G-quadruplex recognition by macrocyclic hexaoxazole (6OTD) dimer: Greater selectivity than monomer

Article abstract of DOI:10.1039/b910242f

Macrocyclic hexaoxazole (6OTD) dimers were designed as candidates for potent G-quadruplex binders and synthesized.

Full text of DOI:10.1039/b910242f

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G-quadruplex recognition by macrocyclic hexaoxazole (6OTD) dimer:
greater selectivity than monomerw
Keisuke Iida,a Masayuki Tera,a Takatsugu Hirokawa,b Kazuo Shin-yac and
Kazuo Nagasawa*a
Received (in Cambridge, UK) 26th May 2009, Accepted 25th August 2009
First published as an Advance Article on the web 14th September 2009
DOI: 10.1039/b910242f
Macrocyclic hexaoxazole (6OTD) dimers were designed as
candidates for potent G-quadruplex binders and synthesized.
length of linkers. These dimers were calculated by molecular
dynamics using AMBER9, and dimer 10 was suggested to
have the appropriate length for binding to the G-quadruplex
(Fig. S1 in ESIw). In this paper, we describe the synthesis
of 6OTD dimers 9–11, and evaluation of their interaction
abilities with telomeric G-quadruplex.
Human telomeres are located at the ends of chromosomes and
consist of repeating sequences of (TTAGGG)n.1 This guanine-
rich DNA sequence forms G-quadruplex structures in the
presence of monovalent cations.2 The G-quadruplex structure
of telomeres promotes dissociation of telomere-related proteins,
such as Pot1 and TRF2, and induces apoptosis of cancer cells.3
Therefore, G-quadruplex binders, which stabilize the telomeric
G-quadruplex structure, are candidate cancer therapeutic
agents.4 In 2001, Seto and Shin-ya reported that the macro-
cyclic polyoxazole-thiazoline telomestatin (TMS; 1) from
Streptomyces annulatus 3533-SV4 is one of the most potent
G-quadruplex binders yet discovered (Fig. 1).5,6 The inter-
action with the telomeric G-quadruplex was reported to
involve two molecules of TMS.7 Based on a docking study,
Hurley et al. proposed an end-stacked binding mode, i.e.,
interaction of the terminal G-quartet flat surfaces through
p–p stacking.8
We recently reported a synthetic TMS derivative, 6OTD,
which has C2-symmetrical macrocyclic hexaoxazole structure,
as a new G-quadruplex binder (Fig. 1).9,10 Taking into
consideration the proposed stacking model of TMS with
telomeric G-quadruplex, we hypothesized that a 6OTD
dimer connected through an appropriate linker would show
cooperative interaction of the two monomer moieties with
telomeric DNA, and would therefore bind more selectively
than the monomer. Since the distance between the terminal
G-quartets in the telomeric G-quadruplex structure is known
to be ca. 8 A (l1 in Fig. 2),11 the distance between two 6OTD
structures in the dimer is required to be ca. 15 A (l2 in Fig. 2)
for the interaction with G-quadruplex as depicted in Fig. 2.
Hence, we designed 6OTD dimers 9–11 having three different
The 6OTD dimers 9–11 were synthesized starting from the
reported macrocyclic bis-amide 4 (Scheme 1).9c Briefly, the
Boc group of 4 was deprotected with 5% TFA to give amine 5.
Reaction of the resulting amine 5 with triphosgene followed by
deprotection of the TIPS ether with TBAF gave an alcohol,
whose hydroxyl group was converted into acetate with acetic
anhydride to afford dimer 9 in 29% yield. The dimers 7 and 8
were also synthesized from the common intermediate of 5.
Thus, the amino group of 5 was reacted with the dipentafluoro-
phenyl adipic diester and 1,12-dodecanedicarboxylate to
afford dimers 7 and 8, respectively.12 The TIPS ether groups
of 7 and 8 were converted into acetate with TBAF followed by
treatment with acetic anhydride to give dimers 10 and 11 in
57 and 61% yield, respectively. LSA2-6OTD (3), which
corresponds to a monomer derivative of 9–11, was also
prepared from 4 in 93% yield.
Induction and structural stabilization of the G-quadruplex
structure of telomeric DNA by the newly synthesized 6OTD
dimers 9–11 and LSA2-6OTD (3) was quantitatively evaluated
by fluorescence resonance energy transfer (FRET) melting
assay using fluorescence-labeled, single-stranded human
telomeric oligonucleotide Flu-ss-telo21.13,14 The DTm values
of 9–11 and 3 at a concentration of 2 mM, which corresponds
to 10 equivalents with respect to labeled oligonucleotide
a Department of Biotechnology and Life Science Faculty of
Technology, Tokyo University of Agriculture and Technology
(TUAT), Koganei, Tokyo 184-8588, Japan.
E-mail: knaga@cc.tuat.ac.jp; Fax: +81-42-388-7295;
Tel: +81-42-388-7295
b Computational Biology Research Center National Institute of
Advanced Industrial Science and Technology, Koto-ku,
Tokyo 135-0064, Japan
c Biological Information Research Center National Institute of
Advanced Industrial Science and Technology, Koto-ku,
Tokyo 135-0064, Japan
w Electronic supplementary information (ESI) available: Further experi-
mental details, NMR spectra, FRET-melting, PCR stop assay and
molecular dynamics simulation experiment. See DOI: 10.1039/b910242f
Fig. 1 Chemical structures of TMS (1) and 6OTDs 2–5.
Chem. Commun., 2009, 6481–6483 | 6481
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Table 1 DTm by FRET melting and IC50 by PCR stop assays
IC50a/mM
Flu-ss-telo21 Flu-ds-26mer ss-telo24
DTm (at 2 mM)/1C
ss-telo24-mut
Ligand
3
9
10
11
25.0
17.2
25.1
8.5
À0.9
À0.1
À0.8
À0.2
2.9 Æ 0.5
243 Æ 13
23.3 Æ 3.7 1150 Æ 36
3.0 Æ 1.2 42500
35.1 Æ 3.8 1000 Æ 41
a
Values represent the means Æ SD of triplicate assays.
over double-stranded DNA, in accordance with the reported
character of 6OTD derivatives.9b These results clearly showed
that the macrocyclic hexaoxazole structure of 6OTD was
important for the interaction with G-quadruplex DNA, even
in the case of the 6OTD dimers 9–11.
Fig. 2 Design concept of 6OTD dimers.
The selectivity of interaction of ligands 3 and 10, which
showed strong interaction with telomeric DNA, with the
single-stranded nucleotide base sequence ss-telo24 and its
mutant sequence ss-telo24-mut was investigated using PCR
stop assay (Table 1 and Fig. S3 in ESIw).14,16 The monomer
ligand of LSA2-6OTD (3) inhibited the extension of ss-telo24
with an IC50 value of 2.9 Æ 0.5 mM, whereas low inhibitory
activity was observed with the mutant sequence of ss-telo24-mut
(IC50 of 243 Æ 13 mM; 83-fold selectivity). In the case of 6OTD
dimer 10, the ss-telo24 extension reaction was inhibited with
an IC50 of 3.0 Æ 1.2 mM, while no inhibitory activity toward
ss-telo24-mut was detectable (IC50 42500 mM; 4800-fold
selectivity).17 Thus, at least 10-fold higher selectivity for
interaction with telomeric DNA was obtained by dimerization
of the 6OTD structure.
The binding stoichiometry of 6OTD dimer 10 with telomeric
DNA was determined by means of ESI-MS spectral analysis.18
The mass spectra of the G-quadruplex DNA (ss-telo24) with
LSA2-6OTD (3) and dimer 10 at a molar ratio of 1 : 4 are
shown in Fig. 3. In the case of LSA2-6OTD (3), the 2 : 1
stoichiometric complex with ss-telo24 was detected as a major
peak (Fig. 3A).19 On the other hand, only the ion signal of the
1 : 1 complex of dimer 10 and ss-telo24 was observed (Fig. 3B).
Thus, dimer 10 was suggested to interact with telo-24 by
utilizing two macrocyclic 6OTD moieties (Fig. S5 in ESIw).20
In summary, we have synthesized 6OTD dimers having a
macrocyclic hexaoxazole structure in which the monomer
units are connected with different lengths of linkers, and
examined the interaction with the telomeric G-quadruplex
structure. Among them, dimer 10 showed potent G-quadruplex
stabilizing activity, and was revealed to interact with ss-telo24
more selectively than the corresponding monomer 3. Since it
forms the 1 : 1 complex with DNA, two macrocyclic moieties
in dimer 10 were suggested to interact with the G-quadruplex.
Further structure development of 6OTD dimers as well as NMR
and X-ray analyses are currently underway to clarify the inter-
acting manner of 6OTD dimers with the G-quadruplex.
Scheme 1 Synthesis of 6OTD dimers 9–11. Reagents and conditions:
(a) TFA, CH2Cl2, r.t. for 2 h; (b) triphosgene, DIPEA, MeCN, r.t. for
18 h (6: 31%); (c) dipentafluorophenyl ester of adipic acid, DIPEA,
MeCN, reflux
3 h (7: 67%); (d) dipentafluorophenyl ester of
1,12-dodecanedicarboxylic acid, DIPEA, MeCN, reflux 3 h (8: 51%);
(e) TBAF, Ac2O, THF, r.t. for 0.5–1 h (29–61%); (f) HCl, MeCN,
0.5 h, then Ac2O, Py, 70 1C for 1 h (93%).
Flu-ss-telo21, are summarized in Table 1. The DTm values
were dependent upon the linker length, i.e., the dimers 9 and
11 showed DTm values of 17.2 and 8.5 1C, respectively. On the
other hand, the DTm value of 10 was found to be 25.1 1C, the
same as that of the monomer 3. Therefore, dimer 10 appears to
afford the same degree of stabilization to the G-quadruplex
structure of telomeric DNA as the monomeric 6OTD derivative
LSA2-6OTD (3) (Fig. S2 in ESIw).
Then, the selectivity of the interactions of ligands 9–11 with
single-stranded telomeric DNA (Flu-ss-telo21) and the duplex
form, Flu-ds-26mer,14 was investigated by the same method
described above (Table 1).15 No significant interaction between
the double-stranded DNA of Flu-ds-26mer and ligands 9–11
was observed even at high concentrations. Thus, these ligands
9–11 were highly selective for single-stranded telomeric DNA
We thank Dr N. Dohmae for helpful discussions. This
work was supported in part by the Novartis Foundation
(Japan) for the Promotion of Science, a Grant-in-Aid for
Exploratory Research (21655060) and a grant under the Industrial
Technology Research Grant Program (01A04006b) from
the New Energy and Industrial Technology Development
Organization (NEDO) of Japan. M. T. is grateful for a JSPS
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Fig. 3 ESI mass spectra of 10 mM ss-telo24 with 40 mM ligand 3 (A)
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Research Fellowship for Young Scientists and a grant under
the education program ‘‘Human Resource Development
Program for Scientific Powerhouse’’ provided through TUAT.
14 The oligonucleotide sequences are as follows:
Notes and references
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20 In the case of 9 and 11, 1 : 1 complexes with DNA were also
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