Synthesis and properties of a novel bridged nucleic acid with a P3′ → N5′ phosphoramidate linkage, 5′-amino-2′,4′-BNA

Article abstract of DOI:10.1039/b307290h

5′-Amino-2′,4′-BNA, a novel analogue of BNA series compounds, was successfully synthesized, and its incorporated oligonucleotides showed potent duplex- and triplex-forming ability and resistance against snake venom phosphodiesterase.

Full text of DOI:10.1039/b307290h

Synthesis and properties of a novel bridged nucleic acid with a P3A ?
N5A phosphoramidate linkage, 5A-amino-2A,4A-BNA†
Satoshi Obika, Osamu Nakagawa, Akiko Hiroto, Yoshiyuki Hari and Takeshi Imanishi*
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
Received (in Cambridge, UK) 25th June 2003, Accepted 11th July 2003
First published as an Advance Article on the web 25th July 2003
5A-Amino-2A,4A-BNA, a novel analogue of BNA series com-
pounds, was successfully synthesized, and its incorporated
oligonucleotides showed potent duplex- and triplex-forming
ability and resistance against snake venom phosphodies-
terase.
further purification, 6 was tritylated into 7, which was
phosphitylated to give amidite 8. By using 8 and natural amidite
building blocks, 5A-amino-2A,4A-BNA modified oligonucleo-
tides 9–15 were successfully prepared according to a standard
phosphoramidite protocol.‡
The duplex-forming ability of 5A-amino-2A,4A-BNA modified
oligonucleotides 9–11 with complementary ssDNA and ssRNA
was evaluated under near-physiological conditions by means of
melting temperature (Tm) experiments ( Table 1). Replacement
of natural nucleotides by 5A-amino-2A,4A-BNA resulted in
significant stabilization of the duplexes formed with both
complementary DNA and RNA, and increases in Tm per
modification by +2 to +5 °C towards DNA and +4 to +5 °C
towards RNA were observed. This increase in Tm is roughly
comparable to that of the corresponding 2A,4A-BNA modified
oligonucleotides 16–18. Note that oligonucleotides containing a
2A,4A-BNA unit and a P3A ? N5A phosphoramidate linkage
showed great affinity for complementary RNA and DNA, in
contrast with the decreasing stability of the duplex in the case of
P3A ? N5A phosphoramidate linked oligonucleotides having no
2A,4A-BNA framework.12
In the last decade the synthesis and evaluation of various nucleic
acid analogues have been developed for their practical antisense
and antigene applications.1–5 One promising chemical deriva-
tive of natural oligonucleotides is involved in the internucleo-
side linkage modifications. N3A ? P5A phosphoramidate linked
oligonucleotides were known to show superior duplex- and
triplex-forming affinity because of the dominant N-type sugar
puckering which decreases the gauche effect between 3A-
nitrogen and 4A-oxygen by replacing from 3A-oxygen to
nitrogen, as well as excellent nuclease resistant ability.6–9 On
the other hand, more easily preparable P3A ? N5A phosphor-
amidate can be cleaved at its phosphoramidate linkage under
mild acidic conditions,10 and this was applied to a novel
technology of DNA sequence-determination.11 However, oligo-
nucleotide P3A ? N5A phosphoramidates were reported to have
only weak binding affinity for complementary nucleic acid
strands, probably due to the difference in preferential sugar
conformation (Fig. 1).12–16
Next, the binding affinity of 5A-amino-2A,4A-BNA modified
oligonucleotides for homopurine–homopyrimidine dsDNA was
studied under neutral conditions with or without MgCl2.
Although the Tm values of 5A-amino-2A,4A-BNA modified
oligonucleotides 12–14 were slightly lower than those of 2A,4A-
BNA modified oligonucleotides 19–21 (Table 2), the DTm
Recently, we achieved the synthesis of a novel nucleoside
with a fixed N-type conformation, 2A-O,4A-C-methylene bridged
nucleic acid (2A,4A-BNA)17 and found that 2A,4A-BNA modified
oligonucleotides exhibited strong hybridizing ability with
complementary strands of RNA and DNA.18–20 Moreover,
these 2A,4A-BNA oligonucleotides appeared to possess high
affinity for dsDNA.19–24
Therefore, we were interested in a combination of 2A,4A-BNA
and P3A ? N5A phosphoramidate linkage to develop P3A ? N5A
phosphoramidate linked oligonucleotides having N-type sugar
puckering. We report here the synthesis, hybridizing properties
and enzymatic stability of novel oligonucleotide analogues with
a 5A-amino-2A,4A-BNA monomer unit (Fig. 1).
The synthesis of the 5A-amino-2A,4A-BNA modified oligonu-
cleotides is shown in Scheme 1. Starting material 125 was
coupled with silylated thymine to give 2. On exposure of 2 to
K2CO3, bicyclic nucleoside 3 was obtained. Desilylation of 3
followed by mesylation provided 4, which underwent catalytic
reduction to afford 5. Reaction of 5 with NaN3, and reduction of
an azide group gave the desired 5A-amino-2A,4A-BNA 6; without
Scheme 1 Reagents and conditions: i, 2TMS-T, TMSOTf, ClCH2CH2Cl,
reflux, 97%; ii, K2CO3, MeOH, r.t., 94%; iii, TBAF, THF, r.t., 92%; iv,
MsCl, pyridine, r.t., 99%; v, Pd(OH)2/C, cyclohexene, EtOH, reflux, 98%;
vi, NaN3, DMF, 100 °C, quant.; vii, PPh3, pyridine, r.t., then NH4OH aq.,
Fig. 1 Structures of the modified oligonucleotides.
† Electronic supplementary information (ESI) available: acid-mediated
cleavage of modified or natural oligonucleotides. See http://www.rsc.org/
suppdata/cc/b3/b307290h/
r.t.; viii, MMTrCl, pyridine, r.t., 87% over 2 steps; ix, (i-
Pr2N)2POCH2CH2CN, diisopropylammonium tetrazolide, MeCN–THF,
r.t., 90%; x, DNA synthesizer (AB Expedite™ 8909).
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CHEM. COMMUN., 2003, 2202–2203
This journal is © The Royal Society of Chemistry 2003

Table 1 Tm values of 5A-amino-2A,4A-BNA modified oligonucleotides with
5A-Amino-2A,4A-BNA modified oligonucleotide 15 was read-
ily cleaved under mild acidic condtions, while the correspond-
ing natural (22) and 2A,4A-BNA modified (23) oligonucleotides
were quite stable under the same conditions (ESI†).
complementary DNA and RNAa
Tm (DTm/modification)/°C
In conclusion, the synthesis of 5A-amino-2A,4A-BNA monomer
and its modified oligonucleotides was successfully accom-
plished. The modified oligonucleotides showed great properties
of hybridization to ssDNA, ssRNA and dsDNA, along with
resistance to enzymatic degradation. We also confirmed that 5A-
amino-2A,4A-BNA modified oligonucleotide can be cleaved
under mild acidic condtions. These properties of 5A-amino-2A,4A-
BNA modified oligonucleotides would be useful not only for
antisense/antigene applications but also for many novel technol-
ogies in the post-genome-sequencing era.
Part of this work was supported by a Grant for Research on
Health Sciences focusing on Drug Innovation from The Japan
Health Sciences Foundation, Industrial Technology Research
Grant Program in ’00 from the New Energy and Industrial
Technology Development Organization (NEDO) of Japan, a
Grant-in-Aid from the Japan Society for the Promotion of
Science, and a Grant-in-Aid from the Ministry of Education,
Science, and Culture, Japan.
Oligonucleotide
DNA
RNA
5A-GCGTTTTTTGCT-3A
47
45
5A-GCGTTTTTTGCT-3A(9)
5A-GCGTTTTTTGCT-3A(10)
5A-GCGTTTTTTGCT-3A(11)
5A-GCGTTtTTTGCT-3A(16)
5A-GCGTTtTtTGCT-3A(17)
5A-GCGtTtTtTGCT-3A(18)
52 (+5)
52 (+3)
53 (+2)
53 (+6)
54 (+4)
56 (+3)
50 (+5)
54 (+5)
58 (+4)
52 (+7)
57 (+6)
62 (+6)
a UV melting profiles 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
concentration of oligonucleotide used was 4 mM for each strand. The
sequence of target DNA or RNA complements is 5A-AGCAAAAAACGC-
3A. T: 5A-amino-2A,4A-BNA with thymine. t: 2A,4A-BNA with thymine.
values per modification of 12–14 were over 7 °C higher than
those of the natural oligonucleotide.
Table 2 Tm values of 5A-amino-2A,4A-BNA modified oligonucleotides with
dsDNAa
Tm (DTm/modification)/°C
Notes and references
‡ The coupling yields for attachment of amidite 8, determined by a trityl
monitor, were > 94%. The synthesized oligonucleotides were purified by
reversed-phase HPLC, and the compositions were determined by MALDI-
TOF-MS: 9 [M 2 H]2 3659.50 (calc. 3659.45); 10 [M 2 H]2 3686.56
(calc. 3686.47); 11 [M 2 H]2 3713.70 (calc. 3713.50); 12 [M 2 H]2
4523.63 (calc. 4523.09); 13 [M 2 H]2 4550.33 (calc. 4550.11); 14 [M 2
H]2 4577.95 (calc. 4577.14); 15 [M 2 H]2 3006.37 (calc. 3006.08).
Oligonucleotide
2MgCl2
+10 mM MgCl2
5A-TTTTTmCTTTmCTmCTmCT-3A
32
42
5A-TTTTTmCTTTmCTmCTmCT-3A (12)
5A-TTTTTmCTTTmCTmCTmCT-3A (13)
5A-TTTTTmCTTTmCTmCTmCT-3A (14)
5A-TTTTTmCTtTmCTmCTmCT-3A (19)
5A-TTTTTmCTtTmCtmCTmCT-3A (20)
5A-TTTTtmCTtTmCtmCTmCT-3A (21)
40 (+8)
45 (+7)
52 (+7)
42 (+10)
48 (+8)
56 (+8)
50 (+8)
57 (+8)
64 (+7)
52 (+10)
60 (+9)
70 (+9)
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Fig. 2 Enzymatic stability of modified oligonucleotides (5A-TTTTTTTTTX-
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