A dinuclear ruthenium(ii) phototherapeutic that targets duplex and quadruplex DNA

Article abstract of DOI:10.1039/c8sc05084h

With the aim of developing a sensitizer for photodynamic therapy, a previously reported luminescent dinuclear complex that functions as a DNA probe in live cells was modified to produce a new iso-structural derivative containing Ru^II(TAP)₂ fragments (TAP = 1,4,5,8-tetraazaphenanthrene). The structure of the new complex has been confirmed by a variety of techniques including single crystal X-ray analysis. Unlike its parent, the new complex displays Ru → L-based ³MLCT emission in both MeCN and water. Results from electrochemical studies and emission quenching experiments involving guanosine monophosphate are consistent with an excited state located on a TAP moiety. This hypothesis is further supported by detailed DFT calculations, which take into account solvent effects on excited state dynamics. Cell-free steady-state and time-resolved optical studies on the interaction of the new complex with duplex and quadruplex DNA show that the complex binds with high affinity to both structures and indicate that its photoexcited state is also quenched by DNA, a process that is accompanied by the generation of the guanine radical cation sites as photo-oxidization products. Like the parent complex, this new compound is taken up by live cells where it primarily localizes within the nucleus and displays low cytotoxicity in the absence of light. However, in complete contrast to [{Ru^II(phen)₂}₂(tpphz)]⁴⁺, the new complex is therapeutically activated by light to become highly phototoxic toward malignant human melanoma cell lines showing that it is a promising lead for the treatment of this recalcitrant cancer.

Full text of DOI:10.1039/c8sc05084h

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Haycock, S. MacNeil and J. A. Thomas, Chem. Sci., 2019, DOI: 10.1039/C8SC05084H.
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A dinuclear ruthenium(II) phototherapeutic that targets duplex and
quadruplex DNA
Stuart A. Archer,a Ahtasham Raza,b Fabian Dröege, a,c Craig Robertson,a Alexander J. Auty,a Dimitri M.
Chekulaev,a Julia A. Weinstein,a Theo Keane,a Anthony J. H. M. Meijer,a John W. Haycock*b, Sheila
MacNeil*b, James A. Thomas*a
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
With the aim of developing a sensitizer for photodynamic therapy, a previously reported luminescent
dinuclear complex that functions as a DNA probe in live cells was modified to produce a new iso-
structural derivative containing RuII(TAP)2 fragments (TAP = 1,4,5,8- tetraazaphenanthrene). The
structure of the new complex has been confirmed by a variety of techniques including single crystal X-ray
analysis. Unlike its parent, the new complex displays RuL-based 3MLCT emission in both MeCN and
water. Results from electrochemical studies and emission quenching experiments involving guanosine
monophosphate are consistent with an excited state located on a TAP moiety. This hypothesis is further
supported by detailed DFT calculations, which take into account solvent effects on excited state
dynamics. Cell-free steady-state and time-resolved optical studies on the interaction of the new complex
with duplex and quadruplex DNA show that the complex binds with high affinity to both structures and
indicate that its photoexcited state is also quenched by DNA, a process that is accompanied by the
generation of the guanine radical cation sites as photo-oxidization products. Like the parent complex, this
new compound is taken up by live cells where it primarily localizes within the nucleus and displays low
cytotoxicity in the absence of light. However, in complete contrast to [{RuII(phen)2}2(tpphz)]4+, the new
complex is therapeutically activated by light to become highly phototoxic toward malignant human
melanoma cell line showing that it is a promising lead for the treatment of this recalcitrant cancer.
sensitizers. 9-18
For similar reasons, this class of compounds has also been
investigated as probes for optical microscopy. A case in point is
Introduction
A promising and emerging therapeutic regime for the treatment
of cancer is photo-dynamic therapy (PDT).1,2 In this approach a
the probe described in previous reports by the Thomas group. We
have
identified
a
dinuclear
RuII
complex,
photo-excitable sensitizer molecule is used to generate highly
reactive species, most commonly singlet oxygen, 1O2, in situ.
Whilst a number of PDT sensitizers have reached clinical trials,
there are still very few successful commercial examples
available; those that are currently employed frequently display
poor water solubility and do not target cancer cells with high
specificity, leading to generalized photosensitivity and off-target
toxic side effects.3 Moreover, these agents are often challenging
to synthesize and so can be costly. Furthermore, whilst the
[{(phen)2Ru}2(tpphz)]4+, 14+,19,20 (phen = 1,10 phenanthroline,
tpphz = tetrapyridophenazine), Scheme 1, whose biophysical
properties are related to the ubiquitous [Ru(bpy)2(dppz)]2+ “light-
switch” complex, (bpy
=
2,2’-bipyridyl, dppz
=
dipyridophenazine).21,22 Like the parent compound, 14+ is
virtually non-emissive in aqueous solution but - on high-affinity
3
binding to DNA – it displays the familiar bright, MLCT-based,
luminescence. Strikingly, the emission of 14+ is dependent on
DNA structure: whereas binding to canonical duplex DNA
induces NIR emission centred at ~680 nm, binding to G-rich,
quadruplex folded, human telomere sequence - (3’-TTAGGG-
5’)n - leads to emission at 650 nm and a greater enhancement of
emission (x150 vs. x60).19,23 Detailed subsequent studies,
including co-localization with nucleic acid stains and TEM
imaging, confirmed that 14+ is taken up by live cells, where it
localizes within the nucleus imaging heterochromatin duplex and
quadruplex structures.24
1
generation of O2 is essentially a photocatalytic process, one of
the drawbacks of this approach is that many tumours are hypoxic,
which can restrict the efficacy of PDT in these circumstances.
One alternative to conventional PDT involves the use of water-
soluble polypyridyl d6-metal complexes as sensitizers as, on
photo-excitation with visible light, these complexes often possess
long-lived triplet states. Because their synthesis from specific
metal ions and ligands is essentially modular, the photophysical
and biophysical properties of these complexes are easily tuned.4-8
and RuII complexes have attracted particular attention as
This observation is particularly significant, as quadruplex
structures have been hypothesized to have roles in a range of
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biological processes, including modulation of gene expression,
epigenetics, nucleating of DNA replication, and genetic
disease.25-30 Consequently, this has led to a plethora of research
into these non-canonical structures, including work on telomeres.
Single stranded telomere sequences are found at the end of
chromosomes and, as outlined above, are known to form
quadruplexes. Telomeres are associated with defining the
Hayflick limits for cell division: each cell division results in
shortening of the telomere sequence, until a critical length is
reached and senescence is triggered. Given these facts, it is
perhaps not surprising that telomere length maintenance, leading
to cell immortalization, is an almost ubiquitous phenomenon in
cancer cells.31,32 For this reason, selective methods to facilitate
the attrition of telomere length in cancer cells, which often
involve stabilization of quadruplex structures, have been
sought.32
As 14+ binds to both quadruplex and duplex structures within
live cells, a phototoxic analogue would function as a sensitizer
for PDT that could additionally enhance telomere attrition rates.
To carry out this transformation we changed the ancillary ligands
attached to the central RuII(tpphz)RuII moiety.
Extensive studies have demonstrated that the substitution of
conventional bpy or phen ancillary ligands with the electron
deficient ligand tetraazaphenanthrene, TAP, produces a complex
with strikingly different photo-excited state properties.33-37 For
sites in both duplex and quadruplex DNA. We also find the
complex is spontaneously taken up by live melanoma cells, where
it localizes in the nucleus and displays potent photo-toxicity,
indicating that it is a readily accessible, highly promising,
photosensitizer for the treatment of
recalcitrant cancer.
a highly malignant
Scheme 1. Structures of 14+ and 24+.
Results and Discussion
We initially set out to synthesize the TAP ligand following
established literature procedures.34,42 The diaminoquinoxaline
precursor was readily obtained using the route described by
Elmes, et al.42 However, in our hands, existing literature
procedures for the final synthesis of TAP from this intermediate
proved to be unreliable and low yielding. We found that by using
isopropanol as the reaction solvent, along with 1.5 equivalents of
glyoxal, the production of intractable side-products could be
eliminated, yielding 80% of pure TAP after work-up.
Complex 24+ was synthesized through an analogous route to
that used for 14+ (see SI for a reaction scheme and experimental
details).19 However, in coupling the two {Ru(TAP)2} cores to the
tpphz bridge, we found it was necessary to use more forcing
reaction conditions that for the related phen or bpy complexes.
This is most likely due to the more strongly electron-deficient
nature of the TAP ligand reducing the donor strength of the
chelating N lone pairs. A microwave-assisted synthesis adapted
from a previous report,39 combined with purification through
alumina column chromatography and then ion-exchange
chromatography on Sephadex-LH25 yielded analytically pure 24+
as a chloride salt in 55% yield. For spectroscopic studies, the
hexafluorophosphate was also isolated by anion metathesis.
example,
unlike
the
parent
light-switch
complex,
[Ru(TAP)2(dppz)]2+ is luminescent in water but is quenched by
binding to DNA. This is due to photo-excitation into a
3
RuIITAP MLCT excited state which photo-oxidizes guanine
sites when intercalated into duplex DNA. In cell free studies, a
related dinuclear complex was shown to be a more efficient at
photo-damaging
oligonucleotides
containing
G
than
[Ru(TAP)2(dppz)]2+ and was particularly effective at creating
photo-adducts when bound to quadruplex folded DNA.38
However, cell studies on such systems are much less developed.
Whilst it is known that, even after 24 hours exposure,
[Ru(TAP)2(dppz)]2+ solely localizes in the cytoplasm of HeLa
39
cells,
the Keyes group has demonstrated that covalent
attachment of a suitable signal peptide to a [Ru(TAP)2(bpy)]2+
unit leads to a construct capable of targeting cell nuclei. Although
this hybrid system possesses considerable phototoxicity, it is
produced on a small scale, using only 10mg of metal complex
starting material.40. Recently, the Elias group have reported
preliminary studies on mononuclear complexes containing the
RuII(TAP)2 moiety coordinated to a quadruplex targeting ligand.
Whilst this complex is phototoxic, so is its RuII(phen)2 analogue,
suggesting that the observed phototoxicity of both complexes
may be due to classic singlet oxygen sensitization.41
This report concerns an essentially iso-structural derivative of
14+ containing RuII(TAP)2 fragments. In this study we describe
the synthesis, characterization, and crystal structure of this new
complex [{Ru(TAP2)}2(tpphz)]4+, 24+, which can be isolated in
good yields.
Experimental and computational studies reveal that 24+ displays a
RuTAP MLCT excited state and cell free studies confirm that
the complex binds to both duplex and quadruplex DNA.
Photophysical studies confirm that the complex is capable of
photo-oxidizing guanosine monophosphate, GMP and guanine
Fig. 1 Thermal ellipsoid diagram showing the Δ,Δ cation of the [2]Cl4
structure. Hydrogen atoms have been omitted for clarity.
Crystals suitable for x-ray crystallography were grown by slow
vapour diffusion of acetone into a saturated solution of 24+ in
methanol. Although the quality of the data (R = 9.27) prevents a
full analysis of bond length and angles, it does confirm
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connectivities. Interestingly, like the corresponding structure of
Two of the chloride counter ions are disordered, and a number
of highly disordered water molecules are present within a solvent
cavity, which were accounted for in the refinement using a
solvent mask.
[{(bpy)2Ru}2(tpphz)]4+,43 the resultant unit cell contains two
stereoisomers of 24+ - the  and  forms - with no evidence
of the meso  form apparent in the crystal data. One of these
cations (the Δ,Δ cation) is shown in Figure 1.
Table 1. Photophysical data for complex 24+
Complex
Absorption
Emission
Quantum Yield
Lifetime (ns)
max (nm)


max (nm)
(L mol-1 cm-1)
41,800
[2].Cl4
423
452
628
0.05 (Water)
0.04 (Phosphate)
0.07 (TRIS)
0.04
650 ± 24
575 ± 37
723± 23
550 ± 30
33,300
b
[2].(PF6)4
423
452
41,800
34,800
613
a Measured in H2O. b Measured in MeCN
The distorted octahedral geometry around each ruthenium (II)
core is essentially identical, with a N1-Ru1-N6 angle of 171.8°
and the average bond length for the 4 TAP ligands of 2.052 A.
The tpphz bridging ligand displays a marked twist –the angle
between the planes of the two “bpy” moieties of the tpphz gives a
twist of 10.4°. As might be expected, the observed bond angles
and lengths are comparable to those of [{(bpy)2Ru}2(tpphz)]4+.
The UV/Vis absorption spectra of [2](PF6)4 and [2]Cl4 were
measured in acetonitrile and water respectively. Apart from the
expected high-energy intraligand bands, the complex possesses
an absorption between 400–500 nm, an energy that is typical for a
RuL 1MLCT transition, see Fig. 2 and Table 1, but it is
substantially more intense than the equivalent transition for 14+.
The energy of the 1MLCT does not significantly change in
acetonitrile compared to aqueous environments, although changes
in relative intensities within the band suggests that it is composed
of at least two intense, overlapping transitions.
conditions, with only a small blue shift in max alongside an
approximately 20% decrease in emission lifetime and quantum
yield. These observations reveal a significant difference in the
lowest excited state of 24+ compared to 14+, suggesting that – as
expected - the emissive state of 24+ is a TAP-based 3MLCT rather
than the tpphz-based 3MLCT of 14+. As expected from these
observations, emission quantum yield are similar, regardless of
solvent environment.
Fig. 3 Cyclic voltammogram for 1 mM [2](PF6)4: in dry DMF with 0.1M
N(n-Bu)4PF6 supporting electrolyte. Red and blue lines show isolated sets
of reductions.
The electrochemical properties of 24+ were investigated in dry
acetonitrile. The complex displays
a single oxidation at
Fig. 2 UV/Vis absorption spectra (left) and emission spectra (right) of
[2]Cl4 and [2](PF6)4 in water, acetonitrile and phosphate buffer (10 mM
KH2PO4, 200 mM KCl, pH 7.2, 20 mM EDTA).
approximately 1.8 V vs. Ag/AgCl, which appears to be
reversible, although it occurs at the edge of the solvent window.
This potential is almost identical to that reported for the
mononuclear [Ru(TAP)2dppz]2+ complex34 (+1.82 V vs.
Ag/AgCl), and is therefore assigned to the expected reversible
RuII/III oxidation. The reduction of the complex in acetonitrile
results in a large stripping peak on the return sweep. This
behaviour has been previously observed for dinuclear complexes
with a tpphz bridge44 and suggests the reduced form of the
The emission spectra of [2](PF6)4 and [2]Cl4 were also
measured in acetonitrile and water respectively (Figure 2, Table
1). The complex displays bright emission between 600-750 nm in
both solvents, which is in direct contrast to the properties of 14+,19
which is quenched in aqueous media.44 The emission wavelength
and lifetimes in acetonitrile were very similar to those in aqueous
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complex displays low solubility in MeCN. Therefore, the
reduction potentials were also measured in dry DMF (Figure 3).
Several reduction waves are observed in these conditions. Two
distinct reversible reductions occur at -0.70 V and -0.86 V, with
further poorly resolved couples occurring between -1.4 to -1.8 V.
Given that the emissive state of [2]Cl4 is not quenched in water,
which implies a TAP-based lowest excited state, the reduction at
-0.70 V is assigned to the first reduction of the TAP ligand, which
is similarly observed in the mononuclear Ru-TAP analogues.33,45
A second reduction process at -0.86 V is also consistent with the
reduction of a TAP ligand coordinated to the second Ru centre.
Through comparison to related complexes, the overlapping redox
processes between -1.4 V to -1.8 V are assigned to a combination
of reductions of the tpphz bridging unit, and further reductions of
TAP ligands.
needed to describe the electronic structure of these compounds in
water correctly, especially since it is known that hydrogen
bonding plays an important role in the excited states dynamics of
complexes such as 24+.
The final optimized structures for the triplet states are given in
Figure 4, with the spin density super-imposed on the final
structure. In these calculations, the singlet (ground-state)
structures are indistinguishable from the triplet structures and can
be found in the SI.
The spin densities and geometries in Figure 4 show very
clearly that, although replacing phen with TAP has little
structural effect, the substitution leads to significant electronic
changes. In particular, the spin density clearly shows that for 24+
(irrespective of the solvent) the triplet excited state has its density
on one of the TAP ligands, whereas for 14+ it is situated on the
tpphz, confirming our experimental observations (vide supra).
Close inspection of the LUMO and LUMO+1 orbitals shows that
these are part of a (near-) degenerate pair, so that small geometric
distortions may alter the specific TAP on which the excited state
localizes.
In the calculations associated with Figure 4(c), water
molecules were placed in hydrogen-bonding positions around 24+.
These interactions will be associated with many orientations of
very similar energies. However, an exhaustive search on this
issue is outside the scope of this paper and will most likely lead
to very similar results. Nevertheless, it is interesting to note that
two water molecules bonding to the central tpphz moiety, which
were originally placed in a hydrogen-bonding position, rotated
and are in a hydrogen-accepting orientation in the final structure.
This observation suggests that the nitrogen atoms of the tpphz
bridge are not accessible to hydrogen-bonding, indicating that
some of the deactivation pathways available to similar complexes
incorporating the RuII(dppz) unit cannot be accessed by 24+.
Using the singlet geometries, UV-VIS spectra were simulated
through TD-DFT calculations, which show qualitative agreement
with the experimental absorption spectrum (see SI Figure S-9).
However, our calculations also show that there are many
transitions underlying absorption bands in the visible region. To
investigate whether the nature of the transitions is affected by
changing the solvent from MeCN to water, a wave function
analysis48-50 was performed on the highest fifty states in the TD-
DFT calculation. The strongest five transitions from this analysis
are also represented graphically in the SI (see Figure S-10).
The wave function analysis indicated that transitions in both
solvents occur at similar energies, resulting in what appear to be
the near-identical experimental UV-VIS spectra shown in Figure
2. However, it is also clear that the nature of each of these
transitions is very different.
Again, the analysis yields results that are consistent with the
experimental data. For example, in MeCN and water, transitions
with similar energy - at 425.8 and 421.0 nm, respectively - are
predicted. Yet, whilst this transition in MeCN is assigned to a
MLCT from both Ru atoms onto the tpphz bridge, in water the
transition is predicted to be a MLCT from the Ru atoms onto the
TAP units. Interestingly, some of the transitions are completely
different in their nature. For example, transitions at 389.1 nm and
386.5 nm in MeCN are again MLCT transitions onto tpphz, but in
water, where these transitions occur at 385.0 nm and 381.0 nm,
Fig. 4 Lowest triplet structures for 14+ in MeCN [panel (a)], 24+ in MeCN
[panel (b)], and 24+ in water [panel (c)]. Blue indicates  spin, whereas
red indicates  spin.
To elucidate our results further a series of density functional
calculations using Gaussian0946 was performed employing
methods and procedures outlined in the supporting information.
Calculations were performed on 14+ in acetonitrile and on 24+ in
both acetonitrile and water. In these calculations, we included
solvent using a continuum model for MeCN. However, from
previous work,47 it is clear that some explicit water molecules are
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respectively, they are inter-ligand transitions from tpphz onto
TAP for the latter transition and a mix of inter-ligand transitions
and an MLCT transition for the former transition.
(which includes geometric relaxation) were at 607 and 637 nm,
respectively. In both cases, there is a correlation with both the
ordering and approximate energies of experimentally measured
spectra as shown in figure 2.
Finally, the behaviour upon reduction of these complexes was
investigated computationally. The differences in electron density
for the first and second reduction of 24+ in both MeCN and water
were calculated and are shown in Figure 5.
Concentrating on the panels (c) and (d) first, the calculations
are in agreement with the experimental data. They clearly
indicate that both reductions are mainly centred on the TAP
ligands. It is also clear that the water molecules do not play a
direct role in the reduction, in that there is no density difference
associated with them. However, water molecules do respond to
the reduction by adapting their orientation with respect to 24+ as is
clear from panel (d), where the water molecules bonded to the
tpphz ligand in 23+ are polarized towards the reduced TAP ligand.
The situation in MeCN as shown in panels (a) and (b) of
Figure 5 is somewhat different. Here, the first reduction is of the
tpphz ligand, whereas the TAP ligands are reduced in the second
reduction. This is in line with the virtual orbitals of 24+ in MeCN,
but is different from what happens upon photo-excitation, where
the triplet state is clearly located on the TAP ligand. Clearly,
these virtual orbitals are close enough to facilitate this re-
arrangement upon reorganization of electron density. It should be
pointed out that this result is slightly different to the observed
electrochemistry for [2](PF6)4; however, the experimental data
were collected in DMF and it the calculations indicate that the
electronic structure of this system is solvent dependent.
Given the interesting redox properties of 24+ revealed by the
experimental data and computational studies, the photo-oxidizing
properties of the complex were investigated.
Fig. 5 Difference density maps for 22+ in acetonitrile [panels (a) and (b)]
and water [panels (c) and (d). Panel (a): Differential density for reduction
from 24+ to 23+ in MeCN. Panel (b): Differential density for reduction
from 23+ to 22+ in MeCN. Panel (c): Differential density for reduction
from 24+ to 23+ in water. Panel (d): Differential density for reduction from
23+ to 22+ in water. Blue indicates increase of electron density, whereas
red indicates loss of electron density. (See SI for the methodology used.).
The calculated triplet geometries and corresponding singlet
states were also used to predict the emission wavelength
assuming no geometrical relaxation. In MeCN, the emission
maximum of 24+ was calculated to be at 647 nm and in water the
corresponding wavelength was predicted to be 677 nm. The
corresponding emission wavelengths for the 0-0 transitions
Fig. 6 Stern-Vollmer plot for the emission of [2]Cl4 quenched by the
addition of guanosine monophosphate, showing change in lifetime and
emission intensity.
It has previously been demonstrated that [Ru(TAP)2(dppz)]2+ and
related complexes photo-oxidize DNA through electron transfer
from guanine sites to the photo-excited complex, quenching its
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33,34,36 35,51
emissive state.
Therefore the effect of increasing
the duplex. This is confirmed by the fact that addition of Poly(A)-
Poly(T) causes a slight increase in emission (see SI).
concentrations of nucleotide monophosphates on the emission
spectrum of 24+ was investigated and as expected these revealed
that only addition of GMP led to emission quenching (Figure 6).
The difference between I0/I and τ0/τ as a function of quencher
concentration indicate that emission changes are caused by both
dynamic and static quenching processes. Such effects often occur
through stacking interactions between nucleotides and
luminophores52,53, therefore this possibility was investigated
using 1H-NMR.
Although distinct changes in the chemical shifts for a number
of protons on 24+ were observed on addition of GMP, these were
difficult to quantify as they were accompanied by significant
signal broadening. However, significant chemical shifts were
simultaneously observed for several GMP signals. Notably, the
purine proton at the 8-position shifted from 7.95 to 8.06, and the
1’ sugar proton 5.60 to 5.75. Fitting these latter changes to a
simple 1:1 guest-host binding model gave a Ka of 8.3 x 102 M-1. It
is known that large anions can interact with dinuclear complexes
by binding into the cleft defined by ancillary and bridging
ligands. Indeed, this has formed the basis of chromatographic
separation of stereoisomers of such complexes.54-56 Thus, the
observed NMR shifts indicate that GMP interacts with 24+ in this
manner.
DNA-induced changes were also observed in the absorption
spectrum of 24+ – Figure 7B. The MLCT transition at 420 nm
shows a significant decrease in intensity throughout the titration.
Approaching the end point of the titration, a new shoulder at 460
nm appears to grow in, suggesting that binding to CT-DNA may
affect the energy of excited states. This is consistent with the
previously described DFT calculations indicating that order of the
close lying excited states of the complex is sensitive to its
environment.
Attempts to fit the titration data to the well-known McGhee-
von Hippel model58 were unsuccessful; it is possible that this is
due to the presence of multiple binding modes. However, the
absorbance data fitted well to the simpler binding model
described by Srishailam, et al,7 Figure 7C, resulting in an
estimated Kb of 2 x 106 M-1, which is of the same order of
magnitude as isostructural 14+,19 implying the addition of the
heteroaromatic nitrogens to the ligands does not significantly
affect binding to duplex DNA.
Previous work within the Thomas group has shown that 14+
binds to quadruplex-folded human telomere sequence (HTS) with
high affinity.19,20,23 Therefore, analogous experiments were
carried out with 24+ using the same experimental conditions.
HTS-induced changes in emission spectra are similar to those
observed on CT-DNA addition; as HTS is G-rich quenching by
G-sites is again expected– Figure 7D. However, a closer
inspection reveals differences in the absorption changes induced
by binding to HTS and CT-DNA – Figure 7E. In particular,
whilst an initial decrease in absorption of the 423 nm band is
observed on addition of HTS, the subsequent grow-in of the band
at 460 nm observed in duplex binding does not occur, suggesting
that binding to quadruplex does not affect the energy of excited
states of 24+ in the same way as duplex binding.
The emission titration data was also best fitted to a one-set-of-
sites binding + non-specific interaction model, – Figure 7F. This
yielded an estimated binding constant of 5.1 x 105 M-1, which is
an order of magnitude lower than the binding constant to the
duplex and twenty-fold lower than the binding of 14+ to HTS.
The difference in quadruplex binding affinities between the
two complexes may be explained by a consideration of our
previously reported NMR–based HTS/14+ structure, which shows
close contacts between the ancillary ligands of 14+ and the
diagonal loops of the quadruplex structure. It seems the lone pairs
of the aromatic nitrogen of the ancillary TAP ligands destabilizes
this interaction through unfavourable interactions with similar
basic sites within the diagonal loop.
The emission quenching data were then fitted using the
relationship: 57
퐼0
(
)(
)
= 1 + 푘푞휏0[푄] 1 + 퐾푎[푄] .
퐼
Where, kq is the quenching constant, 0 is the emission lifetime
of 24+ without a quencher, [Q] is quencher concentration and Ka
the association constant. This analysis gives a quenching constant
of 1.2 x 108 M-1 s-1. No significant emission quenching was
observed on addition of A, T and C monophosphates confirming
that - as expected from previous studies - quenching was
exclusive to GMP
For the interaction of 24+ with both duplex and quadruplex
DNA was the investigated through absorption and luminescence
titrations. To aid comparisons, all titrations in these studies (and
the SI) were carried out in high ionic strength phosphate buffer,
which is consistent with quadruplex folding (10mM KH2PO4,
200mM KCl, 0.1M EDTA, pH 7.0). Absorption measurements
were taken in parallel, both to assess any changes in the
absorption spectrum itself and to correct the emission intensities.
As expected from the GMP experiments and similar studies on
[Ru(TAP)2(dppz)]2+, complex 24+ shows emission quenching on
binding to CT-DNA – Figure 7A.
consistent with a RuIITAP-based 3MLCT excited state and
34,36
Again, this observation is
indicates the expected photoredox quenching by G-sites within
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Fig. 7 CT-DNA and HTS titration data for [2]Cl4 (5 M concentration, in phosphate buffer A) Changes in emission spectra upon addition of CT-DNA. B)
Changes in absorption spectra with CT-DNA. C) Binding curve for the data obtained with CT-DNA based on absorption data– red line shows fit to model
developed by Srishailam, et al.7 D) Changes in emission spectra upon addition of HTS. E) Changes in absorption spectra of [2]Cl4 upon addition of HTS.
F) Binding curve for [2]Cl4 with HTS based on emission data– red curve shows fitting to a one-set-of-sites binding + non-specific interaction mode.
To further investigate the changes in the excited state of DNA
bound 24+ transient absorption experiments were carried out.
Absorption transients were measured in phosphate buffer at a
concentration of 0.1mM complex – Figs 8 and 9. Global lifetime
analysis of the TA data was carried out using Glotaran 1.5.1,
with kinetics of selected processes fitted using Origin 8.0.
Previous studies on Ru complexes of dppz and tpphz have
revealed that intense transient bands in the 500-800 nm region are
produced by charge transfer to the phenazine moiety of the
ligands.36,60-63 However, the computational and steady-state
optical studies described above suggested that the lowest excited
state for complex 24+ is a RuTAP MCLT. These conclusions
are further supported by the transient spectra for [2]Cl4 which
show that the strong ground-state bleach of the MLCT absorption
bands between 400-500 nm is accompanied by the growth of a
weak transient between 500-650nm, changes which occur
immediately following the pump pulse (within 1 ps) – Fig 8A.
These observations are consistent with previous studies on
[Ru(TAP)2(dppz)]2+ in solution - which also shows a weak,
broad, largely featureless transient absorption between 500-650
nm – and confirm that 24+ displays a TAP centred excited
state34,64
59
There are no significant changes in spectral shape throughout
the time-window of the experiment (7900 ps). Global fitting of
the spectra using 4-exponential decay parameters gave the most
reliable fitting of the data. The two fastest components of under
~1 ps, are followed by simultaneous decay of the entire spectrum,
with components of 355 ps and ~100 ns. The slow component is
assigned to the emissive excited state observed above. The 355 ps
component is likely to be due to conversion of a multitude of
initially-formed excited states to the long-lived RuTAP 3MLCT
state, as lifetimes of a similar order of magnitude have been
previously observed for [Ru(TAP)2(dppz)]2+ and related
complexes.
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Fig. 8 Selected transient absorption data for [2]Cl4 following ~40 fs, 400 nm excitation. A) In phosphate buffer. B) In phosphate buffer with 1 mM CT-
DNA. C) In phosphate buffer with 1 mM HTS.
Significantly, in both the CT-DNA and HTS experiments,
but not a distinctive signal at approximately 515 nm emerges –
Fig 9. A very similar transient is observed when DNA is added
to [Ru(TAP)2(dppz)]2+ and this was assigned to the generation
The excited state dynamics of 24+ in the presence of CT-
DNA and HTS are complex, with global lifetime analysis
suggesting a minimum of four distinct excited state processes.
The lifetimes of these states are of similar magnitude in the
presence of both CT-DNA and HTS (Table 2).
of the guanine radical cation as
a photo-oxidization
product.35,36,64,65 Interestingly, this feature is appreciably more
prominent on addition of HTS, which as a sequence possesses a
higher density of G-residues compared to CT-DNA, and is
therefore more likely to undergo photo-oxidation on exposure
to light.
Table 2. Lifetimes obtained from global lifetime analysis of
transient absorption data
Lifetimes (ps)
CT-DNA
3.6 ± 0.4
275 ± 12
2900 ± 150
>30,000
HTS
2.9 ± 0.2
330 ± 17
2930 ± 130
> 30,000




5
Time Delay(ps):
CT-DNA11ps
Phosphate 11ps
G311ps
4
3
2
1
0
The decay-associated spectra (see SI) for each excited state
process feature a grow-in signal at ~510 nm, associated with
the photooxidation of guanine. This implies that this photo-
oxidation process can occur in parallel from multiple excited
states. Unfortunately, there is insufficient detail in the transient
absorption data to accurately model these processes. However,
by isolating and fitting the feature at 510 nm using a 3-
exponential model, the major lifetime component of this
growth is 1808 ± 100 ps for CT-DNA and 1592 ± 90 ps for
HTS. The faster kinetics for HTS is consistent with the
anticipated intimate contact between 24+ and guanine tetrads of
the four-stranded structure.
The intricacies of the dynamics reflect the complexity of
binding modes and the numerous potential photochemical
pathways following photoexcitation. Assuming the lowest
triplet excited state of the complex is a RuTAP 3MLCT state
(as confirmed by the calculations), then subsequent
simultaneous photooxidation of guanine, radiative and non-
radiative decay, as well as c of the oxidised guanine residues
will all contribute to the spectral evolution. Significantly more
500
550
600
650
Wavelength (nm)
5
4
3
2
1
Time Delay(ps):
Phosphate 1489ps
G31489ps
CT-DNA1489ps
0
5
500
550
600
650
Time Delay(ps):
Phosphate 7900ps
4
3
2
1
0
CT-DNA7900ps
G37900ps
500
550
Wavelength (nm)
600
650
Fig. 9 Selected transient absorption spectra for [2]Cl4 in phosphate
buffer with and without CT-DNA and HTS, showing the growth of the
transient band at ~515 nm.
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Chemical Science
detailed analyses, such as time-resolved infrared spectroscopy,
bright intracellular emission. Furthermore, co-staining using
24+ (100 µM, 24hours) and the standard nuclear stain DAPI,
reveals DAPI co-localizes with the new complex, displaying a
Pearson coefficient of 0.51. These observations confirm that -
like its close analogue 14+ - complex 24+ is cell and nuclear
membrane permeant. However, unlike 14+, treatment with 24+
also leads to bright emission from the cytoplasm of the
melanoma cells, therefore analogous co-staining experiments
with commercial stains were carried out, Figure 10.
will be required to fully dissect the subtleties, which will form
the basis of a future report. Nevertheless - although it is clear
that 24+ preferentially binds to duplex over quadruplex DNA -
the cell-free studies described above confirm that this complex
does participate in photo-induced redox reactions with G-sites,
whether they are found in duplex or quadruplex structures.
With these promising results in mind, the live cell uptake
and localization of 24+ was investigated. For reasons of
therapeutic need, malignant human melanoma cells were
specifically chosen for these studies.
Melanoma is one of the most aggressive and therapeutically
resistant cancers. If diagnosed and treated in its early stages it
has an 80% 10-year remission rate. However, if it spreads to
the lymph nodes this figure drops to only 10%.66 In 2017
malignant melanoma was responsible for 72% of skin cancer
deaths in the USA and its incidence continues to rise.67 One of
the difficulties in treating melanoma is that it displays a range
of therapeutic resistance mechanisms,68 so alternative treatment
regimes are being actively sought.69 In this context, it has been
suggested that PDT could provide a novel therapeutic modality
for this cancer.66,70 We chose to investigate the effect of 24+ on
human C8161 melanoma cells as this line is known to be
highly invasive and spontaneously metastatic71, and thus it
represents a relevant and therapeutically challenging treatment
target.
Fig. 11 Phototoxicity of 24+ on melanoma. human melanoma cells
(C8161) were treated at a series concentration of 24+ (0, 10, 50, 100,
200 µM). Photo-toxicity was assessed using a 405 ± 20nm wavelength
light from a ThorLab LED lamp (M405LP1, power output 1500mA) on
exposure to fluences of 6 J cm-2, 12 J cm-2 and 18 J cm-2, respectively.
No cell death was observed with an increase in 24+ compound
concentration. However, cell viability decreased significantly after
exposure to 405 ± 20nm light with pre-treated melanoma cells (e.g. 100
µM with 1 hour (▪) light activation causes > 50% reduction in cell
viability). LD50 value calculated (for each line interpolate sigmoidal)
using Prism software. The error bars denotes SEM (n=3).
These studies revealed some correlation between the
emission of the complex and that of the MitoTracker Green and
LysoTracker Green labels. However, calculated Pearson
coefficients of 0.12 and 0.06, respectively, indicate localization
of 24+ within these organelles is significantly lower than within
nuclei. This is consistent with recent super-resolution and TEM
studies72 that revealed that, while 14+ is only brightly emissive
from the nucleus, it does exhibit significant mitochondrial
localization. The difference in the intracellular luminescence
of the complexes can be attributed to the fact that whilst 14+
displays off/on DNA light-switch emission, 24+ is emissive,
unless quenched by photo-redox processes.
To facilitate a more detailed understanding of cellular
uptake, the concentration- and time-dependent uptake of 24+
was explored – See SI. It was found that intracellular emission
intensity correlates with increased concentrations and exposure
times. These studies confirmed that the complex readily
diffuses into cells, where initially it predominately labels the
nucleus, but then diffuses more generally throughout the cell.
Having established uptake by live melanoma cells and
specific sub-cellular localizations, the cytotoxicity and
phototoxicity of 24+ was then investigated. Cell viabilities after
exposure to different concentrations of the complex in the dark
after 24 hours were quantified using the AlamarBlue method -
Figure 11.58-60
Fig. 10 Intracellular localization and uptake of [2]Cl4 in human C8161
melanoma cells – labelled Ru-Ru TAP in diagrams. Co-staining with
cytoplasm stains LysoTracker Green (Top) and MitoTracker Green
(bottom) show low colocalisation with [2]Cl4. (Pearson coefficient =
0.06±0.01, and 0.12±0.04, respectively, SB 10µm). Co-staining with
nuclear stain DAPI (centre) reveals a significantly higher co-
localisation (Pearson coefficient = 0.51±0.19, scale bar = 10µm).
Encouragingly, it was found that the complex was
spontaneously taken up by live C8161 cells, and it produced
These studies indicated that the initial seeding density of
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Page 10 of 13
1x105 cells/well does not change on treatment with the
complex. Indeed, at all concentrations, no statistical difference
in cell viability was observed compared to cells of non-treated
controls, suggesting the complex displays negligible
cytotoxicity in dark conditions (see SI). Experiments were then
carried out in which melanoma cells treated with various
concentrations of complex were irradiated into the MLCT band
using a 405 ± 20 nm LED laser. Light treated cells were then
incubated for 3 hours, and a change in cell viability measured,
and plotted against various concentrations of 24+.
Irradiation at low fluences, with a maximum of only 18 J
cm-2, showed a dose-response hyperbolic curve, revealing a
radical drop to effectively zero cell-viability at exposure to
100uM of 24+, compared to non-irradiated cells. A similar
pattern of concentration-dependent cell death was also seen on
exposure to a broad-spectrum blue light, a regime commonly
employed by dermatologists in the treatment of topical skin
conditions such as acne.73 We note that this treatment required
longer time exposures, but this is probably due to differences in
power output. Nonetheless, the new complex is therapeutically
active at fluences that are around an order of magnitude lower
than those commonly employed for commercial sensitizers in
the treatment of cancer, including basal cell skin cancer. 74-77
telomere targeted PDT; work on targeting such structures is
currently underway.
Conflicts of Interest
There is no conflict of interest to declare.
Acknowledgements
We are grateful to the EPSRC for postdoctoral funding of SAA
and AR (EP/M015572/1). TK acknowledges support from a U
of S/EPSRC Doctoral Fellowship Prize scheme. We also thank
EPSRC for a Capital Equipment award, which provided the
Lord Porter Laser Laboratory facility used in this study. FD
thanks the EU’s ERASMUS+ scheme for financially
supporting his visit to Sheffield. A license for the OpenEye
tools, obtained via the free academic licensing program, is
gratefully acknowledged.
Notes and references
a Department of Chemistry, University of Sheffield, Brook Hill,
Sheffield, S3 7HF, UK Tel: 44 114 222 9325; E-mail:
james.thomas@sheffield.ac.uk
a Materials Science & Engineering, University of Sheffield, Mappin St,
Sheffield S1 3JD, UK; E-mail: j.w.haycock@sheffield.ac.uk;
s.macneil@sheffield.ac.uk
Conclusions
c Current address: Institute of Physical Chemistry, Helmholtzweg 4, D-
07743 Jena, Germany
Complex 24+ displays similar photophysical properties to
previously reported systems based on the (TAP)2RuII moiety in
that it displays a reactive RuTAP MLCT excited state that
photo-oxidizes guanosine moieties. Steady-state and time-
resolved studies indicate that the complex participates in
similar process when bound to both duplex and G-rich,
quadruplex DNA. Crucially, this new compound is
spontaneously taken up by live melanoma cells, largely
localizing in nuclei. The complex shows no toxicity under dark
condition, even at high concentrations (e.g. 200 M). However,
once sensitized with light, it becomes highly toxic to human
melanoma cells, making it an efficient and promising lead as a
photosensitizer for PDT.1
Complex 24+ binds to a quadruplex structure with affinities
that are around an order of a magnitude weaker than those
displayed for duplex binding. Given that conventional duplex
DNA vastly outnumber putative quadruplex sequences within
the genome,78-81 it seems likely that the photo-cytotoxic effects
of this complex is largely due to damage to duplex sequences.
However, damage to quadruplex structures could still be
disproportionately large. Previous studies using photo-redox
active metal complexes have demonstrated that G-rich
sequences, particularly runs of neighbouring G-sites, are
susceptible to redox damage, 51,82 even when distal to the metal
complex binding site 83-86. Furthermore, experimental evidence
† Electronic Supplementary Information (ESI) available, which
includes additional references to relevant primary literature for this
research:90-98 Experimental methods and data, cell studies details,
computational
methods,
tables
and
figures,
.
See
DOI: 10.1039/b000000x/
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