Stable oligonucleotide-directed triplex formation at target sites with CG interruptions: Strong sequence-specific recognition by 2′,4′-bridged nucleic-acid-containing 2-pyridones under physiological conditions

Article abstract of DOI:10.1002/1521-3765(20021018)8:20<4796::AID-CHEM4796>3.0.CO;2-O

A sequence of double-stranded DNA (dsDNA) which can be recognized by a triplex-forming oligonucleotide (TFO) is limited to a homopurine-homopyrimidine sequence. To develop novel nucleoside analogues which recognize CG interruption in homopurine-homopyrimi

Full text of DOI:10.1002/1521-3765(20021018)8:20<4796::AID-CHEM4796>3.0.CO;2-O

FULL PAPER
Stable Oligonucleotide-Directed Triplex Formation at Target Sites with CG
Interruptions: Strong Sequence-Specific Recognition by 2',4'-Bridged
Nucleic-Acid-Containing 2-Pyridones under Physiological Conditions
Satoshi Obika, Yoshiyuki Hari, Mitsuaki Sekiguchi, and Takeshi Imanishi*[a]
Abstract: A sequence of double-strand-
ed DNA (dsDNA) which can be recog-
nized by a triplex-forming oligonucleo-
tide (TFO) is limited to a homopurine
homopyrimidine sequence. To develop
novel nucleoside analogues which rec-
ognize CG interruption in homopur-
ine homopyrimidine dsDNA, we syn-
thesized a novel 2'-O,4'-C-methyleneri-
bonucleic acid (2'-O,4'-C-methylene
bridged nucleic acid; 2',4'-BNA) that
bears the unnatural nucleobases, 2-pyr-
idone (PB) or its 5-methyl congener
(mPB); these analogues were introduced
into pyrimidine TFOs using a DNA
synthesizer. A TFOwith a 2 '-deoxy-b-
d-ribofuranosyl-2-pyridone (P) or 2',4'-
BNA abasic monomer (HB) was also
synthesized. The triplex-forming ability
of various synthesized 15-mer TFOs and
the corresponding homopurine homo-
pyrimidine dsDNA, which contained a
single pyrimidine purine (PyPu) inter-
ruption, was examined in UV melting
experiments. It was found that PB and
mPB in the TFOs successfully recognized
CG interruption under physiological
conditions (7mm sodium phosphate,
140mm KCl, 5mm spermine, pH 7.0).
Furthermore, triplex formation between
the dsDNA target which contained three
CG interruptions and the TFOwith
three PB units was also confirmed. Addi-
tional four-point 2',4'-BNA modifica-
B
tions of the TFOcontaining three P
units significantly enhanced its triplex-
forming ability towards the dsDNA and
had a Tm value of 438C under physio-
logical conditions. These results indicate
that a critical inherent problem of TFOs,
namely, the sequence limitation of the
dsDNA target, may be overcome to a
large extent and this should promote
antigene applications of TFOs in vitro
and in vivo.
Keywords: molecular recognition ¥
nucleic acids
¥
nucleobases
¥
oligonucleotides
fore, the development of nucleoside analogues is a prereq-
uisite for effective recognition of a pyrimidine purine (PyPu)
base pair (a CG or TA base pair) in dsDNA. In order to
overcome the restriction of the sequence of a dsDNA target, a
large number of synthetic homopyrimidine TFOs containing
unnatural nucleobases have been reported.[5] However, de-
velopment of a modified TFOthat has selective and strong
binding affinity with a PyPu base pair has not yet been
achieved. Although T or C bases have been known to bind to
CG interruptions through a single hydrogen bond,[6, 7] these
nucleobases recognize either ATor GC base pairs much more
strongly. Results from a molecular modeling study suggested
that the 2-carbonyl group in T or C play a crucial role in the
recognition of a CG base pair (Figure 1b).[8] Therefore, we
assumed that 2-pyridone, which lacks suitable functional
groups for ATor GC base pair recognition, could succeed as a
nucleobase for selective interaction with CG interruptions
(Figure 1c).
Introduction
Triplex-forming oligonucleotides (TFOs) can interact with
double-stranded DNA (dsDNA) in a sequence-specific man-
ner. These TFOs have been largely studied as novel reagents
for the control of gene expression in vitro and in vivo
4]
(antigene strategy).[1
Recognition of dsDNA by TFO
follows two patterns that is a pyrimidine or purine. In a
pyrimidine motif triplex, homopyrimidine TFObinds to the
homopurine tract of the dsDNA target by the formation of
‡
Hoogsteen hydrogen bonds to form T¥ AT and C ¥ GC triads
(Figure 1a). In a purine motif triplex, homopurine TFOforms
A ¥ AT (or T¥ AT) and G ¥ GC triads with the homopurine
tract of the dsDNA target through reverse-Hoogsteen hydro-
gen bonds. In other words, the dsDNA target in both motifs is
restricted to a homopurine homopyrimidine duplex. There-
[a] Prof. Dr. T. Imanishi, Dr. S. Obika, Dr. Y. Hari, M. Sekiguchi
Graduate School of Pharmaceutical Sciences
Osaka University, 1-6 Yamadaoka, Suita
Osaka 5650871 (Japan)
We recently achieved the first synthesis of the novel
nucleosides, 2'-O,4'-C-methyleneribonucleic acid (2'-O,4'-C-
methylene bridged nucleic acid, 2',4'-BNA)[9, 10], which has a
fixed N-type conformation, and 3'-O,4'-C-methyleneribonu-
Fax : (‡81)668-798-204
E-mail: imanishi@phs.osaka-u.ac.jp
4796
¹ 2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
0947-6539/02/0820-4796 $ 20.00+.50/0
Chem. Eur. J. 2002, 8, No. 20

4796 4802
Me
N
R
O
H
a)
H
N
Me
N
O
N
H
O
H
R
H
N
R
N
HO
N
O
H
H
N
O
N
H
N
H
N
N
N
H
N
N
O
R
O
R
H
N
O
(PB)
N
N
R = H
O
(
mPB)
N
R
Me
HO
O
N
H
N
R
T•AT
C+•GC
Me
H
N
b)
HO
HO
N
O
H
O
H
O
O
H
N
N
N
N
R
N
N
N
R
HB
P
N
N
O
O
O
H
O
HO
HO
O
R
R
H
H
N
H
N
N
N
N
N
H
N
Figure 2. Structure of 2',4'-BNA monomers and a nucleoside bearing
H
H
N
N
2-pyridone.
H
H
N
H
N
R
O
N
R
O
T•CG
C•CG
c)
the 2',4'-BNA monomers exhibited singlet signals for the C1'-,
C2'- and C3'-hydrogens of the sugar moiety. This indicated
that the sugar in their 2',4'-BNA monomers was locked in
N-type conformation.[22] Furthermore, X-ray crystallographic
analysis[19, 23] of the structure of 4a confirmed that the
conformation of the sugar moiety was in a 3'-endo (N-type)
conformation, in which the sugar pseudorotation phase angle
(P) was 18.08.[24] Protection of the primary alcohol of 4 with
4,4'-dimethoxytrityl chloride (DMTrCl) in pyridine gave
compound 5 in 96 97% yields. The phosphoramidite 6a, a
suitable building block for DNA synthesis, was obtained in
98% yield by phosphitylation of 5a. The yield of phosphor-
amidite 6b was 91%. Then the phosphoramidites 6a and 6b
were incorporated into TFOs by standard phosphoramidite
protocol in a DNA synthesizer (Figure 3). TFOs that con-
tained P[25, 26] or HB[16, 27] were also synthesized. The purity of
the modified TFOs was verified using reversed-phase high
performance liquid chromatography (HPLC) and the compo-
sitions were determined by matrix-assisted laser desorption
ionization time-of-flight MS (MALDI-TOF-MS) (see Exper-
imental Section).
N
R
N
O
O
R
H
H
N
N
N
H
P•CG
H
N
N
H
N
R
O
‡
Figure 1. a) Canonical T¥ AT and C ¥ GC triads in a pyrimidine motif
triplex; b) structure of T¥ CG and C ¥ CG triads; c) proposed structure of a
P ¥ CG triad.
cleic acid (3',4'-BNA)[11, 12] which has a restricted S-type
conformation. Moreover, we have found that oligonucleotides
with a 2',4'-BNA modification leads to a marked increase in
their triplex formation with dsDNA at neutral pH as well as in
their duplex formation with single-stranded RNA (ssRNA).[10, 13 18]
We report here the synthesis of the novel 2',4'-BNA monomers,
2-pyridone derivative and its 5-methyl-2-pyridone congener
(Figure 2), and the effective recognition of one or more CG
base pairs in homopurine homopyrimidine dsDNA by
pyrimidine TFOs containing these 2',4'-BNA monomers.[19]
The triplex-forming ability of PB and mPB-containing TFO
with dsDNA (single interruption): Initially, UV-melting
experiments were used to investigate whether the nucleoside
analogue P, which bears 2-pyridone, could recognize a CG
interruption in dsDNA when incorporated into TFOs. The
Results
Synthesis of 2',4'-BNA containing 2-pyridone and 5-methyl-2-
pyridone: The synthetic route to the phosphoramidites 6a and
6b is shown in Scheme 1. According to method of Vorbr¸g-
gen,[20] the starting material 1,[21]
which was easily prepared from
d-glucose, was treated with
2-pyridone, N,O-bis(trimethyl-
silyl)acetamide and trimethyl-
silyl trifluoromethanesulfonate
(TMSOTf) in 1,2-dichloro-
R
O
R
O
BnO
N
O
BnO
BnO
TsO
N
O
O
a
c
b
OAc
TsO
BnO
O
OBn OAc
OBn OAc
2a : R = H
2b : R = Me
3a : R = H
3b : R = Me
1
ethane to give 2a as the sole
b-anomer in 74% yield. By
using 5-methyl-2-pyridone, 2b
R
R
O
R
DMTrO
DMTrO
N
O
O
N
O
HO
N
O
e
O
d
was produced in 87% yield in a
similar manner. The exposure
of 2 to potassium carbonate in
methanol gave the bicyclic nu-
cleosides 3 in yields of 96
100%. Subsequent hydrogenol-
ysis of 3 afforded the diols 4 in
91 95% yields. 1H NMR anal-
ysis of 4a and 4b showed that
HO
O
O
P
O
HO
O
4a : R = H
4b : R = Me
5a : R = H
5b : R = Me
iPr2N
OCH2CH2CN
6a : R = H
6b : R = Me
Scheme 1. a) 2-Pyridone or 5-methyl-2-pyridone, N,O-bis(trimethylsilyl)acetamide, 1,2-dichloroethane, reflux,
then TMSOTf, reflux, 74% (2a) or 87% (2b); b) potassium carbonate, methanol, RT, 100% (3a), 96% (3b);
c) 20% palladium hydroxide on carbon powder, cyclohexene, ethanol, reflux, 95% (4a), 91% (4b); d) 4,4'-
dimethoxytrityl chloride, pyridine, RT, 96% (5a), 97% (5b); e) 2-cyanoethyl-N,N,N',N'-tetraisopropylphos-
phorodiamidite, diisopropylammonium tetrazolide, CH3CN/THF, RT, 95% (6a), 91% (6b).
Chem. Eur. J. 2002, 8, No. 20
¹ 2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
0947-6539/02/0820-4797 $ 20.00+.50/0
4797

T. Imanishi et al.
FULL PAPER
Table 1. Tm values [8C] of TFOs 7 18 with their corresponding duplexes.[a]
TFOs sequence
XY
7
8
9
5'-TTTTTmCTTTmCTmCTmCT-3'
5'-TTTTTmCTmCTmCTmCTmCT-3'
5'-TTTTTmCTPTmCTmCTmCT-3'
TFOTarget duplex
CG
GC
TA
AT
33
35
34
36
35
44
29
29
33
35
nd[b,c]
nd[b]
7 (T)
23
23
23
23
23
23
24
24
25
25
26
26
44
42
45
55
56
44
39
49
34
43
15[c]
34
35
58
33
37
42
35
33
33
33
33
17[c]
21
59
35
34
44
44
35
53
37
55
34
15[c]
23
8 (mCB)
9 (P)
10 5'-TTTTTmCTPBTmCTmCTmCT-3'
11 5'-TTTTTmCTmPBTmCTmCTmCT-3'
12 5'-TTTTTmCTHBTmCTmCTmCT-3'
13 5'-TTTTTmCTTmCmCTmCTmCT-3'
14 5'-TTTTTmCTPBmCmCTmCTmCT-3'
15 5'-TTTTTmCmCTTmCTmCTmCT-3'
16 5'-TTTTTmCmCPBTmCTmCTmCT-3'
17 5'-TTTTTmCmCTmCmCTmCTmCT-3'
18 5'-TTTTTmCmCPBmCmCTmCTmCT-3'
19 5'-TTTTTTTTTTTmCTmCT-3'
10 (PB)
11 (mPB)
12 (HB)
13 (T)
14 (PB)
15 (T)
16 (PB)
17 (T)
18 (PB)
[a] Conditions: 7mm sodium phosphate buffer (pH 7.0) containing 140mm
KCl and 5mm spermine, concentration of triplex ˆ 1.5mm. [b] A typical
transition in the UV-melting curve was not observed. [c] All the Tm values
were extremely low, and that might mean incomplete formation of the
corresponding triplexes, perhaps due to sequence-peculiarity of the TFO
used though the details in this respect are not clear.
20 5'-TTTTTPBTPBTPBTmCTmCT-3'
21 5'-TTBTTBTPBTPBTPBTmCBTmCBT-3'
22 5'-TTBTTBTmPBTmPBTmPBTmCBTmCBT-3'
target dsDNA
5'-GCTAAAAAGAXAGAGAGATCG-3'
3'-CGATTTTTCTYTCTCTCTAGC-5'
23
5'-GCTAAAAAGAXGGAGAGATCG-3'
3'-CGATTTTTCTYCCTCTCTAGC-5'
24
25 5'-GCTAAAAAGGXAGAGAGATCG-3'
3'-CGATTTTTCCYTCTCTCTAGC-5'
5'-GCTAAAAAGGXGGAGAGATCG-3'
3'-CGATTTTTCCYCCTCTCTAGC-5'
26
27 5'-GCTAAAAACACACAGAGATCG-3'
3'-CGATTTTTGTGTGTCTCTAGC-5'
XY = CG, GC, TAorAT (23-26)
Figure 3. The TFOs sequence and the sequence of the DNA targets.
melting temperature (Tm) was measured in 7mm sodium
phosphate buffer (pH 7.0) that also contained 140mm KCl and
5mm spermine to approach physiological conditions in the
nucleus.[28] The Tm value of the triplex 9 ¥ 23 (XYˆ CG) was
458C, while those of the triplexes 9 ¥ 23 (XYˆ GC, TA and
AT), which contained a P ¥ GC, P ¥ TA and P ¥ AT triad, were
much lower (33, 34 and 348C, respectively). This indicated
that the nucleoside analogue, P, effectively recognized the CG
interruption in dsDNA (Table 1, Figure 4). The thermal
stabilization effect of a T¥ CG and mC ¥ CG triad (mC:
5-methylcytosine) was comparable to that of a P ¥ CG triad,
Figure 4. UV-melting profiles (260 nm) for the triplexes 9 ¥ 23 (XYˆ CG,
red; GC, green; TA, blue; AT, cyan). The presented melting curves were
normalized.
‡
while T and mC formed stable triads, T¥ AT and mC ¥ GC with
Tm values of 59 and 588C, respectively (Table 1). This result
suggests that the 2-carbonyl oxygen in P (or T and mC) plays
an important role in the binding with a CG base pair and that
the absense of a functional group at the 3- and 4-position in a
pyrimidine nucleobase effectively prevents hybridization with
PyPu base pairs.
Next, the effect of the 2',4'-BNA modification of P was
examined. The triplex formation of PB-containing TFO 10 was
also found to be sequence-selective, and the Tm value of the
triplex 10 ¥ 23 (XYˆ CG) was 10 138C higher than those of
the triplexes 7 ¥ 23 (XYˆ CG), 8 ¥ 23 (XYˆ CG), 9 ¥ 23 (XYˆ
CG) or 12 ¥ 23 (XYˆ CG) (Table 1, Figure 5). Moreover, the
thermal stability of the triplex 10 ¥ 23 (XYˆ CG) was almost
comparable to that of the canonical triplexes 7 ¥ 23 (XYˆ AT)
Figure 5. UV-melting profiles (260 nm) for the triplexes which were
formed with the duplex 23 (XYˆ CG) by TFOs 7 (blue), 9 (green), 10
(red), 11 (cyan) and 12 (black) triads. These melting curves have been
normalized.
‡
and 8 ¥ 23 (XYˆ GC), which contained a T¥ AT and mC ¥ GC
4798
¹ 2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
0947-6539/02/0820-4798 $ 20.00+.50/0
Chem. Eur. J. 2002, 8, No. 20

Triplex Formation
4796 4802
triad, respectively. The 5-methyl substitution of U or C in
TFOis known to cause relatively large enhancement of triplex
stability,[29] but notable improvement in recognition of a CG
interruption was not observed in the mPB-containing TFO
(Table 1). Possibly the difference in the spatial position of the
5-methyl group between T¥ AT (mC ¥ GC) and mPB ¥ CG triads
may explain why the 5-methyl group in mPB did not give the
desired effect.
Tm value of 438C, even though the target duplex 27 had three
CG interruptions in its 15-bp TFO-binding region. The TFO
22, the 5-methyl congener of TFO 21, was also found to form a
stable triplex with the target duplex 27 with Tm value of 358C.
Discussion
To confirm the general apllicability of this result, we also
studied the ability of PB to recognize CG interruption in other
Extensive research on TFOs, which involve nucleoside
analogues for the recognition of CG interruption in dsDNA,
‡
dsDNA targets (Table 1). Due to the low stability of the mC ¥
35]
has been reported to date.[8, 32
Most of the nucleoside
GC triad under physiological conditions,[30] the replacement
of an AT base pair by a GC base pair at the neighboring sites
of the target CG interruption caused a decrease in the Tm
values. However, the TFOs 14, 16 or 18 maintained reason-
able triplex-forming ability towards the corresponding target
duplexes 24, 25 or 26 (XYˆ CG) with almost no effect on a
variety of base pairs at the neighboring sites (Table 1). The
increase in Tm values of TFOs 14, 16 and 18 for a CG
interruption ranged from ‡9 198C with respect to those of
TFOs 13, 15 and 17.
analogues have complicated structures that consist of highly
fused-aromatic ring and many functional groups in order to
form hydrogen bonds with the 4-amino hydrogen in C and the
6-carbonyl oxygen and 7-nitrogen atoms in G. However, their
mode of recognition of a CG base pair remained vague, and
the development of an ideal nucleoside analogue to interact
with a CG interruption was not been achieved. However, the
simple structure of 2-pyridone, which we selected as a
nucleobase, has only one functional group that can form a
hydrogen bond in a triplex. By using the 2-pyridone as a
nucleobase, successful recognition of CG interruption in
dsDNA was demonstrated (Table 1, Figure 5), and this
indicates the importance of the hydrogen bond between the
2-carbonyl oxygen in P and the 4-amino hydrogen in C
(Figure 1c). Moreover, the stability of a triplex that contained
a P ¥ CG triad was comparable to that of triplexes containing a
T¥ CG or C ¥ CG triad (Table 1). Again this demonstrates the
requirement of the 2-carbonyl oxygen not only in P but also in
T and C.[8]
Triplex-forming ability of TFO containing PB and mPB with
dsDNA including three CG interruptions: It is of considerable
interest as to whether the TFOthat bears P B or mPB can form a
stable triplex with dsDNA that includes multiple CG inter-
ruptions. Therefore, the triplex-forming ability of TFOs 19
22, which have three-point PB or mPB modifications, with the
target duplex 27 was evaluated (Figure 6).[31] In the case of
On the other hand, Jayaraman and co-workers investigated
triplex-forming ability of TFOs that contained 2-pyridone or
4-pyridone nucleobases in order to confirm the hydrogen
bond arrangement of an antiparallel T¥ CG triad.[26] Conse-
quently, the 4-carbonyl oxygen in T was shown to play an
important role in an antiparallel triplex in the recognition of
the 4-amino hydrogen in C. Their results also support the
requirement of the 2-carbonyl oxygen in P, C or T for
recognition of CG interruption in a parallel triplex formation
since the 4-carbonyl oxygen at T in an antiparallel motif is
considered to be spatially identical with the 2-carbonyl
oxygen at T in a parallel motif. A 2',4'-BNA modification of
the P nucleoside in TFOretained the sequence-selectivity and
promoted a significant increase in the binding affinity with
CG interruption (Table 1). However, 2',4'-BNA abasic ana-
logue (HB) did not show particular specific recognition of a
CG base pair (Table 1). The excellent binding properties of PB
is likely to arise from a combination of the sequence-
selectivity of the 2-pyridone nucleobase moiety and of the
potent triplex-forming ability by the 2',4'-methylene bridged
sugar moiety. As described by Dervan et al.,[30a, 32] their
nucleoside analogue D3 achieved a shape-selective recogni-
tion of PyPu base pairs in dsDNA when the D3 ¥ PyPu triad
was flanked by two T¥ AT triads but its binding affinity was
drastically decreased when the T¥ AT triad in the adjacent 3'
Figure 6. UV-melting profiles (260 nm) for the triplexes 19 ¥ 27 (blue), 20 ¥
27 (cyan), 21 ¥ 27 (red) and 22 ¥ 27 (green). Their Tm values were 228C (20 ¥
27), 438C (21 ¥ 27) and 358C (22 ¥ 27). Typical hyperchromicity of triplex 19 ¥
27 was not observed. These melting curves have been normalized.
natural TFO 19, the formation of a triplex with three T¥ CG
triads was not observed. However, under physiological
conditions, the TFO 20 was found to form a triplex containing
three PB ¥ CG triads with a Tm value of 228C. As we previously
reported, 2',4'-BNA modification of T or mC in homopyrimi-
[16 18]
‡
dine TFOsignificantly promoted the triplex formation.
direction was replaced by a C ¥ GC triad. In the case of the
Therefore, to enhance the triplex stability, an additional four
2',4'-BNA modifications were introduced into TFO 20. This
new TFO, 21, exhibited a potent triplex-forming ability with
interaction of a TA base pair with G,[36 38] the thermal stability
of TFOs containing a G ¥ TA triad was deeply influenced by a
variety of neighboring base triads.[30a] Moreover, in the case of
Chem. Eur. J. 2002, 8, No. 20
¹ 2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
0947-6539/02/0820-4799 $ 20.00+.50/0
4799

T. Imanishi et al.
FULL PAPER
triplexes that contain a T¥ CG triad, UV-melting experiments
affinity with a CG base pair without loss of selectivity. The
triplexes were formed between the TFO, which contained PB
or mPB, and the target duplex, which included three CG
interruptions. Moreover, the additional four-point 2',4'-BNA
‡
showed that the triplex 17 ¥ 26 (XYˆ CG), which has a mC ¥
GC triad at the adjacent 5' direction, was the most destabi-
lized although the destabilization was not large. The triplexes
with a PB ¥ CG triad also displayed similar tendencies. These
results suggest that the nearest 5'-sided, rather than the
nearest 3'-sided, GC base pair of CG interruption in a dsDNA
target causes greater destabilization of a formed triplex. In
any event, it was clearly shown that PB interacts strongly with
CG interruption in a sequence-specific manner, regardless of
the triads adjacent to the PB ¥ CG triad (Table 1). In a triplex
between TFO 20 and dsDNA 27, which included three CG
interruptions (Figure 6), the Tm value was 228C and the
triplex was showed to decrease in DTm per PB modification by
approximately 98C with respect to the fully matched triplex
7 ¥ 23 (XYˆ AT), whereas the triplex 10 ¥ 23 (XYˆ CG) with a
PB ¥ CG triad was 48C less stable in Tm value than the full-
matched triplex. As we have previously reported,[14, 16 18] in the
case of 2',4'-BNA (TB or mCB), which has natural nucleobases,
similar results were also obtained. For example, a triplex,
which contained a TB ¥ AT triad, dramatically increased in Tm
value by 138C with respect to the corresponding natural
triplex, while the Tm values of triplexes containing multiple
TB ¥ AT triads showed an increase by 5 68C per 2',4'-BNA
modification. These results are probably due to a slight warp
of the sugar moiety by 2',4'-BNA modification. However, it
was shown that 20, which contains three PB modifications,
successfully recognized the dsDNA target 27 with three CG
interruptions and formed a triplex that included three PB ¥ CG
triads.
B
modifications of the TFOto involve three P units led to
dramatic enhancement of the triplex stability. We believe that
the novel 2',4'-bridged nucleic acid analogue, PB, should
eventually lead to the advancement of practical applications
of the antigene strategy.
Experimental Section
1-[2-O-Acetyl-3,5-di-O-benzyl-4-(p-toluenesulfonyloxymethyl)-b-d-ribo-
furanosyl]-2-pyridone (2a): Under a nitrogen atmosphere at room temper-
ature, 2-pyridone (52 mg, 0.52 mmol) and N,O-bis(trimethylsilyl)acetamide
(0.16 mL, 0.57 mmol) were added to a solution of compound 1 (154 mg,
0.26 mmol) in anhydrous 1,2-dichloroethane (4 mL) and the mixture was
heated under reflux for 2 h. Trimethylsilyl trifluoromethanesulfonate
(51 mL, 0.28 mmol) was added to the reaction mixture at 08C. The mixture
was then heated under reflux for 4 h. After the addition of a saturated
aqueous solution of sodium hydrogen carbonate, the organic phase was
extracted with ethyl acetate, washed with water and brine, dried over
sodium sulfate and concentrated under reduced pressure. The residue was
purified by silica gel column chromatography (n-hexane/ethyl acetate 1:1)
to give compound 2a (169 mg, 74%) as a colorless oil. [a]2D6 ˆ ‡55.9 (c ˆ
1
1.00 in CHCl3); H NMR (270 MHz, CDCl3, 258C, TMS): d ˆ 7.75 (d, J ˆ
8 Hz, 2H), 7.65 (dd, J ˆ 7, 2 Hz, 1H), 7.38 7.18 (m, 13H), 6.43 (d, J ˆ 9 Hz,
1H), 6.00 (d, J ˆ 3 Hz, 1H), 5.83 (m, 1H), 5.37 (dd, J ˆ 6, 3 Hz, 1H), 4.53,
4.34 (AB, J ˆ 12 Hz, 2H), 4.45 (d, J ˆ 6 Hz, 1H), 4.45, 4.38 (AB, J ˆ 11 Hz,
2H), 4.27, 4.17 (AB, J ˆ 11 Hz, 2H), 3.83, 3.54 (AB, J ˆ10 Hz, 2H), 2.42 (s,
3H), 2.03 (s, 3H); 13C NMR (68 MHz, CDCl3, 258C): d ˆ 169.26, 161.78,
144.94, 137.05, 136.93, 133.31, 129.67, 128.35, 128.32, 128.00, 127.95, 127.86,
120.74, 120.41, 105.67, 88.63, 85.48, 76.64, 75.03, 74.15, 73.53, 69.74, 69.42,
An additional four 2',4'-BNA modifications of the TFO 21
further stabilized the triplex, which included three PB ¥ CG
triads, and its Tm value was 438C. On the other hand, a triplex
consisting of the TFO(5 '-TTBTTBTTTTTTTmCBTmCBT-3'),
with T instead of the PB in 21, and a duplex 27 had a Tm value
of 188C. These results showed that the excellent recognition
of CG interruption shown by PB was maintained even if
natural-type 2',4'-BNA such as TB and mCB were introduced
21.72, 20.77; IR (KBr): nÄmax ˆ 1748, 1667, 1591, 1363, 1229, 1180, 1111 cmÀ1
;
‡
MS (EI): m/z (%): 633 (4) [M] , 91 (100); elemental analysis calcd (%) for
C34H35NO9S (633.7): C 64.44, H 5.57, N 2.21, S 5.06; found: C 64.25, H 5.56,
N 2.18, S 4.94.
1-[2-O-Acetyl-3,5-di-O-benzyl-4-(p-toluenesulfonyloxymethyl)-b-d-ribo-
furanosyl]-5-methyl-2-pyridone (2b): Under a nitrogen atmosphere, 5-me-
thy-2-pyridone (161 mg, 1.47 mmol) and N,O-bis(trimethylsilyl)acetamide
(0.44 mL, 1.78 mmol) were added to a solution of compound 1 (590 mg,
0.99 mmol) in anhydrous 1,2-dichloroethane (10 mL) at room temperature
and the mixture was heated under reflux for 0.5 h. Trimethylsilyl
trifluoromethanesulfonate (0.14 mL, 0.77 mmol) was at room temperature
added to the reaction mixture, which was then heated under reflux for 3 h.
After the addition of a saturated aqueous solution of sodium hydrogen
carbonate, the organic phase was extracted with ethyl acetate. The residue
was obtained as in the synthesis of 2a and further purified by flash silica gel
column chromatography (n-hexane/ethyl acetate 2:3) afforded compound
2b (553 mg, 87%) as a white powder. M.p. 42 458C; [a]2D7 ˆ ‡32.9 (c ˆ
0.64 in CHCl3); 1H NMR (270 MHz, CDCl3, 258C, TMS):d ˆ 7.74 (d, J ˆ
8 Hz, 1H), 7.36 7.19 (m, 13H), 7.09 (dd, J ˆ 9, 2 Hz, 1H), 6.39 (d, J ˆ 9 Hz,
1H), 6.03 (d, J ˆ 4 Hz, 1H), 5.40 (dd, J ˆ 6, 4 Hz, 1H), 4.53, 4.38 (AB, J ˆ
12 Hz, 2H), 4.52 4.44 (m, 2H), 4.50 (d, J ˆ 6 Hz, 1H), 4.24, 4.19 (AB, J ˆ
11 Hz, 2H), 3.82, 3.58 (AB, J ˆ 10 Hz, 2H), 2.42 (s, 3H), 2.03 (s, 3H), 1.71
(s, 3H); 13C NMR (68 MHz, CDCl3, 258C): d ˆ 169.46, 161.17, 144.93,
142.444, 137.14, 137.04, 132.22, 130.43, 129.70, 128.41, 128.33, 127.99, 127.95,
127.87, 127.84, 127.54, 120.13, 115.00, 88.40, 85.41, 77.13, 75.12, 74.30, 73.58,
70.17, 69.36, 21.74, 20.79, 16.96; IR (KBr): nÄmax ˆ 1748, 1674, 1602, 1363,
B
into the TFOwith P to enhance the stability of a triplex.
To the best of our knowledge, this is the first example of a
modified TFOto have achieved effective recognition of the
dsDNAwhich contained three CG interruptions.[39] The use of
PB should substantially overcome one of the critical problems
in pyrimidine motif triplex formation, that is, a limitation of
the target sequence, and these results should afford a vital clue
to the development of practical antigene strategy.
Conclusion
The results presented here demonstrate that the 2-carbonyl
oxygen of pyridone or pyrimidine nucleobases plays an
important role in the recognition of a CG base pair in a
pyrimidine motif triplex. The 2-pyridone derivative (P) has
sufficient CG selectivity due to the lack of a 3-nitrogen atom
and a 4-carbonyl group which are crucial for the formation of
hydrogen bonds with AT base pairs. The combination of
2-pyridone or 5-methyl-2-pyridone and the 2'-O,4'-C-methyl-
ene bridged sugar moiety significantly enhances the binding
‡
1229, 1180, 1105 cmÀ1; MS (EI): m/z (%): 647 (2) [M] , 91 (100); elemental
analysis calcd (%) for C35H37NO9S ¥ 1³3H2O(C 35H37NO9S, 647.7): C 64.30, H
5.81, N 2.14, S 4.90; found: C 64.23, H 5.75, N 2.18, S 4.86.
1-(3,5-Di-O-benzyl-2-O,4-C-methylene-b-d-ribofuranosyl)-2-pyridone
(3a): Potassium carbonate (72 mg, 0.52 mmol) was added to a solution of
compound 2a (110 mg, 0.17 mmol) in methanol (3 mL) at room temper-
ature and the mixture was stirred for 17 h. The solvent was concentrated
under reduced pressure. Ethyl acetate and water were added to the residue
4800
¹ 2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
0947-6539/02/0820-4800 $ 20.00+.50/0
Chem. Eur. J. 2002, 8, No. 20

Triplex Formation
4796 4802
and the organic phase was extracted with ethyl acetate. The residue was
obtained as in the synthesis of 2a and further purified by silica gel column
chromatography (n-hexane/ethyl acetate 3:2 to 1:2) to give compound 3a
(73 mg, 100%) as a white powder. M.p. 117 1188C; [a]2D6 ˆ ‡202.9 (c ˆ
1.00 in CHCl3); 1H NMR (270 MHz, CDCl3, 258C, TMS):d ˆ 7.79 (dd, J ˆ7,
2 Hz, 1H), 7.39 7.20 (m, 11H), 6.51 (d, J ˆ 9 Hz, 1H), 6.10 (m, 1H), 5.90 (s,
1H), 4.67 (s, 1H), 4.65, 4.63 (AB, J ˆ 12 Hz, 2H), 4.59, 4.44 (AB, J ˆ 12 Hz,
2H), 4.05, 3.90 (AB, J ˆ 8 Hz, 2H), 3.97 (s, 1H), 3.86, 3.84 (AB, J ˆ 11 Hz,
2H); 13C NMR (68 MHz, CDCl3, 258C): d ˆ 161.78, 139.50, 137.53, 136.84,
132.03, 128.35, 128.35, 128.27, 127.83, 127.73, 127.53, 127.31, 120.30, 105.64,
88.00, 87.18, 76.29, 75.60, 73.60, 72.15, 72.04, 64.49; IR (KBr): nÄmax ˆ 1661,
purified by silica gel column chromatography (n-hexane/ethyl acetate/
triethylamine 20:100:1) to afford compound 5a (109 mg, 96%). Repreci-
pitation from n-hexane/ethyl acetate gave the analytical specimen as a
white powder. M.p. 114 1158C; [a]2D5 ˆ ‡56.5 (c ˆ 1.00 in CHCl3);
1H NMR (270 MHz, CD3COCD3, 258C, TMS): d ˆ 8.07 (dd, J ˆ 7, 2 Hz,
1H), 7.56 7.26 (m, 10H), 6.92 (d, J ˆ 8 Hz, 4H), 6.38 (d, J ˆ 9 Hz, 1H), 6.23
(s, 1H), 5.77 (s, 1H), 4.78 (d, J ˆ 5 Hz, 1H), 4.38 (d, J ˆ 5 Hz, 1H), 4.34 (s,
1H), 3.94 (d, J ˆ 8 Hz, 1H), 3.82 3.80 (m, 7H), 3.59, 3.52 (AB, J ˆ 11 Hz,
2H); 13C NMR (68 MHz, CD3COCD3, 258C): d ˆ 161.85, 159.40, 145.71,
140.38, 140.36, 136.39, 136.19, 130.77, 130.75, 128.74, 128.46, 127.48, 120.54,
113.76, 105.72, 105.70, 88.56, 88.48, 87.06, 79.78, 72.30, 70.15, 59.44, 55.40;
IR (KBr): nÄmax ˆ 3279, 2952, 1657, 2574, 1508, 1252 cmÀ1; MS (EI): m/z (%):
‡
1584 cmÀ1; MS (EI): m/z (%): 419 (10) [M] , 91 (100); elemental analysis
‡
‡
calcd (%) for C25H25NO5 (419.5): C 71.58, H 6.01, N 3.34; found: C 71.53, H
6.03, N 3.37.
303 (100) [C21H19 (DMTr )]; elemental analysis calcd (%) for C32H31NO7 ¥
1
³2H2O(C 32H31NO7, 541.6): C 69.81, H 5.86, N 2.54; found: C 69.90, H 5.78,
N 2.55.
1-(3,5-Di-O-benzyl-2-O,4-C-methylene-b-d-ribofuranosyl)-5-methyl-2-
pyridone (3b): Potassium carbonate (399 mg, 2.45 mmol) was added to a
solution of compound 2b (530 mg, 0.82 mmol) in methanol (15 mL) at
room temperature and the mixture was stirred for 11 h. The solvent was
concentrated under reduced pressure. Ethyl acetate and water were added
to the residue and the organic phase extracted with ethyl acetate. The
residue was obtained as in the synthesis of 2a and further purified by silica
gel column chromatography (n-hexane/ethyl acetate 4:7) to give compound
3b (340 mg, 96%) as a colorless oil. [a]2D3 ˆ ‡159.9 (c ˆ 0.70 in CHCl3);
1H NMR (270 MHz, CDCl3, 258C, TMS): d ˆ 7.54 (m, 1H), 7.37 7.16 (m,
11H), 6.46 (d, J ˆ 9 Hz, 1H), 5.89 (s, 1H), 4.66 (s, 1H), 4.64, 4.63 (AB, J ˆ
12 Hz, 2H), 4.60, 4.46 (AB, J ˆ 12 Hz, 2H), 4.06, 3.89 (AB, J ˆ 8 Hz, 2H),
4.00 (s, 1H), 3.88, 3.86 (AB, J ˆ 11 Hz, 2H), 1.86 (s, 3H); 13C NMR
(68 MHz, CDCl3, 258C): d ˆ 161.03, 142.15, 137.51, 136.90, 129.18, 128.34,
128.23, 127.77, 127.73, 127.54, 127.43, 119.82, 114.67, 87.91, 87.04, 76.30, 75.44,
73.67, 72.11, 71.96, 64.77, 17.33; IR (KBr): nÄmax ˆ 1668, 1596, 1094,
1-[5-O-(4,4'-Dimethoxytrityl)-2-O,4-C-methylene-b-d-ribofuranosyl]-5-
methyl-2-pyridone (5b): Under a nitrogen atmosphere, 4,4'-dimethoxytri-
tyl chloride (151 mg, 0.45 mmol) was added to a solution of compound 4b
(87 mg, 0.34 mmol) in anhydrous pyridine (1 mL) at room temperature and
the mixture was stirred for 1 h. After the addition of water, the organic
phase was extracted with ethyl acetate. The residue was obtained as in the
synthesis of 2a and further purified by silica gel column chromatography
(n-hexane/ethyl acetate 1:5) to give compound 5b (186 mg, 97%).
Reprecipitation from n-hexane/ethyl acetate gave the analytical sample
as a white powder. M.p. 118 1218C; [a]2D3 ˆ ‡34.8 (c ˆ 1.11 in CHCl3);
1H NMR (270 MHz, CDCl3, 258C, TMS): d ˆ 7.68 (s, 1H), 7.50 7.19 (m,
10H), 6.85 (d, J ˆ 8 Hz, 4H), 6.47 (d, J ˆ 9 Hz, 1H), 5.85 (s, 1H), 4.50 (s,
1H), 4.28 (s, 1H), 3.87 (s, 2H), 3.79 (s, 6H), 3.48, 3.58 (AB, J ˆ 11 Hz, 2H),
1.93 (s, 3H); 13C NMR (68 MHz, CDCl3, 258C): d ˆ 161.15, 158.49, 144.47,
142.37, 135.44, 135.42, 129.99, 129.96, 129.04, 127.99, 127.90, 126.90, 119.93,
115.08, 113.23, 113.22, 87.83, 87.66, 86.59, 79.18, 71.73, 70.25, 58.63, 55.25,
17.60; IR (KBr): nÄmax ˆ 3327, 2951, 1664, 1581, 1508, 1252, 1179, 1081,
‡
1051 cmÀ1; MS (EI): m/z (%): 433 (12) [M] , 91 (100); elemental analysis
calcd (%) for C26H27NO5 ¥ 1/4H2O(C 26H27NO5, 433.5): C 71.30, H 6.33, N
3.20; found: C 71.39, H 6.41, N 3.19.
1046 cmÀ1; MS (FAB): m/z: 556 [M‡H] ; elemental analysis calcd (%) for
‡
C33H33NO7 ¥ 1³3H2O(C 33H33NO7, 555.6): C 70.57, H 6.04, N 2.49; found: C
1-(2-O,4-C-Methylene-b-d-ribofuranosyl)-2-pyridone (4a): Compound 3a
(68 mg, 0.16 mmol), 20% palladium hydroxide on carbon powder (60 mg),
cyclohexene (0.82 mL, 8.10 mmol) and ethanol (2 mL) were heated under
reflux for 1.5 h. The reaction mixture was filtered and silica gel (0.2 g) was
added to the solution. The mixture was concentrated under reduced
pressure. The residue was purified by silica gel column chromatography
(CHCl3/methanol 20:1) to give compound 4a (37 mg, 95%). Crystallization
from acetone gave the analytical specimen as plate crystals. M.p. 206
2078C; [a]2D6 ˆ ‡162.8 (c ˆ 1.00 in CH3OH); 1H NMR (270 MHz, CD3OD,
258C, TMS):d ˆ 8.01 (dd, J ˆ 7, 1 Hz, 1H), 7.59 7.53 (m, 1H), 6.53 6.45
(m, 2H), 5.79 (s, 1H), 4.33 (s, 1H), 4.04 (s, 1H), 3.99, 3.82 (AB, J ˆ 8 Hz,
2H), 3.94 (s, 2H); 13C NMR (68 MHz, CD3OD, 258C): d ˆ 163.83, 142.15,
133.68, 120.24, 108.10, 90.42, 89.09, 80.59, 72.49, 69.92, 57.76; IR (KBr):
70.55, H 6.14, N 2.45.
1-[3-O-[2-Cyanoethoxy(diisopropylamino)phosphino]-5-O-(4,4'-dimeth-
oxytrityl)-2-O-4-C-methylene-b-d-ribofuranosyl]-2-pyridone (6a): Under
a nitrogen atmosphere, 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphoro-
diamidite (52 mL, 0.16 mmol) was added to compound 5a (20 mg, 37 mmol)
and diisopropylammonium tetrazolide (17 mg, 98 mmol) in anhydrous
acetonitrile/tetrahydrofuran (3:1, 0.8 mL) at room temperature and the
mixture was stirred for 9 h. The solvent was removed under reduced
pressure and residue purified by flash silica gel column chromatography (n-
hexane/ethyl acetate/triethylamine 50:50:1) to give compound 6a (26 mg,
95%) as a white powder. M.p. 68 698C; 31P NMR (81 MHz, CDCl3,
‡
258C): d ˆ 149.3, 148.69; MS (FAB): m/z: 742 [M‡H] ; HRMS (FAB):
‡
nÄmax ˆ 3293, 2952, 1655, 1567, 1051 cmÀ1; MS (EI): m/z (%): 239 (30) [M] ,
‡
calcd for C41H48N3O8PNa: 764.3077; found: 764.3077 [M‡Na] .
96 (100); elemental analysis calcd (%) for C11H13NO5 (239.2): C, 55.23, H
5.48, N 5.86; found: C 55.05, H 5.44, N 5.82.
1-[3-O-[2-Cyanoethoxy(diisopropylamino)phosphino]-5-O-(4,4'-dimeth-
oxytrityl)-2-O-4-C-methylene-b-d-ribofuranosyl]-5-methyl-2-pyridone
(6b): Under a nitrogen atmosphere, 2-cyanoethyl-N,N,N',N'-tetraisopro-
pylphosphorodiamidite (33 mL, 0.10 mmol) was added to compound 5b
(34 mg, 61 mmol) and diisopropylammonium tetrazolide (10 mg, 58 mmol)
in anhydrous acetonitrile/tetrahydrofuran (3:1, 2 mL) at room temperature
and the mixture was stirred for 10 h. The solvent was removed under
reduced pressure and the residue purified by flash silica gel column
chromatography (n-hexane/ethyl acetate 50:50) to give compound 6b
(42 mg, 91%) as a white powder. M.p. 72 758C; 31P NMR (81 MHz,
5-Methyl-1-(2-O,4-C-methylene-b-d-ribofuranosyl)-2-pyridone (4b):
A
mixture of compound 3b (265 mg, 0.61 mmol), 20% palladium hydroxide
on carbon powder (140 mg), cyclohexene (3.10 mL, 30.6 mmol), and
ethanol (6 mL) was heated under reflux for 2 h, then filtered. The filtrate
was concentrated under reduced pressure. The residue was purified by
silica gel column chromatography (CHCl3/methanol 13:1) to give com-
pound 4b (141 mg, 91%) as a white powder. M.p. 209 2118C; [a]D28
ˆ
1
‡114.8 (c ˆ 0.93 in CH3OH); H NMR (270 MHz, CD3OD, 258C, TMS):
d ˆ 7.82 (d, J ˆ 2 Hz, 1H), 7.45 (dd, J ˆ 9, 2 Hz, 1H), 6.46 (d, J ˆ 9 Hz, 1H),
5.76 (s, 1H), 4.31 (s, 1H), 4.05 (s, 1H), 3.79, 3.97 (AB, J ˆ 8 Hz, 2H), 3.93 (s,
2H), 2.15 (s, 3H); 13C NMR (68 MHz, CD3OD, 258C): d ˆ 162.93, 144.59,
131.01, 119.79, 117.77, 90.38, 89.04, 80.58, 72.46, 69.83, 57.66, 17.49; IR
‡
CDCl3, 258C): d ˆ 149.09, 148.77; MS (FAB): m/z: 756 [M‡H] ; HRMS
‡
(FAB): calcd for C42H50N3O8PNa: 778.3233; found: 778.3236 [M‡Na] .
Syntheses of modified oligonucleotides: Oligonucleotides were synthesized
on a 0.2 mmol scale on an Applied Biosystems DNA synthesizer by using
standard phosphoramidite chemistry. The 5'-terminal DMTr group-pro-
tected oligonucleotides were treated with concentrated ammonia hydrox-
ide for 18 h at 608C and the solvents were removed. The residue was
purified with NENSORP PREP to give the purposed oligonucleotides. The
purity of the modified oligonucleotides was verified using reversed-phase
HPLC (ChemcoPak CHEMCOSORB 300–5C18, 4.6 mm  250 mm),
and the compositions determined by MALDI-TOF-MS (Voyager-DE).
(KBr): nÄmax ˆ 3406, 2925, 1658, 1567 cmÀ1; MS (EI): m/z (%): 253 (38) [M] ,
‡
109 (100); elemental analysis calcd (%) for C12H15NO5 ¥ 1³5H2O(C 12H15NO5,
253.3): C 56.11, H 6.04, N 5.45; found: C 56.15, H 5.95, N 5.44.
1-[5-O-(4,4'-Dimethoxytrityl)-2-O,4-C-methylene-b-d-ribofuranosyl]-2-
pyridone (5a): Under a nitrogen atmosphere, 4,4'-dimethoxytrityl chloride
(113 mg, 0.33 mmol) was added to a solution of compound 4a (50 mg,
0.21 mmol) in anhydrous pyridine (1 mL) and the mixture was stirred at
room temperature for 1 h. After the addition of saturated aqueous solution
of sodium hydrogen carbonate, the organic phase was extracted with ethyl
acetate. The residue was obtained as in the synthesis of 2a and further
MALDI-TOF-MS data: 9, calcd for [M À H]À: 4465.04; found: 4463.87; 10,
calcd for [M À H]À: 4493.05; found: 4493.37; 11, calcd for [M À H]À: 4507.08;
Chem. Eur. J. 2002, 8, No. 20
¹ 2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
0947-6539/02/0820-4801 $ 20.00+.50/0
4801

T. Imanishi et al.
FULL PAPER
found: 4507.76; 12, calcd for [M À H]À: 4399.97; found: 4400.41; 14, calcd
for [M À H]À: 4492.07; found: 4491.60; 16, calcd for [M À H]À: 4492.07;
found: 4492.83; 18, calcd for [M À H]À: 4491.08; found: 4490.48; 20, calcd
for [M À H]À: 4489.01; found: 4489.89; 21, calcd for [M À H]À: 4601.06;
found: 4600.91; 22, calcd for [M À H]À: 4643.14; found: 4642.37.
UV-melting experiments: UV-melting experiments were carried out on
Beckman DU–650 spectrometer. The profiles were measured at a scan
rate of 0.58CminÀ1 at 260 nm in sodium phosphate buffer (7mm, pH 7.0)
which also contained KCl (140mm) and spermine (5mm). The final
concentration of each oligonucleotide used was 1.5mm. The Tm value was
designated as the maximum of the first derivative calculated from the UV-
melting profile.
[11] a) S. Obika, K. Morio, D. Nanbu, T. Imanishi, Chem. Commun. 1998,
1643 1644; b) S. Obika, K. Morio, Y. Hari, T. Imanishi, Chem.
Commun. 1999, 2423 2424.
[12] S. Obika, K. Morio, D. Nanbu, Y. Hari, H. Itoh, T. Imanishi,
Tetrahedron 2002, 58, 3051 3063.
[13] S. Obika, D. Nanbu, Y. Hari, J. Andoh, K. Morio, T. Doi, T. Imanishi,
Tetrahedron Lett. 1998, 39, 5401 5404.
[14] T. Imanishi, S. Obika, J. Syn. Org. Chem. Jpn. 1999, 57, 969 980.
[15] S. Obika, Y. Hari, K. Morio, T. Imanishi, Tetrahedron Lett. 2000, 41,
221 224.
[16] S. Obika, Y. Hari, T. Sugimoto, M. Sekiguchi, T. Imanishi, Tetrahedron
Lett. 2000, 41, 8923 8927.
[17] a) H. Torigoe, Y. Hari, M. Sekiguchi, S. Obika, T. Imanishi, J. Biol.
Chem. 2001, 276, 2354 2360; b) H. Torigoe, S.Obika, T. Imanishi,
Nucleosides Nucleotides Nucleic Acids, 2001, 20, 1235 1238.
[18] S. Obika, T. Uneda, T. Sugimoto, D. Nanbu, T. Minami, T. Doi, T.
Imanishi, Bioorg. Med. Chem. 2001, 9, 1001 1011.
[19] A part of this work appeared in a preliminary communication: S.
Obika, Y. Hari, M. Sekiguchi, T. Imanishi, Angew. Chem. 2001, 113,
2149 2151; Angew. Chem. Int. Ed. 2001, 40, 2079 2081.
[20] U. Niedballa, H. Vorbr¸ggen, Nucleic Acid Chemistry, Part 1, Wiley,
New York, 1978, pp. 481 484.
Acknowledgement
Part of this work was supported by Industrial Technology Research Grant
Program in 2000 from New Energy and Industrial Technology Develop-
ment Organization (NEDO) of Japan, a Grant-in-Aid from Japan Society
for the Promotion of Science, and a Grant-in-Aid from the Ministry of
Education, Science, and Culture, Japan. We thank Research Fellowships of
the Japan Society for the Promotion of Science for Young Scientists (Y.H.).
[21] A. A. Koshkin, V. K. Rajwanshi, J. Wengel, Tetrahedron Lett. 1998, 39,
4381 4384.
[22] C. Altona, M. Sundaralingam, J. Am. Chem. Soc. 1973, 95, 2333 2344.
[23] CCDC-152458 contains the supplementary crystallographic data for
this paper. These data can be obtained free of charge via www.ccdc.
cam.ac.uk/conts/retrieving.html (or from the Cambridge Crystallo-
graphic Data Centre, 12 Union Road, Cambridge CB2 1EZ (UK);
fax: (‡44)1-223-336-033; or deposit@ccdc.cam.ac.uk).
[24] C. Altona, M. Sundaralingam, J. Am. Chem. Soc. 1972, 94, 8205 8212.
[25] F. Seela, U. Bindig, Liebigs Ann. Chem. 1989, 895 901.
[26] R. H. Durland, T. S. Rao, G. R. Revankar, J. H. Tinsley, M. A. Myrick,
D. M. Seth, J. Rayford, P. Singh, K. Jayaraman, Nucleic Acids Res.
1994, 22, 3233 3240.
[27] L. KvÒrn˘, R. Kumar, B. M. Dahl, C. E. Olsen, J. Wengel, J. Org.
Chem. 2000, 65, 5167 5176.
[28] The concentrations of various cations in the nucleus have been
estimated as 140mm KCl, 1mm MgCl2 and up to 5mm spermine: V. N.
Soyfer, V. N. Potaman, Triple-Helical Nucleic Acids, Springer Verlag,
New York, 1996, pp. 194 219.
[1] For reviews on antigene strategy by using TFOs: a) N. T. Thuong, C.
¬ ¡
Helene, Angew. Chem. 1993, 105, 697 723, Angew. Chem. Int. Ed.
¬ ¡
Engl. 1993, 32, 666 690; b) C. Giovannangeli, C. Helene, Antisense
Nucleic Acid Drug Dev. 1997, 7, 413 421; c) S. M. Gryaznov, Biochim.
Biophys. Acta 1999, 1489, 131 140; d) D. Praseuth, A. L. Guieysse, C.
¬ ¡
Helene, Biochim. Biophys. Acta 1999, 1489, 181 206.
[2] K. M. Vasquez, L. Narayanan, P. M. Glazer, Science 2000, 290, 530
533.
[3] E. M. McGuffie, D. Pacheco, G. M. R. Carbone, C. V. Catapano,
Cancer Res. 2000, 60, 3790 3799.
¬ ¡
[4] M. Faria, C. D. Wood, M. R. H. White, C. Helene, C. Giovannangeli, J.
Mol. Biol. 2001, 306, 15 24.
[5] For reviews on various nucleoside analogues for recognition of PyPu
interruption: a) S. O. Doronina, J.-P. Behr, Chem. Soc. Rev. 1997, 63
71; b) I. Luyten, P. Herdewijn, Eur. J. Med. Chem. 1998, 33, 515 576;
c) D. M. Growers, K. R. Fox, Nucleic Acids Res. 1999, 27, 1569 1577.
[29] T. J. Povsic, P. B. Dervan, J. Am. Chem. Soc. 1989, 111, 3059 3061.
‡
‡
[30] Contiguous C ¥ GC (or mC ¥ GC) triads lead to a marked decrease in
the stability of the DNA triplex: a) L. L. Kiessling, L. C. Griffin, P. B.
Dervan, Biochemistry 1992, 31, 2829 2834; b) D. Leitner, W. Schrˆd-
er, K. Weisz, Biochemistry 2000, 39, 5886 5892.
¬
[6] J.-L. Mergny, J.-S. Sun, M. Rougee, T. Montenay-Garestier, F. Barcelo,
¬ ¡
J. Chomilier, C. Helene, Biochemistry 1991, 30, 9791 9798.
[7] K. Yoon, C. A. Hobbs, J. Koch, M. Sardaro, R. Kutny, A. L. Weis,
Proc. Natl. Acad. Sci. USA 1992, 89, 3840 3844.
[8] It has been proposed that 3-nitrogen in pyrimidine nucleobases
contributed to a hydrogen bond with 4-amino hydrogen in C: a) D. A.
Gianolio, L. W. McLaughlin, Nucleosides Nucleotides 1999, 18, 1751
[31] The UV-melting curves were a little broad, probably due to restriction
of flexibility of TFOs by BNA units which were introduced in every
other nucleotide, but the details are not clear. Triplex formation with
target duplex 27 was also confirmed by UV-melting experiments at
284 nm as a second wavelength.
¬
1769; b) I. Prevot-Halter, C. J. Leumann, Bioorg. Med. Chem. Lett.
1999, 9, 2657 2660.
[9] S. Obika, D. Nanbu, Y. Hari, K. Morio, Y. In, T. Ishida, T. Imanishi,
Tetrahedron Lett. 1997, 38, 8735 8738.
[32] L. C. Griffin, L. L. Kiessling, P. A. Beal, P. Gillespie, P. B. Dervan, J.
Am. Chem. Soc. 1992, 114, 7976 7982.
[33] C.-Y. Huang, G. Bi, P. S. Miller, Nucleic Acids Res. 1996, 24, 2606
2613.
[34] T. E. Lehmann, W. A. Greenberg, D. A. Liberles, C. K. Wada, P. B.
Dervan, Helv. Chim. Acta 1997, 80, 2002 2022.
[35] N. Guzzo-Pernell, G. W. Tregear, J. Haralambidis, J. M. Lawlor,
Nucleosides Nucleotides 1998, 17, 1191 1207.
[36] L. C. Griffin, P. B. Dervan, Science 1989, 245, 967 971.
[37] E. Wang, S. Malek, J. Feigon, Biochemistry 1992, 31, 4838 4846.
[38] I. Radhakrishnan, D. J. Patel, J. M. Veal, X. Gao, J. Am. Chem. Soc.
1992, 114, 6913 6915.
[39] A few attempts to form triplexes with dsDNA containing three CG
interruptions by using T¥ CG triads were performed: a) D. M. Gowers,
K. R. Fox, Nucleic Acids Res. 1997, 25, 3787 3794; b) D. M. Gowers, J.
Bijapur, T. Brown, K. R. Fox, Biochemistry 1999, 38, 13747 13758.
[10] We defined BNA as a novel class of nucleic acid analogues containing
2'-O,4'-C or 3'-O,4'-C-methylene bridged structure (2',4'-BNA or 3',4'-
BNA, respectively). The 2',4'-BNA has been also called ™LNA∫:
a) S. K. Singh, P. Nielsen, A. A. Koshkin, J. Wengel, Chem. Commun.
1998, 455 456; b) S. K. Singh, J. Wengel, Chem. Commun. 1998,
1247 1248; c) A. A. Koshkin, S. K. Singh, P. Nielsen, V. K. Rajwan-
shi, R. Kumar, M. Meldgaard, C. E. Olsen, J. Wengel, Tetrahedron
1998, 54, 3607 3630; d) J. Wengel, Acc. Chem. Res. 1999, 32, 301
310; e) L. KvÒrn˘, J. Wengel, Chem. Commun. 1999, 657 658; f) K.
Bondensgaard, M. Petersen, S. K. Singh, V. K. Rajwanshi, R. Kumar,
J. Wengel, J. P. Jacobsen, Chem. Eur. J. 2000, 6, 2687 2695; g) C.
Wahlestedt, P. Salmi, L. Good, J. Kela, T. Johnsson, T. Hˆkfelt, C.
Broberger, F. Porreca, J. Lai, K. Ren, M. Ossipov, A. Koshkin, N.
Jakobsen, J. Skouv, H. Oerum, M. H. Jacobsen, J. Wengel, Proc. Natl.
Acad. Sci. USA 2000, 97, 5633 5638.
Received: March 18, 2002 [F3960]
4802
¹ 2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
0947-6539/02/0820-4802 $ 20.00+.50/0
Chem. Eur. J. 2002, 8, No. 20