An octahedral cobalt(III) complex with axial NH3 ligands that … · 2020. 5. 14. · An octahedral...
Transcript of An octahedral cobalt(III) complex with axial NH3 ligands that … · 2020. 5. 14. · An octahedral...
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An octahedral cobalt(III) complex with axial NH3 ligands that
templates and selectively stabilises G-quadruplex DNA
Carmen L. Ruehl,1 Aaron H. M. Lim,1,2 Timothy Kench,1 David J. Mann,2 Ramon Vilar1*
1Department of Chemistry, Imperial College London, White City, London W12 0BZ
2Department of Life Sciences, Imperial College London, South Kensington, London SW7 2AZ
Corresponding author: [email protected]
Abstract
Guanine-rich sequences of DNA are known to readily fold into tetra-stranded helical structures
known as G-quadruplexes (G4). Due to their biological relevance, G4s are potential anticancer
drug targets and therefore there is significant interest in molecules with high affinity for these
structures. Most G4 binders are polyaromatic planar compounds which π-π stack on the G4’s
guanine tetrad. However, many of these compounds are not very selective since they can also
intercalate into duplex DNA. Herein we report a new class of binder based on an octahedral
cobalt(III) complex that binds to G4 via a different mode involving hydrogen-bonding,
electrostatic interactions and π-π stacking. We show that this new compound binds selectivity
to G4 over duplex DNA (particularly to the G-rich sequence of the c-myc promoter). This new
octahedral complex also has the ability to template the formation of G4 DNA from the unfolded
sequence. Finally, we show that upon binding to G4, the complex prevents helicase Pif1-p from
unfolding the c-myc G4 structure.
mailto:[email protected]
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Introduction
Guanine-rich sequences of DNA can fold into tetra-stranded structures known as G-
quadruplexes (G4). These structures form due to the ability of guanines to display Hoogsteen
hydrogen bonding and are stabilised by electrostatic interactions between mono-cations (e.g.
K+ and Na+) and the oxygen atoms of the guanine bases (see Figure 1). There is significant
experimental evidence showing that G4s form transiently in cells and are involved in a number
of biological processes such as transcription, telomere function and replication.1-3 While the
detailed molecular mechanisms by which G4s perform their biological functions is still far
from complete, these structures have been identified as potential targets for anticancer drugs.4-
7 For example, it has been shown that molecules which stabilise G4s in the telomere induce
cell death by a number of mechanisms which include inhibition of telomerase (an enzyme over
expressed in 85% of cancer cells), triggering DNA damage and disrupting the interactions
between proteins and the telomeric DNA.6,7 There is also evidence that small molecules can
stabilise G4 structures in promoter regions of oncogenes (e.g. c-myc, kit, KRAS) and in doing
so downregulate expression of the corresponding oncoproteins.5,8 Therefore, there is continued
interest in developing small molecules which can selectively target G4s and in doing so act as
potential drugs – particularly for cancer. G4s have structural features that make them unique
and different to the canonical duplex DNA structure and hence small molecules can in principle
be rationally designed to interact with them selectively. Some of these features are: i) the
guanine tetrads located at the ends of G4s display a uniquely large planar area for targeting; ii)
the ion channel – normally occupied by K+ ions – at the centre of the quadruplex; iii) unlike
duplexes, G4s display a wide range of different topologies depending on the exact sequence;
therefore small molecules can be tailored to target a specific topology.
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A large proportion of G4 DNA binders reported to date have been designed to interact with the
external tetrads of G4s via a combination of π-π stacking and electrostatic interactions.9,10
Therefore, most G4 binders are planar molecules containing two or more aromatic rings that
display strong π-π interactions with the guanine tetrads. In addition, most of these compounds
also feature substituents with positive charges which increase their affinity for the negatively-
charged DNA. A subclass of this type of G4 DNA binders are metal complexes containing at
least one planar face (i.e. square planar or square-based pyramidal complexes).11-13 Metal
complexes with these geometries have demonstrated to be particularly well suited as G4
binders since the electropositive metal withdraws electron density from the planar ligand
making it more suitable for π-π stacking with the guanine tetrad. Furthermore, the metal centre
can be positioned on top of the ion channel where a K+ ion would normally reside facilitating
the interaction of the molecule with the G4 structure.14
Another type of G4 binders – although far less prominent than those that bind via π-π end-
stacking – are compounds designed to interact with the loops and grooves of the
quadruplexes.15,16 Since the exact topology of a G4 structure is highly dependent on its
sequence, it is in principle possible to design molecules that can, not only differentiate G4s
from duplex DNA, but even between two different G4s.
One of the unique structural features of G4s that has been least exploited in the development
of binders is their ion channel. To the best of our knowledge there are only two reports where
molecules have been designed to interact with this structural feature. Balasubramanian reported
a planar organic molecule tethered with a tri-amine which induced the formation of a parallel
G4 structure for a human telomeric DNA sequence.17 It was proposed that the molecule binds
to the G4 structure by a combination of π-π stacking and threading the polyamine into the ion
channel – substituting the K+ ions that would naturally occupy this position. Also, Shao
reported an octahedral ruthenium complex coordinated to a planar di-phenanthroline ligand
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and to NH3 groups on the axial positions.18 This complex was shown to bind with high affinity
to HTelo G4 DNA (over c-myc and c-kit2) and the binding mode was proposed to be via a
combination of π-π stacking and replacement of one of the external K+ ions from the ion
channel.
We and others have previously shown that square planar metal salphen complexes can be
excellent G4 binders.14,19-26 While in some cases these complexes have shown good selectivity,
this is still not high enough to bind to G4s specifically in the presence of a large excess of
duplex DNA – as is the case in a cellular environment. This is partly due to the fact that planar
compounds can potentially intercalate in-between base pairs of duplex DNA or even bind to
its grooves. Therefore, we rationalised that an octahedral metal-salphen complex which can
display π-π interactions with the G-tetrads and contains NH3 ligands in the axial positions to
replace the K+ from the G4’s ion channel, could in principle be more selective. Herein we
report the synthesis of such a compound (1) and show that it binds with very good affinity to
G4 DNA and, more importantly, with excellent selectivity over duplex DNA. We also show
that upon binding to G4 DNA of the c-myc promoter sequence, complex 1 prevents the
unfolding of this G4 structure by the Pif1-p helicase.
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Figure 1. (A) Schematic representation of the proposed interaction of an octahedral metal complex with G4 DNA. (B) Chemical structure of Co(III)-salphen complex 1 with axial NH3 ligands.
Results and Discussion
Synthesis of cobalt(III) complexes 1 and 2.
Cobalt(III) complexes 1 and 2 were prepared in two steps following the synthetic protocol
shown in Scheme 1. As discussed above, the coordination of NH3 groups to the axial positions
of complex 1 was intended to provide a group that could substitute K+ from the G4’s ion
channel. On the other hand, complex 2 – with the bulkier NH2Me axial ligand – was prepared
as a control compound, which was not expected to bind well to G4s due to steric constrains as
has been previously suggested for other complexes.18 For both complexes, salphen ligand 3
was synthesised by reacting 1,2-phenylendiamine with aldehyde 4. The isolated ligand was
reacted with Co(OAc)2 under a nitrogen atmosphere. Subsequently a solution of the
corresponding axial ligand (either aqueous NH4OH or ethanolic NH2Me) was added and the
reaction exposed to air to oxidise the cobalt centre from Co2+ to Co3+. The resulting octahedral
cobalt(III) complexes 1 and 2 were isolated and fully characterised by 1H and 13C NMR
spectroscopy, mass spectrometry and elemental analyses (see Experimental Details). The 1H
NMR spectra of both these complexes show the right number and integration of signals for the
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salphen ligand (including the imine, aromatic protons and the ethyl-trimethylammonium
substituents). In addition, a sharp signal for the coordinated NH3 groups at 2.67 ppm
(integrating to six protons) was observed for 1 confirming the presence of the two axial ligands.
Similarly, the 1H NMR spectrum of complex 2 showed the expected signals for the salphen
ligand as well as a triplet at 1.36 ppm and a multiplet at 3.56 ppm corresponding to the methyl
and amino protons of the NH2Me coordinated to the Co(III) centre of this complex. The
assignment of all protons was corroborated by 2D NMR experiments (see supplementary
information). 1H-1H Selective ROESY experiments were performed to confirm the
coordination of NH3 and NH2Me to the cobalt(III) centre (see Supplementary Information).
The formulation of both complexes was also confirmed by elemental analyses and mass
spectrometry ([M-PF6]+ at 901 and 929 a.m.u. for 1 and 2 respectively).
Scheme 1. Two step synthetic protocol for the preparation of cobalt(III) complexes 1 and 2.
Prior to studying the DNA binding properties of these two complexes, it was of interest to
establish their stability in solution. Thus, the corresponding complex was dissolved in
1 M TRIS – 100 mM KCl (pH 7.2) prepared in D2O/H2O (1:9) (due to solubility issues of
complex 1, 50% DMSO-d6 was added) and the corresponding 1H NMR spectra recorded over
time. DOSY (for complex 1 and 2) and 1H-1H Selective ROESY experiments (for complex 2)
confirmed the absence/presence of free axial ligand over time. This showed both complexes to
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be stable (≥ 90%) during 24 hours at 25 °C, with complex 2 showing some changes after 4
hours while complex 1 remaining unaffected up to 24 hours.
DNA binding assays of complexes 1 and 2.
To establish the affinity of complex 1 towards different topologies of G-quadruplex and duplex
DNA three different biophysical assays were performed. We first screened the compound
against four different G4s (HTelo (K) and HTelo (Na), c-myc, c-kit2 and bcl-2) and duplex
DNA (ds26) using the fluorescent indicator displacement (FID) assay which has been
previously used to establish semi-quantitatively the affinity of compounds against a panel of
DNA structures.27 The assay is based on the emission of thiazole orange (TO) which is
quenched in solution but not when bound to DNA. Compounds with a tendency to bind to G4
can displace the TO resulting in a decrease in its fluorescence. The compound concentration at
which 50% TO is assumed to be displaced (G4DC50, fluorescence signal reduced by 50%) is
used to compare the potential G4 binders. The results clearly showed (see Figure 2) that
complex 1 has excellent affinity for c-myc and HTelo (K) (DC50 < 0.4 µM) and good affinity
for HTelo (Na), c-kit2 and bcl-2 (DC50 values between 0.5 and 2.3 µM for three of the G4
structures). The data also shows that 1 is highly selective for G4s over duplex DNA – for which
it was not possible to reach a 50% displacement of TO even after addition of a large excess of
the binder. The interaction of complex 2 (with the NH2Me axial ligand) towards c-myc G4 and
ds26 was also studied as a control. This compound did not show any significant affinity (i.e.
DC50 > 2.5 µM – see Supplementary Information) for neither of the DNA structures under
study.
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Figure 2. (A) %TO Displacement plotted against concentration of complex 1 up to 2.5 µM. (B) DC50 values calculated from the titration of complex 1 to solutions of five different G4s (HTelo (K), HTelo (Na), c-myc, c-kit2, bcl-2) and duplex DNA (ds26). All values are average from three independent experiments with consistent results throughout.
To confirm the ability of complex 1 to bind and stabilise G4 DNA structures, we then
performed FRET melting assays (see Experimental Details for the sequences used). As can be
seen in Figure 3, complex 1 induced thermal stabilisation for the different G4 structures under
study, particularly for c-myc for which a ΔTm = 20.0 ± 0.2 °C was observed. Interestingly, the
ΔTm for c-kit2, bcl-2 and HTelo (K+) was lower than for c-myc and in the case of HTelo (Na+)
ΔTm = 4.5 ± 0.3 °C suggesting that the complex has very good selectivity for c-myc G4 DNA.
Furthermore, the compound did not induce any thermal stabilisation for ds26 indicating that it
has no affinity towards duplex DNA under these conditions. It should be noted that analogous
FRET melting assays were not performed with the control compound 2 since at high
temperature this complex shows some decomposition and hence it is not possible to record
reliably a melting curve.
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Figure 3. ΔTm (°C) values for six different DNA sequences (including G4 and duplex DNA) in the presence of complex 1. The ΔTm values were determined (in triplicate) by FRET melting assays using 0.2 µM of oligonucleotide and 1 µM of 1.
To assess the selectivity of complex 1 for G4s in the presence of excess duplex DNA, FRET
melting competition assays were carried out. In this assay the FRET melting temperature of
the doubly labelled G4 of interest was recorded in the presence of a fixed concentration of the
compound of interest and in the presence of increasing amounts of unlabelled duplex DNA.
The results obtained for 1 (Figure 4) clearly show that this complex has very high selectivity
for G4 DNA structures (particularly c-myc and HTelo (K)) over duplex DNA since even upon
addition of 600-fold (per base pair) excess CT-DNA, the melting temperature of the G4
remained unaffected.
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Figure 4. ΔTm values (°C) obtained when performing FRET competition assays with five different G4s (all 1 µM) at increasing concentrations of CT-DNA (0 – 120 µM). Results shown are average from three experiments.
To gain more insights into the interaction between the Co(III) complexes and G4 DNA, circular
dichroism (CD) spectroscopic studies were performed. As has been extensively documented,
CD spectroscopy is very useful in providing information about the topology of a DNA
structure.28-30 For G4s, a number of key spectroscopic features are well established for the
different possible topologies: parallel G4s display a positive band at ca. 265 nm and a negative
one at ca. 245 nm while antiparallel G4s show a positive band at ca. 295 nm and a negative
one at ca. 260 nm. Hybrid (or 3+1) structures display more complex spectra with positive bands
at ca. 295 and 260 nm and a negative band at ca. 245 nm.31
To determine the effect that complexes 1 and 2 have on the G4 DNA topology, we first titrated
increasing amounts of the corresponding complex into a solution containing either c-myc
(parallel) or HTelo (hybrid) G4 DNA. As can be seen in Figure 5a, complex 1 did not induce
any changes in the CD spectrum of the c-myc indicating that the interaction does not lead to
topological changes in its structure. In contrast, upon addition of complex 1 to HTelo DNA in
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K+, clear changes in the CD spectrum were observed (Figure 5b): the shoulder at ca. 265 nm
initially present in the spectrum, decreases as compound 1 is added. This is accompanied by an
increase in intensity of the peak centred at ca. 290 nm. Both these changes are consistent with
complex 1 inducing a change in topology from hybrid to antiparallel. In contrast, the control
complex 2 (with the bulkier NH2Me ligand) did not induce any significant changes upon
addition to c-myc or HTelo, which is consistent with our previous observations that it does not
bind to G4 structures.
CD spectroscopy was also used to study the ability of 1 and 2 to template the formation of
HTelo G4 from an unfolded sequence in the absence of K+ ions (or any other added cation). As
shown in Figure 5c, the unfolded sequence showed a characteristic signal at 250 nm, but upon
addition of increasing amounts of 1 to unfolded HTelo DNA, the expected CD pattern for an
antiparallel G4 structure emerged i.e. with signals at ca. 295 nm (positive ellipticity) and ca.
260 nm (negative ellipticity). The presence of an isoelliptic point suggests that the transition to
the antiparallel G4 does not involve other intermediate topologies. On the other hand, addition
of 2 to the same unfolded sequence did not lead to significant changes in the initial spectrum
(only a small increase in the signal at ca. 290 nm) which is consistent with the FID and FRET
melting data discussed above.
It is interesting to note that addition of complex 1 to unfolded HTelo in the absence of K+ or
Na+, templates the formation of an antiparallel structure. This is in contrast to the FRET results
which show that 1 induces a higher thermal stabilisation for a parallel structure (c-myc) than
for an antiparallel one (HTelo (Na)). However, clearly the oligonucleotide sequences of c-myc
and HTelo are different and therefore one should be cautious when comparing directly between
the two. Another important difference between the two observations is that in the FRET melting
experiments the G4 structures are pre-annealed in the presence of either Na+ or K+. Therefore,
it is not unfeasible that the interaction of the complex with each of these structures differs from
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its ability to template a given G4 structure from an unfolded sequence in the absence of the
metal ions. This highlights the complex dynamics of DNA’s folding process when templated
by a small molecule as compared to the interaction of the molecule with a pre-folded structure
that contains metal ions differently positioned with respect to the G-tetrad plane (nearly in the
plane for Na+ and above the plane for K+).
Figure 5. CD spectra recorded upon addition of increasing amounts of complex 1 to pre-annealed c-myc (A) and HTelo (K) (C). (B) and (D) show CD spectra for the titration of complex 2 to pre-annealed c-myc and HTelo (K). For experiments (E) and (F) complex 1 and 2 were added successively to single-strand HTelo in absence of salts. Experiments A – D were performed in 10 mM TRIS buffer with 100 mM KCl (pH 7.2), E – F in 10 mM TRIS buffer (pH 7.2) without salts. DNA- and complex concentrations of 5 µM and 0 – 10 µM were used. The mean of triplicate experiments is plotted.
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Helicase activity assay.
The FID, FRET melting and CD spectroscopic data discussed above all indicated that complex
1 has high affinity for G4 DNA structures – particularly for c-myc – and excellent selectivity
over duplex DNA. We therefore investigated whether 1 would be able to inhibit a helicase from
unfolding c-myc G4 DNA. For this, we used a previously reported FRET assay that monitors
the unwinding of G4 by Pif1-p helicase in real time.32 The assay uses a Dabcyl-/FAM-labelled
DNA sequence (see Table 3) with the potential to form G4 DNA (i.e. with the c-myc promoter
G-rich sequence). Pif1-p helicase is added to the labelled DNA in the presence of a TRAP
oligonucleotide (to capture free single stranded DNA) and ATP to power the reaction. The
activity of the helicase (as %unwound G4 DNA) can then be monitored by recording changes
in the FAM emission over time. In addition to complexes 1 and 2, we carried out this assay in
the presence of BRACO19 (a well-known G4 DNA binder) and DAPI (which does not interact
with G4 DNA) as positive and negative controls respectively. As can be seen in Figure 6a,
addition of complex 1 prevented the helicase from unfolding the G4 structure. A similar result
was observed for BRACO19 (positive control). In contrast, complex 2 had very little effect in
the ability of the helicase to unfold the G4 structure; as can be seen in Figure 6a, this complex
displayed an analogous trend to that observed with DAPI (negative control). To confirm that 1
was halting the activity of the helicase via interaction with G4 DNA (rather than duplex DNA)
we carried out the same assay using a mutated c-myc sequence which is not able to form a G4
structure. As shown in Figure 6b, in this case the addition of 1 did not prevent the helicase from
unfolding the (duplex) DNA structure confirming that the results described above are due to
its selective interaction with the G4 structure in the promoter of the c-myc oncogene.
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Figure 6. (A) and (C) Percentage of S-cmyc and S-mut unwound (%Unwound) by helicase Pif1 over time in presence of no G4 binder, Braco-19, DAPI as well as complexes 1 and 2. Maximal %Unwound is shown for both DNA sequences under investigation in (B) and (D). Results represent an average of three experiments.
Computer modelling of interaction between 1 and G4 DNA.
Having established that complex 1 interacts strongly and selectively with G4 DNA, we were
interested in gaining further insights into the binding mode of the complex. As described in the
introduction, this compound is unusual – as compared to most other G4 DNA metallo-binders
– since its octahedral geometry prevents it from binding by simple π-π stacking interactions.
Instead, our initial hypothesis was that the NH3 ligands in the axial positions of 1 would replace
the K+ in the terminal tetrads of the G4 and position the complex above the G4 structure. This
in turn should allow for π-π stacking interactions with the tetrad.
Molecular docking studies were therefore performed using Autodock 4.2 in order to provide
validation for the proposed binding mode.33 A range of G4s were studied including c-myc (Fig
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7) and HTelo (antiparallel basket type and hybrid type, shown in SI). As expected, the docking
procedure positioned complex 1 with the ammonia ligand directly over the central ion channel
of the three G4 structures under study, with the aromatic rings of the salphen ligand above and
parallel to the guanine bases (see Fig. 7 and S31). The average NH···O distance between the
coordinated NH3 and the closest guanines’ oxygens is shortest for the antiparallel HTelo-basket
(1.97 Å) followed by the parallel c-myc (2.18 Å) and finally by HTelo-hybrid (2.25 Å). A
similar trend is observed for the distances between the centre of each of the three phenyl rings
in 1 to the centre of the closest guanine on the G-tetrad. The distances are consistent with π-π
interactions: closest contacts with HTelo-basket (3.9, 3.5 and 3.8 Å) followed by c-myc (4.3,
3.9 and 3.6 Å) and finally by HTelo-hybrid (3.4, 4.8 and 4.8 Å). The trimethylammonium
substituents of the complex are positioned close to the loop and groove bases, consistent with
the expected electrostatic interactions. This model is in accordance with previous docking
studies conducted on octahedral G4 binders18 and provides further corroboration of G4
stabilisation through an octahedral binding mode.
It is interesting to note that the observed trend in non-covalent interactions from the docking
studies, is consistent with the CD spectroscopic data which shows that, in the absence of K+ or
Na+, complex 1 templates the assembly of the HTelo sequence into an antiparallel topology.
As briefly indicated above, this does not seem consistent with the FRET melting results which
showed a much higher thermal stabilisation for a parallel topology (c-myc) than the antiparallel
one (HTelo (Na)). But, as indicated above, it is important to highlight that one should be
cautious when comparing this data. The FRET melting data measured the interaction of 1 with
a preformed G4 annealed in the presence of either K+ or Na+, while the CD data, reports the
ability of 1 to template the formation of G4 from an unfolded sequence in the absence of metal
ions.
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Figure 7. (A) Full structure of the cobalt(III)-salphen complex with c-myc G4. G bases are emphasised in bold to illustrate the site of complex interaction. (B) Simplified top view of the interactions between the cobalt(III)-salphen and top G-tetrad, showing the position of NH3 ligand and (C) side view showing the parallel positioning of the salphen with the top G-tetrad.
Conclusions
A new octahedral cobalt(III)-salphen complex (1) with axial NH3 ligands has been successfully
synthesised and fully characterised. FID and FRET melting assays have shown that this
complex binds to c-myc G4 DNA with high affinity and selectivity over other G4 structures as
well as duplex DNA. We also demonstrate that this interaction is strong enough to prevent the
Pif1-p helicase from unwinding c-myc G4 DNA. Docking studies indicate that the complex
binds to the external tetrad of the G4 by a combination of non-covalent interactions involving
hydrogen bonding, electrostatic interactions and π-π stacking. This confirms our initial
hypothesis that one of the NH3 ligands of the complex should take the position of the external
K+ ion (from the ion channel) while the salphen ligand would still be close enough to display
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strong π-π stacking interactions. This binding mode (proposed only once before18) provides
great scope to design a wide range of new octahedral G4 DNA binders where the axial ligands
can provide further functionalities and an even higher affinity for the target G4 structure.
Experimental Details
General.
Chemicals were purchased from commercial sources and used without further purification. For
reactions performed under deoxygenated conditions, nitrogen was bubbled through the solvent
of choice for an appropriate amount of time. Oligonucleotides were purchased from Kaneka
Eurogentec S.A. (Belgium) as lyophilised solids (RP-Cartridge GoldTM purification) and
dissolved in MilliQ water or suitable buffer. Deoxyribonucleic acid sodium salt from calf
thymus DNA (CT-DNA) was bought from Merck KGaA (Germany). Concentrations (by
strand) were determined by measuring the absorbance at 260 nm using UV/Vis spectroscopy
and using the extinction coefficient ε (in L mol-1 cm-1) given by the manufacturer. For CT-
DNA only the concentration was determined by base pair (ε = 13 200 L mol-1 cm-1). To form
G-quadruplex structures, DNA sequences were annealed in the buffer of choice for 5 Min at
95 °C, followed by slowly cooling the samples to room temperature over several hours.
Compounds were dissolved in molecular biology grade DMSO. All solutions were stored
at -20 °C. Prior to the experiment, solutions were thawed and diluted in the solvent/buffer of
choice.
Oligonucleotides.
Unlabelled DNA-sequences used for this work are listed in Table 1.
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Table 1. List of DNA sequences used for CD and FID experiments.
DNA Sequence 5’ – 3’ ε (L mol-1 cm-1)
HTelo AGG-GTT-AGG-GTT-AGG-GTT-AGG-G 228500
c-myc TGA-GGG-TGG-GTA-GGG-TGG-GTA-A 228700
c-kit2 CGG-GCG-GGC-GCG-AGG-GAG-GGG 205600
bcl-2 GGG-CGC-GGG-AGG-AAG-GGG-GCG-GG 231300
ds26 CAA-TCG-GAT-CGA-ATT-CGA-TCC-GAT-TG 253200
For FRET melting and competition experiments 5’-FAM (6-carboxyfluorescein) and
3’-TAMRA (6-carboxy-tetramethylrhodamine) labelled sequences in Table 2 were applied.
CT-DNA was used for FRET competition assays.
Table 2. List of DNA sequences used for FRET melting and competition experiments.
DNA Sequence 5’ FAM – TAMRA 3’ ε (L mol-1 cm-1)
FTHTelo GG-GTT-AGG-GTT-AGG-GTT-AGG-G 268300
FTc-myc TGA-GGG-TGG-GTA-GGG-TGG-GTA-A 282000
FTc-kit2 CGG-GCG-GGC-GCG-AGG-GAG-GGG 258900
FTbcl-2 GGG-CGC-GGG-AGG-AAG-GGG-GCG-GG 284600
FTds26 CAA-TCG-GAT-CGA-ATT-CGA-TCC-GAT-TG 306500
5’-FAM- and 3’-Dabcyl-labelled DNA sequences were used for the helicase assay (Table 3).
Table 3. List of DNA sequences used for the helicase assay.
DNA Sequence
S-c-myc 5’-(A)11-GGGTGGGTAGGGTGGGTATTCCGTTGAGCAGAG-3’-Dabcyl
3’-AAGGCAACTCGTCTC-5’-FAM
S-mut 5’-(A)11-TGGTGTGTAGTGTGGTTTATTCCGTTGAGCAGAG-3’-Dabcyl
3’-AAGGCAACTCGTCTC-5’-FAM
TRAP 5’-TTCCGTTGAGCAGAG-3’
C-c-myc 5’-CTCTGCTCAACGGAATACCCACCCTACCCACCC-(T)11-3’
C-mut 5’-CTCTGCTCAACGGAATAACCACACTACACACCA-(T)11-3’
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Fluorescent Intercalator displacement (G4-FID) assay.
Measurements were performed on a Cary Eclipse Fluorescence Spectrophotometer (Agilent
Technologies) following the protocol reported by Teulade-Fichou et al..27 100 µM solutions of
unlabelled annealed DNA sequences in 10 mM lithium cacodylate (Licac) + 100 mM
KCl/NaCl buffer (pH 7.2) were used for the experiment. A 2 mM stock solution of thiazole
orange (TO; Fluka; > 98% purity) in DMSO was stored at -20 °C. Prior to the experiment, the
TO solution was defrosted and diluted to 200 µM in buffer. Ligand dilutions of 100 µM in the
appropriate buffer were prepared. For the experiment, first a 0.25 µM DNA solution was
prepared in 10 mM Licac + 100 mM KCl/NaCl buffer (pH 7.2) and the emission spectrum
(Fmax,0) measured at room temperature. All measurements were taken after an equilibration
time of 5 min using the following parameter: excitation wavelength: 501 nm; data collection:
510 to 750 nm; slit width: 5 nm. The spectrum before addition of TO was used as baseline and
subtracted from all following spectra. 2 or 3 eq TO were added to the DNA sample depending
on the DNA used. The measured emission spectrum reflects the fully bound state of TO to
DNA. TO displacement is determined by gradually adding increasing amounts of compound
to the DNA/TO sample. Measurements were taken after addition of 0.5, 1.0, 1.5, 2.0, 2.5, 3.0,
4.0, 5.0, 6.0, 8.0, 10.0 eq of the corresponding compound. The %Displacement of TO (%DTO)
was calculated for every titration point using the equation: %DTO=100-((Fmax,n/Fmax,0)*100)
with Fmax,n = em. max. for every titration point n (1
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Sample preparation for FRET assays.
An Agilent Stratagene Mx3005P RT-qPCR machine was used for all FRET melting
experiments. The protocol described by Mergny et al. was followed. Doubly-labelled DNA as
well as calf thymus DNA (CT-DNA) was used for the experiments.34 Samples were prepared
in either 96-PCR plates or PCR tubes. Starting with 25 °C a gradient of 0.5 °C/30 sec was
applied up to 95 °C. After every step FAM emission was recorded. Data analysis was
performed using GraphPad Prism 8. Raw data were normalised and the melting curves fitted
to a biphasic function. T1/2 is defined as the temperature at a normalised emission of 0.5. ΔTm
is calculated using the equation ΔTm=T1/2,Com,n-T1/2,DNA with T1/2,Com,n being the melting
temperature of DNA with different concentrations n of the compound studied and T1/2,DNA
being the melting temperature of DNA without compound present. Samples with a ligand
concentration of 1 µM were used for comparison purposes.
For both experiments 20 µM DNA solutions in MilliQ water were prepared which were diluted
further in the appropriate buffer to give a 0.4 µM DNA working solution. Annealing was
performed as described above. Buffer choice depends on DNA sequence used (HTelo and c-
kit2: 10 mM Li cacodylate, 10 mM K+, 90 mM Li+ or 10 mM Li cacodylate, 10 mM Na+,
90 mM Li+; c-myc and ds26: 10 mM Li cacodylate, 1 mM K+, 99 mM Li+; bcl-2: 10 mM Li
cacodylate, 100 mM K+).
FRET melting assay.
10 µM ligand solutions in buffer were prepared from DMSO stock solutions, followed by
further dilution to 0.4, 0.8, 2.0, 4.0, 8.0 µM. For the sample preparation 20 µL 0.4 µM DNA
were added in the PCR tube, followed by 20 µL ligand dilution with increasing concentrations.
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One sample with ligand-free buffered solution was used as a control. After thorough mixing,
samples were measured as described above.
FRET competition assay.
CT-DNA stocks were diluted to 24 µM and 480 µM, ligand stocks to 4 µM in the appropriate
buffer. Six solutions containing no ligand, ligand but no CT-DNA and increasing
concentrations of CT-DNA (0.6 to 120 µM) were prepared. After gentle mixing 20 µL of those
solutions were added to 20 µL of labelled DNA into the PCR tube. Final concentration of
labelled DNA was 0.2 µM and ligand 1.0 µM. Measurements were performed in the same way
as the FRET melting.
CD titrations.
CD spectra were recorded on a JASCO J-810 CD spectrophotometer using a 1 cm quartz
cuvette. A Peltier module controlled the temperature of 25 °C for the measurements. Spectra
were recorded from 400 to 600 nm with a scanning speed of 100 nm/min and a band width of
2.0 nm. Data were collected as an accumulation of three measurements. All spectra were
baseline corrected with the CD spectrum corresponding to the buffer used.
CD titrations of unlabelled, annealed DNA were performed in 10 mM TRIS + 100 mM
KCl/NaCl (pH 7.2) buffer. Experiments studying the templation of G4 formation by compound
1 and 2 were carried out in salt-free conditions using 10 mM TRIS buffer (pH 7.2) and not
annealed DNA. For the experiment 5 µM DNA dilutions in buffer were prepared and the CD
spectrum recorded. Followed by stepwise addition of the studied compound (2 mM stock
solution in DMSO) or salt, e.g. KCl or NaCl. After adding 0.5, 1.0, 2.0 and 5.0 eq of compound
the CD spectra were recorded. Spectra were overlaid to analyse the observed trend.
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Helicase assay.
A previously reported helicase FRET assay was used.32 Prior to the experiment Pif1-p was
overexpressed in bacteria and purified following literature protocols. 10% SDS-Page was used
to determine the purity of the protein. Samples were stored at -20 °C in 25 mM HEPES buffer
(pH 8.0), 100 mM NaCl, 25 mM MgOAc, 50 mM (NH4)2SO4, 1 mM DTT, and 50% glycerol
and thawed prior the experiment. Pif1-p helicase was kept on ice whenever possible. G4
forming DNA sequences were annealed prior to the experiments at a concentration of 1 µM
Dabcyl- and 0.85 µM FAM-labelled oligonucleotides in 20 mM TRIS buffer (pH 7.2), 5 mM
MgCl2, 1 mM KCl, and 99 mM NaCl. Experiments were performed on 96-well plates at room
temperature using a Clariostar (BMG Labtech) microplate reader.
Every well held 50 µL of a solution containing 40 nM S-c-myc/S-mut, 125 nM Pif1 helicase,
and 200 nM TRAP. After adding 5 µL 25 mM ATP solution (20 mM TRIS buffer, pH 7.2) to
each well the emission was recorded until a plateau was reached (ca. 40 Mins). To evaluate
potential G4 binders the same procedure was followed extended by the addition of 1 µM G4
binder/well.
Molecular docking.
Molecular docking studies were performed using Autodock 4.2 with the Lamarckian genetic
algorithm.33 The ligand structure was minimised in Gaussian at the PM6 level and then docked
into c-myc (PDB: 5w77),35 basket type (PDB: 2mcc)36 and hybrid type (PDB: 2mb3)37
quadruplexes. In each case the structures were stripped of any existing counteranions, water
molecules or ligands. The structures were then imported into Autodock 4.2 and hydrogen atoms
were added. A grid box encompassing the entire quadruplex was used in order for blind docking
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to be carried out. In each case, the lowest energy solution was taken. The docked structures
were visualised and hydrogen bond distances measured using Chimera.38
Synthesis.
N,N'-bis[4-[[2-(trimethylammonio)ethyl]oxy]salicylidene]-o-phenyldiamine dibromide
(3). 4-(4-Formyl-3-hydroxyphenoxy)-N,N,N,-trimethylethan-1-ammonium bromide (4,
synthesized as previously reported39) (800 mg, 2.63 mmol, 2 eq) was dissolved in 50 mL
absolute ethanol. 1,2-Phenylenediamine (142.1 mg, 1.31 mmol, 1 eq) was added and the
reaction refluxed for 5 hours under an inert atmosphere. The precipitated product was filtered
and washed with different solvents (EtOAc, DCM and Et2O). A yellow solid could be obtained.
Yield: 739.9 mg, 83%. 1H-NMR (400 MHz, DMSO-d6): δ = 3.19 (s, 18H, NMe3), 3.80 - 3.82
(m, 4H, -CH2N-), 4.53 - 4.55 (m, 4H, -OCH2-), 6.57 (d, 2H, 3JHH = 8.0 Hz, ArH), 6.62 (dd, 2H,
3JHH = 8 Hz, 4JHH = 4.0 Hz, ArH), 7.36 - 7.39 (m, 2H, ArH), 7.44 - 7.46 (m, 2H, ArH), 7.61 (d,
2H, 3JHH = 8.0 Hz, ArH), 8.89 (s, 2H, -CH=N-), 13.57 (s, 2H, OH). 13C-NMR (400 MHz,
DMSO-d6): δ = 53.1, 61.8, 63.8, 101.8, 107.2, 113.8, 119.5, 127.4, 134.2, 141.6, 161.7, 162.9,
163.4. ESI(+)-MS m/z calcd for C30H40BrN4O4+ (M+): 599.22; found: 599.22.
Synthesis of [Co(3)(NH3)2)]PF6 (1). N,N'-bis[4-[[2-(trimethylammonio)ethyl]oxy]
salicylidene]-o-phenyldiamine dibromide (3) (100 mg, 0.15 mmol, 1 eq) was dissolved in 15
mL deoxygenated methanol, followed by the addition of Co(OAc)2·4 H2O (36.5 mg, 0.15
mmol, 1 eq). An aqueous solution of NH4OH (33 wt% aqueous, 1.47 mmol, 177.4 µL, 10 eq)
was added dropwise and the reaction mixture opened to air. An aqueous saturated solutions of
NH4PF6 (> 10 eq) was added to the reaction mixture to precipitate a solid which was filtered
and washed with EtOAc, DCM and Et2O. The resulting brown solid was dried under reduced
pressure and characterised as compound 1. Yield: 99.9 mg, 65%. 1H-NMR (400 MHz, dmso-
-
d6): δ = 2.67 (s, 6H, NH3), 3.20 (s, 18H, NMe3), 3.82 (s, 4H, -OCH2-), 4.52 (s, 4H, -CH2N-),
6.37 (dd, 2H, 3JHH = 8.0 Hz, 4JHH = 4.0 Hz, ArH), 6.68 (s, 2H, ArH), 7.41-7.43 (m, 2H, ArH),
7.58 (d, 2H, 3JHH = 8.0 Hz, ArH), 8.31-8.33 (m, 2H, ArH), 8.73 (s, 2H, -CH=N-). 13C-NMR
(500 MHz, dmso-d6): δ = 53.2, 61.6, 64.0, 104.5, 105.7, 113.8, 116.8, 127.0, 137.2, 143.7,
158.8, 163.6, 169.1. ESI(+)-MS m/z calcd for C30H44CoF12N6O4P2 (M+-PF6): 901.20; found:
901.20. Anal. calcd (%) for C30H44CoF18N6O4P3·0.5 C3H6O: C 35.18, H 4.40, N 7.81; found:
C 35.34, H 4.41, N 8.14.
Synthesis of [Co(3)(methylamine)2)]PF6 (2). This compound was prepared following the
same procedure than that described for complex 1 with the exception that an ethanolic solution
of NH2Me (33 wt% ethanolic, 1.47 mmol, 183.0 µL, 10 eq) was added instead of NH4OH.
Yield: 83.7 mg, 53%. 1H-NMR (400 MHz, dmso-d6): δ = 1.36 (t, 6H, 3JHH = 8.0 Hz, -NCH3-
), 3.20 (s, 18H, NMe3), 3.55-3.57 (m, 4H, NH2), 3.81 (s, 4H, -OCH2-), 4.53 (s, 4H, -CH2N-),
6.39 (dd, 2H, 3JHH = 8.0 Hz, 4JHH = 4.0 Hz, ArH), 6.74 (s, 2H, ArH), 7.43-7.46 (m, 2H, ArH),
7.58 (d, 2H, 3JHH = 8.0 Hz, ArH), 8.32-8.35 (m, 2H, ArH), 8.79 (s, 2H, -CH=N-). 13C-NMR
(500 MHz, dmso-d6): δ = 27.1, 53.2, 61.6, 64.0, 104.3, 106.1, 113.4, 116.8, 127.3, 137.2, 143.3,
158.8, 164.0, 169.1. ESI-MS (M+) m/z calcd for C32H48CoF12N6O4P2 (M+-PF6): 929.24; found:
929.24. Anal. calcd (%) for C32H48CoF18N6O4P3·H2O: C 35.18, H 4.61, N 5.39; found: C 34.96,
H 4.46, N 7.52.
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Acknowledgement
We thank the Chemistry Department, Imperial College for PhD studentships (C.L.R. and
T.K.) and the Singaporean Government for funding (A.H.M.L.).
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Table of Contents – Text and Graphics
G-quadruplex DNA structures have been identified as potential anticancer drug targets and
therefore there is significant interest in molecules with high affinity for these structures. Most
G-quadruplex binders are polyaromatic planar compounds which π-π stack on the G4’s guanine
tetrad. Herein we report a new class of binder based on an octahedral cobalt(III) complex that
interacts with G-quadruplexes via a different mode involving hydrogen-bonding, electrostatic
interactions and π-π stacking. We show that this new compound binds selectivity to G4 over
duplex DNA and has the ability to template the formation of G4 DNA from the unfolded
sequence.
G-quadruplexDNA binder