Triplex-forming enhancement with high sequence selectivity by single 2'-O,4'-C-methylene bridged nucleic acid (2',4'-BNA) modification

Article abstract of DOI:10.1016/S0040-4039(00)01607-5

Triplex-forming ability of the oligonucleotides containing one 2'-O,4'-C-methyleneribonucleic acid (2',4'-BNA) unit was investigated by measurement of the melting temperature (T(m)), and the 2',4'-BNA modification promoted the marked triplex stabilization

Full text of DOI:10.1016/S0040-4039(00)01607-5

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 41 (2000) 8923–8927
Triplex-forming enhancement with high sequence selectivity
by single 2%-O,4%-C-methylene bridged nucleic acid
(2%,4%-BNA) modification
Satoshi Obika, Yoshiyuki Hari, Tomomi Sugimoto, Mitsuaki Sekiguchi and
Takeshi Imanishi*
Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita 565-0871, Osaka, Japan
Received 23 August 2000; revised 11 September 2000; accepted 14 September 2000
Abstract
Triplex-forming ability of the oligonucleotides containing one 2%-O,4%-C-methyleneribonucleic acid
(2%,4%-BNA) unit was investigated by measurement of the melting temperature (Tm), and the 2%,4%-BNA
modification promoted the marked triplex stabilization in a highly sequence-selective manner. © 2000
Elsevier Science Ltd. All rights reserved.
Keywords: nucleic acid analogues; triplex; antigene; melting temperature (Tm).
Pyrimidine oligodeoxyribonucleotides are able to bind to homopurine sequences in dsDNA to
form triplex DNA.1 During the past few years, interest in the possibility of regulation of gene
expression by triplex-forming oligonucleotides (TFOs) has grown because of their widely usable
application as molecular biological tools and as a direct way to treat serious diseases, such as
cancer and genetic disorders. Since the binding affinity of the natural pyrimidine oligonucle-
otides towards dsDNA is relatively low under physiological conditions, extensive efforts have
been directed towards developing novel DNA and RNA analogues for the practical use of the
antigene technologies and for clarification of the triplex structure.2 However, only a few
modified TFOs with sufficient binding affinity under physiological conditions have been
reported.2 It occurred to us that restriction of the oligonucleotide conformation in a suitable
form would be quite effective for triplex formation, as well as binding with complementary
ssDNA or ssRNA.3 Despite their importance, there is only limited information available on the
triplex-forming ability of the conformationally restricted or locked oligonucleotides.3–5 Recently,
we have achieved the first synthesis of a novel nucleoside with a fixed N-type conformation,
2%-O,4%-C-methyleneribonucleic acids (2%-O,4%-C-methylene bridged nucleic acid; 2%,4%-BNA) (1)6
* Corresponding author. Fax: +81 6-6879-8204; e-mail: imanishi@phs.osaka-u.ac.jp
0040-4039/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)01607-5

8924
(see Fig. 1) and found that the BNA has extraordinary hybridizing ability towards complemen-
tary natural nucleic acids, especially towards complementary RNA.7,8 In addition, we also found
that partial 2%,4%-BNA modification of pyrimidine oligonucleotides promotes stable triplex
formation at neutral pH.4 Now, we deal with the triplex-forming ability and sequence selectivity
of the oligonucleotides containing only one BNA monomer unit, and as a result, N-form sugar
puckering was shown to play an important role in stable triplex formation.
Figure 1. Structure of 2%,4%-BNA (2%-O,4%-C-methylene bridged nucleic acid)
As shown in Scheme 1, 2%-O,4%-C-methyleneuridine (1a)6 was effectively converted to its
5-methyl congener 1b† via 5-bromination and subsequent Pd-catalyzed cross-coupling reaction
with trimethylaluminum.11 As a control unit to investigate the effects of the fixed sugar moiety
Scheme 1. (a) NBS, NaN3, 1,2-dimethoxyethane, 20°C, 84%. (b) (i) (NH4)2SO4, 1,1,1,3,3,3-hexamethyldisilazane,
reflux; (ii) PdCl2, Ph3P, Me3Al, THF, reflux; (iii) NH4Cl, MeOH–H2O, reflux, 57%. (c) (i) DMTrCl, DMAP, pyridine,
20°C, 95%; (ii) 2-cyanoethyl N,N,N%,N%-tetraisopropylphosphorodiamidite, diisopropylammonium tetrazolide,
MeCN–THF, 20°C, 99%. (d) 1,2,4-Triazole, POCl3, Et3N, MeCN, 0°C, 91%. (e) NaBH4, MeOH, 20°C, 95%. (f)
1,1%-azobis(N,N-Dimethylformamide), n-Bu3P, CH2Cl2, 20°C, 95%. (g) 20% Pd(OH)2ꢀC, cyclohexene, EtOH, reflux,
68%. (h) (i) DMTrCl, pyridine, 20°C, 99%; (ii) 2-cyanoethyl N,N,N%,N%-tetraisopropylphosphorodiamidite, diiso-
propylammonium tetrazolide, MeCN–THF, 20°C, 86%
† Recently, Wengel et al. reported the synthesis of 1b from 4-C-hydroxymethylribofuranose derivative.8,9 Indepen-
dently, we also achieved the synthesis of 1 in a similar manner.10

8925
on triplex formation, abasic analogue 1c was also synthesized according to the literature12 with
some modifications. The aldehyde 213,14 was reduced with NaBH4 to afford the diol 3, which was
then cyclized under the Mitsunobu conditions to yield 4. The abasic analogue 1c was readily
obtained by the hydrogenolysis of 4.
The phosphoramidites 5 and 7, suitable building blocks for DNA synthesis, were prepared by
dimethoxytritylation and subsequent phosphitylation of 1b and 1c. According to Xu’s proce-
dure,15 the amidite 5 was transformed into the 4-triazolo derivative 6, which was easily
converted into the 5-methylcytidine derivative by treatment with conc. ammonia after oligonu-
cleotide synthesis. TFOs I and II containing one 2%,4%-BNA monomer were effectively prepared
by the standard phosphoramidite protocol on a DNA synthesizer.‡
The triplex-forming property of TFOs I and II towards the target duplex 5%-d(GCTAAAAA-
GAYAGAGAGATCG)-3%/3%-d(CGATTTTTCTZTCTCTCTAGC)-3%, in which Y·Z are all four
natural base pairs, was studied by an analysis of the UV melting curve. At first, Tm measure-
ments for TFO I were carried out in 7 mM sodium phosphate buffer (pH 6.0) containing 140
mM potassium chloride and 0.5 mM magnesium chloride (Table 1).16 The Tm values for the
modified TFOs I containing one BNA thymine monomer (TB) or one BNA cytidine monomer
(CB) were compared with those for the TFO I having one BNA abasic monomer (HB) or the
unmodified reference sequences. The incorporation of BNA monomer TB or CB showed a
significant enhancement of triplex-stability towards a matched duplex sequence with the DTm
values of +10 and +7°C [Tm values of triplexes were 54°C (X·Y·Z=TB·A·T) and 44°C (T·A·T);
57°C (CB·G·C) and 50°C (C·G·C)]. Furthermore, the sufficient sequence selectivity of the
Table 1
Tm values (°C) of triplexes involving TFO Ia
Y·Z
X
A·T
G·C
T·A
C·G
TB
CB
HB
T
54
22
17
44
18
30
57
21
21
50
17
15
21
17
16
36
34
25
27
26
C
a UV melting profiles were measured in 7 mM sodium phosphate buffer (pH 6.0) containing 140 mM KCl and 0.5
mM MgCl2 at a scan rate of 0.5°C/min at 260 nm. The first derivative was calculated from the UV melting profile,
and the peak temperatures in the first derivative curve were designated as the melting temperature, Tm. The
oligonucleotide concentration used was 1.5 mM for each strand.
‡ The BNA containing 2%-O,4%-C-methylenecytidine was prepared as described earlier.7 The BNAs were synthesized
on the DNA synthesizer (Gene Assembler® Plus, Pharmacia, 0.2 mmol scale, 5%-dimethoxytrityl on). After treatment
with conc. ammonia, removal of the 5%-dimethoxytrityl group and purification were performed on NENSORB™
PREP reversed-phase columns. The purity of the BNAs was verified using reversed-phase HPLC and the composi-
tions were determined by MALDI-TOF-MS.

8926
modified TFOs I containing a BNA monomer was also confirmed from an observation of a
large decrease in the Tm value when they hybridized with mismatched duplex sequences.
On the other hand, the Tm values of TFOs II, in which the cytosine nucleobases were replaced
with 5-methylcytosine, were also elucidated in 7 mM sodium phosphate buffer (pH 7.0)
containing 140 mM potassium chloride and 10 mM magnesium chloride. All Tm data are
summarized in Table 2, and selected melting profiles are shown in Fig. 2. In analogy with the
results of TFOs I, it was found that the incorporation of a BNA monomer shows remarkable
triplex-stabilizing ability with sequence selectivity. Especially, TB·A·T triad increased the Tm
Table 2
Tm values (°C) of triplexes involving TFO IIa
Y·Z
X
A·T
G·C
T·A
C·G
TB
57
27
16
44
18
31
53
20
20
43
16
15
20
17
16
35
33
24
25
25
CB
6
HB
T
Cb
6
a UV melting profiles were measured in 7 mM sodium phosphate buffer (pH 7.0) containing 140 mM KCl and 10
mM MgCl2.
b C
=2%-deoxy-5-methylcytidine.
6
Figure 2. UV melting profiles (260 nm) for the triplexes involving TFO II (X·Y·Z=TB·A·T, closed square; T·A·T,
open circle) and first derivative of the melting curves (X·Y·Z=TB·A·T, solid line; T·A·T, dashed line). The triplexes
were melted at a scan rate of 0.5°C/min with detection at 260 nm. The normalized absorbance was obtained simply
by dividing the observed absorbance by the absorbance at 80°C

8927
value by +13 and +41°C, compared with T·A·T and HB·A·T triads, respectively, which was an
unprecedented result of triplex stabilization by an oligonucleotide analogue. This clearly
indicates that both the bridged sugar moiety and a nucleobase were required for the efficient
triplex stabilization, the same as for duplex stabilization.12
In conclusion, incorporation of single BNA unit in TFOs significantly enhanced triplex-form-
ing ability with high sequence selectivity, which reveal that the BNA is a promising candidate
not only for an antisense molecule7,8,17 but also for a novel and practical antigene molecule.
Acknowledgements
A part of this work was supported by a Grant-in-Aid for Scientific Research (B), No.
11470469 and 12557201, from Japan Society for the Promotion of Science.
References
1. Moser, H. E.; Dervan, P. B. Science 1987, 238, 645–650; Le Doan, T.; Perrouault, L.; Praseuth, D.; Habhoub,
N.; Decout, J. L.; Thuong, N. T.; Lhomme, J.; He´le`ne, C. Nucleic Acids Res. 1987, 15, 7749–7760; Lyamichev,
V. I.; Mirkin, S. M.; Frank-Kamenetskii, M. D.; Cantor, C. R. Nucleic Acids Res. 1988, 16, 2165–2178.
2. Thuong, N. T.; He´le`ne, C. Angew. Chem., Int. Ed. Engl. 1993, 32, 666–690; Luyten, I.; Herdewijn, P. Eur. J. Med.
Chem. 1998, 33, 515–576; Gowers, D. M.; Fox, K. R. Nucleic Acids Res. 1999, 27, 1569–1577; Fox, K. R. Curr.
Med. Chem. 2000, 7, 17–37.
3. Herdewijn, P. Liebigs Ann. Chem. 1996, 1337–1348; Kool, E. T. Chem. Rev. 1997, 97, 1473–1487; Wengel, J. Acc.
Chem. Res. 1999, 32, 301–310.
4. Imanishi, T.; Obika, S. J. Synth. Org. Chem. Jpn. 1999, 57, 969–980.
5. Bolli, M.; Leumann, C. Angew. Chem., Int. Ed. Engl. 1995, 34, 694–696; Steffens, R.; Leumann, C. J. Am. Chem.
Soc. 1999, 121, 3249–3255.
6. Obika, S.; Nanbu, D.; Hari, Y.; Morio, K.; In, Y.; Ishida, T.; Imanishi, T. Tetrahedron Lett. 1997, 38, 8735–8738.
7. Obika, S.; Nanbu, D.; Hari, Y.; Andoh, J.; Morio, K.; Doi, T.; Imanishi, T. Tetrahedron Lett. 1998, 39,
5401–5404.
8. Singh, S. K.; Nielsen, P.; Koshkin, A. A.; Wengel, J. Chem. Commun. 1998, 455–456; Koshkin, A. A.; Singh, S.
K.; Nielsen, P.; Rajwanshi, V. K.; Kumar, R.; Meldgaard, M.; Olsen, C. E.; Wengel, J. Tetrahedron 1998, 54,
3607–3630.
9. Koshkin, A. A.; Rajwanshi, V. K.; Wengel, J. Tetrahedron Lett. 1998, 39, 4381–4384.
10. Obika, S. Yakugaku Zasshi 2000, 120, 147–158.
11. Kumar, R.; Wiebe, L. I.; Knaus, E. E. Can. J. Chem. 1994, 72, 2005–2010; Hirota, K.; Kitade, Y.; Kanbe, Y.;
Isobe, Y.; Maki, Y. Synthesis 1993, 213–215.
12. Kværnø, L.; Wengel, J. Chem. Commun. 1999, 657–658.
13. Obika, S.; Hari, Y.; Morio, K.; Imanishi, T. Tetrahedron Lett. 2000, 41, 215–219.
14. Nielsen, P.; Wengel, J. Chem. Commun. 1998, 2645–2646.
15. Xu, Y.-Z.; Zheng, Q.; Swann, P. F. J. Org. Chem. 1992, 57, 3839–3845.
16. Obika, S.; Hari, Y.; Morio, K.; Imanishi, T. Tetrahedron Lett. 2000, 41, 221–224.
17. Wahlestedt, C.; Salmi, P.; Good, L.; Kela, J.; Johnsson, T.; Ho¨kfelt, T.; Broberger, C.; Porreca, F.; Lai, J.; Ren,
K.; Ossipov, M.; Koshkin, A.; Jakobsen, N.; Skouv, J.; Oerum, H.; Jacobsen, M. H.; Wengel, J. Proc. Natl.
Acad. Sci. USA 2000, 97, 5633–5638.
.