Nucleosides and nucleotides. Part 226: Alternate-strand triple-helix formation by 3′-3′-linked oligodeoxynucleotides composed of asymmetrical sequences

Article abstract of DOI:10.1016/j.bmcl.2004.03.062

In this paper, we describe the synthesis of the 3′-3′-linked oligonucleotides connected with pentaerythritol composed of asymmetrical sequences. Stability of the triplexes between these oligonucleotides and the DNA targets involving the adjacent oligopuri

Full text of DOI:10.1016/j.bmcl.2004.03.062

Bioorganic & Medicinal Chemistry Letters 14 (2004) 3333–3336
Nucleosides and nucleotides. Part 226: Alternate-strand triple-helix
formation by 30-30-linked oligodeoxynucleotides composed of
asymmetrical sequencesq
Shuichi Hoshika, Yoshihito Ueno,ꢀ Hiroyuki Kamiya and Akira Matsuda*
Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan
Received 20 February 2004;accepted 18 March 2004
Abstract—In this paper, we describe the synthesis of the 30-30-linked oligonucleotides connected with pentaerythritol composed of
asymmetrical sequences. Stability of the triplexes between these oligonucleotides and the DNA targets involving the adjacent oli-
gopurine domains on alternate strands was investigated using the electrophoretic mobility shift assay (EMSA) and DNase I
footprinting experiment. It was found that the 30-30-linked oligonucleotides composed of asymmetrical sequences formed the stable
antiparallel triplexes with the DNA targets as compared with the unlinked oligonucleotides. Thus, oligonucleotides linked with
pentaerythritol would be useful as antigene oligonucleotides for DNA targets consisting of the alternating oligopyrimidine–oli-
gopurine sequences.
Ó 2004 Elsevier Ltd. All rights reserved.
Synthetic oligonucleotide-directed triple-helix (triplex)
formation represents a promising way to interfere with
gene expression and has become an area of intense
investigation.1 Triplex-forming oligonucleotides (TFOs)
bind in the major groove of duplex DNA containing
oligopurine/oligopyrimidine stretches and form hydro-
gen bonds with the target oligopurine sequence.
Pyrimidine-rich TFOs bind parallel to the purine strand
of the duplex and form TÆAT and CþÆGC base triplets by
Hoogsteen hydrogen bonds,2;3 while purine-rich TFOs
bind antiparallel to the purine strand of the duplex and
form AÆAT (or TÆAT) and GÆGC base triplets by reverse
Hoogsteen hydrogen bonds.4;5 However, target se-
quences in the triplex strategy are quite restricted. Since
the thermal stability of the triplexes is generally lower
than that of the duplexes under physiological condi-
tions, an oligopurine cluster with long chain lengths is
required for stable triplex formation.
purine sequences appear adjacently and alternately on
the two strands of the DNA target, the alternate se-
quences can be recognized by TFOs that simultaneously
and cooperatively bind to the adjacent oligopurine do-
mains by cross-over in the major groove (Fig. 1). Re-
cently, we reported the synthesis of the 30-30-linked
TFOs connected with pentaerythritol, that were com-
posed of symmetrical sequences.25;26 We found that the
30-30-linked TFOs formed the thermally stable parallel
(Fig. 1a) and antiparallel triplexes (Fig. 1b) with the
DNA targets. Additionally, we showed that the 30-30-
linked TFOs inhibited the cleavages of the DNA targets
by restriction enzymes more effectively than the unlinked
decamers. These results prompted us to examine the
synthesis and properties of the 30-30-linked TFOs con-
nected with pentaerythritol, that were composed of
asymmetrical sequences (Fig. 1c).
In this paper, we report the synthesis of the 30-30-linked
TFOs consisting of asymmetrical sequences that can
form the antiparallel triplexes with a DNA target (Fig.
1c). The stabilities of the alternate-strand triplexes be-
tween these TFOs and the DNA targets were investi-
gated with the electrophoretic mobility shift assay
(EMSA) and DNase I footprinting experiment.
Several approaches have been attempted to expand the
repertory of potential DNA targets.6–24 If short oligo-
Keywords: Alternate-strand triple-helix.
q For Part 225 in this series, see Ref. 29.
* Corresponding author. Tel.: +81-11-706-3228;fax: +81-11-706-4980;
e-mail: matuda@pharm.hokudai.ac.jp
ꢀ
Synthesis. The controlled pore glass (CPG) with penta-
erythritol was synthesized as shown in Scheme 1.
Present address: Faculty of Engineering, Gifu University, Yanagido,
Gifu 501-1193, Japan.
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.03.062

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S. Hoshika et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3333–3336
Figure 1. Schematic presentation of ‘alternate-strand triplex’ formation by the 30-30-linked TFOs.
RO
HO
a
OTBDPS
OTBDPS
DMTrO
LevO
OTBDPS
OTBDPS
DMTrO
LevO
OH
OH
b
c
4
3
1: R = H
2: R = DMTr
d
DMTrO
LevO
OBz
O
DMTrO
LevO
OBz
DMTrO
LevO
OBz
OH
f
e
O
O
O
O
N CPG
H
OH
O
5
7
6
CPG = controlled pore glass
Scheme 1. Reagents and conditions: (a) (1) Bu2SnO, benzene, 80 °C;(2) DMTrCl, benzene, rt;(b) levulinic acid, DMAP, 1-ethyl-3-(3-dimethyl-
aminopropyl)carbodiimide hydrochloride (EDCÆHCl), CH2Cl2, rt;(c) TBAF, AcOH, THF, rt, 46% from 1;(d) (1) Bu 2SnO, benzene, 80 °C;(2) BzCl,
benzene, rt, 84%;(e) succinic anhydride, Et 3N, DMAP, CH3CN, rt, 63%;(f) aminopropyl controlled pore glass, EDC ÆHCl, DMF, rt.
2,2-Bis[[(tert-butyldiphenylsilyl)oxy]methyl]-1,3-propane-
diol (1)27 was treated with Bu2SnO in benzene and then
reacted with 4,40-dimethoxytrityl chloride (DMTrCl) to
produce the O-DMTr derivative 2. After protection of
the other hydroxyl group of 2 with a levulinyl (Lev)
group, the tert-butyldiphenylsilyl (TBDPS) groups were
removed by treatment with tetrabutylammonium fluo-
ride (TBAF) to give 4 in 46% yield from 1. One of the
two hydroxyl groups of 4 was protected with a benzoyl
(Bz) group to afford the O-mono(Bz) derivative 5 in 84%
yield. Compound 5 was succinated to give 6, which was
further reacted with CPG to afford the solid support 7
bearing 6 (32 lmol/g).
protected TFOs 10–13, in 40, 24, 29, and 36 OD260 units,
respectively. These TFOs were analyzed by matrix-as-
sisted laser desorption/ionization time-of-flight mass
spectrometry (MALDI-TOF/MS), and the observed
molecular weights supported their structures.28
Studies of triplex formation by the electrophoretic
mobility shift assay. The stabilities of the triplexes were
investigated using the electrophoretic mobility shift as-
say (EMSA). The EMSA detects the difference in elec-
trophoretic mobility of the double-stranded DNA and
the triplex formed.
The 32P-labeled DNA 14 was incubated in the presence
of increasing concentrations of a 1:1 mixture of the un-
linked decamers 8 and 9 or the 30-30-linked TFO 10 in a
buffer of 20 mM Tris–HCl (pH 7.5) containing 10 mM
MgCl2. The solutions were analyzed by non-denaturing
20% polyacrylamide gel electrophoresis (PAGE) at 4 °C.
As shown in Figure 3, the less mobile bands corre-
sponding to the triplexes were observed for the TFO 10,
whereas the band corresponding to the triplex was not
detected up to 100 lM for the mixture of the TFOs 8
and 9. Table 1 summarizes the dissociation constants
(Kds) of the TFOs bound to the duplex DNA target. The
Kd value of the TFO 10 to the DNA 14 was 160 10 nM,
whereas that of the unlinked decamers 8 and 9 was more
The 30-30-linked TFOs 10–13 (Fig. 2a), which consist of
20-deoxyguanosine (dG) and thymidine (T), dG and 20-
deoxyadenosine (dA), or dG, T and dA were synthesized
on a DNA synthesizer using the CPG 7 by the phos-
phoramidite method. One strand was elongated in the
usual manner. After capping the 50-end of the strand and
removing the Lev group by treatment with 0.5 M
hydrazine in pyridine–CH3CO2H (4:1, v/v), the other
strand was elongated. The fully protected TFOs (each
1 lmol) linked to the solid support were treated with
concentrated NH4OH at 55 °C for 16 h. The released
TFOs were purified by denaturing with 20% polyacryl-
amide gel electrophoresis (20% PAGE) to give the de-

S. Hoshika et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3333–3336
3335
(a)
TFO 8
TFO 9
5'-TGGGTTGGGT-3'
5'-GGGTTGGGTT-3'
TFO 10 5'-TGGGTTGGGT
TTGGGTTGGG-5'
OH
HO
TFO 11
TFO 12
TFO 13
5'-TGGGTTGGGT
HO
TAGGGAAGGG-5'
OH
5'-TGGGTTGGGT
HO
AAGGGAAGGG-5'
OH
5'-TGGGTTGGGT
HO
GGGTTGGGTT-5'
OH
(b) DNA 14 5'-CTTGGTCCCTTCCCTAAGGGAAGGGTTCGG-3'
3'-GAACCAGGGAAGGGATTCCCTTCCCAAGCC-5'
DNA 15 5'-CTTGGTCCCTTCCCTGGGAAGGGAATTCGG-3'
3'-GAACCAGGGAAGGGACCCTTCCCTTAAGCC-5'
5'-CTTGGCTTGGAAGCTTCCCTTCCCTAAGGGAAGGGAATTCTTCGGTTCGG-3'
3'-GAACCGAACCTTCGAAGGGAAGGGATTCCCTTCCCTTAAGAAGCCAAGCC-5'
DNA 16
Hind III
EcoR I
Figure 2. Sequences of TFOs and the target duplexes. The binding sites of TFOs are written in boldface.
Figure 3. Quantitative EMSA to detect binding of TFOs 8 and 9, TFO 10, and TFO 11 to the DNA target 14. DNA concentration: 10 nM. TFO
concentrations are indicated.
Table 1. Summary of dissociation constants (Kd) of the TFOs bound
to the DNA targets
linked TFO 11 comprised of T, dG, and dA to bind to
the DNA 14 was also examined. The Kd value of the
TFO 10 to the DNA 14 was 160 10 nM, whereas that
of the TFO 11 was 260 20 nM. Thus, it was found that
the TFO consisting of T and dG stabilized the alternate-
strand triplex slightly more efficiently than that involv-
ing dA.
TFO
Duplex
Kd (nM)
TFO 8 + 9
TFO 10
TFO 11
TFO 12
TFO 13
DNA 14
DNA 14
DNA 14
DNA 14
DNA 15
>100,000
160 ( 10)
260 ( 20)
1100 ( 100)
>100,000
It has been reported that the sequences of the junction
regions of the alternate-strand triplexes critically influ-
ence the stabilities of the triplexes.13 Thus, the ability of
the TFO 12 with dA and the TFO 13 with dG at the
junction regions to bind to the DNAs 14 and 15 was also
examined. The Kd values of the TFOs 12 and 13 to the
DNAs 14 and 15 were 1100 100 nM and more than
100 lM, respectively. Thus, it turned out that the TFO
with T at the junction region stabilized the alternate-
strand triplex more efficiently than those with dA or dG.
than 100 lM. Thus, this result proved that the triplex
was significantly stabilized by connecting the TFO
fragments with pentaerythritol.
The antiparallel triplexes are formed by the AÆAT,
TÆAT, or GÆGC base triplet. In order to examine the
effect of the composition of a third strand on the alter-
nate-strand triplex formation, the ability of the 30-30-

3336
S. Hoshika et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3333–3336
for the Promotion of Science (JSPS-RFTF97I00301).
We thank Y. Misawa (Hokkaido University) for tech-
nical assistance.
References and notes
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DNase I footprinting experiment. We next performed a
DNase I footprinting experiment to analyze the struc-
ture of the triplexes. The 32P-labeled DNA 16, which has
the oligopurine domains between the Hind III and EcoR
I recognition sites, was digested by DNase I at 25 °C in
the presence of increasing concentrations of the mixture
of the unlinked decamers 8 and 9 or the 30-30-linked TFO
10 in a buffer of 20 mM Tris–HCl (pH 7.5) containing
10 mM MgCl2. The solutions were analyzed by dena-
turing 20% PAGE. As shown in Figure 4, the 30-30-
linked TFO 10 gave a clear footprint between the Hind
III and EcoR I recognition sites, whereas the unlinked
decamers 8 and 9 did not exhibit the binding to the
DNA target. Thus, it was revealed that the 30-30-linked
TFO 10 binds simultaneously to both the adjacent oli-
gopurine domains of the DNA 16.
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In conclusion, we have synthesized the 30-30-linked
TFOs connected with pentaerythritol, that were com-
posed of asymmetrical sequences. We found that 30-30-
linked TFOs form stable antiparallel triplexes with the
DNA target consisting of the adjacent oligopurine do-
mains on alternate strands as compared with the un-
linked TFOs. Thus, the TFOs linked with
pentaerythritol would be useful as antigene oligonucle-
otides for DNA targets consisting of the alternating
oligopyrimidine–oligopurine sequences.
28. Data of MALDI-TOF/MS. TFO 10: calcd mass, 6519.17;
obsd mass, 6517.93. TFO 11: calcd mass, 6546.25;obsd
mass, 6547.91. TFO 12: calcd mass, 6555.26;obsd mass,
6555.60. TFO 13: calcd mass, 6519.17;obsd mass, 6518.24.
29. Ichikawa, S.;Matsuda, A. Nucleos. Nucleot. Nucl. Acids
2004, 23, 239–253.
Acknowledgements
This work was supported in part by a Grant from the
‘Research for the Future’ Program of the Japan Society