Synthesis of oligodiaminosaccharides having α-glycoside bonds and their interactions with oligonucleotide duplexes

Article abstract of DOI:10.1021/jo200951p

Syntheses of the novel oligodiaminosaccharides, α-(1→4)-linked- 2,6-diamino-2,6-dideoxy-d-glucopyranose oligomers, and their interactions with nucleic acid duplexes DNA-DNA, RNA-RNA, and DNA-RNA are described. Monomers to tetramers of oligodiaminoglucose

Full text of DOI:10.1021/jo200951p

FEATURED ARTICLE
pubs.acs.org/joc
Synthesis of Oligodiaminosaccharides Having r-Glycoside Bonds
and Their Interactions with Oligonucleotide Duplexes
Rintaro Iwata, Masafumi Sudo, Kenta Nagafuji, and Takeshi Wada*
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
S Supporting Information
b
ABSTRACT: Syntheses of the novel oligodiaminosaccharides, R-(1f4)-
linked-2,6-diamino-2,6-dideoxy-D-glucopyranose oligomers, and their in-
teractions with nucleic acid duplexes DNAꢀDNA, RNAꢀRNA, and
DNAꢀRNA are described. Monomers to tetramers of oligodiaminoglu-
cose derivatives having R-glycosyl bonds were successfully synthesized
using a chain elongation cycle including glycosylation reactions of a
6-phthalimide glycosyl donor. UV melting experiments for a variety of
nucleic acid duplexes in the absence and presence of the oligodiamino-
saccharides were performed. The synthesized oligodiaminosaccharides
exhibited notable thermodynamic stabilization effects on A-type RNAꢀ
RNA and DNAꢀRNA duplexes, whereas B-type DNAꢀDNA duplexes
were not stabilized by the synthesized oligodiaminosaccharides. Among the oligodiaminosaccharides, the tetramer exhibited the
highest ability to stabilize A-type duplexes, and the increase in Tm values induced by the tetramer were higher than those induced by
neomycin B and tobramycin, which are known aminoglycosides having ability to bind and stabilize a variety of RNA molecules. CD
spectrometry experiments revealed that the oligodiaminosaccharides caused small structural changes in RNAꢀRNA duplexes,
whereas no appreciable changes were observed in the structure of DNAꢀDNA duplexes. ITC (isothermal titration calorimetry)
experiments demonstrated that the amount of heat generated by the interaction between RNAꢀRNA duplexes and the
tetradiaminosaccharides was approximately double that generated by that between DNAꢀDNA duplexes and the tetradiamino-
saccharides. These results strongly suggested the existence of an A-type nucleic acid specific-binding mode of the oligodiamino-
saccharides, which bind to these duplexes and cause small structural changes.
’ INTRODUCTION
proposed.18 Another strategy to stabilize siRNAs is the use of
molecules that can noncovalently bind to RNA and protect
them from nucleases. For example, cationic liposomes, such as
Lipofectamine, were broadly used as transfection reagents of
nucleic acids.19 An excess usage of cationic molecules including
cationic liposomes and aminoglycosides, in many cases, causes
cytotoxicity.13,20 In contrast, if an RNA-interactive molecule
can bind to the target in a stoichiometric manner, it would be
useful as a less toxic carrier of RNA-drugs in vivo. On the basis of
these considerations, we have designed “R-(1f4)-linked-2,6-
diamino-2,6-dideoxy-D-glucopyranose oligomers (Figure 1, 1)”
which are expected to efficiently interact with double stranded
RNA. Compound 1, which possesses the properly arranged
amino groups at the 2- and 6-positions in the repeating glucose
units, can interact with ambilateral phosphate groups at the major
groove of RNA duplexes because the major groove width of an
A-type RNAꢀRNA duplex (7ꢀ9 Å21,22) is similar to the distance
between 2-N and 6-N nitrogen atoms of 1 (approximately 6 Å).
Moreover, because the R-(1f4)-linked glucose derivative is
expected to form a curved structure similar to that of amylose,
Nucleic acids are vital biomolecules that can form a wide variety
of higher order structures.1,2 Numerous applications of nucleic
acids as disease-specific drugs, highly functional devices, and
components for molecular architecture have been reported.3ꢀ6
Moreover, a wide variety of nucleic acid binding molecules such as
intercalators7 and groove binders8 have been known. Among these
nucleic-acid-binding molecules, naturally occurring aminoglyco-
sides are known as RNA-binding molecules and they thermo-
dynamically stabilize the higher order structures of RNA.9ꢀ13
Concerning these cationic molecules, it has been reported that
interactions between the protonated amino groups of these
molecules and phosphate anions of nucleic acids are crucial to
the stability of the RNA duplexes.12 Among a variety of higher
order structures of RNA, double-stranded RNA molecules re-
cently have received attention because of the discovery of “RNA
interference (RNAi)”.14 RNAi is a gene-regulating system that is
controlled by external double-stranded RNAs (siRNAs). These
siRNAs have been widely studied for therapeutic applications.
However, such RNA molecules are not sufficiently stable under
physiological conditions,15,16 it is difficult to administer these
RNAi-based drugs orally or intravenously.17 To increase the
stability of siRNAs, a number of chemical modifications have been
Received: May 10, 2011
Published: June 20, 2011
r
2011 American Chemical Society
5895
dx.doi.org/10.1021/jo200951p J. Org. Chem. 2011, 76, 5895–5906
|

The Journal of Organic Chemistry
FEATURED ARTICLE
synthetic cycle by recursively using a properly functionalized
glycosyl donor recursively (Scheme 1). To make this strategy
possible, the glycosyl donor should have a nonparticipating azide
group at the 2-position, and the 4- and 6-hydroxyl groups should
be protected by orthogonal protecting groups.
The glycosyl donors 4 and 6 were synthesized from a known
2-azidoglucose derivative 2 according to the procedure described
in the literature.26 Compound 2 was converted to 4 and 6
through a regioselective azidation27 or phthalimidation28 at the
6-position followed by acetylation of their 4-hydroxyl groups
(Scheme 2). The starting glycosyl acceptor 12 was separately
synthesized from a glucosamine derivative 7 that was synthesized
following previously published procedures.29 The 2-amino group
of 7 was converted to an azido group,30,31 and the 3-hydroxy
group was protected by a benzyl group to produce 9.32 After
acidic cleavage of 4,6-O-benzylidene acetal33 of 9, a 6-hydroxyl
selective mesylation followed by a azidation afforded the glycosyl
acceptor 12 (Scheme 3).
Figure 1. R-(1f4)-Linked 2,6-diamino-2,6-dideoxy-D-glucopyranose
oligomer.
Extension of Sugar Chains. In the glycosylation reaction,
commonly used N-iodosuccinimide and trifluoromethanesulfo-
nic acid were used for activation of the thiophenyl glycoside 4
and 634 (Table 1). The glycosylation reactions of both the
glycosyl donors 4 and 6 with the glycosyl acceptor 12 were
performed to obtain the disaccharides 13 and 14 in a similar
stereoselectively. In the case of 14, the R-isomer was easily
separated from the R/β mixture by using silica gel column
chromatography and pure 14r was isolated with 65% yield
(53% from the glycosyl acceptor 12). In contrast, the R-isomer
of 13 could not be isolated from the anomeric isomers.
The disaccharide 14 can be used as a glycosyl acceptor toward the
synthesis of trisaccharides after deacetylation of the 4-hydroxyl
group at the nonreducing terminus. However, deacetylation under
basic conditions by using sodium methoxide or potassium carbo-
nate, simultaneously caused the decomposition of phthaloyl groups.
Meanwhile, deacetylation of 14 was successful under the anhydrous
and acidic conditions using acetyl chloride35 (Scheme 4).
Figure 2. Molecular model of 4mer of 1 binding to A-type RNA duplex
(12mer).
1 is entropically favored to bind to the nucleic acid duplexes. On
the basis of molecular mechanics calculations with a GB/SA
water solution model,23 a 4mer of 1 can bind to the major groove
of an A-type RNAꢀRNA duplex in which all the protonated
amino groups of 1 form hydrogen bonds to the phosphate anions
of the duplex (Figure 2). In a similar fashion, DNAꢀRNA
hybrids that have an A-like duplex structure, can also be target
molecules. To the best of our knowledge, only a few molecules
are known to have an ability to bind and stabilize DNA-RNA
duplexes.24,25
’ RESULT AND DISCUSSION
The glycosylation and deacetylation established above were
performed repeatedly to generate a protected trisaccharide and
tetrasaccharide (Scheme 5). At these glycosylation steps, surprisingly,
Synthesis of Monomer Building Blocks. Toward the synth-
eses of the R-linked oligodiaminoglucoses, we planned a
Scheme 1. Synthetic Cycle of r-Linked Oligodiaminoglucose 1
5896
dx.doi.org/10.1021/jo200951p |J. Org. Chem. 2011, 76, 5895–5906

The Journal of Organic Chemistry
FEATURED ARTICLE
Scheme 2. Syntheses of the Glycosyl Donors
Scheme 3. Synthesis of the Glycosyl Acceptor
Table 1. Synthesis of r-Linked Disaccharides
entry
glycosyl donor
equiv of 4 or 6
activator
temp (°C)
yield (%)
R:βa
time (h)
1
2
3
4
5
6
4
6
6
6
1.5
2.0
2.0
2.0
2.0
NIS (2.5 equiv)/TfOH (0.5 equiv)
NIS (5.0 equiv)/TfOH (1.0 equiv)
NIS (5.0 equiv)/TfOH (1.0 equiv)
NIS (5.0 equiv)/TfOH (1.0 equiv)
NIS (5.0 equiv)/TfOH (1.0 equiv)
rt
rt
52
81:19
79:21
86:14
85:15
1
1
1
2
n.d.
82
0
ꢀ35
ꢀ78
80
n.r.
a Anomeric ratio was determined by 1H NMR.
the R-isomers could be separated more easily than in the synthesis of
the disaccharide.
Next, the simultaneous reduction of benzyl groups and azide
groups was performed using a palladium carbon or palladium
5897
dx.doi.org/10.1021/jo200951p |J. Org. Chem. 2011, 76, 5895–5906

The Journal of Organic Chemistry
FEATURED ARTICLE
Scheme 4. Deacetylation under Anhydrous Acidic Conditions
Scheme 5. Syntheses of the Protected Oligodiaminosaccharides
Scheme 6. Synthesis of Mono-diaminosaccharide 20
melting curves and Table 2 shows the Tm values for a self-
complementary RNA 12mer (50-rCGCGAAUUCGCG-30)2 in
the absence or presence of oligodiaminosaccharides. It is appar-
ent that tri- and tetradiaminosaccharides markedly stabilize the
RNAꢀRNA duplex by 3.4 and 7.9 °C, respectively. Conversely,
mono- and di diaminosaccharides did not stabilize the RNAꢀR-
NA duplexes. Table 2 also shows the results obtained using
different equivalents of tetradiaminosaccharides 29. In these
experiments, Tm values increased as the number of equivalents
increased. Furthermore, the Tm values increased drastically until
2 equivalents of tetradiaminosaccharides were added. For com-
parison, Table 2 also shows the results of UV melting analyses of
the duplexes in the absence and presence of the natural amino-
glycosides neomycin B and tobramycin (Figure 4) that are widely
studied and known to bind and stabilize a variety of RNA
molecules including RNAꢀRNA duplexes. In particular, neo-
mycin B is reported to have high ability to stabilize RNAꢀRNA
duplexes among a variety of aminoglycosides.40 In this study, it is
clear that tetradiaminosaccharide 29 increased the melting
temperature of RNAꢀRNA duplexes more strongly than the
neomycin B.
hydroxide catalyst after the dephthaloylation. However, in this
case, multiple products were produced (Scheme 6), while 12 was
converted into 20 via a palladium-catalyzed reduction.
The protected 2ꢀ4mers were dephthaloylated by treatment
with hydrazine monohydrate36,37 followed by reduction of azido
groups by the Staudinger reaction.38 Thus, the obtained oligo-
diamino-3-O-benzylated saccharides 22, 25, and 28 were purified
by reversed-phase HPLC. Finally, 23, 26, and 29 were success-
fully obtained by the reductive cleavage of the benzyl groups by
using a palladium carbon catalyst (Scheme 7). In addition, a trial
to perform simultaneous deacetylation and dephthaloylation by
hydrazine monohydrate was failed because of the rearrangement
of the acetyl group to an amino group. These amido linkages are
difficult to cleavage under mild conditions.39
Melting Temperature (Tm) Analysis. The ratio of an amino
sugar to a duplex was based on the number of amino groups in
the oligodiaminosaccharide and the number of phosphate groups
in the nucleic acid duplex. All experiments were performed near
the physiological conditions with a 10 mM phosphate buffer
containing 100 mM NaCl at pH 6.91. Figure 3 shows the UV
The results of UV melting analyses for a variety of 12mer and
24mer nucleic acid duplexes are summarized in Table 3, and a
typical Tm curve for the 24mer RNAꢀRNA duplex rA24-rU24 is
shown in Figure 5. In the cases of RNAꢀRNA duplexes, both the
oligodiaminosaccharides and aminoglycosides increased the Tm
values for a variety of duplexes. For (50-rAACCCGCGGGUU-30)2,
its melting temperature (77.7 °C) was 14 °C higher than that
5898
dx.doi.org/10.1021/jo200951p |J. Org. Chem. 2011, 76, 5895–5906

The Journal of Organic Chemistry
FEATURED ARTICLE
Scheme 7. Synthesis of Oligodiaminosaccharides 23, 26, and 29
Figure 3. UV melting curves of (50-rCGCGAAUUCGCG-30)2.
of (50-rCGCGAAUUCGCG-30)2, which had the same nucleo-
base composition, but the Tm values were unaffected by the
addition of mono- to tridiaminosaccharides. In this case, tetra-
diaminosaccharide 29 and aminoglycosides increased the Tm
values, although these increases were smaller than those for
(50-rCGCGAAUUCGCG-30)2. In contrast, for rA12-rU12, which
has an innately low Tm of 18.8 °C, a prominent stabilization of
the duplexes was observed by the addition of the amino sugars,
even in the presence of didiaminosaccharides. These results
suggest that the oligodiaminosaccharides and aminoglycosides
stabilize thermodynamically unstable RNA duplexes. Notably,
for almost all RNA duplexes that were used in the experiments,
the ability of oligodiaminosaccharides to stabilize RNA duplexes
increased as the length of the sugar chain increased, and tetra-
diaminosaccharides 29 exhibited the best ability to stabilize the
duplexes among the amino sugars examined.
Table 2. MeltingTemperaturesof(50-rCGCGAAUUCGCG-30)2
entry
aminosaccharide
equiva
Tm(°C)
Δ Tm (°C)
1
2
none
63.9
63.9
64.3
69.0
68.0
70.9
71.8
72.7
69.4
66.2
1mer
12
6
0.0
0.4
5.1
4.1
7.0
7.9
8.8
5.5
2.3
3
2mer
4
3mer
4
5
4mer
1
6
4mer
2
7
4mer
3
8
4mer
4
9
neomycin B
4
10
tobramycin
4
a Equivalent is for the amount of nucleic acid duplex (not for the amount
of nucleic acid molecule).
In clear contrast to the results of RNA duplex of the corre-
sponding sequence (Figure 3, Table 2), all of the oligodiamino-
saccharides did not stabilize DNAꢀDNA duplexes. Next, entries
5ꢀ7 show the results of DNA duplexes. In clear contrast to the
results of RNA duplexes, none of the oligodiaminosaccharides
and aminoglycosides induced substantial changes of Tm values for
all of the DNAꢀDNA duplexes. These results suggest that the
oligodiaminosaccharides and aminoglycosides do not increase the
thermal stability of DNAꢀDNA duplexes. In some of the entries,
however, oligodiaminosaccharides and aminoglycosides strength-
ened the relatively hypochromicity of DNAꢀDNA duplexes (see
the Supporting Information). This may indicate the interactions
between the sugars and the duplexes or single-stranded DNA at
high temperature.
Entries 8ꢀ11 show the results of the UV melting analyses for
the 24mer DNAꢀRNA hybrid duplexes, which have an A-type
duplex structure, in the absence or presence of mono to tetra-
diaminosaccharides or aminoglycosides. For both rA24-dT24 and
dA24-rU24, a certain degree of increase of the Tm values was
observed. These results clearly suggest the oligodiaminosacchar-
ides can also interact to A-like nucleic acid duplexes.
5899
dx.doi.org/10.1021/jo200951p |J. Org. Chem. 2011, 76, 5895–5906

The Journal of Organic Chemistry
FEATURED ARTICLE
Figure 4. Chemical structures of aminoglycosides.
Table 3. Melting Temperatures of a Variety of Sequences of Duplexesa
Δ Tm (°C)
entry
duplex
Tm (°C)
1mer
2mer
3mer
4mer
neomycin
tobramycin
1
r(CGCGAAUUCGCG)2
r(AACCCGCGGGUU)2
rA12-rU12
63.9
77.7
18.8
39.3
55.7
60.4
32.9
11.9
26.6
24.9
45.2
0.0
0.4
ꢀ0.2
3.4
5.1
0.3
7.9
1.9
5.5
2.3
2.3
0.6
2
ꢀ0.3
3
11.7
7.5
16.4
15.2
ꢀ0.3
0.0
14.5
10.4
0.0
6.4
4
rA24-rU24
2.5
5.2
5
d(CGCGAATTCGCG)2
d(AACCCGCGGGTT)2
d(AAAAAATTTTTT)2
dA12-rU12
ꢀ0.3
ꢀ0.4
ꢀ0.3
ꢀ0.1
ꢀ0.1
ꢀ0.4
ꢀ0.3
0.1
ꢀ1.2
ꢀ0.3
0.1
6
ꢀ0.3
0.4
7
0.2
8
8.1
6.3
9
rA12-dT12
2.8
1.9
10
11
dA24-rU24
7.1
0.0
14.7
1.3
21.8
4.0
20.8
2.7
14.8
0.6
rA24-dT24
a Equivalents of aminosaccharides are 12 (1mer 20), 6 (2mer 23), 4 (3mer 26, neomycin, tobramycin), and 3 (4mer 29) for 12mer duplexes and 12
(2mer 23), 8 (3mer 26, neomycin, tobramycin), and 6 (4mer 29) for 24mer duplexes.
Figure 5. UV melting curves of rA24-rU24.
As an extension of this study, stereoselectively synthesized
phosphorothioate DNA (RP-PS-DNA and SP-PS-DNA)-native
RNA duplex was also used as substrates. Because the all-RP-PS-
DNAꢀRNA duplex has negatively charged sulfate atoms on the
inner side of the major groove of the duplexes and the all-SP-PS-
DNAꢀRNA has these on the outer side,41 we expected to
observe differences of ΔTm caused by electrostatic interactions
with cationic oligodiaminosaccharides.
tetradiaminosaccharide 29. Changes of the Tm values of PS-
DNA-native RNA hybrid duplexes were observed, and these
values varied with their stereochemistry. As shown in entries
1ꢀ3, the all RP-PS-DNAꢀRNA duplexes, whose negatively
charged sulfate atoms are located on the inner side of the major
groove of the duplexes, were slightly more stabilized by the
tetradiaminosaccharide. These results did not conflict with our
hypothesis that oligodiaminosaccharides bind to the major
groove of A-type nucleic acid duplexes. Conversely, regarding
the PS-DNA-native DNA duplexes (entry 4ꢀ6), only slight
Table 4 shows the Tm values of PS-DNA-native RNA and PS-
DNA-native DNA duplexes42 in the absence or presence of the
5900
dx.doi.org/10.1021/jo200951p |J. Org. Chem. 2011, 76, 5895–5906

The Journal of Organic Chemistry
FEATURED ARTICLE
Table 4. Melting Temperatures of PS-DNAꢀRNA and PS-
DNAꢀDNA Duplexes in the Absence or Presence of
Tetradiaminosaccharide
tetradiaminosaccharide 29 (Figure 6). In contrast, appreciable
spectral changes were not observed on adding the mono- or
didiaminosaccharides. In the case of DNAꢀDNA duplexes, no
spectral changes were observed under identical experimental
conditions (Figure 7). Spectral changes of the RNAꢀRNA
duplex caused by the addition of 1ꢀ4 equivalents of tetradiami-
nosaccharide 29 are shown in the Supporting Information. The
spectral changes were nearly saturated with 2 equivalents of 29.
All these results correspond to those of Tm analyses described in
the previous section. These facts strongly suggest 26 and 29
stabilize RNAꢀRNA duplexes by causing small structural changes
and binding them to maintain the A-type duplex structure.
For comparison, CD spectra of RNAꢀRNA duplexes in the
presence of neomycin B were measured (see the Supporting
Information). The spectra of RNAꢀRNA duplexes changed with
the peak shift to a longer wavelength and the increase of the peak
intensity around 210 and 265 nm. These spectral changes were
very similar to those for the tri- and tetradiaminosaccharides.
These results suggest that the structural changes of the duplexes
caused by neomycin B and oligodiaminosaccharides are quite
similar.
Isothermal Titration Calorimetry (ITC) Experiments. In the
ITC experiments, the duplex concentration was 2-fold higher
than those in the UV melting and CD spectrometry analyses,
because the amount of heat generated by binding between the
nucleic acid duplexes and the tetradiaminosaccharide was ex-
pected not to be sufficient to calculate thermodynamic param-
eters. Figure 8 shows the results of ITC titration of the
tetradiaminosaccharide with the self-complementary RNAꢀ
RNA duplex (50-rCGCGAAUUCGCG-30)2 and DNAꢀDNA
entry
duplexa
Tm (°C)b
Δ Tm (°C)c
1
2
3
4
5
6
native DNAꢀRNA
RP-PS-DNAꢀRNA
SP-PS-DNAꢀRNA
native DNAꢀDNA
RP-PS-DNAꢀDNA
SP-PS-DNAꢀDNA
48.6
42.1
36.9
49.7
41.2
43.3
4.0
6.9
5.3
0.0
0.8
0.4
a Base sequiences are 50-dCpsApsGpsTpsCpsApsGpsTpsCpsApsGpsT-
30/50-dACTGACTGACTG-30 or 50-dCpsApsGpsTpsCpsApsGpsTps-
CpsApsGpsT-30/50-rACUGACUGACUG-30. b Tm value in the absence
of saccharide. c Δ Tm in the presence of 3 equiv of tetradiaminosacchar-
ide 29.
changes of the Tm values were observed and these results are
similar to that for native DNA duplexes.
CD spectrometry. CD spectra of self-complementary RNAꢀ
RNA and DNAꢀDNA duplexes (50-rCGCGAAUUCGCG-30)2
and (50-dCGCGAATTCGCG-30)2 in the presence or absence of
mono- to tetradiaminosaccharides were measured to detect their
structural changes. Upon the addition of tri- or tetradiaminosac-
charides, some changes of the spectra of RNAꢀRNA duplex were
observed. The positive peak near 265 nm and the negative peak
near 210 nm due to the Cotton effect slightly shifted 1ꢀ2 nm to
the longer wavelength, and their peak intensities increased up to
24% at the positive peak of 265 nm on the addition of the
Figure 6. CD spectra of (50-rCGCGAAUUCGCG-30)2.
Figure 7. CD spectra of (50-rCGCGAATTCGCG-30)2.
5901
dx.doi.org/10.1021/jo200951p |J. Org. Chem. 2011, 76, 5895–5906

The Journal of Organic Chemistry
FEATURED ARTICLE
Figure 8. (A) ITC profiles at 25 °C for the titration of tetradiaminosaccaride 29 into a solution of RNAꢀRNA duplex; Each curve is the result of a 10 μL
injection of 250 μM saccharide. The duplex concentration was 10 μM in a 10 mM phosphate buffer with 100 mM NaCl at pH 6.91; (B) Corrected
injection heats in the case of RNAꢀRNA were plotted.
duplex (50-dCGCGAATTCGCG-30)2. These results indicated
that the tetradiaminosaccharide bound to both the RNAꢀRNA
and DNAꢀDNA duplexes. However, the binding modes were
clearly different. The amount of heat generated by the instillation
of the saccharide solution to the RNA solution is approximately
double that for DNA (See the Supporting Information). These
results indicate that the binding of the saccharide to RNAꢀRNA
is stronger than to DNAꢀDNA, and strongly suggest the
existence of an A-type duplex specific binding mode of the oligo-
diaminosaccharides. In the case of tetradiaminosaccharide-RNA,
binding ratio N was 0.73. ΔH and ΔS were estimated to
Conversely, all of the oligodiaminosaccharides did not stabilize
DNAꢀDNA duplexes or change their structures. This is prob-
ably because the major groove width of well-known DNAꢀDNA
duplexes is 14ꢀ16 Å in solution, which is too wide to be
efficiently bound by the amino groups of the saccharides. On
the basis of these unique properties, 26 and 29 will be useful as
stabilizers of siRNA- and miRNA-based drugs or new materials
for their DDS.
’ EXPERIMENTAL SECTION
be ꢀ7705 (kcal/mol) and 2.67 (kcal/mol K), respectively. In
3
Conditions for Measurement of Tm Analysis. Absorbance
versus temperature profile measurements were carried out with eight-
sample cell changer, in quarts cells of 1 cm path length. The variation of
UV absorbance with temperature was monitored at λ = 260 nm. The
temperature was scanned between 0 and 95 °C, and rate of temperature
increase was 0.2 °C/min. Oligonucleotides were annealed after addition
of amino sugars. The samples containing oligonucleotides and amino
sugars were first rapidly heated to 95 °C, left for 20 min, and then
allowed to cool slowly to room temperature. 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 0.25 °C/min.
contrast, in the case of DNA, precise parameters could not be
calculated because of the large margin of error in the same
experimental conditions. These errors may be attributable to
nonspecific interactions between the tetradiaminosaccharide and
the DNA at the present concentration. With a higher concentra-
tion of nucleic acid duplexes, more than two state binding modes
were observed (data not shown). Under this condition, thermo-
dynamic parameters corresponding to the UV melting analysis
and CD spectrometry experiments were not calculated.
Conditions for Measurement of CD Spectra. All CD spectra
were recorded at 25 °C. The following instrument settings were used:
resolution, 0.1nm; sensitivity, 10 mdeg; response, 4 s; speed, 10 nm/min;
accumulation, 6.
Conditions for ITC Experiments. The tetradiaminosaccharide 29
and the nucleic acid duplexes were in a 10 mM phosphate buffer with
100 mMNaClatpH6.91. The tetradiaminosaccharidesolution (250μM)
was titrated into the nucleic acid duplex solutions (10 μM) at 25 °C. Each
titration of the tetradiaminosaccharide solution consisted of a preliminary
3 μL injection followed by 24 subsequent 10 μL additions, which were
performed over 20 s periods at 210 s intervals.
Phenyl 4-O-Acetyl-2,6-dazido-2,6-dideoxy-3-O-benzyl-1-thio-β-D-
glucopyranoside (4). Under argon atmosphere, 2 (0.194 g, 0.5 mmol)
was coevaporated with dry pyridine (5 mL ꢁ 3) and dissolved in dry
pyridine (10 mL). The solution was stirred and cooled to ꢀ15 °C, and
methanesulfonyl chloride (43 μL, 0.53 mmol, 1.06 equiv) was added.
’ CONCLUSION
We have developed a method to synthesize oligodiaminoglu-
coses, and we successfully synthesized 1- to 4mers of R-(1f4)-
linked-2,6-diamino-2,6-dideoxy-D-glucopyranose
oligomers.
Based on the UV melting analysis, CD spectrometry findings,
and ITC measurements, these oligodiaminosaccharides, particu-
larly the 3mer and 4mer, stabilized only A-type nucleic acid
duplexes such as RNAꢀRNA and DNA-RNA, by causing small
structural changes. The experimental results suggested that the
existence of an A-type duplex specific binding mode of the
oligodiaminosaccharides. In particular, tetradiaminosaccharide
29 had the largest stabilization effect on these duplexes among
the saccharides used in this study, including neomycin B and
tobramycin, which are naturally occurring aminoglycosides
known to bind and stabilize a variety of RNA molecules.
5902
dx.doi.org/10.1021/jo200951p |J. Org. Chem. 2011, 76, 5895–5906

The Journal of Organic Chemistry
FEATURED ARTICLE
After 11 h, the reaction was quenched by the addition of methanol
(2 mL), concentrated, and coevaporated with toluene (7 mL ꢁ 2). The
mixture was dissolved in chloroform, washed with saturated aqueous
solution of NaHCO3 (15 mL ꢁ 3), dried over Na2SO4, and evaporated.
The crude 3wascoevaporatedwithdryN,N-dimethylformamide (8 mL ꢁ 3)
and dissolved in dry N,N-dimethylformamide (5 mL). Sodium azide
(0.324 g, 5.0 mmol, 10 equiv) was added to the solution, and it was
stirred and heated to 80 °C. After 12 h, the mixture was cooled to rt and
concentrated to dryness. The crude product was extracted with ethyl
acetateꢀwater (1:1, v/v, 20 mL), the organic layer was washed with
water (10 mL ꢁ 2), and the aqueous layer was back extracted with ethyl
acetate (10 mL). The organic layer was dried over MgSO4, filtered and
concentrated. The crude azidosugar was dissolved in dry pyridine
(10 mL) under argon atmosphere. The solution was stirred and acetic
anhydride (75 μL, 0.79 mmol, 1.58 equiv) was added. After 28 h, the
mixture was concentrated to dryness and dissolved in chloroform. The
solution was washed with saturated aqueous solution of NaHCO3
(15 mL ꢁ 2), dried over Na2SO4, filtered, and evaporated. The crude
product was purified with silica gel column chromatography (chloro-
form) and 4 was obtained as colorless oil (0.196 g, 0.43 mmol, 86%): 1H
NMR (300 MHz, CDCl3) δ 7.28ꢀ7.62 (m, 10H, SPh, Ph-CH2),
4.80ꢀ4.90 (m, 2H, H-4, Ph-CH2a), 4.62 (d, J = 11.3 Hz, 1H, Ph-
CH2b), 4.43 (d, J = 9.9 Hz, 1H, H-1), 3.47ꢀ3.54 (m, 2H, H-3, H-5),
3.22ꢀ3.39 (m, 3H, H-2, H-6), 1.96 (s, 3H, Ac); 13C NMR (75 MHz,
CDCl3) δ 169.5, 137.2, 134.5, 129.8, 129.1, 129.0, 128.5, 128.1, 128.0,
85.9, 82.3, 75.4, 70.5, 64.5, 51.3, 20.7; ESI-HRMS m/z calcd for
C21H22KN6O4S+ (M + K+) 493.1060, found 493.1075.
Ph-CH2), 4.79ꢀ4.90 (m, 2H, H-4, Ph-CH2a), 4.63 (d, J = 11.3 Hz, 1H,
Ph-CH2b), 4.36(d, J =9.9Hz, 1H, H-1), 4.00 (dd, J= 9.6and14.0Hz, 1H,
H-6a), 3.72 (m, 1H, H-5), 3.57 (dd, J = 2.5 and 14.0 Hz, 1H, H-6b), 3.49
(t, J = 9.3 Hz, 1H, 3-H), 3.35 (t, J = 9.3 Hz, 1H, H-2), 2.00 (s, 3H, Ac); 13
C
NMR (300 MHz, CDCl3) δ 169.9, 167.8, 137.2, 134.3, 134.1, 131.2,
129.9, 128.7, 128.5, 128.1, 128.0, 123.4, 85.7, 82.2, 75.4, 75.0, 71.8, 69.6,
64.6, 39.2, 20.8; ESI-HRMS m/z calcd for C29H26KN4O6S+ (M + K+)
597.1205, found 597.1237.
Methyl O-(2,6-Diazido-2,6-dideoxy-3-O-benzyl-R-D-glucopyranoside)
(12). Under argon atmosphere, 9 (2.53 g, 6.4 mmol) was dissolved to
70% aqueous solution of acetic acid (300 mL) and heated to 70 °C. After
1.5 h, the solution was cooled to rt and concentrated. The solution of
crude product was diluted to ethyl acetate (200 mL) and washed with
saturated aqueous solution of NaHCO3 (100 mL ꢁ 2). The aqueous
layer was back-extracted with ethyl acetate (100 mL). The organic layer
was dried over Mg2SO4, filtered, and evapolated to dryness. The mixture
was purified by silica gel column chromatography (Ethyl acetate/hexane,
1:2, v/v and then ethyl acetate), and crude 10 was obtained. Under argon
atmosphere, the crude 10 was coevaporated with dry pyridine (15 mL ꢁ
3) and dissolved in dry pyridine (130 mL). The solution was stirred and
cooled to ꢀ15 °C, and methanesulfonyl chloride (0.50 mL, 6.46 mmol,
1.02 equiv) was added. After 7 h, the reaction was quenched by addition
of methanol (5 mL) and saturated aqueous solution of NaHCO3 (3 mL)
and the mixture was concentrated to dryness. The mixture was dissolved
in ethyl acetate (100 mL) and washed with saturated aqueous solution of
NaHCO3 (50 mL ꢁ 3), and the water phase was back extracted with
ethyl acetate (50 mL), dried over MgSO4, and evaporated. The crude
mesylate 11 was coevaporated with dry N,N-dimethylformamide
(10 mL ꢁ 3) and dissolved to dry N,N-dimethylformamide (64 mL).
Sodium azide (4.16 g, 64 mmol, 10 equiv) was added to the solution, and
the solution was stirred and heated to 80 °C. After 10.5 h, the mixture
was cooled to rt and concentrated. The crude product was extracted with
ethyl acetateꢀwater (2:1, v/v, 150 mL), the organic layer was washed
with water (50 mL ꢁ 2), and the water layer was back extracted with
ethyl acetate (50 mL). The organic layer was dried over MgSO4, filtered,
and concentrated to dryness. The crude product was purified with silica
gel column chromatography (dichloromethaneꢀhexane = 2:1 to 1:0,
v/v) and 12 was obtained as pale yellow oil (1.76 g, 5.3 mmol, 82%): 1H
NMR (300 MHz, CDCl3) δ 7.31ꢀ7.43 (m, 5H, Ph-CH2), 4.99 (d, J =
11.2 Hz, 1H, Ph-CH2a), 4.83 (d, J = 3.6 Hz, 1H, H-1), 4.67 (d, J = 11.6 Hz,
1H, PhꢀCH2b), 3.73ꢀ3.80 (m, 2H, H-3, H-5 or H-6), 3.36ꢀ3.55
(m, 6H, H-4, OCH3, H-5 or H-6), 2.08 (d, J = 2.8 Hz, 1H, OH-4), 3.35
(t, 1H, H-2); 13C NMR (75 MHz, CDCl3) δ 137.8, 128.6, 128.4, 128.1,
98.7, 80.1, 75.1, 71.0, 70.6, 63.2, 55.5, 51.3; ESI-HRMS m/z calcd for
C14H18KN6O4+ (M + K+) 373.1021, found 373.1025.
Methyl O-((4-O-Acetyl-2-azido-2-deoxy-3-O-benzyl-6-deoxy-6-phth-
alimide-R-D-glucopyranosyl)-(1f4)-2,6-dazido-2,6-dideoxy-3-O-benzyl-
R-D-glucopyranoside) (14). Under argon atmosphere, N-iodosuccini-
mide (0.0562 g, 250 μmol) was added to a mixture of 6 (56.1 mg,
100 μmol, 2.0 equiv) and 12 (50 μmol) after coevaporation with dry
toluene (1 mL ꢁ 6). The mixture was dissolved in a mixed solvent
(1 mL, Et2OꢀCH2Cl2, 4:1, v/v), cooled to 0 °C, and stirred, and then
trifluoromethanesulfonic acid (4.4 μL, 50 μmol) was added to the
solution. After 1 h, water (2 μL) was added and the mixture warmed to
rt. The solution was diluted with chloroform (20 mL) and washed with a
saturated aqueous solution of NaHCO3 (15 mL) and 10% Na2S2O3aq
(15 mL). The organic layer was dried over Na2SO4, filtered, and
concentrated. The crude product purified by silica gel column chroma-
tography (ethyl acetateꢀhexane (3:7, v/v)) to afford the pure R-
glycoside 14 as a pale yellow oil (20.4 mg, 26.5 μmol, 53%): 1H NMR
(300 MHz, CDCl3) δ 7.72ꢀ7.88 (m, 4H, NPhth), 7.28ꢀ7.38 (m, 10H,
Ph-CH2), 5.61 (d, J = 3.9 Hz, 1H, H-10), 4.95 (m, 2H, H-40, Ph-CH2a),
4.80ꢀ4.86 (m, 2H, Ph-CH2b, Ph-CH2c), 4.74 (d, J = 3.3 Hz, H-1), 4.65
(d, J = 11.0 Hz, 1H, Ph-CH2d), 3.89ꢀ4.00 (m, 4H, H-30, H-6a0, H-6b0,
Phenyl 2-Azido-2-deoxy-3-O-benzyl-6-deoxy-6-phthalimide-1-thio-
β-D-glucopyranoside (5). Under argon atmosphere, 2(5.417 g, 14.0 mmol)
was coevaporated with dry pyridine (10 mL ꢁ 3) and dissolved in dry
pyridine (200 mL). The solution was stirred and cooled to ꢀ15 °C, and
methanesulfonyl chloride (1.1 mL, 14.3 mmol, 1.02 equiv) was added.
After 5 h, the reaction was quenched by addition of methanol (10 mL),
concentrated and coevaporated with toluene (10 mL ꢁ 2). The mixture was
dissolved in chloroform (100 mL), washed with saturated aqueous solution
of NaHCO3 (100 mL ꢁ 3), dried over Na2SO4 and evaporated. The crude
mesylate 3 was coevaporated with dry N,N-dimethylformamide (15 mL ꢁ
3) and dissolved in dry N,N-dimethylformamide (140 mL). Potassium
phthalimide (4.89 g, 26.4 mmol, 1.9 equiv) was added to it, and the solution
wasstirredandheatedto100°C. After 13 h, the mixture was cooled to rt and
concentrated. The solution containing the crude product was diluted with
chloroform (100 mL) and washed with 10% brine (100 mL). After further
washing of the organic layer with 10% brine (100 mL), the organic layer
was dried over Na2SO4, filtered, and evaporated. The crude product was
recrystallized from ethanol (400 mL), and 5 was obtained as a colorless solid
1
(5.28 g, 10.2 mmol, 73%): H NMR (300 MHz, CDCl3) δ 7.74ꢀ7.88
(m, 4H, NPhth), 7.02ꢀ7.51 (m, 10H, SPh, Ph-CH2), 4.81ꢀ4.86 (dd, J =
10.7 and 16.5 Hz, 2H, PhꢀCH2), 4.32 (d, J = 10.2 Hz, 1H, H-1), 4.16 (dd,
J = 3.3 and 14.6 Hz, 1H, H-6a), 3.97 (dd, J = 4.1 and 14.6 Hz, 1H, H-6b),
3.48ꢀ3.54 (m, 1H, 5-H), 3.38 (t, J = 9.0 Hz, 1H, 3-H), 3.25 (m, 1H, 4H),
3.10 (dd, J = 9.1 and 10.2 Hz, 1H, 2H); 13C NMR (75 MHz, CDCl3) δ
168.7, 137.6, 135.3, 134.4, 131.7, 129.4, 128.8, 128.6, 128.3, 128.1, 123.6,
85.3, 83.4, 75.7, 71.4, 64.2, 38.1; ESI-HRMS m/z calcd for C27H24KN4O5S+
(M + K+) 555.1099, found 555.1106.
Phenyl 4-O-Acetyl-2-azido-2-deoxy-3-O-benzyl-6-deoxy-6-phthali-
mide-1-thio-β-D-glucopyranoside (6). Under argon atmosphere, 5
(5.28 g, 10.2 mmol) was dissolved in dry pyridine (100 mL). The solution
was stirred, and acetic anhydride (1.5 mL, 15.9 mmol, 1.56 equiv)
was added. After 1 d, the mixture was concentrated and dissolved in
chloroform. The solution was washedwith a saturated aqueous solution of
NaHCO3 (30 mL ꢁ 3), dried over Na2SO4, filtered, and evaporated. The
crude product was recrystallized from ethanol (50 mL), and 6 was
1
obtained as a colorless solid (5.29 g, 9.5 mmol, 93%): H NMR (300
MHz, CDCl3) δ 7.72ꢀ7.86(m, 4H, NPhth), 6.98ꢀ7.42 (m, 10H, SPh,
5903
dx.doi.org/10.1021/jo200951p |J. Org. Chem. 2011, 76, 5895–5906

The Journal of Organic Chemistry
FEATURED ARTICLE
H-3), 3.75 (m, 1H, H-5), 3.60ꢀ3.66 (m, 2H, H-50, H-4), 3.27ꢀ3.39 (m,
6H, H-2, H-6a, H-20, OCH3), 3.01 (dd, J = 4.4 and 13.2 Hz, 1H, H-6b),
2.05 (s, 3H, Ac); 13C NMR (75 MHz, CDCl3) δ 169.9, 168.0, 137.4,
137.2, 134.0, 131.9, 128.5, 128.0, 127.9, 127.6, 123.4, 123.2, 98.4, 97.4,
80.1, 74.9, 74.5, 74.4, 72.1, 69.8, 69.6, 63.8, 62.4, 55.5, 51.0, 39.1, 20.9;
ESI-HRMS m/z calcd for C37H38KN10O10+ (M + K+) 821.2404, found
821.2413.
CDCl3) δ 7.59ꢀ7.88 (m, 12H), 7.30ꢀ7.36 (m, 20H, Ph-CH2), 5.55
(d, J = 3.9 Hz, 1H), 5.44ꢀ5.48 (2d, 3.6 Hz, 3.9 Hz, 2H), 4.73ꢀ5.03
(m, 10H), 4.08ꢀ5.03 (m, 4H), 3.71ꢀ3.96 (m, 7H), 3.15ꢀ3.98 (m, 13H),
3.09 (dd, J=3.6and10.3Hz,1H,H-20 or H-200),3.00(dd,J=4.4and13.5Hz,
1H), 1.97 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 170.0, 167.8, 167.6, 137.4,
137.3, 137.1, 134.1, 133.9, 132.0, 131.5, 131.4, 128.5, 127.9, 127.8, 127.5, 127.4,
127.3, 123.4, 123.3, 123.2, 98.3, 98.1, 97.4, 80.0, 79.2, 78.9, 77.7, 76.8, 75.1, 74.3,
74.1, 74.0, 72.3, 69.7, 69.5, 69.2, 68.9, 63.8, 63.2, 63.1, 62.6, 55.5, 50.1, 39.7,
39.4, 39.1, 20.8; ESI-HRMS m/z calcd for C79H74N18NaO20+ (M + Na+)
1617.5219, found 1617.5205.
Compound 19. The same method for 15 was applied for the synthesis
of 19, except for using 18 (53.3 mg, 33 μmol) as a starting material.
Purification by silica gel column chromatography was performed with
ethyl acetateꢀtoluene (1:6, v/v) as an eluent. Compound 19 was
obtained as colorlessoil(36.6 mg, 23.6 μmol, 71%): 1H NMR(300 MHz,
CDCl3) δ 7.64ꢀ7.91 (m, 12H), 7.31ꢀ7.42 (m, 20H), 5.33ꢀ5.53 (3d,
J = 3.9 Hz, J = 3.6 Hz, J = 3.9 Hz, 3H), 4.72ꢀ5.14 (m, 9H), 4.43 (t, 1H),
4.11ꢀ4.29 (m, 4H), 3.54ꢀ4.04 (m, 11H), 3.26ꢀ3.46 (m, 10H), 3.11
(dd, J = 1H, J = 3.6 and 10.2 Hz), 3.01 (dd, J = 3.9 and 13.5 Hz, 1H); 13C
NMR (75 MHz, CDCl3) δ 168.8, 168.0, 167.9, 137.9, 137.8, 137.4,
137.2, 134. 2, 133.9, 132.1, 131.6, 128.6, 128.5, 128.4, 128.2, 127.9,
127.8, 127.6, 127.5, 123.5, 123.4, 123.2, 98.9, 98.3, 97.6, 80.2, 79.8, 79.1,
78.6, 75.6, 74.7, 74.5, 74.1, 73.9, 72.6, 71.3, 70.0, 69.7, 69.6, 63.8, 63.2,
63.1, 63.0, 55.5, 50.9, 39.7, 38.0; ESI-HRMS m/z calcd for
C77H72N18NaO19+ (M + Na+) 1575.5113, found 1575.5097.
Methyl O-(2-Azido-2-deoxy-3-O-benzyl-6-phthalimide-6-deoxy-
R-D-glucopyranosyl)-(1f4)-2,6- dazido-2,6-dideoxy-3-O-benzyl-R-D-
glucopyranoside) (15). Under argon atmosphere, 14 (0.148 g, 0.19
mmol) was dissolved in a mixed solvent (18 mL, MeOHꢀCH2Cl2, 5:1,
v/v) and stirred at rt. Acetyl chloride (450 μL) was added to the solution,
and it was refluxed. After 19 h, the solution was cooled to rt, and a
saturated aqueous solution of NaHCO3 was added. The mixture was
concentrated, dissolved in chloroform (20 mL), and washed with
saturated aqueous solution of NaHCO3 (15 mL ꢁ 3). The organic
was dried over Na2SO4, filtered, and concentrated. The residue was
purified by silica gel column chromatography (ethyl acetateꢀtoluene
(1:9, v/v)) to give 15 as a pale yellow oil (0.123 g, 0.165 mmol, 88%): 1H
NMR (300 MHz, CDCl3) δ7.72ꢀ7.88 (m, 4H, NPhth), 7.24ꢀ7.42
(m, 10H, Ph-CH2), 5.51 (d, J = 3.9 Hz, 1H, H-10), 4.83ꢀ4.97 (m, 5H,
H-1, Ph-CH2), 4.10 (dd, J = 3.3 and 14.6 Hz, 1H, H-6a0), 3.85ꢀ4.04 (m,
4H, H-30, H-50, H-60, H-3), 3.71ꢀ3.78 (m, 2H, H-3, H-5), 3.35ꢀ3.56
(m, 8H, H-40, H-6b, H-2, OCH3, H-6a, OH-40), 3.21 (dd, J = 4.1
and 10.2 Hz, 1H, H-2); 13C NMR (75 MHz, CDCl3) δ 168.9, 137.7,
137.5, 134.3, 131.7, 128.6, 128.4, 128.2, 128.0, 127.8, 127.7, 123.6, 98.5,
97.9, 80.4, 78.8, 75.5, 74.6, 74.1, 72.4, 71.3, 69.8, 69.6, 63.9, 62.6, 55.5,
Methyl O-(2,6-Diamino-2,6-dideoxy-R-D-glucopyranoside) hydro-
chloride (20). Compound 12 (8.0 mg, 24 μmol) was dissolved in a
mixed solvent (1.5 mL, waterꢀmethanol, 1:1, v/v, 0.1 M HCl), 5% Pd/
C (0.01 g) was added and stirred. After 5 h of hydrogen bubbling, Pd/C
was removed byfiltration with filter paper and membrane filter (0.45μm),
and the solution was concentrated and coevaporated with water (2 mL ꢁ
4). The crude product was dissolved in methanol (0.1 mL), reprecipitated
from acetone (1.5 mL), and washed with acetone (1.5 mL ꢁ 2). The
precipitate was dissolved in water and lyophilized from water to give 20 as
colorless solid (19.1 μmol, 80%): 1H NMR (300 MHz, D2O) δ 5.07 (d,
J = 3.9 Hz, 1H, H-1), 3.86ꢀ3.90 (m, 2H, H-3, H-5), 3.22ꢀ3.51 (m, 6H,
51.2, 38.2; ESI-HRMS m/z calcd for C35H36KN10O9 (M + K+)
+
779.2298, found 779.2314.
Compound 16. The same method for 14 was applied for the synthesis
of 16, except that 6 (0.117 g, 0.209 mmol, 1.5 equiv) and 15 (0.139 g,
0.103 mmol) were used as starting materials. Purification by silica gel
column chromatography was performed with ethyl acetateꢀtoluene
(1:9, v/v) and ethyl acetateꢀhexane (7:13, v/v) as eluent. 16 was
obtained as colorless oil (0.105 g, 88 μmol, 63%). 1H NMR (300 MHz,
CDCl3) δ 7.57ꢀ7.90 (m, 8H), 7.31ꢀ7.57 (m, 15H), 5.59 (d, J = 3.9 Hz,
1H), 5.49 (d, J = 3.6 Hz, 1H), 4.71ꢀ5.05 (m, 8H), 4.34 (t, J = 8.7 Hz,
1H), 4.09ꢀ4.17 (m, 3H), 3.87ꢀ3.98 (m, 3H), 3.54ꢀ3.75 (m, 5H),
3.23ꢀ3.43 (m, 8H), 3.14 (dd, J = 3.6 and 10.2 Hz, 1H), 2.98 (dd, J = 4.1
and 13.5 Hz, 1H), 2.07 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 170.2,
167.8, 137.5, 137.3, 137.2, 134.2, 133.9, 132.0, 131.4, 128.6, 128.5, 128.0,
127.9, 127.7, 127.6, 127.5, 123.4, 123.2, 98.4, 98.3, 97.6, 80.1, 79.2, 77.3,
75.2, 74.4, 74.1, 74.0, 72.5, 69.7, 69.4, 63.8, 63.2, 62.5, 55.5, 50.9, 39.6,
H-2, H-4, OCH3, H-6a), 3.17 (dd, J = 10.4 and 13.5 Hz, 1H, H-6b); 13
C
NMR (75 MHz, D2O) δ 96.91, 71.82, 70.11, 68.47, 56.19, 54.29, 40.88;
+
ESI-HRMS m/z calcd for C7H17N2O4 (M + H+) 193.1183, found
193.1187.
Methyl O-((2,6-Diamino-2,6-dideoxy-3-O-benzyl-R-D-glucopyranosyl)-
(1f4)-2,6-diamino-2,6-dideoxy-3-O-benzyl-R-D-glucopyranoside) hy-
drochloride (22). Under argon atmosphere, 15 (13.9 mg, 18.8 μmol),
ethanol (1 mL), and hydrazine monohydrate (25 μL) were added to a
round-bottom flask, stirred, and refluxed. After 3 h, the mixture was
cooled to rt and concentrated. The crude product was dissolved in
chloroform (2 mL), and insoluble phthalyl hydrazide was removed by
filtration. The chloroform solution was washed with water (10 mL ꢁ 3)
and the organic layer was dried over Na2SO4, filtered, and concentrated.
The crude sugar was dissolved in a mixed solvent (2.4 mL, dioxaneꢀ
methanol, 5:1, v/v) and stirred. Triphenylphosphin (0.0800 g,
305 μmol, 16.2 equiv) was added to the solution and refluxed over 3 h.
After cooling to rt, 25% aqueous solution of ammonia (2.4 mL) was
added to the solution and the flask was sealed. After the solutionw as
stirred overnight, the mixture was concentrated. Then water (5 mL) and
0.1 M hydrochloric acid (1 mL) were added and the mixture sonicated
for 1 min. The aqueous solution was filtered, washed with chloroform
(10 mL ꢁ 3), concentrated, and coevaporated with water (3 mL ꢁ 3).
The crude product was purified with C18 reversed-phase HPLC (0.05%
TFA, waterꢀacetonitrile). After HPLC purification, fraction was con-
centrated and 0.02 M hydrochloric acid (5 mL) was added. The solution
was concentrated and coveaporated with water (1 mL ꢁ 4) to give a
benzyl-protected 22 as colorless oil (6.8 μmol, 36%): 1H NMR (300 MHz,
D2O) δ 7.38ꢀ7.45 (m, 10H, Ph-CH2), 5.64 (d, J = 3.3 Hz, 1H, H-10), 5.14
+
39.1, 20.9; ESI-HRMS m/z calcd for C58H56KN14O15 (M + K+)
1227.3681, found 1227.3706.
Compound 17. The same method for 15 was applied for the synthesis
of 17, except for using 16 (81.8 mg, 69 μmol) as a starting material.
Purification by silica gel column chromatography was performed with
ethyl acetate-toluene (3:17, v/v) as an eluent. 17 was obtained as a
colorless oil (69.2 mg, 60 μmol, 88%): 1H NMR (300 MHz, CDCl3) δ
7.65ꢀ7.91 (m, 8H), 7.29ꢀ7.46 (m, 15H), 5.57 (d, J = 3.3 Hz, 1H), 5.47
(d, J = 4.1 Hz, 1H), 4.79ꢀ5.07 (m, 6H), 4.80 (d, J = 10.2 Hz, 1H), 4.27
(m, 1H), 3.91ꢀ4.09 (m, 6H), 3.25ꢀ3.84 (m, 14H), 3.03 (J = 3.9 and
10.5 Hz, dd, 1H); 13C NMR (75 MHz, CDCl3) δ 168.7, 168.1, 137.9,
137.3, 134.3, 134.0, 132.0, 131.6, 128.5, 128.2, 128.0, 127.9, 127.6, 123.5,
123.3, 98.8, 98.4, 97.5, 80.2, 79.2, 78.9, 75.5, 74.5, 74.3, 73.9, 72.9, 71.2,
70.5, 69.7, 63.8, 63.1, 62.6, 55.5, 50.9, 39.8, 38.4; ESI-HRMS m/z calcd
for C56H54N14NaO14+ (M + Na+) 1169.3836, found 1169.3860.
Compound 18. The same method for 14 was applied for the synthesis
of 18, except for using 6 (50.3 mg, 90 μmol, 1.45 equiv) and 17 (71.0 mg,
62 μmol) as starting materials. Purification by silica gel column
chromatography was performed with ethyl acetateꢀtoluene (1:6, v/v)
and ethyl acetateꢀhexane (2:3, v/v) as an eluent. Compound 16 was
obtained as colorless oil (53.3 mg, 33.4 μmol, 54%): 1H NMR (300 MHz,
5904
dx.doi.org/10.1021/jo200951p |J. Org. Chem. 2011, 76, 5895–5906

The Journal of Organic Chemistry
FEATURED ARTICLE
(d, J = 3.9 Hz, 1H, H-1), 4.68ꢀ4.89 (m, 4H, Ph-CH2), 4.38 (dd, J = 8.0 and
10.5 Hz, 1H), 4.13ꢀ4.19 (m, 2H), 3.78ꢀ3.94 (m, 3H), 3.70 (t, J = 8.0 Hz,
1H), 3.31ꢀ3.56 (m, 8H); 13C NMR (75 MHz, D2O) δ137.4, 136.6, 129.7,
129.4, 129.2, 129.1, 128.4, 96.9, 93.8, 78.0, 76.1, 75.2, 72.7, 72.2, 71.4, 70.0,
’ AUTHOR INFORMATION
Corresponding Author
*E-mail: wada@k.u-tokyo.ac.jp.
+
66.9, 56.6, 52.4, 41.1, 40.0; ESI-HRMS m/z calcd for C27H41N4O7
(M + H+) 533.2970, found 533.3015.
’ ACKNOWLEDGMENT
Methyl O-((2,6-Diamino-2,6-dideoxy-R-D-glucopyranosyl)-(1f4)-
2,6-diamino-2,6-dideoxy-R-D- glucopyranoside Hydrochloride (23).
The same method for 20 was applied for the synthesis of 23, except
that 22 (6.8 μmol) was used as a starting material. Compound 23 was
We thank Professor Kazuhiko Saigo (Kochi University of
Technology) for helpful suggestions, Professor Kohei Tsumoto
(University of Tokyo) for ITC measurements and helpful discus-
sions, and Professor Natsuhisa Oka (Gifu University) and Dr.
Naoki Iwamoto for helpful discussions and the synthesis of PS-
DNAs. We also thank the Japan Student Services Organization for
the scholarship. And this work was supported by National Institute
1
obtained as a colorless solid (6.5 μmol, 95%): H NMR (300 MHz,
D2O) δ 5.78 (d, J = 3.3 Hz, 1H, H-10), 5.09 (d, J = 3.6 Hz, 1H, H-1),
4.03ꢀ4.18 (m, 2H, H-5, H-3), 3.76ꢀ3.88 (m, 3H, H-4, H-50, H-30),
3.38ꢀ3.53 (m, 8H, H-20, H-6a, H-6a0, H-2, OCH3, H-40), 3.24ꢀ3.48
(m, 2H, H-6b, H-6b0); 13C NMR (75 MHz, D2O) δ 96.8, 96.6, 77.1,
71.1, 70.6, 69.9, 69.2, 66.7, 56.5, 54.6, 54.2, 41.1, 40.6; ESI-HRMS m/z
calcd for C13H29N4O7+ (M + H+) 353.2031, found 353.2049.
of Biomedical Innovation (NIBIO), KAKENHI (22 5612), and
Japan Society for the Promotion of Science (JSPS).
3
Compound 25. The same method for 22 was applied for the synthesis
of 25, except 17 (10.7 mg, 9.3 μmol) was used as a starting material.
Compound 25 was obtained as a colorless oil (2.79 μmol, 29%): H
’ REFERENCES
(1) Neidle, S. Oxford Handbook of Nucleic Acid Structure; Oxford
University Press: Oxford, 1999.
(2) Wolfram, S. Principles of Nucleic Acid Structure; Springer:
New York, 1983.
(3) Uhlmann, E.; Peyman, A. Chem. Rev. 1990, 90, 543.
(4) Helene, C.; Toulme, J. J. Biochim. Biophys. Acta 1990, 1049, 99.
(5) Delaney, S.; Barton, J. K. J. Org. Chem. 2003, 68, 6475.
(6) Kuzuya, A.; Komiyama, M. Nanoscale 2010, 2, 310.
(7) Brana, M. F.; Cacho, M.; Gradillas, A.; de Pascual-Teresa, B.;
Ramos, A. Curr. Pharm. Des. 2001, 7, 1745.
(8) Baraldi, P. G.; Bovero, A.; Fruttarolo, F.; Preti, D.; Tabrizi, M. A.;
Pavani, M. G.; Romagnoli, R. Med. Res. Rev. 2004, 24, 475.
(9) Kaul, M.; Pilch, D. S. Biochemistry 2002, 41, 7695.
(10) Zapp, M. L.; Stern, S.; Green, M. R. Cell 1993, 74, 969.
(11) Mei, H. Y.; Galan, A. A.; Halim, N. S.; Mack, D. P.; Moreland,
D. W.; Sanders, K. B.; Truong, H. N.; Czarnik, A. W. Bioorg. Med. Chem.
Lett. 1995, 5, 2755.
1
NMR (300 MHz, D2O) δ 7.20ꢀ7.42 (m, 15H), 5.48 (d, J = 1.9 Hz, 1H),
5.35 (d, J = 3.6 Hz, 1H), 5.10 (d, J = 3.6 Hz, 1H), 4.71ꢀ4.99 (m, 6H),
4.51ꢀ4.57 (m, 3H), 4.07ꢀ4.28 (m, 6H), 3.80ꢀ3.88 (m, 1H), 3.74 (t, J =
8.5 Hz, 1H), 3.36ꢀ3.60 (m, 10H), 3.15ꢀ3.22 (m, 2H); 13C NMR
(75 MHz, D2O) δ 137.09, 136.98, 129.81, 129.71, 129.57, 129.49,
129.38, 128.87, 128.47, 97.10, 96.57, 91.81, 78.37, 76.93, 75.19, 75.80,
73.64, 72.93, 71.16, 70.94, 70.42, 67.94, 67.36, 56.57, 53.96, 53.01, 49.78,
41.06, 39.99, 38.82; ESI-HRMS m/z calcd for C40H59N6O10+ (M + H+)
783.4287, found 783.4271.
Compound 26. The same method for 20 was applied for the synthesis
of 26, except 25 (2.79 μmol) was used as a starting material. Compound 26
was obtained as a colorless solid (2.42 μmol, 87%): 1H NMR (300 MHz,
D2O) δ5.78ꢀ5.83 (2d, J = 3.9 Hz, 3.9 Hz, 2H), 5.10 (d, J = 3.6 Hz, 1H),
4.03ꢀ4.24 (m, 4H), 3.80ꢀ3.94 (m, 4H), 3.28ꢀ3.55 (m, 13H); 13C NMR
(75 MHz, D2O) δ 97.08, 96.74, 96.22, 77.45, 77.24, 70.92, 70.52, 69.93,
69.51, 69.12, 68.34, 66.61, 56.50, 54.57, 54.44, 54.27, 41.18, 40.45; ESI-
HRMS m/z calcd for C19H41N6O10+ (M + H+) 513.2879, found 513.2883.
Compound 28. The same method for 22 was applied for the synthesis
of 28, except 19 (13.7 mg, 8.8 μmol) was used as a starting material.
(12) Jin, E.; Katritch, V.; Olson, W. K.; Kharatisvili, M.; Abagyan, R.;
Pilch, D. S. J. Mol. Biol. 2000, 298, 95.
(13) Forge, A.; Schacht, J. Audiol. Neurootol. 2000, 5, 3.
(14) Fire, A.; Xu, S. Q.; Montgomery, M. K.; Kostas, S. A.; Driver,
S. E.; Mello, C. C. Nature 1998, 391, 806.
(15) Czauderna, F.; Fechtner, M.; Dames, S.; Aygun, H.; Klippel, A.;
Pronk, G. J.; Giese, K.; Kaufmann, J. Nucleic Acids Res. 2003, 31, 2705.
(16) Holen, T.; Amarzguioui, M.; Wiiger, M. T.; Babaie, E.; Prydz,
H. Nucleic Acids Res. 2002, 30, 1757.
(17) Shim, M. S.; Kwon, Y. J. FEBS J. 2010, 277, 4814.
(18) Watts, J. K.; Deleavey, G. F.; Damha, M. J. Drug Discov. Today
2008, 13, 842.
(19) de Lima, M. C. P.; Neves, S.; Filipe, A.; Duzgunes, N.; Simoes,
S. Curr. Med. Chem. 2003, 10, 1221.
(20) Sioud, M.; Sorensen, D. R. Biochem. Biophys. Res. Commun.
2003, 312, 1220.
(21) Gyi, J. I.; Lane, A. N.; Conn, G. L.; Brown, T. Biochemistry 1998,
37, 73.
(22) Wang, F.; DeMuro, N. E.; Elmquist, C. E.; Stover, J. S.; Rizzo,
C. J.; Stone, M. P. J. Am. Chem. Soc. 2006, 128, 10085.
(23) Mohamadi, F.; Richards, N. G. J.; Guida, W. C.; Liskamp, R.;
Lipton, M.; Caufield, C.; Chang, G.; Hendrickson, T.; Still, W. C.
J. Comput. Chem. 1990, 11, 440.
1
Compound 28 was obtained as a colorless oil (2.82 μmol, 32%): H
NMR (300 MHz, D2O) δ 7.31ꢀ7.46 (m, 20H), 5.46ꢀ5.62 (3d, J = 2.8 Hz,
J = 3.0 Hz, J = 2.5 Hz, 3H), 5.16 (d, J = 3.9 Hz, 1H), 4.69ꢀ4.94 (8H),
4.33ꢀ4.49 (m, 4H), 4.02ꢀ4.26 (m, 7H), 3.67ꢀ3.80 (m, 4H), 3.32ꢀ
3.57 (m, 12H); 13C NMR (75 MHz, D2O) δ 137.46, 136.90, 136.76,
129.80, 129.68, 129.62, 129.49, 129.31, 129.22, 128.56, 128.43, 128.39,
96.97, 95.51, 93.76, 92.96, 78.27, 76.28, 75.51, 75.22, 74.46, 73.62, 72.99,
72.62, 71.91, 67.10, 56.57, 53.24, 52.53, 51.00 41.20, 39.82; ESI-HRMS m/z
calcd for C53H77N8O13+ (M + H+) 1033.5605, found 1033.5637.
Compound 29. The same method for 20 was applied for the synthesis
of 29, except 28 (2.82 μmol) was used as a starting material. Compound
1
29 was obtained as a colorless solid (2.59 μmol, 92%): H NMR (300
MHz, D2O) δ 5.81ꢀ5.87 (m, 3H), 5.11 (d, J = 3.6 Hz, 1H), 4.10ꢀ4.34
(m, 6H), 3.82ꢀ3.96 (m, 5H), 3.31ꢀ3.60 (m, 16H); 13C NMR (75 MHz,
D2O) δ 97.03, 96.72, 96.58, 96.16, 77.40, 77.27, 70.89, 70.51, 69.92, 69.38,
69.10, 68.43, 68.23, 66.59, 56.50, 54.57, 54.42, 54.28, 41.34, 41.25, 41.11,
+
40.42; ESI-HRMS m/z calcd for C25H53N8O13 (M + H+) 673.3727,
found 673.3705.
(24) Shaw, N. N.; Xi, H. J.; Arya, D. P. Bioorg. Med. Chem. Lett. 2008,
18, 4142.
(25) West, C.; Francis, R.; Friedman, S. H. Bioorg. Med. Chem. Lett.
2001, 11, 2727.
(26) Benakli, K.; Zha, C. X.; Kerns, R. J. J. Am. Chem. Soc. 2001, 123,
9461.
(27) Venot, A.; Swayze, E. E.; Griffey, R. H.; Boons, G. J. ChemBio-
Chem 2004, 5, 1228.
’ ASSOCIATED CONTENT
Supporting Information. 1H and 13C NMR spectra of
S
b
4ꢀ6, 12, 14ꢀ20, 22, 23, 25, 26, 28, and 29, methods of
experiments, UV melting curves, and ITC profiles. This material
is available free of charge via the Internet at http://pubs.acs.org.
5905
dx.doi.org/10.1021/jo200951p |J. Org. Chem. 2011, 76, 5895–5906

The Journal of Organic Chemistry
FEATURED ARTICLE
(28) Heitmann, W.; Liepmann, H.; Matzel, U.; Zeugner, H.; Fuchs,
A. M.; Krahling, H.; Ruhland, M.; Mol, F.; Tulp, M. T. M. Eur. J. Med.
Chem. 1988, 23, 249.
(29) Emmerson, D. P. G.; Hems, W. P.; Davis, B. G. Tetrahedron:
Asymmetry 2005, 16, 213.
(30) Lindberg, J.; Ohberg, L.; Garegg, P. J.; Konradsson, P. Tetra-
hedron 2002, 58, 1387.
(31) Pozsgay, V.; Coxon, B. Carbohydr. Res. 1994, 257, 189.
(32) Tabeur, C.; Machetto, F.; Mallet, J. M.; Duchaussoy, P.;
Petitou, M.; Sinay, P. Carbohydr. Res. 1996, 281, 253.
(33) Trumtel, M.; Tavecchia, P.; Veyrieres, A.; Sinay, P. Carbohydr.
Res. 1990, 202, 257.
(34) Konradsson, P.; Mootoo, D. R.; McDevitt, R. E.; Frasereid, B.
J. Chem. Soc., Chem. Commun. 1990, 270.
(35) Ma, Z. C.; Zhang, J. J.; Kong, F. Z. Tetrahedron: Asymmetry
2003, 14, 2595.
(36) Santhanam, B.; Worfent, M. A.; Moore, J. N.; Boons, G. J.
Chem.—Eur. J. 2004, 10, 4798.
(37) Tsvetkov, Y. E.; Backinowsky, L. V. Carbohydr. Res. 1985,
135, 312.
(38) Fernandez, J. M. G.; Mellet, C. O.; Blanco, J. L. J.; Mota, J. F.;
Gadelle, A.; Costesarguet, A.; Defaye, J. Carbohydr. Res. 1995, 268, 57.
(39) Rakotondramasy, V. C.; Laschiazza, R.; Lecso-Bornet, M.;
Koch, M.; Tillequin, F.; Deguin, B. J. Nat. Prod. 2007, 70, 19.
(40) Chen, Q.; Shafer, R. H.; Kuntz, I. D. Biochemistry 1997, 36,
11402.
(41) Kawahara, S.; Wada, T.; Sekine, M. J. Am. Chem. Soc. 1996, 118,
9461.
(42) Oka, N.; Yamamoto, M.; Sato, T.; Wada, T. J. Am. Chem. Soc.
2008, 130, 16031.
5906
dx.doi.org/10.1021/jo200951p |J. Org. Chem. 2011, 76, 5895–5906