7, K2.0–K4.5 (br) ppm; HR FAB MS (positive): [MC
Na]C Found m/z 927.5095, C47H84O9NaSi4 requires m/z
927.5090 IR (film) n: 1751, 1510 cmK1; [a]D25 K25.48 (c
1.0, CHCl3).
7.34 (5H, m), 7.02 (2H, d, JZ8.3 Hz), 6.65 (2H, d, JZ
8.3 Hz), 4.61 (1H, t, JZ6.3 Hz), 4.55 (2H, s), 4.29 (1H, d,
JZ7.8 Hz), 3.78 (1H, d, JZ3.4 Hz), 3.65–3.74 (3H, m),
3.64 (3H, s), 3.55 (1H, dd, JZ7.8, 9.8 Hz), 3.45 (1H, dd,
JZ3.4, 9.8 Hz), 3.02 (2H, d, JZ6.3 Hz) ppm; 13C NMR
(100 MHz, CD3OD, rt): 174.0, 157.1, 139.5, 131.5, 129.3,
128.8, 128.6, 128.2, 115.9, 104.1, 79.2, 75.4, 74.7, 74.4,
72.4, 70.9, 70.5, 52.4, 39.5 ppm; HR FAB MS (positive):
[MCH]C Found m/z 449.1786, C23H29O9 requires m/z
449.1812 IR (film) n: 3398, 1736, 1614, 1516 cmK1; [a]D25
K1.98 (c 1.0, MeOH).
Compound 12b. 1H NMR (400 MHz, CD3OD, rt): 7.21K7/
37 (5H, m), 7.04 (2H, d, JZ8.3 Hz), 6.67 (2H, d, JZ
8.3 Hz), 4.52 (2H, s), 4.30 (1H, t, JZ6.8 Hz), 4.27 (1H, d,
JZ7.8 Hz), 3.76 (1H, d, JZ3.4 Hz), 3.53–3.72 (4H, m),
3.52 (3H, s), 3.42 (1H, dd, JZ3.4, 10.3 Hz), 3.01 (1H, dd,
JZ6.8, 13.7 Hz), 2.91 (1H, dd, JZ6.8, 13.7 Hz) ppm; 13C
NMR (100 MHz, CD3OD, rt): 174.4, 157.2, 139.5, 131.5,
129.3, 128.7, 128.6, 128.0, 116.0, 104.7, 80.8, 75.2, 74.6,
74.3, 72.2, 70.6, 52.3, 38.8 ppm; HR FAB MS (positive):
[MCH]C Found m/z 449.1841, C23H29O9 requires m/z
449.1812 IR (film) n: 3585, 1738, 1614, 1518 cmK1; [a]D25
K22.08 (c 1.0, MeOH).
4.1.8. Methyl 2-(20,30,40-tri-t-butyldimethylsilyloxy-b-D-
galactopyranosyloxy)-3-p-t-butyldimethylsilyloxyphenyl
propionate (14). Compound 13a (86.3 mg, 95.3 mmol) was
dissolved in THF (1.0 mL). To this solution, a catalytic
amount of Pd(OH)2 (10 mg) was added, and the reaction
mixture was stirred overnight under hydrogen atmosphere.
Then the reaction mixture was filtered with Celite,
concentrated in vacuo, and purified by silica gel column
chromatography (n-hexane/ethyl acetate 5:1) to give 14a
(61.3 mg, 79%).
Similarly, 13b (23.5 mg, 26.0 mmol) was dissolved in THF
(0.25 mL). To this solution, a catalytic amount of Pd(OH)2
(2.5 mg) was added, and the reaction mixture was stirred
overnight under hydrogen atmosphere. Then the reaction
mixture was filtered with Celite, concentrated in vacuo, and
purified by silica gel column chromatography (n-hexane/
ethyl acetate 5:1) to give 14b (21.9 mg, quant.).
4.1.7. Methyl 2-(60-benzyl-20,30,40-tri-t-butyldimethyl-
silyloxy-b-D-galactopyranosyloxy)-3-p-t-butyldimethyl-
silyloxyphenyl propionate (13). Compound 12a (47.6 mg,
0.106 mmol) was dissolved in CH2Cl2 (1.0 mL). To this
solution, 2,6-lutidine (0.18 mL, 1.55 mmol) and
TBDMSOTf (0.24 mL, 1.05 mmol) were added at 0 8C.
the reaction mixture was stirred for an hour, washed with
water and then brine, dried over abs Na2SO4, and
concentrated in vacuo. The residue was then purified by
silica gel column chromatography (n-hexane/ethyl acetate
10:1) to give 13a (86.3 mg, 90%).
1
Compound 14a. H NMR (400 MHz, acetone-d6, 40 8C):
7.14 (2H, d, JZ8.8 Hz), 6.75 (2H, d, JZ8.8 Hz), 4.74 (1H,
br s), 4.51 (1H, d, JZ4.4 Hz), 4.20 (1H, br s), 3.96 (1H, dd,
JZ4.9, 7.4 Hz) 3.64–3.79 (3H, m), 3.56 (3H, s), 3.42 (1H,
br s), 3.06 (1H, dd, JZ6.4, 14.2 Hz), 2.99 (1H, dd, JZ7.3,
14.2 Hz), 0.99 (9H, s), 0.974 (9H, s), 0.965 (9H, s), 0.93
(9H, s), 0.18–0.19 (15H, m), 0.14 (3H, s), 0.12 (3H, s), 0.11
(3H, s) ppm; 13C NMR (100 MHz, acetone-d6, rt): 171.9,
154.8, 131.0, 130.4, 120.3, 102.3, 76.8, 76.6, 73.3, 72.8,
61.7, 51.5, 38.8, 30.6, 27.1, 26.5, 26.0, 19.3, 19.1, 18.72,
18.68, K3.2, K3.7, K.3.9, K4.3, K4.4 ppm; HR FAB MS
(positive): [MCNa]C Found m/z 837.4606, C40H78O9NaSi4
requires m/z 837.4621 IR (film) n: 3585, 1751, 1610,
1510 cmK1; [a]2D5 K18.88 (c 1.0, CHCl3).
Similarly, 12b (41.0 mg, 91.5 mmol) was dissolved in
CH2Cl2 (1.0 mL). To this solution, 2,6-lutidine (0.18 mL,
1.55 mmol) and TBDMSOTf (0.24 mL, 1.05 mmol) were
added at 0 8C. the reaction mixture was stirred for an hour,
washed with water and then brine, dried over abs Na2SO4,
and concentrated in vacuo. The residue was then purified by
silica gel column chromatography (n-hexane/ethyl acetate
10:1) to give 13b (23.5 mg, 28%).

T. Fujii et al. / Tetrahedron 61 (2005) 7874–7893
7883
1
Compound 14b. H NMR (400 MHz, acetone-d6, 40 8C):
of 15a and 15b (124 mg, 0.145 mmol) was dissolved in
1,4-dioxane (1.5 mL), and DDQ (165 mg, 0.727 mmol) was
added to this solution. After additional DDQ (65.8 mg,
0.290 mmol) was added to this solution, the reaction
mixture was refluxed for 24 h under argon atmosphere.
After filtration, the filtrate was mixed with Et2O, washed
with satd NaHCO3 aq, dried over abs Na2SO4, and
concentrated in vacuo. The residue was separated by silica
gel column chromatography (n-hexane/ethyl acetate 10:1),
and then by silica gel TLC (n-hexane/ethyl acetate 5:1) to
give (Z)-16 (76.0 mg, 61%) and (E)-16 (9.6 mg, 8%) with
recovered 15a and 15b as a mixture (9.2 mg, 7%).
7.09 (2H, d, JZ8.8 Hz), 6.77 (2H, d, JZ8.8 Hz), 4.49 (1H,
br s), 4.29 (1H, m), 4.14 (1H, br s), 3.92 (1H, m), 3.68–3.79
(2H, m), 3.50–3.63 (5H, m), 3.10 (1H, m), 2.95 (1H, dd, JZ
8.8, 14.2 Hz), 0.98 (9H, s), 0.97 (9H, s), 0.96 (9H, s), 0.95
(9H, s), 0.11–0.21 (24H, m) ppm; 13C NMR (100 MHz,
acetone-d6, rt): 172.8, 155.2, 131.1, 120.7, 120.4, 104.0,
80.8, 77.0, 76.7, 76.5, 72.8, 61.4, 51.6, 38.3, 27.1, 26.8,
26.5, 26.0, 19.5, 19.1, 18.74, 18.69, K2.3, K3.3, K3.6,
K3.9, K4.3, K4.35, K4.37 ppm; HR FAB MS (positive):
[MCNa]C Found m/z 837.4604, C40H78O9Si4Na requires
m/z 837.4621 IR (film) n: 3585, 1738, 1610, 1510 cmK1
[a]2D5 K25.18 (c 1.0, CHCl3).
;
Compound (Z)-16. 1H NMR (400 MHz, acetone-d6, rt): 7.77
(2H, d, JZ8.3 Hz), 6.95 (1H, s), 6.79 (2H, d, JZ8.3 Hz),
5.32 (1H, d, JZ7.3 Hz), 4.18 (1H, dd, JZ7.3, 9.7 Hz),
4.00–4.09 (2H, m), 3.96 (1H, m), 3.76–3.85 (2H, m), 3.72
(3H, s), 1.90 (3H, s), 1.00 (9H, s), 0.96 (18H, s), 0.85 (9H, s),
0.25 (3H, s), 0.19 (9H, s), 0.17 (3H, s), 0.10 (3H, s), 0.07
(3H, s), 0.02 (3H, s) ppm; 13C NMR (100 MHz, acetone-d6,
rt): 170.4, 164.4, 157.2, 139.9, 133.0, 127.8, 126.0, 120.6,
101.8, 79.2, 76.5, 73.6, 73.3, 63.5, 52.1, 27.3, 26.8, 26.7,
26.0, 20.6, 19.6, 19.3, 19.0, 18.8, K2.3, K3.0, K3.46,
K3.51, K3.8, K4.21, K4.23, K4.6 ppm; HR FAB MS
(positive): [MCNa]C Found m/z 877.4579, C42H78O10-
NaSi4 requires m/z 877.4570 IR (film) n: 1720, 1603,
1508 cmK1; [a]2D6 C79.58 (c 1.0, CHCl3).
4.1.9. Methyl 2-(60-O-acetyl-20,30,40-tri-t-butyldimethyl-
silyloxy-b-D-galactopyranosyloxy)-3-p-t-butyldimethyl-
silyloxyphenyl propionate (15). Compound 14a (61.3 mg,
75.2 mmol) was dissolved in pyridine (0.5 mL). To this
solution, acetic anhydride (21 mL, 0.222 mmol) was added,
and the reaction mixture was stirred overnight under argon
atmosphere. Then, the reaction mixture was mixed with
toluene and evaporated to remove pyridine as an azeotropic
mixture to give 15a (58.2 mg, 90%).
Similarly, 14b (21.9 mg, 26.9 mmol) was dissolved in
pyridine (0.2 mL). To this solution, acetic anhydride
(7 mL, 0.72 mmol) was added, and the reaction mixture
was stirred overnight under argon atmosphere. Then, the
reaction mixture was mixed with toluene and evaporated to
remove pyridine as an azeotropic mixture to give 15b
(23.8 mg, quant.).
4.1.11. Methyl (Z)-2-(20,30,40-tri-t-butyldimethylsilyloxy-
b-D-galactopyranosyloxy)-3-p-t-butyldimethylsilyloxy-
phenyl acrylate (17). Compound (Z)-16 (22.1 mg,
25.8 mmol) was dissolved in MeOH (0.5 mL). To this
solution was added sodium methoxide (2.8 mg, 51.9 mmol)
at K35 8C, and this reaction mixture was stirred for an hour
at K20 8C. After neutralized with Amberlite IR-120B (HC),
the reaction mixture was filtered and separated by silica gel
TLC (n-hexane/ethyl acetate 3:1) to give 17 (17.0 mg,
81%).
1
Compound 15a. H NMR (400 MHz, acetone-d6, 40 8C):
7.06 (2H, d, JZ8.8 Hz), 6.73 (2H, d, JZ8.8 Hz), 4.70 (1H,
br s), 4.60 (1H, dd, JZ5.4, 7.8 Hz), 4.52 (1H, m), 4.39 (1H,
br s), 4.23 (1H, br s), 3.94 (2H, m), 3.80 (1H, br s), 3.51 (3H,
s), 3.01 (1H, dd, JZ5.9, 13.7 Hz),4.14 (1H, br s), 3.92 (1H,
m), 3.79–3.68 (2H, m), 3.63–3.50 (5H, m), 3.10 (1H, m),
2.91 (1H, m), 2.01 (3H, s), 0.85–0.96 (36H, m), 0.04–0.18
(24H, m) ppm; 13C NMR (100 MHz, acetone-d6, 40 8C):
172.2, 170.9, 155.3, 131.2, 130.2, 120.6, 100.7, 77.3, 75.0,
73.7, 68.3, 65.8, 51.6, 39.0, 26.6, 26.5, 26.3, 26.1, 21.0,
19.0, 18.8, 18.6, K4.0, K4.2, K4.6 ppm; HR FAB MS
(positive): [MCNa]C Found m/z 879.4697, C42H80O10Si4-
Na requires m/z 879.4726 IR (film) n: 1747, 1610,
1512 cmK1; [a]2D0 K12.68 (c 1.0, CHCl3).
1
Compound 17. H NMR (400 MHz, acetone-d6, rt): 7.79
(2H, d, JZ8.3 Hz), 6.94 (1H, s), 6.79 (2H, d, JZ8.3 Hz),
5.29 (1H, d, JZ7.3 Hz), 4.20 (1H, dd, JZ7.3, 9.3 Hz), 4.14
(1H, d, JZ2.0 Hz), 3.77 (1H, dd, JZ2.0, 9.3 Hz), 3.72 (3H,
s), 3.46–3.57 (3H, m), 1.01 (9H, s), 0.96 (18H, s), 0.85 (9H,
s), 0.23 (3H, s), 0.19 (9H, s), 0.16 (3H, s), 0.11 (3H, s), 0.06
(3H, s), 0.01 (3H, s) ppm; 13C NMR (100 MHz, acetone-d6,
rt): 164.2, 156.9, 139.8, 132.9, 127.7, 125.9, 120.4, 101.9,
76.8, 76.5, 73.3, 72.6, 60.9, 52.0, 27.3, 26.83, 26.76, 26.0,
19.6, 19.4, 19.0, 18.8, K2.2, K3.1, K3.40, K3.42, K3.7,
K4.18, K4.21, K4.3 ppm; HR FAB MS (positive): [MC
Na]C Found m/z 835.4450, C40H76O9Si4Na requires m/z
1
Compound 15b. H NMR (400 MHz, acetone-d6, 40 8C):
7.04 (2H, d, JZ8.8 Hz), 6.73 (2H, d, JZ8.8 Hz), 4.62–4.07
(5H, m), 3.96–3.69 (3H, m), 3.49 (3H, s), 3.01 (1H, m), 2.93
(1H, dd, JZ7.8, 13.7 Hz), 1.93 (3H, s), 0.94 (9H, s), 0.93
(9H, s), 0.91 (9H, s), 0.90 (9H, s), 0.17–0.07 (24H, m) ppm;
13C NMR (100 MHz, acetone-d6, 40 8C): 171.7, 170.7,
155.3, 131.2, 120.7, 120.6, 101.6, 80.8, 79.2, 77.3, 75.0,
73.6, 65.2, 52.0, 38.5, 26.8, 26.6, 26.5, 26.1, 20.8, 19.0,
18.8, 18.7, K4.1, K4.2, K4.6 ppm; HR FAB MS
(positive): [MCNa]C Found m/z 879.4697, C42H80O10Si4-
Na requires m/z 879.4726 IR (film) n: 1743, 1610,
1510 cmK1; [a]2D5 K13.8 8C (c 1.0, CHCl3).
835.4464; IR (film) n: 3448, 1722, 1637, 1603, 1508 cmK1
[a]2D5 C57.08 (c 1.0, CHCl3).
;
4.1.12. Methyl (Z)-2-(60-formyl-30,40,50-tri-t-butyldi-
methylsilyloxy-b-D-galactopyranosyloxy)-3-p-t-butyl-
dimethylsilyloxyphenyl acrylate (18). Compound 17
(73.0 mg, 89.8 mmol) was dissolved in DMSO (1.0 mL),
and TEA (1.0 mL), SO3.pyr (71.0 mg, 447 mmol) were
added to this solution. The reaction mixture was stirred for
an hour under argon atmosphere, mixed with water,
extracted with Et2O. After washed with satd NH4Cl aq,
satd NaHCO3, and brine, the organic layer was dried over
4.1.10. Methyl (Z)-2-(60-O-acetyl-20,30,40-tri-t-butyl-
dimethylsilyloxy-b-D-galactopyranosyloxy)-3-p-t-butyl-
dimethylsilyloxyphenyl acrylate (16a). A diastereomixture

7884
T. Fujii et al. / Tetrahedron 61 (2005) 7874–7893
abs Na2SO4, and concentrated in vacuo. The residue was
then separated by silica gel column chromatography
(n-hexane/ethyl acetate 5:1) to give 18 (43.9 mg, 60%).
4.1.14. t-Butyl 3-p-benzyloxyphenyl-2-acetyloxypropio-
nate (21). Compound 5 (1.2 g, 4.2 mmol) was dissolved in
33 mL of H2O–MeOH (2: 31), and 8.4 mL of 1 M KOH aq
was added to this solution. After 1 h stirring, the solution
was neutralized with Amberlite IR-120B (HC) and filtered.
Evaporation of the filtrate gave a white crystal. This crystal
was dissolved in pyridine (20 mL), and acetic anhydride
(10 mL) was added to this solution. After 4 h stirring, the
reaction mixture was mixed with 20 mL of toluene and
concentrated in vacuo. The residue was washed with
saturated NaHCO3 aq, 1 M HCl aq and then extracted
with chloroform. The organic layer was evaporated and the
residue was dissolved in abs CH2Cl2 (10 mL). t-Butanol
(0.62 mL, 6.54 mmol), dimethylaminopyridine (DMAP;
53 mg, 0.44 mmol), and then dicyclohexylcarbodiimide
(DCC; 990 mg, 4.80 mmol) were added to this solution.
After 2 days stirring, the reaction mixture was mixed with
water, and extracted with chloroform, dried over abs
Na2SO4, and evaporated to dryness. Then the residue was
purified by silica gel column chromatography (n-hexane/
ethyl acetate 5:1) to give 21 (1.26 mg, 78%).
1
Compound 18. H NMR (400 MHz, acetone-d6, rt): 9.43
(1H, br s), 7.85 (2H, d, JZ8.8 Hz), 7.04 (1H, s), 6.81 (2H, d,
JZ8.8 Hz), 5.41 (1H, m), 4.37 (1H, br s), 4.26 (1H, dd, JZ
6.8, 8.8 Hz), 4.10 (1H, d, JZ2.0 Hz), 3.89 (1H, dd, JZ2.0,
8.8 Hz), 3.73 (3H, s), 0.95 (18H, s), 0.93 (9H, s), 0.86 (9H,
s), 0.194 (6H, s), 0.186 (6H, s), 0.17 (3H, s), 0.08 (3H, s),
0.04 (3H, s), K0.01 (3H, s) ppm; 13C NMR (100 MHz,
acetone-d6, rt): 201.4, 164.2, 157.2, 139.7, 133.0, 127.5,
126.5, 120.5, 101.8, 80.4, 79.0, 75.8, 73.1, 52.1, 27.0, 26.6,
26.4, 25.8, 19.3, 19.1, 18.8, 18.7, K2.6, K3.5, K3.6, K3.7,
K4.0, K4.35, K4.38, K4.8 ppm; HR FAB MS (positive):
[MCNa]C Found m/z 833.4303, C40H74O9NaSi4 requires
m/z 833.4308; IR (film) n: 1739, 1720, 1637, 1602,
1508 cmK1; [a]2D5 C56.28 (c 1.0, CHCl3).
4.1.13. Potassium (Z)-2-[60-[2-[2-[2-(2-biotinylamino-
ethoxy)-ethoxy]-ethoxy]-4-(3-tri-fluoromethyl)-3H-dia-
zirin-3-yl]benzyloxyimino]-b-D-galactopyranosyloxy]-3-
p-hydroxyphenyl acrylate (2). Compound 18 (43.9 mg,
54.1 mmol) was dissolved in MeOH (0.5 mL). Affilight-
CHOe (19, 15 mg, 21.7 mmol) was deprotected by TFA in
CH2Cl2, and resulting 20 was added to methanolic solution
of 18. After adjusting pH at 5 by acetate buffer (10 mL), the
reaction mixture was stirred overnight at 35 8C, and
concentrated in vacuo. The residue was purified by silica
gel TLC (CHCl3/MeOH 10:1) to give coupling product
(26.3 mg, 87%). 5.7 mg (4.7 mmol) of coupling product was
dissolved in THF (0.5 mL), and deprotected by 3 h
treatment with TBAF (1 M THF solution, 30 mmol) under
argon atmosphere. The reaction mixture was concentrated
and purified by silica gel TLC (CHCl3/MeOH 5:1) to give a
free acid form of 2 (3.5 mg, 95%). Free acid form of 2
(14.8 mg, 16.3 mmol) was dissolved in MeOH–H2O (1:1,
1.4 mL). This solution was mixed with 1 M KOH aq
(32.6 mL, 32.6 mmol), and stirred for 2 days at rt.
21: 1H NMR (270 MHz, CDCl3, rt): 7.43–7.28 (5H, m), 7.13
(2H, d, JZ8.6 Hz), 6.98 (2H, d, JZ8.6 Hz), 5.03 (2H, s),
5.03 (1H, m), 3.09 (1H, dd, JZ5.0, 13.9 Hz), 3.09 (1H, dd,
JZ8.2, 13.9 Hz), 2.07 (3H, s), 1.39 (9H, s) ppm; 13C NMR
(68 MHz, CDCl3 rt): 170.1, 168.5, 157.5, 136.8, 130.2,
128.3, 128.2, 127.7, 127.2, 114.5, 82.0, 73.4, 69.8, 36.4,
27.8, 20.7 ppm; HR FAB MS (positive): [MCNa]C Found
m/z 393.1706, C22H26O5Na requires m/z 393.1678; IR (film)
n: 1741, 1612, 1512, 1454 cmK1
.
4.1.15. t-Butyl 3-p-benzyloxyphenyl-2-hydroxypropio-
nate (22). Compound 21 (1.26 g, 3.40 mmol) was dissolved
in methanol (30 mL), and sodium methoxide (220 mg,
4.1 mmol) was added to this solution at 0 8C. After 1 h
stirring, the reaction mixture was neutralized by Amberlite
IR-120B (HC) and filtered. The filtrate was evaporated and
purified by silica gel column chromatography (n-hexane/
ethyl acetate 5:1) to give 22 (1.07 g, 98%).
After neutralization with Amberlite IR-120B (HC), the
reaction mixture was filtered, mixed with 0.1 M K2CO3 (81.
3 mL, 8.13 mmol), and purified by HPLC[Develosil C8-UG-
5 (f20!250 mm), 40% MeCN aq] to give (E)-2 (5.4 mg,
34%) and (Z)-2 (4.0 mg, 25%), respectively.
1
22: H NMR (270 MHz, CD3OD, rt): 7.42–7.24 (5H, m),
7.14 (2H, d, JZ8.7 Hz), 6.88 (2H, d, JZ8.7 Hz), 5.04 (2H,
s), 4.17 (1H, dd, JZ5.6, 7.1 Hz), 2.92 (1H, dd, JZ5.6,
13.9 Hz), 2.82 (1H,) ppm; 13C NMR (68 MHz, CDCl3 rt):
170.1, 168.5, 157.5, 136.8, 130.2, 128.3, 128.2, 127.7,
127.2, 114.5, 82.0, 73.4, 69.8, 36.4, 27.8, 20.7 ppm; HR
FAB MS (positive): [MCNa]C Found m/z 393.1706,
C22H26O5Na requires m/z 393.1678; IR (film) n: 3502,
1
Compound (E)-2. H NMR (400 MHz, CD3OD, rt): 7.76
(2H, d, JZ8.8 Hz), 7.33 (1H, d, JZ7.8 Hz), 6.85 (1H, s),
6.82 (1H, d, JZ7.8 Hz), 6.72 (2H, d, JZ8.8 Hz), 6.71 (1H,
d, JZ13.2 Hz), 5.13 (2H, s), 4.65 (1H, dd, JZ1.4, 4.9 Hz),
4.44 (1H, dd, JZ4.4, 7.3 Hz), 4.25 (1H, dd, JZ4.4, 7.8 Hz),
4.10–4.17 (3H, m), 3.78–3.87 (3H, m), 3.54–3.70 (3H, m),
3.51 (2H, t, JZ5.8 Hz), 3.15 (1H, m), 2.88 (1H, dd, JZ4.9,
12.7 Hz), 2.66 (1H, d, JZ12.7 Hz), 2.18 (2H, t, JZ7.3 Hz),
1.48–1.75 (4H, m), 1.31–1.44 (2H, m) ppm; 13C NMR
(100 MHz, CD3OD, rt): 176.2, 175.5, 158.8, 158.1, 149.7,
133.2, 131.0, 130.7, 130.5, 130.1, 127.0, 123.6 (JZ275 Hz),
122.8, 120.1, 115.9, 110.6, 105.0, 75.2, 74.7, 72.6, 71.9,
71.5, 71.3, 70.6, 69.6, 63.3, 61.6, 57.0, 41.1, 40.3, 36.7,
29.7, 29.5, 26.8 ppm; HR FAB MS (negative): [MKK]K
Found m/z 925.2872, C40H48O14F3N6S requires m/z
1741, 1612, 1512, 1454 cmK1
.
4.1.16. 30,40,60-Tri-O-acetyl-20-deoxy-20-phthalimide-a-
D-galactopyranosyl bromide (23). D-Galactosamine hydro-
chloride (1 g, 4.65 mmol, Sigma-Aldrich Co., Ltd) was
suspended in DMF (30 mL), and to this solution, was added
triethylamine (1.7 mL, 11.6 mmol). After 20 min-stirring at
rt, phtalic anhydride (688 mg, 4.65 mmol) was added to this
solution, and the mixture was stirred for 2 h at 50 8C under
argon atmosphere. After the reaction mixture was allowed
to stand until its temperature dropped to rt, excess amounts
of triethylamine (5 mL) and acetic anhydride (5 mL) were
added. After 2-day stirring at rt under argon atmosphere, the
reaction mixture was concentrated in vacuo, mixed with
925.2901; IR (film) n: 3309, 1685, 1650, 1608, 1578 cmK1
[a]2D2 C66.18 (c 0.62, MeOH).
;

T. Fujii et al. / Tetrahedron 61 (2005) 7874–7893
7885
water (150 mL), extracted three times with n-hexane/ethyl
acetate1:1, and dried over abs Na2SO4. The organic layer
was separated by silica gel column chromatography
(n-hexane/ethyl acetate 1:1) to give 32 (1.82 g, 82%).
Resulting 32 (1.395 g, 2.92 mmol) was mixed with acetic
anhydride (0.69 mL, 7.31 mmol), and then treated with
HBr–AcOH (30%, 16 mL, excess) under ice-cooling. After
stirred for an hour at rt under argon atmosphere, the mixture
was concentrated in vacuo. The reaction mixture was mixed
with saturated NaHCO3 aq, extracted three times with
CHCl3, and dried over abs Na2SO4 to give 23 (1.41 g, 97%,
Crude).
pionate (25). Diastereomixture of 24 (1.13 g, 1.52 mmol)
was dissolved in ethyl acetate (15 mL) and 10% Pd–C was
added to this solution as catalyst. This solution was stirred
overnight under hydrogen atmosphere and filtered with
Celite. After evaporation, the residue was purified by silica
gel column chromatography (n-hexane/ethyl acetate 1:1) to
give 25 (1.08 g, quant.) as a mixture of diastereomers.
Similarly, 24a (79 mg, 0.1 mmol) was dissolved in ethyl
acetate (1 mL) and 10% Pd–C was added to this solution as
catalyst. This solution was stirred overnight under hydrogen
atmosphere and filtered with Celite. After evaporation, the
residue was purified by silica gel column chromatography
(n-hexane/ethyl acetate 1:1) to give 25a (69 mg, quant.).
4.1.17. t-Butyl (30,40,60-tri-O-acetyl-20-deoxy-20-phthali-
mide-b-D-galactopyranosyloxy)-3-p-benzyloxyphenyl
propionate (24). Crude 30,40,60-tri-O-acetyl-20-deoxy-20-
phthalimide-b-D-galacto-pyranosyloxy bromide 23
(215 mg, 655 mmol), 22 (379 mg, 762 mmol), dried MS
4A (200 mg), and abs CH2Cl2 (6 mL) was mixed. To this
solution, was added diisopropylethylamine (DIPEA)
(0.11 mL, 657 mmol), and then AgOTf (207 mg,
788 mmol) with stirring at 0 8C under argon atmosphere.
After 2 h stirring, reaction mixture was filtered with Celite,
and insoluble was washed with CHCl3. The filtrate was
washed with satd NaHCO3 aq, and dried over abs Na2SO4.
After concentration, the residue was separated with silica
gel column chromatography (toluene/acetone 10:1) to give
24 (420 mg, 86%) as a mixture of diastereomers. A part of
the mixture was then separated by silica gel TLC to give 24a
(79 mg) and 24b (72 mg).
Compound 24b (72 mg, 0.1 mmol) was dissolved in ethyl
acetate (1 mL) and 10% Pd–C was added to this solution as
catalyst. This solution was stirred overnight under hydrogen
atmosphere and filtered with Celite. After evaporation, the
residue was purified by silica gel column chromatography
(n-hexane/ethyl acetate 1:1) to give 25b (68 mg, quant.).
Compound 25a. 1H NMR (270 MHz, CDCl3, rt): 7.85–7.67
(4H, m), 6.77 (2H, d, JZ8.4 Hz), 6.17 (2H, d, JZ8.4 Hz),
5.64 (1H, m), 5.64 (1H, dd, JZ3.4, 11.4 Hz), 5.41 (1H, d,
JZ3.1 Hz), 5.30 (1H, d, JZ8.4 Hz), 4.58 (1H, dd, JZ8.4,
11.4 Hz), 4.23–3.95 (4H, m), 2.75 (2H, d, JZ7.1 Hz), 2.17
(3H, s), 2.05 (3H, s), 1.81 (3H, s), 1.45 (9H, s) ppm; 13C
NMR (68 MHz, CDCl3, rt): 170.5, 170.3, 170.3, 169.7,
167.7, 167.1, 154.0, 134.1, 134.0, 131.0, 129.7, 127.8,
123.4, 123.3, 114.7, 99.2, 81.8, 81.5, 70.8, 68.0, 66.5, 61.2,
51.1, 37.9, 27.9, 27.8, 20.7, 20.5 ppm; HR FAB MS
Compound 24a. 1H NMR (270 MHz, CDCl3, rt): 7.84–7.68
(4H, m,), 7.38–7.33 (5H, m), 7.03 (2H, d, JZ8.3 Hz), 6.74
(2H, d, JZ8.3 Hz), 5.90 (1H, dd, JZ3.0, 11.2 Hz), 5.47
(1H, d, JZ3.0 Hz), 5.23 (1H, d, JZ8.2 Hz), 4.98 (2H, s),
4.56 (1H, dd, JZ8.2, 11.2 Hz), 4.37 (1H, dd JZ8.6
11.5 Hz), 4.20–4.02 (3H, m), 2.92 (2H, d, JZ5.8 Hz),
2.21 (3H, s), 2.05 (3H, s), 1.86 (3H, s), 1.11 (9H, s) ppm;
13C NMR (100 MHz, CDCl3, rt): 170.3, 170.3, 169.7, 169.4,
168.1,167.7, 157.3, 137.0, 133.9, 133.7, 132.0, 131.5, 130.6,
128.4, 128.2, 127.8, 127.3, 123.3, 123.1, 114.2, 97.2, 81.5,
70.6, 69.8, 67.5, 66.4, 61.2, 51.0, 37.9, 27.6, 20.7, 20.6,
20.5 ppm; HR FAB MS (positive): [MCNa]C Found m/z
768.2625, C40H43O13NNa requires m/z 768.2632; IR (film)
n: 1749, 1718, 1512, 1389 cmK1; [a]D24 29.08 (c 0.5, CHCl3).
(positive):
[MCNa]C
Found
m/z
678.2148,
C33H37O13NNa requires m/z 678.2163: IR (film) n: 3460,
1751, 1718, 1614, 1517, 1390, 1369 cmK1; [a]D26 5.08 (c 1.0,
CHCl3).
Compound 25b. 1H NMR (270 MHz, CDCl3, rt): 7.85–7.68
(4H, m), 6.96 (2H, d, JZ8.4 Hz), 6.60 (2H, d, JZ8.4 Hz),
6.11 (1H, m), 5.92 (1H, dd, JZ3.4, 11.4 Hz), 5.48 (1H, d,
JZ3.4 Hz), 5.24 (1H, d, JZ8.4 Hz), 4.57 (1H, dd, JZ8.4,
11.4 Hz), 4.38 (1H, t, JZ6.1 Hz), 4.25–4.02 (3H, m), 2.90
(2H, d, JZ6.3 Hz), 2.20 (3H, s), 2.06 (3H, s), 1.87 (3H, s),
1.14 (9H, s) ppm; 13C NMR (68 MHz, CDCl3, rt): 170.4,
170.3, 169.7, 169.5, 168.1, 167.7, 133.8, 131.8, 131.3,
130.6, 127.4, 123.3, 123.1, 114.7, 97.2, 81.7, 77.4, 70.6,
67.6, 66.5, 61.2, 51.1, 38.0, 27.9, 27.7, 20.8, 20.8, 20.6 ppm;
HR FAB MS (positive): [MCNa]C Found m/z 678.2150,
C33H37O13NNa requires m/z 678.2163: IR (film) n: 3460,
1749, 1718, 1614, 1516, 1390, 1371 cmK1; [a]D26 K25.38 (c
1.0, CHCl3).
Compound 24b. 1H NMR (270 MHz, CDCl3, rt): 7.81–7.64
(4H, m), 7.44–7.37 (5H, m), 6.84 (2H, d, JZ8.6 Hz), 6.34
(2H, d, JZ8.6 Hz), 5.62 (1H, dd, JZ3.3, 11.5 Hz), 5.41
(1H, d, JZ3.3 Hz), 5.31 (1H, d, JZ8.6 Hz), 4.79 (2H, s),
4.58 (1H, dd, JZ8.6, 11.5 Hz), 4.23–3.97 (4H, m), 2.75
(2H, d, JZ6.6 Hz), 2.19 (3H, s), 2.05 (3H, s), 1.81 (3H, s),
1.44 (9H, s) ppm; 13C NMR (100 MHz, CDCl3, rt): 170.4,
170.3, 170.3, 169.7, 167.8, 167.1, 156.8, 136.9, 134.0,
133.9, 131.1, 129.7, 128.6, 128.5, 127.8, 127.1, 123.3,
114.1, 99.2, 81.7, 81.3, 70.8, 69.3, 67.9, 66.4, 61.2, 60.3,
51.0, 37.8, 27.8, 20.7, 20.6, 20.4 ppm; HR FAB MS
4.1.19. t-Butyl (30,40,60-tri-O-acetyl-20-deoxy-20-phthali-
mide-b-D-galactopyranosyloxy)-3-p-t-butyldimethylsilyl-
oxyphenyl-propionate (26). Diastereomixture of 25
(1.08 g, 1.65 mmol) was dissolved in dimethylformamide
(DMF; 15 mL). To this solution, imidazole (450 mg,
6.62 mmol), t-butyldimethylsilyl chloride (625 mg,
4.13 mmol), and a catalytic amount of DMAP were added
to this solution. This solution was stirred overnight under
argon atmosphere, mixed with water (20 mL), extracted
with ethyl acetate. The organic layer was then dried over abs
Na2SO4 and evaporated to dryness. The residue was then
(positive):
[MCNa]C
Found
m/z
768.2603,
C40H43O13NNa requires m/z 768.2632: IR (film) n: 1751,
1718, 1511 1389 cmK1; [a]D24 K22.58 (c 0.5, CHCl3).
4.1.18. t-Butyl (30,40,60-tri-O-acetyl-20-deoxy-20-phthali-
mide-b-D-galactopyranosyloxy)-3-p-hydroxyphenyl pro-

7886
T. Fujii et al. / Tetrahedron 61 (2005) 7874–7893
purified by silica gel column chromatography (n-hexane/
ethyl acetate 2:1) to give 26 (1.20 g, 95%).
filtrate was concentrated in vacuo, and the residue was
dissolved in diethyl ether (50 mL). The organic layer was
washed with saturated NaHCO3 aq and then brine, dried
over abs Na2SO4, and concentrated in vacuo. The residue
was then separated by silica gel column chromatography
(n-hexane/ethyl acetate 3:1) to give 27 (490 mg, 41%).
Compound 25a (69 mg, 0.1 mmol) was dissolved in
dimethylformamide (DMF; 1 mL). To this solution,
imidazole (26 mg, 377 mmol), t-butyldimethylsilyl chloride
(36 mg, 235 mmol) and catalytic amount of DMAP were
added. This solution was stirred overnight under argon
atmosphere, mixed with water (20 mL), extracted with ethyl
acetate. The organic layer was then dried over abs Na2SO4
and evaporated to dryness. The residue was then purified by
silica gel column chromatography (n-hexane/ethyl acetate
2:1) to give 26a (64 mg, 83%).
1
Compound 27. H NMR (270 MHz, CDCl3, rt): 7.84–7.62
(4H, m), 7.56 (2H, d, JZ8.6 Hz), 6.75 (1H, s), 6.70 (2H, d,
JZ8.6 Hz), 5.93 (2H, m), 5.45 (1H, d, JZ3.6 Hz), 4.72
(1H, dd, JZ8.6, 11.5 Hz), 4.03 (3H, m), 2.19 (3H, s), 1.97
(3H, s), 1.84 (3H, s), 1.41 (9H, s), 0.97 (9H, s), 0.20 (6H,
s) ppm; 13C NMR (68 MHz, CDCl3, rt): 170.0, 169.9, 169.4,
167.8, 167.3, 162.2, 156.1, 139.4, 133.8, 133.6, 131.9,
131.6, 131.2, 125.9, 124.4, 123.2, 123.1, 119.5, 97.0, 81.5,
70.8, 67.6, 66.3, 60.9, 51.4, 28.0, 25.6, 25.5, 20.7, 20.5,
20.5, 18.2, K4.4 ppm; HR FAB MS (positive): [MCNa]C
Found m/z 792.2875, C39H49O13NSiNa requires m/z
792.2871: IR (film) n: 1753, 1720, 1601, 1508, 1389,
1369 cmK1; [a]2D5 K88.28 (c 1.0, CHCl3).
Compound 25b (68 mg, 0.1 mmol) was dissolved in
dimethylformamide (DMF; 1 mL). To this solution,
imidazole (28 mg, 408 mmol), t-butyldimethylsilyl chloride
(38 mg, 255 mmol) and catalytic amount of DMAP were
added. This solution was stirred overnight under argon
atmosphere, mixed with water (20 mL), extracted with ethyl
acetate. The organic layer was then dried over abs Na2SO4
and evaporated to dryness. The residue was then purified by
silica gel column chromatography (n-hexane/ethyl acetate
2:1) to give 26b (67 mg, 87%).
4.1.21. t-Butyl (Z)-2-(30,40,60-tri-O-acetyl-20-deoxy-20-
phthalimide-b-D-galactopyranosyloxy)-3-p-hydroxy-
phenyl-2-acrylate t-butylester (28). Compound 27
(250 mg, 0.33 mmol) was dissolved in THF (3 mL), and
tetrabutyl ammonium fluoride (1.0 M in THF, 0.39 mL,
0.39 mmol) was added to this solution at 0 8C. After 5 min
stirring, this solution was mixed with water (5 mL) and
extracted by ethyl acetate. This organic layer was washed
with brine, dried over abs Na2SO4, and evaporated in vacuo.
The residue was then separated by silica gel column
chromatography (n-hexane/ethyl acetate 1:1) to give 28
(215 mg, quant.).
Compound 26a. 1H NMR (270 MHz, CDCl3, rt): 7.81–7.65
(4H, m), 6.76 (2H, d, JZ8.4 Hz), 6.19 (2H, d, JZ8.4 Hz),
5.64 (1H, dd, JZ3.3, 11.4 Hz), 5.42 (1H, d, JZ3.3 Hz),
5.31 (1H, d, JZ8.6 Hz), 4.59 (1H, dd, JZ8.6, 11.4 Hz),
4.20–3.94 (4H, m), 2.74 (2H, d, JZ6.7 Hz), 2.19 (3H, s),
2.05 (3H, s), 1.81 (3H, s), 1.42 (9H, s) 0.95 (9H, s), 0.09
(6H, s) ppm; 13C NMR (100 MHz, CDCl3, rt): 170.3, 170.2,
170.2, 169.6, 167.7, 167.0, 153.6, 134.0, 133.9, 131.2,
131.0, 129.6, 128.9, 123.4, 123.3, 119.5, 99.1, 81.5, 81.2,
70.8, 67.9, 66.4, 61.2, 51.1, 37.9, 27.9, 27.8, 25.7, 20.8,
20.7, 20.5, 18.1, K4.5 ppm; HR FAB MS (positive): [MC
Na]C Found m/z 792.3040, C39H51O13NSiNa requires m/z
792.3027: IR (film) n: 1753, 1718, 1610 1510, 1389,
1369 cmK1; [a]2D4 11.48 (c 1.0, CHCl3).
1
Compound 28. H NMR (270 MHz, CDCl3, rt): 7.87–7.68
(4H, m), 7.58 (2H, d, JZ8.7 Hz), 6.80 (1H, s), 6.75 (2H, d,
JZ8.7 Hz), 5.95 (2H, m), 5.48 (1H, d, JZ3.1 Hz), 4.75
(1H, dd, JZ8.4, 11.4 Hz), 4.08 (3H, m), 2.21 (3H, s), 2.00
(3H, s), 1.88 (3H, s), 1.44 (9H, s) ppm; 13C NMR (68 MHz,
CDCl3, rt): 170.4, 170.3, 169.7, 168.0, 167.7, 162.5, 156.8,
139.2, 134.1, 134.0, 133.8, 132.2, 131.6, 131.1, 124.9,
123.4, 123.2, 115.0, 97.3, 81.8, 70.9, 67.8, 66.5, 61.1, 51.6,
28.0, 28.0, 20.7, 20.6, 20.6 ppm; HR FAB MS (negative):
[MKH]K Found m/z 654.2214, C33H36O13N requires m/z
654.2187: IR (film) n: 3413, 1751, 1718, 1606, 1514, 1389,
1369 cmK1; [a]2D4 K101.58 (c 1.0, CHCl3).
Compound 26b. 1H NMR (270 MHz, CDCl3, rt): 7.86–7.69
(4H, m), 6.96 (2H, d, JZ8.4 Hz), 6.59 (2H, d, JZ8.4 Hz),
5.93 (1H, dd, JZ3.1, 11.5 Hz), 5.48 (1H, d, JZ3.1 Hz),
5.24 (1H, d, JZ8.4 Hz), 4.58 (1H, dd, JZ8.4, 11.5 Hz),
4.39 (1H, t, JZ6.0 Hz), 4.26–4.03 (3H, m), 2.92 (2H, d, JZ
6.7 Hz), 2.22 (3H, s), 2.07 (3H, s), 1.87 (3H, s), 1.11 (9H, s),
0.95 (9H, s), 0.11 (6H, s) ppm; 13C NMR (100 MHz, CDCl3,
rt): 170.2, 170.2, 169.6, 169.3, 168.1, 167.6, 154.0, 133.8,
133.7, 131.9, 131.4, 130.5, 128.5, 123.3, 123.1, 119.4, 97.2,
81.5, 77.3, 70.6, 67.6, 66.5, 61.2, 51.1, 38.1, 27.7, 25.7,
20.8, 20.7, 20.6, 18.2, K4.4 ppm; HR FAB MS (positive):
[MCNa]C Found m/z 792.3053, C39H51O13NSiNa requires
m/z 792.3027: IR (film) n: 1753, 1720, 1610, 1510, 1389,
1369 cmK1; [a]2D4 K27.38 (c 1.0, CHCl3).
4.1.22. t-Butyl (Z)-2-(20-deoxy-20-phthalimide-b-D-galac-
topyranosyl-oxy)-3-p-hydroxyphenyl-2-acrylate t-butyl-
ester (29). Compound 28 (215 mg, 0.33 mmol) was
dissolved in methanol (3.3 mL) and sodium methoxide
(57 mg, 1.06 mmol) was added to this solution at 0 8C. After
overnight stirring, sodium methoxide (57 mg, 1.06 mmol)
was added to this solution. This reaction mixture was then
neutralized with Amberlite IR-120B (HC), filtered, and
evaporated to dryness. The residue was then separated by
silica gel column chromatography (CHCl3–MeOHZ5:1) to
give 29 (168 mg, 97%).
4.1.20. t-Butyl (Z)-2-(30,40,60-tri-O-acetyl-20-deoxy-20-
phthalimide-b-D-galactopyranosyl-oxy)-3-p-t-butyldi-
methylsilyloxyphenyl-2-acrylate t-butylester (27). Dia-
stereomixture of 26 (1.20 g, 1.60 mmol) was dissolved in
dioxane (35 mL), and 2,3-dichloro-5,6-dicyano-1,
4-benzoquinone (DDQ; 1.42 g, 6.20 mmol) was added to
this solution. This reaction mixture was refluxed under
argon atmosphere for 2 days, and then filtered by Celite. The
Compound 29. 1H NMR (270 MHz, CD3OD, rt): 7.92–7.75
(4H, m), 7.58 (2H, d, JZ8.6 Hz), 6.67 (1H, s), 6.67 (2H, d,
JZ8.6 Hz), 5.78 (1H, d, JZ8.0 Hz), 4.62 (1H, dd, JZ8.0,
11.0 Hz), 4.58 (1H, dd, JZ2.9, 11.0 Hz), 3.98 (1H, d, JZ

T. Fujii et al. / Tetrahedron 61 (2005) 7874–7893
7887
2.9 Hz), 3.81–3.62 (3H, m), 1.46 (9H, s) ppm; 13C NMR
(68 MHz, CD3OD, rt): 169.9, 169.5, 164.4, 159.1, 140.7,
135.1, 134.9, 133.2, 133.0, 132.6, 125.5, 124.7, 124.0,
123.7, 116.0, 98.6, 82.8, 77.2, 69.4, 69.1, 61.8, 55.5, 28.3,
28.2 ppm; HR FAB MS (negative): MKH]K Found m/z
526.1714, C27H28O10N requires m/z 526.1713: IR (film) n:
3402, 1774, 1710, 1606, 1512, 1390 cmK1; [a]D23 K152.78
(c 1.0, MeOH).
8.2 Hz), 6.80 (2H, s), 6.66 (2H, d, JZ8.6 Hz), 5.32 (1H, d,
JZ8.6 Hz), 4.43 (2H, m), 4.25 (3H, m), 3.92–3.78 (5H, m),
3.73–3.45 (11H, m), 2.90 (1H, dd, JZ4.9, 12.9 Hz), 2.67
(1H, d, JZ12.9 Hz), 2.18 (2H, t, JZ7.5 Hz), 1.66–1.41 (6H,
m) ppm; 13C NMR (100 MHz, DMSO-d6, rt): 172.3, 165.8,
165.8, 162.8, 157.2, 156.5, 131.6, 131.4, 131.0, 128.4,
125.9, 125.4, 123.1, 120.4 (1JCFZ273 Hz), 118.9, 115.0,
111.3, 99.5, 77.6, 75.8, 75.5, 73.2, 69.8, 69.6, 69.2, 68.6,
67.2, 61.1, 60.1, 59.2, 55.4, 53.8, 38.4, 35.1, 29.0, 28.2,
28.0 (2JCFZ16 Hz), 25.3 ppm; HR FAB MS (negative):
[MKK]K Found m/z 925.2899, C40H48O14N6F3S requires
m/z 925.2901; IR (film) n: 3329, 1693, 1651, 1608, 1549,
1512 cmK1; [a]2D4 K21.28 (c 0.5, MeOH).
4.1.23. t-Butyl (Z)-2-[20-[2-[2-[2-(2-biotinylamino-
ethoxy)-ethoxy]-ethoxy]-4-(3-trifluoro-methyl)-3H-dia-
zirin-3-yl]benzyloxyimino]-amino-b-D-galactopyranosyl-
oxy)-3-p-hydroxyphenyl-2-acrylate (31). Compound 29
(21.3 mg, 53.7 mmol) was dissolved in ethanol (1 mL), and
hydrazine monohydrate (10 mL) was added to this solution.
After overnight stirring, the reaction mixture was evapo-
rated to dryness. The residue was then separated repeatedly
by ODS TLC (H2O/CH3CN 2:1 containing 1% acetic acid)
to give crude amine (21 mg). Crude amine was dissolved
in dimethylformacmide (1.5 mL), and 30 (20.0 mg,
33.2 mmol), hydroxybenzotriazole (HOBt; 8.7 mg,
64.3 mmol), and dicyclohexylcarbodiimide (DCC; 13 mg,
64.3 mmol) were added to this solution. After stirring for
2 days under light-shading conditions, the reaction mixture
was evaporated to dryness. The residue was then separated
by ODS TLC (H2O/CH3CNM 1:1) to give 31 (10.2 mg,
19%).
4.1.25.
2-(60-Amino-b-D-galactopyranosyloxy)-3-
hydroxyphenyl-2-acrylolactam (35). Methyl ester of 29
(34) (3 mg, 5 mmol) was dissolved in MeOH (0.5 mL).
Hydrazine monohydrate (80% solution, 5 mL) was added to
this solution. After overnight stirring, the reaction mixture
was concentrated in vacuo, and separated by ODS-TLC
(RP-18W, H2O/CH3CN/AcOH 66:33:1) to give 32 (1.6 mg,
5 mmol).
Compound 35. 1H NMR (400 MHz, CD3OD, rt): 7.64 (2H,
d, JZ8.8 Hz), 6.74 (2H, d, JZ8.8 Hz), 6.71 (1H, s), 4.86
(1H, d, JZ7.1 Hz), 3.91 (1H, dd, JZ8.3, 12.5 Hz), 3.87
(1H, dd, JZ0.7, 2.7 Hz), 3.81 (1H, dd, JZ4.6, 12.5 Hz),
3.80 (1H, dd, JZ0.7, 4.6 Hz), 3.66 (1H, dd, JZ2.9,
10.7 Hz), 3.63 (1H, dd, JZ7.1, 10.7 Hz) ppm; FAB MS
(positive) m/z 324 [MCH]C: IR (film) n: 1670, 1606, 1512,
Compound 31. 1H NMR (270 MHz, CD3OD, rt): 7.96 (1H,
d, JZ8.0 Hz), 7.64 (2H, d, JZ8.5 Hz), 6.99 (1H, d, JZ
8.0 Hz), 6.81 (1H, s), 6.73 (1H, s), 6.67 (2H, d, JZ8.5 Hz),
5.36 (1H, d, JZ8.1 Hz), 4.42 (2H, m), 4.25 (3H, m), 3.92–
3.85 (5H, m), 3.78–3.46 (11H, m), 2.91 (1H, dd, JZ3.6,
12.7 Hz), 2.67 (1H, d, JZ12.7 Hz), 2.15 (2H, t, JZ7.2 Hz),
1.72–1.57 (6H, m), 1.51 (9H, s) ppm; 13C NMR (100 MHz,
DMSO-d6, rt): 172.3, 164.2, 162.8, 162.6, 157.9, 156.6,
139.5, 132.2, 131.8, 131.3, 125.4, 124.4, 124.1, 123.1,
121.1, 120.4, 119.1, 117.7 (1JCHZ273 Hz), 115.2, 111.4,
98.7, 80.9, 76.0, 71.4, 69.8, 69.7, 69.6, 69.2, 68.6,
67.1, 61.1, 59.8, 59.3, 55.5, 52.8, 48.6, 38.4, 35.1,
28.2, 28.1 (2JCHZ16 Hz), 27.9, 27.8, 25.3 ppm; HR FAB
MS (negative): [MKH]K Found m/z 981.3524,
C44H56O14N6F3S requires m/z 981.3527; IR (film) n: 3340,
1697, 1649, 1608, 1549, 1512, 1458 cmK1; [a]D24 K43.08 (c
1.0, MeOH).
1441 cmK1
.
4.1.26. t-Butyl (Z)-2-(20-deoxy-20-phthalimide-b-D-galac-
topyranosyl-oxy)-3-p-t-butyldimethysilyloxyphenyl-2-
acrylate(36). Compound 27 (1.01 g, 1.32 mmol) in metha-
nol (13 mL) was mixed with sodium methoxide (142 mg,
2.64 mmol) at K30 8C, and the mixture was stirred under
argon atmosphere. After 1 h stirring, Amberlite IR-120 B
was added to this solution to adjust pH at 7.0. After
filtration, the filtrate was concentrated in vacuo, and purified
by silica gel column chromatography (CHCl3/MeOH 20:1)
to give 36 (719 mg, 88%).
1
Compound 36. H NMR (400 MHz, CDCl3, rt): 7.83–7.79
(2H, m), 7.69–7.67 (2H, m), 7.53 (2H, d, JZ8.8 Hz), 6.83
(1H, s), 6.65 (2H, d, JZ8.8 Hz), 5.71 (1H, d, JZ8.3 Hz),
4.71 (1H, dd, JZ8.3, 10.7 Hz), 4.52 (1H, dd, JZ2.5,
10.7 Hz), 4.12 (1H, d, JZ2.5 Hz), 3.84 (1H, dd, JZ4.4,
5.3 Hz), 3.83 (1H, dd, JZ4.8, 5.3 Hz), 3.66 (1H, dd, JZ4.4,
4.8 Hz), 1.45 (9H, s), 0.97 (9H, s), 0.19 (6H, s) ppm;13C
NMR (68 MHz, CDCl3, rt): 168.1, 163.3, 155.9, 140.1,
133.5, 131.6, 129.8, 123.8, 123.2, 122.6, 119.6, 97.5, 81.8,
74.8, 68.5, 68.1, 61.3, 54.3, 27.9, 25.5, 18.0, K4.5 ppm; HR
FAB MS (negative): [MKH]K Found m/z 640.2570,
C33H42O10NSi requires 640.2578: IR (film) n: 3419, 1774,
1716, 1637, 1600, 1508, 1471, 1394 cmK1; [a]D20 K4.28 (c
1.0, CHCl3).
4.1.24. Potassium (Z)-2-[20-[2-[2-[2-(2-biotinylamino-
ethoxy)-ethoxy]-ethoxy]-4-(3-trifluoro-methyl)-3H-dia-
zirin-3-yl]benzyloxyimino]-amino-b-D-galactopyranosyl-
oxy)-3-p-hydroxyphenyl-2-acrylate (3). TFA (purityO
98%, 40 mL) was added to the 5 mL round-bottom flask in
which compound 31 (2.2 mg, 2.24 mmol) was dried. This
flask was set to the vacuum line immediately, and dried in
vacuo to remove TFA. After drying in vacuo for 1 h under
light-shading conditions, the residue was then separated by
ODS TLC (H2O–CH3CNZ3:2) to 3 as free carboxylic acid
form, which was then dissolved in H2O–MeOH (3:2) and
neutralized by 0.1 M K2CO3 aq. The solution was
evaporated to dryness, and the residue was separated by
ODS TLC (H2O/CH3CN 3:2) to give 3 (0.7 mg, 32%).
4.1.27. t-Butyl (Z)-2-(20-deoxy-20-phthalimide-60-O-p-
toluenesulfonyl-b-D-galactopyranosyloxy)-3-p-t-butyl-
dimetylsilyloxyphenyl-2-acrylate (37). p-Toluene sulfonyl
chloride (12.3 mg 66 mmol) was added to the solution of 36
(35.3 mg, 55 mmol) in pyridine (0.5 mL) at 0 8C, and the
Compound 3. 1H NMR (270 MHz, CD3OD, rt): 7.88 (1H, d,
JZ8.2 Hz), 7.65 (2H, d, JZ8.6 Hz), 6.96 (1H, d, JZ

7888
T. Fujii et al. / Tetrahedron 61 (2005) 7874–7893
mixture was stirred for 25 h under argon atmosphere. To
remove pyridine, the mixture was evaporated with toluene,
and the residue was purified by silica gel column
chromatography (n-hexane/EtOAc 1:1) to give 37
(14.3 mg, 33%) with recovered 36 (15.9 mg, 45%).
dd, JZ8.0, 11.6 Hz), 3.24 (1H, dd, JZ3.6, 11.6 Hz), 1.45
(9H, s) ppm; 13C NMR (100 MHz, CDCl3, rt): 168.8, 163.2,
156.6, 145.0, 139.4, 134.0, 132.1, 131.5, 125.0, 124.6,
115.4, 96.8, 82.0, 74.6, 69.2, 68.2, 54.4, 51.1, 28.1 ppm; HR
FAB MS (negative) [MKH]K Found m/z 551.1769,
C27H27O9N4 requires 551.1778; IR (film) n: 3397, 2102,
1710, 1606, 1511, 1390 cmK1; [a]D20 K10.78 (c 1.0, CHCl3).
1
Compound 37. H NMR (400 MHz, CDCl3, rt): 7.81–7.79
(2H, m), 7.74 (2H, d, JZ8.3 Hz), 7.69–7.67 (2H, m), 7.51
(2H, d, JZ8.5 Hz), 7.29 (2H, d, JZ8.3 Hz), 6.75 (1H, s),
6.69 (2H, d, JZ8.5 Hz), 5.79 (1H, d, JZ7.1 Hz), 4.57 (2H,
m), 4.20 (1H, dd, JZ7.1, 10.4 Hz), 4.05 (1H, d, JZ6.0 Hz),
3.97 (1H, dd, JZ6.0, 10.4 Hz), 3.87 (1H, t, JZ6.6 Hz), 2.41
(3H, s), 1.45 (9H, s), 0.99 (9H, s), 0.21 (6H, s) ppm; 13C
NMR (68 MHz, CDCl3, rt): 168.2, 163.0, 156.1, 144.8,
140.0, 133.7, 131.8, 129.8, 129.5, 127.7, 127.3, 126.0,
124.0, 123.2, 119.8, 97.0, 82.0, 72.6, 68.0, 67.7, 54.2, 28.1,
25.7, 21.6, 18.2, K4.3 ppm; HR FAB MS (positive) [MC
Na]C Found m/z 818.2672, C40H49NO12SSiNa requires
818.2642: IR (film) n: 3484, 1776, 1716, 1600, 1508, 1389,
1368 cmK1; [a]2D0 K7.98 (c 1.0, CHCl3).
4.1.30. N-Biotinylglycylglycylglycine (40). Compound 46
(212.7 mg, 0.50 mmol) was dissolved in 1 M KOH aq
(0.6 mL) and the solution was stirred for an hour at rt. After
the solution was adjusted to pH 7.0 by Amberlite IR-120B,
the resulting precipitate was dissolved in MeOH, and dried
in vacuo to give 40 (180.8 mg, 88%).
Compound 40. 1H NMR (400 MHz, DMSO-d6, 30 8C): 8.12
(1H, t, JZ5.6 Hz), 8.07 (1H, t, JZ5.6 Hz), 8.03 (1H, t, JZ
5.6 Hz), 6.38 (2H, s), 4.30 (1H, dd, JZ4.9, 7.9 Hz), 4.12
(1H, dd, JZ4.9, 7.9 Hz), 3.12–3.07 (1H, m), 2.81 (1H, dd,
JZ5.4, 12.2 Hz), 2.57 (1H, d, JZ12.2 Hz), 2.13 (2H, t, JZ
7.3 Hz) ppm; 13C NMR (100 MHz, DMSO-d6, 30 8C):
172.4, 170.8, 169.2, 168.9, 162.5, 60.9, 59.2, 55.2, 42.0,
41.7, 40.5, 39.8, 34.9, 28.1, 28.0, 25.0 ppm: HR FAB MS
(negative) [MKH]K Found m/z 414.1461 C16H24O6N5S
requires 414.1447 IR (film) n: 3285, 3085, 1735, 1718,
1685, 1560, 1508, 1420 cmK1; [a]D20 K3.58 (c 1.0, DMSO).
4.1.28. t-Butyl (Z)-2-(20-deoxy-20-phthalimide-60-O-p-
toluenesulfonyl-b-D-galactopyranosyloxy)-3-p-hydroxy-
phenyl-2-acrylate (38). TBAF (1.0 M in THF, 0.13 mL,
0.13 mmol) was added to the solution of 37 (85.8 mg,
0.11 mmol) in THF (1.0 mL) at 0 8C, and the mixture was
stirred for 5 min under argon atmosphere. The reaction
mixture was mixed with water, and extracted with ethyl
acetate, and the organic layer was washed with brine, dried
over abs Na2SO4. After evaporation, the residue was
purified by silica gel column chromatography (n-hexane/
EtOAc 2:3) to give 38 (49.0 mg, quant.).
4.1.31. t-Butyl (Z)-2-(20-deoxy-20-phthalimide-60-[N-bio-
tinylglycyl-glycylglycyl]-amino-b-D-galactopyranosyl-
oxy)-3-p-hydroxyphenyl-2-acrylate (41). To the solution
of 39 (40.5 mg, 73 mmol) in MeOH (1 mL) was added 5%
Pd–CaCO3, and the mixture was stirred for 2 h under
hydrogen atmosphere at rt. The reaction mixture was filtered
with Celite and dried in vacuo to give crude amine
(39.8 mg).
1
Compound 38. H NMR (400 MHz, CDCl3, rt): 7.74–7.58
(6H, m), 7.41 (2H, d, JZ7.8 Hz), 7.21 (2H, d, JZ8.4 Hz),
6.78 (1H, s), 6.61 (2H, d, JZ8.4 Hz), 5.77 (1H, d, JZ
5.8 Hz), 4.61 (1H, dd, JZ8.4, 10.5 Hz), 4.55 (1H, d, JZ
5.4 Hz), 4.22 (1H, dd, JZ5.4, 10.5 Hz), 4.05–4.01 (2H, m),
3.83 (1H, t, JZ6.3 Hz), 2.33 (3H, s), 1.46 (9H, s) ppm; 13C
NMR (100 MHz, CDCl3, rt): 168.8, 163.1, 156.8, 145.0,
139.5, 133.9, 132.0, 131.5, 129.9, 127.8, 124.8, 124.4,
123.5, 115.3, 96.9, 82.1, 72.7, 68.1, 68.0, 67.9, 54.3, 28.1,
21.6 ppm; HR FAB MS (negative) [MKH]K Found m/z
680.1780, C34H34O12NS requires 680.1802; IR (film) n:
3458, 1774, 1714, 1606, 1511, 1392 cmK1; [a]D22 K8.48 (c
1.0, CHCl3).
Resulting crude amine (39.8 mg, ca.73 mmol) in DMF
(1.0 mL) was mixed with HOBt (14.8 mg, 0.11 mmol) and
40 (31.5 mg, 75 mmol). After cooling to 0 8C, DCC
(17.8 mg, 86 mmol) was added to this solution and stirred
for 41 h. After filtration by hyflosupercell, the filtrate was
dried up in vacuo to remove DMF and the residue was
purified by ODS-TLC (H2O/CH3CN 5:4) to give 41
(12.9 mg, 27% in two steps).
1
Compound 41. H NMR (400 MHz CD3OD, rt): 7.83–7.73
4.1.29. t-Butyl (Z)-2-(20-deoxy-20-phthalimide-60-azido-
b-D-galactopyranosyloxy)-3-p-hydroxyphenyl-2-acrylate
(39). 15-crown-5 (0.15 mL, 0.72 mmol) and sodium azide
(47 mg, 0.72 mmol) was added to the solution of 38
(49.0 mg, 72 mmol) in DMF (0.7 mL) and stirred for a day
under argon atmosphere. The reaction mixture was mixed
with water, extracted with ethyl acetate. And the organic
layer was washed with brine, dried over abs Na2SO4. After
evaporation, the residue was purified by silica gel column
chromatography (CHCl3/MeOH 10:1) to give 39 (40.5 mg,
quant.).
(4H, m), 7.54 (2H, d, JZ8.8 Hz), 6.68 (1H, s), 6.67 (2H, d,
JZ8.8 Hz), 5.71 (1H, d, JZ8.1 Hz), 4.59 (1H, dd, JZ8.1,
11.0 Hz), 4.52 (1H, dd, JZ6.6, 11.0 Hz), 4.44 (1H, dd, JZ
4.4, 7.9 Hz), 4.28 (1H, dd, JZ4.4, 7.9 Hz), 3.93–3.70 (7H,
m), 3.73 (2H, m), 3.47 (1H, dd, JZ7.6, 14.0 Hz), 3.39 (1H,
dd, JZ6.6, 14.0 Hz), 3.20–3.15 (1H, m), 2.89 (1H,dd, JZ
4.7, 12.7 Hz), 2.67 (1H, d, JZ12.7 Hz), 2.35 (2H, t, JZ
7.3 Hz), 1.75–1.49 (6H, m), 1.45 (9H, s) ppm; 13C NMR
(100 MHz, CD3OD, rt): 176.9, 172.9, 172.1, 170.2, 169.7,
166.0, 164.6, 159.4, 141.0, 135.2, 135.0, 133.4, 132.9,
125.6, 125.2, 124.2, 123.8, 99.1, 82.9, 74.4, 69.1, 69.0, 63.2,
61.6, 56.9, 55.6, 44.0, 41.1, 40.4, 36.4, 29.6, 29.4, 28.5,
26.7, 26.6 ppm; HR FAB MS (negative) [MKH]K Found
m/z 922.3298, C43H52N7O14S requires 922.3293; IR (film)
n: 3328, 1704, 1515, 1375, 1259, 1153 cmK1; [a]D21 K55.18
(c 1.0, MeOH).
1
Compound 39. H NMR (400 MHz, CDCl3, rt): 7.75–7.60
(4H, m), 7.44 (2H, d, JZ8.4 Hz), 6.78 (1H, s), 6.64 (2H, d,
JZ8.4 Hz), 5.85 (1H, d, JZ8.0 Hz), 4.68–4.50 (3H, m),
3.92 (1H, d, JZ5.6 Hz), 3.65 (1H, dd, JZ5.6 Hz), 3.52 (1H,

T. Fujii et al. / Tetrahedron 61 (2005) 7874–7893
7889
4.1.32. t-Butyl (Z)-2-(20-deoxy-20-[4-[methyl-phenyl]-
phenyl-methanone]-amino-60-[N-biotinyl glycylglycyl-
glycyl]-amino-b-D-galactopyranosyloxy)-3-p-hydroxy-
phenyl-2-acrylate (43). Hydrazine monohydrate (10 mL)
was added to the solution of 41 (12.9 mg, 14 mmol) in EtOH
(1.0 mL) and the solution was stirred for 23 h at rt. The
reaction mixture was evaporated and dried in vacuo to
remove hydrazine, and then purified by ODS-TLC (H2O/
CH3CNZ5:4 containing 1% AcOH) to give crude amine
(9.9 mg).
4.1.34. N-tert-Butoxycarbonyl glycylglycylglycine methyl
ester (45). To the solution of N-tert-Butoxycarbonyl
glycylglycylglycine (44) (200 mg, 0.69 mmol) in
DMF(7.0 mL) was added CaCO3 (143 mg, 1.03 mmol)
and CH3I (0.13 mL, 0.83 mmol), and the mixture was
stirred for an hour. Then, the reaction mixture was mixed
with water, extracted with ethyl acetate. And the organic
layer was washed with brine, dried over abs Na2SO4. After
dried in vacuo, 45 (234 mg, quant.) was obtained.
Compound 45. 1HNMR (270 MHz CD3OD, rt): 3.92 (2H, s),
3.89 (2H, s), 3.71 (2H, s), 3.67 (3H, s), 1.40 (9H, s) ppm;
13C NMR (68 MHz, CD3OD, rt): 173.2, 172.3, 171.8, 158.7,
81.0, 52.7, 44.9, 43.3, 41.9, 28.8 ppm; HR FAB MS
(positive): [MCH]C Found m/z 304.1516 C12H22O6N3
requires 304.1509; IR (film) n: 3315, 1747, 1668, 1533,
1438, 1369, 1216 cmK1; [a]2D5 K22.28 (c 1.0, MeOH).
TEA(3.5 mL, 25 mmol) and 4-Bromomethylbenzophenone
(42, 3.75 mg, 13.7 mmol) were added to the solution of
crude amine (9.9 mg, crude) in DMF(0.5 mL), and the
mixture was stirred for 10 h at rt. After dried in vacuo to
remove DMF, the residue was purified by ODS-TLC (H2O/
CH3CNZ2:3 containing 1% AcOH) to give 43 (6.3 mg,
52% in two steps).
4.1.35. N-Biotinylglycylglycylglycine methyl ester (46).
Compound 45 (161.1 mg 0.53 mmol) was dissolved in
CH2Cl2 (2.5 mL) and TFA(2.5 mL), and the mixture was
stirred for 20 min at rt. After careful evaporation to remove
TFA completely, the residue was dissolved in DMF
(5.0 mL). After adjusted to pH 7.0 by the addition of TEA
at 0 8C, the reaction mixture was mixed with N-Hydroxyl
succinimidobiotin (341 mg, 0.532 mmol) and stirred for
35 h. After careful evaporation to remove DMF, the residue
was washed with MeOH, and dried in vacuo to give 46
(222.5 mg, 88%).
Compound 43. 1H NMR (400 MHz, CD3OD, rt): 7.80
(2H,d, JZ7.6 Hz), 7.78 (2H, d, JZ8.8 Hz), 7.72 (2H, d, JZ
8.4 Hz), 7.67–7.62 (4H, m), 7.56 (1H, t, JZ7.6 Hz), 7.51
(2H, t, JZ8.0 Hz), 7.14 (1H, s), 6.76 (2H, d, JZ8.8 Hz),
5.20 (1H, d, JZ8.4 Hz), 4.47 (1H, m), 4.27 (1H, dd, JZ4.4,
8.6 Hz), 4.08 (1H, dd, JZ4.4, 8.6 Hz), 3.88 (2H, s), 3.84
(4H, s), 3.73 (1H, d, JZ2.8 Hz), 3.60–3.39 (4H, m), 3.21–
3.14 (1H, m), 2.90 (1H, dd, JZ4.4, 12.8 Hz), 2.67 (1H, d,
JZ12.8 Hz), 2.30 (2H, t, JZ7.2 Hz), 1.68–1.34 (6H, m),
1.28 (9H, s) ppm; 13C NMR (100 MHz CD3OD, rt): 197.7,
177.1, 173.1, 172.3, 172.2, 167.1, 162.7, 160.9, 139.9,
139.5, 138.4, 134.5, 134.1, 131.5, 131.4, 130.9, 129.9,
129.6, 124.8, 116.3, 101.1, 85.0, 74.6, 70.6, 68.8, 63.2, 61.6,
56.9, 54.8, 52.4, 44.0, 43.7, 43.6, 41.0, 40.1, 36.4, 29.6,
29.5, 28.4, 28.0, 26.6, 8.1 ppm; HR FAB MS (negative)
[MKH]K Found m/z 986.3984 C49H60O13N7S requires
986.3970: IR (film) n: 3290, 1668, 1612, 1454, 1279, 1203,
1147 cmK1; [a]2D2 K9.58 (c 0.6, MeOH).
Compound 46. 1H NMR (400 MHz, DMSO-d6, 30 8C): 8.18
(1H, t, JZ5.6 Hz), 8.05 (1H, t, JZ5.6 Hz), 7.98 (1H, t, JZ
5.6 Hz), 6.71 (1H, s), 6.64 (1H, s), 4.62 (1H, dd, JZ5.3,
7.4 Hz), 4.44 (1H, dd, JZ5.3, 7.4 Hz), 4.16 (2H, d, JZ
5.6 Hz), 4.05 (2H, d, JZ5.6 Hz), 4.02 (2H, d, JZ5.6 Hz),
3.13 (1H, dd, JZ4.8, 10.6 Hz), 2.89 (1H, d, JZ10.6 Hz),
2.42 (1H, t, JZ7.4 Hz) 1.98–1.56 (6H, m) ppm; 13C NMR
(100 MHz, DMSO-d6, 30 8C): 172.4, 170.0, 169.2, 169.1,
162.5, 61.0, 59.1, 55.3, 51.6, 48.6, 45.8, 42.0, 40.5, 34.9,
28.1, 28.0, 25.0 ppm; HR FAB MS (negative) [MKH]K
Found m/z 428.1597 C17H26O6N5S requires 428.1604 IR
4.1.33. (Z)-2-(20-Deoxy-20-[4-[methyl-phenyl]-phenyl-
methanone]-amino-60-[N-biotinylglycylglycylglycyl]-
amino-b-D-galactopyranosyloxy)-3-p-hydroxyphenyl-2-
acrylate (4). Compound 43 (3.6 mg, 3.65 mmol) in TFA
(100 mL) was stirred for 25 min. After dried in vacuo to
remove TFA immediately, the residue was purified by
ODS-TLC (H2O/CH3CN 1:5 containing 1% AcOH) to give
4 (2.4 mg, 71%).
(film) n: 3267, 1751, 1709, 1637, 1560, 1421, 1205 cmK1
[a]2D0 K2.78 (c 1.0, DMSO).
;
4.1.36. t-Butyl (Z)-2-(20-deoxy-20-phthalimide-60-O-p-
toluenesulfonyl-b-D-galactopyranosyloxy)-3-p-methoxy-
phenyl-2-acrylate (47). Compound 38 (72 mg, 106 mmol)
was dissolved in MeOH/CH3CN 1:9. To this solution,
diisopropylethylamine (DIPEA)(22 mL, 127 mmol), and
then TMSCHN2 (2 M, 69 mL, 137 mmol) were added. The
reaction mixture was stirred for 5 h at rt under argon
atmosphere, concentrated in vacuo, mixed with water and
then extracted three times with ethyl acetate. The organic
layer was washed with brine, dried over abs Na2SO4.,
and separated with silica gel column chromatography
(n-hexane/ethyl acetate 1:3) to give 47 (59 mg, 81%).
Compound 4. 1H NMR (400 MHz, CD3OD, rt): 7.79 (2H, d,
JZ8.0 Hz), 7.77 (2H, d, JZ8.0 Hz), 7.69 (4H, m), 7.63
(1H, t, JZ7.6 Hz), 7.47 (2H, t, JZ7.6 Hz), 7.22 (1H, s),
6.75 (2H, d, JZ8.8 Hz), 5.18 (1H, d, JZ8.8 Hz), 4.73 (1H,
d, JZ13.2 Hz), 4.53 (1H, d, JZ13.2 Hz), 4.46 (1H, dd, JZ
4.4, 7.6 Hz), 4.28 (1H, dd, JZ4.4, 7.6 Hz), 4.05–3.42 (12H,
m), 3.17 (1H, m), 2.89 (1H, dd, JZ5.2, 8.8 Hz), 2.68 (1H, d,
JZ8.8 Hz), 2.31 (2H, m), 1.79–1.58 (6H, m) ppm; 13C
NMR (100 MHz CD3OD, rt): 181.6, 175.3, 172.8, 172.6,
172.3, 166.7, 162.0, 138.4, 137.3, 133.9, 133.6, 131.5,
131.4, 130.9, 129.6, 129.5, 120.5, 116.1, 98.2, 79.5, 76.3,
74.9, 73.0, 71.6, 64.2, 63.3, 61.7, 56.9, 53.6, 43.9, 43.7,
41.0, 36.4, 33.1, 30.7, 29.6, 26.6, 23.7, 8.0 ppm; HR FAB
MS (negative): [MKH]K Found m/z 930.3368,
C45H52O13N7S requires m/z 930.3344; IR (film) n: 1680,
1205, 1140 cmK1; [a]2D5 C5.008 (c 0.5, MeOH).
1
Compound 47. H NMR (300 MHz, CDCl3, rt): 7.80–7.54
(8H, m), 7.29 (2H, d, JZ9.2 Hz), 6.78 (1H, s), 6.75 (2H, d,
JZ9.2 Hz), 5.80 (1H, d, JZ8.1 Hz), 4.57 (2H, dd, JZ3.3,
3.6 Hz), 4.24 (1H, dd, JZ6.8, 10.3 Hz), 4.08–3.70 (3H, m),
3.82 (3H, s), 2.41 (3H, s), 1.46 (9H, s) ppm; HR ESI MS

7890
T. Fujii et al. / Tetrahedron 61 (2005) 7874–7893
(positive): [MCNa]C Found m/z 718.1935, C35H37-
NNaO12S requires m/z 718.1929.
TLC (RP-18W, Merck Co., Ltd) (water/acetonitrile 5:4,
containing 1% acetic acid). Resulting crude product was
dissolved in DMF (0.5 mL), and 4-(bromomethyl)benzo-
phenone (2.0 mg, 7.3 mmol) and triethylamine (1.7 mL,
12.2 mmol) were added to this solution. The reaction
mixture was stirred for 24 h under light-shielded condition.
After removal of DMF using vacuum line, the residue was
separated by ODS-preparative TLC (RP-18W, Merck Co.,
Ltd) (water/acetonitrile 1:7, containing 1% TFA) to give 50
(1.2 mg, 20% in two steps).
4.1.37. t-Butyl (Z)-2-(20-deoxy-20-phthalimide-60-azido-
b-D-galactopyranosyloxy)-3-p-methoxyphenyl-2-acryl-
ate (48). Compound 47 (61 mg, 88 mmol), sodium azide
(57 mg, 880 mmol), and 15-crown-5 (175 mL, 880 mmol)
were dissolved in DMF (1 mL), and this mixture was stirred
overnight at 70 8C under argon atmosphere. After the
reaction mixture was allowed to stand at rt, the reaction
mixture was mixed with water, extracted with ethyl acetate,
and separated by silica gel column chromatography
(toluene/acetone 2:1) to give 48 (45 mg, 90%).
1
Compound 50. H NMR (300 MHz CD3OD, rt): 7.88–7.63
(10H, m), 7.50 (2H, d, JZ9.1 Hz), 7.17 (1H, s), 6.89 (2H, d,
JZ9.1 Hz), 5.24 (1H, d, JZ8.4 Hz), 4.46 (1H, dd, JZ4.7,
7.7 Hz), 4.32 (1H, dd, JZ4.0, 7.7 Hz), 3.90–3.45 (12H, m),
3.81 (3H, s), 2.90 (1H, dd, JZ5.1, 12.8 Hz), 2.67 (1H, d,
JZ12.8 Hz), 2.29 (2H, t, JZ7.0 Hz), 1.72–1.62 (6H, m),
1.58 (9H, s) ppm; HR ESI MS (positive): [MCNa]C Found
m/z 1024.4105, C50H63N7NaO13S, requires m/z 1024.4097.
1
Compound 48. H NMR (300 MHz, CDCl3, rt): 7.85–7.67
(4H, m), 7.60 (2H, d, JZ8.7 Hz), 6.84 (1H, s), 6.77 (2H, d,
JZ8.7 Hz), 5.82 (1H, d, JZ6.4 Hz), 4.64 (2H, m), 3.97
(1H, d, JZ4.0 Hz), 3.79–3.65 (3H, m), 3.79 (3H, s), 1.48
(9H, s) ppm; HR ESI MS (positive): [MCNa]C Found m/z
589.1905, C28H30N4NaO9 requires m/z 589.1905.
4.1.40. (Z)-2-(20-Deoxy-20-[4-benzoylphenyl]methyl-
amino-60-[N-biotinylglycylglycylglycyl]-amino-b-D-
galactopyranosyloxy)-3-p-methoxyphenyl-2-acrylate
(51). TFA (neat, 150 mL) was added to 50 (1.2 mg,
1.2 mmol). This mixture was stirred for 15 min under
argon atmosphere in complete light-shielded condition.
After stirring, residual TFA was removed with vaccum line,
and the residue was separated by ODS-preparative TLC
(RP-18W, Merck Co., Ltd) (water/acetonitrile 1:5, contain-
ing 1% TFA) to give 51 (0.5 mg, 44%) with recovered 50
(0.6 mg, 50%).
4.1.38. t-Butyl (Z)-2-(20-deoxy-20-phthalimide-60-[N-bio-
tinylglycylglycylglycyl]-amino-b-D-galactopyranosyl-
oxy)-3-p-methoxy-phenyl-2-acrylate (49). Compound 48
(20 mg, 34.6 mmol) was dissolved in MeOH (1 mL). After
addition of catalytic amount of Pd–CaCO3, the solution was
stirred overnight at rt under hydrogen atmosphere. The
reaction mixture was filtered with Celite, concentrated in
vacuo, and dissolved in DMF (1 mL). To this solution,
biotin unit 40 (17 mg, 41.6 mmol), HOBt (7 mg, 51.9 mmol),
and then DCC (9 mg, 41.6 mmol) were added. After
overnight stirring, the reaction mixture was filtered with
Hyflo-Super Cel (Wako Pure chemical Industry Co., Ltd),
and the filtrate was concentrated to dryness using vacuum
line, and the residue was separated with ODS-preparative
TLC (RP-18W, Merck Co., Ltd) (water/acetonitrile 10:1) to
give 49 (6.8 mg, 18% in two steps).
1
Compound 51. H NMR (600 MHz CD3OD, rt): 7.86–7.63
(10H, m), 7.49 (2H, d, JZ8.1 Hz), 6.96 (1H, s), 6.88 (2H, d,
JZ8.1 Hz), 5.09 (1H, d, JZ9.0 Hz), 4.58 (1H, d, JZ
12.2 Hz), 4.50 (1H, dd, JZ4.0, 8.0 Hz), 4.43 (1H, d, JZ
12.2 Hz), 4.32 (1H, dd, JZ4.5, 7.9 Hz), 3.95–3.65 (6H m),
3.82 (3H, s), 3.48 (3H, m), 3.42 (3H, m), 2.92 (1H, dd, JZ
5.1, 12.9 Hz), 2.72 (1H, d, JZ12.9 Hz), 2.33 (2H, m),1.75–
1.60 (6H, m) ppm; HR ESI MS (positive): [MCNa]C
Found m/z 968.3478, C46H55N7NaO13S, requires m/z
968.3471.
1
Compound 49. H NMR (300 MHz CD3OD, rt): 7.88–7.76
(4H, m), 7.65 (2H, d, JZ8.8 Hz), 6.82 (2H, d, JZ8.8 Hz),
6.72 (1H, s), 5.73 (1H, d, JZ8.1 Hz), 4.61 (1H, dd, JZ8.0,
11.0 Hz), 4.53 (1H, dd, JZ3.0, 9.0 Hz), 4.46 (1H, dd, JZ
4.8, 7.7 Hz), 4.32 (1H, dd, JZ5.4, 7.7 Hz), 3.70–3.15 (12H,
m), 3.58 (3H, s), 2.65 (1H, dd, JZ4.8, 12.9 Hz), 2.43 (1H, d,
JZ12.9 Hz), 2.09 (2H, t, JZ7.3 Hz), 1.61–1.31 (6H, m),
1.24 (9H, s) ppm; 13C NMR (400 MHz, CD3OD, rt): 177.1,
173.1, 172.2, 172.1, 170.3, 169.9, 166.1, 164.7, 161.7,
141.8, 135.3, 135.2, 133.3, 126.9, 124.8, 124.3, 123.9,
114.9, 99.2, 83.1, 74.5, 69.1, 69.0, 63.2, 61.7, 56.9, 55.8,
55.6, 44.0, 43.6, 41.0, 40.4, 36.4, 34.8, 29.6, 29.4, 28.4 ppm;
4.1.41. t-Butyl 2-(20-deoxy-20-phthalimide-b-D-galacto-
pyranosyl-oxy)-3-p-benzyloxyphenylpropanate (52).
Compound 24 (117 mg, 0.157 mmol) in methanol
(1.5 mL) was mixed with sodium methoxide (17.1 mg,
0.316 mmol) at K20 8C, and the mixture was stirred under
argon atmosphere. After 4 h stirring, Amberlite IR-120 B
was added to this solution to adjust pH at 7.0. After
filtration, the filtrate was concentrated in vacuo, and purified
by silica gel column chromatography (CHCl3/MeOHZ
15:1) to give 52 (91.6 mg, 0.148 mmol, 94%).
IR (film) n: 33 23, 1772, 1718, 1680, 1640, 1604, 1510 cmK1
;
[a]2D0 K55.18 (c 1.0, MeOH); ESI MS (positive): [MCNa]
Found m/z 960.3422, C44H55N7NaO14S requires m/z
960.3420.
1
Compound 52. H NMR (300 MHz, CDCl3, rt): 7.79 (2H,
4.1.39. t-Butyl (Z)-2-(20-deoxy-20-[4-benzoylphenyl]
methylamino-60-[N-biotinylglycylglycylglycyl]-amino-b-
D-galactopyranosyloxy)-3-p-methoxyphenyl-2-acrylate
(50). Compound 49 (5.7 mg, 6.1 mmol) and hydrazine
monohydrate (5 mL) were dissolved in MeOH (0.5 mL),
and this solution was stirred for 18 h at rt. After stirring,
excess hydrazine was removed completely by using vacuum
line, and the residue was purified with ODS-preparative
br), 7.71–7.65 (2H, m), 7.42–7.30 (5H, m), 7.05 (2H, d, JZ
8.8 Hz), 6.76 (2H, d, JZ8.8 Hz), 5.15 (1H, d, JZ8.1 Hz),
4.98 (2H, s), 4.51–4.27 (3H, m), 4.10 (1H, br), 3.85 (2H, br),
3.60 (1H, t, JZ4.6 Hz), 2.87 (2H, d, JZ6, 4 Hz), 1.14 (9H,
s) ppm; 13C NMR (75 MHz, CDCl3, rt): 170.3, 168.9, 157.7,
137.2, 134.0, 132.2, 131.1, 129.0, 128.7, 128.0, 127.6,
123.5, 114.4, 97.3, 81.6, 77.5, 73.5, 70.0, 69.9, 68.3, 63.1,
54.1, 38.0, 27.7 ppm; HR ESI MS (positive): [MCNa]C

T. Fujii et al. / Tetrahedron 61 (2005) 7874–7893
7891
1
Found m/z 642.2309, C34H37NNaO10 requires m/z
642.2310; IR (film) n: 3470, 2978, 2934, 1774, 1717,
1512 cmK1; [a]1D8 K23.68 (c 0.1, MeOH).
Compound 55. H NMR (300 MHz, MeOH, rt): 7.91–7.87
(1H, m), 7.80 (3H, br), 6.92 (2H, d, JZ8.4 Hz), 5.12 (1H, d,
JZ8.4 Hz), 4.55 (1H, dd, JZ3.3, 11.0 Hz), 4.46–4.37 (2H,
m), 3.86–3.69 (3H, m), 3.26 (1H, dd, JZ3.3, 12.8 Hz), 2.82
(2H, ddd, JZ5.9, 14.0, 25.5 Hz), 1.12 (9H, s) ppm; 13C
NMR (75 MHz, MeOH, rt): 171.8, 170.3, 170.0, 157.1,
135.3, 135.2, 133.7, 133.2, 131.9, 128.0, 124.2, 123.9,
115.7, 98.9, 82.6, 78.6, 76.4, 70.4, 68.8, 55.2, 52.5, 39.2,
28.0 ppm; HR ESI MS (positive): [MCNa]C Found m/z
577.1903, C27H30N4NaO12S requires m/z 577.1905; IR
(film) n: 3445, 2106, 1776, 1709, 1516, 1394 cmK1; [a]D19
K40.28 (c 0.1,MeOH).
4.1.42. t-Butyl 2-(20-deoxy-20-phthalimide-60-O-p-tolu-
enesulfonyl-b-D-galactopyranosyloxy)-3-p-benzyloxy-
phenylpropanate (53). p-Toluene sulfonyl chloride
(9.9 mg 52 mmol) was added to the solution of 52
(26.9 mg, 43 mmol) in dichloromethane (0.5 mL) at
K20 8C, and the mixture was stirred for 5 h under argon
atmosphere. The mixture was evaporated with toluene, and
the residue was purified by preparative TLC using Merck 60
F254 (CHCl3/MeOHZ10:1) to give 53 (18.5 mg, 55%) with
recovered 52 (4.9 mg, 18%).
4.1.45. t-Butyl 2-(20-deoxy-20-phthalimide-60-[N-biotinyl-
glycylglycylglycyl]amino-b-D-galactopyranosyloxy)-3-
hydroxyphenylpropanate (56). Compound 55 (41.5 mg,
74.9 mmol) was dissolved in MeOH (1.5 mL). After
addition of catalytic amount of Pd–CaCO3, the solution
was stirred for 3 h at rt under hydrogen atmosphere. The
reaction mixture was filtered with Celite, concentrated in
vacuo to give crude amine (38.6 mg). The resulting amine
(38.6 mg, 73.1 mmol, crude) was dissolved in DMF (1 mL).
To this solution, biotin unit 40 (46.3 mg, 112 mmol), HOBt
(22.5 mg, 164 mmol) were added, and then 6.6 mL of 1 M
DCC in DMF was added 10-times in every 20 min. After
24 h stirring, the reaction mixture was filtered with Hyflo-
Super Cel (Wako Pure chemical Industry Co., Ltd), and the
filtrate was concentrated to dryness using vacuum line, and
the residue was separated with ODS-preparative TLC
(RP-18W, Merck Co., Ltd) (water/acetonitrile 5:4) to give
56 (40.1 mg, 58% in two steps).
1
Compound 53. H NMR (300 MHz, CDCl3, rt): 7.82–7.79
(4H, m), 7.69–7.65 (2H, m), 7.40–7.30 (7H, m), 7.00 (2H, d,
JZ8.4 Hz), 6.73 (2H, d, JZ8.4 Hz), 5.10 (1H, d, JZ
8.4 Hz), 4.96 (2H, s), 4.53 (1H, br), 4.33–4.28 (3H, m), 4.14
(1H, dd, JZ6.4, 10.5 Hz), 4.01 (1H, br), 3.87 (1H, t, JZ
6.2 Hz), 2.85 (2H, d, JZ6.1 Hz), 2.44 (3H, s), 1.11 (9H,
s) ppm; 13C NMR (75 MHz, CDCl3, rt): 169.6, 168.5, 157.3,
145.2, 137.0, 133.8, 132.3, 131.9, 130.7, 130.0, 128.5,
128.4, 128.0, 127.8, 127.4, 123.2, 114.2, 97.2, 81.5, 72.0,
69.8, 67.9, 67.7, 67.6, 45.9, 38.0, 29.7, 27.7, 21.7 ppm; HR
ESI MS (positive): [MCNa]C Found m/z 796.2396,
C41H43NNaO12S requires m/z 796.2398; IR (film) n: 3474,
2978, 2930, 1717, 1661, 1512, 1392, 1367 cmK1; [a]D18
K30.78 (c 0.1,MeOH).
4.1.43. t-Butyl 2-(20-deoxy-20-phthalimide-60-O-p-tolu-
enesulfonyl-b-D-galactopyranosyloxy)-3-hydroxyphenyl-
propanate (54). Compound 53 (220 mg, 0.284 mmol) was
dissolved in THF (3 mL) and catalytic amount of 20%
Pd(OH)2 was added to this solution. This solution was
stirred for 30 h under hydrogen atmosphere and filtered with
Celite. After evaporation, the residue was purified by silica
gel column chromatography (CHCl3–MeOHZ23:1) to give
54 (161 mg, 83%.).
1
Compound 56. H NMR (300 MHz, MeOH, rt): 7.91–7.88
(1H, m), 7.81–7.78 (3H, m), 6.95 (2H, d, JZ8.5 Hz), 6.54
(2H, d, JZ8.5 Hz), 5.06 (1H, d, JZ8.4 Hz), 4.56–4.45 (3H,
m), 4.42–4.30 (2H, m), 3.97–3.80 (7H, m), 3.72 (1H, t, JZ
6.7 Hz), 3.51 (2H, d, JZ6.8 Hz), 3.25–3.12 (1H, m), 2.94–
2.76 (3H, m), 2.69 (1H, d, JZ12.8 Hz), 2.32 (2H, t, JZ
7.4 Hz), 1.77–1.31 (6H, m) ppm; 13C NMR (75 MHz,
MeOH, rt): 177.1, 173.1, 172.3, 172.2, 171.9, 170.3,
170.1, 166.2, 157.0, 135.2, 135.1, 133.7, 133.2, 131.9,
128.1, 124.2, 123.9, 115.7, 98.9, 82.6, 78.5, 74.1, 69.6, 68.8,
63.2, 61.7, 56.9, 55.3, 44.1, 43.7, 43.6, 41.1, 41.0, 39.2,
36.4, 29.6, 29.4, 28.0, 26.6 ppm; HR ESI MS (positive):
[MCNa]C Found m/z 948.3427, C43H55N7NaO14S requires
m/z 948.3420; IR (film) n: 3306, 2932, 1713, 1663, 1542,
1516, 1393 cmK1; [a]1D9 12.88 (c 0.1,MeOH).
1
Compound 54. H NMR (300 MHz, MeOH, rt): 7.85–7.78
(6H, m), 7.42 (2H, d, JZ8.1 Hz), 6.92 (2H, d, JZ8.4 Hz),
6.55 (2H, d, JZ8.4 Hz), 5.04 (1H, d, JZ8.4 Hz), 4.49 (1H,
dd, JZ3.1, 11.2 Hz), 4.37–4.12 (4H, m), 3.87–3.81 (2H, m),
2.79 (2H, ddd, JZ6.2, 14.7, 21.1 Hz), 2.44 (3H, s), 1.13
(9H, s) ppm; 13C NMR (75 MHz, MeOH, rt): 171.6, 170.2,
170.0, 157.1, 146.7, 135.2, 134.2, 133.6, 133.2, 131.9,
131.2, 131.1, 129.1, 128.1, 124.2, 123.9, 98.9, 82.7, 78.6,
74.2, 70.8, 69.7, 68.7, 55.1, 40.1, 28.0, 21.7 ppm; HR ESI
MS (positive): [MCNa]C Found m/z 706.1932, C34H37-
NNaO12S requires m/z 706.1929; IR (film) n: 3464, 1776,
1713, 1516, 1392, 1366 cmK1; [a]D19 30.98 (c 0.1,MeOH).
4.1.46. t-Butyl 2-(20-deoxy-20-[4-benzoylphenyl]methyl-
amino-60-[N-biotinylglycylglycylglycyl]amino-b-D-
galactopyranosyloxy)-3-hydroxyphenylpropanate (57).
Compound 56 (26.7 mg, 28.7 mmol) was dissolved in
0.2 M hydrazine monohydrate in EtOH (2.0 mL), and this
solution was stirred for 25 h at 30 8C, and then stirred for 4 h
at 40 8C. After stirring, excess hydrazine was removed
completely by using vacuum line, and the residue was
purified with ODS-preparative TLC (RP-18W, Merck Co.,
Ltd) (water/acetonitrile 3:2, containing 5% acetic acid).
Resulting crude amine (25.4 mg) was dissolved in DMF
(0.7 mL), and 4-(bromomethyl)benzophenone (9.7 mg,
35.1 mmol) and triethylamine (8.9 mL, 63.9 mmol) were
added to this solution at 0 8C. The reaction mixture
was stirred for 3.5 h at rt under argon atmosphere in
4.1.44. t-Butyl 2-(20-deoxy-20-phthalimide-60-azido-b-D-
galactopyranosyloxy)-3-hydroxyphenylpropanate (55).
Compound 54 (35.3 mg, 51.5 mmol), sodium azide (5.1 mg,
77 mmol) were dissolved in DMF/H2O 10:1 (0.6 mL), and
this mixture was stirred for 11 h at 100 8C. After the reaction
mixture was allowed to stand at rt, the reaction mixture was
mixed with water, extracted with ethyl acetate, and then the
organic layer was dried over abs Na2SO4. The organic layer
was separated by preparative TLC using Merck 60F254
(CHCl3/MeOH 7:1) to give 55 (26.4 mg, 93%).

7892
T. Fujii et al. / Tetrahedron 61 (2005) 7874–7893
light-shielded condition. After removal of DMF using
vacuum line, the residue was separated by ODS-preparative
TLC (RP-18W, Merck Co., Ltd) (water/acetonitrile 2:3,
containing 5% TFA) to give 57 (18.4 mg, 64% in two steps).
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.
1
Compound 57. H NMR (600 MHz, MeOH, rt): 7.81–7.70
(5H, m), 7.59 (2H, t, JZ7.8 Hz), 7.31 (2H, d, JZ8.2 Hz),
7.21 (2H, d, JZ8.5 Hz), 6.75 (2H, d, JZ8.5 Hz), 4.88 (1H,
t, JZ4.6 Hz), 4.76 (1H, d, JZ8.5 Hz), 4.57–4.53 (2H, m),
4.40–4.35 (2H, m), 3.94–3.86 (7H, m), 3.71–3.69 (2H, m),
3.56 (1H, dd, JZ5.6, 13.7 Hz), 3.48 (1H, dd, JZ7.7,
13.7 Hz), 3.38 (1H, m), 3.28–3.23 (1H, m), 3.19 (1H, d, JZ
4.6 Hz), 2.98 (1H, dd, JZ5.0, 12.8 Hz), 2.75 (1H, d, JZ
12.7 Hz), 2.35 (2H, m), 1.83–1.65 (4H, m), 1.58–1.48 (11H,
m) 1.10 (9H, s) ppm; 13C NMR (150 MHz, MeOH, rt):
197.6, 177.2, 174.7, 173.2, 172.4, 172.3, 166.1, 157.9,
139.8, 138.4, 136.7, 134.2, 132.5, 131.4, 131.3, 131.1,
129.7, 127.6, 116.1, 99.3, 85.6, 78.4, 74.8, 71.0, 69.5, 63.3,
62.4, 61.7, 57.0, 53.8, 44.3, 43.9, 43.7, 41.1, 41.0, 38.7,
36.5, 29.7, 29.5, 28.4, 26.6 ppm; HR ESI MS (positive):
[MCH]C Found m/z 990.4283, C49H64N7O13S requires m/z
4.3. Photoaffinity labeling of membrane protein in plant
motor cell
A large amount of plant motor cells was collected by cutting
off a large number of sections of plant pulvini (size; ca. 0.
5 mm!0.5 mm) containing the motor cell one by one from
plant leaves under a stereoscopic microscope. Collected
pulvini were immersed in the extraction buffer (0.25 M
sucrose, 3 mM EDTA-2K, 2.5 mM DTT, 25 mM Tris–
MES, 1 tablet of completee/50 mL, pH 7.2) immediately.
One cross-linking experiment needed about nine hundred
plant sections.
Successive homogenization (15,000 rpm, 10 min) in extrac-
tion buffer (0.25 M sucrose, 3 mM EDTA, 2.5 mM DTT,
25 mM Tris–MES, pH 7.8) at 4 8C, filtration with nylon
mesh (50 mm), and twice centrifugation (Beckman Coulter
Optima TLX, 1st; 3000!g, 15 min, 4 8C, 2nd; 100,000!g,
60 min, 4 8C) gave a pellet of the crude membrane fraction.
The content of total protein in that fraction was determined
to be 68.7 mg by the Bradford method with BSA as a
reference. The membrane ATPase activity was determined
to be 0.67 mmol/mg protein$min by Sandstom’s method.15
The crude membrane fractions were suspended in the 50 mL
of extraction buffer, and 14 mL of this solution containing
10 mg of crude membrane protein was incubated with probe
2 or 3 (3 mM concentration) for 20 min at rt, respectively.
After cross-linking by irradiation of UV-light (365 nm) for
60 min at 4 8C. Electrophoresis buffer (0.3 M Tris–Cl, 10%
SDS, 30% glycerol, 9.3% DTT, pH 6.8) was added to this
reaction mixture and the solution was heated at 90 8C for
5 min. The reaction mixture was analyzed by SDS-PAGE
(Ready Gel J 7.5%, BIO-RAD Co. Ltd) with molecular
weight marker (Biotinylated SDS-PAGE standard, High-
Range, BIO-RAD Co. Ltd). After western blotting using
PVDF membrane (Hybond-P, Amersham Bioscience Co.
Ltd), detection of the bands of the potential receptor for leaf-
opening substance was carried out by chemiluminescence
detection with ECL Advance Western Blotting Detection
Kit (Amersham Bioscience Co. Ltd) with ECL mini-camera
(Amersham Bioscience Co. Ltd).
990.4277; IR (film) n: 3290, 2932, 1670, 1202, 1136 cmK1
[a]1D8 21.98 (c 0.1, MeOH).
;
4.1.47. (Z)-2-(20-Deoxy-20-[4-benzoylphenyl]methyl-
amino-60-[N-biotinylglycylglycylglycyl]-amino-b-D-
galactopyranosyloxy)-3-p-methoxyphenyl-2-acrylate
(58). TFA (neat, 700 mL) was added to 57 (4.8 mg,
48.4 mmol). This mixture was srirred for 1 h under argon
atmosphere in complete light-shielded condition. After
stirring, residual TFA was removed with vaccum line, and
the residue was separated by ODS-preparative TLC
(RP-18W, Merck Co., Ltd) (water/acetonitrile 1:1, contain-
ing 5% TFA) to give 58 (2.3 mg, 51%) with recovered 57
(2.2 mg, 46%).
Compound 58. 1H NMR (600 MHz, MeOH, rt): 7.81 (2H, d,
JZ8.1 Hz), 7.75–7.70 (3H, m), 7.59 (2H, t, JZ7.8 Hz),
7.25 (2H, d, JZ8.1 Hz), 7.19 (2H, d, JZ8.4 Hz), 6.73 (2H,
d, JZ8.4 Hz), 4.90 (1H, m), 4.77 (1H, d, JZ8.6 Hz), 4.55–
4.53 (1H, m), 4.41 (1H, JZ12.8 Hz), 4.37–4.35 (1H, m),
4.24 (1H, d, JZ12.8 Hz), 3.97–3.84 (7H, m), 3.67–3.66
(2H, m), 3.57 (1H, dd, JZ5.4, 13.8 Hz), 3.46 (1H, dd, JZ
7.8, 13.8 Hz), 3.30–3.24 (2H, m), 3.20–3.18 (2H, m), 2.97
(1H, dd, JZ5.0, 12.7 Hz), 7.74 (1H, d, JZ12.7 Hz), 2.39–
2.30 (2H, m), 1.83–1.47 (6H, m) ppm; 13C NMR (150 MHz,
MeOH, rt): 197.7, 177.2, 173.1, 172.4, 172.3, 171.1, 165.1,
157.7, 139.6, 138.4, 136.8, 134.1, 132.3, 131.3, 131.3,
131.1, 129.6, 128.1, 116.1, 99.8, 74.8, 71.2, 69.5, 63.3, 62.7,
61.7, 57.0, 54.3, 44.2, 43.8, 43.7, 41.1, 41.0, 38.6, 36.5,
30.8, 29.7, 29.5, 26.6 ppm; HR ESI MS (positive): [MC
H]C Found m/z 934.3658, C45H56N7O13S requires m/z
934.3651; IR (film) n: 3315, 1663, 1202 cmK1; [a]D18 24.28
(c 0.1, MeOH).
Acknowledgements
We thank Experimental Station for Medicinal Plant Studies,
Graduate School of Pharmaceutical Science, Tohoku
University for the supply of Cassia mimosoides L. And
we also thank to Dr. Shosuke Yamamura (Keio University)
for the pioneering work of this area and useful discussion
about this work. This work was financially supported by the
Ministry of Education, Science, Sports and Culture (Japan)
Grant-in-Aid for Scientific Research (No. 16073216 and
No. 15681013) and JSPS Grant for TS. This work was also
supported by Grant-in-Aid for the 21st Century COE
program of Tohoku University from the Ministry of
4.2. 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 25–35 8C and
allowed to stand overnight. The leaves, which opened again
the next morning (around 10:00 am.) were used for the

T. Fujii et al. / Tetrahedron 61 (2005) 7874–7893
7893
8. Shigemori, H.; Sakai, N.; Miyoshi, E.; Shizuri, Y.; Yamamura,
S. Tetrahedron 1990, 46, 383–394.
Education, Culture, Sports, Science, and Technology,
Japan.
9. Ueda, M.; Sawai, Y.; Yamamura, S. Tetrahedron Lett. 1999,
40, 3757–3760.
10. Hatanaka, Y.; Hashimoto, M.; Kurihara, H.; Nakayama, H.;
Kanaoka, Y. J. Org. Chem. 1994, 59, 383–395.
11. Dorman, G.; Prestwich, G.D. Biochemistry 1994, 33, 5661–
5673.
References and notes
1. Hung, D. T.; Jamison, T. F.; Screiber, S. L. Chem. Biol. 1996,
3, 623–639.
12. Sugimoto, T.; Fujii, T.; Hatanaka, Y.; Yamamura, S.; Ueda, M.
Tetrahedron Lett. 2002, 43, 6529–6532.
2. Darwin, C. The Power of Movement in Plants. Third
Thousand; John Murray: London, 1882.
13. Hatanaka, Y.; Hashimoto, M.; Kanaoka, Y. Bioorg. Med.
Chem. 1994, 12, 1367–1371.
3. Satter, R. L.; Gorton, H. L.; Vogelmann, T. C. The Pulvinus:
Motor Organ for Leaf Movement; American Society of Plant
Physiologists: Rockville, 1990.
14. Xia, J.; Srikrishnan, T.; Alderfer, J. L.; Jain, R. K.; Piskorz, C.
F.; Matta, K. L. Carbohydr. Res. 2000, 329, 561.
15. Fujii, T.; Sugimoto, T.; Yamamura, S.; Ueda, M. Tetrahedron
Lett. 2003, 44, 2497–2499.
4. Ueda, M.; Yamamura, S. Angew. Chem., Int. Ed. 2000, 39,
1400–1414.
16. Wong, T. C.; Townsend, R. R.; Lee, Y. C. Carbohydr. Res.
1987, 170, 27–46.
5. Ueda, M.; Wada, Y.; Yamamura, S. Tetrahedron Lett. 2001,
42, 3869–3872. Nagano, H.; Kato, E.; Yamamura, S.; Ueda,
M. Org. Biomol. Chem. 2003, 1, 3186–3192.
17. Sugimoto, T.; Fujii, T.; Idutu, Y.; Yamamura, S.; Ueda, M.
Tetrahedron Lett. 2004, 45, 335–338.
6. Kotzyba-Hilbert, F.; Kapfer, I.; Goeldner, M. Angew. Chem.,
Int. Ed. Engl. 1995, 34, 1296–1312. Dorman, G.; Prestwich,
G. D. Trends Biotechnol. 2000, 18, 64–77.
18. Sanderson, R. P.; de Boer, A. H.; Lomax, T. L.; Cleland, R. E.
Plant Physiol. 1987, 85, 693.
19. http://www.cbs.umn.edu/arabidopsis/
7. Shigemori, H.; Miyoshi, E.; Shizuri, Y.; Yamamura, S.
Tetrahedron Lett. 1989, 30, 6389–6392.
20. Matsubayashi, Y.; Sakagami, Y. J. Biol. Chem. 2000, 275,
15520–15525.