A click chemistry approach to C3 symmetric, G-quadruplex stabilising ligands

Article abstract of DOI:10.1039/c005055e

Described is the structure-based design and synthesis of a series of tris-triazole G-quadruplex binding ligands utilising the copper catalysed azide-alkyne 'click' reaction. The results of G-quadruplex stabilisation by the ligands are reported and discussed.

Full text of DOI:10.1039/c005055e

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www.rsc.org/obc | Organic & Biomolecular Chemistry
A click chemistry approach to C3 symmetric, G-quadruplex stabilising
ligands†
John E. Moses,*a Dougal J. Ritson,a Fengzhi Zhang,a Caterina Maria Lombardo,b Shozeb Haider,b
Neil Oldhama and Stephen Neidle*b
Received 13th April 2010, Accepted 12th May 2010
First published as an Advance Article on the web 25th May 2010
DOI: 10.1039/c005055e
Described is the structure-based design and synthesis of a
series of tris-triazole G-quadruplex binding ligands utilising
the copper catalysed azide–alkyne ‘click’ reaction. The results
of G-quadruplex stabilisation by the ligands are reported and
discussed.
The maintenance of telomere integrity in the majority of human
cancer cells is achieved by the telomerase enzyme complex, which
acts as a reverse transcriptase and catalyses the synthesis of telom-
eric DNA repeats,1 and is preferentially expressed in cancer cells.2
This process is regulated by several other telomere-associated
Fig. 1 G-quadruplex stabilising ligands.
proteins in a cell cycle-dependent manner. Telomerase inhibition
can be achieved in several ways;3 one of particular current interest
involves the sequestration of the 3¢ end of telomeric DNA, to
which extra telomeric DNA units would be attached by telomerase,
into a higher-order quadruplex structure that is then inaccessible
for attachment.4 Quadruplex folding and stabilisation can be
achieved with small-molecule ligands,5 of which a large number
have been examined.6 Almost all share common features of a
planar heteroaromatic chromophore (or equivalent) together with
cationic side-chains. Selectivity for human telomeric quadruplexes
over duplex DNA is a key requirement, which has been challenging
to achieve for such polycyclics since their structural and electronic
features are shared, at least in part, by many conventional duplex-
binding agents.
pyrimidine-based frameworks.9 The melting temperature (DTm)
of the bis-triazole compound 28 was comparable to the best of
other previously reported synthetic G-quadruplex binding◦ligands,
such as the acridine derivative BRACO-1910 (DTm = 28 C) and
telomestatin itself (DTm = 27 ◦C). The bis-triazole 2 showed
superior selectivity for the quadruplex structure over duplex DNA
compared to BRACO-19. This trisubstituted acridine compound
was designed on the basis of all three (3, 6 and 9- position)
substituents being able to interact in a groove of a quadruplex
structure.11 A subsequent X-ray crystallographic study has shown
that this is indeed the case.12
Several other non-planar and non-aromatic G-quadruplex
ligands have been reported, notably two steroidal molecules.13 As
yet there is no structural data, or even a model for their quadruplex
interactions, but findings in other contexts,14 that there can be
attractive interactions between an aromatic system and a hydrogen
atom perpendicular to it, suggests that the concept of an array of
hydrogen atoms presented to a G-quartet surface might result
in a stabilised complex. The present study examines this concept
with a series of symmetric phenyl derivatives containing three
triazole moieties linked by sp3 carbon atoms to the benzene core
(3, Fig. 2), i.e. potentially presenting a total of six hydrogen atoms
to a G-quartet surface. The three symmetric substituents could
perhaps maximise quadruplex groove interactions, with each one
residing in a groove – analogous to the binding of the BRACO-19
substituents.
As the synthesis of 28 was achieved using the CuAAC reaction,15
it was thought that this methodology could be extended to give 3 in
a one pot reaction starting from 1,3,5-tris(azidomethyl)benzene 5,
itself readily available from triester 4 (Scheme 1). The complemen-
tary alkyne coupling partners were assembled via two routes, as
the 3-(dialkyl)amino-propyl derivatives 32–36 tended to undergo
elimination during the CuAAc reaction (Scheme 1). Hence,
4-ethynylaniline 7 was coupled to either chloroacetyl chloride
The polyoxazole compound telomestatin 1, a natural metabolite
extracted from Streptomyces annulatus, represents a contrasting
and non-polycyclic aromatic class of G-quadruplex ligand.7 It
has a high level of G-quadruplex stabilising ability and selectivity
for quadruplex versus duplex DNA, and is a potent inhibitor of
telomerase. We recently reported the synthesis of a first generation
of rather simpler ligands (2)8 based on the non-polycyclic aromatic
concept, which showed excellent selectivity and good quadruplex-
stabilising properties (Fig. 1).
Other molecules of this general non-polycyclic type that
have been reported include macrocyclic hexaoxazole-, urea- and
aThe School of Chemistry, University of Nottingham, University Park,
Nottingham, UK NG7 2RD. E-mail: john.moses@nottingham.ac.uk;
Fax: (+44) 115-951-3555; Tel: (+44) 115-951-3533
bCRUK Biomolecular Structure Group, The School of Pharmacy, University
of London, 29-39 Brunswick Square, London, UK, WC1N 1AX. E-mail:
stephen.neidle@pharmacy.ac.uk; Fax: (+44) 20-7753-5970; Tel: (+44) 20-
7753-5969
† Electronic supplementary information (ESI) available: Experimental
procedures for the preparation of compounds 5, 6, 8–36, full characteri-
sation of all new compounds, copies of H1 and C13 NMR spectra for final
compounds, ESI-MS data and HPLC traces of all final compounds. See
DOI: 10.1039/c005055e
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Table 1 Thermal stabilization (DTm in ◦C) for ligands interacting with
a human telomeric DNA sequence (h-tel) and a duplex DNA. The
FRET method was used to obtain values, which are the mean of three
determinations. Average esds are 0.1 ◦C
G4 h-tel DNA
1 mM
ds DNA
Compound
2 mM
1 mM
20
21
22
23
24
27
28
29
32
33
34
35
36
1
0
0
2
0
1
4
0
2
1
2
1
0
1
1
0
2
0
7
11
0
7
2
0
0
0
0
0
0
0
0
0
0
0
0
0
5
1
0
Fig. 2 C3 Symmetric ligand design.
or 4-chlorobutyryl chloride to give the acylated products 8 and
9, which were then reacted with the appropriate dialkylamine to
afford trialkylamines 10–19.
match those of the first generation for their ability to stabilise
G-quadruplexes.16 The ligands with acetylamide appendages are
uniformly poor quadruplex stabilisers, and in all cases the mor-
pholino analogues (24, 29, 36) show no significant G-quadruplex
stabilisation, which is likely to result from reduced electrostatic
interactions in the backbone loops and grooves by the uncharged
morpholine moiety. The butanamide analogues 27 and 28 show the
greatest quadruplex stabilisation. The analogous propanamides 34
and 35 show far less stabilisation, which suggests that the longer
side-chains of 27 and 28 enable more effective positioning of the
protonated amines in the phosphate backbone loops, or simply
provide more surface area for the ligand to bind to the quadruplex.
A general trend is that longer side-chains of the tris-triazole
The alkynes were then ‘clicked’ together with 6 to arrive at the
C3 symmetric ligands 20–29. For reasons as yet unknown, the
dimethyl and diethyl derivatives 25 and 26 gave complex mixtures
after the CuAAc reaction and could not be obtained in appreciable
amounts. To arrive at the propyl analogues, 7 was added to
acryloyl chloride, and then subjected to the CuAAc reaction with
6. Michael addition of the desired dialkyl amines to 31 in DMSO
led to the corresponding ligands 32–36 in varying yields.
The DTm values for the compounds 20–24 and 27–36, deter-
mined by a FRET method,16 are given in Table 1. The data
show that these second generation tris-triazole ligands do not
Scheme 1 Synthesis of the azido coupling partner 6 and click reactions to form the tris-triazole ligands. Reagents and conditions: (i) LiAlH4, THF, 85 ◦C,
45%; (ii) PBr3, Et2O, rt; (iii) NaN3, DMF, 80 ◦C, 92% (2 steps); (iv) chloroacetyl chloride or 4-chlorobutyryl chloride, Et3N, THF, 0 ◦C to rt; (v) HNR2,
MeOH; (vi) 6, CuSO4·5H2O, sodium ascorbate, DMF–H2O, 120 ◦C, mWave; (vii) acryloyl chloride, Et3N, CH2Cl2, 0 ◦C to rt, 85%; (viii) 6, CuSO4·5H2O,
sodium ascorbate, DMF–H2O, 120 ◦C, mWave; (ix) HNR2, DMSO, 40 to 70 ◦C.
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Fig. 3 ESI-MS of h-tel (10 mM) alone (A) and in the presence of 28 (25 mM) (B) showing the presence of one and two binding events.
compound lead to improved quadruplex stabilisation, in accord
with the original findings.8 It should be noted that for all ligands
there is no increase in duplex (ds) DNA DTm at concentrations of
1 mM. At higher concentrations the DTm values generally increase,
although the selectivity is not compromised e.g. at a concentration
of 3 mM of 27 the quadruplex DTm is 17 ◦C, and the ds DNA DTm
is 2 ◦C.
Further analysis of the quadruplex binding of 28 by electro-
spray ionisation-mass spectrometry (ESI-MS) was then conducted
(Fig. 3), which revealed the presence of one and two binding events
(as previously seen for the bis-triazole ligands).17 An estimated
apparent KD of 23 mM was obtained for the first binding event
and a small degree of positive cooperativity was evident (see
SI). However, considering that the first generation of bis-triazole
ligands gave DTm of up to 19 ◦C for quadruplex DNA with
complete selectivity over ds DNA at a concentration of 1 mM,
further investigation of this new series of ligands is not being
pursued.
bilising ability, together with the parallel telomeric G-quadruplex19
as the quadruplex structure. Other quadruplex folds such as
(3 + 1) hybrids are likely to be present, at least in dilute solution,20
but the results found here are not dependent on the fold itself, and
so interactions with these other folded structures have not been
explored further.
The modelling clearly shows that the three methylene bridges
perturb the planarity of the aromatic region in 28 to such an
extent, that effective p–p stacking of the ligand onto the terminal
G-quartet is no longer possible (Fig. 4). This lack of planarity
and loss of ability to p-stack would also prevent intercalation in
duplex DNA, hence the observed selectivity of the tris-triazole
ligands for quadruplex DNA over duplex DNA. The observed
(though low) affinity of some members of the present family of
ligands for the quadruplex structure, thus arises in large part from
interactions between the side-chains and the grooves/loops of the
quadruplex. The details of these interactions will depend on the
nature of these binding sites i.e. on the particular fold adopted
by the quadruplex. These results also suggest that much of the
observed high degree of stabilisation found for compound 2 and
its analogues is a consequence of p–p stacking interactions and
that groove/loop non-bonded interactions are relatively minor
contributors to the overall stabilisation.
The key difference between the bis-triazole and tris-triazole
series is the presence of the sp3 methylene groups in the latter.
Molecular modelling has been used18 to better understand the
ligand–quadruplex interactions of these two series. Compound 28
was selected for this study as it has the greatest G-quadruplex sta-
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a concentration of 1 mM and good stabilisation at a concentration
of 2 mM whilst displaying excellent selectivity for quadruplex DNA
over duplex DNA.
Acknowledgements
JEM thanks EPSRC, the Association for International Cancer
Research (AICR), and The University of Nottingham for financial
support. SN is grateful to CRUK for support. Work at the School
of Pharmacy was supported by Cancer Research UK (programme
grant to S. N.) and a studentship to support C. L. (grant to J.E.M
and S.N.).
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