Adjustment of the γ dihedral angle of an oligonucleotide P3′→N5′ phosphoramidate enhances its binding affinity towards complementary strands

Article abstract of DOI:10.1002/anie.200461942

(Chemical Equation Presented) Bridge to the right angle: A methylene bridge between the C3′ and N5′ atoms restricts the γ dihedral angle of an oligonucleotide P3′→N5′ phosphoramidate within the +sc orientation (see scheme), which improves duplex-forming a

Full text of DOI:10.1002/anie.200461942

Angewandte
Chemie
Communications
A methylene bridge between the C3’ and N5’ atoms adjusts the g
dihedral angle in 5’-amino-3’,5’-BNA. The bridged structure of this new
type of oligonucleotide P3’!N5’ phosphoramidate greatly enhances
duplex-forming ability with complementary strands and accelerates
cleavage of the phosphoramidate linker under mild acidic conditions.
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DOI: 10.1002/anie.200461942
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Bridged Nucleic Acids
Adjustment of the g Dihedral Angle of an
Oligonucleotide P3’!N5’ Phosphoramidate
Enhances Its Binding Affinity towards
Complementary Strands**
Satoshi Obika, Mitsuaki Sekiguchi, Roongjang Somjing,
and Takeshi Imanishi*
Chemical modification of oligodeoxynucleotides (ODNs) has
been receiving increasing attention in the fields of gene
therapeutics and genetic diagnosis.[1,2] One promising
approach is an internucleoside linkage modification of the
ODNs. An N3’!P5’-phosphoramidate-linked ODN, in which
the 3’-oxygen atom is replaced with a nitrogen atom, forms a
stable duplex structure with its DNA or RNA complement.[3]
On the other hand, P3’!N5’-phosphoramidate-linked ODNs
(5’-amino-DNA, Scheme 1a), with a 5’-nitrogen atom instead
of an oxygen atom, can be hydrolyzed at the phosphorami-
date linkage under mild acidic conditions.[4] This property of
5’-amino-DNA has attracted much attention and has been
applied to a DNA-sequence determination.[5,6] However, the
5’-amino-DNA modification of ODNs decreases the hybrid-
izing ability with its complementary strand.[7,8] This disad-
vantage of 5’-amino-DNA may be caused by an inappropriate
g dihedral angle (N5’–C5’–C4’–C3’). 1H NMR analysis of a 5’-
amino-DNA dimer revealed that the orientation of the C4’–
C5’ bond is predominantly + ap (g ꢀ 1808) or ꢁsc (g ꢀ ꢁ608),
which is different from that in a typical DNA/DNA or RNA/
RNA duplex (+ sc, g ꢀ 608).[9]
Scheme 1. a) Structures of 5’-amino-DNA, 5’-amino-2’,4’-BNA, and 5’-
amino-3’,5’-BNA. b) Structures and g dihedral angle orientation of
bicyclo-DNA, tricyclo-DNA, and the 5’-amino-3’,5’-BNA monomer.
One promising strategy for restricting the conformational
flexibility of the nucleoside sugar moiety is to increase the
binding affinity of the ODNs. We have developed a series of
novel nucleic acid analogues bearing a conformationally
restricted sugar moiety, bridged nucleic acids (BNAs), and
have found that ODNs containing some kinds of BNA
acquired extremely high binding affinity for their DNA or
RNA complements.[10–12] One such nucleic acid analogue, 5’-
amino-2’,4’-BNA (Scheme 1a), in which the sugar puckering
is exactly restricted to the C3’-endo conformation (a typical
N-type conformation), exhibited high binding affinity with
complementary strands, although this nucleic acid analogue
has a P3’!N5’ phosphoramidate linkage.[12] Thus, the 5’-
amino-2’,4’-BNA may be one example of how to overcome
the drawback of 5’-amino-DNA; however, the effect of the g
dihedral angle of 5’-amino-DNA on its hybridizing properties
is still unclear.
In this study, we have focused on the adjustment of the g
dihedral angle of 5’-amino-DNA. As DNA derivatives having
a restricted g dihedral angle, bicyclo-DNA[13] and tricyclo-
DNA,[14] developed by Leumann et al., are well known. These
DNA analogues showed interesting duplex- and triplex-
forming properties, and the tricyclo-DNA was found to be
useful even as an antisense oligonucleotide.[15] However, the g
dihedral angles of bicyclo-DNA and tricyclo-DNA were
observed to be 1498 and 1188, respectively (Scheme 1b).
These g angles are beyond the range of those for typical
DNA/DNA and RNA/RNA duplexes. To adjust the g angle of
5’-amino-DNA to an appropriate value for stable duplex
formation, we have designed a novel bridged nucleic acid, 5’-
amino-3’,5’-BNA, which has a methylene linkage between the
3’-carbon and 5’-nitrogen atoms (Scheme 1a). The orientation
of the C4’–C5’ bond of the 5’-amino-3’,5’-BNA was fully
expected to be + sc by comparison with the structure of
bicyclo-DNA (Scheme 1b). Herein we describe the synthesis
[*] Dr. S. Obika, M. Sekiguchi, R. Somjing, Prof. Dr. T. Imanishi
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
[**] This research was partially supported by a Grant-in-Aid from the
Japan Society for the Promotion of Science, a Grant-in-Aid from the
Ministry of Education, Culture, Sports, Science, and Technology of
Japan, and a SUNBOR Grant from the Suntory Institute for
Bioorganic Research. We gratefully acknowledge a JSPS Research
Fellowship for Young Scientists (M.S.).
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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and conformation of the 5’-amino-3’,5’-BNA monomer and
some interesting properties of its ODN derivatives.
For the synthesis of the 5’-amino-3’,5’-BNA, we chose 3’-
deoxy-3’-C-methylene-5’-O-triphenylmethylthymidine (2)[16]
as the starting material (Scheme 2). Stereoselective oxidation
Figure 1. X-ray crystal structure of 5’-amino-3’,5’-BNA
monomer 1.
The phosphoramidite 9 was incorporated into 12-
mer DNA strands 11–13 by using an automated DNA
synthesizer.[18] To improve coupling and oxidizing
efficiency, 4,5-dicyanoimidazole[19] and tBuOOH[20,21]
were used instead of 1H-tetrazole and I2/pyridine/
H2O, respectively. The coupling efficiency for 9,
estimated from monitoring the free trityl groups, was
approximately 90%.
Duplex-forming ability of the ODNs with com-
plementary DNA and RNA was evaluated by means
of UV melting experiments. The differences in melt-
ing temperatures (DTm values) between the duplexes
containing 5’-amino-3’,5’-BNA or 5’-amino-DNA[22]
and the natural DNA/DNA or DNA/RNA duplexes
are summarized in Figure 2.[23] As previously
reported,[7,8] the duplexes comprising the 5’-amino-
DNA, 14–16, showed a decrease in Tm value compared
with the corresponding DNA/DNA and DNA/RNA
duplexes. ODN 16, in particular, containing six con-
Scheme 2. Synthesis of 5’-amino-3’,5’-BNA-thymine monomer 1 and its phosphoramidite
derivative 9. a) OsO4 (cat.), NMO, pyridine, H2O, tBuOH, 768C, 69%; b) TsCl, nBu2SnO,
Et3N, CH2Cl2, RT, 72%; c) K2CO3, MeOH, RT; d) NaN3, DMF, 908C, 98% from 4; e) CSA,
CH2Cl2/MeOH, RT; f) TsCl, pyridine, 508C, 53% from 5; g) 10% Pd/C (wet), H2, MeOH,
RT; h) MMTrCl, pyridine, RT, 69% from 6; i) 2-cyanoethyl N,N-diisopropylchlorophosphor-
amidite, iPr2NEt, CH2Cl2, RT, 86%; j) DNA synthesizer (ABI Expedite 8909); k) Et3N,
MeOH, RT, then purified by reversed-phase HPLC. CSA=(+)-camphorsulfonic acid,
DMF=N,N-dimethylformamide, MMTr=monomethoxytrityl, NMO=4-methylmorpholine
N-oxide, Thy=thymin-1-yl, Tr=triphenylmethyl=trityl, Ts=toluene-4-sulfonyl=tosyl.
of 2 by using osmium tetroxide gave diol 3. A p-toluenesul-
fonyl group was introduced at the primary hydroxy group of 3
to afford 4. Epoxidation with potassium carbonate and
subsequent treatment with sodium azide gave 5. After
removing the 5’-O-triphenylmethyl (trityl) group, the
obtained diol was treated with p-toluenesulfonyl chloride to
give 6. The azide group of 6 was reduced with palladium on
carbon under a hydrogen atmosphere and subsequent pyrro-
lidine ring formation afforded the desired 5’-amino-3’,5’-BNA
monomer 7 as a salt of p-toluenesulfonic acid. Without any
purification at this stage, 7 was treated with monomethoxy-
trityl chloride to give 8, and the phosphoramidite building
block 9 was obtained by phosphitilation of 8. Alternatively,
the salt 7 was treated with triethylamine and was then purified
by reversed-phase HPLC to afford the 5’-amino-3’,5’-BNA
monomer 1 in a free form. X-ray crystallograpic analysis of 1
(Figure 1) showed that the g dihedral angle of 1 is 28.38 (+ sc
orientation).[17] This value is quite different from that found in
the 5’-amino-DNA dimer[9] and would be appropriate for
stable duplex formation. It was also observed that the
furanose ring of 1 has the C1’-exo-O4’-endo conformation
(pseudorotational phase angle P = 115.48), which is neither an
N-type nor an S-type conformation, but an in-between
conformation.
Figure 2. Differences in melting temperatures (DTm values) between
the modified oligonucleotides 11–16 and the reference oligonucleotide
10, 5’-d(GCGTTTTTTGCT)-3’. The Tm values of duplexes of 10 with
complementary DNA and RNA were 508C and 458C, respectively. The
Tm values obtained from the maxima of the first derivatives of the melt-
ing curves (absorbance at 260 nm versus temperature) were recorded
in a medium salt buffer (10 mm sodium phosphate, 100 mm NaCl,
pH 7.2) with 4 mm complementary strands. The sequence of target
DNA and RNA complement is 5’-AGCAAAAAACGC-3’.
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secutive 5’-amino-DNA monomers, displayed a drastically
decreased binding affinity with its DNA and RNA comple-
ments. On the other hand, the 5’-amino-3’,5’-BNA ODNs 11–
13 achieved stable duplex formation with complementary
strands. An increase in the Tm values by 1–38C and 4–108C
was observed when the ODNs 11–13 formed duplexes with
complementary DNA and RNA, respectively. It is note-
worthy that the difference between the Tm values of 13/RNA
and 16/RNA hybrids was over 308C. Thus, the 5’-amino-3’,5’-
BNAs have strong duplex-forming ability. This result indi-
cates that the methylene bridge between the C3’ and N5’
atoms successfully restricts the conformation around the g
dihedral angle in an appropriate form for duplex formation.
Next, we investigated the effect of the methylene bridge
on acid-mediated hydrolysis of the P3’!N5’-phosphorami-
date linkage. The 5’-amino-3’,5’-BNA ODN 11 was treated
with buffer (pH 3.0 or pH 7.0) to be hydrolyzed, and the
amount of intact 11 was determined by reversed-phase HPLC
analysis (Figure 3). Under pH 3.0 conditions, 50% of 11 was
Keywords: conformation analysis · nucleic acids · nucleosides ·
oligonucleotides · phosphoramidates
.
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m.ac.uk/data_request/cif.
Figure 3. Hydrolytic cleavage of the P3’!N5’-phosphoramidate bond
~
&
in modified ODNs: : 5’-amino-3’,5’-BNA ODN 11 at pH 3.0; : 5’-
amino-3’,5’-BNA ODN 11 at pH 7.0; &: 5’-amino-DNA ODN 14 at
pH 3.0. The reaction was carried out at 308C with 1 nmol of ODN in
buffer (100 mL). The sequence of oligonucleotides 11 and 14 is shown
in Figure 2.
[18] The composition of the oligonucleotides 11–13 was confirmed by
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[MꢁH]ꢁ was detected in all cases, except for oligonucleotide 12.
See the Supporting Information.
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hydrolyzed at the P3’!N5’-phosphoramidate linkage within
30 min and 95% was cleaved at 120 min, while no hydrolysis
was observed at pH 7.0. The 5’-amino-DNA 16 was also
cleaved at pH 3.0; however, hydrolysis is much slower than
for 5’-amino-3’,5’-BNA. Thus, the additional methylene
bridge between N5’ and C3’ atoms accelerates the hydrolysis
of phosphoramidate linkage probably due to its electron-
donating property.
We have synthesized a novel 5’-amino-DNA analogue, 5’-
amino-3’,5’-BNA, with a g dihedral angle that is well adjusted
by the methylene bridge between the C3’ and N5’ atoms. We
have also found that the methylene bridge effectively elicits
not only a strong hybridizing ability but also the rapid
hydrolysis of the P3’!N5’-phosphoramidate linkage of 5’-
amino-3’,5’-BNA. This feature of 5’-amino-3’,5’-BNA would
be applicable to a variety of genome technologies, such as a
novel sequence determination or single-nucleotide-poly-
morphism analysis.
[22] Synthesis of 5’-amino-DNA oligonucleotides 14–16 was per-
formed according to ref. [4].
[23] The Tm profiles of duplexes containing the oligonucleotides 11–
13 can be found in the Supporting Information.
Received: September 10, 2004
Published online: January 28, 2005
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