3′-amino-2′,4′-BNA: Novel bridged nucleic acids having an N3′→P5′ phosphoramidate linkage

Article abstract of DOI:10.1039/b105640a

Novel oligonucleotide analogues, containing a 3′-amino-2′,4′-BNA unit, were successfully synthesized, and they showed superior duplex and triplex forming ability as well as BNA itself, along with remarkable enzymatic stability.

Full text of DOI:10.1039/b105640a

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3A-Amino-2A,4A-BNA: novel bridged nucleic acids having an N3A P5A
phosphoramidate linkage
Satoshi Obika,a Mayumi Onoda,a Koji Morita,b Jun-ichi Andoh,a Makoto Koizumib and Takeshi
Imanishi*a
a Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka
565-0871, Japan. E-mail: imanishi@phs.osaka-u.ac.jp; Fax: (+81) 6-6879-8204;
Tel: (+81) 6-6879-8200
b Exploratory Chemistry Research Laboratories, Sankyo Co., Ltd, 1-2-58 Hiromachi, Shinagawa, Tokyo
140-8710, Japan.
Received (in Cambridge, UK) 27th June 2001, Accepted 24th August 2001
First published as an Advance Article on the web 18th September 2001
Novel oligonucleotide analogues, containing a 3A-amino-
2A,4A-BNA unit, were successfully synthesized, and they
showed superior duplex and triplex forming ability as well as
BNA itself, along with remarkable enzymatic stability.
ssRNA were elucidated under physiological conditions on the
basis of melting temperatures (Tms) (Table 1). Replacement of
a natural 2A-deoxyribonucleotide by a 3A-amino-2A,4A-BNA
monomer in the 12-mer oligonucleotide 9 resulted in significant
Oligonucleotides for practical antisense and/or antigene mole-
cules should fulfil some requirements, such as a high binding
affinity for the target ssRNA or dsDNA, high sequence
selectivity, and sufficient enzymatic stability. During the past
decade, various chemically modified oligonucleotides have
been synthesized, and their properties have been investi-
gated;1–5 however, an ideal antisense or antigene molecule is
still lacking. We recently achieved the first synthesis of a novel
nucleoside with a fixed N-type conformation,6 2A-O,4A-C-
methylene bridged nucleic acid (2A,4A-BNA)7,8 (Fig. 1) and
found that 2A,4A-BNA modified oligonucleotides exhibited
strong hybridization ability with complementary strands of
RNA and DNA.9,10 Moreover, these 2A,4A-BNA oligonucleo-
tides appeared to possess high affinity for dsDNA forming a
stable DNA triplex.10–15
On the other hand, Gryaznov et al. reported in numerous
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papers that oligonucleotide analogues with an N3A P5A
phosphoramidate linkage show superior properties of hybrid-
ization to ssDNA, ssRNA and dsDNA, along with high
resistance to enzymatic degradation.16–19
Therefore, we became interested in a combination of a BNA
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structure and an N3A P5A phosphoramidate linkage.† Here, we
report the synthesis, hybridization properties and enzymatic
stability of novel oligonucleotide analogues with a 3A-amino-
2A,4A-BNA monomer unit (Fig. 1).
The synthesis of 3A-amino-2A,4A-BNA heterodimer building
block 1 was accomplished as shown in Scheme 1. After
protection of the 5A-hydroxy group in 2,20 the 3A-azido group
was successfully reduced to give the 3A-amino derivative 3.
Condensation of 3 with a 5A-phosphoramidite 421 was per-
formed according to the reported procedure.17,21 The obtained
heterodimer 5 was converted to the deisred compound 1 by
treatment with tetrabutylammonium fluoride and subsequent
phosphitylation. The 3A-amino-2A,4A-BNA oligonucleotides 6–8
were prepared using the phosphoramidite 1.‡
Scheme 1 Reagents and conditions: i 4,4A-dimethoxytrityl chloride, DMAP,
Py, room temp., 76%; ii Ph3P, Py, room temp., then NH4OH aq., room
temp., 97%; iii CCl4, Et3N, MeCN, room temp., 39%; iv TBAF, THF, room
temp., 78%; v 2-cyanoethyl N,N,NA,NA-tetraisopropylphosphorodiamidite,
diisopropylammonium tetrazolide, MeCN–THF, room temp., 61%; vi DNA
synthesizer (AB Expedite™ 8909). CE = 2-cyanoethyl. DMTr = 4,4A-
dimethoxytrityl.
Table 1 Tm values for 3A-amino-2A,4A-BNA with complementary DNA and
RNAa
The duplex-forming abilities of the 3A-amino-2A,4A-BNA
modified oligonucleotide 6 with complementary ssDNA and
T
m (DTm)/°C
Oligonucleotides
DNA
RNA
5A-GCGTTTaBTTTGCT-3A (6)
5A-GCGTTTBTTTGCT-3A (10)
5A-GCGTTTTTTGCT-3A (9)
51 (+4)
53 (+6)
47
52 (+7)
52 (+7)
45
a UV melting profiles were measured in 10 mM sodium phosphate buffer
(pH 7.2) containing 100 mM NaCl at a scan rate of 0.5 °C min21 at 260 nm.
The oligonucleotide concentration used was 4 mM for each strand. The
sequence of target DNA or RNA complements is 5A-AGCAAAAAACGC-
3A.
Fig. 1 Structure and conformation of 2A,4A-BNA and 3A-amino-2A,4A-BNA.
1992
Chem. Commun., 2001, 1992–1993
This journal is © The Royal Society of Chemistry 2001
DOI: 10.1039/b105640a

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Table 2 Tm values of 3A-amino-2A,4A-BNA with dsDNAa
m (DTm)/°C
sufficiently resistant to enzymatic hydrolysis in other cases.10 In
contrast, more than 98% of full length 3A-amino-2A,4A-BNA
oligonucleotide 8 was unchanged after 40 min, while 83% of 15
was intact after the same time period. It is noteworthy that
enzymatic stability of the 3A-amino-2A,4A-BNA 8 was superior to
that of the phosphorthioate modified oligonucleotide 15.22
The results presented here clearly demonstrate that the novel
nucleic acid analogue, 3A-amino-2A,4A-BNA, was a good candi-
date for a practical antisense and antigene molecule, due to its
potent hybridization ability with DNA and RNA complements
and homopurine•homopyrimidine dsDNA, and remarkable
enzymatic stability surpassing that of phosphorthioate oligonu-
cleotides. Further investigation of properties of the 3A-amino-
2A,4A-BNA is currently under way.
T
+10 mM
MgCl2
Oligonucleotides
2MgCl2
5A-TTTTTmCTTaBTmCTmCTmCT-3A (7)
5A-TTTTTmCTTBTmCTmCTmCT-3A (12)
5A-TTTTTmCTTTmCTmCTmCT-3A (11)
55 (+11)
57 (+13)
44
44 (+11)
44 (+11)
33
a UV melting profiles were measured in 7 mM sodium phosphate buffer (pH
7.0) containing 140 mM KCl without or with additional 10 mM MgCl2. The
oligonucleotide concentration used was 1.5 mM for each strand. The
sequence of target dsDNA is 5A-GCTAAAAAGAAAGAGAGATCG-3A/3A-
CGATTTTTCTTTCTCTCTAGC-5A. mC means 5-methylcytidine.
Notes and references
† Recently Wang and Stoisavljevic reported a combination of a phosphor-
amidate linkage and another type of bicyclic nucleoside, and their
modification of oligonucleotides decreased the duplex-forming ability. See
ref. 21.
‡ The obtained 3A-amino-2A,4A-BNA oligonucleotides were purified by
reversed-phase HPLC, and the compositions were determined by MALDI-
TOF-MS. MALDI-TOF-MS data: 6 [M 2 H]2 3024.58 (calc. 3024.05); 7
[M 2 H]2 4523.32 (calc. 4523.13); 8 [M 2 H]2 3615.11 (calc.
3614.47).
1 E. Uhlmann and A. Peyman, Chem. Rev., 1990, 90, 543.
2 S. L. Beaucageand R. P. Iyer, Tetrahedron, 1993, 49, 6123.
3 N. T. Thoung and C. Hélène, Angew. Chem., Int. Ed. Engl., 1993, 32,
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4 S. O. Doronina and J.-P. Behr, Chem. Soc. Rev., 1997, 63.
5 I. Luyten and P. Herdewijn, Eur. J. Med. Chem., 1998, 33, 515.
6 W. Saenger, Principles of Nucleic Acid Structure, Springer-Verlag,
New York, 1984, p. 17.
7 S. Obika, D. Nanbu, Y. Hari, K. Morio, Y. In, T. Ishida and T. Imanishi,
Tetrahedron Lett., 1997, 38, 8735.
Fig. 2 Enzymatic stability of T8TaBT (8) (closed circle), T10 (13) (cross),
T8TBT (14) (open circle) and T8TST (15) (triangle). Hydrolysis of the
oligonucleotides (10 mg) was carried out at 37 °C in a buffer (320 ml)
containing 50 mM Tris–HCl (pH 8.0), 10 mM MgCl2 and SVPDE (0.2 mg).
‘S’ means phosporthioate linkage.
8 We defined BNA as a novel class of nucleic acid analogues containing
2A-O,4A-C- or 3A-O,4A-C-methylene bridged structure (e.g. 2A,4A-BNA or
3A,4A-BNA). The 2A,4A-BNA was also called ‘LNA’ by Wengel et al. See:
S. K. Singh, P. Nielsen, A. A. Koshkin and J. Wengel, Chem. Commun.,
1998, 455.
9 S. Obika, D. Nanbu, Y. Hari, J. Andoh, K. Morio, T. Doi and T.
Imanishi, Tetrahedron Lett., 1998, 39, 5401.
10 T. Imanishi and S. Obika, J. Syn. Org. Chem., Jpn., 1999, 57, 969.
11 S. Obika, Y. Hari, K. Morio and T. Imanishi, Tetrahedron Lett., 2000,
41, 221.
12 S. Obika, Y. Hari, T. Sugimoto, M. Sekiguchi and T. Imanishi,
Tetrahedron Lett., 2000, 41, 8923.
13 H. Torigoe, Y. Hari, M. Sekiguchi, S. Obika and T. Imanishi, J. Biol.
Chem., 2001, 276, 2354.
14 S. Obika, T. Uneda, T. Sugimoto, D. Nanbu, T. Minami, T. Doi and T.
Imanishi, Bioorg. Med. Chem., 2001, 9, 1001.
15 S. Obika, Y. Hari, M. Sekiguchi and T. Imanishi, Angew. Chem., 2001,
40, 2079; S. Obika, Y. Hari, M. Sekiguchi and T. Imanishi, Angew.
Chem., Int. Ed., 2001, 113, 2149.
stabilization of the duplexes formed with both complementary
DNA and RNA. The increases in Tms were up to 4 and 7 °C for
the duplexes formed with the DNA and RNA strands,
respectively. Next, the binding affinity of the oligonucleotide 7
to a homopurine•homopyrimidine dsDNA was also studied and
shown to be much higher than that of the natural oligonucleotide
11. The Tm values of triplex formation for 7 were 55 and 44 °C
under neutral conditions with or without 10 mM MgCl2,
respectively, which is over 10 °C higher than the corresponding
value for 11 as summarized in Table 2.
Thus, the oligonucleotides with one-point modification by 3A-
amino-2A,4A-BNA, a combination of the 2A,4A-BNA backbone
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and N3A P5A phosphoramidate linkage, exhibited enhanced
hybridizing properties towards complementary ssDNA, ssRNA
and dsDNA, compared to the corresponding natural oligonu-
cleotides. These DTm values were almost comparable to those
for 2A,4A-BNA singly modified congeners 10 and 12, and seem
to reach the utmost level of hybridization.
16 S. M. Gryaznov, Biochim. Biophys. Acta, 1999, 1489, 131.
17 J.-K. Chen, R. G. Schultz, D. H. Lloyd and S. M. Gryaznov, Nucleic
Acids Res., 1995, 23, 2661.
The nuclease-resistance of the 3A-amino-2A,4A-BNA modified
oligonucleotide 8 was investigated by using snake venom
phosphodiesterase (SVPDE), compared with natural and one
point modified (2A,4A-BNA or phosphorthioate22) oligothymidi-
late 10-mers 13, 14 and 15, respectively. The reaction mixtures
were analyzed at several time points by reversed-phase HPLC
to monitor the percentage of full length oligonucleotides (Fig.
2). Under the conditions used, the natural T 10-mer control 13
and the 2A,4A-BNA 14 were immediately digested. In both cases,
no full length oligomer was detected after 10 min, although the
2A,4A-BNA modification of oligonucleotides was found to be
18 R. G. Schultz and S. M. Gryaznov, Nucleic Acids Res., 1996, 24,
2966.
19 S. M. Gryaznov and H. Winter, Nucleic Acids Res., 1998, 26, 4160.
20 S. Obika, J. Andoh, T. Sugimoto, K. Miyashita and T. Imanishi,
Tetrahedron Lett., 1999, 40, 6465.
21 G. Wang and V. Stoisavljevic, Nucleosides Nucleotides, Nucleic Acids,
2000, 19, 1413.
22 The phosphorthioate oligonucleotide used in this study was an SP-
isomer which is known to be more resistant to degradation by SVPDE
than an RP-isomer. See: P. M. J. Burgers, B. K. Sathyanarayana, W.
Saenger and F. Eckstein, Eur. J. Biochem., 1979, 100, 585.
Chem. Commun., 2001, 1992–1993
1993