Synthesis and properties of double-stranded RNA-bindable oligodiaminogalactose derivatives conjugated with vitamin e

Article abstract of DOI:10.1016/j.bmc.2013.12.060

RNA interference (RNAi) is a gene-regulating system that is controlled by external short interfering RNAs (siRNAs). Sequence selective gene silencing by siRNA shows promise in clinical research. However, there have been few efficient methods for delivering siRNAs to target cells. In this study, we propose a novel type of RNA duplex-bindable molecule with an oligodiaminosaccharide structure. These 2,6-diamino-2,6-dideoxy-(1-4)-β-d-galactopyranose oligomers (oligodiaminogalactoses; ODAGals) conjugated with α-tocopherol (vitamin E; VE) or a VE analog were designed as novel siRNA-bindable molecules that can be utilized to deliver RNAi drugs to the liver. Among these compounds, the VE analog-bound ODAGal was suggested to bind to RNA duplexes without inhibiting RNAi activity.

Full text of DOI:10.1016/j.bmc.2013.12.060

Bioorganic & Medicinal Chemistry 22 (2014) 1394–1403
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and properties of double-stranded RNA-bindable
oligodiaminogalactose derivatives conjugated with vitamin E
Rintaro Iwata a,b, Kazutaka Nishina c, Takanori Yokota c, Takeshi Wada a,b,
⇑
a Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan
b Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Bioscience Building 702, 5-1-5 Kashiwanoha, Kashiwa,
Chiba 277-8562, Japan
c Department of Neurology and Neurological Science, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
RNA interference (RNAi) is a gene-regulating system that is controlled by external short interfering RNAs
(siRNAs). Sequence selective gene silencing by siRNA shows promise in clinical research. However, there
have been few efficient methods for delivering siRNAs to target cells. In this study, we propose a novel
type of RNA duplex-bindable molecule with an oligodiaminosaccharide structure. These 2,6-diamino-
Received 10 October 2013
Revised 24 December 2013
Accepted 27 December 2013
Available online 4 January 2014
2,6-dideoxy-(1-4)-b-D-galactopyranose oligomers (oligodiaminogalactoses; ODAGals) conjugated with
a
-tocopherol (vitamin E; VE) or a VE analog were designed as novel siRNA-bindable molecules that
Keywords:
Oligodiaminogalactose
Vitamin E
RNA interference
RNA duplex
can be utilized to deliver RNAi drugs to the liver. Among these compounds, the VE analog-bound ODAGal
was suggested to bind to RNA duplexes without inhibiting RNAi activity.
Ó 2014 Elsevier Ltd. All rights reserved.
Drug delivery system
1. Introduction
be used in large doses to form nanoparticles, and suffer from cyto-
toxicity because of the increased quantities of cations.9–11
Since the discovery of RNA interference (RNAi),1 short interfer-
ing RNAs (siRNAs) have been receiving a lot of attention as candi-
dates for next-generation drugs.2–4 SiRNAs are composed of
double-stranded RNA. Because they target complementary se-
quences of mRNAs, siRNAs promise high target specificity. Further-
more, RNA molecules are smaller than antibody drugs and can
cross cell membranes using a suitable drug delivery system
(DDS). In principle, therefore, siRNAs can be delivered to any tissue
and target mRNA in any cell. However, DDSs for nucleic acid drugs
are far from established.
For the effective transfection and DDS of siRNAs, a variety of
methods have previously been reported, including viral and non-
viral delivery methods. Viral delivery is much effective, but there
are problems of cytotoxicity and immunological response.5 Non-
viral delivery strategies have included the use of a variety of
RNA-conjugates and carriers, such as cationic lipids, polymers,
and other molecules.6–8 Among these strategies, cationic carriers
are one of the dominant methods, and cationic polymers, repre-
sented by Lipofectamine™, have been widely used for in vivo
experiments. However, these cationic carriers generally need to
We previously reported that ‘oligodiaminosaccharides,’ which
have amino groups at the 2- and 6-positions of -1-4 linked oli-
go- -glucose, exhibit A-type nucleic acid duplex-binding proper-
a
D
ties.12 These ‘oligodiaminoglucoses (ODAGlcs)’ can specifically
interact with RNA duplexes rather than B-type DNA duplexes.
The 4mer of an ODAGlc can bind to the 12mer of an RNA duplex
in approximately a stoichiometric ratio. Thus, these RNA duplex-
specific-binding oligodiaminosaccharides can be useful as a com-
ponent of siRNA carriers.
Herein we report the synthesis of a novel type of oligodiamino-
saccharide conjugated with vitamin E or its analogs that can be
useful as a carrier of siRNA drugs. Vitamin E (VE), known as
a-tocopherol, is a fat-soluble vitamin. Such lipophilic compounds
are transported to liver cells with chylomicrons after absorption
from the small intestine. The liver-endocytosed vitamin E is drawn
to the cytosol of liver cells by
-TTP).13
Previously, a vitamin E-conjugated siRNA was reported to be
a-tocopherol transfer protein
(a
efficiently transported to mouse liver cells. Importantly, vitamin
E had very low cytotoxicity,14 and the vitamin E-conjugated siRNA
did not show any side effects.15
In the present study, we attempted to construct vitamin E-siR-
NA conjugates through noncovalent interaction with oligodiami-
nosaccharides. The noncovalent approach can prevent vitamin E
⇑
Corresponding author. Tel./fax: +81 4 7121 3671.
E-mail address: twada@rs.tus.ac.jp (T. Wada).
0968-0896/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.12.060

R. Iwata et al. / Bioorg. Med. Chem. 22 (2014) 1394–1403
1395
from the sterically hindering the RNAi process if the binding
molecules become disassociated from the siRNA in cytoplasm.
Conjugates of vitamin E and its analogs with 2,6-diamino-2,6-dide-
The PMB groups of trisaccharide 7 were removed in good yield
under acidic conditions using trifluoroacetic acid, although a minor
amount of cleavage of the glycoside bond occurred as a side reac-
tion. It is noteworthy that in the case of 3-O-benzyl trisaccharide,
the benzyl groups were not efficiently removed. In reductive reac-
tions, such as catalytic reduction using palladium on carbon, the
reaction rate was very slow, and two of the benzyl groups re-
mained even after one day. Under acidic conditions, the desired
compound was not obtained because nealy all of the glycoside
bonds were cleaved.
As shown in Schemes 4–6, the trisaccharide bromide 8 was azi-
dated and conjugated with a propargylated vitamin E (11), vitamin
E analog (1617), and the 4-methoxytriphenylmethyl (MMTr) group
(19) via the Huisgen reaction21 to obtain triazole-linked trisaccha-
rides 13, 17 and 20, respectively. The phthalimide groups of 13 and
17 were then removed by treatment with hydrazine monohydrate
and the products were purified by reverse-phase HPLC to afford the
VE and VE analog conjugated ODAGal derivatives 14 and 18. Sepa-
rately, the MMTr group was removed following dephthaloylation
of 20 to obtain the non-conjugated ODAGal 21.
oxy-b-D-oligogalactopyranoside are expected to have similar struc-
tures and functions as those of ODAGlcs. We thus propose these
‘oligodiaminogalactoses (ODAGals)’ as novel RNA duplex-bindable
molecules.
It has been previously reported that vitamin E is recognized by
a
-TTP at the chroman ring, rather than at the alkyl chain.16,17 Thus,
we synthesized two types of ODAGal derivatives: one ODAGal
derivative was conjugated with native vitamin E, whose phenolic
hydroxy group was used to covalently bind vitamin E to the ODA-
Gal moiety, and the other ODAGal derivative was conjugated with
a VE analog whose phenolic hydroxyl group was preserved.
2. Results and discussion
2.1. Preparation of the glycosyl donor
To construct the b-linked oligodiaminogalactose structure via
glycosylation, an adequately designed glycosyl donor was synthe-
sized from the known galactosamine derivative 1, which was
synthesized by the procedure described in the literature.18 Next,
the 6-OH group of 1 was converted to a phthalimide group via
the Mitsunobu reaction to afford 2.19 The 3-OH group of 2 was then
selectively protected with a p-methoxybenzyl (PMB) group using
dibutyltin oxide to obtain 3.20 Finally, the 4-OH group of 3 was
chloroacetylated to afford the glycosyl donor 4 (Scheme 1).
2.3. Evaluation of the interactions between the ODAGal
derivatives and RNA–RNA duplexes
To evaluate whether ODAGals can interact with and induce
structural changes or thermodynamic stabilization of RNA du-
plexes, UV melting, CD spectrometry, and fluorescence anisotropy
measurements were carried out. All of the experiments were per-
formed under near to physiological conditions with a 10 mM phos-
phate buffer containing 100 mM NaCl at pH 7.0. Figures 1 and 2
present the results of the UV melting analyses of the non-conju-
gated ODAGal 21 complexed with the RNA duplexes
(50-rCGCGAAUUCGCG-30)2 (RNA-I) and (50-rAAAAAAUUUUUU-30)2
(RNA-II) (1 equiv of 21 was added to the RNA duplex solution)
respectively. The melting temperatures (Tm) for the two systems
increased by 2.0 °C and 2.2 °C, respectively (Table 1). These results
suggest that the ODAGal moiety did interact with the RNA du-
plexes, and thermodynamically stabilized them. On the other hand,
when 4 equiv of 21 were added to the RNA duplex solutions, the Tm
values were slightly decreased. These results suggest that an ex-
cess amount of ODAGal thermodynamically destabilizes the du-
plexes, or aggregation of 21 occurs at such a high concentration.
The UV melting curves for the complexes of VE-bound ODAGal
14 with RNA-I and RNA-II are shown in Figures 3 and 4, respec-
tively. Although the curves for both RNA-I and RNA-II were signif-
icantly changed, an increase in the Tm values was not observed in
either case. In addition, when 4 equiv of 14 were added, the Tm val-
ues for RNA-I decreased and the UV melting curve showed an
2.2. Elongation of sugar chains
In the glycosylation reaction, commonly used N-iodosuccini-
mide and trifluoromethanesulfonic acid were employed for activa-
tion of the thiophenyl glycoside 4. However, when using
dichloromethane or dichloromethane–diethyl ether (1:1, v/v) as
the solvent, partial removal and iodination of the PMB group were
observed (Scheme 3). Furthermore, the iodinated PMB group was
more stable and difficult to remove than the unmodified PMB
group under acidic conditions (data not shown). In this study,
these side reactions were inhibited using dichloromethane-diethyl
ether (1:3, v/v) as the solvent, and the glycosylation reaction
proceeded in good yield. Dechloroacetylation was then carried
out under weak basic conditions, as shown in Scheme 1. When
the 4-O-acetylated glycosyl donor was used, however, the acetyl
group could not be selectively removed under basic, acidic, or
enzymatic conditions. Thus, by repeating the synthetic cycle
including glycosylation and dechloroacetylation, the trimer of
ODAGal 7 was obtained (Scheme 2).
NPhth
NPhth
HO
OH
HO
HO
HO
HO
b
a
O
O
O
SPh
NPhth
SPh
NPhth
SPh
PMBO
NPhth
1
2
3
c
NPhth
ClAcO
PMBO
O
SPh
NPhth
4
Scheme 1. Synthesis of the glycosyl donor 4. Reagents and conditions: (a) DIAD, phthalimide, PPh3, THF, rt, 1 h, 71%; (b) (i) Bu2SnO, toluene, reflux, 12.5 h, (ii) PMBCl, TBAI,
toluene, reflux, 5.5 h, 93% over two steps; (c) ClAcCl, pyridine, 0 °C, 30 min, 93%.

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R. Iwata et al. / Bioorg. Med. Chem. 22 (2014) 1394–1403
NPhth
NPhth
NPhth
HO
PMBO
ClAcO
PMBO
O
PMBO
a
b
O
O
H
O
O
Br
O
SPh
NPhth
Br
3
3
NPhth
NPhth
2
5
4
6
c
NPhth
O
H
O
O
Br
PMBO
3
NPhth
3
7
d
NPhth
O
H
O
O
Br
HO
3
NPhth
3
8
Scheme 2. Synthesis of the protected tri-diaminogalactose 8. Reagents and conditions: (a) (i) NIS, TfOH, 3-bromo-1-propanol, CH2Cl2, 0 °C, 20 min, (ii) NaOMe, CH2Cl2–
MeOH, 0 °C, 12.5 h, 85% over 2 steps; (b) (i) 4, NIS, TfOH, CH2Cl2–Et2O, 0 °C, 1.5 h, (ii) NaOMe, CH2Cl2–MeOH, 0 °C, 13 h, 79% over two steps; (c) (i) 4, NIS, TfOH, CH2Cl2–Et2O,
0 °C, 1.5 h, (ii) NaOMe, CH2Cl2–MeOH, 0 °C, 14 h, 77% over two steps; (d) 10%TFA in CH2Cl2, 0 °C, 2 h, 71%.
of RNA-I in the presence and absence of 18. Upon addition of VE
analog-bound ODAGal 18, changes in the spectra of the
RNA–RNA duplex were observed. The positive peak near 265 nm
NPhth
NPhth
HO
O
H
O
O
a
O
Br
O
Br
PMBO
RO
3
3
shifted 1–2 nm to
a longer wavelength, its peak intensity
NPhth
5
NPhth
2
increased, and the molar ellipticity near 230 nm continuously
changed. These changes in the peak at 265 nm are very similar to
those observed in our previous study upon addition of ODAGlcs
to RNA duplexes.12 On the basis of the combined results for UV
melting and CD analyses, it can be concluded that the VE analog-
bound ODAGal 18 interacts with RNA duplexes in a manner similar
to that for ODAGlcs.
Finally, we measured the binding affinity of the VE derivative-
bound ODAGals for RNA duplexes using direct fluorescence
anisotropy titration. A fluorophore-labeled RNA duplex (50-FAM-
CGCGAAUUCGCG)2 was used in this experiment. Figure 8 clearly
shows that the VE analog-bound ODAGal 18 binds to the RNA du-
plex with Kd 3.8 1.2 (ꢀ10ꢁ8 M). On the other hand, the affinity of
VE-bound ODAGal 14 for the the RNA duplex could not be
confirmed from the titration (see SI). In addition, with 14, the ob-
served values did not converge and anisotropy could not be mea-
9
not isolated
R= PMB and IPMB
H2
C
H3C O
IPMB =
I
Scheme 3. Side reaction in glycosylation reaction.
abnormal shape, while the Tm value for RNA-II could not be
determined. These results indicate that, unlike the non-conjugated
ODAGal 21, the VE-bound 14 did not bind efficiently to the RNA
duplexes.
In contrast, the VE analog-bound ODAGal 18 stabilized the RNA
duplexes. As shown in Figures 5 and 6, although the addition of
1 equiv of 18 induced no significant change in the UV melting
curves, an increase in the melting temperatures was observed
when 4 equiv of 18 were added to the RNA solutions. In this case,
the Tm values increased by 2.7 °C for RNA-I and 9.4 °C for RNA-II.
These results suggest that the VE analog-bound ODAGal 18 inter-
acts with the RNA duplexes in a manner similar to that of ODAGlc
and non-conjugated ODAGal. However, for RNA-II, a second flexion
point was observed at 50–60 °C. This phenomenon can be
attributed to other events rather than dissociation of the duplex.
This difference in the properties of the VE-bound ODAGal 14
and VE analog-bound ODAGal 18 likely result from steric hin-
drance near the oligodiaminogalactose moiety. In the case of VE-
bound ODAGal 14, the methyl groups on the aromatic ring are in
proximity to the ODAGal moiety and prevent it from interacting
with the RNA duplexes. On the other hand, the VE analog-bound
ODAGal 18 is less sterically hindered and advantageous for RNA
binding.
sured at more than 0.5 lM 14.
2.4. Evaluation of RNAi activity in the presence of ODAGal
derivatives
On the basis of the above results, it can be concluded that at
least a portion of the ODAGal derivatives interact and form com-
plexes with RNA duplexes. Therefore, experiments designed to
evaluate RNA interference in the presence of the ODAGal deriva-
tives were performed using an siRNA that targets apoB1 mRNA.22
In these experiments, only the VE analog-bound ODAGal 18 was
used because it was determined that the VE-bound ODAGal 14
had no or only a weak binding ability with RNA duplexes. Before
the RNAi experiments were conducted, UV melting analyses of
the siRNA r(50-GUCAUCACACUGAAUACCAAU-3)-r(30-CACAGUAGU
GUGACUUAUGGU-50) with added 18 were carried out. Interest-
ingly, it was observed that the Tm value of the RNA was only
slightly increased upon addition of 18 (see SI). These results are
likely to derive from the high Tm value of the siRNA (71 °C). It
has been previously shown that the oligodiaminosaccharides
Next, to detect the structural changes in the RNA duplexes, CD
spectra were measured for RNA-I and VE analog-bound ODAGal 18,
which possibly binds to the duplex. Figure 7 shows the CD spectra

R. Iwata et al. / Bioorg. Med. Chem. 22 (2014) 1394–1403
1397
HO
O
a
O
O
10
11
NPhth
NPhth
O
O
b
H
H
O
O
O
c
Br
O
N3
HO
HO
3
3
NPhth
NPhth
3
3
8
12
NPhth
O
N
N
H
O
O
O
N
HO
3
NPhth
O
3
13
d
RP-HPLC
NH2
O
N
N
H
O
O
O
N
HO
3
NH2
O
3
6TFA
14
Scheme 4. Synthesis of the VE bound ODAGal 14. Reagents and conditions: (a) 3-bromopropyne, NaH, DMF, rt, 20 min, 82%; (b) NaN3, DMF, 80 °C, 15 h; (c) Cu powder, t-
BuOH–water, 80 °C, 19 h, 66% over two steps from 8; (d) 3%NH2NH2ꢂH2O, EtOH, 80 °C, 4.5 h, 29%.
OH
OTBDMS
a
O
O
8
O
HO
8
15
16
NPhth
NPhth
O
O
b
H
H
O
O
O
Br
O
c
N3
HO
HO
3
3
NPhth
NPhth
3
3
8
12
NPhth
OH
O
N
N
H
O
O
O
N
O
HO
3
NPhth
3
17
d
RP-HPLC
NH2
OH
O
N
N
H
O
O
O
N
O
HO
3
NH2
3
6TFA
18
Scheme 5. Synthesis of the VE analog bound ODAGal 18. Reagents and conditions: (a) (i) 3-bromopropyne, NaH, THF, 60 °C, 6 h, (ii) 1 M TBAF, THF, rt, 30 min, 75% over two
steps; (b) NaN3, DMF, 80 °C, 12 h; (c) Cu powder, t-BuOH–water, 80 °C, 3.5 h, 91% over two steps from 8; (d) 3%NH2NH2ꢂH2O, EtOH, 90 °C, 4.5 h, 59%.

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R. Iwata et al. / Bioorg. Med. Chem. 22 (2014) 1394–1403
a
HO
MMTrCl
MMTrO
19
NPhth
NPhth
O
O
H
b
H
O
O
O
Br
O
c
N3
HO
HO
3
3
NPhth
NPhth
3
3
8
12
NPhth
O
N
N
H
O
OMMTr
O
N
HO
3
NPhth
3
20
d
NH2
O
N
N
H
O
OH
O
N
HO
3
NH2
3
6TFA
21
Scheme 6. Synthesis of the VE unbound ODAGal 21. Reagents and conditions: (a) pyridine, rt, 3 d, 82%; (b) NaN3, DMF, 80 °C, 12 h; (c) Cu powder, t-BuOH–water, 80 °C, 3.5 h,
quant over two steps from 8; (d) (i) 3%NH2NH2ꢂH2O, EtOH, 90 °C, 4 h, (ii) 80%AcOHaq, 11% over two steps.
Table 1
Melting temperatures of RNA duplexes in the presence of ODAGal derivatives
RNA-I
RNA-II
Tm (°C)
D
Tm (°C)
Tm (°C)
DTm (°C)
RNA only
1 equiv
4 equiv
1 equiv
4 equiv
1 equiv
4 equiv
19.7
21.9
18.8
19.8
17.6
20.1
29.1
—
2.2
63.2
65.2
62.4
62.5
n.d.
—
2.0
ꢁ0.8
ꢁ0.7
—
21
14
18
ꢁ0.9
0.1
ꢁ2.1
0.4
9.4
63.8
65.9
0.6
2.7
Figure 1. UV melting curves of (50-rCGCGAAUUCGCG-30)2 in the presence of VE
unbound ODAGal 21.
Figure 3. UV melting curves of (50-rCGCGAAUUCGCG-30)2 in the presence of VE
bound ODAGal 14.
Figure 2. UV melting curves of (50-rAAAAAAUUUUUU-30)2 in the presence of VE
unbound ODAGal 21.
The level of apoB1 mRNA was evaluated using a quantitative re-
verse transcriptase-polymerase chain reaction (qRT-PCR). The cell
lines showed nearly the same RNAi activity regardless of the
addition of the ODAGal derivative. This result confirms that the
ODAGal did not affect the RNAi activity. Although there is room
for further discussion as to whether the ODAGal derivative 18
increase the Tm values of RNA duplexes that have low Tm values,
and not high Tm values.12 Next, RNAi activity in the presence and
absence of the ODAGal derivative 18 was determined, and the re-
sults are shown in Figure 9.

R. Iwata et al. / Bioorg. Med. Chem. 22 (2014) 1394–1403
1399
Figure 4. UV melting curves of (50-rAAAAAAUUUUUU-30)2 in the presence of VE
unbound ODAGal 14.
Figure 8. Fluorescence anisotropy of 100 nM of 50-FAMlabeled RNA duplex was
titrated by increaseing concentration of VE analog-bound ODAGal 18 at 20 °C. The
formation of the 18-RNA complex is reflected an increase in the observed anistropy
values.
Figure 5. UV melting curves of (50-rCGCGAAUUCGCG-30)2 in the presence of VE
analog bound ODAGal 18.
Figure 9. Evaluation of siRNA activity in the presence of ODAGal derivatives; Cell
line name: McA-RH7777; Transfection reagent: Lipofectamine 2000 in culture cells
of McA-RH7777.
m
SEM Sense Sequence: 50-GUCAUCACACUGAAUACCAAU-30
Antisense Sequence: 30-CACAGUAGUGUGACUUAUGGU-50.
binds to siRNAs, it appears that the compound does not inhibit the
process of RNA interference, such as RISC formation.
Figure 6. UV melting curves of (50-rAAAAAAUUUUUU-30)2 in the presence of VE
analog bound ODAGal 18.
3. Conclusion
Novel oligodiaminosaccharides, 2,6-diamino-2,6-dideoxy-b-D-
oligogalactopyranoside derivatives conjugated with VE and its
analog, were synthesized. The analysis of UV melting and CD
spectral observations suggests that, like ODAGlcs, the ODAGal
moiety efficiently interacts with and thermodynamically stabi-
lizes RNA duplexes with small structural changes, although ste-
ric hindrance likely affects their binding ability. RNA
interference experiments also demonstrated that the addition
of the VE analog-bound ODAGal 18 to an siRNA did not affect
the RNAi activities. This VE analog has recognition sites for
a-TTP, and thus, the ODA derivative 18 may have potential as
a useful carrier of RNAi drugs with the ability to bind to RNA
duplexes without inhibiting RNAi activity. Further in vitro and
in vivo experiments, including siRNA delivery to liver cells are
now in progress.
Figure 7. UV melting curves of (50-rCGCGAAUUCGCG-30)2 in the presence of VE
analog bound ODAGal 18.

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R. Iwata et al. / Bioorg. Med. Chem. 22 (2014) 1394–1403
and the solvent was evaporated. The crude product was recrystal-
4. Experimental section
4.1. General methods and materials
lized from methanol (50 mL) and then from ethyl acetate-hexane
(1:5, v/v, 180 mL), and 2 was obtained as a colorless solid (6.28 g,
11.8 mmol, 71%).
1H NMR (CDCl3) d 7.91–7.69 (m, 8H, NPhth), 7.39–7.34 (m, 2H,
SPh), 7.20–7.10 (m, 3H, SPh), 5.59 (d, J = 10.2, 1H, H-1), 4.48–4.32
(m, 2H, H-2, H-3), 4.19–4.12 (m, 1H), 4.00–3.83 (m, 3H, 4-H),
3.62 (d, J = 5.1, 1H, 4-OH), 2.68 (d, J = 10.8, 1H, 3-OH).
13C NMR (CDCl3) d 168.7, 134.6, 134.1, 132.7, 132.0, 131.8,
131.7, 131.6, 128.8, 127.9, 123.8, 123.7, 123.3, 84.0, 75.1, 68.6,
67.8, 53.1, 37.3.
All reactions were carried out under an atmosphere of argon. 1H
NMR spectra were obtained at 300 MHz on a Varian MERCURY 300
spectrometer with tetramethylsilane (TMS) as an internal standard
in CDCl3. 13C NMR spectra were obtained at 75.45 MHz on a Varian
MERCURY 300 spectrometer with CDCl3 as an internal standard (d
77.0) in CDCl3. Mass spectra were recorded on Voyager System
4327 (Applied Biosystem). Analytical TLC was performed on Merck
Kieselgel 60-F254 plates. Melting curves of nucleic acid duplexes
were recorded on a UV-1650PC UV–visible spectrophotometer
(Shimadzu). CD spectra were measured on a J-725 spectropolarim-
MALDI-TOF MS: m/z calcd for C36H30N2NaO8S [M+Na]+: 553.10
Found: 553.11.
eter (JASCO). Fluorescence anisotropies were recorded on
FP-6500 spectrofluorometer (JASCO). Silica gel column chromatog-
raphy was carried out using Silica gel 60 N (63–210 m or
40–50 m). Reversed phase HPLC was carried out using a Bonda-
sphere 5-
m C4 or C18, 100 Å, 4 mm ꢀ 150 mm (Waters) with a
a
4.3. Phenyl 2,6-dideoxy-2,6-diphthalimimde-3-O-p-
methoxybenzyl-1-thio-b-D-galactopyranoside (3)
l
l
l
Compound 2 (6.28 g, 11.8 mmol) was coevaporated with dry
toluene (10 mL ꢀ 4) and dissolved in dry toluene (200 mL). Dibu-
tyltin oxide (3.25 g, 13.0 mmol, 1.1 equiv) was added to the solu-
tion and it was refluxed for 12.5 h. Tetrabutylammonium iodide
(4.81 g, 13.0 mmol, 1.1 equiv) and p-methoxybenzyl chloride
(1.55 mL, 13.0 mmol, 1.1 equiv) was added. After 5.5 h, the mixture
was cooled to rt and concentrated to dryness. The crude product
was purified by silica gel column chromatography (dichlorometh-
ane (1.5% methanol)). The purified product was dissolved in tolu-
ene–dichloromethane–ethyl acetate (8:1:1, v/v/v, 500 mL) and
washed with water (200 mL ꢀ 3). The product was further purified
by silica gel column chromatography (dichloromethane (1.5%
methanol)) and 3 was obtained as a colorless foam (7.35 g,
11.0 mmol, 93%).
l
gradient of 0–100% acetonitrile in water at 25 °C at a rate of
0.5 mL/min and acetonitrile and water is buffered by TFA (trifluo-
roacetic acid) with 0.05% v/v. Organic solvents were purified and
dried by the appropriate procedure. RNA oligomers were pur-
chased from Hokkaido System Science Co., Ltd and Japan Bio
Services Co., Ltd. The yields of compounds were calculated from
their dry weights except for 14, 18 and 21. The yields of 14 and
18 were estimated from absorbance compared with that of corre-
sponding vitamin E derivatives. The yield of 21 was estimated from
absorbance of MMTr+ generated by deprotection.
4.1.1. Conditions for the UV melting analyses
Absorbance versus temperature profile measurements were
carried out with eight-sample cell changer, in quarts cells of
1 cm pathlength. The variation of the difference of UV absorbance
at between 260 nm and 320 nm with temperature was monitored.
The temperature was scanned between 0 °C and 95 °C, and the rate
of temperature increase was 0.2 °C/min. Oligonucleotides were an-
nealed after addition of amino sugars. The samples containing oli-
gonucleotides and amino sugars were first rapidly heated to 95 °C,
left for 20 min, and then allowed to cool slowly to room tempera-
ture. These samples were furthermore cooled to 0 °C and left for
1 h, and then the dissociation was recorded by heating to 95 °C
at rate of 0.2 °C/min.
1H NMR (CDCl3) d 7.88–7.63 (m, 8H, NPhth), 7.23–7.16 (m, 2H,
Ar), 6.97–6.82 (m, 5H, Ar), 6.45 (d, J = 8.7, 2H, Ar), 5.36 (d, J = 8.7,
1H, H-1), 4.57–4.50 (m, 2H, ArCH2-a, H-2), 4.43–4.36 (m, 1H, H-
5), 4.27–4.21 (m, 2H, ArCH2-b, H-3), 4.08 (s, 1H, 4-H), 4.01–3.97
(m, 1H, H-6a), 3.82–3.76 (m, 1H, H-6b), 3.65 (s, 3H, CH3OPh),
2.74 (d, J = 0.9, 1H, 4-OH).
13C NMR (CDCl3) d 168.1, 167.9, 167.3, 159.1, 134.0, 133.9,
133.8, 132.9, 131.9, 131.8, 131.6, 131.5, 129.6, 129.0, 128.3,
127.6, 123.4, 123.1, 113.6, 83.7, 75.0, 74.9, 71.0, 66.0, 54.9, 50.7,
38.9.
MALDI-TOF MS: m/z calcd for C36H30N2NaO8S [M+Na]+: 673.16
Found: 673.08.
4.1.2. Conditions for the CD spectrometry
4.4. Phenyl 4-O-chloroacetyl-2,6-dideoxy-2,6-diphthalimimde-
3-O-p-methoxybenzyl-1-thio-b-D-galactopyranoside (4)
All of the CD spectra were recorded at 25 °C. The following
instrument settings were used: resolution, 0.1 nm; sensitivity,
10 mdeg; response, 4 s; speed, 10 nm/min; accumulation, 6.
Compound 3 (7.35 g, 11.0 mmol) was dissolved in a mixed sol-
vent of pyridine-dichloromethane (1:20, v/v, 126 mL) and cooled
to 0 °C. To the solution was added chloroacetyl chloride (1.75 mL,
22.0 mmol, 2.0 equiv). After 25 min, the solution was diluted with
toluene and the solvent was evaporated. The crude product was
purified by silica gel column chromatography (chloroform (0.5%
4.1.3. Conditions for the fluorescence anisotropy measurement
The all titrations were measured at 20 °C. The following instru-
ment settings were used: Ex/Em = 490 nm/520 nm; response, 2 s;
Band width (Ex), 5 nm; Band width (Em), 5 nm; PMT voltage,
450 V; No. of cycle, 4;
ethanol)) and
4 was obtained as a colorless foam (7.39 g,
10.2 mmol, 93%).
4.2. Phenyl 2,6-dideoxy-2,6-diphthalimimde-1-thio-b-D-
1H NMR(CDCl3) d 7.88–7.60 (m, 8H, NPhth), 7.29–7.26 (m, 2H,
Ar), 7.07–6.94 (m, 3H, Ar), 6.83 (d, J = 7.2, 2H), 6.38 (d, J = 8.4,
2H), 5.54 (d, J = 2.7, 1H, 4-H), 5.46 (d, J = 10.5, 1H, H-1),
4.45–4.23 (m, 6H, ClAc, ArCH2-a, H-2, H-3, H-5), 4.12–3.98 (m,
2H, ArCH2-b, H-6a), 3.88–3.81 (m, 1H, H-6b), 3.64 (s, 3H, CH3OPh).
13C NMR (CDCl3) d 167.9, 167.6, 167.4, 167.2, 159.1, 134.2,
133.9, 133.8, 133.0, 131.7, 131.5, 131.4, 129.8, 128.9, 128.5,
127.9, 123.5, 123.4, 123.1, 113.5, 83.9, 73.4, 72.8, 70.9, 68.0, 54.9,
51.1, 41.1, 38.1.
galactopyranoside (2)
Compound 1 (6.74 g, 16.8 mmol), triphenylphosphine (8.81 g,
33.6 mmol, 2.0 equiv), and phthalimide (4.94 g, 33.6 mmol,
2.0 equiv) were dissolved in dry tetrahydrofurane (168 mL) and
cooled to 0 °C. To this mixture, diisopropyl azodicarboxylate
(6.5 mL, 33.6 mmol, 2.0 equiv) was added dropwise over 20 min.
After 40 min, further diisopropyl azodicarboxylate (3.0 mL) was
added. After 30 min, methanol (20 mL) was added to the mixture

R. Iwata et al. / Bioorg. Med. Chem. 22 (2014) 1394–1403
1401
MALDI-TOF MS: m/z calcd for C38H31ClN2NaO9S [M+Na]+:
MALDI-TOF MS: m/z calcd for C93H79BrN6NaO25 [M+Na]+:
749.13 Found: 749.12.
1241.26 Found: 1241.26.
4.7. Compound 7
4.5. Compound 5
The same procedure for the synthesis of 6 was applied to that of
Compound 4 (0.1452 g, 200
toluene (2 mL ꢀ 4). N-Iodosuccinimide (0.1127 g, 500
2.5 equiv) and 3-bromo-1-propanol (36 L, 400 mol, 2.0 equiv)
l
mol) was coevaporated with dry
7, except for using
6 (0.0942 g, 77 lmol) and 4 (0.1056 g,
lmol,
146 mol, 1.9 equiv) as starting materials, and ethyl acetate–tolu-
l
l
l
ene (9:11, v/v) as eluting solvent in silica gel column chromatogra-
were added and the mixture was dissolved in dichloromethane
(1.0 mL). The solution was cooled and then 0.2% trifluorometh-
anesulfonic acid in diethyl ether (1.0 mL) was added. After stir-
ring for 20 min, a saturated aqueous solution of NaHCO3 (1 mL)
was added and the mixture was warmed to rt. The solution was
diluted with chloroform (30 mL), washed with a saturated aque-
ous solution of NaHCO3 (30 mL) and 10% Na2S2O3aq (30 mL).
The organic layer was dried over Na2SO4, filtered, and concen-
trated. After the crude product was cooled to 0 °C, 7.5 mM so-
dium methoxide solution in methanol (10 mL) and
dichloromethane (5 mL) was added to the mixture. After
12.5 h, Dowex 50 W ꢀ 8 (2 g) was added, and stirred for
30 min and the Dowex resin was removed by filtration. The
crude product was purified by silica gel column chromatography
(ethyl acetate–toluene (1:4, v/v)) to afford the pure b-glycoside
phy. Compound 7 was obtained as a colorless oil (0.1044 mg,
59
lmol, 77%).
1H NMR (CDCl3) d 8.35 (d, J = 7.2, 1H, Ar), 7.91–7.05 (m, 27H,
Ar), 6.52–6.07 (m, 8H, Ar), 5.24 (d, J = 8.7, 1H, H-1), 4.94 (d,
J = 7.8, 1H, H-1), 4.76–4.51 (m, 4H), 4.37–4.00 (m, 10H), 3.93–
3.43 (m, 21H), 3.31–3.08 (m, 4H), 1.95–1.73 (m, 2H).
13C NMR (CDCl3) d 169.2, 168.4, 168.1, 168.0, 167.9, 167.6,
167.3, 166.1, 165.8, 159.0, 158.5, 158.4, 133.9, 133.7, 133.6,
133.3, 133.1, 133.0, 132.8, 132.4, 132.2, 132.0, 131.8, 131.6,
131.3, 129.7, 129.6, 129.4, 129.1, 129.1, 125.2, 123.8, 123.3,
123.2, 123.1, 122.6, 122.3, 122.0, 121.7, 113.5, 113.0, 112.9, 99.6,
99.5, 97.5, 75.1, 74.3, 74.0, 73.8, 72.8, 71.7, 71.6, 71.0, 70.8, 70.6,
66.4, 65.3, 55.0, 54.8, 54.7, 52.1, 51.8, 51.3, 40.5, 40.4, 39.4, 32.4,
30.8.
MALDI-TOF MS: m/z calcd for C93H79BrN6NaO25 [M+Na]+:
1781.42 Found: 1781.39.
5 as a colorless oil (0.1156 g, 170 lmol, 85%).
1H NMR (CDCl3) d 7.91–7.65 (m, 8H, NPhth), 6.95 (d, J = 8.7, 2H,
Ar), 6.46 (d, J = 8.4, 2H, Ar), 5.04 (d, J = 8.4, 1H, H-1), 4.54 (d,
J = 12.6, 1H), 4.48–4.41 (m, 1H), 4.32–4.22 (m, 3H), 4.01–3.92 (m,
3H), 3.73–3.65 (m, 4H), 3.48–3.40 (m, 1H), 3.18–3.14 (m, 1H),
2.83 (s, 1H), 1.95–1.74 (m, 2H).
4.8. Compound 8
Compound 7 (21.9 mg, 12.5 lmol) was dissolved in dichloro-
13C NMR (CDCl3) d 168.2, 167.7, 159.0, 134.1, 133.8, 133.7,
131.8, 131.5, 129.4, 129.1, 123.4, 123.3, 122.9, 113.4, 98.4, 74.3,
71.5, 71.1, 66.5, 65.6, 54.9, 52.0, 38.5, 32.1, 30.2.
methane (5 mL) and cooled to 0 °C. To the solution, 20% trifluo-
roacetic acid in dichloromethane (5 mL) was added and stirred
for 2 h. The reaction was quenched by addition of a saturated
aqueous solution of NaHCO3 (5 mL) and diluted with dichloro-
methane (60 mL). The solution was washed with a saturated
aqueous solution of NaHCO3 (100 mL) and the aqueous layer
was back extracted with dichloromethane (20 mL ꢀ 3). The or-
ganic layer was dried over Na2SO4, filtered and concentrated.
The crude product was purified by silica gel column chromatog-
raphy (dichloromethane (3% methanol)) and 8 was obtained as a
4.6. Compound 6
A mixture of 5 (50.4 mg, 71
1.5 equiv) was coevaporatated with dry toluene (2 mL ꢀ 4). N-
Iodosuccinimide (59.9 mg, 265 mol, 3.75 equiv) was added
lmol) and 4 (77.6 mg, 106 lmol,
l
and the mixture was dissolved in dichloromethane (0.5 mL).
The solution was cooled, and then 0.04% trifluoromethanesulfo-
nic acid in diethyl ether (1.5 mL) was added to the solution.
colorless oil (12.4 mg, 8.9 lmol, 71%).
1H NMR (CDCl3) d 8.03–7.60 (24H, NPhth), 5.69 (d, J = 8.1, 1H,
H-1), 5.46 (d, J = 7.8, 1H, H-1), 4.81 (d, J = 8.1, 1H, H-1), 4.62–4.56
(m, 1H), 4.34–3.59 (m, 18H), 3.51–3.44 (m, 1H), 3.25–3.17
(m, 2H), 3.04–2.93 (m, 4H), 1.95–1.73 (m, 2H).
After 1.5 h,
a saturated aqueous solution of NaHCO3 (1 mL)
was added and the mixture was warmed to rt. The solution
was diluted with chloroform (30 mL), washed with a saturated
aqueous solution of NaHCO3 (30 mL) and 10% Na2S2O3aq
(30 mL). The organic layer was dried over Na2SO4, filtered, and
concentrated. After the crude product was cooled to 0 °C,
7.5 mM sodium methoxide solution in methanol (10 mL) and
dichloromethane (5 mL) was added to the mixture. After 13 h,
Dowex 50 W ꢀ 8 (1 g) was added, stirred for 30 min and the
Dowex resin was removed by filtration. The crude product
was purified by silica gel column chromatography (ethyl ace-
tate–toluene (7:13, v/v)) to afford the pure b-glycoside 6 as a
13C NMR (CDCl3) d 168.5, 168.2, 167.9, 134.4, 133.9, 133.7,
132.2, 131.9, 131.7, 131.6, 124.1, 123.8, 123.5, 123.1, 99.3, 99.1,
98.5, 75.4, 75.3, 72.6, 72.4, 72.3, 71.2, 69.6, 68.9, 68.5, 66.7, 54.4,
54.2, 53.6, 39.8, 37.6, 31.9, 30.3.
MALDI-TOF MS: m/z calcd for C69H55BrN6NaO22 [M+Na]+:
1421.24 Found: 1420.91.
4.9. Compound 11
a
-Tocopherol 10 (0.2495 g, 0.58 mmol) was dissolved in dry
colorless oil (68.7 mg, 56.3 lmol, 79%).
1H NMR (CDCl3) d 8.10 (d, J = 7.8, 1H, Ar), 7.88–7.00 (m, 17H,
Ar),.6.51–6.17 (m, 6H, Ar), 5.24 (d, J = 8.1, 1H, H-1), 4.74 (d,
J = 8.4, H-1), 4.68–4.58 (m, 2H), 4.47–4.25 (m, 4H), 4.15–3.45 (m,
18H), 3.33–3.25 (m, 1H), 3.11–3.06 (m, 2H), 2.84 (d, J = 3.0, 1H),
1.87–1.67 (m, 2H).
N,N-dimethylformamide (6 mL). To the solution, sodium hydride
(60% purity, 91.6 mg, 2.3 mmol, 4 equiv) and propargyl bromide
(200 lL, 2.3 mmol, 4 equiv) were added. After 2 h, methanol was
added and the mixture was concentrated to dryness. The mixture
was dissolved in dichloromethane (50 mL) and washed with water
(30 mL ꢀ 2). The crude product was purified by silica gel column
chromatography (dichloromethane-hexane (1:9, v/v)) to give 11
as a pale yellow oil (0.2229 g, 0.474 mmol, 82%).
13C NMR (CDCl3) d 168.8, 168.1, 168.0, 167.8, 167.4, 166.1,
159.1, 158.6, 133.9, 133.8, 133.3, 133.2, 132.9, 132.7, 132.2,
131.8, 131.6, 131.4, 129.5, 129.3, 124.5, 123.5, 123.3, 123.2,
122.7, 122.5, 122.0, 113.6, 113.4, 113.1, 99.8, 97.7, 75.1, 74.4,
73.8, 72.1, 71.6, 71.0, 66.2, 65.4, 55.0, 54.8, 52.0, 51.4, 40.1, 39.4,
32.3, 30.6.
1H NMR (CDCl3) d 4.36 (d, s = 2.7, 2H, HC„CCH2O), 2.57 (t,
J = 6.6, 2H, ArCH2), 2.48 (d, J = 2.1, 1H, HC„C), 2.20–2.08 (s ꢀ 3,
9H, ArCH3), 1.81–0.83 (m, 38H).

1402
R. Iwata et al. / Bioorg. Med. Chem. 22 (2014) 1394–1403
13C NMR (CDCl3) d 127.9, 126.0, 122.9, 117.5, 74.9, 74.5, 60.5,
(6:4, v/v)) to afford the pure 16 as a pale yellow oil (26.2 mg,
40.0, 39.3, 37.4, 37.3, 32.8, 32.7, 31.2, 28.0, 24.8, 24.4, 23.9, 22.7,
22.6, 21.0, 20.6, 19.7, 19.6, 13.0, 12.2, 11.8.
62 lmol, 75%).
1H NMR (CDCl3) d 4.25 (s, 1H, ArOH), 4.13 (d, J = 2.7, 2H,
HCꢃCCH2O), 3.50 (t, J = 6.6, 2H, OCH2CH2), 2.59 (t, J = 6.9, ArCH2),
2.41 (t, J = 2.4, HC„C), 2.15–2.10 (s ꢀ 2, 9H, ArCH3), 1.81–1.22
(m, 21H).
4.10. Compound 13
Compound 8 (10.5 mg, 7.5
toluene (1 mL ꢀ 3). Sodium azide (5.0 mg, 75
l
mol) was coevaporated with dry
mol, 10 equiv)
13C NMR (75.5 MHz, CDCl3) d 145.5, 144.5, 122.5, 121.0, 118.5,
117.3, 80.0, 74.4, 74.0, 70.3, 58.0, 39.4, 31.5, 30.1, 29.5, 29.5, 29.4,
26.0, 23.8, 23.6, 20.7, 12.2, 11.7, 11.3.
l
was added and the mixture was dissolved in dry N,N-dimethyl-
formamide (2 mL). The suspending solution was heated to 80 °C,
stirred for 19 h and then cooled to rt. The solution was concen-
trated, diluted with ethyl acetate (20 mL), and washed with water
(20 mL ꢀ 3). The aqueous layer was back extracted with toluene
(20 mL) and the combined organic layer was dried over MgSO4,
filtered, and concentrated. To the crude product including 12,
MALDI-TOF MS: m/z calcd for C25H39O3 [M+H]+: 387.3 Found:
386.8.
4.13. Compound 17
Compound 8 (4.5 mg, 3.2
lmol) was coevaporated with dry tol-
11 (14.2 mg, 30
l
mol, 4 equiv) and copper powder (9.1 mg) were
uene (1 mL ꢀ 4), sodium azide (8.6 mg, 132
l
mol, 41 equiv) was
added. The mixed solvent (t-butanol–water (2:1, v/v, 3.0 mL)) was
added to the mixture and the solution was stirred and heated to
65 °C. After 14 h, the solution was cooled to rt, filtered and
purified by silica gel column chromatography (dichloromethane
(3% to 4% methanol)) to afford the pure 13 as a colorless oil
added and the mixture was dissolved in dry N,N-dimethylformam-
ide (2.0 mL). The suspending solution was heated to 80 °C, stirred
for 15 h and then cooled to rt. The solution was concentrated, dis-
solved in ethyl acetate (20 mL), and washed with water
(20 mL ꢀ 3). The aqueous layer was back extracted with toluene
(10 mL) and the combined organic layer was dried over MgSO4, fil-
tered, and concentrated. To the crude product including 12, 16
(9.0 mg, 4.9 lmol, 66%).
1H NMR (CDCl3) d 8.02–7.60 (24H, NPhth), 7.49 (s, 1H, triazole-
H), 5.69 (d, J = 8.7, 1H, H-1), 5.43 (d, J = 7.8, 1H, H-1), 4.83 (d, J = 7.8,
1H, H-1), 4.72 (s, 2H), 4.60 (m, 1H), 4.35–3.40 (m, 21H), 3.14–3.10
(m, 3H), 2.95 (d, J = 11.1, 1H), 2.58 (t, J = 6.6, 2H), 2.18–2.09 (s ꢀ 3,
9H), 1.88–0.80 (m, 40H).
(2.7 mg, 7.0 lmol, 2.2 equiv) and copper powder were added.
Mixed solvent (t-butanol–water (2:1, v/v, 1.5 mL)) was added to
the mixture and the solution was stirred and heated to 80 °C. After
3.5 h, the solution was cooled to rt, filtered and purified by silica
gel column chromatography (dichloromethane (2.5–3.5% metha-
13C NMR (75.5 MHz, CDCl3) d 168.5, 168.2, 168.0, 148.0, 147.9,
144.4, 134.4, 133.9, 133.8, 132.1, 131.9, 131.7, 131.6, 131.5,
127.9, 126.0, 123.8, 123.5, 123.1, 122.9, 117.6, 99.5, 99.2, 98.5,
75.6, 74.8, 72.6, 72.2, 70.8, 69.6, 69.0, 68.5, 66.3, 65.6, 54.5, 54.1,
53.5, 47.0, 40.1, 39.8, 39.3, 37.5, 37.4, 37.3, 32.8, 32.7, 31.2, 30.2,
29.7, 28.0, 24.8, 24.4, 23.8, 22.7, 22.6, 21.0, 20.6, 19.7, 19.6, 12.9,
12.0, 11.8.
nol)) to afford the pure 13 as a colorless oil (5.1 mg, 2.9 lmol, 91%).
1H NMR (CDCl3) d 8.01–7.61 (24H, NPhth), 7.31 (s, 1H, triazole-
H), 5.69 (d, J = 8.7, 1H, H-1), 5.44 (d, J = 8.4, 1H, H-1), 4.82 (d, J = 7.8,
1H, H-1), 4.63–4.56 (m, 1H), 4.50 (s, 2H), 4.40–3.37 (m, 26H), 3.13–
3.07 (m, 3H), 2.91 (d, J = 11.1, 1H), 2.58 (t, J = 6.6, 2H), 2.14–2.10
(s ꢀ 3, 9H), 1.83–1.22 (m, 21H).
MALDI-TOF MS: m/z calcd for C101H107N9NaO24 [M+Na]+:
1853.74 Found: 1852.79.
13C NMR (75.5 MHz, CDCl3) d 168.6, 168.2, 168.0, 145.4, 144.8,
144.5, 134.5, 133.8, 132.1, 131.9, 131.5, 123.8, 123.5, 123.2,
122.5, 121.1, 118.6, 117.3, 99.4, 99.2, 98.4, 75.6, 74.5 72.6, 72.2,
70.7, 69.6, 69.0, 68.4, 65.4, 64.0, 54.5, 54.1, 53.5, 46.9, 39.9, 39.2,
37.5, 31.5, 30.1, 29.6, 29.5, 29.4, 26.1, 23.9, 23.5, 20.7, 12.3, 11.8,
11.3.
4.11. Compound 14
Compound 13 (3.9 mg, 2.1
(2.0 mL) and hydrazine monohydrate (60
lmol) was dissolved in ethanol
l
L) were added and the
MALDI-TOF MS: m/z calcd for C94H93N9NaO25 [M+Na]+: 1771.62
Found: 1770.43.
mixture was stirred and heated to 80 °C. After 4.5 h, the mixture
was cooled to rt and concentrated. Eighth part of the entire crude
product was purified by C4 reversed-phase HPLC (0.05% TFA,
water-acetonitrile) and lyophilized from water to give 14 as a col-
orless solid (78.5 nmol, 29%).
4.14. Compound 18
Compound 17 (5.1 mg, 2.9
lmol) was dissolved in ethanol
MALDI-TOF MS: m/z calcd for C53H95N9NaO12 [M+Na]+: 1072.70
Found: 1072.52.
(2.0 mL) and hydrazine monohydrate (60
lL) were added and the
mixture was stirred and heated to 90 °C. After 4 h, the mixture
was cooled to rt and concentrated. Four-twenty ninth of the entire
crude product was purified by C18 reversed-phase HPLC (0.05%
TFA, water–acetonitrile) and lyophilized from water to give 18 as
colorless solid (235 nmol, 59%).
4.12. Compound 16
Compound 15 (38.5 mg, 83 lmol) was coevaporated with dry
toluene (10 mL ꢀ 4) and dissolved in dry tetrahydrofurane
MALDI-TOF MS: m/z calcd for C46H81N9NaO13 [M+Na]+: 990.59
Found: 990.49.
(1.5 mL). To the solution, sodium hydride (60% purity, 15.1 mg,
377 lmol, 4.5 equiv) and propargyl bromide (15 lL, 188 lmol,
2.3 equiv) were added. The mixture was heated to 60 °C and stirred
for 16 h. After the solution was cooled to rt, methanol (1 mL) was
added and concentrated. The crude product was dissolved in dichlo-
romethane (30 mL) and washed with saturated brine (10 mL ꢀ 3)
and the organic layer was dried over Na2SO4, filtered and concen-
trated. The crude product was dissolved in dry tetrahydrofurane
4.15. Compound 19
4-Monomethoxytrityl chloride (0.6416 g, 2.0 mmol) was dis-
solved in dry pyridine (20 mL) and stirred. To the solution, propar-
gyl alcohol (150 lL, 2.54 mmol, 1.27 equiv) was added and stirred
for 3 d. The solvent was evaporated. And the mixture was dissolved
in dichloromethane (30 mL) and washed with saturated aqueous
solution of NaHCO3 (20 mL ꢀ 2). The aqueous layer was back ex-
tracted with dichloromethane (10 mL ꢀ 4). The combined organic
layer was dried over Na2SO4, filtered and concentrated. The crude
product was purified by silica gel column chromatography
(1.5 mL) and tetrabutylammonium fluoride (37.2 mg, 150 lmol)
was added. After the solution was stirred for 20 min, the reaction
was quenched by addition of saturated aqueous solution of NaHCO3
and the solution was concentrated. The crude product was purified
by silica gel column chromatography (dichloromethane-hexane

R. Iwata et al. / Bioorg. Med. Chem. 22 (2014) 1394–1403
1403
(dichloromethane–hexane (3:1, v/v)) to afford the pure 19 as a
colorless solid (0.5989 g, 1.82 mmol, 91%).
4.18.2. In vitro siRNA activity assay
To determine in vitro activity of siRNAs, McA-RH7777 cells
(kindly gifted from Hiroyuki Arai, PhD) were transfected with 10
nM siRNAs in absence and presence of equally amount of com-
pound 18 respectively using Lipofectamine™ 2000 (Invitrogen).
The cells were harvested 24 h after transfection. Total RNA was ex-
tracted and the amount of endogenous apoB mRNA was measured
by quantitative real-time polymerase chain reaction (qRT-PCR).
1H NMR (CDCl3) d 7.49–7.45 (m, 4H, Ar), 7.37–7.20 (m, 8H, Ar),
6.87–6.82 (m, 2H, Ar), 3.80 (s, 3H, CH3OAr), 3.75 (d, J = 2.4,
HC„CH2), 2.39 (d, J = 2.4, HC„CH2).
13C NMR (CDCl3) d 158.7, 143.8, 135.0, 130.2, 128.2, 127.9,
127.0, 113.2, 87.2, 80.5, 55.2, 52.7.
4.16. Compound 20
4.18.3. Quantitative RT-PCR assay
Compound 8 (4.2 mg, 3.0
uene (1 mL ꢀ 3), sodium azide (6.4 mg, 100
l
mol) was coevaporated with dry tol-
mol, 33 equiv) was
DNase-treated 2 lg of RNAs were reverse transcribed with
l
Super Script III and Random Hexamers (Life Technologies, Carls-
bad, CA). The cDNAs were amplified by the quantitative TaqMan
system using the Light Cycler 480 Real-Time PCR Instrument
(Roche Diagnostics, Mannheim, Germany). The primers and probes
for rat apoB (NM_019287) and rat glyceraldehyde-3-phosphate
dehydrogenase (gapdh; NM_017008) were designed by Applied Bio-
systems (Foster City, CA).
added and the mixture was dissolved in dry N,N-dimethylformam-
ide (1.0 mL). The suspending solution was heated to 80 °C, stirred
for 18 h and then cooled to rt. The solution was concentrated, dis-
solved in ethyl acetate (20 mL), and washed with water
(20 mL ꢀ 3). The aqueous layer was back extracted with toluene
(10 mL) and the combined organic layer was dried over MgSO4, fil-
tered, and concentrated. To the crude product including 12, 19
(6.4 mg, 20
l
mol, 6.7 equiv) and copper powder (3.8 mg) were
4.18.4. Statistical analysis
added. Mixed solvent (t-butanol–water (2:1, v/v, 1.0 mL)) was
added to the mixture and the solution was stirred and heated to
65 °C. After 12 h, the solution was cooled to rt, filtered and purified
by silica gel column chromatography (dichloromethane (3% meth-
Student’s two-tailed t-test was used to determine the signifi-
cance of differences between control and transfected groups in
quantitative RT-PCR assay. Data are presented as means standard
error of the means (SEMs); P <0.05 was considered significant.
anol)) to afford the pure 20 as a colorless oil (5.1 mg, 3.0 lmol,
quant).
Acknowledgements
1H NMR (CDCl3) d 8.04–7.23 (m, 37H, Ar), 6.84 (d, J = 9.0, 2H,
Ar), 5.72 (d, J = 8.1, 1H, H-1), 5.49 (d, J = 8.1, 1H, H-1), 4.86 (d,
J = 8.1, 1H, H-1), 4.62–4.55 (m, 1H), 4.35–3.42 (m, 26H),
3.21–3.03 (m, 3H), 2.80 (d, J = 11.7, 1H), 1.83–1.78 (m, 2H).
13C NMR (75.5 MHz, CDCl3) d 168.6, 168.2, 167.9, 158.6, 145.5,
144.2, 135.4, 134.5, 134.0, 133.8, 132.1, 131.9, 131.7, 131.5,
130.3, 128.4, 127.9, 126.9, 124.1, 123.8, 123.6, 123.2, 122.3,
113.2, 99.3, 99.1, 98.5, 86.9, 78.5, 75.3, 72.7, 72.6, 72.3, 71.1,
69.7, 69.2, 68.3, 65.7, 58.6, 55.2, 54.5, 54.3, 53.6, 46.9, 39.8, 37.3,
30.1.
We thank the Japan Student Services Organization for the schol-
arship. And this work was supported by National Institute of Bio-
medical Innovation (NIBIO), KAKENHI (22ꢂ5612) and Japan
Society for the Promotion of Science (JSPS).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.12.060.
MALDI-TOF MS: m/z calcd for C92H75N9NaO24 [M+Na]+: 1712.48
Found: 1712.16.
References and notes
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4.17. Compound 21
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(3.0 mL) and hydrazine monohydrate (90
l
mol) was dissolved in dry ethanol
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l
mixture was stirred and heated to 80 °C. After 4 h, the mixture
was cooled to rt and concentrated. Eighth part of the entire crude
product was purified by C18 reversed-phase HPLC (0.05% TFA,
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was evaporated after 1.5 h. The product was dissolved to water
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MALDI-TOF MS: calcd for C24H47N9NaO11 m/z [M+Na]+: 660.33
Found: 660.05.
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4.18. Biological experiments
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4.18.1. Cell culture
Rat hepatocellular carcinoma (McA-RH7777) cells were main-
tained in Dulbecco’s modified Eagle’s medium (Sigma–Aldrich, St
Louis, MO) only or supplemented with 10% fetal bovine serum
(Invitrogen, Carlsbad, CA), 100 units/ml penicillin, and 100 lg of
streptomycin at 37 °C in 5% CO2.