DOTASQ as a prototype of nature-inspired G-quadruplex ligand

Article abstract of DOI:10.1039/c0cc04960c

DOTASQ (for DOTA-templated Synthetic G-quartet) is the first prototype of nature-inspired G-quadruplex ligand: its design, founded on a possible intramolecular G-quartet formation, enables it to interact with G-quadruplex DNA via an unprecedented nature-m

Full text of DOI:10.1039/c0cc04960c

View Article Online / Journal Homepage / Table of Contents for this issue
Dynamic Article Links
ChemComm
Cite this: Chem. Commun., 2011, 47, 4992–4994
www.rsc.org/chemcomm
COMMUNICATION
DOTASQ as a prototype of nature-inspired G-quadruplex ligandw
a
b
b
b
a
´
Loic Stefan, Aurore Guedin, Samir Amrane, Nicole Smith, Franck Denat,
Jean-Louis Mergnyb and David Monchaud*a
Received 15th November 2010, Accepted 1st March 2011
DOI: 10.1039/c0cc04960c
DOTASQ (for DOTA-templated Synthetic G-quartet) is the
first prototype of nature-inspired G-quadruplex ligand: its design,
founded on a possible intramolecular G-quartet formation,
enables it to interact with G-quadruplex DNA via an unprece-
dented nature-mimicking binding mode, based on the association
between two G-quartets, one being native (quadruplex) and the
other one artificial (ligand).
quartets. Given that the G-quartet is the common building
block of all quadruplex-structures, a G-quartet-based ligand
should interact with virtually all quadruplex-types (assuming a
sterically possible access), notwithstanding the nature of the
quadruplex-forming sequence.
The ability of guanines to self-assemble is known for almost
half a century now.8 An elegant approach has been recently
described by the group of Sherman,9 in which four guanine
residues (in blue, Fig. 1A) were grafted on a calixarene moiety
(in purple, Fig. 1A) that acts as a template to enable an
intramolecular G-quartet formation even in the absence of a
cation. This class of compounds was termed TASQ, for
Template-Assembled Synthetic G-Quartet. However, while
it is indisputable that the lipophilicity of TASQs (provided
by the four alkyl chains grafted on the calixarene, Fig. 1A)
was advantageous for performing spectroscopic studies, it
precludes their use in aqueous media and, a fortiori, in
physiological context. One way round this water-solubility
problems consists in replacing lipophilic calixarene by a
water-soluble and C4-symmetrical template: we selected
DOTA-type macrocycles,z for their ease of synthesis, their
structural plasticity and their wide use in biomedical context.10
Given that the formation of the synthetic G-quartet will be
templated by a DOTA-like macrocycle (in purple, Fig. 1B), the
compounds were named DOTASQ (for DOTA-templated
Synthetic G-Quartet).
G-quadruplex-DNA is currently considered as a highly valuable
anticancer therapeutic target;1 quadruplex-binding small molecules
(also called G-quadruplex ligands)2 are intensively studied both
in vitro, to gain insights into their ability to interact with their
DNA target, and in vivo, to evaluate their ability to halt cancer
cells progression.3 The most recent studies strongly suggest of a
multi-level mode of action,4 bolstered by the identification of
putative quadruplex forming sequences distributed throughout
(and in key regions of) the human genome.5 A current trend in
devising G-quadruplex ligands aims at identifying molecules that
are both quadruplex-selective (i.e. interacting with quadruplex-
DNA while avoiding binding to duplex-DNA) and intra-quad-
ruplex selective (i.e. able to interact with one among many
quadruplex structures); a contrario, we believe that, to decipher
their actual in vivo mechanism of action, it could be valuable
to devise a ligand able to interact with most G-quadruplex
types, while, of course, avoiding interaction with unintended
duplex-DNA or other nucleic acid structures.
The synthesis of DOTASQ was straightforward and
convergent (see Fig. 2 and ESIw), since it was realized in
5 steps along the longest synthetic pathway, from commercially
available materials. The diversity in DOTASQ structures was
introduced by playing on the length of the acidic arm of the
purine base (n = 1 or 5, Fig. 2), via an extension of a reported
methodology for PNA bases preparation.11 These two lengths
Herein, we would like to report on a novel series of
G-quadruplex ligands. We devised this series of molecules on
the basis of an artificial G-quartet: this approach steps on the
observation that (i) the stability of a G-quadruplex is related
to the number of its constitutive G-quartets,6 and (ii) two
G-quadruplexes can interact strongly via G-quartets stacking.7
In this way, a G-quartet-based molecule should mimic nature,
binding to G-quadruplex via both stacking interaction with the
accessible quadruplex’s quartet and chelation of biologically
relevant cations (K+, Na+). The process efficiency thus relies
on shape recognition between artificial and naturally occurring
a
´
´
Institut de Chimie Moleculaire, CNRS UMR5260, Universite de
Bourgogne, Dijon, France. E-mail: david.monchaud@u-bourgogne.fr;
Fax: +33 380 396 098; Tel: +33 380 399 043
b
´ ´
INSERM U869, Institut Europeen de Chimie et Biologie, Universite
de Bordeaux, Pessac, France
Fig. 1 Chemical formulae of TASQ (A, from Sherman et al., ref. 9) and
DOTASQ (B, studied herein); guanines appear in blue and the templating
scaffold (a calixarene in A and a DOTA-like macrocycle in B) in purple.
w Electronic supplementary information (ESI) available: Synthesis,
NMR, luminescence, FRET-melting results. See DOI: 10.1039/
c0cc04960c
c
4992 Chem. Commun., 2011, 47, 4992–4994
This journal is The Royal Society of Chemistry 2011

View Article Online
Fig. 2 Synthesis of DOTASQ: (i) H2NCH2CH2NH2, 4 d, 20 1C, 91%; (ii–iv) (adapted from ref. 11): (ii) Br-(CH2)n-CO2CH3, NaH, DMF, 16 h,
20 1C, n = 1: 62%, n = 5: 80%; (iii) LiOH, dioxane, 3 h, 20 1C, n = 1: 67%, n = 5: 44%; (iv) PhCH2OH, K2CO3, DABCO, 16 h, 85 1C, n = 1: 83%,
n = 5: 49%; (v) EDCI, HOBt, DMF, 4 d, 20 1C, then (vi) MeOH/HCl, 1 h, 20 1C, n = 1: 63%, n = 5: 95% (over two steps).
were selected since preliminary in silico investigations have
shown that they both enable the formation of the intramolecular
G-quartet (see ESIw). Two DOTASQs were thus synthesized,
DOTASQ-C1 and DOTASQ-C5 (n = 1 and 5, respectively,
Fig. 2). They are soluble in water (as desired) or in highly
dissociative solvents (like DMSO); we thus tried to demon-
strate the existence of the closed conformation of DOTASQ by
performing NMR studies in water: a weak signal is obtained
for the DOTASQ alone (see ESIw), implying that the closed
conformation may exist in solution; when performed in the
presence of a quadruplex-DNA (1 : 1 ratio), obtained spectra
clearly indicate that a ligand/DNA interaction takes place, and
the presence of an additional peak (at B11.8 ppm, see ESIw)
infers that this interaction is mediated by an additional
G-quartet, thereby supporting again the existence of the closed
conformation in solution. To confirm this, we decided to study
their ability to interact with quadruplex-DNA by the FRET-
12
melting assay,z wishing to indirectly demonstrate the intra-
molecular G-quartet formation by the DOTASQs’ ability t
o stabilize quadruplex-DNA. Results obtained with both
DOTASQs are disappointing (with DT1/2 o 2.5 1C in both
instances, Fig. 3A and ESIw). This low quadruplex-stabilization
was attributed to the weak propensity of DOTASQ to adopt
the desired ‘closed’ conformation (Fig. 2). This may originate
from structural flexibility problems that may be two fold: (i)
the DOTA macrocycle is too flexible to permit the ad hoc
coplanar arrangement of the four guanines, or (ii) it is too
rigid to enable the four guanine arms to be concomitantly
present on the same side of the molecule (thus precluding the
quartet formation). One way round these problems is to force
the DOTA macrocycle to adopt a well-defined conformation.
This can be achieved by the introduction of a metal within the
DOTA cavity: indeed, as demonstrated by solid state studies,
the coordination of a metal—like terbium—by a DOTA
requires eight ligand-to-metal bonds, the four ring nitrogen
atoms (defining the N4 plane, Fig. 4) and the four carbonyl
oxygen atoms.10 Interestingly, the metal coordination forces
the four oxygen atoms to be on the same side of the DOTA
ring (defining the O4 plane) in that way making the N4 and
O4 planes virtually planar and parallel. The presence of a
metal in the cavity of the DOTASQ will thus fulfil satisfyingly
all the structural requirements for the G-quartet (so called
‘G4 plane’ in Fig. 4) to be formed: its coordination will place
the four guanine arms on the same side of the ring and will
freeze structurally this conformation, thereby offering a stable
three-layer NOG sandwich (Fig. 4). The terbium coordination
Fig.
3
FRET-melting results of experiments carried out with
F21T (0.2 mM) in 10 mM lithium cacodylate buffer plus 100 mM
NaCl, without (black curve) or with 1 mM DOTASQ-C1 (A) and
TbꢀDOTASQ-C1 (B), in the absence (dark blue curve) or presence of
ds26 (3 mM, light blue curve or 10 mM, green curve).
was thus realized following previously reported procedures
(see ESIw),13 and provides the two complexes TbꢀDOTASQ-C1
and TbꢀDOTASQ-C5. Both of them were subjected to FRET-
melting evaluation: results obtained with the two complexes
are drastically better than the parent molecules, since DT1/2 are
B10 1C for both complexes (Fig. 3B and ESIw). It cannot be ruled
out that this improvement is due to the cationic nature (3+) of the
final complexes; however, in the case of ligands displaying a
moderate affinity, high cationic charges generally lead to random
associations with DNA, i.e. low quadruplex selectivity. Herein,
the complexes display both affinity and very high selectivity
(the stabilization is >85% maintained in the presence of a 50-fold
excess of ds26 (see ESIw)), thereby making us confident as for the
responsibility of the metal-mediated conformational switch for the
quadruplex recognition. TbꢀDOTASQs thus compete with some
of the recently reported ligands, displaying good stabilizations but
exquisite selectivities, thoroughly studied for their activity in cells.14
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 4992–4994 4993

View Article Online
Fig. 4 Schematic representation of the structural rearrangements related to the coordination of terbium by a DOTASQ: guanines (G) are
represented as grey squares, nitrogen and oxygen atoms as blue and red circles, and N4, O4 and G4 planes as blue, red and grey squares
respectively (terbium-bound water has been omitted for clarity).
To further investigate the TbꢀDOTASQ quadruplex recog-
nition, competitive FRET-melting experiments were also
performed with the tetramolecular G-quadruplex [TG5T]4.
As depicted in the ESIw, the presence of competitive [TG5T]4
leads to a complete loss of F21T-stabilization for both Tbꢀ
DOTASQs: this indicates that TbꢀDOTASQs are sensitive to
the presence of competitive G-quartets, thereby supporting
that their association with G-quadruplex is mainly driven by
G-quartets recognition. It was finally of interest to demon-
strate that DOTASQs can also recognize quadruplex-DNA
from other sequences, since their original design makes them
theoretically able to interact with all accessible G-quartets. To
this end, FRET-melting experiments are performed with four
different quadruplex-forming oligonucleotides (QFOs), all
corresponding to biologically relevant sequences, viz. the
promoter region of c-myc, c-kit and K-ras oncogenes (see
ESIw for complete sequences).15 As depicted in Fig. 5 and
ESIw, both TbꢀDOTASQs have significant influence on the
melting of all above-mentioned QFOs (DT1/2 comprised
between 6.7–12.1 1C and 7.3–11.0 1C for TbꢀDOTASQ-C1
and TbꢀDOTASQ-C5, respectively, at 1 mM dose), in a dose-
dependent manner (with DT1/2 up to 19.5 and 17.8 1C for Tbꢀ
DOTASQ-C1 and TbꢀDOTASQ-C5, respectively, at 3 mM
dose, see ESIw). These results indicate that DOTASQ complexes
are able to stabilize a broad variety of quadruplex architectures,
thereby supporting the validity of their original design.
terbium complexes represent a novel class of quadruplex-
interacting metallo-organic complexes, whose interest keeps
on increasing exponentially.17 Efforts are currently undertaken
to benefit from the exquisite quadruplex selectivity of Tbꢀ
DOTASQ complexes for performing further in vitro and in
cells investigations, and also to furnish their scaffold with
an antenna to sensitize terbium ion,18 since very preliminary
results (see ESIw) make us confident as for their use as
quadruplex-selective luminescent probes.
The authors thank L. Lacroix for fruitful discussions,
F. Cuenot for project help, CheMatechs company for the kind
gift of DOTAEt, the CNRS, the Universite
the Conseil Regional de Bourgogne (3MIM project (FD,DM)),
the Agence Nationale de la Recherche (ANR-10-JCJC-0709
(DM)) and the Conseil General d’Aquitaine (JLM).
´
de Bourgogne and
´
´
´
Notes and references
z DOTA:
1,4,7,10-tetraazacyclododecane-N,N0,N00,N0 0 0-tetraacetic
acid; FRET: fluorescence resonance energy transfer.
1 S. Balasubramanian and S. Neidle, Curr. Opin. Chem. Biol., 2009,
13, 345.
2 D. Monchaud and M.-P. Teulade-Fichou, Org. Biomol. Chem.,
2008, 6, 627.
3 D. Yang and K. Okamoto, Future Med. Chem., 2010, 2, 619.
4 A. De Cian, L. Lacroix, C. Douarre, N. Temime-Smaali,
C. Trentesaux, J.-F. Riou and J.-L. Mergny, Biochimie, 2008, 90, 131.
5 A. K. Todd, M. Johnston and S. Neidle, Nucleic Acids Res., 2005,
33, 2901; J. L. Huppert and S. Balasubramanian, Nucleic Acids
Res., 2005, 33, 2908; J. Eddy and N. Maizels, Nucleic Acids Res.,
2006, 34, 3887.
6 J.-L. Mergny, A.-T. Phan and L. Lacroix, FEBS Lett., 1998, 435, 74.
7 G. N. Parkinson, M. P. H. Lee and S. Neidle, Nature, 2002, 417, 876;
S. Haider, G. N. Parkinson and S. Neidle, Biophys. J., 2008, 95, 296.
8 M. Gellert, M. N. Lipsett and D. R. Davis, Proc. Natl. Acad. Sci.
U. S. A., 1962, 48, 2013; J. T. Davis, Angew. Chem., Int. Ed., 2004,
43, 688; J. T. Davis and G. P. Spada, Chem. Soc. Rev., 2007, 36, 296.
9 N. Nikan and J. C. Sherman, Angew. Chem., Int. Ed., 2008, 47,
4900; N. Nikan and J. C. Sherman, J. Org. Chem., 2009, 74, 5211.
10 R. E. Mewis and S. J. Archibald, Coord. Chem. Rev., 2010, 254, 1686.
11 C. Schwergold, G. Depecker, C. Di Giorgio, N. Patino, F. Jossinet,
B. Ehresmann, R. Terreux, D. Cabrol-Bass and R. Condom,
Tetrahedron, 2002, 58, 5675.
DOTASQs are the first prototype of nature-inspired G-quad-
ruplex ligand, which found the ligand/quadruplex recognition
on a biomimetic process. DOTASQ as a free base is relatively
ineffectual in stabilizing quadruplex-DNA; however, we demon-
strate that, reminiscently of what we demonstrated with the
bisquinolinium series,16 a metal-mediated conformational switch
controls the G-quadruplex binding affinity. The corresponding
12 A. De Cian, L. Guittat, M. Kaiser, B. Sacca, S. Amrane,
A. Bourdoncle, P. Alberti, M.-P. Teulade-Fichou, L. Lacroix
and J.-L. Mergny, Methods, 2007, 42, 183.
13 F. Kielar, G.-L. Law, E. J. New and D. Parker, Org. Biomol.
Chem., 2008, 6, 2256.
14 For a recent example, see: F. Cuenca, M. J. B. Moore, K. Johnson,
B. Guyen, A. De Cian and S. Neidle, Bioorg. Med. Chem. Lett.,
2009, 19, 5109.
15 Y. Qin and L. Hurley, Biochimie, 2008, 90, 1149.
16 D. Monchaud, P. Yang, L. Lacroix, M.-P. Teulade-Fichou and
J.-L. Mergny, Angew. Chem., Int. Ed., 2008, 47, 4858.
17 S. N. Georgiades, N. H. Abd Karim, K. Suntharalingam and
R. Vilar, Angew. Chem., Int. Ed., 2010, 49, 4020.
Fig. 5 FRET-melting results of experiments carried out with 0.2 mM
F21T (black star), FmycT (red circle), Fkras35B1T (green triangle)
and Fkit2T (blue square), with increasing amount (from 0 to 3 mM) of
TbꢀDOTASQ-C5 in 10 mM lithium cacodylate buffer plus 1 mM KCl/
99 mM LiCl.
18 J. P. Leonard and T. Gunnlaugsson, J. Fluoresc., 2005, 15, 585.
c
4994 Chem. Commun., 2011, 47, 4992–4994
This journal is The Royal Society of Chemistry 2011