Synthesis and properties of a novel bridged nucleic acid analogue, 5′-amino-3′,5′-BNA

Article abstract of DOI:10.1081/NCN-200061836

An oligonucleotide P3′→ N5′ phosphoramidate (5′-amino-DNA) attracts much attention because of its potential for application to DNA sequencing; however, its ability to hybridize with complementary strands is low. To overcome this drawback of the 5′-amino-D

Full text of DOI:10.1081/NCN-200061836

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SYNTHESIS AND PROPERTIES OF A NOVEL BRIDGED
NUCLEIC ACID ANALOGUE, 5′-AMINO-3′,5′-BNA
Mitsuaki Sekiguchi a , Satoshi Obika a , Roongjang Somjing a & Takeshi Imanishi a
a Graduate School of Pharmaceutical Sciences, Osaka University , Osaka, Japan
Published online: 15 Nov 2011.
To cite this article: Mitsuaki Sekiguchi , Satoshi Obika , Roongjang Somjing & Takeshi Imanishi (2005) SYNTHESIS AND
PROPERTIES OF A NOVEL BRIDGED NUCLEIC ACID ANALOGUE, 5′-AMINO-3′,5′-BNA, Nucleosides, Nucleotides and Nucleic Acids,
24:5-7, 1097-1100, DOI: 10.1081/NCN-200061836
To link to this article: http://dx.doi.org/10.1081/NCN-200061836
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Nucleosides, Nucleotides, and Nucleic Acids, 24 (5–7):1097–1100, (2005)
Copyright D Taylor & Francis, Inc.
ISSN: 1525-7770 print/ 1532-2335 online
DOI: 10.1081/NCN-200061836
SYNTHESIS AND PROPERTIES OF A NOVEL BRIDGED NUCLEIC ACID
ANALOGUE, 5’-AMINO-3’,5’-BNA
Mitsuaki Sekiguchi, Satoshi Obika, Roongjang Somjing, and Takeshi Imanishi
5
Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan
5
An oligonucleotide P3’!N5’ phosphoramidate (5’-amino-DNA) attracts much attention because of
its potential for application to DNA sequencing; however, its ability to hybridize with complementary
strands is low. To overcome this drawback of the 5’-amino-DNA, we have designed and successfully
synthesized a novel nucleic acid analogue having a P3’!N5’ phosphoramidate linkage and a
constrained sugar moiety, 5’-amino-3’-C,5’-N-methylene bridged nucleic acid (5’-amino-3’,5’-BNA).
The binding affinity of the 5’-amino-3’,5’-BNA towards complementary DNA and RNA strands was
investigated by UV melting experiments. The melting temperature (Tm) of the duplex comprising the 5’-
amino-3’,5’-BNA and its complementary strand was much higher than that of the duplex containing the
corresponding 5’-amino-DNA.
INTRODUCTION
A nucleic acid analogue having a P3’!N5’ phosphoramidate linkage (5’-
amino-DNA) is of great interest in genome science, because the P3’!N5’
phosphoramidate linkage is readily hydrolyzed under mild acidic conditions. In
fact, several applications of the 5’-amino-DNA for a DNA sequence-determining
technology have been reported to date.[1,2] However, the ability of the 5’-amino-
DNA to hybridize with the complementary strand is decreased, probably due to an
inappropriate g dihedral angle (N5’-C5’-C4’-C3’). 1H NMR analysis of the 5’-amino-
DNA dimer revealed that the +ap and ꢀsc orientations for g dihedral angle are
favorable, which is quite different from the +sc orientation for g in a typical DNA/
DNA or RNA/RNA duplex.[3] We supposed that restriction of the g dihedral angle
into a suitable +sc orientation promotes stable duplex formation of the 5’-amino-
DNA. We therefore designed and synthesized a novel 5’-amino-DNA analogue,
5’-amino-3’-C,5’-N-methylene bridged nucleic acid (5’-amino-3’,5’-BNA) depicted in
Figure 1.
We thank JSPS Research Fellowships for Young Scientists (M.S.).
Address correspondence to 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
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M. Sekiguchi et al.
FIGURE 1 Structure of the oligonucleotide P3’!N5’ phosphoramidate analogues used in this work.
The synthetic route to the 5’-amino-3’,5’-BNA thymidine monomer (5’-amino-
BNA-T) and its amidite derivative is outlined in Scheme 1. Starting from thymidine
1, 3’-C-hydroxymethylthymidine derivative 2 was obtained in 26% overall yield
according to the literature.[4] Introduction of the azido group to the epoxide, and
the following conversion of the 5’-O-trityl group to a p-toluenesulfonyl group,
afforded 3 in 38% yield. Next, the desired compound, 5’-amino-BNA-T 4 was
1
successfully synthesized by Pd-mediated hydrogenation. Both H NMR and X-ray
crystallographic analysis revealed that the g dihedral angle of 4 is in the appropriate
+sc orientation. Monomethoxytritylation of the secondary amine followed by
phosphitilation gave phosphoramidite building block 5. The obtained 5 was in-
corporated into 12-mer ODN 6 [5’-d(GCGTTTTTTGCT)-3’, T = 5’-amino-BNA-
T] using an automated DNA synthesizer. The corresponding 5’-amino-DNA
modified ODN 7 [5’-d(GCGttttttGCT)-3’, t = 5’-amino-DNA-T] was also pre-
pared according to the literature.[5]
To evaluate the hybridization property of the 5’-amino-3’,5’-BNA, we have
carried out UV melting experiments by using the 5’-amino-3’,5’-BNA ODN 6,
the 5’-amino-DNA ODN 7, and the corresponding natural DNA ODN
8 [5’-d(GCGTTTTTTGCT)-3’]. The melting temperature (Tm) of the duplex
SCHEME 1 (a) pTsCl, nBu2SnO, Et3N, CH2Cl2, rt; (b) K2CO3, MeOH, rt; (c) NaN3, DMF, 90°C; (d) CSA,
CH2Cl2 –MeOH, rt; (e) pTsCl, pyridine, 50°C, 37% for 5 steps; (f) wet.10%Pd/C, MeOH, H2, rt, 52%; (g) MMTCl,
pyridine, rt, 56%; (h) 2-cyanoethyl N,N-diisopropylchlorophosphoramidite, iPr2NEt, CH2Cl2, rt, 86%; (i) DNA
synthesizer; Thy; thymin-1-yl.

M. Sekiguchi et al.
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FIGURE 2 Tm profiles of the duplexes comprising ODNs 6--8 and DNA complement (A) or RNA
complement (B). UV melting experiments were carried out in 10 mM NaH2PO4 (pH7.0) containing 100 mM NaCl
at a scan rate of 0.5°C/min at 260 nm. The concentration of each strand was 4.0 mM. Target sequence: 5’-
AGCAAAAAACGC-3’.
ODN 6/DNA was similar to that of the ODN 8/DNA and much higher than that of
the ODN 7/DNA (Figure 2A). Furthermore, it is noteworthy that the 5’-amino-3’,5’-
BNA ODN 6 formed a significantly stable duplex with its RNA complement
(Figure 2B). The Tm value of the ODN 6/RNA was higher than those of the ODN
7/RNA and ODN 8/RNA. These results clearly indicate that an adjustment of the g
dihedral angle to the +sc orientation by a methylene bridge between the N5’ and
C3’ atoms enhances the duplex-forming ability of the 5’-amino-DNA.
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