Hybrid stereoisomers of a compact molecular probe based on a jasmonic acid glucoside: Syntheses and biological evaluations

Article abstract of DOI:10.1016/j.bmc.2012.08.003

12-O-β-d-glucopyranosyl jasmonic acid (JAG) shows unique biological activities, including leaf-closing of Samanea saman. It is expected that the mode of action for such regulation is distinct from that of other jasmonates. We developed high-performance compact molecular probes (CMPs) based on JAG that can be used for the FLAG-tagging of JAG target. We synthesized four hybrid-type JAG-CMP stereoisomers (7, ent-7, 8, and ent-8), which are composed of (-)-12-OH-JA (2)/d-galactopyranoside, (-)-2/l-galactopyranoside, (+)-ent-2/d-galactopyranoside, and (+)-ent-2/l-galactopyranoside moieties, respectively, and we examined their biological features, such as the stereospecific induction of shrinkage, rate of the cellular response, and dependence on potassium channel activity. These features of the JAG-CMPs were completely consistent with those of the original JAG. These results indicate the biological equivalence of JAG and the JAG-CMPs. During the course of such biological evaluations, it was revealed that the biological activity of the CMPs is greatly dependent on the d/l-stereochemistry of a glycon moiety. To the best of our knowledge, this is the first study suggesting that the d/l-stereochemistry of the glycon moiety significantly affects the biological activity of the associated glycoside.

Full text of DOI:10.1016/j.bmc.2012.08.003

Bioorganic & Medicinal Chemistry 20 (2012) 5832–5843
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Hybrid stereoisomers of a compact molecular probe based on a jasmonic
acid glucoside: Syntheses and biological evaluations
Minoru Ueda a, , Gangqiang Yang a, Yasuhiro Ishimaru a, Tetsuya Itabashi a, Satoru Tamura a,
⇑
Hiromasa Kiyota b, Shigefumi Kuwahara b, Sho Inomata a, Mitsuru Shoji c, Takeshi Sugai c
a Department of Chemistry, Tohoku University, 6-3 Aramaki-aza Aoba, Aoba-ku, Sendai 980-8578, Japan
b Department of Applied Bioorganic Chemistry, Division of Bioscience & Biotechnology for Future Bioindustry, Graduate School of Agricultural Science, Tohoku University, 1-1
Tsutsumidori-Amamiya, Aoba-ku, Sendai 981-8555, Japan
c Faculty of Pharmaceutical Sciences, Keio University, 1-5-30 Shibakoen, Minato-ku, Tokyo 105-8512, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
12-O-b-D-glucopyranosyl jasmonic acid (JAG) shows unique biological activities, including leaf-closing of
Received 18 July 2012
Revised 3 August 2012
Accepted 4 August 2012
Available online 13 August 2012
Samanea saman. It is expected that the mode of action for such regulation is distinct from that of other
jasmonates. We developed high-performance compact molecular probes (CMPs) based on JAG that can
be used for the FLAG-tagging of JAG target. We synthesized four hybrid-type JAG-CMP stereoisomers
(7, ent-7, 8, and ent-8), which are composed of (ꢀ)-12-OH-JA (2)/
D
-galactopyranoside, (ꢀ)-2/
L-galactopy-
ranoside, (+)-ent-2/ -galactopyranoside, and (+)-ent-2/ -galactopyranoside moieties, respectively, and we
D
L
Keywords:
Molecular probe
Jasmonic acid glucoside
Enatiodifferential approach
Ion channel activation
examined their biological features, such as the stereospecific induction of shrinkage, rate of the cellular
response, and dependence on potassium channel activity. These features of the JAG-CMPs were com-
pletely consistent with those of the original JAG. These results indicate the biological equivalence of
JAG and the JAG-CMPs. During the course of such biological evaluations, it was revealed that the biolog-
ical activity of the CMPs is greatly dependent on the
our knowledge, this is the first study suggesting that the
icantly affects the biological activity of the associated glycoside.
Ó 2012 Elsevier Ltd. All rights reserved.
D/
L
-stereochemistry of a glycon moiety. To the best of
D/L
-stereochemistry of the glycon moiety signif-
1. Introduction
However, it is widely recognized that the identification of the
protein target of a bioactive metabolite remains a difficult task.9,10
We and Uesugi et al. have proposed that the stereospecific nature of
ligand recognition by a target protein provides a powerful strategy
for identifying the target of a bioactive metabolite. For this strategy,
the stereoisomer of the bioactive metabolite can be used as the
ideal control.11,12 Differences in the affinity profile among the ster-
eoisomers of a ligand suggest the specific binding of the ligand
based on the stereospecificity of ligand recognition by the specific
target. We applied an ‘enantio-differential’ approach (EDA) in
which an enantiomer was adopted as the control for investigating
the target of 3. EDA using a pair of probes (5 and 6) enabled the dis-
covery of a membrane target protein, referred to as membrane tar-
get protein of jasmonate glucoside (MTJG), in the motor cells of S.
saman.11 However, the trace amounts of MTJG present in the cells
limit its accessibility, making identification difficult. Thus, it is
essential to develop a high-performance molecular probe for the
efficient tagging of target proteins having low-abundance.
Jasmonic acid (JA, 1) and its derivatives, collectively referred to
as jasmonates, play important roles in controlling growth and
development and regulating plant responses to environmental
changes in higher plants. Recently, it was revealed that (+)-7-iso-
jasmonoyl isoleucine (JA-Ile) functions as a trigger for a JA signal-
ing module. Accordingly, (+)-7-iso-JA-Ile binds to the co-receptor
complex COI1-JAZ1 and triggers the degradation of the JAZ repres-
sor by 26S-proteasome, thereby releasing the transcription factor
MYC2 and allowing the activation of gene expression.2,3
On the other hand, 12-O-b-D-glucopyranosyl jasmonic acid (JAG,
3) shows unique biological activities, such as inducing leaf-closing
of Samanea saman4–6 as well as tuber induction if Solanaceous
species,7 but no involvement in the above-mentioned COI1-JAZ
signaling module.8 This result implies the existence of an unknown
mode of action mediated by jasmonates. Therefore, studies to
determine the protein target of 3 would provide important clues
to help identify this new mechanism.
Recently, we reported the advantages of a method for designing
a high-performance compact molecular probe (CMP) for target
identification in which the retention of the original bioactivity of
the ligand was given the highest priority.13 An azide-functionalized
ligand equipped with a carbon elecrophile/photoreactive functional
⇑
Corresponding author.
E-mail address: ueda@m.tohou.ac.jp (M. Ueda).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.08.003

M. Ueda et al. / Bioorg. Med. Chem. 20 (2012) 5832–5843
5833
group represents a very simple design for a molecular probe to
2. Results and discussion
which a large tag unit, such as FITC, biotin, or FLAG epitope peptide,
can be attached after crosslinking with the target protein. The use of
stepwise tagging enables the incorporation of a large FLAG peptide
tag, which is an octapeptide antigen with the sequence DYKDDDDK
and known to give only low levels of non-specific binding.14 The
CMP method was highly successful for the detection and purifica-
tion of the cytosolic target for isolespedezate, CoMetE.
Protein labeling using a probe with an azide handle and subse-
quent on-the-cell click chemistry15–17 are employed as a powerful
method for tagging a membrane protein. In this method, a target
protein on living cells is labeled by a sterically inconsequential
azide handle that is subsequently used to introduce a tag by Hüs-
gen [3+2] cycloaddition employing CuAAC18,19 or copper-free click
chemistry20–23 (Fig. 1). The different results obtained using stereo-
isomeric probes, such as enantiomers and diastereomers, provide
robust information concerning the stereospecific recognition of a
ligand by its target protein. Furthermore, a related co-dependence
of both target affinity and bioactivity on ligand stereochemistry af-
fords strong support for target validation.
We already have probe 7 in hand,24 then we synthesized other
three hybrid-type JAG-CMP stereoisomers (ent-7, 8, and ent-8),
which are composed of (+)-ent-2/
L
-galactopyranoside, (ꢀ)-2/
L-
galactopyranoside, and (+)-ent-2/ -galactopyranoside moieties,
D
respectively. Additionally, the related co-dependence of both tar-
get affinity and bioactivity on the stereochemistry of 7, ent-7, 8,
and ent-8 would afford strong support for target validation.
The synthesis of ent-7 requires synthetic supply of commer-
cially unavailable
L-galactosamine derivative. We planned to syn-
thesize -galactosamine from
L
a
commercially available
L-
galactose derivative. Some methods to convert hexose into 20-
aminohexose have been reported.25,26 Among them, Lemieux’s azi-
donitration27 is recognized as a very useful one and represents a
unique approach involving the azidonitration via glycals. Using
this method, we synthesized L-galactosamine derivative 16. The
stereoselective addition of an azido radical to the galactal28 inter-
mediate gave a 10:1:5 mixture of the desired 20S derivative (10)
and undesired 20R derivative (11) as well as the 1
a-acetamide ana-
log (12) (Scheme 1). The mixture was then reduced by catalytic
hydrogenation and protected by phthalimide using the acid chlo-
ride29 to give 15.
Galactosamine derivative 15 was brominated to form 16 which
was used as a glycosyl donor. The glycosylation of 1711,30 by glycosyl
donor16gave18, andsubsequentmodificationsgaveprobe ent-7, an
enantiomerof probe 7 (Scheme 2). Thus, we obtained an enatiomeric
pair of CMPs (7 and ent-7). Similarly, we synthesized two
diastereomeric CMPs (8 and ent-8), which were composed of (ꢀ)-
On the basis of these positive results, we planned to integrate
EDA with the CMP method for the chemical tagging of the target
protein. The use of a JAG-CMP combined with the subsequent
introduction of an alkyne-linked FLAG tag by Hüsgen [3+2] cyclo-
addition is expected to enable the highly efficient tagging of the
low-abundant MTJG (Fig. 1). In this report, we describe the synthe-
ses of four hybrid stereoisomeric JAG-CMPs (7, ent-7, 8, and ent-8)
and their biological evaluations, which substantiated their reliabil-
ity as probes for tagging the target protein of JAG. The results also
showed an intriguing importance of the specific glycoside moiety
for the bioactivity and target affinity of 3.
2/L-galactosamine or (+)-ent-2/D-galactosamine moieties, respec-
tively (Scheme 3a and b).
Next, we performed the biological evaluations of the resulting
four stereoisomeric CMPs (7, ent-7, 8, and ent-8). Nyctinastic
leaf-folding of S. saman has been attributed to volume changes in
specific motor cells located on the opposite side of the vascular
bundle: the extensor cells that reside on the adaxial side of the ter-
tiary pulvinus shrink, whereas the flexor cells that reside on the
abaxial side swell during leaf-folding.31,32 We have recently re-
ported that JAG 3 is able to selectively induce the shrinking of pro-
toplasts isolated from extensor motor cells, whereas flexor motor
cell protoplasts prove to be insensitive to JAG treatment.8 Thus,
the leaf-folding of S. saman is attributed to cell shrinking in exten-
sor motor cells. This unique feature can be employed for the bio-
logical evaluation of the four stereoisomeric CMPs (7, ent-7, 8,
and ent-8). Each CMP was incubated with the freshly prepared pro-
toplasts of extensor motor cells, and then, the change in the size of
the cells was measured and plotted in Figure 2a and b. The cell-
shrinking activity was evaluated using the radii of the protoplasts
surviving the preparation and 40-min incubation (Fig. 2d). CMP 7
as well as 3 induced the shrinking of extensor protoplasts, whereas
the other stereoisomers, ent-7, 8, and ent-8, did not induce any
shrinking. Thus, the stereospecific induction of cell shrinkage was
found among the JAG-based CMPs. It should also be emphasized

5834
M. Ueda et al. / Bioorg. Med. Chem. 20 (2012) 5832–5843
N
N
CF3
OH
SDS-PAGE
ent-
JAG-CMP (7)
COOH
1) cross-linking
2) cycloaddition
TAG
HO
O
O
O
JAG-N3
HN
JAG-N3
O
N3
N3
mirror
N3
O
O
OH
OH
HN
COOH
motor cell
N
ent-JAG-CMP (ent-7)
N
: tagged-MTJG
CF3
TAG
N
N
N
Figure 1. Overview of CMP-CuAAC strategy using stereoisomeric CMPs for the tagging of target protein.
In addition, the biological evaluation also revealed an intriguing
feature of JAG 3, namely, the influence of the glycon moiety on its
biological activity. Previous SAR studies8 unequivocally demon-
strated that the glycon moiety of 3 is indispensable for its bioactiv-
ity because (ꢀ)-12-OH-JA ((ꢀ)-2), the aglycon of JAG 3, cannot
induce leaf-folding of S. saman as well as shrinking of the motor
cells. However, biological activity of 3 was retained when its
glucopyranoside was replaced by the 40-epimer,
-galactopyrano-
side (4),34 or a -galactosaminopyranoside derivative (in 7).11 This
D-
D
D
implies that structural modification of the glycon moiety of 3, such
as that exemplified in 5 and 7, will not severely affect its biological
activity. On the other hand, the importance of the stereochemistry
of the 12-OH-JA moieties11,30,34 was suggested by EDA studies using
probes 5 and 6 in which probe 6 with the enantiomeric 12-OH-JA
moiety cannot induce leaf-folding and bind to MTJG. Thus, it was
very intriguing that diastereomeric CMP 8, which is composed of
(ꢀ)-2/
it has the naturally occurring stereoisomer found with 12-OH-JA.
The biological activity of 3 was lost when -glucopyranoside was
replaced by an -galactosaminopyranoside derivative (in 8). These
L-galactopyranoside, cannot induce shrinking even though
Scheme 1. Synthesis of L-galactosamino unit (16).
D
L
results suggest that the naturally occurring stereochemistry in both
the aglycon and glycon moieties is indispensable for the leaf-fold-
ing/motor-cell-shrinking activity of JAG-related compounds.
Glycosylation dramatically changes the physical properties of a
substrate. For example, it causes reduced lipophilicity leading to a
decrease in cell permeability. Therefore, the glycosylation of JA 1
may lower its cell permeability and thereby decrease its binding
to the cytosolic COI1-JAZ receptor complex. However, the above-
mentioned results strongly suggested that the glycon moiety of 3
may be involved in or related to its recognition by MTJG rather
than simply functioning as a hydrophilic anchor decreasing the cell
permeability of 3.
Next, we examined the viability of the motor cells during the
course of each step, which include the photoaffinity-labeling
(PAL) and CuAAC reaction (Fig. 3a). Cell viability under the two
conditions used for PAL were compared (Fig. 3b): Freshly prepared
protoplasts11,32 were (1) incubated with probe 7 at 4 °C for 20 min
and then photo-crosslinked at 4 °C for 5 min or (2) incubated with
probe 7 at 4 °C for 5 min and photo-crosslinked at 4 °C for 20 min.
‘Condition 1’ (Fig. 3b-C) gave better viability than ‘condition 2’
(Fig. 3b-F). Surprisingly, longer UV-irradiation (20 min) improved
the viability of the protoplasts (Fig. 3(b), C and F, D and G). Consid-
ering the fact that photo-crosslinking by trifluoromethyldiazirine
(TFMD) is completed within 30 min, it was concluded that condi-
tion 1 is superior to condition 2. The longer irradiation time pro-
vides a better cross-linking result as well as increased cell viability.
that CMP 7 was as effective as the original JAG 3 in the cell-shrink-
ing assay (Fig. 2a), suggesting that 7 has an affinity for MTJG com-
parable to the original JAG 3. In addition, we examined the time-
course profiles of the cellular response to the JAG-based CMPs
against those to JAG 3. The response of protoplasts to CMP 7 was
as quick as that to JAG 3; the shrinking started within 10 min after
the addition of each compound. This rapid response suggests that
the shrinking of motor cell protoplasts upon CMP application is
the result of an ion channel-mediated process. Regarding the cell
shrinking in response to JAG 3, the involvement of a potassium
channel was confirmed by the inhibition of this response using a
potassium channel blocker, namely, tetraethyl ammonium chlo-
ride (TEA).8 This is based on Moran’s experiment in which the out-
ward-directed K+ current from extensor motor cells was blocked by
TEA.33 We also examined the effect of TEA on the cell shrinking in-
duced by CMP 7. As shown in Figure 2c, the addition of TEA com-
pletely inhibited cell shrinking in response to CMP 7 as well as JAG,
which suggested that regulation through potassium channels is in-
volved in the shrinking response induced by both compounds.
Next, we examined the biological features of the JAG-CMPs,
such as the stereospecific induction of shrinkage, rate of the cellu-
lar response, and dependence on potassium channel activity, using
motor cells. These features were completely consistent with those
of original JAG. These results support the biological equivalence of
JAG 3 and probe 7.

M. Ueda et al. / Bioorg. Med. Chem. 20 (2012) 5832–5843
5835
Scheme 2. Synthesis of enantiomeric CMP (ent-7).
(a)
(b)
Scheme 3. (a) Synthesis of diastereomeric CMP (8). (b) Synthesis of diastereomeric CMP (ent-8).
Subsequently, we examined the conditions for the CuAAC reac-
tion using the FLAG-linked alkyne unit 35.13,32 Two conditions
were examined for the CuAAC reaction (Fig. 3c): (I) one based on
the reports by Finn et al35,36 and (II) another based on our previous
reports.37 Under both conditions, a tris(3-hydroxypropyltriazolylm-
ethyl)amine (THPTA) ligand was added as a catalyst.35 However,
under the first condition, CuAAC was used without the addition
of aminoguanidine35 because the motor cells were treated at a
pH less than 5.5, a level at which aminoguanidine cannot function
efficiently to intercept the byproducts of ascorbate oxidation that
can react with proteins. Better cell viability was observed using
condition I (Fig. 3c-C) than using condition II (Fig. 3c-D). The in-
crease in copper (I) ion concentration (and equivalence against
THPTA) caused a severe decrease in the viability of the protoplasts.
This may be because of the toxicity of copper (I) ion, which was re-
ported in Ref. 36. This combination of mild conditions (1) and (I)
was selected for the application of CMP to pull-down the purifica-
tion of the target (MTJG, Fig. 1).
3. Conclusion
Jasmonate glucoside is a metabolite of an important plant hor-
mone, jasmonic acid (JA).38 The mode of action of JA has been ver-
ified through the identification of the COI1-JAZ signaling
module39,40 in which a complex of cytosolic COI1 and JAZ proteins
directly functions as a JA receptor.40,41 Then MTJG8,11 became the

5836
M. Ueda et al. / Bioorg. Med. Chem. 20 (2012) 5832–5843
(a)
(b)
1.03
1.01
0.99
0.97
0.95
0.93
0.91
0.89
0.87
1.03
b
b
b
b
b
b
b
b
b
b
b
b
b
b
1.01
b
b
b
b
a
a
0.99
b
b
a
0.97
a
a
a
0.95
a
a
a
a
0.93
a
8
CMP (7)
0.91
ent-8
ent-7
a
3
JAG (3)
Water
0.89
Water
0.87
0
10
20
30
40 (min)
0
10
20
30
40 (min)
(c)
(d)
Fluorescence by fluorescein
diacetate (FDA)
1.03
1.01
0.99
0.97
0.95
0.93
0.91
0.89
0.87
Transmission view
b
b
b
b
b
b
b
b
b
b
b
b
a
a
a
a
7
7 + tetraethyl ammonium chloride (TEA)
3 + TEA
Water
0
10
20
30
40 (min)
Figure 2. CMP induced shrinkage of extensor motor cell protoplast in S. saman (a) Extensor protoplasts were treated with 100
compared to the treatments with 100 M ent-7 (purple triangles), 100 M JAG (blue circles), or water (green triangles). (b) Extensor protoplasts were treated with 100
(red circles) or 100 M ent-8 (purple triangles), and the responses were compared to the treatments with 100 M JAG (blue circles), or water (green triangles). (c) The
response of extensor protoplasts to treatment with 7and TEA (purple triangles) is compared to the ones caused by 7 (red circles), JAG and TEA (blue circles), or water (green
triangles). (d) Transmission view and FDA fluorescence in extensor protoplast after 40 min incubation. Scale bar = 20 m. Error bars represent the SD. The values followed by
lM CMP (7) (red circles), and the response was
l
l
l
M 8
l
l
l
different letters are statistically different according to analysis of variance followed by SNK test (a, p <0.01; 7, n = 10; ent-7, n = 12; JAG, n = 13; Water, n = 13; b, p <0.01; 8,
n = 12; ent-8, n = 11; JAG, n = 12; Water, n = 12; c, p <0.01; 7, n = 9; 7+TEA, n = 11; JAG + TEA, n = 8; Water, n = 7).
first example of a membrane target protein that binds to jasmo-
nate-related compounds. Our CMP probes were confirmed to have
the same biological features as those of the original JAG 3, that is,
they can induce a rapid shrinking response on motor-cells through
the activation of a potassium channel (Fig. 3). Thus, it was con-
cluded that JAG 3 and CMP 7 were involved in the same signaling
path for inducing the shrinkage of S. samanea motor cells, which
supports the suitability of using CMP 7 for the exploration of the
JAG 3 target.
To date, many secondary metabolites with unique bioactivities
have been identified from nature. However, in many cases, their
mode of action remains unknown. Thus, studies aimed at identify-
ing these unknown targets bridge the fields of chemical biology
and natural products chemistry.44 CMPs employing stereoisomers
of bioactive metabolites provide a powerful method for target
identification.
4. Experimental section
It is also interesting that a unique feature of ligand recognition
using JAG-related compounds was found, that is, the stereochem-
istry of their glycon moieties strongly influences the induced bio-
logical response. In SAR studies, attention is usually focused on
the biological consequences of the stereochemistry of the aglycon,
whereas there are no reports of those of the glycon moiety. In some
cases, hydrogen bonds to the glycon moiety of a glycoside provide
an important contribution to target affinity. For example, the
rhamnose moiety of ouabain can form hydrogen bonds with its tar-
get, an Na/K-ATPase, thereby conferring a much higher affinity to
ouabain than that of the corresponding aglycon, ouabagenin.42,43
Similar involvement of the glycon moiety can be anticipated in
our case.
Unless otherwise stated, reactions were performed in flame-
dried glassware under an argon or a nitrogen atmosphere using
dry solvents. Solvents were dried over activated molecular sieves
under an argon atmosphere. All the starting materials were pur-
chased from commercial sources and used as received, unless other-
wise stated. Liquids and solutions were transferred via syringe or
positive-pressure cannula. Brine solutions refer to saturated aque-
ous sodium chloride solutions. Thin-layer chromatography (TLC)
was performed using silica gel 60 F254 precoated plates (0.25 mm)
and visualized by UV fluorescence quenching, anisaldehyde, or
H3(PMo12O40) staining. Silica gel 60 N (particle size 63–210 mm)
was used for column chromatography. HPLC purifications were

M. Ueda et al. / Bioorg. Med. Chem. 20 (2012) 5832–5843
5837
(a)
(b)
STEP2:
CuAAC with
STEP1:
PAL by
CMP (7)
35
(c)
Figure 3. (a) FLAG-tagging by PAL-CuAAC (STEP1 and STEP2) and subsequent fluorescence-labeling by suing anti-FLAG-antibody. Cell viabilities during STEP1 and STEP2
were observed. (b) Viability of protoplasts in STEP1: The viability of prepared protoplasts (= basal) was evaluated by the ratio of alive cells in total cells, and that of each
condition was calculated on the basis of the number of alive protoplasts in basal. A: basal, B–D: ‘condition 1’ (incubation at 4 °C for 5 min, then irradiated with UV light at 4 °C
for 20 min), E–G: ‘condition 2’ (incubation at 4 °C for 20 min, then irradiated with UV light at 4 °C for 5 min). B and E: intact (protoplast with buffer), C and F: control
(protoplast in 5% DMSOaq), D and G (protoplast with 500
protoplasts in STEP 2: The viability of prepared protoplasts (= basal) was evaluated by the ratio of alive cells in total cells, and that of each condition was calculated on the
basis of the number of alive protoplasts in basal. A: basal, B: intact (protoplast with buffer), C: control (protoplast with DMSO), D: condition I (50 M CuSO4, 250 M THPTA
M THPTA ligand, 0.5 mM sodium ascorbate, 0.5 mM alkyne-FLAG
lM 7 in 5% DMSOaq). The data shows average and SD of quadruplicate individual experiments. (c)Viability of
l
l
ligand, 2.5 mM sodium ascorbate, 0.5 mM alkyne-FLAG 35, 7.5% DMSO), E: condition II (500
35, 7.5% DMSO). The data shows average and SD of triplicate individual experiments.
lM CuSO4, 500 l
carried out using PU-2089 with UV-2075 detector (Jasco Ltd.)
concentrated in vacuo. The obtained crude mixture (2.81 g, con-
taining 10, 11 and 12 was subjected to the next reaction without
further purification.
equipped with Cosmosil 5C18AR (
u
20 ꢁ 250 mm, Nakalai. Tesque
Ltd.) at a flow rate of 4.0 mL/min. 1H and 13C NMR spectra were re-
corded on ECA400 and Alpha 500 spectrometers (Jeol Ltd.). Chemi-
cal shifts are given in parts per million (ppm) relative to Me4Si
(0.0 ppm). Data for 1H NMR spectra are reported as follows: chem-
ical shift in ppm (integration, multiplicity, coupling constant in Hz).
Multiplicity and qualifier abbreviations are as follows: s = singlet,
d = doublet, t = triplet, q = quartet, quint = quintet, m = multiplet,
br = broad. IR spectra were recorded on FT/IR-410 spectrometer
(Jasco Co., Ltd.) and are reported in frequency of absorption (cm
ꢀ1). LR and HR MS were recorded on an ESI-mode by using MicrO-
TOF-II and APEX-III spectrometers (Bruker Daltonics Ltd.). Optical
rotation was recorded on a DIP-360 spectrometer using 100-mm
cell.
4.2. Compound 13
The crude mixture of 10, 11 and 12 (2.76 g) in AcOH (18 mL)
was added NaOAc (1.20 g, 14.6 mmol) at RT under N2. After being
stirred for 3 h at 100 °C, the reaction mixture was cooled to 0 °C,
and diluted with CHCl3 and sat. NaHCO3 aq, extracted with CHCl3.
The combined organic layer was washed with sat. NaHCO3 aq and
brine, dried over Na2SO4, filtered and concentrated in vacuo. Puri-
fication by silica gel column chromatography (n-hexane/EtOAc = 4/
1–3/1) gave 13 (
colorless viscous oil.
13-
1H NMR (400 MHz, CDCl3) d 6.33 (d, J = 3.6 Hz, 1 H, 1-H),
a: b = 7: 4, 1.46 g, 3.91 mmol, 51% in 2 steps) as a
a
,
4.1. Compound 10, 11 and 12
5.48 (dd, J = 3.2 Hz, 1.2 Hz, 1 H, 4-H), 5.32 (dd, J = 11.2 Hz, 3.2 Hz, 1
H, 3-H), 4.29 (td, J = 6.8 Hz, 1.2 Hz, 1 H, 5-H), 4.07 (dd, J = 11.2 Hz,
6.8 Hz, 1 H, 6-Ha), 4.03 (dd, J = 11.2 Hz, 6.8 Hz, 1 H, 6-Hb), 3.94 (dd,
J = 11.2 Hz, 3.6 Hz, 1 H, 2-H), 2.18 (s, 3 H, 1-OAc), 2.17 (s, 3 H, 4-
OAc), 2.08 (s, 3 H, 3-OAc), 2.04 (s, 3 H, 6-OAc); 13C NMR
(100 MHz, CDCl3) d 170.2 (1 C, 6-OCOCH3), 169.9 (1 C, 4-OCOCH3),
169.7 (1 C, 3-OCOCH3), 168.6 (1 C, 1-OCOCH3), 90.4 (1 C, 1-C), 68.7
(1 C, 3-C), 68.7 (1 C, 5-C), 66.9 (1 C, 4-C), 61.0 (1 C, 6-C), 56.9 (1 C,
Compound 927 (2.12 g, 7.79 mmol) in dehydrated MeCN
(39 mL) was added to a mixture of CAN (15.84 g, 28.9 mmol) and
NaN3 (763 mg, 11.7 mmol) at ꢀ15 °C under N2. After being stirred
for 7 h at ꢀ15 °C, the reaction mixture was diluted with cold Et2O
and H2O, extracted with Et2O. The combined organic layer was
washed with H2O and brine, dried over Na2SO4, filtered and

5838
M. Ueda et al. / Bioorg. Med. Chem. 20 (2012) 5832–5843
2-C), 20.8 (1 C, 1-OCOCH3), 20.5(3 C, 3, 4, 6-OCOCH3); IR (film)
2964, 2938, 2116, 1754, 1434, 1372, 1217, 1133, 1110, 1077,
1045, 1013, 938, 899, 762; HRMS (ESI, positive) m/z [M+Na]+ calcd
for C14H19N3O9Na 396.1019, found 396.1017. HR MS was observed
123.7 (2 C, 2-N(CO)2C6H4), 90.3 (1 C, 1-C), 71.8 (1 C, 5-C), 67.7 (1 C,
3-C), 66.5 (1 C, 4-C), 61.2 (1 C, 6-C), 50.3 (1 C, 2-C), 20.7 (2 C, 1, 4-C),
20.6 (1 C, 6-C), 20.4 (1 C, 3-C); IR (film) 3026, 1752, 1721, 1636,
1614, 1540, 1469, 1434, 1387, 1220, 1173, 1134, 1117, 1074,
1044, 1013, 948, 756, 723.
as a mixture of a- and b- isomers.
13-b, 1H NMR (400 MHz, CDCl3) d 5.55 (d, J = 8.4 Hz, 1 H, 1-H),
5.38 (dd, J = 3.2 Hz, 0.8 Hz, 1 H, 4-H), 4.90 (dd, J = 10.4 Hz, 3.2 Hz,
1 H, 3-H), 4.12 (dd, J = 11.2 Hz, 6.8 Hz, 1 H, 6-Ha), 4.07 (dd,
J = 11.2 Hz, 6.8 Hz, 1 H, 6-Hb), 4.02 (td, J = 6.8 Hz, 0.8 Hz, 1 H, 5-
H), 3.84 (dd, J = 10.4 Hz, 8.4 Hz, 1 H, 2-H), 2.21 (s, 3 H, 1-OAc),
2.17 (s, 3 H, 4-OAc), 2.07 (s, 3 H, 3-OAc), 2.04 (s, 3 H, 6-OAc); 13C
NMR (100 MHz, CDCl3) d 170.2 (1 C, 6-OCOCH3), 169.8 (1 C, 4-
OCOCH3), 169.5 (1 C, 3-OCOCH3), 168.4 (1 C, 1-OCOCH3), 92.8 (1
C, 1-C), 71.7 (1 C, 5-C), 71.3 (1 C, 3-C), 66.2 (1 C, 4-C), 60.9 (1 C,
6-C), 59.7 (1 C, 2-C), 20.8(1 C, 1-OCOCH3), 20.5(3 C, 3, 4, 6-
OCOCH3); IR (film) 2964, 2938, 2116, 1754, 1434, 1372, 1217,
1133, 1110, 1077, 1045, 1013, 938, 899, 762.
4.5. Compound 16
To a solution of 15 (675.8 mg, 1.42 mmol) in Ac2O (266 lL) was
added 33% HBr-AcOH (7.1 mL) at 0 °C. The reaction mixture was di-
luted with CHCl3, quenched with sat. NaHCO3 aq and extracted
with CHCl3. The combined organic layer was washed with sat.
NaHCO3 aq and brine, dried over Na2SO4, filtered and concentrated
in vacuo. Resultant bromide 16 (695.7 mg) was subjected to the
next reaction without further purification.
4.6. Compound 18
4.3. Compound 14
To a suspension of crude 16 (695.7 mg), 1711 (203.0 mg,
0.72 mmol), Ag2CO3 (476.5 mg, 1.73 mmol) and MS4Å (2.03 g) in
dehydrated CH2Cl2 (7.2 mL) was slowly added AgOTf (221.7 mg,
0.86 mmol) in dehydrated toluene (7.2 mL) at 0 °C under N2. After
being stirred for 1 h at RT in the dark, the reaction mixture was fil-
tered through a pad of Celite. The filtrate was washed with sat.
NaHCO3 aq and brine, dried over Na2SO4, filtered and concentrated
in vacuo. Purification by silica gel column chromatography (tolu-
To a solution of 13 (1.46 g, 3.91 mmol) in MeOH (39 mL) were
added 10% w/w Pd/C (416.8 mg, 0.392 mmol) and 10 M HCl aq
(0.43 mL, 4.3 mmol) at 0 °C. After being stirred for 1 h at RT under
H2, the reaction mixture was filtered through a pad of Celite and
concentrated in vacuo. Resultant crude amine 14 (1.37 g) was sub-
jected to the next reaction without further purification.
ene/acetone = 50/1–40/1–30/1–20/1–15/1–10/1)
(478.7 mg, 0.68 mmol, 95%) as a pale yellow viscous oil.
1H NMR (400 MHz, CDCl3) d 7.86 (br, 2 H), 7.76 (dd, J = 6.0 Hz,
2.8 Hz, H), 5.79 (dd, J = 11.6 Hz, 3.2 Hz, H), 5.48 (dd,
gave
18
4.4. Compound 15
To a suspension of 14 (1.37 g) and acid chloride (2.41 g,
12.1 mmol) in dehydrated THF (71.5 mL) was added NEt3
(3.9 mL, 28.0 mmol) at 0 °C under N2. After the reaction mixture
was stirred for 1 h at RT, DBU (1.17 mL, 7.84 mmol) was added to
the reaction mixture at 0 °C. After being stirred for 8 h at 40 °C,
the reaction mixture was cooled to 0 °C, diluted with CHCl3,
quenched with sat. NaHCO3 aq and extracted with CHCl3. The com-
bined organic layer was washed with brine, dried over Na2SO4, fil-
tered and concentrated in vacuo. Purification by silica gel column
2
1
J = 3.2 Hz, 0.4 Hz, 1 H), 5.32 (d, J = 8.8 Hz, 1 H), 5.19-5.13 (m, 1
H), 5.06–5.00 (m, 1 H), 4.53 (dd, J = 11.6 Hz, 8.4 Hz, 1 H), 4.23
(dd, J = 11.2 Hz, 6.8 Hz, 1 H), 4.19 (dd, J = 11.2 Hz, 6.8 Hz, 1 H),
4.09 (td, J = 6.8 Hz, 0.8 Hz, 1 H), 3.89 (dt, J = 9.6 Hz, 6.4 Hz, 1 H),
3.43 (ddd, J = 9.6 Hz, 7.6 Hz, 6.8 Hz, 1 H), 2.50 (dd, J = 20.0 Hz,
8.0 Hz, 1 H), 2.35–1.98 (m, 15 H), 1.86 (s, 3 H), 1.79–1.74 (m, 1
H), 1.48–1.43 (m, 1 H), 1.45 (s, 9 H); 13C NMR (100 MHz, CDCl3) d
218.8, 171.3, 170.3, 170.3, 169.8, 168.2, 167.4, 134.2 (2 C), 131.4
(2 C), 127.8, 127.2, 123.6, 123.4, 98.6, 80.6, 70.7, 69.5, 68.0, 66.7,
61.3, 53.7, 51.3, 40.1, 38.0, 37.6, 28.0 (3 C), 27.5, 26.9, 25.2, 20.7,
20.6, 20.5; IR (film) 2977, 2933, 1776, 1750, 1718, 1389, 1369,
1239, 1154, 1131, 1107, 1076, 1047, 1017, 952, 761, 723;
chromatography (n-hexane/EtOAc = 3/1–2/1–1/1) gave 15
b = 2:1, 1.64 g, 3.44 mmol, 88% in 2 steps) as a colorless viscous oil.
15-
1H NMR (400 MHz, CDCl3) d 7.88–7.83 (m, 2 H, 2-NPhth),
(a:
a
7.78–7.74 (m, 2 H, 2-NPhth), 6.53 (dd, J = 12.0 Hz, 3.2 Hz, 1 H, 3-H),
6.34 (d, J = 3.2 Hz, 1 H, 1-H), 5.67 (dd, J = 3.2 Hz, 1.2 Hz, 1 H, 4-H),
4.91 (dd, J = 12.4 Hz, 3.2 Hz, 1 H, 2-H), 4.50 (td, J = 6.8 Hz, 1.2 Hz,
1 H, 5-H), 4.17 (dd, J = 11.2 Hz, 6.8 Hz, 1 H, 6-Ha), 4.13 (dd,
J = 11.2 Hz, 6.8 Hz, 1 H, 6-Hb), 2.19 (s, 3 H, 4-OAc), 2. 06 (s, 6 H,
½
a 2D6 + 38.4 (c 1, MeOH); HRMS (ESI, positive) m/z [M+Na]+ calcd
for C36H45N1O13Na 722.2789, found 722.2770.
ꢂ
4.7. Compound 19
13
1, 6-OAc), 1.89 (s, 3 H, 3-OAc); C NMR (100 MHz, CDCl3) d 170.3
(1 C, 6-OCOCH3), 170.1 (1 C, 4-OCOCH3), 169.4 (2 C, 1, 3-OCOCH3),
167.7 (1 C, 2-N (CO)2C6H4), 167.5 (1 C, 2-N(CO)2C6H4), 134.4 (2 C,
2-N(CO)2C6H4), 131.3 (2 C, 2-N(CO)2C6H4), 123.5 (2 C, 2-
N(CO)2C6H4), 91.2 (1 C, 1-C), 69.2 (1 C, 5-C), 67.0 (1 C, 4-C), 64.6
(1 C, 3-C), 61.3 (1 C, 6-C), 49.5 (1 C, 2-C), 20.9 (1 C, 1 or 6-OCOCH3),
20.6 (2 C, 1 or 6, 4-OCOCH3), 20.5 (1 C, 3-OCOCH3); IR (film) 3026,
1752, 1721, 1636, 1614, 1540, 1469, 1434, 1387, 1220, 1173, 1134,
1117, 1074, 1044, 1013, 948, 756, 723; HRMS (ESI, positive) m/z
[M+Na]+ calcd for C22H23N1O11Na 500.1169, found 500.1155. HR
To a solution of 18 (457.8 mg, 0.65 mmol) in MeOH (13 mL)
was added NaOMe (38.9 mg, 0.72 mmol) at 0 °C under N2. After
being stirred for 1 h at 0 °C, the reaction mixture was neutralized
with Amberlite IR120B, filtered and concentrated in vacuo. Purifi-
cation by silica gel column chromatography (CHCl3/MeOH = 50/1–
25/1–10/1) gave 19 (330.2 mg, 0.58 mmol, 88%) as a colorless
viscous oil.
1H NMR (400 MHz, CD3OD) d 7.88–7.83 (m, 2 H), 7.81 (dd,
J = 5.6 Hz, 3.2 Hz, 2 H), 5.19–4.95 (m, 2 H), 5.12 (d, J = 8.0 Hz, 1
H), 4.42 (dd, J = 11.2 Hz, 3.2 Hz, 1 H), 4.33 (dd, J = 11.2 Hz, 8.4 Hz,
1 H), 3.95 (dd, J = 2.8 Hz, 0.8 Hz, 1 H), 3.86 (dt, J = 10.0 Hz, 6.0 Hz,
1 H), 3.82 (dd, J = 11.2 Hz, 6.8 Hz, 1 H), 3.78 (dd, J = 11.2 Hz,
5.6 Hz, 1 H), 3.65 (ddd, J = 6.8 Hz, 5.6 Hz, 0.8 Hz, 1 H), 3.42 (ddd,
J = 9.6 Hz, 7.6 Hz, 6.0 Hz, 1 H), 2.50 (dd, J = 14.4 Hz, 3.2 Hz, 1 H),
2.31–1.96 (m, 9 H), 1.82–1.77 (m, 1 H), 1.50–1.44 (m, 1 H), 1.43
(s, 9 H); 13C NMR (100 MHz, CD3OD) d 221.4, 173.4, 170.3, 169.8,
135.5 (2 C), 133.2, 133.2, 128.9, 128.8, 124.4, 124.0, 100.4, 81.8,
77.0, 70.2, 70.0, 69.6, 62.5, 55.5, 55.0, 41.1, 39.3, 38.5, 28.7, 28.4
(3 C), 28.0, 26.2; IR (film) 3484, 2955, 1714, 1391, 1365, 1177,
MS was observed as a mixture of a- and b- isomers.
15-b 1H NMR (400 MHz, CDCl3) d7.88–7.83 (m, 2 H, 2-NPhth),
7.78–7.74 (m, 2 H, 2-NPhth), 6.45 (d, J = 8.8 Hz, 1 H, 1-H), 5.94
(dd, J = 11.6 Hz, 3.2 Hz, 1 H, 3-H), 5.52 (d, J = 3.6 Hz, 1 H, 4-H),
4.67 (dd, J = 11.6 Hz, 8.8 Hz, 1 H, 2-H), 4.26-4.17 (m,3 H, 5, 6-H),
2.22 (s, 3 H, 4-OAc), 2.07 (s, 3 H, 6-OAc), 2.01 (s, 3 H, 1-OAc),
1.86 (s, 3 H, 3-OAc); 13C NMR (100 MHz, CDCl3) d 170.3 (1 C, 6-
OCOCH3), 170.1 (1 C, 4-OCOCH3), 169.5 (1 C, 3-OCOCH3), 168.6 (1
C, 1-OCOCH3), 167.7 (1 C, 2-N (CO)2C6H4), 167.5 (1 C, 2-
N(CO)2C6H4), 134.4 (2 C, 2-N(CO)2C6H4), 131.3 (2 C, 2-N(CO)2C6H4),

M. Ueda et al. / Bioorg. Med. Chem. 20 (2012) 5832–5843
5839
1153, 1078, 722; ½a D26
ꢂ
+ 39.5 (c 0.5, MeOH); HRMS (ESI, positive) m/
1 H), 2.65–2.58 (m, 1 H), 2.46–2.04 (m, 9 H), 1.99–1.94 (m, 1 H),
1.58–1.48 (m, 1 H), 1.46 (s, 9 H).
z [M+Na]+ calcd for C30H39N1O10Na 596.2472, found 596.2466.
4.8. Compound 20
4.11. Compound 23
To a solution of 19 (303.8 mg, 0.53 mmol) in dehydrated CH2Cl2
(10.6 mL) were added NEt3 (177 lL, 1.27 mmol) and TsCl
To a suspension of 22 (24 mg) and MS4Å (130.0 mg) in dehy-
drated DMF (0.4 mL) was added Cs2CO3 (25.2 mg, 0.077 mmol) at
RT under an Ar atmosphere. After being stirred for 1.5 h at RT, 4-
(bromomethyl)trifluoromethyldiazirine24 (11.8 mg, 0.042 mmol)
was added at 0 °C in dark. After being stirred for further 3 h at
RT in dark, the reaction mixture was cooled to 0 °C, quenched with
AcOH (3 d), filtered through a pad of Celite and concentrated in va-
cuo. Purification by silica gel column chromatography (CHCl3/
MeOH = 1/0–100/1–80/1–60/1–40/1–20/1–10/1–5/1) gave 23
(14.2 mg, 0.021 mmol, 42% in two steps, 76% brsm) as a pale yellow
viscous oil. The start material 22 (10.7 mg, 0.023 mmol, 45%) was
recovered.
(121.0 mg, 0.63 mmol) at 0 °C under N2. After being slowly
warmed from 0 °C to RT, the reaction mixture was quenched with
MeOH and concentrated in vacuo. Purification by silica gel column
chromatography (CHCl3/MeOH = 100/1–50/1–20/1–10/1) gave 20
(297.5 mg, 0.41 mmol, 77%, 94% brsm) as a colorless viscous oil.
The start material 19 (53.2 mg, 0.093 mmol, 17%) was recovered.
1H NMR (400 MHz, CD3OD) d 7.83–7.79 (m, 6 H), 7.46 (d,
J = 8.0 Hz, 2 H), 5.18–4.98 (m, 2 H), 5.05 (d, J = 8.4 Hz, 1 H), 4.38
(dd, J = 11.2 Hz, 3.2 Hz, 1 H), 4.27 (dd, J = 11.2 Hz, 8.4 Hz, 1 H),
4.27 (dd, J = 10.8 Hz, 4.8 Hz, 1 H), 4.23 (dd, J = 10.8 Hz, 6.8 Hz, 1
H), 3.87–3.84 (m, 2 H), 3.72 (dt, J = 9.6 Hz, 6.0 Hz, 1 H), 3.36 (ddd,
J = 9.6 Hz, 7.2 Hz, 6.4 Hz, 1 H), 2.51 (dd, J = 15.2 Hz, 3.2 Hz, 1 H),
2.46 (s, 3 H), 2.31–1.97 (m, 9 H), 1.84–1.79 (m, 1 H), 1.53–1.46
(m, 1 H), 1.43 (s, 9 H); 13C NMR (100 MHz, CD3OD) d 221.4,
173.4, 170.1, 169.7, 146.6, 135.5 (2 C), 134.3, 133.1 (2 C), 131.2
(2 C), 129.1 (2 C), 128.9, 128.8, 124.4, 124.0, 100.2, 81.8, 74.1,
70.8, 70.3, 69.7, 69.1, 55.2, 54.9, 41.1, 39.3, 38.5, 28.7, 28.4 (3 C),
28.0, 26.2, 21.7; IR (film) 3484, 2955, 1714, 1391, 1365, 1177,
1H NMR (500 MHz, CD3OD) d 7.47 (d, J = 8.5 Hz, 2 H), 7.21 (d,
J = 8.5 Hz, 2 H), 5.54–5.39 (m, 2 H), 4.33 (d, J = 8.0 Hz, 1 H), 4.10
(d, J = 13.5 Hz, 1 H), 3.98–3.93 (m, 2 H), 3.67–3.60 (m, 3 H), 3.55
(dt, J = 9.5 Hz, 7.0 Hz, 1 H), 3.43 (dd, J = 10.5 Hz, 3.0 Hz, 1 H), 3.19
(dd, J = 11.5 Hz, 2.5 Hz, 1 H), 2.73 (dd, J = 10.5 Hz, 8.0 Hz, 1 H),
2.59 (dd, J = 13.5 Hz, 3.0 Hz, 1 H), 2.47–2.14 (m, 8 H), 2.09–2.02
(m, 1 H), 1.97–1.93 (m, 1 H), 1.55–1.46 (m, 1 H), 1.44 (s, 9 H);
13C NMR (125 MHz, CD3OD) d 221.4, 173.4, 143.8, 130.4 (2 C),
129.4, 129.1, 128.6, 127.7 (2 C), 123.7 (q, J = 274 Hz), 105.9, 81.8,
75.9, 73.5, 70.1, 70.0, 59.9, 55.1, 53.4, 52.6, 41.2, 39.5, 38.5, 29.5
(q, J = 40 Hz), 29.1, 28.4 (3 C), 28.0, 26.5; IR (film) 3433, 2972,
2931, 2878, 2100, 1730, 1613, 1519, 1457, 1368, 1343, 1233,
1153, 976, 722; ½a D25
ꢂ
+ 39.4 (c 0.5, MeOH); HRMS (ESI, positive)
m/z [M+Na]+ calcd for C37H45N1O12SNa 750.2560, found 750.2551.
4.9. Compound 21
1155, 1080, 1054, 938, 881, 844, 812, 739; ½a D24
ꢂ
+ 32.4 (c 0.3,
To a solution of 20 (12.2 mg, 0.017 mmol) in DMF (0.34 mL) was
added NaN3 (10.8 mg, 0.17 mmol) at RT under N2. After being stir-
red for 6 h at 100 °C, the reaction mixture was cooled to RT, diluted
with H2O and extracted with EtOAc. The combined organic layer
was washed with H2O and brine, dried over Na2SO4, filtered and
concentrated in vacuo. Purification by silica gel column chroma-
tography (CHCl3/MeOH = 300/1–100/1–50/1) gave 21 (9.6 mg,
0.016 mmol, 96%) as a pale yellow viscous oil.
MeOH); HRMS (ESI, positive) m/z [M+H]+ calcd for C31H42F3N6O7
667.3067, found 667.3061.
4.12. Compound ent-7
TFA (0.5 mL) was added to 23 (14.2 mg, 0.021 mmol) at 0 °C in
dark. After being stirring for 1 h at RT in dark, the reaction mixture
was concentrated in vacuo. Purification by HPLC (38% MeCN aq
containing 0.1% TFA, UV 227 nm) gave ent-7 (12.1 mg, 0.020 mmol,
93%) as a pale yellow viscous oil.
1H NMR (400 MHz, CD3OD) d 7.89–7.83 (m, 2 H), 7.81 (dd,
J = 5.2 Hz, 3.2 Hz, 2 H), 5.20–4.98 (m, 2 H), 5.16 (d, J = 8.4 Hz, 1
H), 4.44 (dd, J = 11.2 Hz, 3.2 Hz, 1 H), 4.34 (dd, J = 10.8 Hz, 8.4 Hz,
1 H), 3.89–3.82 (m, 3 H), 3.72 (dd, J = 12.8 Hz, 8.4 Hz, 1 H), 3.44
(ddd, J = 9.6 Hz, 7.6 Hz, 6.0 Hz, 1 H), 3.29 (dd, J = 12.8 Hz, 3.6 Hz,
1 H), 2.51 (dd, J = 14.4 Hz, 3.2 Hz, 1 H), 2.32–1.99 (m, 9 H), 1.84–
1.78 (m, 1 H), 1.48–1.44 (m, 1 H), 1.44 (s, 9 H); 13C NMR
(100 MHz, CD3OD) d 221.5, 173.4, 170.2, 169.8, 135.5 (2 C),
133.2, 133.1, 128.9, 128.8, 124.4, 124.0, 100.3, 81.8, 76.2, 70.5,
70.2, 69.3, 55.3, 55.0, 52.5, 41.1, 39.3, 38.5, 28.7, 28.4 (3 C), 28.0,
26.2; IR (film) 3465, 3006, 2977, 2933, 2101, 1774, 1714, 1391,
1368, 1335, 1280, 1257, 1154, 1116, 1072, 883, 793, 757, 722;
1H NMR (500 MHz, CD3OD) d 7.61 (d, J = 8.5 Hz, 2 H), 7.36 (d,
J = 8.0 Hz, 2 H), 5.53–5.45 (m, 2 H), 4.73 (d, J = 8.5 Hz, 1 H), 4.47
(d, J = 13.5 Hz, 1 H), 4.43 (d, J = 13.5 Hz, 1 H), 3.97 (dt, J = 9.5 Hz,
6.7 Hz,
1 H), 3.83 (dd, J = 11.0 Hz, 3.0 Hz, 1 H), 3.78 (dd,
J = 3.0 Hz, 1.0 Hz, 1 H), 3.74 (ddd, J = 8.5 Hz, 4.0 Hz, 1.0 Hz, 1 H),
3.65 (dt, J = 9.5 Hz, 6.7 Hz, 1 H), 3.64 (dd, J = 13.0 Hz, 8.5 Hz, 1 H),
3.24 (dd, J = 13.0 Hz, 4.0 Hz, 1 H), 3.16 (dd, J = 11.0 Hz, 8.5 Hz, 1
H), 2.65 (dd, J = 14.5 Hz, 3.5 Hz, 1 H), 2.56–2.19 (m, 8 H), 2.09–
1.99 (m, 2 H), 1.57–1.49 (m, 1 H); 13C NMR (125 MHz, CD3OD) d
221.7, 176.0, 134.3, 132.3 (2 C), 131.4, 129.6, 128.8, 128.4 (2 C),
123.5 (q, J = 274 Hz), 100.3, 76.1, 70.3, 70.3, 69.8, 59.6, 55.1, 52.2,
51.1, 39.7, 39.2, 38.6, 29.4 (q, J = 40 Hz), 28.7, 28.2, 26.4; IR (film)
3390, 3018, 2935, 2894, 2103, 1733, 1677, 1523, 1429, 1411,
1347, 1232, 1189, 1145, 1058, 940, 884, 840, 801, 759, 723;
½
a 2D7 + 51.8 (c 0.5, MeOH); HRMS (ESI, positive) m/z [M+Na]+ calcd
for C30H38N4O9Na 621.2536, found 621.2530.
ꢂ
4.10. Compound 22
½
a 2D2 + 17.0 (c 0.2, MeOH); HRMS (ESI, positive) m/z [M+H]+ calcd
for C27H34F3N6O7 611.2441, found 611.2442.
ꢂ
0.8 M Ethylenediamine in MeOH (1.1 mL) was added to 21
(30.3 mg, 0.51 mmol) at RT under N2. After being stirred for 26 h
at 40 °C, the reaction mixture was cooled to 0 °C, quenched with
AcOH (0.1 mL), diluted with EtOAc and sat. NaHCO3 aq, extracted
with EtOAc. The combined organic layer was washed with brine,
dried over Na2SO4, filtered and concentrated in vacuo. Crude amine
22 (24.0 mg) was obtained as a pale yellow viscous oil.
4.13. Compound 24
To a suspension of ent-1711 (43.6 mg, 0.15 mmol), 16 (142.5 mg,
0.29 mmol), Ag2CO3 (105.2 mg, 0.38 mmol) and MS4Å (440.0 mg) in
dehydrated CH2Cl2 (1.55 mL) was slowly added AgOTf (47.6 mg,
0.19 mmol) in dehydrated toluene (7.2 mL) at 0 °C under N2. After
being stirred for 1 h at RT in the dark, the reaction mixture was fil-
tered through a pad of Celite. The filtrate was washed with sat. NaH-
CO3 aq and brine, dried over Na2SO4, filtered and concentrated in
1H NMR (400 MHz, CD3OD) d 5.54–5.39 (m, 2 H), 4.22 (d,
J = 8.0 Hz, 1 H), 3.90 (dt, J = 9.6 Hz, 6.8 Hz, 1 H), 3.69–3.62 (m, 3
H), 3.55 (dt, J = 9.6 Hz, 6.8 Hz, 1 H), 3.40 (dd, J = 10.4 Hz, 3.2 Hz, 1
H), 3.21 (dd, J = 10.4 Hz, 1.2 Hz, 1 H), 2.90 (dd, J = 10.4 Hz, 8.0 Hz,

5840
M. Ueda et al. / Bioorg. Med. Chem. 20 (2012) 5832–5843
vacuo. Purification by silica gel column chromatography (toluene/
acetone = 50/1–40/1–30/1–20/1–15/1–10/1) gave 24 (86.5 mg,
0.12 mmol, 80%, 93% brsm) as a pale yellow viscous oil. The starting
material ent-17 (6 mg, 0.02 mmol, 14%) was recovered.
H), 1.83–1.78 (m, 1 H), 1.50–1.45 (m, 1 H), 1.43 (s, 9 H); 13C
NMR (100 MHz, CD3OD) d 221.4, 173.4, 170.1, 169.7, 146.6, 135.5
(2 C), 134.3, 133.1 (2 C), 131.2 (2 C), 129.1 (2 C), 128.9, 128.7,
124.3, 124.0, 100.2, 81.8, 74.1, 70.8, 70.3, 69.7, 69.1, 55.2, 54.9,
41.1, 39.3, 38.5, 28.7, 28.4 (3 C), 27.9, 26.3, 21.6; IR (film) 3478,
3009, 2970, 2929, 1774, 1714, 1391, 1366, 1177, 1152, 1087,
1H NMR (400 MHz, CDCl3) d 7.86 (br, 2 H), 7.76 (dd, J = 5.6 Hz,
3.2 Hz,
2 H), 5.78 (dd, J = 11.4 Hz, 3.0 Hz, 1 H), 5.48 (dd,
J = 3.4 Hz, 0.6 Hz, 1 H), 5.32 (d, J = 8.8 Hz, 1 H), 5.20–5.14 (m, 1
H), 5.08–5.02 (m, 1 H), 4.53 (dd, J = 11.2 Hz, 8.4 Hz, 1 H), 4.24
(dd, J = 11.2 Hz, 6.4 Hz, 1 H), 4.19 (dd, J = 11.2 Hz, 6.8 Hz, 1 H),
4.09 (ddd, J = 6.8 Hz, 6.4 Hz, 0.8 Hz, 1 H), 3.89 (dt, J = 9.6 Hz,
6.4 Hz, 1 H), 3.43 (ddd, J = 9.6 Hz, 7.6 Hz, 6.8 Hz, 1 H), 2.50 (dd,
J = 18.8 Hz, 8.0 Hz, 1 H), 2.35–1.99 (m, 15 H), 1.86 (s, 3 H), 1.79–
1.74 (m, 1 H), 1.47–1.40 (m, 1 H), 1.45 (s, 9 H); 13C NMR
(100 MHz, CDCl3) d 218.8, 171.3, 170.4, 170.3, 169.8, 168.2,
167.4, 134.3, 131.5, 129.0, 128.2, 127.9, 127.2, 123.6, 123.5, 98.6,
80.7, 70.8, 69.6, 68.1, 66.7, 61.4, 53.8, 51.4, 40.1, 38.1, 37.6, 28.1
(3 C), 27.5, 26.9, 25.3, 20.7, 20.7, 20.5; IR (film) 3478, 3016, 2975,
2933, 2895, 1776, 1750, 1723, 1469, 1431, 1389, 1370, 1338,
1239, 1216, 1154, 1131, 1107, 1073, 1047, 1016, 952, 915, 876,
979, 836, 816, 754, 722, 663, 554, 532; ½a D24
ꢀ 11.7 (c 1.0, MeOH);
ꢂ
HRMS (ESI, positive) m/z [M+Na]+ calcd for C37H45N1O12SNa
750.2560, found 750.2564.
4.16. Compound 27
To a solution of 26 (61.5 mg, 0.084 mmol) in DMF (1.7 mL) was
added NaN3 (55.1 mg, 0.85 mmol) at RT under N2. After being stir-
red for 7 h at 100 °C, the reaction mixture was cooled to RT, diluted
with H2O and extracted with EtOAc. The combined organic layer
was washed with H2O and brine, dried over Na2SO4, filtered and
concentrated in vacuo. Purification by silica gel column chroma-
tography (CHCl3/MeOH = 100/0–100/1–50/1–20/1) gave 27 (50.0
mg, 0.84 mmol, 99%) as a colorless viscous oil.
844, 760, 722, 633, 531; ½a D20
ꢀ 14.3 (c 1, MeOH); HRMS (ESI, posi-
ꢂ
tive) m/z [M+Na]+ calcd for C36H45N1O13Na 722.2789, found
1H NMR (400 MHz, CD3OD) d 7.88–7.84 (m, 2 H), 7.81 (dd,
J = 5.2 Hz, 3.2 Hz, 2 H), 5.20–5.14 (m, 1 H), 5.15 (d, J = 8.4 Hz, 1
H), 5.04–4.97 (m, 1 H), 4.43 (dd, J = 11.2 Hz, 3.2 Hz, 1 H), 4.34
(dd, J = 10.8 Hz, 8.4 Hz, 1 H), 3.89–3.82 (m, 3 H), 3.71 (dd,
J = 12.8 Hz, 8.4 Hz, 1 H), 3.44 (ddd, J = 10.0 Hz, 7.2 Hz, 6.0 Hz, 1
H), 3.29 (dd, J = 12.8 Hz, 4.0 Hz, 1 H), 2.50 (dd, J = 14.0 Hz, 3.2 Hz,
1 H), 2.30–1.98 (m, 9 H), 1.83–1.78 (m, 1 H), 1.51–1.42 (m, 1 H),
1.45 (s, 9 H); 13C NMR (100 MHz, CD3OD) d 221.4, 173.4, 170.2,
169.7, 135.5, 135.5, 133.2, 133.1, 128.9, 128.8, 124.3, 124.0,
100.3, 81.8, 76.2, 70.5, 70.2, 69.3, 55.3, 54.9, 52.5, 41.1, 39.4,
38.5, 28.6, 28.4 (3 C), 27.9, 26.3; IR (film) 3461, 3011, 2977,
2933, 2889, 2101, 1774, 1714, 1391, 1368, 1335, 1280, 1257,
722.2788.
4.14. Compound 25
To a solution of 24 (83.1 mg, 0.12 mmol) in MeOH (2.4 mL) was
added NaOMe (7.1 mg, 0.13 mmol) at 0 °C under N2. After being
stirred for 1.9 h at 0 °C, the reaction mixture was neutralized with
Amberlite IR120B, filtered and concentrated in vacuo. Purification
by silica gel column chromatography (CHCl3/MeOH = 50/1–20/1–
10/1) gave 25 (62.4 mg, 0.11 mmol, 92%) as a colorless viscous oil.
1H NMR (400 MHz, CD3OD) d 7.88–7.83 (m, 2 H), 7.81 (dd,
J = 5.6 Hz, 3.2 Hz, 2 H), 5.19–5.14 (m, 1 H), 5.11 (d, J = 8.0 Hz, 1
H), 5.01–4.95 (m, 1 H), 4.41 (dd, J = 11.2 Hz, 3.2 Hz, 1 H), 4.35
(dd, J = 11.2 Hz, 8.0 Hz, 1 H), 3.95 (dd, J = 2.8 Hz, 0.8 Hz, 1 H),
3.87 (dt, J = 10.0 Hz, 6.0 Hz, 1 H), 3.82 (dd, J = 11.2 Hz, 6.8 Hz, 1
H), 3.78 (dd, J = 11.2 Hz, 5.6 Hz, 1 H), 3.65 (ddd, J = 6.8 Hz, 5.6 Hz,
0.8 Hz, 1 H), 3.42 (ddd, J = 9.6 Hz, 7.2 Hz, 6.4 Hz, 1 H), 2.49 (dd,
J = 14.0 Hz, 3.2 Hz, 1 H), 2.30–1.97 (m, 9 H), 1.82–1.77 (m, 1 H),
1.50–1.40 (m, 1 H), 1.43 (s, 9 H); 13C NMR (100 MHz, CD3OD) d
221.4, 173.4, 170.2, 169.8, 135.5, 135.4, 133.2, 133.1, 128.8 (2 C),
124.3, 124.0, 100.4, 81.8, 77.0, 70.2, 70.0, 69.6, 62.5, 55.5, 54.9,
41.1, 39.3, 38.5, 28.7, 28.4 (3 C), 27.9, 26.2; IR (film) 3473, 3006,
2972, 2939, 2900, 1773, 1713, 1391, 1368, 1337, 1256, 1154,
1153, 1115, 1072, 883, 793, 756, 722; ½a D21
ꢀ 0.2 (c 1.0, MeOH);
ꢂ
HRMS (ESI, positive) m/z [M+Na]+ calcd for C30H38N4O9Na
621.2536, found 621.2521.
4.17. Compound 28
0.8 M Ethylenediamine in MeOH (2 mL) was added to 27
(50.0 mg, 0.84 mmol) at RT under N2. After being stirred for 40 h
at 40 °C, the reaction mixture was cooled to 0 °C, quenched with
AcOH (0.2 mL), diluted with EtOAc and sat. NaHCO3 aq extracted
with EtOAc. The combined organic layer was washed with brine,
dried over Na2SO4, filtered and concentrated in vacuo. Crude amine
28 (36.3 mg) was obtained as an yellow viscous oil.
1115, 1073, 757, 723; ½a D22
ꢂ
–25.3 (c 1.0, MeOH); HRMS (ESI, posi-
tive) m/z [M+Na]+ calcd for C30H39N1O10Na 596.2472, found
596.2463.
4.18. Compound 29
4.15. Compound 26
To a suspension of 28 (36.3 mg) and MS4Å (200.0 mg) in dehy-
drated DMF (0.7 mL) was added Cs2CO3 (30 mg, 0.092 mmol) at RT
under an Ar atmosphere. Then, 4-(bromomethyl)trifluoromet
hyldiazirine (22.0 mg, 0.079 mmol) in dehydrated DMF (0.7 mL)
was added at 0 °C in dark. After being stirred for 10 h at RT in dark,
the reaction mixture was cooled to 0 °C, quenched with AcOH
(0.2 mL), filtered through a pad of Celite and concentrated in vacuo.
Purification by silica gel column chromatography (CHCl3/
MeOH = 1/0–100/1–80/1–60/1–40/1–20/1–10/1–5/1) gave 29
(25.0 mg, mixture) as a brown viscous oil. The starting material
28 (15.3 mg, 0.033 mmol, 42%) was recovered.
To a solution of 25 (62.4 mg, 0.11 mmol) in dehydrated CH2Cl2
(2.2 mL) were added NEt3 (36.4 lL, 0.26 mmol) and TsCl
(24.9 mg, 0.13 mmol) at 0 °C under an Ar atmosphere. After being
stirred for 11 h when it was slowly warmed from 0 °C to RT, the
reaction mixture was quenched with MeOH and concentrated in
vacuo. Purification by silica gel column chromatography (CHCl3/
MeOH = 100/1–50/1–20/1–10/1) gave 26 (59.4 mg, 0.082 mmol,
75%, 90% brsm) as a colorless viscous oil. The starting material 25
(10.3 mg, 0.018 mmol 17%) was recovered.
1H NMR (400 MHz, CD3OD) d 7.86–7.79 (m, 6 H), 7.45 (d,
J = 8.0 Hz, 2 H), 5.18–5.10 (m, 1 H), 5.05 (d, J = 8.4 Hz 1 H), 5.05–
4.98 (m, 1 H), 4.37 (dd, J = 11.2 Hz, 3.2 Hz, 1 H), 4.28 (dd,
J = 11.2 Hz, 8.4 Hz, 1 H), 4.28 (dd, J = 10.4 Hz, 4.8 Hz, 1 H), 4.24
4.19. Compound 8
TFA (1.5 mL) was added to 29 (25.0 mg, mixture) at 0 °C in dark.
After being stirring for 2 h at RT in dark, the reaction mixture was
concentrated in vacuo. Purification by HPLC (38% MeCN aq
(dd, J = 10.4 Hz, 7.6 Hz, 1 H), 3.87–3.84 (m,
J = 10.0 Hz, 6.0 Hz, 1 H), 3.36 (ddd, J = 9.6 Hz, 7.2 Hz, 6.4 Hz, 1 H),
2.50 (dd, J = 13.6 Hz, 2.8 Hz, 1 H), 2.46 (s, 3 H), 2.30–1.98 (m, 9
2 H), 3.73 (dt,

M. Ueda et al. / Bioorg. Med. Chem. 20 (2012) 5832–5843
5841
containing 0.1% TFA, UV 227 nm) gave 8 (9.0 mg, 0.015 mmol, 18%
in 3 steps, 29% brsm) as a pale yellow viscous oil.
135.5, 135.5, 133.2, 133.1, 128.9, 128.8, 124.3, 124.0, 100.2, 81.8,
76.2, 70.5, 70.2, 69.3, 55.3, 54.9, 52.5, 41.1, 39.3, 38.5, 28.6, 28.4,
27.9, 26.3; IR (film) 3464, 3011, 2977, 2933, 2889, 2101, 1774,
1714, 1391, 1368, 1335, 1280, 1257, 1153, 1115, 1073, 883, 793,
1H NMR (500 MHz, CD3OD) d 7.61 (d, J = 8.5 Hz, 2 H), 7.35 (d,
J = 8.0 Hz, 2 H), 5.53–5.45 (m, 2 H), 4.74 (d, J = 8.5 Hz, 1 H), 4.48
(d, J = 13.0 Hz, 1 H), 4.43 (d, J = 13.0 Hz, 1 H), 3.97 (dt, J = 9.0 Hz,
7.0 Hz, 1 H), 3.84 (dd, J = 11.0 Hz, 3.0 Hz, 1 H), 3.77 (dd, J = 3 Hz,
0.5 Hz, 1 H), 3.75 (ddd, J = 8.5 Hz, 4.0 Hz, 1.0 Hz, 1 H), 3.67 (dt,
J = 9.0 Hz, 7.0 Hz, 1 H), 3.64 (dd, J = 13.0 Hz, 8.5 Hz, 1 H), 3.24
(dd, J = 13.0 Hz, 4.0 Hz, 1 H), 3.16 (dd, J = 11.0 Hz, 8.5 Hz, 1 H),
2.65 (dd, J = 14.0 Hz, 3.5 Hz, 1 H), 2.55–2.27 (m, 7 H), 2.24–2.18
(m, 1 H), 2.08–1.97 (m, 2 H), 1.57–1.48 (m, 1 H); 13C NMR
(125 MHz, CD3OD) d 221.6, 176.0, 134.3, 132.3 (2 C), 131.4,
129.6, 128.9, 128.4 (2 C), 123.5 (q, J = 274 Hz), 100.3, 76.2, 70.3,
70.3, 69.8, 59.6, 55.1, 52.2, 51.1, 39.7, 39.2, 38.6, 29.4 (q,
J = 40 Hz), 28.9, 28.2, 26.5; IR (film) 3356, 3019, 2960, 2938,
2895, 2103, 1732, 1671, 1436, 1347, 1232, 1190, 1153, 1058,
758, 722; ½a 2D3
ꢂ
+ 2.0 (c 1.0, MeOH); HRMS (ESI, positive) m/z
[M+Na]+ calcd for C30H38N4O9Na 621.2536, found 621.2523.
4.22. Compound 33
0.8 M Ethylenediamine in MeOH (3.2 mL) was added to 32
(96.4 mg, 0.16 mmol) at RT under N2. After being stirred for 40 h
at 40 °C, the reaction mixture was cooled to 0 °C, quenched with
AcOH (0.3 mL), diluted with EtOAc and sat. NaHCO3 aq, extracted
with EtOAc. The combined organic layer was washed with brine,
dried over Na2SO4, filtered and concentrated in vacuo. Crude amine
33 (71.0 mg) was obtained as a yellow viscous oil.
940, 839, 799, 759, 722; ½a D23
ꢀ 16.9 (c 0.5, MeOH); HRMS (ESI, po-
ꢂ
sitive) m/z [M+H]+ calcd for C27H34F3N6O7 611.2441, found
4.23. Compound 34
611.2415.
To a suspension of 33 (57.7 mg) and MS4Å (620.0 mg) in dehy-
4.20. Compound 31
drated DMF (1.2 mL) was added Cs2CO3 (61.0 mg, 0.18 mmol) at RT
under
an
Ar
atmosphere.
Then,
4-(bromomethyl)trif-
To a suspension of 17 (92.2 mg, 0.33 mmol), 3024 (339.8 mg,
0.71 mmol), Ag2CO3 (260.0 mg, 0.94 mmol) and MS4Å (1.2 g) in
dehydrated CH2Cl2 (3.5 mL) was slowly added AgOTf (114 mg,
0.44 mmol) in dehydrated toluene (3.5 mL) at 0 °C under N2. After
being stirred for 0.5 h at RT in the dark, the reaction mixture was
filtered through a pad of Celite. The filtrate was washed with sat.
NaHCO3 aq and brine, dried over Na2SO4, filtered and concentrated
in vacuo. Purification by silica gel column chromatography (tolu-
luoromethyldiazirine (33.0 mg, 0.12 mmol) was added at 0 °C in
dark. After being stirred for 25 h at RT in dark, the reaction mixture
was cooled to 0 °C, quenched with AcOH (4 d), filtered through a
pad of Celite and concentrated in vacuo. Purification by silica gel
column chromatography (CHCl3/MeOH = 1/0–100/1–80/1–60/1–
40/1–20/1–10/1–5/1) gave 34 (28.2 mg, mixture) as a yellow vis-
cous oil. The starting material 33 (28.3 mg, 0.060 mmol, 49%)
was recovered.
ene/acetone = 50/1–40/1–30/1–20/1–15/1–10/1)
(212.6 mg, 0.31 mmol, 95%) as a pale colorless viscous oil.
1H NMR (400 MHz, CDCl3) d 7.86 (br, 2 H), 7.76 (dd, J = 5.2 Hz,
3.2 Hz, H), 5.77 (dd, J = 11.2 Hz, 3.2 Hz, H), 5.43 (dd,
gave
31
4.24. Compound ent-8
2
1
TFA (1.0 mL) was added to 35 (28.2 mg, mixture) at 0 °C in dark.
After being stirring for 2 h at RT in dark, the reaction mixture was
concentrated in vacuo. Purification by HPLC (35% MeCN aq con-
taining 0.1% TFA, UV 227 nm) gave ent-8 (14.1 mg, 0.023 mmol,
18% in 3 steps) as a pale yellow viscous oil.
J = 3.2 Hz, 0.4 Hz, 1 H), 5.34 (d, J = 8.8 Hz, 1 H), 5.17 (dt, J = 10.8,
7.2 Hz, 1 H), 5.06 (dt, J = 10.4, 7.6 Hz, 1 H), 4.54 (dd, J = 11.2,
8.8 Hz, 1 H), 4.03 (ddd, J = 8.4, 4.0, 0.4 Hz, 1 H), 3.93 (dt, J = 9.6,
6.4 Hz, 1 H), 3.61 (dd, J = 12.8, 8.0 Hz, 1 H), 3.46 (dt, J = 9.6,
7.2 Hz, 1 H), 3.17 (dd, J = 12.8, 4.0 Hz, 1 H), 2.50 (dd, J = 18.8 Hz,
8.4 Hz, 1 H), 2.35–1.99 (m, 12 H), 1.85 (s, 3 H), 1.79–1.74 (m, 1
H), 1.49–1.41 (m, 1 H), 1.45 (s, 9 H); 13C NMR (100 MHz, CDCl3) d
218.8, 171.3, 170.2, 169.7, 168.2, 167.4, 134.2 (2 C), 131.4 (2 C),
127.9, 127.1, 123.5, 123.4, 98.5, 80.6, 72.9, 69.5, 68.0, 67.6, 53.7,
51.2, 50.6, 40.0, 38.0, 37.5, 28.0 (3 C), 27.4, 26.8, 25.3, 20.7, 20.4;
IR (film) 3484, 3013, 2977, 2935, 2891, 2013, 1777, 1752, 1711,
1388, 1368, 1237, 1154, 1123, 1107, 1069, 1052, 1011, 952, 760,
1H NMR (400 MHz, CD3OD) d 7.61 (d, J = 8.0 Hz, 2 H), 7.35 (d,
J = 8.4 Hz, 2 H), 5.54–5.44 (m, 2 H), 4.74 (d, J = 8.0 Hz, 1 H), 4.48
(d, J = 13.2 Hz, 1 H), 4.43 (d, J = 13.2 Hz, 1 H), 3.98 (dt, J = 9.2 Hz,
6.8 Hz, 1 H), 3.84 (dd, J = 11.2, 3.2 Hz, 1 H), 3.78 (dd, J = 3.2,
0.8 Hz, 1 H), 3.75 (ddd, J = 8.8, 3.6 Hz, 0.8 Hz, 1 H), 3.66 (dt,
J = 9.2, 6.8 Hz, 1 H), 3.64 (dd, J = 12.8, 8.8 Hz, 1 H), 3.24 (dd,
J = 12.8, 4.0 Hz, 1 H), 3.15 (dd, J = 11.2, 8.0 Hz, 1 H), 2.65 (dd,
J = 14.0 Hz, 3.2 Hz, 1 H), 2.54–2.26 (m, 7 H), 2.24–2.18 (m, 1 H),
2.08–1.96 (m, 2 H), 1.57–1.47 (m, 1 H); 13C NMR (100 MHz, CD3OD)
d 221.6, 175.9, 134.4, 132.3, 131.4, 129.6, 128.9, 128.4, 123.5 (q,
J = 273 Hz), 100.3, 76.1, 70.3, 70.3, 69.8, 59.6, 55.1, 52.3, 51.1,
39.8, 39.2, 38.6, 29.4 (q, J = 40 Hz), 28.9, 28.2, 26.5; IR (film)
3383, 3017, 2933, 2895, 2103, 1732, 1673, 1408, 1347, 1232,
722; ½a 2D5
ꢂ
+ 8.4 (c 1.0, MeOH); HRMS (ESI, positive) m/z [M+Na]+
calcd for C34H42N4O11Na 705.2748, found 705.2722.
4.21. Compound 32
To a solution of 31 (201.8 mg, 0.30 mmol) in MeOH (6.0 mL)
was added NaOMe (16.0 mg, 0.30 mmol) at 0 °C under N2. After
being stirred for 1.8 h at 0 °C, the reaction mixture was neutralized
with Amberlite IR120B, filtered and concentrated in vacuo. Purifi-
cation by silica gel column chromatography (n-Hexane/EtOAc = 5/
1–4/1–3/1–2/1–1/1) gave 32 (149.6 mg, 0.25 mmol, 85%) as a col-
orless viscous oil.
1188, 1154, 1058, 940, 845, 800, 759, 721; ½a D22
ꢂ
+ 17.0 (c 1.0,
MeOH); HRMS (ESI, positive) m/z [M+H]+ calcd for C27H34F3N6O7
611.2441, found 611.2431.
4.25. Cell-shrinking assay using motor cell protoplasts of S.
saman
1H NMR (400 MHz, CD3OD) d 7.88–7.84 (m, 2 H), 7.81 (dd,
J = 5.6 Hz, 3.2 Hz, 2 H), 5.20–5.13 (m, 1 H), 5.15 (d, J = 8.4 Hz, 1
H), 5.04–4.97 (m, 1 H), 4.43 (dd, J = 11.2 Hz, 3.2 Hz, 1 H), 4.34
(dd, J = 11.2 Hz, 8.4 Hz, 1 H), 3.89–3.82 (m, 3 H), 3.71 (dd,
J = 12.8 Hz, 8.0 Hz, 1 H), 3.42 (td, J = 10.0 Hz, 6.8 Hz, 1 H), 3.29
(dd, J = 12.8 Hz, 3.6 Hz, 1 H), 2.50 (dd, J = 13.6 Hz, 3.2 Hz, 1 H),
2.30–1.98 (m, 9 H), 1.83–1.78 (m, 1 H), 1.51–1.40 (m, 1 H), 1.44
(s, 9 H); 13C NMR (100 MHz, CD3OD) d 221.4, 173.4, 170.2, 169.7,
The protoplasts were prepared from S. saman using the extensor
(adaxial) part of the tertiary pulvini on the 2nd or 3rd branch from
the short apex according to the previously reported method (Gor-
ton and Satter, 1984; Moran et al., 1990; Nakamura et al., 2008).
They were suspended in a wash solution (0.57 M sorbitol, 10 mM
KCl, 1 mM CaCl2, 20 mM MES-Tris, pH 5.5), loaded on a histopaque
solution (0.5 M sorbitol, 2 mM CaCl2, 90 v/v%), and centrifuged at
1500ꢁg for 5 min. The purified protoplasts found in the middle

5842
M. Ueda et al. / Bioorg. Med. Chem. 20 (2012) 5832–5843
green layer were collected, suspended in wash buffer, centrifuged
at 110ꢁg for 5 min,and the washing step was then repeated. The
freshly prepared protoplasts were suspended in 1.5 mL wash buf-
fer and maintained on ice in the dark. The protoplasts were centri-
fuged at 110ꢁg for 5 min and resuspended in a small volume of
wash buffer before use.
Acknowledgments
This work was supported by a Grant-in-Aid for Scientific Re-
search on Innovative Areas ‘Chemical Biology of Natural Products’
from The Ministry of Education, Culture, Sports, Science and Tech-
nology, Japan, and Grants-in-Aid for Scientific Research (No.
23310147), JSPS Bilateral Programs from JSPS, Japan.
The prepared protoplasts in 50
glass-bottom petri dish (
Milli Q, placed under an inverted microscope (IX-71, Olympus, To-
l
L wash solution were sealed in a
L of
u
35 mm ꢁ 12 mm) coated with 300
l
Supplementary data
kyo, Japan), and monitored at 24 1 °C under continuous irradiation
with light (50
(43IF550-W45, Olympus, Tokyo, Japan), as previously reported.8
Following incubation on the microscope for 6 min, 50 L of a
l
mol mꢀ2 sꢀ1 PAR) passed through a green filter
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.08.003.
l
solution of the different compounds (each at 1 ꢁ 10ꢀ8 mol in wash
solution, azide compounds include an additional 2% DMSO) was
added to the protoplast suspension. In the experiment using a K+
channel blocker, the protoplasts were incubated with 1 mM tetra-
ethyleneammonium chloride (TEA) for 30 min before exposure to 7
or JAG. The status of the protoplasts was recorded with time-lapse
photography (10 min intervals) for 40 min using a digital camera
(DP 72, Olympus, Tokyo, Japan) and DP2-BSW analysis software
References and notes
1. Yan, J.; Zhang, C.; Gu, M.; Bai, Z.; Zhang, W.; Qi, T.; Cheng, Z.; Peng, W.; Luo, H.;
Nan, F.; Wang, Z.; Xie, D. Plant Cell 2009, 21, 2220.
2. Wasternack, C.; Kombrink, E. ACS Chem. Biol. 2010, 5, 63.
3. Fonseca, S.; Chini, A.; Hamberg, M.; Adie, B.; Porzel, A.; Kramell, R.; Miersch, O.;
Wasternack, C.; Solano, R. Nat. Chem. Biol. 2009, 5, 344.
4. Ueda, M.; Okazaki, M.; Ueda, K.; Yamamura, S. Tetrahedron 2000, 56,
8101.
5. Ueda, M.; Nakamura, Y. Nat. Prod. Rep. 2006, 23, 548.
6. Ueda, M.; Nakamura, Y. Plant Cell Physiol. 2007, 48, 900.
7. Yoshihara, T.; Omer, E.-S. A.; Koshino, H.; Sakamura, S.; Kikuta, Y.; Koda, Y.
Agric. Biol. Chem. 1989, 53, 2835.
8. Nakamura, Y.; Mithofer, A.; Kombrink, E.; Boland, W.; Hamamoto, S.; Uozumi,
N.; Tohma, K.; Ueda, M. Plant Physiol. 2011, 155, 1226.
9. Lomenick, B.; Olsen, R. W.; Huang, J. ACS Chem. Biol. 2010, 6, 34.
10. Bottcher, T.; Pitscheider, M.; Sieber, S. A. Angew. Chem., Int. Ed. Engl. 2010, 49,
2680.
11. Nakamura, Y.; Miyatake, R.; Ueda, M. Angew. Chem., Int. Ed. Engl. 2008, 47, 7289.
We used galactoside instead of glucoside as a molecular probe to supress its
hydrolytic degradation in vivo by glycosidase.
12. Sato, S.; Murata, A.; Orihara, T.; Shirakawa, T.; Suenaga, K.; Kigoshi, H.; Uesugi,
M. Chem. Biol. 2011, 18, 131.
13. Ueda, M.; Manabe, Y.; Otsuka, Y.; Kanzawa, N. Chem., Asian J. 2011, 6,
3286.
14. Ueda, M.; Manabe, Y.; Mukai, M. Bioorg. Med. Chem. Lett. 2011, 21,
1359.
15. Speers, A. E.; Adam, G. C.; Cravatt, B. F. J. Am. Chem. Soc. 2003, 125, 4686.
16. Ballell, L.; Alink, K. J.; Slijper, M.; Versluis, C.; Liskamp, R. M.; Pieters, R. J.
ChemBioChem 2005, 6, 291.
17. Best, M. D. Biochemistry (Mosc). 2009, 48, 6571.
18. Tornøe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002, 67, 3057.
19. Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed. Engl. 2004,
2001, 40.
20. Codelli, J. A.; Baskin, J. M.; Agard, N. J.; Bertozzi, C. R. J. Am. Chem. Soc. 2008, 130,
11486.
21. Ning, X.; Temming, R. P.; Dommerholt, J.; Guo, J.; Ania, D. B.; Debets, M. F.;
Wolfert, M. A.; Boons, G. J.; van Delft, F. L. Angew. Chem., Int. Ed. Engl. 2010, 49,
3065.
22. Sletten, E. M.; Bertozzi, C. R. Angew. Chem., Int. Ed. Engl. 2009, 48, 6974.
23. Jewett, J. C.; Bertozzi, C. R. Chem. Soc. Rev. 2010, 39, 1272.
24. Nakamura, Y.; Inomata, S.; Ebine, M.; Manabe, Y.; Iwakura, I.; Ueda, M. Org.
Biomol. Chem. 2011, 9, 83.
(DP 72, Olympus, Tokyo, Japan). Finally, 100 lL of 0.02% fluorescein
diacetate (FDA) was added to enable the selection of living protop-
lasts. The semidiameter for each magnified image of living protop-
lasts was measured by DP2-BSW analysis software (DP 72,
Olympus, Tokyo, Japan).
4.26. Viability of protoplasts under the UV irradiation condition
A suspension of protoplasts was prepared at the concentration
of 5 ꢁ 105 cells/mL in buffer A (20 mM MES-Tris (pH 5.5), 0.57 M
D-sorbitol, 10 mM KCl, and 1 mM CaCl2). A solution of probe 8 in
DMSO (10 mM, 1 lL) was added to 19 lL of the protoplast suspen-
sion. The mixture was incubated under different conditions, as fol-
lows. Condition 1: the mixture was incubated at 4 °C for 5 min, and
then, irradiated with black light at 4 °C for 20 min. Condition 2: the
mixture was incubated at 4 °C for 20 min, and then, irradiated with
black light at 4 °C for 5 min. After irradiation under each condition,
living protoplasts were counted under a microscope after staining
with 20 lL of 0.4% (w/v) Trypan Blue solution (Wako Pure Chemi-
cal Industries, Ltd.). The probe solution was replaced with DMSO or
buffer A for the control or intact condition, respectively. The basal
viability of the prepared protoplasts was calculated from the ratio
of living cells to total cells. Then, the viability for each condition
was calculated from the ratio of the number of living protoplasts
under a given condition to that under basal conditions. Fig. 3 shows
the average and SD of Individual experiments performed in
quadruplicate.
25. Guazzelli, L.; Catelani, G.; D’Andrea, F.; Giannarelli, A. Carbohydr. Res. 2009, 344,
298.
26. Bongat, A. F.; Demchenko, A. V. Carbohydr. Res. 2007, 342, 374.
27. Lemieux, R. U.; Ratcliffe, R. M. Can. J. Chem.: Revue Canadienne De Chimie 1979,
57, 1244.
4.27. Viability of protoplasts under the CuAAC condition
28. Lafont, D.; Boullanger, P. Tetrahedron-Asymmetry 2006, 17, 3368.
29. Raposo, M. M. M.; Sampaio, A. M. B. A.; Kirsch, G. J. Heterocycl. Chem. 2005, 42,
1245.
30. Nakamura, Y.; Miyatake, R.; Matsubara, A.; Kiyota, H.; Ueda, M. Tetrahedron
2006, 62, 8805.
A suspension of protoplasts was prepared at the concentration
of 5 ꢁ 105 cells/mL in buffer A (20 mM MES-Tris (pH 5.5), 0.57 M
D-sorbitol, 10 mM KCl, 1 mM CaCl2). The suspension was incu-
bated at 4 °C for 30 min under different conditions, as follows. Con-
dition 1 was based on the reports by Finn et al.35,36 and included
31. Coté, G. G. Plant Physiol. 1995, 109, 729.
32. Gorton, H. L.; Satter, R. L. Plant Physiol. 1984, 76, 680.
33. Moran, N.; Ehrenstein, G.; Iwasa, K.; Mischke, C.; Bare, C.; Satter, R. L. Plant
Physiol. 1988, 88, 643.
34. Nakamura, Y.; Miyatake, R.; Inomata, S.; Ueda, M. Biosci. Biotechnol. Biochem.
2008, 72, 2867.
35. Hong, V.; Presolski, S. I.; Ma, C.; Finn, M. G. Angew. Chem., Int. Ed. Engl. 2009, 48,
9879.
36. Hong, V.; Steinmetz, N. F.; Manchester, M.; Finn, M. G. Bioconjug. Chem. 2010,
21, 1912.
37. Manabe, Y.; Mukai, M.; Ito, S.; Kato, N.; Ueda, M. Chem. Commun. (Camb) 2010,
46, 469.
38. Wasternack, C. Ann. Bot. 2007, 100, 681.
39. Thines, B.; Katsir, L.; Melotto, M.; Niu, Y.; Mandaokar, A.; Liu, G.; Nomura, K.;
He, S. Y.; Howe, G. A.; Browse, J. Nature 2007, 448, 661.
the following reagents: 50 lM CuSO4, 250 lM THPTA ligand,
2.5 mM sodium ascorbate, 0.5 mM alkyne-FLAG 35, 7.5% DMSO.
Condition II was based on our previous report13 and included the
following reagents: 500 lM CuSO4, 500 lM THPTA ligand,
0.5 mM sodium ascorbate, 0.5 mM alkyne-FLAG 35, 7.5% DMSO.
The effectiveness of each condition was calculated from the num-
ber of living protoplasts in the same manner as described in ‘viabil-
ity of protoplasts under the UV irradiation condition.’ Fig. 3 shows
the average and SD of individual experiments performed in
triplicate.

M. Ueda et al. / Bioorg. Med. Chem. 20 (2012) 5832–5843
5843
40. Chini, A.; Fonseca, S.; Fernandez, G.; Adie, B.; Chico, J. M.; Lorenzo, O.; Garcia-
Casado, G.; Lopez-Vidriero, I.; Lozano, F. M.; Ponce, M. R.; Micol, J. L.; Solano, R.
Nature 2007, 448, 666.
42. Paula, S.; Tabet, M. R.; Ball, W. J., Jr. Biochemistry 2005, 44, 498.
43. Ogawa, H.; Shinoda, T.; Cornelius, F.; Toyoshima, C. Proc. Natl. Acad. Sci. U.S.A.
2009, 106, 13742.
41. Sheard, L. B.; Tan, X.; Mao, H.; Withers, J.; Ben-Nissan, G.; Hinds, T. R.;
Kobayashi, Y.; Hsu, F. F.; Sharon, M.; Browse, J.; He, S. Y.; Rizo, J.; Howe, G. A.;
Zheng, N. Nature 2010, 468, 400.
44. Ueda, M. Chem. Lett. 2012, 41, 658.