Synthesis of p-amino-WNA derivatives to enhance the stability of the anti-parallel triplex

Article abstract of DOI:10.1016/j.tet.2008.05.096

We have previously developed W-shaped nucleoside analogs (WNAs) having a nucleobase and an aromatic ring for the formation of the unnatural triplex DNA. Modification of an aromatic ring of WNA is highly effective regarding the stability of the triplex DNA. In this study, we designed new WNA analogs having the p-aminobenzene as an aromatic ring, which were synthesized via the Curtius-Yamada rearrangement. Based on the evaluation of the triplex formation with p-amino-WNA-TFO, it has been shown that the amino group may produce a non-selective interaction with the phosphate backbone of the target duplexes. These results indicate that the amino modification is useful to overcome the sequence-dependence of the TFO containing WNA analogs.

Full text of DOI:10.1016/j.tet.2008.05.096

Tetrahedron 64 (2008) 7164–7170
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis of p-amino-WNA derivatives to enhance the stability
of the anti-parallel triplex
,
,
,b,
*
Yosuke Taniguchi a b, Mieko Togo a, Eriko Aoki a b, Yuko Uchida a, Shigeki Sasaki a
a Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
b CREST, Japan Science and Technology Agency, Kawaguchi, Center Building, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 30 April 2008
Received in revised form 21 May 2008
Accepted 21 May 2008
Available online 24 May 2008
We have previously developed W-shaped nucleoside analogs (WNAs) having a nucleobase and an aro-
matic ring for the formation of the unnatural triplex DNA. Modification of an aromatic ring of WNA is
highly effective regarding the stability of the triplex DNA. In this study, we designed new WNA analogs
having the p-aminobenzene as an aromatic ring, which were synthesized via the Curtius–Yamada re-
arrangement. Based on the evaluation of the triplex formation with p-amino-WNA-TFO, it has been
shown that the amino group may produce a non-selective interaction with the phosphate backbone of
the target duplexes. These results indicate that the amino modification is useful to overcome the
sequence-dependence of the TFO containing WNA analogs.
Keywords:
Nucleoside analogs
Non-natural type triplex DNA
Rearrangement
Antigene
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
in sequences, which could not be stabilized by the original WNA-
b
T.7–9 On the other hand, the para-bromo-substituted WNA analogs
Triplex DNA is useful for gene research, therapies, and biological
tools by the gene inhibition1 or activation2 of the gene expression,
gene recombination,3 etc. In an anti-parallel triplex DNA, the pu-
rine-rich triplex forming oligonucleotides (TFOs) can bind to only
the homopurine strand by two reverse Hoogsteen hydrogen bonds
within the major groove of the duplex DNA in a sequence specific
manner (G/GC and A/AT).4 However, pyrimidine nucleosides, which
insert into the purine strand, such as TA and CG base pairs, inhibit
the stable triplex formation using the natural TFOs. These base pairs
are called interrupting sites.
had no effect on the triplex stability. These results suggested that
the triplex DNA was inhibited by the steric hindrance between the
p-bromo group of WNA and the phosphate group of the target
duplex DNA. Therefore, a functional group, such as an amino group,
at the para position of the benzene ring may produce an attractive
interaction with the phosphate group to enhance the stability of the
triplex.10 Thus, a new p-amino-substituted analog of WNA (p-
amino-WNA) was designed. It is suggested by molecular modeling
O
O
O
O
Recently, we developed nucleoside analogs (WNA: W-shaped
nucleoside analogs) having a nucleobase as a recognition part and
an aromatic ring as a stacking part.5,6 It was demonstrated that the
O
O
WNA- C
WNA- T
O
O
WNA-bT and WNA-bC exhibited selective recognition of a TA or
a CG interrupting site, respectively (Fig. 1). Our previous studies
have also shown that the recognition of the interrupting sites is
dependent on the neighboring nucleobase of the WNA analogs in
the TFOs.9 In this study, we focused on the modification of the
aromatic ring to solve this sequence dependency and synthesized
a variety of WNA analogs having a modified aromatic ring. It was
N
H
N
H
O
H
O
N
N
H
N
O
H
H
N
H
N
O
O
N
N
H
shown that bromo-substituted WNA-
bT analogs, oBr-WNA-bT and
N
N
N
O
H
N
N
O
N
dR
dR
mBr-WNA- T, could stabilize a TA interrupting site with selectivity
b
N
N
N
dR
N
H
N
H
dR
C
G
T
A
* Corresponding author. Tel./fax: þ81 92 642 6615.
E-mail address: sasaki@phar.kyushu-u.ac.jp (S. Sasaki).
Figure 1. Speculated recognition model of WNA-bT/TA and WNA-bC combinations
(WNA W-shaped nucleoside analogs).
0040-4020/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2008.05.096

Y. Taniguchi et al. / Tetrahedron 64 (2008) 7164–7170
7165
of the triplex with the base triplet of p-amino-WNA-
b
T and a TA
allylation reaction, the anomeric isomers were separated by col-
umn chromatography to obtain the -allyl 3b in 40% and the -allyl
base pair that the amino group is in close proximity to the phos-
phate group (Fig. 2). We now describe the synthesis and evaluation
of the triplex forming ability of the new WNA analogs, p-amino-
a
b
compound in 56% yield. These isomers were determined by the 1H
COSY and NOESY spectra. The hydrolysis of the desired isomer 3b
gave the carboxylic acid 4 in 95% yield in three steps.12,13 Next, we
investigated the Yamada procedure using DPPA14 for the Curtius
rearrangement of 4 (Table 1). As an amino protecting group needs
to be tolerable to acidic conditions included in the later synthesis
(Scheme 2), the reaction was performed in the absence of alcohol to
obtain the amino compound. The rearranged product 5a was
obtained, but in low yield together with the dimerized urea com-
pound (Table 1, entry 1). In the presence of ethanol, the ethoxy-
carbonylamino compound (5b) was obtained in good yield (Table 1,
entry 2), but this protecting group was not removed under basic or
acidic conditions. The rearrangement was also successful in the
presence of tert-butanol (Table 1, entry 4) and not in the presence of
trifluoroethanol (Table 1, entry 3). Thus, an acid-labile t-Boc pro-
tecting group was converted to the acid-stable trifluoroacetyl group
(Scheme 2). The Boc group was removed by TFA in CH2Cl2 at 0 ꢁC to
produce the corresponding amino compound, which was again
protected with the trifluoroacetyl group to give the intermediate 6
in 91% yield in two steps. The trifluoroacetyl group is compatible to
reagents and conditions used in an automated DNA synthesizer.15
Subsequently, oxidative cleavage of the vinyl group of 6, the cycli-
zation reaction under the acidic condition, followed by protection
of the hydroxyl group afforded the bicyclic intermediate 7 in a 64%
overall yield. Nucleobases, thymine or N-benzoylcytosine, were
WNA-a, bT and -a, bC.
Figure 2. The predicted structure of triplex formation with TFO having p-amino-WNA-
T by MD calculation. The complex between p-amino-WNA- T and TA interrupting site
b
b
(left). TFO is shown with Space-Filling Model and p-aminobenzene shows green color
(right).
2. Results and discussion
2.1. Synthesis of TFOs containing p-amino-WNA
coupled with this intermediate 7 using Lewis acids to give 8b-1 (
bT)
in 50%, 8a-1 ( T) in 47%, 9b-1 ( C) in 47%, and 9a-1 ( C) in 50%
a
b
a
According to our early method, in which the WNA analogs were
synthesized through the Lewis acid-catalyzed allylation of the 10-
phenyl substituted ribose intermediate (2), the 2,5-dimethylpyrole
substituted derivative (2a) was chosen as an intermediate (Scheme
yield. These N-glycosylated compounds were separated by silica-
gel column chromatography and their stereochemistries were de-
termined by the 1H COSY and NOESY spectra. Deprotection of
TBDPS and the acetyl groups of the 50- and 30-hydroxyl groups gave
the corresponding diol compounds (8a,b-2, 9a,b-2) in 69–97%
yield. These diol compounds were converted to the phosphor-
1).
D
-Ribonolacton 16 was treated with 1-(4-bromophenyl)-2,5-
dimethylpyrrole11 and n-BuLi at ꢀ78 ꢁC to afford 2a in 86% yield.
However, in spite of attempts such as the reactions at higher
temperature or in the presence of other Lewis acids, the allylation
of 2a was not successful. Alternatively, it was expected that the
already-reported p-cyano derivative (3b)8 might be transformed
into the amino derivative via the Curtius rearrangement following
amidite precursor (bT: 8b-3 in 49%; aT: 8a-3 in 39%; bC: 9b-3 in
59%; and C: 9a-3 in 35% yield). The TFOs (TFO1–4) containing p-
a
amino-WNA derivatives were obtained in good yield using an au-
tomated DNA synthesizer. The TFO1 in the sequence of 30-AZG-50
containing p-amino-WNA-aT at the position of Z was called
hydrolysis.
D-Ribonolacton 1 was treated with p-bromocyano-
TFO1( T). After purification by HPLC, the purity and structure of
a
benzene and n-BuLi at ꢀ100 ꢁC to afford 2b in 79% yield. After the
these TFOs were identified by MALDI-TOF MS measurements.
R
2.2. Triplex ability of TFOs containing WNA analogs
R'O
R'O
O
O
O
a or b
The ability of the triplex formations was evaluated by gel-shift
OH
assay using the 32P-labeled TFO as a tracer. The association
O
O
O
O
2a R = N
2b R = CN
Table 1
c
Conditions of Curtius–Yamada rearrangement
R' = TBDPS
1
R
COOH
NH
c
N
R
R'O
R'O
O
O
DPPA, Et3N,
alcohol, reflux
R'O
R'O
O
O
O
O
O
O
H
O
H
3b R = CN
R = COOH
H
O
O
O
d
R' = TBDPS
4
5a-d
4
NOE
3a
Entry
–R
Yield (%)
Scheme 1. Reagents and conditions: (a) 1-(4-bromophenyl)-2,5-dimethylpyrrole, n-
BuLi, THF, ꢀ78 ꢁC (86%). (b) 4-Bromobenzonitrile, n-BuLi, THF, ꢀ100 ꢁC (79%). (c) Al-
1
2
3
4
–H
50
87
10
85
–COOCH2CH3
–COOCH2CF3
–COOC(CH3)3
lyltrimethylsilane, ZnBr2, CH3NO2, rt, (a-allyl: 40%, b-allyl: 56%). (d) (1) KOH, H2O2,
EtOH, H2O, reflux; (2) TBDPSCl, Et3N, DMAP, CH2Cl2, rt; (3) K2CO3, H2O, MeOH, THF, rt
(95% for three steps).

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Y. Taniguchi et al. / Tetrahedron 64 (2008) 7164–7170
O
The parent WNA-bT exhibited selective stabilization to the TA
interrupting site in the sequence of TFO1 and 2 (30-AZG-50 and 30-
O
HN
CF3
GZG-50, Z¼WNA-
b
T).9 Therefore, it was expected that the TFO
T would also recognize
F3C
NH
containing the p-amino-substituted WNA-
b
the TA interrupting site. However, the p-amino-substituted WNA-
R'O
b
T series showed quite different recognition profiles, in which there
O
a
b
O
R'O
was no stabilization by TFO1 (30-AZG-50), CG stabilization by TFO2
(30-GZG-5), and stabilization to the AT and GC sites by TFO3 (30-
AZA-50) and 4 (30-GZA-5) (Fig. 3A and B). It was speculated in our
5d
OAc
O
O
O
AcO
R' = TBDPS
O
6
7
previous study that the thymine of WNA-bT might interact with the
O
junction of the T–A base pair.6 As the site for the formation of the
base triplet is in a crowded environment as shown in Figure 2,
the amino-phosphate interaction might distort this complex
structure and induce a drastic change in the recognition profiles. A
preferable complex structure formed between a TA site and the
HN
CF3
HN
CF3
c or d
α
R
O
parent WNA-bT in TFO1 might suffer from a subtle change, resulting
O
R'O
R'O
in loss of overall affinity. The parent WNA-bC stabilized a CG site
O
β
O
R
only in the sequence TFO1(30-AZG-50),9 in contrast, the p-amino
derivatives of WNA-C showed non-specific stabilization (Fig. 3C and
D). It was reported that the sequence dependency of non-natural
nucleobases in a parallel triplex was attributable to stacking in-
teractions between the non-natural nucleobase and the bases of the
TFO and the target DNA strands.16 Similarly, stacking interactions
may contribute to the sequence dependency of the WNA analogs,
because WNA-H, lacking a nucleobase, showed sequence dependent
triplex stabilization.6,9 Surprisingly, although the recognition base of
the WNA analogs and the magnitude of the Ks value are different,
R"O
R"O
8b, 9b
8a, 9a
R = Thymine (8)
R = N-Bz-cytosine (9)
8a,b-1, 9a,b-1: R' = TBDPS, R" = Ac
8a,b-2, 9a,b-2: R', R" = H
e
f
8a,b-3, 9a,b- : R' = DMTr, R" = iPr NPOC H CN
3
2
2 4
DNA synthesis
and purification
both stereochemistries
a and b produced a similar selectivity to the
5'
5'
5'
TFO1( , T), ( , C) 3' GGAAGG AZG GAGGAGGGA
TFO2( , T), ( , C) 3' GGAAGG GZG GAGGAGGGA
TFO3( , T), ( , C) 3' GGAAGG AZA GAGGAGGGA
TFO4( , T), ( , C) 3' GGAAGG GZA GAGGAGGGA
Z = p-amino-WNA
target base pair (Fig. 3A vs B and Fig. 3C vs D). Triplex stabilization is
made by harmonization of various factors such as hydrogen bonds,
stacking interactions, shape complementarity, and so on. The results
of this study have clearly indicated that the interaction of the amino
group of the WNA analogs with the phosphate backbone of the
target DNA can be an additional stabilizing effect of the triplex.
5'
Scheme 2. Reagents and conditions: (a) (1) TFA, CH2Cl2, 0 ꢁC; (2) (CF3CO)2O, pyridine,
0 ꢁC; (3) acetone, TsOH, rt (91% for three steps). (b) (1) OsO4 (aq), NaIO4 (aq), pyridine,
rt; (2) H2SO4 (aq), THF, 60 ꢁC; (3) Ac2O, pyridine, rt (64% for three steps). (c) Thymine,
TMSOTf, CH3CN, rt (97%). (d) Benzoylcytosine, BSA, TMSOTf, CH3CN, rt (97%). (e) (1)
TBAF, THF, rt; (2) 0.2 M NaOH (aq), MeOH, THF, 0 ꢁC (69–97% for two steps). (f) (1)
3. Conclusion
In conclusion, we designed and synthesized p-amino modified
WNA analogs. The evaluation of the triplex forming ability showed
that the p-amino-WNA analogs provide a non-selective stabilizing
effect for the triplex DNA despite the different recognition bases
such as a thymine and a cytosine and a different stereochemistry.
These results indicated that the amino group might interact with
the phosphate group of the target duplex DNA in close proximity.
Therefore, the modification of the para position at the aromatic part
of the WNA analogs has a potential enhancement effect on the
stability of the triplex DNA. Further studies involving other modi-
fications of the para position are now ongoing.
i
DMTrCl, pyridine; (2) Pr2NP(Cl)OCH2CH2CN, DIPEA, CH2Cl2, 0 ꢁC (35–59% for two
steps). The naming of the TFOs: for example, TFO1(
aT) represents the TFO1 sequence
incorporating p-amino-WNA- T at the Z position.
a
constants (Ks values) of the triplex formations were calculated from
the intensity of the radioactive bands, as previously described.6,9
The results of the Ks values of TFO1-4(
amino-WNA- and p-amino-WNA-
Figure 3A and B, respectively. TFO1( T) having the p-amino-WNA-
T in the sequence of 30-AZG-50 had no stabilizing effect for the four
target duplex DNAs (Fig. 3A, purple bar). TFO2( T) consisting of G
at the two neighboring sides of the p-amino-WNA- T showed
a stabilizing effect for the CG and GC base pairs (Fig. 3A, green bar).
TFO3(
T) (30-AZA0) and TFO4( T) (30-GZA-50) also exhibited a non-
selective stabilizing effect (Fig. 3A, yellow and pink bars). In
Figure 3B, interestingly, TFO1–4( T) having the p-amino-WNA-
showed a similar selectivity with TFO1–4( T) in each combination,
although the magnitude of the Ks values was different.
The Ks values of p-amino-WNA-C are summarized in Figure 3C
b
,a
T) containing Z¼p-
b
T
aT
are summarized in
b
b
b
b
4. Experimental section
4.1. General
b
b
a
aT
b
The 1H NMR (400, 500 MHz) and 13C NMR (100, 125 MHz)
spectra were recorded by Varian UNITY-400 and INOVA-500
spectrometers, respectively. 31P NMR (161 MHz) spectrum was
recorded using 10% phosphoric acid in D2O for the internal standard
at 0 ppm. IR spectra were obtained using a PerkinElmer FTIR-
SpectrumOne. High-resolution mass spectra were recorded on an
Applied Biosystems Mariner System 5299 spectrometer.
and D. TFO1(bC), TFO2(bC), and TFO4(b
C) having a sequence of 30-
AZG-50, 30-GZG-50, and 30-GZA-50 showed a non-selective stabiliz-
ing effect (Fig. 3C, purple, green, and pink bars). On the other hand,
TFO3(
formations for the AT and GC base pairs (Fig. 3C, yellow bar).
Similarly, TFO1( C), TFO2( C), and TFO4( C) containing p-amino-
WNA- showed the non-selective triplex formations, and
TFO3(
C) (30-AZA-50) stabilized the AT and GC base pairs (Fig. 3D).
Therefore, the selectivity of the -isomer is similar to that of the
isomer of p-amino-WNA-C (Fig. 3C and D).
b
C) in the sequence of 30-AZA-50 showed selective triplex
a
a
a
4.1.1. (1R,5R,6S,8R)-3,3-Dimethyl-6-(p-cyanophenyl)-6-(20-
propenyl)-8-(tert-butyldiphenylsilyloxymethyl)-2,4,7-
trioxabicyclo[3.3.0]octane (3b)
To a solution of 4-bromobenzonitrile (0.85 g, 4.69 mmol) in THF
(18 mL) was added n-BuLi (1.6 M in hexane solution, 3.0 mL,
a
C
a
b
a
-

Y. Taniguchi et al. / Tetrahedron 64 (2008) 7164–7170
7167
Figure 3. Bar graph of the association constant (Ks) by TFOs containing p-amino-WNA derivatives. Conditions: triplex formation was done for 12 h at 22 ꢁC in the buffer containing
20 mM Tris–HCl, 5 mM MgCl2, 2.5 mM spermidine, and 10% sucrose at pH 7.5. Electrophoresis was done at 10 ꢁC with 15% non-denatured polyacrylamide gel. TFO (10 nM, 18mer)
containing the 32P-labeled one as the tracer was used. The concentration of the target duplex (30 bp) was increased (0–100 nM). The Ks values of the natural G/GC triplet using the
natural TFO (Z¼dG) are 0.086ꢂ109 (30-AGG-50), 0.66ꢂ109 (30-GGG-50), 0.58ꢂ109 (30-AGA-50), and 0.69ꢂ109 (30-GGA-50).9
4.69 mmol) at ꢀ100 ꢁC. This mixture was stirred at the same tem-
perature for 1 h. A solution of 1 (1.0 g, 2.34 mmol) in THF (18 mL)
was added to this mixture. After stirring for 2.5 h, the reaction was
quenched with a satd NH4Cl solution and extracted with EtOAc. The
organic solvent was washed with water and brine, dried over
Na2SO4, and evaporated. The residue was purified by silica-gel
column chromatography (hexane/EtOAc¼9:1) to give 2b as a col-
orless foam (0.98 g, 1.86 mmol, 79%; HRMS (ESI) m/z: calcd for
HRMS (ESI) m/z: calcd for C34H39NO4SiNa (MþNa)þ 576.2541, found
576.2519.
4.1.2. (1R,5R,6S,8R)-3,3-Dimethyl-6-(p-carboxyphenyl)-6-(20-
propenyl)-8-(tert-butyldiphenylsilyloxymethyl)-2,4,7-
trioxabicyclo[3.3.0]octane (4)
A solution of 3b (3.92 g, 7.09 mmol) in CH3CH2OH (30 mL), 40%
KOH solution (35 mL) and 30% H2O2 (5 mL) were refluxed for 21 h.
The reaction mixture was cooled at 0 ꢁC and acidified using a 23%
HCl solution. This solution was extracted with EtOAc. The organic
solvent was washed with water and brine, dried over Na2SO4, and
evaporated. The residue dissolved in CH2Cl2 (25 mL), Et3N (2.37 mL,
17 mmol), tert-butylchlorodiphenylsilane (4.36 mL, 17 mmol), and
DMAP (0.21 g, 1.7 mmol) were stirred for 3 h. This reaction mixture
was diluted with EtOAc, and washed with a satd NH4Cl solution,
water, and brine. The organic layer was dried over Na2SO4 and
evaporated. The residue dissolved in THF (30 mL), CH3OH (10 mL),
and a K2CO3 solution (2.94 g in water (15 mL), 21.3 mmol) were
stirred for 13 h. The reaction was quenched with a 10% HCl solution
and extracted with EtOAc. The organic solvent was washed with
water and brine, dried over Na2SO4, and evaporated. The residue
was purified by silica-gel chromatography (hexane/EtOAc¼5:1 to
2:1) to give 4 as a colorless foam (3.84 g, 6.70 mmol, 95%). 1H NMR
C
31H35NO5SiNa (MþNa)þ 552.2177, found 552.2173). A solution of
2b (0.92 g, 1.73 mmol) in CH3CN (4.6 mL) and allyltrimethylsilane
(1.67 mL, 10.38 mmol) was added to a suspension of zinc bromide
(1.33 g, 5.88 mmol) at 0 ꢁC. After stirring for 3 h at room tempera-
ture, the reaction was quenched with a satd NaHCO3 solution and
extracted with EtOAc. The organic solvent was washed with water
and brine, dried over Na2SO4, and evaporated. The residue was
purified by silica-gel column chromatography (hexane/
EtOAc¼17:1) to give 3b as a colorless oil (
40%) and the other isomer as a colorless oil (
56%). Compound of
-allyl. 1H NMR (400 MHz, CDCl3):
a
-allyl, 0.38 g, 0.69 mmol,
-allyl, 0.97 mmol,
7.62–7.59
b
a
d
(4H, m), 7.52 (2H, d, J¼8.2 Hz), 7.46 (2H, d, J¼8.2 Hz), 7.43–7.39 (2H,
m), 7.36–7.32 (4H, m), 5.53–5.43 (1H, m), 4.89–4.80 (2H, m), 4.71
(1H, dd, J¼7.0, 3.4 Hz), 4.62 (1H, d, J¼7.0 Hz), 4.25 (1H, q, J¼4.3 Hz),
3.77 (2H, d, J¼4.3 Hz), 2.77 (1H, dd, J¼14.7, 7.0 Hz), 2.52 (1H, dd,
J¼14.7, 7.3 Hz), 1.61 (3H, s), 1.35 (3H, s), 0.98 (9H, s). 13C NMR
(400 MHz, CDCl3):
d
8.00 (2H, d, J¼8.6 Hz), 7.65–7.60 (4H, m), 7.48
(125 MHz, CDCl3):
d
149.9, 135.6, 133.22, 132.5, 131.6, 129.8, 127.7,
(2H, d, J¼8.6 Hz), 7.43–7.32 (6H, m), 5.57–5.47 (1H, m), 4.89–4.83
(2H, m), 4.72–4.68 (2H, m), 4.26 (1H, q, J¼4.6 Hz), 3.79 (1H, dd,
J¼11.0, 4.6 Hz), 3.75 (1H, dd, J¼11.0, 4.6 Hz), 2.79 (1H, dd, J¼14.7,
126.4, 118.9, 118.2, 114.9, 110.6, 87.6, 87.5, 82.2, 82.1, 64.1, 40.5, 26.8,
26.0, 24.9, 19.2. FTIR (neat): 3074, 2931, 2858, 2229, 1739 cmꢀ1
.

7168
Y. Taniguchi et al. / Tetrahedron 64 (2008) 7164–7170
7.0 Hz), 2.57 (1H, dd, J¼14.7, 7.3 Hz), 1.62 (3H, s), 1.36 (3H, s), 0.99
(9H, s). 13C NMR (125 MHz, CDCl3):
171.4, 150.7, 135.6, 133.2, 132.8,
0.73 mmol) in CH3CN (7.0 mL) was added to the above mixture.
After stirring for 3 h, the reaction mixture was quenched with
a satd NaHCO3 solution and extracted with EtOAc. The organic layer
was washed with water and brine, dried over Na2SO4, and evapo-
rated. The isomers were separated by flash silica-gel column
chromatography (hexane/CHCl3/acetone¼5:5:2 to 2:2:1) to give
d
129.8,129.7, 127.7, 125.7,118.0,114.7, 87.7, 82.2, 64.2, 40.7, 26.8, 26.0,
25.0, 19.2. FTIR (neat): 2931, 1689 cmꢀ1. HRMS (ESI) m/z: calcd for
C
34H40O6SiNa (MþNa)þ 595.2486, found 595.2533.
4.1.3. (1R,5R,6S,8R)-3,3-Dimethyl-6-(p-trifluoroacetyl-
aminophenyl)-6-(20-propenyl)-8-(tert-butyldiphenylsilyl-
oxymethyl)-2,4,7-trioxabicyclo[3.3.0]octane (6)
each isomer in a total of 97% yield. p-Amino-WNA-
a white powder (276 mg, 0.37 mmol, 50%). 1H NMR (400 MHz,
CDCl3):
bT (8b-1):
d
9.62 (1H, s), 8.46 (1H, s), 7.71 (2H, d, J¼8.5 Hz), 7.63 (2H,
The solution of 4 (6.0 g, 10.5 mmol) in tert-BuOH (220 mL), EtN3
(2.9 mL, 21 mmol), and DPPA (diphenylphosphoryl azide, 4.5 mL,
21 mmol) was refluxed for 20 h. The reaction mixture was diluted
with EtOAc. The organic layer was washed with water and brine,
dried over Na2SO4, and evaporated. The residue was purified by
silica-gel chromatography (hexane/Et2O¼10:1) to give 5d as a col-
orless oil (5.8 g, 8.93 mmol, 85%). This residue (5.7 g, 8.88 mmol)
was dissolved in CH2Cl2 (76 mL) and trifluoroacetic acid (19 mL) at
0 ꢁC. After stirring for 2.5 h, this reaction mixture was quenched
with satd NaHCO3 solution and extracted with EtOAc. This organic
layer was washed with water and brine, dried over Na2SO4, and
evaporated. The solution of this residue in pyridine (145 mL) and
trifluoroacetic anhydride (5.0 mL, 35.6 mmol) was stirred for 1.5 h
at 0 ꢁC. The reaction mixture was diluted with satd NaHCO3 and
extracted with EtOAc. This organic layer was washed with water
and brine, dried over Na2SO4, and evaporated. This residue was
dissolved in acetone (40 mL) and p-toluenesulfonic acid mono-
hydrate (50.8 mg, 0.26 mmol). After stirring for 90 h, this reaction
mixture was quenched with NaHCO3 (80 mg, 0.98 mmol), the solids
were filtered off, and then the filtrate was evaporated. The residue
was purified by silica-gel chromatography (hexane/Et2O¼6:1) to
give 6 as colorless oil (5.16 g, 8.06 mmol, 91%). 1H NMR (400 MHz,
dd, J¼7.9, 1.2 Hz), 7.58 (2H, dd, J¼7.9, 1.2 Hz), 7.51 (2H, d, J¼8.5 Hz),
7.44–7.30 (6H, m), 7.11 (1H, d, J¼1.2 Hz), 6.01 (1H, t, J¼6.7 Hz), 5.27
(1H, d, J¼3.7 Hz), 5.10 (1H, dd, J¼9.2, 3.7 Hz), 4.27 (1H, dt, J¼9.2,
3.4 Hz), 4.00 (1H, dd, J¼12.0, 3.1 Hz), 3.72 (1H, dd, J¼12.0, 3.7 Hz),
2.85 (1H, dd, J¼14.0, 6.7 Hz), 2.77 (1H, dd, J¼14.0, 6.7 Hz), 2.05 (3H,
s), 1.94 (3H, s), 0.99 (9H, s). 13C NMR (100 MHz, CDCl3):
d 170.9,
164.2, 150.7, 137.2, 135.6, 135.5, 135.4, 133.0, 132.9, 129.8, 127.8,
127.7, 126.4, 120.3, 111.1, 92.9, 87.4, 80.2, 73.4, 62.7, 48.1, 29.7, 26.7,
20.7, 19.2, 12.4. FTIR (neat): 3262, 3070, 2929, 2856, 2336,
1688 cmꢀ1. HRMS (ESI) m/z: calcd for C38H41F3N3O8Si (MþH)þ
752.2610, found 752.2595. p-Amino-WNA-aT (8a-1): a white
powder (260 mg, 0.35 mmol, 47%). 1H NMR (400 MHz, CDCl3):
d
8.24 (1H, s), 7.91 (1H, s), 7.63–7.60 (4H, m), 7.55 (1H, d, J¼1.22 Hz),
7.49 (4H, s), 7.45–7.33 (6H, m), 6.52 (1H, dd, J¼7.9, 5.8 Hz), 4.99 (1H,
dd, J¼8.9, 4.0 Hz), 4.77 (1H, d, J¼4.0 Hz), 4.33 (1H, quintet,
J¼4.0 Hz), 3.94 (1H, dd, J¼11.6, 3.4 Hz), 3.76 (1H, dd, J¼11.6, 4.3 Hz),
2.83 (1H, dd, J¼15.3, 7.9 Hz), 2.61 (1H, dd, J¼15.3, 5.8 Hz), 2.03 (3H,
s), 1.95 (3H, s), 1.01 (9H, s). 13C NMR (125 MHz, CDCl3):
d 169.9,
163.3, 150.5, 138.4, 135.6, 135.5, 134.9, 133.0, 132.8, 129.9, 129.8,
127.8, 126.2, 120.7, 112.0, 92.2, 85.9, 85.5, 80.3, 72.7, 63.0, 48.0, 29.7,
26.8, 20.6, 19.2, 12.8. FTIR (neat): 2958, 2923, 2853, 2331,
1694 cmꢀ1. HRMS (ESI) m/z: calcd for C38H41F3N3O8Si (MþH)þ
752.2610, found 752.2631.
CDCl3):
d 7.74 (1H, s), 7.64–7.61 (4H, m), 7.45–7.32 (10H, m), 5.58–
5.47 (1H, m), 4.89–4.83 (2H, m), 4.71–4.65 (2H, m), 4.23 (1H, q,
J¼4.6 Hz), 3.77 (1H, dd, J¼11.0, 4.9 Hz), 3.73 (1H, dd, J¼11.0, 4.6 Hz),
2.76 (1H, dd, J¼14.7, 7.0 Hz), 2.54 (1H, dd, J¼14.6, 7.3 Hz), 1.61 (3H,
s), 1.35 (3H, s), 1.00 (9H, s). FTIR (neat): 3302, 2933, 2859,
1709 cmꢀ1. HRMS (ESI) m/z: calcd for C35H40F3NO5SiNa (MþNa)þ
662.2520, found 662.2531.
4.1.6. (10S,30R,40R,50R,70S and R)-N4-Benzoyl-1-{40-acetoxy-10-(p-
trifluoroacetylaminophenyl)-30-(tert-butyldiphenylsilyloxymethyl)-
20,60-dioxabicyclo-[3.3.0]oct-70-yl}-cytosines (9b-1 and 9a-1)
N,O-Bis(trimethylsilyl)acetamide (0.54 mL, 2.19 mmol) was
added to a suspension of N-benzoylcytosine (235 mg, 1.1 mmol) in
CH3CN (12 mL). A solution of TMSOTf (0.2 mL, 1.1 mmol) and 7
(500 mg, 0.73 mmol) in CH3CN (7.0 mL) was added to the above
mixture. After stirring for 3 h, the reaction mixture was quenched
with satd NaHCO3 solution and extracted with EtOAc. The organic
layer was washed with water and brine, dried over Na2SO4, and
evaporated. The isomers were separated by flash silica-gel column
chromatography (hexane/CHCl3/acetone¼2:2:1 to 1:1:1) to give
4.1.4. (1R,3R,4R,5R,7RS)-1-(p-Trifluoroacetylaminophenyl)-3-(tert-
butyldiphenylsilyloxymethyl)-4,7-diacetoxy-2,6-dioxabicyclo-
[3.3.0]octane (7)
This detailed procedure was described in Ref. 6. 1H NMR
(400 MHz, CDCl3): d 7.83 (0.5H, s), 7.77 (0.5H, s), 7.69–7.60 (5H, m),
7.55 (1H, d, J¼8.55 Hz), 7.49 (1H, d, J¼8.55 Hz), 7.47 (1H, d,
J¼8.9 Hz), 7.45–7.31 (6H, m), 6.62 (0.5H, d, J¼4.3 Hz), 6.50 (0.5H, d,
J¼5.8 Hz), 5.03 (0.5H, dd, J¼9.2, 5.2 Hz), 5.02 (0.5H, dd, J¼9.5,
4.3 Hz), 4.89 (0.5H, d, J¼5.2 Hz), 4.77 (0.5H, d, J¼4.3 Hz), 4.51 (0.5H,
dt, J¼9.2, 3.1 Hz), 4.20 (0.5H, dt, J¼9.5, 2.4 Hz), 4.06 (0.5H, dd,
J¼11.9, 2.4 Hz), 4.05 (0.5H, dd, J¼11.9, 2.7 Hz), 3.76 (0.5H, dd, J¼11.6,
3.2 Hz), 3.73 (0.5H, dd, J¼11.6, 3.2 Hz), 2.83 (0.5H, dd, J¼15.0,
5.8 Hz), 2.71 (0.5H, dd, J¼15.0, 5.8 Hz), 2.60 (0.5H, dd, J¼15.3,
1.5 Hz), 2.57 (0.5H, d, J¼15.0 Hz), 2.13 (1.5H, s), 2.07 (1.5H, s), 2.02
each isomer in a total of 97% yield. p-Amino-WNA-
a white powder (287 mg, 0.34 mmol, 47%). 1H NMR (400 MHz,
CDCl3): 8.75 (1H, s), 7.98 (1H, s), 7.94–7.88 (3H, m), 7.66–7.59 (8H,
bC (9b-1):
d
m), 7.52–7.33 (10H, m), 6.28 (1H, t, J¼7.0 Hz), 5.16 (1H, d, J¼4.0 Hz),
5.05 (1H, dd, J¼9.2, 4.0 Hz), 4.27 (1H, dt, J¼9.2, 3.4 Hz), 4.03 (1H, dd,
J¼11.6, 2.8 Hz), 3.75 (1H, dd, J¼11.6, 3.4 Hz), 3.17 (1H, dd, J¼14.5,
7.0 Hz), 2.59 (1H, dd, J¼14.5, 7.0 Hz), 2.04 (3H, s), 1.01 (9H, s). 13
C
NMR (125 MHz, CDCl3):
d 170.3, 162.5, 154.9, 154.6, 144.5, 137.7,
(3H, s), 1.02 (9H, s). 13C NMR (125 MHz, CDCl3):
d
170.0, 169.7, 139.9,
135.6, 135.0, 133.3, 133.1, 132.9, 129.9, 129.8, 129.1, 127.8, 127.6,
126.5, 120.5, 92.4, 91.1, 87.4, 80.4, 73.0, 62.4, 49.4, 29.7, 29.3, 26.8.
FTIR (neat): 2928, 2855, 1731, 1703, 1660 cmꢀ1. HRMS (ESI) m/z:
calcd for C44H44F3N4O8Si (MþH)þ 841.2875, found 841.2925. p-
135.6, 134.4, 133.1, 132.9, 129.8, 127.8, 127.7, 126.4, 126.3, 120.4,
120.3, 99.5, 91.4, 88.8, 87.2, 80.1, 78.7, 72.3, 71.1, 62.7, 61.9, 50.4, 49.0,
29.7, 26.8, 21.3, 20.6, 20.5, 19.2. FTIR (neat): 3302, 2932, 1731 cmꢀ1
.
HRMS (ESI) m/z: calcd for C35H48F3NO8SiNa (MþNa)þ 708.2211,
Amino-WNA-
aC (9a-1): a white powder (304 mg, 0.36 mmol, 50%).
found 708.2222.
1H NMR (400 MHz, CDCl3):
d
8.74 (1H, s), 8.22 (1H, d, J¼7.63 Hz),
8.03 (1H, s), 7.90 (2H, d, J¼7.33 Hz), 7.63–7.57 (6H, m), 7.53–7.39
(8H, m), 7.37–7.33 (4H, m), 6.36 (1H, dd, J¼7.3, 5.0 Hz), 5.05 (1H, dd,
J¼8.2, 4.6 Hz), 4.94 (1H, d, J¼4.6 Hz), 4.21 (1H, dt, J¼8.2, 4.3 Hz),
3.82 (1H, dd, J¼11.6, 4.3 Hz), 3.73 (1H, dd, J¼11.6, 4.3 Hz), 3.05 (1H,
dd, J¼15.3, 7.3 Hz), 2.66 (1H, dd, J¼15.3, 5.0 Hz), 2.08 (3H, s),1.00
4.1.5. (10S,30R,40R,50R,70S and R)-{40-Acetoxy-10-(p-trifluoro-
acetylaminophenyl)-30-(tert-butyldiphenylsilyloxymethyl)-20,60-
dioxabicyclo[3.3.0]oct-70-yl}-thymines (8b-1 and 8a-1)
N,O-Bis(trimethylsilyl)acetamide (0.54 mL, 2.19 mmol) was
added to a suspension of thymine (138 mg, 1.1 mmol) in CH3CN
(12 mL). A solution of TMSOTf (0.2 mL, 1.1 mmol) and 7 (500 mg,
(9H, s). 13C NMR (125 MHz, CDCl3):
d 169.8,162.4,155.0,154.7,143.8,
138.3, 135.6, 135.5, 135.1, 133.3, 132.9, 132.8, 129.9, 129.8, 129.1,

Y. Taniguchi et al. / Tetrahedron 64 (2008) 7164–7170
7169
127.8, 127.6, 126.1, 120.7, 92.4, 88.5, 87.0, 80.4, 73.6, 63.0, 53.9, 49.5,
HRMS (ESI) m/z: calcd for C26H24F3N4O7 (MþH)þ 561.1592, found
29.7, 29.3, 26.8. FTIR (neat): 2924, 2860, 1703, 1656, 1618 cmꢀ1
.
561.1631.
HRMS (ESI) m/z: calcd for C44H44F3N4O8Si (MþH)þ 841.2875, found
841.2907.
4.1.12. General procedure for the synthesis of phosphoramidite
DMTrCl (2.3 equiv) was added to a solution of the diol com-
pound in pyridine. After stirring for 1 h, the reaction mixture was
diluted with EtOAc, and then washed with water and brine. The
organic layer was washed with water and brine, dried over Na2SO4,
evaporated, and then the residue was purified by flash silica-gel
column chromatography (CHCl3/CH3OH¼99:1 containing 0.5%
pyridine) to give the corresponding DMTr-protected WNA. iPr2NEt
(6 equiv) was added to the solution of the above DMTr–WNA and
iPr2NP(Cl)OC2H4CN (3 equiv) in CH2Cl2 at 0 ꢁC. After stirring for 1 h,
the reaction mixture was diluted with a satd NaHCO3 solution and
extracted with EtOAc. The organic layer was dried over Na2SO4 and
evaporated, and then the residue was purified by flash silica-gel
column chromatography (hexane/EtOAc¼1:1) to give the purified
materials.
4.1.7. General procedure of deprotection reaction
A THF solution of the above compound and TBAF (1.0 M THF
solution, 3 equiv) were stirred for 3.5 h at room temperature.
MeOH and a 0.2 M NaOH solution (4 equiv) were then added to this
mixture at 0 ꢁC. After stirring for 2.5 h at 0 ꢁC, the reaction was
quenched with acetic acid and diluted with MeOH. The solvents
were removed under reduced pressure. The residue was purified by
flash silica-gel column chromatography (CHCl3/CH3OH¼13:1 to
9:1) to give the corresponding diol compound.
4.1.8. (10S,30R,40R,50R,70S)-{10-(p-Trifluoroacetylaminophenyl)-40-
hydroxy-30-hydroxymethyl-20,60-dioxabicyclo[3.3.0]oct-70-yl}-
thymine (8b-2)
A colorless foam (97%). 1H NMR (400 MHz, CD3OD):
d 7.74
(2H, d, J¼8.3 Hz), 7.69 (1H, d, J¼1.0 Hz), 7.64 (2H, d, J¼8.3 Hz),
6.27 (1H, t, J¼8.3 Hz), 4.84 (1H, d, J¼3.7 Hz), 4.03–3.99 (1H, m),
3.91–3.88 (2H, m), 3.68 (1H, dd, J¼12.1, 5.5 Hz), 2.75–2.73 (2H,
4.1.13. (10S,30R,40R,50R,70S)-{10-(p-Trifluoroacetylaminophenyl)-30-
dimethoxytrityloxymethyl-40-O-(N,N-diisopropyl-
b
-cyanoethyl-
phosphoramidyl)-20,60-dioxabicyclo[3.3.0]oct-70-yl}-thymine (8b-3)
A colorless foam (49%). 1H NMR (400 MHz, CDCl3):
7.92 (1H, s),
m), 1.93 (3H, d, J¼1.0 Hz). 13C NMR (125 MHz, CD3OD):
d
166.4,
d
152.3, 139.3, 139.0, 137.1, 127.4, 122.0, 111.8, 93.0, 90.5, 90.3, 84.7,
7.65 (2H, d, J¼8.9 Hz), 7.46 (2H, dd, J¼8.9, 3.1 Hz), 7.43–7.39 (2H, m),
7.31–7.19 (8H, m), 6.79 (4H, dd, J¼9.2, 2.8 Hz), 6.32 (0.5H, dd, J¼8.2,
5.8 Hz), 6.13–6.10 (0.5H, m), 5.02 (0.5H, d, J¼3.7 Hz), 4.91 (0.5H, d,
J¼3.4 Hz), 4.37–4.31 (0.5H, m), 4.24–4.22 (1H, m), 4.17–4.11 (0.5H,
m), 3.83–3.62 (1H, m), 3.78 (3H, s), 3.77 (3H, s), 3.55–3.37 (4H, m),
3.20 (1H, dd, J¼10.7, 4.6 Hz), 2.96–2.90 (1H, m), 2.69–2.64 (0.5H,
m), 2.60–2.54 (1H, m), 2.52–2.46 (0.5H, m), 2.37–2.32 (1H, m), 1.98
(1.5H, s), 1.94 (1.5H, s), 1.12 (3H, d, J¼6.7 Hz), 1.07–1.04 (6H, m), 0.90
73.6, 63.3, 12.3. FTIR (neat): 3267, 3074, 2922, 1686 cmꢀ1. HRMS
(ESI) m/z: calcd for
472.1364.
C
20H21F3N3O7 (MþH)þ 472.1326, found
4.1.9. (10S,30R,40R,50R,70R)-{10-(p-Trifluoroacetylaminophenyl)-40-
hydroxy-30-hydroxymethyl-20,60-dioxabicyclo[3.3.0]oct-70-yl}-
thymine (8a-2)
A colorless foam (69%). 1H NMR (400 MHz, CD3OD):
d
7.92 (1H,
(3H, d, J¼6.7 Hz). 31P NMR (161 MHz, CDCl3):
d
150.0, 149.3. 13C
NMR (125 MHz, CDCl3): d 163.5, 163.4, 158.5, 150.0, 149.8, 144.6,
d, J¼1.0 Hz), 7.67–7.63 (4H, m), 6.58 (1H, dd, J¼8.0, 5.3 Hz), 4.53
(1H, d, J¼3.9 Hz), 4.15–4.11 (1H, m), 3.88 (1H, dd, J¼8.7, 3.9 Hz), 3.85
(1H, dd, J¼12.1, 2.5 Hz), 3.68 (1H, dd, J¼12.1, 6.6 Hz), 2.90 (1H, dd,
J¼15.1, 8.0 Hz), 2.68 (1H, dd, J¼15.1, 5.3 Hz), 1.94 (3H, d, J¼1.0 Hz).
144.5, 138.3, 138.1, 137.0, 136.3, 136.0, 135.9, 134.7, 134.6, 130.3,
130.2, 128.4, 127.8, 126.8, 126.6, 120.6, 118.0, 117.5, 113.1, 111.4, 110.9,
92.0, 91.9, 90.6, 88.8, 88.0, 86.2, 81.3, 81.2, 74.2, 74.0, 72.7, 72.5, 63.0,
62.0, 58.8, 58.7, 58.0, 57.8, 55.3, 55.2, 48.3, 47.9, 43.4, 43.3, 43.2, 24.7,
24.6, 24.5, 24.4, 24.3, 20.4, 20.3, 20.1, 12.5. FTIR (neat): 3289, 2964,
2932, 1690 cmꢀ1. ESI-MS m/z: 974 (MþH)þ, 996 (MþNa)þ.
13C NMR (125 MHz, CD3OD):
d 166.3, 152.7, 140.3, 138.3, 137.1, 127.2,
122.2, 112.2, 93.1, 90.3, 87.1, 84.1, 73.3, 63.7, 12.6. FTIR (neat): 2924,
2860, 1703, 1656, 1618 cmꢀ1
C
. HRMS (ESI) m/z: calcd for
20H21F3N3O7 (MþH)þ 472.1326, found 472.1361.
4.1.14. (10S,30R,40R,50R,70R)-{10-(p-Trifluoroacetylaminophenyl)-3’-
4.1.10. (10S,30R,40R,50R,70S)-N4-Benzoyl-1-{10-(p-trifluoroacetyl-
aminophenyl)-40-hydroxy-30-hydroxymethyl-20,60-
dioxabicyclo[3.3.0]oct-70-yl}-cytosine (9b-2)
dimethoxytrityloxymethyl-4’-O-(N,N-diisopropyl-
phosphoramidyl)-20,60-dioxabicyclo[3.3.0]oct-70-yl}-thymine (8a-3)
A colorless foam (39%). 1H NMR (400 MHz, CDCl3):
7.97 (1H, s),
b-cyanoethyl-
d
A colorless foam (92%). 1H NMR (400 MHz, CD3OD):
d
8.40 (1H,
7.75 (0.5H, s), 7.71 (0.5H, s), 7.53–7.39 (6H, m), 7.31–7.20 (8H, m),
6.81–6.78 (4H, m), 6.51 (1H, dd, J¼7.9, 5.2 Hz), 4.68 (0.5H, d,
J¼4.0 Hz), 4.59 (0.5H, d, J¼3.1 Hz), 4.36–4.27 (1.5H, m), 4.13–4.08
(0.5H, m), 3.78–3.77 (6H, m), 3.75–3.65 (1H, m), 3.62–3.35 (4H, m),
3.27–3.22 (1H, m), 2.88–2.81 (1H, m), 2.69–2.62 (1H, m), 2.56–2.53
(1H, m), 2.36–2.32 (1H, m), 2.00 (1.5H, s), 1.98 (1.5H, s), 1.12 (3H, d,
J¼6.7 Hz), 1.09–1.06 (6H, m), 0.92 (3H, d, J¼6.7 Hz). 31P NMR
d, J¼7.6 Hz), 7.99 (2H, d, J¼7.6 Hz), 7.74 (2H, d, J¼8.9 Hz), 7.69–7.62
(4H, m), 7.56–7.52 (2H, m), 6.35–6.32 (1H, m), 4.94 (1H, d,
J¼3.7 Hz), 4.07–4.04 (1H, m), 3.95–3.91 (2H, m), 3.70 (1H, dd,
J¼12.2, 5.5 Hz), 3.00 (1H, dd, J¼14.0, 5.8 Hz), 2.72 (1H, dd, J¼14.0,
7.9 Hz). 13C NMR (125 MHz, CD3OD):
d 169.2, 165.0, 157.7, 150.1,
147.2, 139.3, 137.1, 134.7, 134.1, 129.8, 129.2, 127.4, 122.1, 98.7, 93.1,
92.4, 91.4, 84.4, 73.5, 63.2, 50.2. FTIR (neat): 3275, 2931, 1705,
1651 cmꢀ1. HRMS (ESI) m/z: calcd for C26H24F3N4O7 (MþH)þ
561.1592, found 561.1582.
(161 MHz, CDCl3):
163.3, 158.5, 150.3, 144.6, 144.5, 139.0, 135.8, 134.8, 134.7, 130.2,
d
150.3, 149.7. 13C NMR (125 MHz, CDCl3):
d
128.3, 127.8, 126.8, 126.3, 126.2, 120.7, 117.6, 117.4, 113.1, 111.5, 111.4,
92.1, 92.0, 87.8, 87.3, 86.2, 85.8, 85.7, 81.1, 73.7, 73.6, 72.7, 72.6, 63.3,
62.5, 58.7, 58.6, 58.2, 58.1, 55.3, 55.2, 48.6, 48.4, 43.5, 43.4, 43.3,
31.6, 24.6, 24.5, 24.4, 22.6, 22.4, 20.3, 20.2, 14.1, 12.8. FTIR (neat):
2967, 1691 cmꢀ1. ESI-MS m/z: 996 (MþNa)þ.
4.1.11. (10S,30R,40R,50R,70R)-N4-Benzoyl-1-{10-(p-trifluoroacetyl-
aminophenyl)-40-hydroxy-30-hydroxymethyl-20,60-
dioxabicyclo[3.3.0]oct-70-yl}-cytosine (9a-2)
A colorless foam (95%). 1H NMR (400 MHz, CD3OD):
d 8.66 (1H,
d, J¼7.7 Hz), 7.99 (2H, d, J¼7.7 Hz), 7.69–7.62 (6H, m), 7.56–7.52 (2H,
m), 6.49 (1H, dd, J¼7.6, 4.7 Hz), 4.71 (1H, d, J¼4.3 Hz), 4.11–4.07
(1H, m), 3.97 (1H, dd, J¼8.6, 4.3 Hz), 3.82 (1H, dd, J¼11.9, 2.4 Hz),
3.65 (1H, dd, J¼11.9, 6.4 Hz), 3.07 (1H, dd, J¼15.0, 7.6 Hz), 2.71 (1H,
4.1.15. (10S,30R,40R,50R,70S)-N4-Benzoyl-1-{10-(p-trifluoroacetyl-
aminophenyl)-30-dimethoxytrityloxymethyl-40-O-(N,N-diisopropyl-
b
-cyanoethyl-phosphoramidyl)-20,60-dioxabicyclo[3.3.0]oct-70-yl}-
cytosine (9b-3)
dd, J¼15.0, 4.7 Hz). 13C NMR (125 MHz, CD3OD):
d
169.0, 164.7,
A colorless foam (59%). 1H NMR (400 MHz, CDCl3):
d 8.63 (1H, s),
146.2, 140.3, 134.8, 133.9, 129.7, 129.2, 127.1, 122.1, 98.3, 93.0, 91.4,
8.13 (0.5H, d, J¼7.3 Hz), 7.94–7.90 (3H, m), 7.68 (1H, d, J¼8.6 Hz),
89.7, 84.5, 73.5, 63.6, 50.5. FTIR (neat): 3318, 2925, 1702, 1633 cmꢀ1
.
7.63–7.59 (2H, m), 7.52–7.41 (6H, m), 7.32–7.17 (8H, m), 6.81–6.78

7170
Y. Taniguchi et al. / Tetrahedron 64 (2008) 7164–7170
(4H, m), 6.36 (0.5H, t, J¼7.0 Hz), 6.23 (0.5H, t, J¼6.7 Hz), 5.06 (0.5H,
d, J¼3.4 Hz), 4.98 (0.5H, d, J¼3.4 Hz), 4.41–4.35 (0.5H, m), 4.31–4.25
(1H, m), 4.20–4.15 (0.5H, m), 3.78 (3H, s), 3.77 (3H, s), 3.76–3.67
(1H, m), 3.60–3.36 (4H, m), 3.29–3.18 (2H, m), 2.65–2.55 (1.5H, m),
2.49 (0.5H, dd, J¼14.3, 7.6 Hz), 2.39–2.29 (1H, m), 1.12 (3H, d,
J¼6.7 Hz), 1.07 (4H, d, J¼6.7 Hz), 0.92–0.85 (5H, m). 31P NMR
found 5857.75. TFOs having p-amino-WNA-
5858.05, found 5856.82; TFO2( C): calcd for 5874.04, found
5873.32; TFO3( C): calcd for 5842.06, found 5841.70; TFO4( C):
aC. TFO1(aC): calcd for
a
a
a
calcd for 5858.05, found 5857.67.
4.3. Gel-shift assay
(161 MHz, CDCl3):
158.5, 144.7, 144.6, 138.4, 138.2, 138.0, 136.0, 135.9, 134.6, 133.2,
d
150.3, 149.6. 13C NMR (125 MHz, CDCl3):
Triplex formation was done for 12 h at 22 ꢁC in the buffer con-
taining 20 mM Tris–HCl, 5 mM MgCl2, 2.5 mM spermidine, and 10%
sucrose at pH 7.5. Electrophoresis was done at 10 ꢁC with a non-
denatured polyacrylamide gel. TFO (10 nM) containing the 32P-la-
beled one as the tracers was used.6,9 The association constant was
calculated using the concentration of each component and the
following equation: Ks¼[triplex]/[duplex][TFO].
d
130.3, 129.1, 128.4, 127.7, 127.5, 126.8, 126.7, 126.6, 120.6, 117.9, 117.4,
113.1, 92.1, 91.9, 90.7, 88.8, 88.6, 86.1, 81.1, 81.0, 62.9, 62.0, 58.8, 58.6,
58.0, 57.9, 55.2, 49.9, 49.1, 43.4, 43.3, 43.2, 24.6, 24.5, 20.4, 20.1. FTIR
(neat): 2964, 2930, 1706, 1660, 1609 cmꢀ1. ESI-MS m/z: 974
(MþH)þ, 1086 (MþNa)þ.
4.1.16. (10S,30R,40R,50R,70R)-N4-Benzoyl-1-{10-(p-trifluoroacetyl-
aminophenyl)-30-dimethoxytrityloxymethyl-40-O-(N,N-
Acknowledgements
diisopropyl-b
-cyanoethyl-phosphoramidyl)-20,60-
dioxabicyclo[3.3.0]oct-70-yl}-cytosine (9a-3)
S.S. and Y.T. are grateful for the support by a Grant-in-Aid for
Scientific Research (A) and Young Scientists (Start-up) from the
Japan Society for the Promotion of Science (JSPS) and CREST from
Japan Science and Technology Agency (JST).
A colorless foam (35%). 1H NMR (400 MHz, CDCl3):
d 8.68 (1H, s),
8.49–8.47 (1H, m), 8.09 (1H, s), 7.88 (2H, d, J¼7.3 Hz), 7.62–7.58
(1.5H, m), 7.52–7.46 (6H, m), 7.37–7.35 (2H, m), 7.26–6.17 (7.5H, m),
6.81–6.75 (4H, m), 6.38–6.31 (1H, m), 4.92–4.89 (0.5H, m), 4.81
(0.5H, d, J¼4.6 Hz), 4.53–4.47 (0.5H, m), 4.19–4.14 (1.5H, m), 3.77–
3.76 (6H, m), 3.60–3.33 (5H, m), 3.27–3.02 (2H, m), 2.85–2.71 (1.5H,
m), 2.60–2.56 (0.5H, m), 2.36–2.32 (1H, m), 1.17–1.12 (6H, m), 1.05–
References and notes
1. (a) Ye, Z.; Guntaka, R. V.; Mahato, R. I. Biochemistry 2007, 46, 11240–11252; (b)
Alam, M. R.; Majumbar, A.; Thazhathveetil, A. K.; Liu, S.-T.; Liu, J.-L.; Puri, N.;
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33, 3492–3502.
1.04 (2H, m), 0.88–0.84 (4H, m). 31P NMR (161 MHz, CDCl3):
d 150.0,
149.9. 13C NMR (125 MHz, CDCl3):
d
158.5, 144.6, 135.8, 133.3, 133.2,
130.2, 129.1, 128.3, 128.2, 127.8, 127.5, 126.9, 126.8, 126.3, 126.2,
120.8, 120.7, 113.2, 113.1, 88.6, 62.2, 55.3, 55.2, 49.9, 46.5, 45.3, 43.5,
43.4, 24.7, 24.6, 23.0, 22.9, 22.6, 22. FTIR (neat): 2967, 2930, 1703,
1660, 1609 cmꢀ1. ESI-MS m/z: 1064 (MþH)þ.
4.2. Synthesis of TFOs containing p-amino-WNA derivatives
4. Soyfer, V. N.; Potaman, V. N. Triple-Helical Nucleic Acids; Springer: New York, NY,
1996; and references cited therein.
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2006, 71, 2115–2122.
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6973–6976.
Triplex forming oligonucleotides (TFOs) incorporating the p-
amino-WNA analogs were synthesized using an automated DNA
synthesizer (Applied Biosystems 394 DNA/RNA Synthesizer). After
cleavage from CPG, they were purified by HPLC (HPLC conditions:
COSMOSIL 5C18-MS-II (10ꢂ250 mm), buffer A: 0.1 M TEAA, buffer
B: CH3CN, 10–40%/20 min, 40–100%/ 30 min linear gradient, flow
rate: 4.0 mL/ min, UV-detector: 254 nm, column oven: 35 ꢁC). All
synthesized TFOs were identified by MALDI-TOF MS measurement.
MALDI-TOF MASS results: TFOs having p-amino-WNA-
calcd for 5873.05, found 5873.68; TFO2( T): calcd for 5889.04,
found 5889.83; TFO3( T): calcd for 5857.06, found 5856.29;
TFO4( T): calcd for 5873.05, found 5873.88. TFOs having p-amino-
WNA- T. TFO1( T): calcd for 5873.05, found 5869.52; TFO2( T):
calcd for 5889.04, found 5889.53; TFO3( T): calcd for 5857.06,
found 5856.19; TFO4( T): calcd for 5873.05, found 5872.54. TFOs
having p-amino-WNA- C. TFO1( C): calcd for 5858.05, found
5857.70; TFO2( C): calcd for 5874.04, found 5873.78; TFO3( C):
calcd for 5842.06, found 5841.18; TFO4( C): calcd for 5858.05,
bT. TFO1(bT):
b
b
b
a
a
a
a
a
b
b
16. (a) Griffin, L. C.; Kiessling, L. L.; Beal, P. A.; Gillepie, P. B.; Dervan, P. B. J. Am.
Chem. Soc. 1992, 114, 7976–7982; (b) Wang, E.; Koshlap, K. M.; Gillespie, P.;
Dervan, P. B.; Feigon, J. J. Mol. Biol. 1996, 257, 1052–1069.
b
b
b