Fluorescence study on the nyctinasty of Phyllanthus urinaria L. using novel fluorescence-labeled probe compounds

Article abstract of DOI:10.1016/j.tet.2006.05.022

We report the synthesis of fluorescence-labeled probes based on phyllanthurinolactone 1, which is a leaf-closing substance of Phyllanthus urinaria L. The fluorescence study using biologically active probe 2 and inactive probes (epi-2 and 31) revealed that

Full text of DOI:10.1016/j.tet.2006.05.022

Tetrahedron 62 (2006) 7307–7318
Fluorescence study on the nyctinasty of Phyllanthus urinaria L.
using novel fluorescence-labeled probe compounds
Nobuki Kato, Masayoshi Inada, Hirotaka Sato, Ryoji Miyatake, Tsutomu Kumagai
*
and Minoru Ueda
Tohoku University, Department of Chemistry, Aoba-ku, Sendai 980-8578, Japan
Received 31 March 2006; revised 10 May 2006; accepted 11 May 2006
Available online 5 June 2006
Abstract—We report the synthesis of fluorescence-labeled probes based on phyllanthurinolactone 1, which is a leaf-closing substance of
Phyllanthus urinaria L. The fluorescence study using biologically active probe 2 and inactive probes (epi-2 and 31) revealed that the target
cell for 1 is a motor cell and suggested that some receptors, which recognize the aglycon of 1 exist on the plasma membrane of the motor cell,
as with leaf-opening substances. Moreover, binding of probe 2 was specific to the plant motor cell contained in the plants belonging to the
genus Phyllanthus. These results showed that the binding of probe 2 with a motor cell is specific to the plant genus and suggested that the
genus-specific receptor for the leaf-closing substance would be involved in nyctinasty.
Ó 2006 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 a biological clock.1 Nyc-
tinastic 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
Figure 1.
in plant leaf-movement. The 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.2 We have revealed that nyctinasty is controlled
by a pair of leaf-movement factors: leaf-opening and leaf-
closing substances.3 It is already clarified by using fluores-
cence-labeled leaf-opening substances that the target cell
of the leaf-opening substance is a motor cell.4 On the other
hand, no attempt has been carried out to clarify the target
cell of the leaf-closing substances because the structure of
most leaf-closing substances is too simple3 to develop a
molecular probe, such as a fluorescence-labeled one. This is
because large fluorescence dye would cause serious decrease
in the bioactivity of the synthetic probe.
that is essential for a synthetic probe. Syntheses of 1 and its
analogs were completed by Mori and Audran6 and our
group.7 The structure–activity relationship studies using
them showed that the stereochemistry in aglycon of natural
product 1 is important for its bioactivity and the structure
modification in the sugar moiety has no effect on the bio-
activity. Recently, we developed the fluorescence-labeled
probe 2 based on the result of structure–activity relationship
studies, and revealed that probe 2 binds to a motor cell spe-
cifically.8 However, in order to prove the existence of a recep-
tor on a motor cell, an appropriate control experiment using
biologically inactive probe compounds such as C-4 epi-2
and unsaturated derivative 31 that cannot bind to the target
cell is essential. In this paper, we carried out the direct obser-
vation of the target cell for 1 using biologically active
and inactive fluorescence-labeled probes and revealed that
the receptor of 1 exists in a motor cell. Additionally, we
found by using the biologically active probe 2 that genus-
specific receptors of leaf-closing substances are involved
in nyctinasty.
Phyllanthurinolactone 1 is a glycoside-type leaf-closing sub-
stance of Phyllanthus urinaria L.5 (Fig. 1). The structure of 1
is comparatively large enough for the structure modification
Keywords: Nyctinasty; Leaf-closing substance; Fluorescence; Probe com-
pound; Motor cell; Mitsunobu reaction; Glycosidation.
* Corresponding author. Tel./fax: +81 22 795 6553; e-mail: ueda@mail.
tains.tohoku.ac.jp
0040–4020/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2006.05.022

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N. Kato et al. / Tetrahedron 62 (2006) 7307–7318
2. Results and discussion
2.1. The design and synthetic plan of fluorescence-
labeled probe compounds
Based on the previously obtained result6,7 that the structure
modification of the sugar moiety did not affect the bioactiv-
ity, the big fluorescence dye was introduced into the hydroxy
group at the C60 position of the sugar moiety of 1. Since 1 is
easily hydrolyzed by b-glucosidase, the sugar moiety was
changed from D-glucose into D-galactose.9 Moreover, in
order to increase the resistance against the esterase involved
in a plant body, an amide bond was selected instead of ester
bond connecting the fluorescence dye with 1. The retrosyn-
thesis of 2 is shown in Scheme 1. The fluorescence-labeled
probe 2 could be synthesized from the intermediate 3, which
was bisected into the sugar moiety 4 and aglycon 5. The lac-
tone in aglycon 5 could be constructed by the intramolecular
Horner–Emmons reaction10 of phosphonoacetate 6, which
could be synthesized from the ketoalcohol 7 via Mitsunobu
inversion11 of the C4 stereocenter using phosphonoacetic
acid. The ketoalcohol 7 could be synthesized from D-glu-
cose.
Scheme 2. (i) TBSOTf, lutidine, CH2Cl2, 99%; (ii) t-BuOK, THF, 95%; (iii)
cat. Hg(OCOCF3)2, acetone/H2O; (vi) MsCl, Et3N, CH2Cl2, 70% (two
steps); (v) TBAF, AcOH, THF, quant.; (vi) see Table 1; (vii) t-BuOK,
THF, 0 ꢀC, 63%; (viii) DDQ, CH2Cl2/H2O, 71%.
Scheme 1.
2.2. Synthesis of fluorescence-labeled probe compounds
the Mitsunobu reagents (entry 3). However, the purification
of 6 was very difficult because of a large amount of triphe-
nylphosphine oxide, which was produced as a byproduct.
Therefore, we examined phosphines 14 and 15, which are
easily removed by acid treatment (entry 4 and 5).13 Phos-
phine 14 gave lower yield and stereoselectivity (R:S ¼ 3:1).
However, phosphine 15 afforded phosphonoacetate 6 in
moderate yield with higher stereoselectivity (R:S ¼ 10:1)
than triphenylphosphine. Additionally, the phosphine oxide
from 15 was easily removed by simple acid treatment.
The synthesis of aglycon is shown in Scheme 2.8 Intermedi-
ate 8, prepared according to the previous paper,7 was pro-
tected by the TBS group, and then subjected to elimination
of iodide to afford the enol ether 9. A catalytic amount of
Hg(OCOCF3)2-mediated Ferrier’s carbocyclization12 of 9
and following treatment of the resulting alcohol 10 with
mesyl chloride and triethylamine, that is, a one-pot reaction
involving mesylation and b-elimination, afforded the
cyclohexanone derivative 11 in 70% overall yield in two
steps. Deprotection of the TBS group gave the alcohol 7.
We considered that epimerization of the C4 stereocenter
and introduction of diethyl phosphonoacetate could be car-
ried out simultaneously using the Mitsunobu reaction11
(Table 1). At first, the reaction did not proceed under the
standard Mitsunobu reaction condition (entry 1). Tributyl-
phosphine gave many products (entry 2). The phosphono-
acetate 6 was obtained as a mixture of C4 stereoisomers
(R:S ¼ 5:1) in moderate yield by using excess amount of
Intramolecular Horner–Emmons reaction of the resulting 6
using t-BuOK as the base afforded lactone 12. Finally, de-
protection of the PMB group by DDQ gave the aglycon 5
as a 7:1 diastereomeric mixture.
Next, we examined the conditions of the glycosidation reac-
tion. In our previous synthesis, b-D-galactopyranosyl bro-
mide was used for glycosidation according to the method
reported by Mori and Audran.6 In this method, the desired

N. Kato et al. / Tetrahedron 62 (2006) 7307–7318
7309
Table 1. Mitsunobu reaction of alcohol 7
Table 2. Glycosidation
Entry 20 or 21 Condition (equiv)
(equiv)
22 or 23
5
(%)
Entry Phosphine DEAD 13
(equiv)
Temp Yield %
Ratio
(6+epi-6) (6:epi-6)
(equiv) (equiv) (ꢀC)
1
2
3
4
20 (2.0)
20 (2.0)
21 (1.2)
21 (1.6)
Cp2HfCl2 (3.0), AgClO4 (3.0) 22 (trace) 32
SnCl2 (5.0), AgClO4 (5.0) 22 (trace) 45
Cp2HfCl2 (2.0), AgClO4 (2.0) 23 (26%) 40
Cp2HfCl2 (2.0), AgClO4 (2.0) 23 (52%) 15
1
2
3
4
5
PPh3 (2.0) 2.0
PBu3 (2.0) 2.0
PPh3 (8.0) 8.0
2.0
2.0
8.0
8.0
8.0
rt
rt
0
0
0
0
—
—
5:1
3:1
Messy
42
16
14 (2.0)
15 (2.0)
8.0
8.0
40
10:1
The synthesis of fluorescence-labeled probe 2 was then
examined (Scheme 4). Acetyl groups of the glycoside
23 were removed with KCN6,17 to give the triol 24 as a 4:1
mixture of C4 stereoisomers. At this stage, further epimeri-
zation occurred. Because the C4 proton in 24 could be easily
abstracted by KCN, the resulting stable furan-type inter-
mediate gave a mixture of 24 and its epimer by protona-
tion. Deprotection of the Fmoc group and introduction
of AMCA-X gave fluorescence-labeled probe 2 as a 4:1
diastereomeric mixture. Diastereomerically pure 2 and its
epimer epi-2 were obtained after purification by HPLC
(COSMOSIL 5C18-AR, 20% CH3CN aq, 260 nm). The de-
sired 2 was confirmed by coupling constants in the 1H NMR
spectra (Scheme 4). The relation between H4 and H6 was de-
termined to be syn by the coupling constants between H4 and
H5ax, H4 and H5eq, H5ax and H6, and H5eq and H6.
coupling product was obtained in low yield (28% yield) with
a large amount of acetyl aglycon. Therefore, we examined
the use of glycosyl fluoride as a glycosyl donor. The synthe-
sis of b-D-galactopyranosyl fluoride is shown in Scheme 3.
Acetate 16, prepared from D-galactose according to the pro-
cedure in Ref. 14, was treated with hydrazine monohydrate
and fluorination of the resulting hydroxy group with DAST
afforded fluoride 17. Removal of acetates and reduction of
azide were followed by protection of amine with a Boc or
Fmoc group to give the triols 18 and 19. Finally, acetylation
of the triols 18 and 19 yielded the 1-fluorosugars 20 and 21.
Scheme 3. (i) See Ref. 14; (ii) H2NNH2/AcOH, DMF, 0 ꢀC, 74%; (iii)
DAST, CH2Cl2, 92%; (iv) MeONa, MeOH, 0 ꢀC; (v) H2, Pd/C, MeOH;
(vi) (Boc)2O, pyridine or FmocCl, DIPEA, THF, 0 ꢀC; (vii) Ac2O pyridine,
65% (four steps) (R ¼ Boc), 28% (four steps) (R ¼ Fmoc).
Glycosidation using b-D-galactopyranosyl fluoride is sum-
marized in Table 2. In the case of N-Boc protected galacto-
pyranosyl fluoride 20, both Suzuki15 and Mukaiyama
methods16 gave only a trace amount of glycoside 22 (entry
1 and 2). Under these conditions, N-Boc protected sugar
was decomposed and the aglycon 5 was recovered. On the
other hand, glycosidation of N-Fmoc protected galactopyra-
nosyl fluoride 21 via the Suzuki method gave glycoside 23 in
moderate yield (entry 3). Using 1.6 equiv of the sugar 21, the
yield was improved to 52% (entry 4). In this reaction condi-
tion, aglycon 5 was recovered in 15% yield with no produc-
tion of acetyl aglycon.
Scheme 4. (i) KCN, MeOH, 0 ꢀC, 47%; (ii) piperidine, DMF, 0 ꢀC; (iii)
AMCA-X, SE, DMF, 0 ꢀC, quant. (two steps); (iv) HPLC separation.
To prove the existence of a receptor, it is essential to demon-
strate that biologically inactive probe compounds cannot

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N. Kato et al. / Tetrahedron 62 (2006) 7307–7318
bind to the target cell for active compounds. Structure modi-
fication in the aglycon moiety of 2 reduces its bioactivity.
Thus, we planned synthesis of fluorescence-labeled com-
pound 31, which has the reduced C7–C8 double bond, as
a biologically inactive probe for the control fluorescence
experiment.
2.3. Bioassay of fluorescence-labeled probes
With the fluorescence-labeled probes 2, epi-2 and 31 were
now available. Then, we examined their bioactivities. The
young leaves detached from the stem of the plant P. urinaria
with a sharp razor blade were used for bioassay. One leaf was
placed in H2O (ca. 300 ml) using a 5-ml glass tube in the bio-
tron kept at 32 ꢀC and allowed to stand overnight. The
leaves, which opened again in the morning were used for
the bioassay. The test solution was carefully poured into
test tubes with a microsyringe at around 10:00 a.m. The bio-
active fraction was judged by the leaf-closing activity before
the leaf-closing of the plant leaf in the blank solution. Probe
Synthesis of 7,8-dihydro analog 27 is shown in Scheme 5.
The hydrogenation of aglycon 5 using the Lindlar catalyst
gave 7,8-dihydro aglycon 25. At this stage, the reduced agly-
con and its C4 epimer were easily separated by silica gel col-
umn chromatography. Glycosidation of 7,8-dihydro aglycon
25 and 2,3,4,6-tetra-O-acetyl-a-D-glucopyranosyl bromide
˚
Scheme 5. (i) H2, Lindlar catalyst, AcOEt, 68%; (ii) 26, AgOTf, Ag2CO3, MS4A, CH2Cl2, 20%; (iii) KCN, MeOH, 31%.
26 was carried out with Ag2CO3 and AgOTf to afford the
glycoside and subsequent deprotection of acetyl groups
yielded 7,8-dihydro phyllanthurinolactone 27.
2 was effective for the leaf-closing of P. urinaria at
1ꢁ10ꢂ5 M, that is, one-hundredth as effective as natural
product 1. On the other hand, epi-2, which is the C4 epimer
of 2, and 7,8-dihydro probe 31 were biologically inactive
(>1ꢁ10ꢂ4 M).
The 7,8-dihydro analog 27 showed no leaf-closing activity
against P. urinaria L. even at 1ꢁ10ꢂ4 M. From this result,
we judged that the fluorescence-labeled compound with
7,8-dihydro aglycon could be used as a biologically inactive
probe.
2.4. Fluorescence study using fluorescence-labeled
probes
We used fluorescence-labeled probes to seek the target cell
for 1. For this purpose, the binding experiment using a plant
section was carried out. The leaf of P. urinaria opening in the
daytime was cut into an appropriate size and fixed in agar.
The agar was sliced perpendicular to the petiole by a micro-
slicer (Dousaka EM Co., Ltd) to a thickness of 30 mm and the
sections containing the pulvini were floated on distilled
water. The sections were immersed in a solution containing
1ꢁ10ꢂ4 M of the probes (2, epi-2, and 31), and allowed to
stand for staining under shaded condition at room tempera-
ture for 6 h. After staining, the stained section was incubated
for 15 min in equilibrium buffer (Slow FadeÔ Gold Anti-
fade Reagent, Molecular Probes Inc.) to remove excess fluo-
rescence probes. Then, the stained section was placed on
a slide glass and covered by a cover glass after adding
a drop of antifade reagent (Slow FadeÔ Gold Antifade Re-
agent, Molecular Probes Inc.). The observation of these
sections was carried out by a fluorescence microscope
(ECLIPSE E800, Nicon Co., Ltd) with an appropriate filter
(B-2A, Nicon Co., Ltd; excitation wavelength 450–
490 nm). At this time, the use of an antifade reagent was es-
sential to prevent photobleaching (fading of fluorescence).
Figure 2 shows photographs of plant pulvini, which contains
a motor cell, under a fluorescence microscope. No stain was
observed in the control section, which was treated with an
aqueous solution containing no fluorescence-labeled probe
(upper center). Red stains seen in the fluorescence images
are due to the porphyrin in the plant tissue. The staining pat-
tern for the fluorescence of the biologically active probe 2
was observed on the surface of the motor cell (upper right).
We also carried out fluorescence studies of the interaction
Synthesis of the biologically inactive probe 31 is shown
in Scheme 6. Glycosidation of 7,8-dihydro aglycon 25 and
glucopyranosyl bromide 28 via Koenigs–Knorr reaction
afforded glycoside 29. Acetyl groups of glycoside 29 were
removed with KCN to give triol 30 as a 4:1 mixture of C4
stereoisomers. At this stage, epimerization of the C4 stereo-
center was observed as in the case of 23, which is the inter-
mediate of the biologically active probe 2. Deprotection
of the Fmoc group and introduction of AMCA-X gave fluo-
rescence-labeled probe 31 as a 4:1 diastereomeric mixture.
Diastereomerically pure 31 was obtained after purification
by HPLC (COSMOSIL 5C18-AR, 40% CH3CN aq, 260 nm).
˚
Scheme 6. (i) 28, AgOTf, Ag2CO3, MS4A, CH2Cl2, 48%; (ii) KCN, MeOH
61%; (iii) piperidine, DMF, 0 ꢀC; (iv) AMCA-X, SE, DMF, 0 ꢀC, 62% (two
steps); (v) HPLC separation.

N. Kato et al. / Tetrahedron 62 (2006) 7307–7318
7311
Figure 2. Fluorescence study using plant pulvini containing motor cell with fluorescence-labeled probes; upper left: Nomarskii image of plant section, upper
center: fluorescence image of control section, upper right: fluorescence image of a section treated with 2, lower left: fluorescence image of a section treated with
epi-2, lower center: fluorescence image of a section treated with 31, lower right: fluorescence image of a section treated with 2 in the presence of 100 molar
excess of phyllanthurinolactone 1.
between biologically inactive probes (epi-2 and 31) and the
plant motor cell. Stains were not observed in the section
treated with epi-2 and 31 (lower left and lower center).
Thus, it was proved that biologically inactive probe com-
pounds cannot bind to the plant motor cell. Also, binding
of probe 2 was inhibited by the coexistence of 100-fold con-
centration of the natural product 1. When the section was
treated by 1ꢁ10ꢂ4 M of 2 together with 1ꢁ10ꢂ2 M of 1,
no staining was observed in the plant section (lower right).
Results of the structure–activity relationship were consistent
with that of binding experiments. Also, it was clearly shown
that the binding of biologically active fluorescence-labeled
probe 2 with a motor cell is due to the specific binding of
the aglycon moiety, which is the active site of this molecule,
and is not a nonspecific binding due to the hydrophobicity of
the fluorescence dye group (AMCA group). These results
strongly suggested that some receptor for 1 exists in the
motor cell, which plays a central role in the plant leaf-
movement,18 as with leaf-opening substances.4 Along with
the previous result,6,7 some properties were revealed in a
receptor molecule of 1, that is, this receptor recognizes the
precise structure of the aglycon moiety, whereas it does not
recognize the sugar moiety at all.
We examined the genus-specificity and the bioactivity of
probe 2. Probe 2 did not show leaf-closing activity against
the leaves of C. mimosoides, Mimosa pudica, and Leucaena
leucocephala at 1ꢁ10ꢂ4 M. From these results, the binding
of probe 2 is expected to be specific to the section of plants
belonging to the genus Phyllanthus and no binding would be
observed in the experiment using the section of other plants.
Then, we used probe 2 for the binding experiment with the
sections of M. pudica, C. mimosoides, and L. leucocephala.
The binding experiments were carried out according to the
same method used in the case of P. urinaria; however, these
sections gave no staining pattern resulting from 2 (Fig. 3).
These results showed that the binding of probe 2 with a motor
cell is specific to the genus Phyllanthus and suggested that
a genus-specific receptor molecule for the leaf-movement
factor, which is located on a motor cell would be involved
in nyctinasty.
3. Conclusion
The binding of probes can be strongly correlated with leaf-
closing activity. Biologically inactive epi-2 and 31 did not
bind to a motor cell at all. Additionally, the staining pattern
resulting from the binding of probe 2 disappeared by the co-
existence of an excess amount of the natural product 1.
These results strongly suggested that some receptors for 1,
which specifically recognize the stereochemistry of aglycon
of 1 would be involved in the leaf-closing movement of
P. urinaria. Moreover, sinceprobe2didnotshowleaf-closing
activity against plants belonging to other genus, it was
revealed that the binding of a leaf-closing substance with a
motor cell is specific to the plant genus as well as the leaf-
opening substance and suggested that the genus-specific
receptor for the leaf-closing substance in a motor cell would
be involved in nyctinasty. In conclusion, we have succeeded
invisualization of the target cell of the leaf-closing substance
in the plant body by using the biologically active probe 2
2.5. Specific binding ability of fluorescence-labeled
probe compound to P. urinaria L.
From our previous studies, it was revealed that each nycti-
nastic plant has a different leaf-movement factor whose bio-
activity is specific to the plant genus.19 Recently, we proved
that the leaf-opening substances do not bind with motor cells
of plants belonging to other genus by using the fluorescence-
labeled probes based on the leaf-opening substances of
Cassia mimosoides L.4 and Albizzia julibrissin Durazz.20
Thus, fluorescence-labeled probe 2 is expected to show spe-
cific leaf-closing activity to genus Phyllanthus, and not to be
effective for other plants as well as leaf-opening substances.

7312
N. Kato et al. / Tetrahedron 62 (2006) 7307–7318
Figure 3. Photographs of plant sections in the binding experiments, which show specific binding of probe 2 with the motor cells of Phyllanthus plants (upper:
Nomarski image of the plant section, lower: fluorescent image of the plant section treated with probe 2.
and inactive probes epi-2 and 31, and revealed that the bind-
ing between a leaf-closing substance and its receptor in a
motor cell is genus-specific. Some receptor for 1 would be
involved in the leaf-closing movement in P. urinaria L. To
reveal a receptor protein of 1, the synthesis of photoaffinity
labeling probes based on 1 is now in progress.
was slowly added TBSOTf (72.4 ml, 320 mmol) at 0 ꢀC un-
der Ar atmosphere. After stirring for 30 min at 0 ꢀC, to the
reaction mixture was added H2O (2 ml). The aqueous layer
was extracted with CHCl3. The combined organic layer
was washed with brine, dried over Na2SO4, and concentrated
in vacuo. The residue was purified by silica gel column chro-
matography (hexane/AcOEt ¼ 2:1) to afford a TBS ether
(110 mg, 99%) as a colorless oil: [a]2D1 +75.5 (c 1.00,
4. Experimental
MeOH). IR (film) n 2856, 2835, 1612, 1514 cmꢂ1 1H
.
NMR (300 MHz, CDCl3, rt) d 7.25 (2H, d, J¼8.6 Hz),
6.90 (2H, d, J¼8.6 Hz), 4.60 (1H, s), 4.55 (2H, s), 3.82
(3H, s), 3.62 (1H, ddd, J¼4.0, 8.5, 11.0 Hz), 3.50 (1H, dd,
J¼2.2, 10.1 Hz), 3.45 (1H, d, J¼2.2 Hz), 3.40 (3H, s),
3.20 (1H, dd, J¼8.5, 10.1 Hz), 1.90 (1H, ddd, J¼3.6, 4.0,
13.2 Hz), 1.65 (1H, ddd, J¼3.0, 11.0, 13.2 Hz), 0.95 (9H,
s), 0.15 (3H, s), 0.12 (3H, s). 13C NMR (75 MHz, CDCl3,
rt) d 159.2, 130.1, 129.1, 113.8, 98.5, 74.6, 73.2, 70.7,
67.6, 55.2, 54.9, 33.3, 25.7, 7.7, ꢂ4.1, ꢂ4.6. ESI-HRMS
(positive-ion) calcd for C21H35IO5SiNa: (M+Na)+
545.1196; found: 545.1191.
4.1. General procedures
NMR spectra were recorded on a Jeol JNM-A600 spectro-
meter [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 spec-
trometer [1H (270 MHz) and 13C (67.5 MHz)] using TMS
in CDCl3, CD2HOD in CD3OD (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
FABMS and HR-FABMS spectra were recorded on a Jeol
JMS-700 or JMS-SX102 spectrometer, using glycerol or
m-nitrobenzylalcohol as a matrix. The ESI-HRMS spectra
were recorded on a Bruker APEX-III spectrometer. The IR
spectra were recorded on a JASCO FT/IR-410 spectrometer.
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 (f20ꢁ250 mm) (Na-
kalai 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 d.) before use. Silica gel column
chromatography was performed on silica gel 60 K070 (Ka-
tayama Chemical Co., Ltd) or silica gel 60N (Kanto Chemi-
cal Co., Ltd). Reversed-phase open-column chromatography
was performed on Cosmosil 75C18-OPN (Nakalai Tesque
Co., Ltd). TLC was performed on silica gel F254 (0.25 or
0.5 mm, MERCK) or RP-18F254S (0.25 mm, MERCK).
To a solution of the above TBS ether (55.9 mg, 107 mmol) in
THF (1.0 ml) was added t-BuOK (39.3 mg, 321 mmol) at
room temperature under Ar atmosphere. After stirring for
15 h, to the reaction mixture was added 1 N HCl (2 ml).
The aqueous layer was extracted with AcOEt. The combined
organic layer was washed with satd NaHCO3 aq and brine,
dried over Na2SO4, and concentrated in vacuo. The residue
was purified by silica gel column chromatography (hex-
ane/AcOEt ¼ 2:1) to afford 9 (70.8 mg, 99%) as a colorless
oil: [a]2D2 +11.8 (c 0.42, MeOH). IR (film) n 2832, 2858,
1
2837, 1660, 1612 cmꢂ1. H NMR (300 MHz, CDCl3, rt)
d 7.25 (2H, d, J¼8.6 Hz), 6.90 (2H, d, J¼8.6 Hz), 4.68
(1H, s), 4.70 (1H, d, J¼1.5 Hz), 4.62 (1H, d, J¼11.2 Hz),
4.59 (1H, d, J¼11.2 Hz), 4.55 (1H, d, J¼1.5 Hz), 4.59
(1H, d, J¼11.2 Hz), 4.41 (1H, m), 3.82 (3H, s), 3.62 (1H,
s), 3.45 (3H, s), 2.05 (1H, ddd, J¼2.9, 4.0, 13.2 Hz), 1.85
(1H, ddd, J¼2.9, 11.4, 13.2 Hz), 0.95 (9H, s), 0.15 (3H, s),
0.12 (3H, s). 13C NMR (75 MHz, CDCl3, rt) d 159.2,
159.0, 130.0, 129.3, 113.9, 100.4, 94.0, 74.5, 70.9, 64.2,
55.2, 55.0, 34.0, 25.7, 25.6, 25.6, ꢂ4.9, ꢂ5.0. ESI-HRMS
(positive-ion) calcd for C21H34O5SiNa: (M+Na)+
417.2073; found: 417.2068.
4.1.1. tert-Butyl-[6-methoxy-5-(4-methoxy-benzyloxy)-2-
methylene-tetrahydro-pyran-3-yloxy]-dimethyl-silane 9.
To a solution of 8 (86.9 mg, 213 mmol) in CH2Cl2 (2.4 ml)

N. Kato et al. / Tetrahedron 62 (2006) 7307–7318
7313
4.1.2. 2-(tert-Butyl-dimethyl-silanyloxy)-5-hydroxy-4-(4-
methoxy-benzyloxy)-cyclohexanone 10. To a solution of
9 (42.4 mg, 108 mmol) in acetone/H2O (3:1, 600 ml) was
slowly added Hg(OCOCF3)2 (2.3 mg, 5.4 mmol) at 0 ꢀC un-
der Ar atmosphere. After stirring for 9 h at 0 ꢀC, the reaction
mixture was diluted with AcOEt and washed with 10% KI
aq, 20% Na2S2O3 aq, and brine. The organic layer was dried
over Na2SO4, and concentrated in vacuo. The residue was
purified by silica gel column chromatography (hexane/
AcOEt ¼ 1:1) to afford 10 (28.8 mg, 70%) as a colorless
oil: [a]1D9 +27.0 (c 1.00, MeOH). IR (film) n 3422, 2856,
4.1.5. (Diethoxy-phosphoryl)-acetic acid 5-(4-methoxy-
benzyloxy)-2-oxo-cyclohex-3-enyl ester 6. To a solution
of 7 (67.2 mg, 271 mmol) in benzene (20 ml) were slowly
added DEAD (40% toluene solution, 1.0 ml, 2.17 mmol),
15 (662 mg, 2.17 mmol), and 13 (425 mg, 2.17 mmol) at
0 ꢀC under Ar atmosphere. After stirring for 1.5 h at room
temperature, the reaction mixture was diluted with AcOEt
and washed with 0.5 N HCl, satd NaHCO3 aq, and brine.
The organic layer was dried over Na2SO4, and concentrated
in vacuo. The residue was purified by silica gel column chro-
matography (hexane/AcOEt ¼ 1:5) to afford 6 (46.3 mg,
40%. R:S ¼ 10:1) as a colorless oil: [a]2D0 ꢂ6.8 (c 1.00,
1H NMR (270 MHz, CDCl3, rt) d 7.28 (2H, d, J¼8.2 Hz),
6.97 (1H, d, J¼10.6 Hz), 6.91 (2H, d, J¼8.2 Hz), 6.06
(1H, d, J¼10.6 Hz), 5.31 (1H, dd, J¼5.0, 13.7 Hz), 4.62
(1H, d, J¼11.5 Hz), 4.56 (1H, d, J¼11.5 Hz), 4.43 (1H,
m), 4.21 (2H, q, J¼6.9 Hz), 4.18 (2H, q, J¼6.9 Hz), 3.82
(3H, s), 3.14 (1H, d, J¼5.0 Hz), 3.06 (1H, d, J¼5.0 Hz),
2.75–2.67 (1H, m), 2.17 (1H, dt, J¼8.6, 11.2 Hz), 1.36
(6H, t, J¼6.9 Hz). 13C NMR (75 MHz, CDCl3, rt) d 192.2,
164.9, 159.4, 151.8, 129.4, 129.0, 127.4, 113.6, 72.3, 72.2,
70.9, 62.9, 55.3, 36.0, 34.0. ESI-HRMS (positive-ion) calcd
for C20H28O8P: (M+H)+ 427.1522; found: 427.1540.
1
1719 cmꢂ1. H NMR (300 MHz, CDCl3, rt) d 7.25 (2H,
d, J¼8.6 Hz), 6.90 (2H, d, J¼8.6 Hz), 4.65 (1H, d,
J¼11.5 Hz), 4.57 (1H, d, J¼11.5 Hz), 4.27 (1H, ddd,
J¼1.0, 4.8, 7.7 Hz), 4.09 (1H, m), 3.90 (3H, s), 2.76 (1H,
dd, J¼4.3, 13.8 Hz), 2.65 (1H, dd, J¼7.0, 13.8 Hz), 2.25
(1H, m), 1.95 (1H, m), 0.95 (9H, s), 0.15 (3H, s), 0.12
(3H, s). 13C NMR (75 MHz, CDCl3, rt) d 129.4, 114.0,
73.6, 71.9, 71.3, 55.3, 43.3, 35.0, 25.7, 25.7, 18.2, ꢂ4.8,
ꢂ5.3. ESI-HRMS (positive-ion) calcd for C20H32O5SiNa:
(M+Na)+ 403.1917; found: 403.1911.
CHCl3). IR (film) n 1747, 1703, 1612, 1514, 1250 cmꢂ1
.
4.1.3. 6-(tert-Butyl-dimethyl-silanyloxy)-4-(4-methoxy-
benzyloxy)-cyclohex-2-enone 11. To a solution of 10
(5.8 mg, 15.3 mmol) and Et3N (21.3 ml, 153 mmol) in
CH2Cl2 (1.0 ml) was slowly added MsCl (3.3 ml,
42.8 mmol) at 0 ꢀC under Ar atmosphere. After stirring for
1 h at room temperature, the reaction mixture was diluted
with CHCl3 and washed with 1 N H2SO4, satd NaHCO3
aq, and brine. The organic layer was dried over Na2SO4,
and concentrated in vacuo. The residue was purified by silica
gel column chromatography (hexane/AcOEt ¼ 2:1) to afford
11 (5.5 mg, quant.) as a colorless oil: [a]1D9 ꢂ127.5 (c 1.00,
4.1.6. 6-(4-Methoxy-benzyloxy)-7,7a-dihydro-6H-benzo-
furan-2-one 12. To a solution of 6 (4.7 mg, 11.0 mmol) in
THF (500 ml) was added t-BuOK (1.2 mg, 11.0 mmol) at
0 ꢀC under Ar atmosphere. After stirring for 30 min at
0 ꢀC, the reaction mixture was diluted with AcOEt and
washed with 1 N HCl, satd NaHCO3 aq, and brine. The
organic layer was dried over Na2SO4, and concentrated in
vacuo. The residue was purified by silica gel column
chromatography (hexane/AcOEt ¼ 1:5) to afford 12
(1.9 mg, 63%. R:S ¼ 10:1) as a colorless oil: [a]2D4 ꢂ1.1
(c 1.00, CHCl3). IR (film) n 1741, 1641, 1612, 1585,
MeOH). IR (film) n 3483, 2941, 2116, 1719 cmꢂ1 1H
.
NMR (300 MHz, CDCl3, rt) d 7.25 (2H, d, J¼8.6 Hz), 6.90
(2H, dd, J¼2.0, 8.6 Hz), 6.89 (1H, d, J¼10.3 Hz), 5.97
(1H, d, J¼10.3 Hz), 4.65 (1H, d, J¼11.5 Hz), 4.57 (1H, d,
J¼11.5 Hz), 2.25 (2H, m), 0.95 (9H, s), 0.15 (3H, s), 0.12
(3H, s). 13C NMR (75 MHz, CDCl3, rt) d 197.5, 159.4,
147.8, 129.8, 129.4, 128.1, 113.9, 71.2, 71.0, 70.2, 55.2,
37.6, 25.7, ꢂ4.7, ꢂ5.4. ESI-HRMS (positive-ion) calcd for
C20H30O4SiNa: (M+Na)+ 385.1811; found: 385.1806.
1
1514 cmꢂ1. H NMR (270 MHz, CDCl3, rt) d 7.28 (2H, d,
J¼8.6 Hz), 6.90 (2H, d, J¼8.6 Hz), 6.57 (1H, dd, J¼2.3,
9.9 Hz), 6.34 (1H, d, J¼9.9 Hz), 5.80 (1H, s), 4.82 (1H,
dd, J¼5.0, 13.5 Hz), 4.61 (1H, d, J¼11.5 Hz), 4.55 (1H, d,
J¼11.5 Hz), 4.35–4.31 (1H, m), 3.82 (3H, s), 2.94 (1H, dt,
J¼5.0, 10.9 Hz), 1.71 (1H, dt, J¼10.9, 13.5 Hz). 13C NMR
(75 MHz, CDCl3, rt) d 173.1, 162.8, 159.4, 141.5, 129.4,
129.3, 120.2, 114.0, 111.4, 78.0, 72.5, 70.8, 55.3, 37.0.
ESI-HRMS (positive-ion) calcd for C16H17O4: (M+H)+
273.1127; found: 273.1147.
4.1.4. 6-Hydroxy-4-(4-methoxy-benzyloxy)-cyclohex-2-
enone 7. To a solution of 11 (10.1 mg, 27.9 mmol) and
AcOH (4.8 ml, 83.7 mmol) in THF (500 ml) was slowly added
TBAF (1 M in THF, 83.7 ml, 83.7 mmol) at 0 ꢀC under Ar at-
mosphere. After stirring for 46 h at room temperature, the re-
action mixture was diluted with AcOEt and washed with
H2O and brine. The organic layer was dried over Na2SO4,
and concentrated in vacuo. The residue was purified by silica
gel column chromatography (hexane/AcOEt ¼ 1:1) to afford
7 (6.9 mg, quant.) as a colorless oil: [a]1D9 ꢂ173.1 (c 1.00,
4.1.7. Aglycon 5. To a solution of 12 (10.1 mg, 37.1 mmol) in
CH2Cl2 (500 ml) were added H2O (25 ml) and DDQ
(12.6 mg, 55.7 mmol) at 0 ꢀC under Ar atmosphere. After
stirring for 4.5 h at 0 ꢀC, to the reaction mixture was added
satd NaHCO3 aq. The aqueous layer was extracted with
AcOEt. The combined organic layer was washed with brine,
dried over Na2SO4, and concentrated in vacuo. The residue
was purified by silica gel column chromatography (hex-
ane/AcOEt ¼ 1:5) to afford 5 (4.0 mg, 71%) as a white pow-
der: [a]1D7 ꢂ13.6 (c 0.70, CHCl3). IR (film) n 3441, 1732,
MeOH). IR (film) n 3449, 2837, 1693, 1612, 1514 cmꢂ1
.
1H NMR (300 MHz, CDCl3, rt) d 7.25 (2H, d, J¼8.6 Hz),
6.90 (2H, d, J¼8.6 Hz), 6.89 (1H, dd, J¼5.5, 12.3 Hz),
4.53 (1H, d, J¼11.3 Hz), 4.34 (1H, ddd, J¼2.0, 4.0,
5.1 Hz), 3.90 (3H, s), 2.63 (1H, ddd, J¼2.0, 5.5, 13.5 Hz),
1.98 (1H, ddd, J¼4.0, 12.3, 13.5 Hz). 13C NMR (75 MHz,
CDCl3, rt) d 200.3, 159.4, 146.7, 129.6, 129.4, 127.8,
113.9, 71.5, 69.7, 68.9, 55.2, 35.0. ESI-HRMS (positive-
ion) calcd for C14H16O4Na: (M+Na)+ 271.0946; found:
271.0941.
1
1639, 1105 cmꢂ1. H NMR (300 MHz, CDCl3, rt) d 5.76
(1H, s), 6.57 (1H, dd, J¼1.8, 9.9 Hz), 6.36 (1H, d,
J¼9.9 Hz), 5.81 (1H, s), 4.90 (1H, dd, J¼4.4, 13.2 Hz),
4.70–4.58 (1H, m), 3.40–3.50 (1H, br s, OH), 2.93 (1H, td,
J¼5.2, 10.8 Hz), 1.68 (1H, q, J¼11.0 Hz). 13C NMR
(75 MHz, CDCl3, rt) d 173.8, 163.5, 144.1, 119.6, 111.0,

7314
N. Kato et al. / Tetrahedron 62 (2006) 7307–7318
78.3, 66.5, 39.7. ESI-HRMS (positive-ion) calcd for
C8H8O3Na: (M+Na)+ 175.0371; found: 175.0365.
5.50 (1H, dd, J¼2.2, 55.5 Hz), 4.30 (2H, d, J¼7.0 Hz),
4.21 (1H, t, J¼7.0 Hz), 3.82 (1H, t, J¼6.3 Hz), 3.70–3.50
(2H, m), 3.50 (1H, dd, J¼7.7, 12.9 Hz), 3.20 (2H, m). 13C
NMR (75 MHz, DMSO-d6, rt) d 156.3, 143.9, 140.8,
127.7, 127.1, 125.2, 120.1, 109.7, 106.8, 71.6, 68.9, 68.7,
65.4, 46.7, 41.1. ESI-HRMS (positive-ion) calcd for
C21H22FNO6Na: (M+Na)+ 426.1329; found: 426.1323.
b-anomer: [a]2D2 ꢂ43.0 (c 0.10, MeOH). IR (film) n 3165,
4.1.8. Acetic acid 4,5-diacetoxy-6-azidomethyl-2-fluoro-
tetrahydro-pyran-3-yl ester 17. To a solution of 16
(378 mg, 1.01 mmol) in DMF (10 ml) was slowly added
H2NNH2/AcOH (1:1, 41 ml) at 0 ꢀC under Ar atmosphere.
After stirring for 1.5 h at 0 ꢀC, the reaction mixture was di-
luted with CHCl3 and washed with 1 N HCl, satd NaHCO3
aq, and brine. The organic layer was dried over Na2SO4,
and concentrated in vacuo to afford a crude mixture of
anomeric galactopyranoses (268 mg) as a colorless oil. To
a solution of the mixture (248 mg) in CH2Cl2 (7.5 ml) was
added DAST (98.2 ml, 750 mmol) at 0 ꢀꢀC. After stirring
for 30 min at 0 ꢀC, the reaction mixture was diluted with
CHCl3 and washed with H2O. The organic layer was dried
over Na2SO4, and concentrated in vacuo. The residue was
purified by silica gel column chromatography (hexane/
AcOEt ¼ 1:1) to afford 17 (250 mg, 80% in two steps) as
a white powder. a-anomer: [a]2D0 +90.3 (c 1.00, MeOH).
1643 cmꢂ1 1H NMR (300 MHz, DMSO-d6, rt) d 7.85
.
(2H, d, J¼7.8 Hz), 7.68 (2H, d, J¼7.3 Hz), 7.40 (4H, m),
4.90 (1H, dd, J¼6.9, 54.7 Hz), 4.30 (2H, d, J¼7.0 Hz),
4.21 (1H, t, J¼7.0 Hz), 3.60–3.10 (6H, m). 13C NMR
(75 MHz, DMSO-d6, rt) d 158.5, 145.5, 142.5, 129.5,
128.9, 126.8, 121.7, 113.1, 110.4, 80.0, 74.2, 74.2, 73.1,
73.0, 71.7, 71.4, 69.2, 66.7, 47.7, 41.9. ESI-HRMS (posi-
tive-ion) calcd for C21H22FNO6Na: (M+Na)+ 426.1329:
found: 426.1323.
4.1.10. Acetic acid 4,5-diacetoxy-6-(tert-butoxycarbonyl-
amino-methyl)-2-fluoro-tetrahydro-pyran-3-yl ester 20.
To a solution of 17 (24.0 mg, 72.1 mmol) in MeOH
(700 ml) was slowly added MeONa (12.9 mg, 238 mmol) at
0 ꢀC under Ar atmosphere. After stirring for 30 min at
0 ꢀC, Amberlite IR-120B (H+) was added to this solution
for neutralization. After filtration, the filtrate was concen-
trated in vacuo to afford a crude mixture. To a solution of
the mixture in MeOH (700 ml) was added palladium on acti-
vated carbon (8.7 mg). Hydrogen was admitted via a balloon
and the reaction mixture was stirred for 30 min and the cat-
alyst removed by filtration. The filtrate was concentrated in
vacuo to afford a crude mixture. To a solution of the mixture
and triethylamine (10.1 ml, 71.9 mmol) in MeOH (1.0 ml)
was added Boc2O (16.6 ml, 71.9 mmol) at 0 ꢀC. After stirring
for 2 h at 0 ꢀC, the reaction mixture was concentrated in
vacuo to afford a crude mixture. To a solution of the mixture
in pyridine (1.0 ml) was added Ac2O (0.5 ml) at room tempera-
ture. After stirring for 7 h, the reaction mixture was diluted with
AcOEt and washed with 1 N HCl, satd NaHCO3 aq, and
brine. The organic layer was dried over Na2SO4, and concen-
trated in vacuo. The residue was purified by silica gel column
chromatography (hexane/AcOEt ¼ 1:1) to afford 20
(19.1 mg, 65% in four steps) as a colorless oil: [a]1D8 +51.4
(c 0.40, CHCl3). IR (film) n 2108, 1751, 1371, 1221, 1067,
1
IR (film) n 2941, 2116, 1718 cmꢂ1. H NMR (300 MHz,
CDCl3, rt) d 5.80 (1H, dd, J¼2.2, 53.3 Hz), 5.50 (1H, d,
J¼2.5 Hz), 5.35 (1H, dd, J¼2.5, 11.0 Hz), 5.20 (1H, ddd,
J¼2.2, 11.0, 23.4 Hz), 4.30 (1H, dd, J¼5.1, 7.7 Hz), 3.50
(1H, dd, J¼7.7, 12.9 Hz), 3.25 (1H, d, J¼5.1, 12.9 Hz),
2.20 (3H, s), 2.18 (1H, s), 2.00 (3H, s). 13C NMR
(75 MHz, CDCl3, rt) d 171.5, 171.2, 171.1, 106.5, 103.4,
70.6, 70.6, 68.4, 67.9, 67.6, 67.4, 50.6, 20.8, 20.7. ESI-
HRMS (positive-ion) calcd for C12H16FN3O7Na: (M+Na)+
356.0870; found: 356.0864. b-anomer: [a]2D0 +28.6 (c 1.00,
MeOH). IR (film) n 2837, 2108, 1751 cmꢂ1 1H NMR
.
(300 MHz, CDCl3, rt) d 5.50 (1H, d, J¼3.3, Hz), 5.33 (1H,
ddd, J¼6.9, 9.9, 10.6 Hz), 5.30 (1H, dd, J¼6.9, 52.5 Hz),
5.06 (1H, dd, J¼3.3, 9.9 Hz), 3.95 (1H, dd, J¼4.8,
7.6 Hz), 3.60 (1H, dd, J¼7.6, 12.8 Hz), 3.32 (1H, d,
J¼4.8, 12.8 Hz), 2.20 (3H, s), 2.10 (3H, s), 2.00 (3H, s).
13C NMR (75 MHz, CDCl3, rt) d 171.2, 171.1, 170.5,
109.1, 106.2, 73.0, 72.9, 70.3, 70.1, 69.2, 68.9, 67.5, 50.5,
20.7, 20.6, 20.6. ESI-HRMS (positive-ion) calcd for
C12H16FN3O7Na: (M+Na)+ 356.0870; found: 356.0864.
4.1.9. (6-Fluoro-3,4,5-trihydroxy-tetrahydro-pyran-2-yl-
methyl)-carbamic acid 9H-fluoren-9-ylmethyl ester 19.
To a solution of 17 (59.0 mg, 177 mmol) in MeOH (1.8 ml)
was slowly added MeONa (31.6 mg, 585 mmol) at 0 ꢀC
under Ar atmosphere. After stirring for 30 min at 0 ꢀC,
Amberlite IR-120B (H+) was added to this solution for
neutralization. After filtration, the filtrate was concentrated
in vacuo to afford a crude mixture. To a solution of the mix-
ture in MeOH (1.5 ml) was added palladium on activated
carbon (17.4 mg). Hydrogen was admitted via a balloon
and the reaction mixture was stirred for 1.5 h and the catalyst
removed by filtration. The filtrate was concentrated in vacuo
to afford a crude mixture. To a solution of the mixture
and diidopropylethylamine (30.8 ml, 177 mmol) in THF
(8.0 ml) was added FmocCl (45.8 mg, 177 mmol) at room
temperature. After stirring for 43 h, the reaction mixture
was concentrated in vacuo. The residue was purified by silica
gel column chromatography (CHCl3/MeOH¼5:1) to afford
19 (22.7 mg, 32% in three steps) as a white powder.
a-anomer: [a]2D3 +88.4 (c 0.10, MeOH). IR (film) n 3436,
1
1020 cmꢂ1. H NMR (300 MHz, CDCl3, rt) d 5.78 (1H,
dd, J¼2.2, 53.4 Hz), 5.43 (1H, d, J¼2.7 Hz), 5.36 (1H, dd,
J¼2.7, 11.0 Hz), 5.19 (1H, ddd, J¼2.2, 11.0, 23.5 Hz),
4.20 (1H, t, J¼6.6 Hz), 3.28 (2H, m), 2.19 (3H, s), 2.15
(3H, s), 2.02 (3H, s), 1.45 (9H, s). 13C NMR (75 MHz,
CDCl3, rt) d 170.3, 169.9, 155.6, 104.3 (d, JC–F¼113 Hz),
80.0, 70.1, 67.9, 67.7, 67.4, 67.1, 39.9, 28.3, 20.6. ESI-
HRMS (positive-ion) calcd for C17H26FNO9Na: (M+Na)+
430.1489; found: 430.1484.
4.1.11. Acetic acid 3,5-diacetoxy-2-[(9H-fluoren-9-yl-
methoxycarbonylamino)-methyl]-6-fluoro-tetrahydro-
pyran-4-yl ester 21. To a solution of 19 (22.7 mg, 56.3 mmol)
in pyridine (1.0 ml) was added Ac2O (0.5 ml) at room tem-
perature. After stirring for 7 h, the reaction mixture was di-
luted with AcOEt and washed with 1 N HCl, satd NaHCO3
aq. and brine. The organic layer was dried over Na2SO4, and
concentrated in vacuo. The residue was purified by silica gel
column chromatography (hexane/AcOEt ¼ 1:1) to afford 21
(25.9 mg, 87%) as a colorless oil. a-anomer: [a]2D0 +39.5
1697 cmꢂ1
.
1H NMR (300 MHz, DMSO-d6, rt) d 7.85
(2H, d, J¼7.8 Hz), 7.68 (2H, d, J¼7.3 Hz), 7.40 (4H, m),

N. Kato et al. / Tetrahedron 62 (2006) 7307–7318
7315
(c 0.10, MeOH). IR (film) n 3366, 2961, 1701 cmꢂ1. H
NMR (300 MHz, CDCl3, rt) d 7.78 (2H, d, J¼7.3 Hz),
7.58 (2H, d, J¼7.3 Hz), 7.35 (4H, m), 5.80 (1H, dd,
J¼2.5, 53.9 Hz), 5.50 (1H, d, J¼2.5 Hz), 5.35 (1H, dd,
J¼2.5, 11.0 Hz), 5.18 (1H, ddd, J¼2.5, 11.0, 23.5 Hz),
4.43 (2H, d, J¼7.0 Hz), 4.23 (2H, m), 3.34 (2H, t,
J¼6.6 Hz), 2.20 (3H, s), 2.15 (1H, s), 2.03 (3H, s). 13C
NMR (75 MHz, CDCl3, rt) d 170.3, 169.8, 156.2, 143.7,
141.3, 127.7, 127.1, 125.0, 120.0, 105.7, 102.7, 69.9, 68.1,
67.6, 67.3, 67.0, 47.1, 40.5, 20.6. ESI-HRMS (positive-
ion) calcd for C27H28FNO9Na: (M+Na)+ 552.1646; found:
552.1639. b-anomer: [a]1D7 +7.4 (c 0.49, MeOH). IR (film)
mixture was concentrated in vacuo. The residue was purified
by silica gel column chromatography (CHCl3/MeOH ¼ 1:1)
to afford 24 (0.7 mg, 47%, R:S¼4:1) as a colorless oil:
[a]2D0 ꢂ7.1 (c 0.10, MeOH). IR (film) n 3533, 3308, 1719,
1
1686, 1638, 1535, 1448, 1263, 1078 cmꢂ1 1H NMR
.
(300 MHz, CD3OD, rt) d 7.77 (2H, d, J¼7.3 Hz), 7.66
(2H, t, J¼7.3 Hz), 7.37 (2H, t, J¼7.3 Hz), 7.30 (2H, t,
J¼7.3 Hz), 6.63 (1H, dd, J¼2.0, 10.1 Hz), 6.46 (1H, d,
J¼10.1 Hz), 5.83 (1H, s), 4.80 (1H, dd, J¼4.5, 13.4 Hz),
4.57–4.62 (1H, m), 4.44 (1H, dd, J¼6.5, 10.5 Hz), 4.39
(1H, dd, J¼6.5, 10.5 Hz), 4.37 (1H, d, J¼7.0 Hz), 4.38
(2H, dd, J¼6.4, 10.4 Hz), 4.22 (1H, t, J¼6.4 Hz), 3.70
(1H, d, J¼2.2 Hz), 3.63 (1H, s), 3.52–3.44 (4H, m), 2.94
(1H, dt, J¼4.5, 11.4 Hz), 1.64 (1H, dt, J¼10.6, 13.2 Hz).
13C NMR (125 MHz, CD3OD, rt) d 175.8, 165.9, 159.1,
145.4, 145.3, 143.2, 142.7, 128.8, 128.2, 128.1, 121.1,
120.9, 120.9, 111.7, 104.8, 79.9, 75.3, 74.9, 74.8, 72.2,
70.5, 67.6, 49.6, 42.5, 40.1. ESI-HRMS (positive-ion) calcd
for C29H29NO9Na: (M+Na)+ 558.1740; found: 558.1735.
1
n 3437, 2963, 1637 cmꢂ1. H NMR (300 MHz, CDCl3, rt)
d 7.78 (2H, d, J¼7.3 Hz), 7.58 (2H, d, J¼7.3 Hz), 7.35
(4H, m), 5.40–5.13 (4H, m), 4.43 (2H, d, J¼6.6 Hz), 4.21
(1H, t, J¼6.6 Hz), 3.88 (1H, t, J¼6.6 Hz), 3.39 (2H, t,
J¼6.6 Hz), 2.20 (3H, s), 2.15 (1H, s), 2.03 (3H, s).
13C NMR (75 MHz, CDCl3, rt) d 172.0, 171.4, 171.1,
158.8, 145.4, 145.2, 142.6, 128.8, 128.2, 126.3, 126.2,
121.0, 109.9, 107.1, 79.5, 73.2, 73.1, 71.8, 71.7, 70.6,
70.3, 68.4, 67.9, 48.2, 41.2, 20.6, 20.5. ESI-HRMS (posi-
tive-ion) calcd for C27H28FNO9Na: (M+Na)+ 552.1646:
found: 552.1639.
4.1.14. Biologically active fluorescence-labeled probe 2.
To a solution of 24 (1.6 mg, 3.0 mmol) in DMF (300 ml)
was added piperidine (1.0 ml, 10 mmol) at 0 ꢀC under Ar at-
mosphere. After stirring for 4 h at 0 ꢀC, piperidine was re-
moved in vacuo. To the reaction mixture were added DMF
(200 ml) and AMCA-X, SE (1.4 mg, 3.3 mmol) at room tem-
perature under Ar atmosphere. After stirring for 2 h, the re-
action mixture was concentrated in vacuo. The residue was
purified by ODS TLC (RP-18W, H2O/MeOH ¼ 1:1) and
HPLC with COSMOSIL 5C18-AR column (f20.0ꢁ
250 mm, H2O/CH3CN ¼ 1:1) to afford 2 (0.9 mg, 48%) and
epi-2 (0.2 mg, 11%). Compound 2: white powder. [a]D18
4.1.12. Acetic acid 3,5-diacetoxy-2-[(9H-fluoren-9-yl-
methoxycarbonylamino)-methyl]-6-(2-oxo-2,6,7,7a-tetra-
hydro-benzofuran-6-yloxy)-tetrahydro-pyran-4-yl ester
23. A mixture of 5 (6.6 mg, 43.4 mmol), Cp2HfCl2 (32.9 mg,
86.8 mmol), AgClO4 (18.0 mg, 86.8 mmol) and dried molec-
˚
ular sieves 4A (5.0 mg) in anhydrous CH2Cl2 (500 ml) was
stirred at 0 ꢀC under Ar atmosphere for 1 h. Then a solution
of 4 (36.0 mg, 68.1 mmol) in anhydrous CH2Cl2 (300 ml) was
added to the stirred mixture. After stirring for 6 h, the reac-
tion mixture was diluted with CHCl3 and filtered through
Celite. The filtrate was washed with brine. The organic layer
was dried over Na2SO4, and concentrated in vacuo. The resi-
due was purified by silica gel column chromatography (tol-
uene/acetone ¼ 1:3) to afford 23 (15.0 mg, 52%, R:S ¼ 7:1) as
a colorless oil: [a]1D8 +12.6 (c 0.10, CHCl3). IR (film) n 1747,
+28.0 (c 1.00, MeOH). IR (film) n 3292, 2856, 1734 cmꢂ1
.
1H NMR (500 MHz, CD3OD, rt) d 7.48 (1H, d, J¼8.5 Hz),
6.64 (1H, dd, J¼2.5, 8.5 Hz), 6.63 (1H, dd, J¼3.0,
10.0 Hz), 6.50 (1H, d, J¼2.5 Hz), 6.47 (1H, d,
J¼10.0 Hz), 5.80 (1H, s), 5.00 (1H, ddd, J¼2.0, 5.0,
13.0 Hz), 4.69 (1H, m), 4.40 (1H, d, J¼7.5 Hz), 3.73 (1H,
dd, J¼1.0, 3.0 Hz), 3.60 (1H, ddd, J¼1.0, 5.5, 8.0 Hz),
3.54 (2H, s), 3.50 (1H, dd, J¼2.5, 7.0 Hz), 3.46 (1H, dd,
J¼7.5, 10.0 Hz), 3.45 (1H, dd, J¼5.5, 13.5 Hz), 3.36 (1H,
dd, J¼8.0, 13.5 Hz), 3.18 (2H, dt, J¼2.5, 7.0 Hz), 2.98
(1H, ddd, J¼5.0, 6.0, 10.5 Hz), 2.38 (3H, s), 2.22 (2H, t,
J¼7.5 Hz), 1.69 (1H, ddd, J¼10.5, 11.0, 13.0 Hz), 1.64
(2H, m), 1.52 (2H, m), 1.36 (2H, m). 13C NMR (125 MHz,
CD3OD, rt) d 176.7, 175.8, 172.9, 165.9, 164.8, 155.9,
153.9, 152.9, 143.7, 127.4, 121.1, 114.5, 113.2, 111.8,
111.7, 105.0, 100.5, 79.9, 75.7, 74.8, 74.4, 72.2, 70.6,
41.3, 40.3, 40.2, 37.0, 35.2, 30.0, 27.5, 26.6, 15.4. ESI-
HRMS (positive-ion) calcd for C32H39N3O11Na: (M+Na)+
664.2478; found: 664.2478. Compound epi-2: white powder.
[a]1D7 ꢂ37.3 (c 0.07, MeOH). IR (film) n 3348, 2926, 1741,
1
1221, 1155, 1070, 760 cmꢂ1. H NMR (600 MHz, CDCl3,
rt) d 7.77 (2H, d, J¼7.4 Hz), 7.59 (2H, d, J¼7.4 Hz), 7.41
(2H, t, J¼7.4 Hz), 7.33 (2H, t, J¼7.4 Hz), 6.59 (1H, d,
J¼10.0 Hz), 6.20 (1H, d, J¼10.0 Hz), 5.84 (1H, s), 5.35
(1H, d, J¼2.0 Hz), 5.21 (1H, dd, J¼7.8, 10.0 Hz), 5.12
(1H, t, J¼6.0 Hz), 5.03 (1H, dd, J¼2.0, 10.0 Hz), 4.83
(1H, dd, J¼4.5, 12.0 Hz), 4.62 (1H, d, J¼7.8 Hz), 4.59–
4.54 (1H, br s), 4.44 (2H, d, J¼6.6 Hz), 4.22 (1H, t,
J¼6.6 Hz), 3.78 (1H, t, J¼6.8 Hz), 3.46 (1H, td, J¼6.8,
14.0 Hz), 3.15 (1H, td, J¼6.8, 14.0 Hz), 2.94 (1H, td,
J¼4.5, 12.0 Hz), 2.20 (3H, s), 2.07 (3H, s), 2.02 (3H, s),
1.81 (1H, q, J¼12.0 Hz). 13C NMR (150 MHz, CDCl3, rt)
d 172.9, 171.0, 170.0, 169.3, 162.0, 156.4, 143.7, 141.3,
139.6, 127.8, 127.1, 125.0, 121.1, 120.0, 115.5, 100.8,
77.9, 74.1, 71.7, 70.8, 69.0, 67.8, 67.0, 47.2, 40.4,
38.3, 20.8, 20.6. ESI-HRMS (positive-ion) calcd for
C35H35NO12Na: (M+Na)+ 684.2057; found: 684.2051.
1639, 1601, 1556, 1051 cmꢂ1
.
1H NMR (300 MHz,
CD3OD, rt) d 7.49 (1H, d, J¼7.5 Hz), 6.71 (1H, d, J¼
8.0 Hz), 6.65 (1H, dd, J¼2.0, 7.5 Hz), 6.48 (1H, d,
J¼2.0 Hz), 6.45 (1H, dd, J¼4.0, 8.0 Hz), 5.85 (1H, d,
J¼1.5 Hz), 5.38 (1H, ddd, J¼1.5, 5.5, 11.5 Hz), 4.65 (1H,
m), 4.37 (1H, d, J¼6.0 Hz), 3.62 (1H, dd, J¼1.0, 4.0,
6.5 Hz), 3.52 (2H, s), 3.50 (1H, dd, J¼6.5, 8.5 Hz), 3.48
(1H, dd, J¼4.0, 12.0 Hz), 3.45 (1H, dd, J¼3.0, 8.5 Hz),
3.38 (1H, dd, J¼6.5, 12.0 Hz), 3.17 (1H, t, J¼6.0 Hz),
2.82 (1H, ddd, J¼2.0, 5.5, 11.5 Hz), 2.37 (3H, s), 2.22
(2H, t, J¼7.5 Hz), 1.73 (1H, dt, J¼3.0, 11.5 Hz), 1.62 (2H,
4.1.13. [3,4,5-Trihydroxy-6-(2-oxo-2,6,7,7a-tetrahydro-
benzofuran-6-yloxy)-tetrahydro-pyran-2-ylmethyl]-car-
bamic acid 9H-fluoren-9-ylmethyl ester 24. To a solution
of 23 (1.8 mg, 2.9 mmol) in MeOH (200 ml) was added
KCN (6 mM in MeOH, 200 ml, 1.2 mmol) at 0 ꢀC under Ar
atmosphere. After stirring for 5 h at 0 ꢀC, the reaction

7316
N. Kato et al. / Tetrahedron 62 (2006) 7307–7318
m), 1.51 (2H, m), 1.35 (2H, m). ESI-HRMS (positive-ion)
calcd for C32H39N3O11Na: (M+Na)+ 664.2478: found:
664.2478.
62.8, 42.0, 32.7, 24.8. ESI-HRMS (positive-ion) calcd for
C14H20O8Na: (M+Na)+ 339.1056; found: 339.1049.
4.1.17. Acetic acid 3,5-diacetoxy-2-bromo-6-[(9H-fluo-
ren-9-ylmethoxycarbonyl amino)-methyl]-tetrahydro-
pyran-4-yl ester 28. Synthesis of 33, 34, and 28 is shown
in Scheme 7.
4.1.15. 7,8-Dihydro aglycon 25. To a solution of 5 (6.2 mg,
41.0 mmol) in AcOEt (800 ml) was added Lindlar catalyst
(8.2 mg). Hydrogen was admitted via a balloon and the reac-
tion mixture was stirred for 15 min and the catalyst removed
by filtration. The filtrate was concentrated in vacuo. The resi-
due was purified by silica gel column chromatography
(CHCl3/MeOH ¼ 20:1) to afford 25 (4.3 mg, 68%) as a color-
less oil: [a]2D4 ꢂ52.1 (c 0.20, CHCl3). IR (film) n 3422, 1732,
1647, 1005 cmꢂ1. 1H NMR (300 MHz, CDCl3, rt) d 5.76 (1H,
s), 4.73 (1H, dd, J¼5.7, 10.8 Hz), 3.92 (1H, tt, J¼3.0,
10.8 Hz), 2.84–2.83 (1H, m), 2.79–2.71 (1H, m), 2.33 (1H,
ddd, J¼1.8, 5.7, 14.1 Hz), 2.23–2.19 (1H, m), 1.49–1.36
(1H, m), 1.36 (1H, dd, J¼11.4, 14.1 Hz). 13C NMR
(75 MHz, CDCl3, rt) d 173.2, 169.9, 113.4, 79.5, 66.9, 42.1,
34.7, 24.2. ESI-HRMS (positive-ion) calcd for C8H10O3Na:
(M+Na)+ 177.0528; found: 177.0521.
Scheme 7. (i) See Ref. 15; (ii) TESOTf, 2,6-lutidine, DMF, 92%; (iii) H2/
Pd–C, CH2Cl2; (iv) FmocCl, NaHCO3, H2O, 1,4-dioxane, 80% (two steps);
(v) TFA, THF, H2O; (vi) Ac2O, pyridine, 80% (two steps); (vii) BiBr3,
TMSBr, CH2Cl2.
4.1.16. 7,8-Dihydro phyllanthurinolactone 27. A mixture
of 25 (8.6 mg, 56.0 mmol), AgOTf (7.2 mg, 27.9 mmol),
Ag2CO3 (32.3 mg, 117 mmol) and dried molecular sieves
˚
4A (17.7 mg) in anhydrous CH2Cl2 (200 ml) was stirred at
4.1.17.1. Synthesis of 33. To a solution of 32 (340 mg,
1.66 mmol) and 2,6-lutidine (1.86 ml, 15.9 mmol) in DMF
(16 ml) was slowly added TESOTf (1.79 ml, 8.00 mmol)
at 0 ꢀC under Ar atmosphere. After stirring for 1 h at 0 ꢀC,
to the reaction mixture was added H2O (2 ml). The aqueous
layer was extracted with AcOEt. The combined organic
layer was washed with brine, dried over Na2SO4, and con-
centrated in vacuo. The residue was purified by silica gel col-
umn chromatography (hexane/AcOEt ¼ 9:1) to afford TES
ether (1.03 g, 92%, a:b ¼ 1:1) as a colorless oil: [a]D24
+15.3 (c 0.50, CHCl3). IR (film) n 2878, 2102, 1458, 1414,
0 ꢀC under Ar atmosphere for 1 h. Then a solution of 26
(22.7 mg, 55.0 mmol) in anhydrous CH2Cl2 (500 ml) was
added to the stirred mixture. After stirring for 2 h, the reac-
tion mixture was diluted with CHCl3 and filtered through
Celite. The filtrate was washed with satd NaHCO3 aq and
brine. The organic layer was dried over Na2SO4, and concen-
trated in vacuo. The residue was purified by silica gel column
chromatography (hexane/AcOEt ¼ 1:3) to afford a glycoside
(5.4 mg, 20%) as a colorless oil: [a]1D6 ꢂ15.3 (c 0.10,
CHCl3). IR (film) n 1751, 1225, 1038 cmꢂ1 1H NMR
.
1
(300 MHz, CDCl3, rt) d 5.79 (1H, s), 5.21 (1H, t,
J¼9.6 Hz), 5.08 (1H, t, J¼9.6 Hz), 4.95 (1H, dd, J¼8.1,
9.6 Hz), 4.74 (1H, dd, J¼6.3, 12.3 Hz), 4.65 (1H, d,
J¼8.1 Hz), 4.27 (1H, dd, J¼4.8, 12.0 Hz), 4.14 (1H, dd,
J¼2.7, 12.0 Hz), 3.88 (1H, tt, J¼3.6, 11.1 Hz), 3.71 (1H,
ddd, J¼2.7, 4.8, 9.6 Hz), 2.93–2.83 (2H, m), 2.36–2.19
(2H, m), 2.15 (3H, s), 2.09 (3H, s), 2.03 (3H, s) 2.01 (3H,
s), 1.53–1.38 (2H, m). 13C NMR (75 MHz, CDCl3, rt)
d 172.9, 170.6, 170.3, 169.3, 169.1, 169.0, 113.7, 99.8,
79.2, 74.5, 72.7, 71.9, 71.3, 68.4, 62.0, 40.3, 31.6, 23.9,
20.7, 20.6. ESI-HRMS (positive-ion) calcd for
C22H28O12Na: (M+Na)+ 507.1478; found: 507.1470.
1283, 1240 cmꢂ1. H NMR (300 MHz, CDCl3, rt) d 5.10
(1H, d, J¼2.7 Hz, a), 4.47 (1H, d, J¼6.5 Hz, b), 3.97 (1H,
dd, J¼2.0, 9.4 Hz, a), 3.96–3.91 (1H, m, a), 3.88 (1H, dd,
J¼2.7, 9.4 Hz, b), 3.82 (1H, br s, a), 3.77 (1H, br s, b),
3.63 (1H, dd, J¼6.5, 9.0 Hz, b), 3.49 (1H, dd, J¼7.8,
12.4 Hz, a), 3.46–3.34 (4H, m, b), 3.20 (1H, dd, J¼5.3,
12.4 Hz, a), 1.01–0.92 (72H, m), 0.72–0.62 (48H, m). 13C
NMR (75 MHz, CDCl3, rt) d 103.2, 94.3, 87.3, 84.3, 78.9,
77.2, 73.9, 72.1, 70.9, 70.1, 54.2, 51.6, 7.14, 7.03, 6.99,
6.90, 6.82, 6.78, 6.72, 6.66, 5.29, 5.15, 5.12, 4.97, 4.92,
4.77, 4.67. ESI-HRMS (positive-ion) calcd for C30H67N3O5-
Si4Na: (M+Na)+ 684.4050; found: 684.4052.
To a solution of the above glycoside (5.4 mg, 11 mmol) in
MeOH (600 ml) was added KCN (8 mM in MeOH, 100 ml,
800 nmol) at room temperature under Ar atmosphere. After
stirring for 6 h, the reaction mixture was concentrated in
vacuo. The residue was purified by ODS TLC (RP-18W, H2O/
MeOH ¼ 1:1) to afford 27 (1.1 mg, 31%) as a colorless oil:
[a]1D8 ꢂ26.8 (c 0.05, MeOH). IR (film) n 3393, 1738,
To a solution of the above TES ether (63.7 mg, 94.1 mmol) in
CH2Cl2 (2 ml) was added palladium on activated carbon
(31.7 mg). Hydrogen was admitted via a balloon and the re-
action mixture was stirred for 30 min and the catalyst re-
moved by filtration. The filtrate was concentrated in vacuo
to afford a crude mixture. To a solution of the mixture and
10% NaHCO3 aq (118 ml, 141 mmol) in 1,4-dioxane
(1.5 ml) was added FmocCl (36.5 mg, 141 mmol) at 0 ꢀC.
After stirring for 12 h, to the reaction mixture was added
H2O (2 ml). The aqueous layer was extracted with AcOEt.
The combined organic layer was washed with brine, dried
over Na2SO4, and concentrated in vacuo. The residue was
purified by silica gel column chromatography (hexane/
AcOEt ¼ 9:1) to afford 33 (65.8 mg, 81%, a:b ¼ 3:2) as a
colorless oil: [a]1D9 +41.2 (c 0.50, CHCl3). IR (film) n 2955,
1
1072 cmꢂ1. H NMR (600 MHz, CD3OD, 40 ꢀC) d 5.79
(1H, s), 4.91 (1H, ddd, J¼ 1.2, 6.2, 11.8 Hz), 4.41 (1H, d,
J¼ 7.9 Hz), 4.05 (1H, tt, J¼ 4.0, 11.2 Hz), 3.87 (1H, dd,
J¼ 2.1, 11.8 Hz), 3.66 (1H, dd, J¼5.6, 11.8 Hz), 3.36–2.26
(3H, m), 3.14 (1H, dd, J¼7.9, 9.1 Hz), 2.90–2.84 (2H, m),
2.42–2.34 (2H, m), 1.44–1.38 (1H, m), 1.35 (1H, q,
J¼11.8 Hz). 13C NMR (150 MHz, CD3OD, rt) d 176.0,
173.9, 113.4, 103.2, 81.6, 78.1, 78.0, 75.0, 74.6, 71.6,

N. Kato et al. / Tetrahedron 62 (2006) 7307–7318
7317
2912, 2878, 1724, 1514, 1458, 1414, 1240, 1140, 1105,
CDCl3, rt) d 170.2, 170.1, 169.7, 156.2, 143.9, 141.3,
127.7, 127.1, 125.0, 120.0, 88.2, 72.2, 68.1, 67.9, 37.7,
67.0, 47.1, 40.2, 20.7, 20.6. ESI-HRMS (positive-ion) calcd
for C27H28BrNO9Na: (M+Na)+ 612.0840; found: 612.0842.
1
1055 cmꢂ1. H NMR (300 MHz, CDCl3, rt) d 7.76 (4H, d,
J¼7.4 Hz), 7.59 (4H, d, J¼7.4 Hz), 7.40 (4H, t, J¼7.4 Hz),
7.30 (4H, t, J¼7.4 Hz), 5.11 (1H, d, J¼2.5 Hz, a), 5.06
(1H, br d, a), 4.45 (1H, d, J¼7.5 Hz, b), 4.41 (1H, dd,
J¼6.9, 10.4 Hz, a), 4.37 (2H, d, J¼6.9 Hz, b), 4.30 (1H,
dd, J¼6.9, 10.4 Hz, a), 4.21 (2H, t, J¼6.9 Hz), 3.98 (1H,
dd, J¼1.8, 9.7 Hz, a), 4.00–3.88 (1H, m, b), 3.90 (1H, dd,
J¼2.5, 9.7 Hz, a), 3.85 (1H, br s, a), 3.75 (1H, br s, b),
3.64 (1H, dd, J¼7.5, 8.7 Hz, b), 3.54–3.47 (2H, m), 3.40
(1H, br d, b), 3.29–3.22 (2H, m), 0.99–0.93 (72H, m),
0.71–0.56 (48H, m). 13C NMR (75 MHz, CDCl3, rt)
d 156.5, 144.0, 141.3, 128.7, 127.0, 125.1, 120.0,
99.2, 94.4, 77.2, 75.7, 74.1, 73.9, 73.8, 70.9, 70.6, 70.2,
66.9, 47.3, 42.5, 7.20, 7.07, 6.99, 6.87, 6.73, 6.61, 5.82,
5.43, 5.34, 5.25, 5.18, 4.78. ESI-HRMS (positive-ion) calcd
for C45H79NO7Si4Na: (M+Na)+ 880.4826; found: 880.4828.
4.1.18. Acetic acid 4,5-diacetoxy-2-[(9H-fluoren-9-yl-
methoxycarbonylamino)-methyl]-6-(2-oxo-2,4,5,6,7,7a-
hexahydro-benzofuran-6-yloxy)-tetrahydro-pyran-3-yl
ester 29. A mixture of 25 (4.9 mg, 32.0 mmol), AgOTf
(4.1 mg, 16.0 mmol), Ag2CO3 (18.4 mg, 67.0 mmol) and
˚
dried molecular sieves 4A (15.2 mg) in anhydrous
CH2Cl2 (200 ml) was stirred at 0 ꢀC under Ar atmosphere
for 1 h. Then a solution of 28 (26.5 mg, 44.9 mmol) in anhy-
drous CH2Cl2 (200 ml) was added to the stirred mixture. After
stirring for 1 h, the reaction mixture was diluted with CHCl3
and filtered through Celite. The filtrate was washed with
satd NaHCO3 aq and brine. The organic layer was dried
over Na2SO4, and concentrated in vacuo. The residue was
purified by silica gel column chromatography (hexane/
AcOEt ¼ 1:5) to afford 29 (10.1 mg, 48%) as a colorless
oil: [a]2D0 ꢂ2.25 (c 0.20, CHCl3). IR (film) n 2920, 2851,
4.1.17.2. Synthesis of 34. To a solution of 33 (65.8 mg,
76.6 mmol) in THF/H2O (2:1, 900 ml) was slowly added
TFA (1.5 ml) at 0 ꢀC. After stirring for 1 h at room tempera-
ture, the reaction mixture was concentrated invacuo to afford
a crude mixture. To a solution of the mixture in pyridine
(1.0 ml) was added Ac2O (1.0 ml) at room temperature. Af-
ter stirring for 3 h, the reaction mixture was concentrated in
vacuo. The residue was purified by silica gel column chroma-
tography (hexane/AcOEt ¼ 2:1) to afford 34 (36.6 mg, 84%,
a:b ¼ 1:2) as a colorless oil: [a]1D9 +35.7 (c 0.50, CHCl3). IR
(film) n 3382, 3020, 1751, 1526, 1450, 1369, 1221,
1720, 1649, 1529, 1450, 1369, 1225 cmꢂ1 1H NMR
.
(300 MHz, CDCl3, rt) d 7.75 (2H, d, J¼7.5 Hz), 7.57 (2H,
d, J¼7.5 Hz), 7.39 (2H, t, J¼7.5 Hz), 7.31 (2H, t, J¼
7.5 Hz), 5.76 (1H, s), 5.32 (1H, d, J¼3.0 Hz), 5.15 (1H, t,
J¼8.1 Hz), 5.00 (1H, dd, J¼3.0, 8.1 Hz), 4.69 (1H, dd, J¼
5.9, 11.3 Hz), 4.54 (1H, d, J¼8.1 Hz), 4.40 (2H, d,
J¼6.6 Hz), 4.20 (1H, t, J¼6.6 Hz), 3.84–3.65 (2H, m), 3.47
(1H, td, J¼6.9, 13.7 Hz), 3.09 (1H, td, J¼6.9, 13.7 Hz),
2.89–2.80 (2H, m), 2.32–2.10 (2H, m), 2.18 (3H, s), 2.02
(3H, s), 1.99 (3H, m), 1.45–1.22 (2H, m). 13C NMR
(75 MHz, CDCl3, rt) d 172.9, 171.1, 170.0, 169.2, 169.1,
156.4, 143.7, 141.3, 127.7, 127.1, 125.0, 120.0, 113.6, 100.3,
79.2, 74.3, 71.5, 70.8, 69.1, 67.8, 67.0, 47.1, 40.4, 40.3,
31.4, 23.8, 20.7, 20.6, 20.6. ESI-HRMS (positive-ion) calcd
for C35H37N1O12Na: (M+Na)+ 686.2208; found: 686.2207.
1
1074 cmꢂ1. H NMR (300 MHz, CDCl3, rt) d 7.77 (4H, d,
J¼7.2 Hz), 7.58 (4H, d, J¼7.2 Hz), 7.41 (4H, t, J¼7.2 Hz),
7.32 (4H, t, J¼7.2 Hz), 6.37 (1H, br s, a), 5.70 (1H, d,
J¼8.4 Hz, b), 5.48 (1H, br s, a), 5.40 (1H, d, J¼2.7 Hz, b),
5.34 (1H, dd, J¼8.4, 9.8 Hz, b), 5.37–5.31 (2H, m, a), 5.08
(1H, dd, J¼2.7, 9.8 Hz, b), 5.10–5.06 (1H, m, a), 4.40
(2H, d, J¼6.9 Hz, a), 4.38 (2H, dd, J¼6.9 Hz, b), 4.22
(2H, t, J¼6.9 Hz), 3.89 (1H, t, J¼6.8 Hz, b), 3.40–3.27
(4H, m), 2.19 (6H, s), 2.13 (6H, s), 2.05 (6H, s), 2.03 (3H,
s, a), 2.01 (3H, s, b). 13C NMR (75 MHz, CDCl3, rt)
d 170.5, 170.0, 169.9, 169.8, 169.4, 168.9, 156.2, 143.8,
141.3, 127.7, 127.0, 125.0, 120.0, 92.2, 89.6, 77.2, 72.8,
70.8, 69.8, 68.4, 68.0, 67.6, 67.4, 67.0, 66.5, 47.1, 40.5,
20.9, 20.8, 20.6, 20.5. ESI-HRMS (positive-ion) calcd for
C29H31NO11Na: (M+Na)+ 592.1789; found: 592.1790.
4.1.19. [3,4,5-Trihydroxy-6-(2-oxo-2,4,5,6,7,7a-hexahy-
dro-benzofuran-6-yloxy)-tetrahydro-pyran-2-ylmethyl]-
carbamic acid 9H-fluoren-9-ylmethyl ester 30. To a
solution of 29 (5.1 mg, 7.7 mmol) in MeOH (150 ml) was added
KCN (8 mM in MeOH, 100 ml, 800 nmol) at room tempera-
ture under Ar atmosphere. After stirring for 4.5 h, the reac-
tion mixture was concentrated in vacuo. The residue was
purified by ODS TLC (RP-18W, H2O/MeOH ¼ 1:4) to afford
30 (1.7 mg, 41%) as a colorless oil: [a]1D8 +21.4 (c 0.20,
MeOH). IR (film) n 3358, 2926, 2361, 1705, 1533, 1448,
4.1.17.3. Synthesis of 28. To a solution of 34 (27.2 mg,
47.8 mmol) in CH2Cl2 (500 ml) were added BiBr3 (0.5 mg,
1.20 mmol) and TMSBr (24.7 ml, 191 mmol) at 0 ꢀC under
Ar atmosphere. After stirring for 1 h at 0 ꢀC, to the reaction
mixture was added satd NaHCO3 aq. The aqueous layer
was extracted with CHCl3. The combined organic layer
was washed with brine, dried over Na2SO4, and concentrated
in vacuo. The residue was purified by silica gel column chro-
matography (hexane/AcOEt ¼ 1:1) to afford 28 (24.8 mg,
88%) as an orange oil: [a]1D9 +137.8 (c 0.50, CHCl3). IR
(film) n 3379, 3067, 2950, 1749, 1526, 1450, 1371, 1223,
1
1261, 1069 cmꢂ1. H NMR (600 MHz, CD3OD, rt) d 7.78
(2H, d, J¼7.5 Hz), 7.65 (2H, d, J¼7.5 Hz), 7.38 (2H, t,
J¼7.5 Hz), 7.33 (2H, t, J¼7.5 Hz), 5.78 (1H, s), 4.70 (1H,
dd, J¼6.0, 11.3 Hz), 4.41 (2H, d, J¼6.6 Hz), 4.31 (1H, d,
J¼7.0 Hz), 4.21 (1H, t, J¼6.6 Hz), 3.94 (1H, tt, J¼3.6,
11.2 Hz), 3.69 (1H, d, J¼2.4 Hz), 3.52–3.49 (1H, m),
3.45–3.37 (4H, m), 3.32–3.27 (2H, m), 2.86–2.80 (2H, m),
2.33–2.28 (2H, m), 1.40–1.28 (2H, m). 13C NMR
(150 MHz, CD3OD, rt) d 175.9, 173.7, 159.2, 145.4, 145.3,
142.7, 128.8, 128.2, 126.2, 126.1, 121.4, 120.9, 114.2,
113.4, 112.6, 104.0, 81.5, 75.0, 74.8, 74.7, 72.3, 70.4, 67.7,
49.6, 42.4, 42.0, 32.8, 24.8. ESI-HRMS (positive-ion) calcd
for C29H31N1O9Na: (M+Na)+ 560.1891; found: 560.1892.
1
1078 cmꢂ1. H NMR (300 MHz, CDCl3, rt) d 7.76 (2H, d,
J¼7.4 Hz), 7.57 (2H, d, J¼7.4 Hz), 7.40 (2H, t, J¼7.4 Hz),
7.31 (2H, t, J¼7.4 Hz), 6.70 (1H, d, J¼3.9 Hz), 5.49 (1H,
d, J¼2.8 Hz), 5.40 (1H, dd, J¼2.8, 10.6 Hz), 5.05 (1H,
dd, J¼3.9, 10.6 Hz), 4.46–4.36 (2H, m), 4.33 (1H, t,
J¼6.8 Hz), 4.22 (1H, t, J¼6.9 Hz), 3.46–3.27 (2H, m), 2.16
(3H, s), 2.11 (3H, s), 2.02 (3H, s). 13C NMR (75 MHz,
4.1.20. Biologically inactive fluorescence-labeled probe
31. To a solution of 30 (3.5 mg, 6.5 mmol) in DMF

7318
N. Kato et al. / Tetrahedron 62 (2006) 7307–7318
(720 ml) was added piperidine (4.8 ml, 49 mol) at 0 ꢀC under
Ar atmosphere. After stirring for 2 h at 0 ꢀC, piperidine was
removed in vacuo. To the reaction mixture was added
AMCA-X, SE (3.1 mg, 7.2 mmol) at room temperature un-
der Ar atmosphere. After stirring for 2 h, the reaction mix-
ture was concentrated in vacuo. The residue was purified
by ODS TLC (RP-18W, H2O/MeOH ¼ 1:1) and HPLC with
COSMOSIL 5C18-AR column (f20.0ꢁ250 mm, H2O/
CH3CN ¼ 6:4) to afford 31 (1.3 mg, 62% in two steps) as
a colorless oil: [a]2D1 +6.6 (c 0.05, MeOH). IR (film)
of other nyctinastic plants, M. pudica, C. mimosoides, and
L. leucocephala were prepared and treated with fluores-
cence-labeled probe compound in the same procedure.
Acknowledgements
This work was supported by the Ministry of Education,
Science, Sports and Culture (Japan) Grant-in-Aid for Scien-
tific Research (No. 15681013) and Grant-in-Aid for the 21st
Century COE program of Tohoku University from the
Ministry of Education, Culture, Sports, Science, and Tech-
nology, Japan. We thank Mr. Kazuo Sasaki for his detailed
NMR spectroscopic analysis, and Mr. Hiroyuki Monma for
his assistance in mass spectrometric analysis.
n 3348, 2924, 1734, 1652, 1558, 1057 cmꢂ1 1H NMR
.
(600 MHz, CD3OD, 40 ꢀC) d 7.48 (1H, d, J¼8.8 Hz), 6.65
(1H, dd, J¼2.2, 8.8 Hz), 6.51 (1H, d, J¼2.2 Hz), 5.74 (1H,
s), 4.77 (1H, dd, J¼5.5, 11.1 Hz), 4.33 (1H, d, J¼7.6 Hz),
3.99 (1H, tt, J¼4.0, 11.5 Hz), 3.72 (1H, d, J¼1.4 Hz), 3.58
(1H, dd, J¼5.4, 7.2 Hz), 3.52 (2H, s), 3.49–3.45 (3H, m),
3.35 (1H, dd, J¼7.8, 13.7 Hz), 3.18 (2H, t, J¼7.5 Hz),
2.88–2.84 (2H, m), 2.37 (3H, s), 2.41–2.32 (2H, m), 2.21
(2H, t, J¼7.5 Hz), 1.62 (2H, quintet, J¼7.5 Hz), 1.52 (2H,
quintet, J¼7.5 Hz), 1.42 (1H, dd, J¼5.0, 11.7 Hz), 1.36
(2H, t, J¼7.5 Hz), 1.34 (1H, m). 13C NMR (150 MHz,
CD3OD, 40 ꢀC) d 176.7, 175.9, 173.8, 173.0, 164.8, 155.9,
154.0, 152.9, 127.4, 114.4, 113.4, 113.2, 111.8, 104.1,
100.5, 81.6, 75.4, 74.7, 74.3, 72.3, 70.5, 42.0, 41.3, 40.3,
37.0, 35.2, 32.9, 30.0, 27.5, 26.7, 24.3, 15.4. ESI-HRMS
(positive-ion) calcd for C32H41N3O11Na: (M+Na)+
666.2633; found: 666.2635.
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