A 2′,4′-Bridged Nucleic Acid Containing 2-Pyridone as a Nucleobase: Efficient Recognition of a C · G Interruption by Triplex Formation with a Pyrimidine Motif

Full text of DOI:10.1002/1521-3773(20010601)40:11<2079::AID-ANIE2079>3.0.CO;2-Z

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A 2',4'-Bridged Nucleic Acid
Containing 2-Pyridone as a
Nucleobase: Efficient Recognition of a
C ´ G Interruption by Triplex
Formation with a Pyrimidine Motif**
Satoshi Obika, Yoshiyuki Hari,
Mitsuaki Sekiguchi, and Takeshi Imanishi*
Triplex formation between duplex DNA and a
triplex-forming oligonucleotide (TFO) has been
noted in practical applications of antigene method-
ology and in genomic analysis. In the triplex DNA
formed with the pyrimidine motif, the homopyr-
imidine TFO binds to the homopurine tract of the
target duplex DNA in a sequence-specific manner
through the formation of Hoogsteen hydrogen
‡
bonds to form T´ A ´ T and C ´ G ´ C triads. Inter-
ruption of the homopurine sequence of the target
duplex DNA by a pyrimidine nucleotide causes
destabilization of the triplex. Chemical modifica-
tion of the TFOs in the nucleobase moiety has
been reported to give efficient recognition of a
pyrimidine ´ purine base pair, such as a C ´ G or
T´ A base pair.[1, 2] However, development of
practical TFOs for recognition of general duplex
DNA sequences is still pending.
We recently achieved the first synthesis of a
novel nucleoside with a fixed N-type conformation
(C-3'-endo),[3] namely, 2'-O,4'-C-methyleneribonu-
cleic acid (2'-O,4'-C-methylene Bridged Nucleic
Acid 2',4'-BNA; Scheme 1A)[4, 5] and found that
pyrimidine oligonucleotides with partial 2',4'-BNA
modification exhibited strong triplex-forming abil-
ities at neutral pH values.[6±9]
From the fact that T or C moderately interacts
with a C ´ G base pair in homopurine ´ homopyri-
midine double-stranded DNA (dsDNA),[10±12] we
considered that the 2-carbonyl oxygen atom of Tor
C plays an important role in the recognition of a
C ´ G base pair. Therefore, we selected the 2-pyridone
group,[13, 14] which has only a 2-carbonyl group and lacks the
3-nitrogen atom and a 4-carbonyl or amino group of thymine
or cytosine (Scheme 1B), as a nucleobase to interact with a
C ´ G base pair. Here, we report the synthesis of the novel 2',4'-
BNA monomer 1 bearing a 2-pyridone group, and its
application to the efficient recognition of C ´ G interruption
in homopurine ´ homopyrimidine dsDNA.
Scheme 1. A) Structure and characteristics of 2',4'-BNA. B) The oligonucleotides used in
this study.
treated with 2-pyridone, N,O-bis(trimethylsilyl)acetamide
(BSA), and trimethylsilyl trifluoromethanesulfonate
(TMSOTf) in dichloroethane to give 3. Bicyclic nucleoside 4
was obtained upon exposure of 3 to potassium carbonate in
methanol. Next, hydrogenolysis of 4 afforded the desired
compound 1. The structure of 2',4'-BNA monomer 1 was
confirmed by X-ray crystallographic analysis (Figure 1),[16]
which revealed that the sugar moiety in 1 was fixed in an
N-type conformation (pseudorotation phase angle P is 16.78),
the same as that in the 2',4'-BNA uracil monomer (P is
17.48).[4] The phosphoramidite 6, the suitable building block
for DNA synthesis, was obtained by dimethoxytritylation
(1 !5) and phosphitylation (5 !6), and then incorporated
into TFOs I and I' (Scheme 1B) by a standard phosphorami-
dite protocol on a DNA synthesizer. The purity of the
modified TFOs was verified by using reversed-phase HPLC,
and the compositions were determined by matrix-assisted
laser desorption/ionization time-of-flight (MALDI-TOF)
MS.[17]
The synthetic route to 1 is shown in Scheme 2. The starting
material 2,[15] which was easily prepared from d-glucose, was
[*] Prof. Dr. T. Imanishi, Dr. S. Obika, Y. Hari, M. Sekiguchi
Graduate School of Pharmaceutical Sciences
Osaka University
1-6 Yamadaoka, Suita, Osaka 565-0871 (Japan)
Fax : (‡81)6-6879-8204
E-mail: imanishi@phs.osaka-u.ac.jp
[**] Part of this work was supported by a Grant-in-Aid for Scientific
Research (B) (No. 12557201) from the Japan Society for the Promo-
tion of Science.
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Table 1. Tm values [8C] of the triplex formed between TFO I and the target
duplex II ´ III.[a]
BnO
BnO
TsO
Y´ Z
N
O
X
C ´ G
G ´ C
T´ A
A ´ T
O
O
OAc
a
b
T
25
25
24
33
24
20
43
16
19
20
17
16
15
14
20
44
18
15
23
16
mC
P
TsO
BnO
OBn
OBn
OAc
OAc
PB
HB
2
3
[a] UV melting profiles were measured in 7 mm sodium phosphate buffer
(pH 7.0) containing 140 mm KCl and 10 mm MgCl2 at a scan rate of
0.5 Kmin at 260 nm. The oligonucleotide concentration used was 1.5 mm
HO
N
O
N
O
1
O
O
c
d
for each strand.
OBn
HO
O
O
4
1
DMTrO
N
O
DMTrO
N
O
O
O
e
O
P
O
HO
O
5
iPr2N
OCH2CH2CN
6
Scheme 2. Synthesis of 2',4'-BNA-pyridone monomer 1 and its phosphor-
amidite derivative 6. a) 2-pyridone, BSA, TMSOTf, dichloroethane, reflux,
74%; b) K2CO3, MeOH, RT, 100%; c) 20% Pd(OH)2/C, cyclohexene,
EtOH, reflux, 95%; d) DMTrCl, pyridine, RT, 96%; e) 2-cyanoethyl
N,N,N',N'-tetraisopropylphosphorodiamidite, diisopropylammonium tetra-
zolide, MeCN/THF, RT, 98%. Bn ˆ benzyl, Ac ˆ acetyl, Ts ˆ toluene-4-
sulfonyl, DMTrˆ dimethoxytrityl.
Scheme 3. Proposed structures of the P ´ C ´ G, T´ C ´ G, and mC ´ C ´ G
triads.
Triplex formation between TFO I (X ˆ PB) and the target
duplex II ´ III was also found to be sequence selective, with the
triplex I ´ II ´ III containing a PB ´ C ´ G triad being the most
stable in the triplexes I ´ II ´ III with a PB ´ X ´ Y triad (Fig-
ure 2A). Moreover, the thermal stability of the triplex I ´ II ´
III containing a PB ´ C ´ G triad is preferable to that of the
triplex I ´ II ´ III containing a T´ C ´ G triad (Figure 2B).
Comparison of the Tm values of the triplexes with PB ´ C ´ G
(338C) and P ´ C ´ G triad (248C) demonstates that the level of
triplex stabilization induced by the 2',4'-BNA modification of
the 2-pyridone derivative for recognition of the C ´ G inter-
ruption (DTm ˆ ‡9 K) is comparable to that caused by the
2',4'-BNA modification of thymidine or 5-methylcytidine in
the fully matched triplexes.[7]
Figure 1. X-ray structure of 2',4'-BNA-pyridone monomer 1.
To confirm the general applicability of this result, the ability
of PB to recognize the C ´ G interruption in other dsDNA
targets was also studied (Table 2). Replacement of an A ´ T
base pair by a G ´ C base pair at the neighboring sites of the
C ´ G interruption caused a decrease in the Tm values as a
result of the low stability of a mC ´ G ´ C triad under neutral
conditions. However, the TFOs I' (X ˆ PB) preserved the
reasonable triplex-forming ability towards the corresponding
target duplex (Y´ Z ˆ C ´ G). Furthermore, the triplexes
I' ´ II' ´ III' containing a PB ´ C ´ G triad had much superior
thermal stability than any other triplexes I' ´ II' ´ III' having a
PB ´ T´ A, T´ C ´ G, or T´ T´ A triad.
To the best of our knowledge, PB is one of the best nucleic
acid analogues for recognizing a C ´ G base pair since it gives a
significant increase in binding affinity without loss of selec-
tivity. Considering that the TFO I containing the 2',4'-BNA
abasic monomer (HB) had only a slight effect on the triplex
The melting temperatures (Tm) for the triplexes formed by
TFO I containing the 2',4'-BNA-2-pyridone monomer (PB)
were compared with those of triplexes where TFO I contained
a natural nucleobase (T or mC), a DNA-2-pyridone monomer
(P),[14] or the 2',4'-BNA abasic monomer (HB, Table 1).[7, 18] At
first, it was found that the triplex I ´ II ´ III containing a P ´ C ´ G
triad was as stable as the triplexes I ´ II ´ III containing a
T´ C ´ G or mC ´ C ´ G triad, which indicates the great impor-
tance of the 2-carbonyl oxygen atom of T, mC, and P in the
recognition of a C ´ G base pair.[19] Furthermore, as we
expected, the TFO I containing P showed high sequence
selectivity, while that containing T or mC preferentially
recognizes an A ´ T or G ´ C base pair. The proposed hydro-
gen-bonding pattern for the P ´ C ´ G triad is shown in
Scheme 3.
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Angew. Chem. Int. Ed. 2001, 40, No. 11

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2-pyridone and the 2'-O,4'-C-methylene bridged sugar moiety
significantly enhances the binding affinity with a C ´ G base
pair, without loss of selectivity. The application of the present
findings to the regulation of gene expression is currently
under way.
Received: December 4, 2000
Revised: March 2, 2001 [Z16220]
[1] I. Luyten, P. Herdewijn, Eur. J. Med. Chem. 1998, 33, 515 ± 576, and
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[2] a) S. O. Doronina, J.-P. Behr, Chem. Soc. Rev. 1997, 63 ± 71, and
Â
references therein; b) I. Prevot-Halter, C. J. Leumann, Bioorg. Med.
Chem. Lett. 1999, 9, 2657 ± 2660.
[3] W. Saenger, Principles of Nucleic Acid Structure, Springer, Berlin,
1984, p. 17.
[4] S. Obika, D. Nanbu, Y. Hari, K. Morio, Y. In, T. Ishida, T. Imanishi,
Tetrahedron Lett. 1997, 38, 8735 ± 8738.
[5] We defined BNA as a novel class of nucleic acid analogues containing
the 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 also been called ªLNAº;
see S. K. Singh, P. Nielsen, A. A. Koshkin, J. Wengel, Chem. Commun.
1998, 455 ± 456.
[6] T. Imanishi, S. Obika, J. Synth. Org. Chem. Jpn. 1999, 57, 969 ± 980.
[7] S. Obika, Y. Hari, T. Sugimoto, M. Sekiguchi, T. Imanishi, Tetrahedron
Lett. 2000, 41, 8923 ± 8927.
[8] H. Torigoe, Y. Hari, M. Sekiguchi, S. Obika, T. Imanishi, J. Biol. Chem.
2001, 276, 2354 ± 2360.
[9] S. Obika, T. Uneda, T. Sugimoto, D. Nanbu, T. Minami, T. Doi, T.
Imanishi, Bioorg. Med. Chem., in press.
[10] K. Yoon, C. A. Hobbs, J. Koch, M. Sardaro, R. Kutny, A. L. Weis,
Proc. Natl. Acad. Sci. USA 1992, 89, 3840 ± 3844.
Â
[11] J. L. Mergny, D. Collier, M. Rougee, T. Montenay-Garestier, C.
 Á
Helene, Nucleic Acids Res. 1991, 19, 1521 ± 1526.
[12] B. P. Belotserkovskii, A. G. Veselkov, S. A. Filippov, V. N. Dobrynin,
S. M. Mirkin, M. D. Frank-Kamenetskii, Nucleic Acids Res. 1990, 18,
6621 ± 6624.
[13] U. Niedballa, H. Vorbrüggen in Nucleic Acid Chemistry, Part 1 (Eds.:
L. B. Townsend, R. S. Tipson), Wiley, New York, 1978, pp. 481 ± 484.
[14] 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.
Figure 2. Tm profiles of triplex I ´ II ´ III containing a A) PB ´ C ´ G (ÐÐ ),
P ´ T´ A (- - - -), P ´ G ´ C ( ± ±), or P ´ A ´ T (ÐÐ) triad and B) a P ´ C ´ G
(ÐÐ ) or T´ C ´ G (- - - -) triad.
B
B
B
B
*
*
[15] A. A. Koshkin, V. K. Rajwanshi, J. Wengel, Tetrahedron Lett. 1998, 39,
4381 ± 4384.
Table 2. Tm values [8C] of the triplex formed from the TFO I' and the
target duplex II' ´ III'.[a]
[16] Crystallographic data (excluding structure factors) for the structure
reported in this paper have been deposited with the Cambridge
Crystallographic Data Centre as supplementary publication
no. CCDC-152458. Copies of the data can be obtained free of charge
on application to CCDC, 12 Union Road, Cambridge CB21EZ, UK
(fax: (‡44)1223-336-033; e-mail: deposit@ccdc.cam.ac.uk).
[17] MALDI-TOF-MS data: m/z (calcd): TFO I (X ˆ PB) [M H]
4493.37 (4493.05); TFO I (HB) [M H] 4400.41 (4399.97); TFO I'
(X1-X-X2 ˆ T-PB-mC) [M H] 4491.60 (4492.07); TFO I' (mC-PB-T)
[M H] 4492.83 (4492.07); TFO I' (mC-PB-mC) [M H] 4490.48
(4491.08).
Y´ Z
Y´ Z
X1-X-X2
T-T-mC
C ´ G
T´ A
X1-X-X2
C ´ G
T´ A
[b]
16
27
13
ca. 10
ca. 10
ca. 10
mC-PB-T
mC-T-mC
21
±
±
[b]
[b]
T-PB-m
C
±
[b]
mC-T-T
mC-PB-m
C
16
±
[a] See captions in Table 1. [b] Typical hyperchromicity for the triplex
dissociation was not observed.
stabilization, the unprecedented C ´ G recognition ability of PB
would be attributable to the 2'-O,4'-C-methylene bridged
ribofuranose moiety coupled with an appropriate hydrogen
bond between the 2-carbonyl oxygen atom in PB and the
4-amino group in C.
[18] L. Kvñrnù, J. Wengel, Chem. Commun. 1999, 657 ± 658.
Â
[19] Recently, Prevot-Halter and Leumann proposed that the 3-nitrogen
atom in pyrimidine contributed to the hydrogen bonding of the
4-amino group in C. See ref. [2b].
The results presented here demonstrate that the 2-carbonyl
oxygen atoms of pyridone and pyrimidine nucleobases play an
important role in the recognition of a C ´ G base pair. In
contrast to T or mC, the 2-pyridone derivative (P) has a
reasonable C ´ G selectivity because of the lack of a 3-nitrogen
atom and a 4-carbonyl or amino group which are crucial for
hydrogen bonding with other base pairs. The combination of
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