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: r.vilar@imperial.ac.uk

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:r.vilar@imperial.ac.uk

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.

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

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.

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

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

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.

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.

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.

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

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

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.

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.

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

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.

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

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.

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’

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

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.

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.

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

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.

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