Towards the Development of Photo-Reactive Ruthenium(II) Complexes Targeting Telomeric G-Quadruplex DNA

Article abstract of DOI:10.1002/chem.201804771

The design and characterization of new ruthenium(II) complexes aimed at targeting G-quadruplex DNA is reported. Importantly, these complexes are based on oxidizing 1,4,5,8-tetraazaphenanthrene (TAP) ancillary ligands known to favour photo-induced electron

Full text of DOI:10.1002/chem.201804771

DOI: 10.1002/chem.201804771
Full Paper
&
DNA structures |Hot Paper|
Towards the Development of Photo-Reactive Ruthenium(II)
Complexes Targeting Telomeric G-Quadruplex DNA
Justin Weynand,[a, b] AurØlie Diman,[c] Michal Abraham,[a] Lionel MarcØlis,[a] HØl›ne Jamet,[b]
Anabelle Decottignies,[c] JØrôme Dejeu,[b] Eric Defrancq,*[b] and Benjamin Elias*[a]
Abstract: The design and characterization of new rutheniu-
m(II) complexes aimed at targeting G-quadruplex DNA is re-
ported. Importantly, these complexes are based on oxidizing
1,4,5,8-tetraazaphenanthrene (TAP) ancillary ligands known
to favour photo-induced electron transfer (PET) with DNA.
The photochemistry of complexes 1–4 has been studied by
classical methods, which revealed two of them to be capa-
ble of photo-abstracting an electron from guanine. From
studies of the interactions with DNA through luminescence,
circular dichroism, bio-layer interferometry, and surface plas-
mon resonance experiments, we have demonstrated the se-
lectivity of these complexes for telomeric G-quadruplex DNA
over duplex DNA. Preliminary biological studies of these
complexes have been performed: two of them showed re-
markable photo-cytotoxicity towards telomerase-negative
U2OS osteosarcoma cells, whereas very low mortality was
observed in the dark at the same photo-drug concentration.
Introduction
sponds to the coplanar arrangement of four guanine bases
held together by Hoogsteen hydrogen bonds and stabilized by
physiologically abundant Na+ or K+ cations. G-quartets can
stack to form G-quadruplexes, which can adopt a wide variety
of topologies according to the number of strands involved in
the structure, the strand direction, and variations in loop size
and sequence.[2–4] Sequencing and bioinformatics analyses of
the human genome indicate that it contains as many as
700000 sequences of potential G-quadruplex structures (PQS).
Interestingly, these putative G-quadruplex-forming sequences
are not distributed randomly in the genome. Indeed, a statisti-
cally significant enrichment of PQS has been found in several
relevant domains of the human genome, including the telo-
meric region and promoter regions of a number of genes,
such as the proto-oncogenes c-Myc, c-Kit, bcl-2, and KRAS, as
well as in viruses.[5,6] Strong arguments have recently been pre-
sented in favour of the formation of G-quadruplex DNA struc-
tures within cells by using G-quadruplex antibodies as well as
binding-activated fluorescent G4-targeting ligands.[7–9] Several
G4-binding regulatory proteins have been identified, and G4
formation is now suspected to be involved in numerous patho-
genic processes, including degenerative disorders, oncogene
regulation, and viral infections. Taken together, these data con-
sistently point to a biologically relevant regulatory role for G-
quadruplexes.
DNA is considered as an interesting target for developing
novel classes of therapeutic agents. Until recently, the focus
has been on double-stranded DNA structures (duplex DNA), in
which two sequences of DNA are held together in an antiparal-
lel double-helical architecture through canonical Watson–Crick
A/T and G/C base pairing. Duplex DNA has mainly been target-
ed by using intercalators (i.e., small molecules that interact
with DNA through intercalation between two adjacent base
pairs) or groove binders (i.e., small molecules that interact with
DNA in the minor and/or major groove regions).[1] More recent-
ly, targeting alternative DNA architectures, in particular G-
quadruplexes (G4s), has been increasingly pursued. G-quadru-
plexes are secondary DNA structures found in guanine-rich se-
quences. The basic unit is called a G-quartet, which corre-
[a] J. Weynand, M. Abraham, Dr. L. MarcØlis, Prof. Dr. B. Elias
UniversitØ catholique de Louvain (UCLouvain)
Institut de la Mati›re CondensØe et des Nanosciences (IMCN), Molecular
Chemistry, Materials and Catalysis (MOST)
Place Louis Pasteur 1, bte L4.01.02, 1348 Louvain-la-Neuve (Belgium)
E-mail: Benjamin.Elias@uclouvain.be
[b] J. Weynand, Dr. H. Jamet, Dr. J. Dejeu, Prof. Dr. E. Defrancq
UniversitØ Grenoble-Alpes (UGA)
DØpartement de Chimie MolØculaire, UMR CNRS 5250, CS 40700
38058 Grenoble (France)
E-mail: Eric.Defrancq@univ-grenoble-alpes.fr
In this context, the biological functions of G4s are certainly
the most documented for the telomeric region. Human telo-
[c] Dr. A. Diman, Prof. Dr. A. Decottignies
5’
meric DNA is made of a repeat of the sequence TTAGGG3’ and
UniversitØ catholique de Louvain (UCLouvain) de Duve Institute
Avenue Hippocrate 75 1200 Brussels (Belgium)
it is widely accepted that telomeric DNA plays important roles
in the development of cancer cells.[10,11] In healthy cells, the te-
lomere shortens after each cell division, and when a limit is
reached (i.e., the Hayflick limit), the cell enters into senescence.
In most cancer cells, however, the telomere length is main-
Supporting information and the ORCID identification numbers for the au-
thors of this article can be found under:
https://doi.org/10.1002/chem.201804771: Copies of NMR and mass spectra,
absorption and luminescence spectra, cyclic voltammograms, CD, BLI, and
SPR sensorgrams and microscope images of cells.
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tained, leading to replicative immortality. Two different pro-
cesses are involved in telomere maintenance: (i) overexpression
of the telomerase enzyme, which adds copies of telomeric re-
peats at the chromosome ends,[12] and (ii) the homologous re-
combination-based mechanism, termed alternative lengthen-
ing of telomeres (ALT).[13,14] Telomerase overexpression is ob-
served in almost 85% of cancers, whereas the ALT mechanism
is active in 5–10% of cases (in particular in osteosarcoma and
glioblastoma).
(ii) DNA photo-cleavage (type I photo-oxidation).[43–45] A photo-
reaction process leading to bridging of two guanine bases of a
quadruplex oligonucleotide by the rigid dinuclear complex
[(TAP)2Ru(tpac)Ru(TAP)2]4+ has also been reported.[46]
In the present study, three new photo-oxidizing rutheniu-
m(II) complexes (Scheme 1) that selectively target G-quadru-
Over the past decade, a number of small molecules (termed
G-quadruplex ligands), displaying varying degrees of affinity
and more importantly selectivity (i.e., an ability to interact only
with quadruplex DNA and not with duplex DNA), have been
designed to target G-quadruplex DNA.[15–19] Most of these mol-
ecules have been built from an aromatic core, capable of inter-
acting with G-quadruplex motifs through p-stacking, and deco-
rated with substituents (often positively charged) that can in-
teract with G-quadruplex grooves and/or loops so as to im-
prove affinity for G4s as well as the selectivity over duplex
DNA. The G4 ligands approach is now considered to be a
useful molecular tool to enhance and/or promote quadruplex-
related biological effects in cells and shows high potential for
future therapies.[20] To the best of our knowledge, however,
very few studies have been devoted to the design of photo-re-
active probes targeting G4s that could be interesting for pho-
totherapy development. Freccero and co-workers have modi-
fied the well-known G4 ligand naphthalene diimide (NDI) with
a phenol moiety that produced phenoxyl radicals upon irradia-
tion.[21] They demonstrated the ability of this NDI-phenolate
conjugate to kill MCF7 cancer cells after irradiation.
Scheme 1. Synthesis of A) CPITAP ligand and B) RuII complexes 1–4.
plex DNA over duplex DNA have been synthesized. They are
based on the 2-(4-chlorophenyl)-1H-imidazo[4,5-f][1,10]phenan-
throline (CPIP) ligand, some ruthenium(II),[47] platinum(II),[48] or
iridium(III)[36] complexes of which have previously been report-
ed for selective DNA switch. However, none of these com-
plexes has shown an ability to induce photo-electron transfer
(PET) with guanine units. With the aim of achieving this, the
TAP moiety has been introduced in our ruthenium(II) com-
plexes through different strategies (Scheme 1): (i) by incorpo-
rating two ancillary TAP ligands (complex 3), (ii) by modifying
the phenanthroline imidazole ligand with TAP (complex 2), and
(iii) by combining both strategies (complex 4). Complex 1 bear-
ing phen-based ligands was used as a reference. Steady-state
luminescence, circular dichroism (CD), bio-layer interferometry
(BLI), and surface plasmon resonance (SPR) studies have been
performed, which demonstrated good affinity for G-quadruplex
DNA as well as selectivity over duplex DNA. Preliminary biolog-
ical studies with these complexes have been performed, which
revealed that two of them showed remarkable photo-cytotox-
icity towards U2OS osteosarcoma cells, whereas very low mor-
tality was observed in the dark at the same photo-drug con-
centration.
The design of new metal complexes targeting G-quadruplex
DNA has attracted intense interest with regard to their poten-
tial anticancer properties.[22,23] Indeed, some PtII,[24–28] NiII,[29–31]
RuII,[32–34] and IrIII complexes[35–37] have shown good affinity and
selectivity towards G4s. In comparison to organic compounds,
metal complexes have many advantages, such as a net positive
charge, tunable geometry, and, most interestingly, some of
them display potentially useful photochemical properties. In
this context, polyazaaromatic ruthenium(II) complexes repre-
sent ideal candidates to target genetic material such as G4s. By
virtue of their optical properties, including a large Stokes shift,
good photostability, high quantum yield, and long-lived lumi-
nescence, they have been developed to probe different DNA
sequences,[38] such as mismatches,[39] abasic sites,[40] or G4s.[32]
More recently, two ruthenium(II) complexes, [Ru(phen)2(dph)]2+
and [{Ru(phen)2}2(dph)]4+, based on the dph ligand (dph=di-
pyrazino[2,3-a:2’,3’-h]phenazine), have been reported to show
good selectivity towards G4s.[41] However, none of the reported
compounds is able to photo-induce oxidative damage under
light irradiation. Highly p-deficient ligands, such as 1,4,5,8-tet- Results and Discussion
raazaphenanthrene (TAP), are known to enhance the photo-ox-
idizing power of the resulting complexes.[42] Indeed, in the
Synthesis of complexes 1–4
presence of DNA, photo-induced electron transfer (PET) from a
guanine (G) base to the excited complex has been evidenced
for ruthenium(II) complexes bearing at least two TAP ligands.
This PET leads to dramatic consequences for living cells: (i) for-
mation of an adduct between the complex and the guanine or
The planar ligand CPIP (X=CH) was synthesized by condensa-
tion of 4-chlorobenzaldehyde with 1,10-phenanthroline-5,6-
dione in an ammonium-containing medium as previously re-
ported in the literature.[36] The ligand CPITAP (X=N) was ob-
tained according to a new protocol developed in our laborato-
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ry, using 4-chlorobenzaldehyde and 9,10-diamino-1,4,5,8-tetraa-
zaphenanthrene. The reaction was carried out in a refluxing
acetic acid/ethanol mixture over a period of 60 h. Purification
by preparative chromatography on SiO2 afforded pure CPITAP,
which was characterized by 1H and 13C NMR spectroscopies
and high-resolution mass spectrometry (HRMS) (see the Experi-
mental Section and Figures S1, S2, and S11 in the Supporting
Information). The corresponding RuII complexes were synthe-
sized by direct chelation of the N^N ligand onto an RuII precur-
sor bearing either 1,10-phenanthroline (phen, Y=CH) or
1,4,5,8-tetraazaphenanthrene (TAP, Y=N) moieties (Scheme 1).
The reactions were carried out in the dark and under argon to
avoid photo-dechelation and oxidation of the metal centre.
Complexes 1–4 were characterized by 1H NMR spectroscopy
and HRMS analyses (see the Experimental Section and Figur-
es S3–S10 and S12–S15) as well as UV/Vis, cyclic voltammetry
(CV), and photochemical studies (see below).
the metal-centred HOMO, leading to a more energetic transi-
tion. Comparison of luminescence lifetimes under air and
under argon allows us to conclude that all of the complexes
are capable of photosensitizing oxygen, as previously reported
in the literature for similar compounds.[52,53] It is also noted
that the luminescence quantum yield of complex 1 is lower in
acetonitrile than in water, consistent with data for the complex
[Ru(phen)3]2+ [54]
The relatively low luminescence of complexes
.
2–4 in water is typical of TAP-based complexes and is generally
attributed to increased non-radiative processes, likely due to
interaction of the solvent with non-chelating nitrogen atoms
(light-switch effect). It should also be noted that complex 4
showed a very short excited-state lifetime and a very low
quantum yield of emission in water, thus suggesting poor
photo-induced damaging properties.
Electrochemical study
The oxidation and reduction potentials of complexes 1–4 were
determined by cyclic voltammetry measurements in dry deoxy-
genated acetonitrile (Table 2 and Figures S20–S23). Based on
Absorption and luminescence properties
The absorption and luminescence data for complexes 1–4 are
gathered in Table 1 and in Figures S16–S19. For all of the com-
plexes, the absorption bands in the UV region could be attrib-
uted to ligand-centred (LC) transitions from comparison with
literature data, whereas the absorption maxima between 400
and 500 nm could be ascribed to metal-to-ligand charge-trans-
fer (MLCT) transitions, as previously shown for similar com-
plexes.[49,50] Emission spectra were measured at room tempera-
ture in acetonitrile and water, and at 77 K in EtOH/MeOH (4:1,
v/v). Complexes 1–4 display broad unstructured emissions in
both organic solvents and water.
Table 2. Electrochemical data for complexes 1–4.[a]
[b]
E*ox
[b]
Complex
Eox 1/2
Ered 1/2
E*red
1
2
3
4
+1.37
+1.63
+1.83
>2
À0.74
À0.29
À0.18
>À0.16
À1.30
À0.77
À0.78
À0.77
+0.81
+1.15
+1.23
+1.39
[a] Data were measured at room temperature in MeCN with 0.1m
Bu4NClO4 as the supporting electrolyte (V vs. Ag/AgCl), concentration of
complexes 0.8 mm. [b] Excited-state potentials estimated from the equa-
Positive solvatochromism on going from acetonitrile to
water and a hypsochromic shift of the emission band at 77 K
suggest a more polar excited state with respect to the ground
state, in agreement with the occurrence of charge transfer
upon irradiation, that is, MLCT. The excited-state energy de-
creases on going from 1 with CPIP to 2 with more electron-
withdrawing CPITAP, in agreement with stabilization of the
LUMO localized on the imidazophenanthroline ligand. Similar
observations were made for complexes 2–4, whereby the in-
creased number of p-deficient ligands results in stabilization of
*
*
tions Eox
E =
1
ox À E0À0 and Ered
E =
1
E0À0. The energy of the excit-
red
2
2
ed state, E0-0, was estimated by Franck–Condon line-shape analysis of the
emission spectrum at 298 K in CH3CN (see the Supporting Information,
Tables S1 and S2).
other similar RuII complexes described in the literature, we can
assume that complexes 1–4 display a one-electron oxidation
wave. This corresponds to the oxidation of RuII to RuIII, as the
anodic shift observed on going from 1 to 4 reflects stabiliza-
Table 1. Absorption and luminescence data for complexes 1–4.
[b]
[c]
lAbs (e)[a]
lEm
FEm
t [ns][d]
Complex
CH3CN
CH3CN
597
H2O
77 K
570
CH3CN
H2O
CH3CN
H2O
1
460
(1.61)
488
(0.59)
471
(1.11)
451
603
704
641
600
0.009
(0.058)
0.011
(0.082)
0.037
(0.138)
0.01
0.069
(0.14)
115
(380)
376
(692)
686
(1620)
80
572
(1315)
162
(162)
629
(786)
6
2
3
4
671
623
586
612
598
565
0.0059
(0.007)
0.017
(0.011)
0.0006
(0.0005)
(1.28)
(0.02)
(78)
(6)
[a] l in nm for the most bathochromic transition in MeCN (extinction coefficient, e”104 mÀ1 cmÀ1). [b] l in nm at RT in MeCN and H2O and at 77 K in EtOH/
MeOH (4:1, v/v). [c] Quantum yield of emission measured by comparison with the reference [Ru(bpy)3]2+, under air and under argon (in brackets), excita-
tion at 450 nm, errors are estimated as 10%.[51] [d] Luminescence lifetime (after irradiation at l=400 nm) measured under air and under argon (in brack-
ets); errors are estimated as 5%.
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tion of the metal-centred HOMO due to the presence of highly
p-deficient ligands (TAP), consistent with the spectroscopic
properties. In the other direction, each complex displayed sev-
eral one-electron reduction waves, corresponding to the suc-
cessive addition of electrons to the ligands. The first reduction
wave measured for 1 can be attributed to reduction of phen
moieties of CPIP or the ancillary ligands. For complexes 2–4,
the first reduction wave, anodically shifted with respect to
complex 1, corresponds to reduction of the TAP moiety of
either CPITAP or the ancillary ligand.
All of these data suggest the following photophysical
scheme for this family of complexes. According to literature
data and our results, we can safely conclude that the HOMO is
metal-centred and that its relative energy depends on the re-
spective ligands surrounding the metal. The more p-deficient
ligands that are chelated to the metal centre, the less reducing
the complex will be. As for the LUMO, it is centred on a ligand,
confirming the MLCT nature of the excited state. From the
cyclic voltammetry measurements and the spectroscopic data,
the oxidation and reduction potentials of the excited state can
be roughly estimated. Not surprisingly, complexes 3 and 4 dis-
play strong photo-oxidizing power (+1.23 and +1.39 V vs. Ag/
AgCl, respectively). With these results in hand, we tested
whether a photo-induced electron transfer (PET) would occur
in the presence of the most reducing building block of the
DNA G-quadruplex, that is, a guanine residue (Eox = +1.10 V vs.
Ag/AgCl).[55]
Figure 1. Luminescence quenching of complex 3 in the presence of dGMP:
(a) emission spectra of complex 3 in the presence of increasing concentra-
tions of dGMP; (b) Stern–Volmer plot. Complex concentration: 50 mm in Tris-
HCl buffer (50 mm at pH 7.4). Addition of dGMP from 0 mm to 10 mm. Exci-
tation at l=430 nm.
Luminescence studies in the presence of dGMP
As mentioned in the Introduction, G-quadruplex DNA is a gua-
nine-rich sequence present in the genome. Recently, we have
reported on a new family of Ru complexes exhibiting good af-
finity and selectivity towards G-quadruplex DNA.[41] However,
these complexes proved not to be sufficiently oxidizing in
their excited state to trigger direct oxidative damage (type I
photoreactivity). Ruthenium(II) complexes bearing at least two
TAP ligands are well known in the literature to photoreact with
a guanine moiety upon irradiation. Therefore, the photoreac-
tivities of our complexes towards dGMP were investigated by
Stern–Volmer steady-state luminescence quenching experi-
ments. As is well established in the literature, the luminescence
quenching of an RuII-TAP complex upon addition of dGMP is
due to electron transfer (ET) from dGMP to the complex in its
excited state. According to their estimated E*red values
(Table 2), complexes 1 and 2 should not be sufficiently photo-
oxidizing to undergo ET with dGMP (Eox = +1.10 V vs. Ag/
AgCl). Indeed, as anticipated, no luminescence quenching was
observed for these complexes in the presence of dGMP. In con-
trast, a Stern–Volmer plot obtained with complex 3 (Figure 1)
showed luminescence quenching in the presence of increasing
dGMP concentrations, with a high efficiency close to the diffu-
2
1
C
G
*
1
RuŠ
G ! RuŠ
Considering the oxidizing power of the excited state of 3 and
using the empirical Rehm–Weller equation, it is expected that
the process according to Equation (1) will be exergonic by
about À0.13 eV. In the case of 4, PET should also be favoured
by about À0.29 eV, but the poor luminescence properties of
this complex precluded luminescence quenching experiments.
Luminescence studies in the presence of ODNs
Due to the ability of complexes 1–3 to emit light in aqueous
media, their luminescence upon the addition of increasing
concentrations of oligonucleotides (ODNs) could be monitored.
Experiments were performed with human telomeric wtTel23
(3’TT(GGGATT)3GGG5’) and
a
GC-rich hairpin sequence
(3’(GC)4TTTT(GC)4 ). The experiments were carried out in 10 mm
HEPES (pH 7.4), 35 mm NaCl, 50 mm KCl buffer, in which the
desired DNA structures (i.e., duplex or G-quadruplex) were
formed (Figure S32). According to the redox properties of the
complex, two behaviours were observed upon the addition of
ODNs: (i) an increase in luminescence due to protection of the
ruthenium(II) probe from the solvent in the hydrophobic envi-
ronment of the ODN, or (ii) a decrease in luminescence due to
PET with a neighbouring guanine base. Figure 2a,b show the
5’
sion limit (quenching rate constant 2.63”109 mÀ1 À1). Based on
s
thermodynamic data, this luminescence quenching can be
safely ascribed to PET from the guanine moiety to the excited
state of complex 3 [Eq. (1)].
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DNA binding analysis
Various biophysical techniques, including FRET melting, UV/Vis
spectrophotometry, circular dichroism (CD), NMR, and SPR
have been developed for studying G-quadruplex DNA/ligand
interactions.[17] In the present case, the use of UV/Vis absorb-
ance was found to be unsuitable for determining the binding
affinity. Modifications of the absorption spectra of the com-
plexes were detected upon the addition of increasing concen-
trations of G-quadruplex DNA (Figures S24–S31), but they were
relatively moderate and thus it was tedious to measure a bind-
ing affinity. Binding affinities of complexes 1 and 2 for G-quad-
ruplex and duplex DNA could also be estimated by fitting the
variation in luminescence intensity with the ratio of binding
sites per complex (Figure 2). Complex 1 showed apparent dis-
sociation constants (KD) of 77 mm for duplex DNA and 6.5 mm
for G-quadruplex DNA. The difference in binding affinities for
duplex and G-quadruplex DNA was more drastic for complex
2, with KD values of 123 mm and 2 mm, respectively. Only quali-
tative conclusions can be made about the affinity of complex
3 for wtTel23 G-quadruplex versus GC-rich hairpin DNA, since a
dynamic quenching process is also operative, which should
alter the luminescence intensity. However, it can be observed
that the slope of the curve is steeper with G-quadruplex DNA
than with duplex DNA, indicating that the binding affinity is
certainly stronger for the former. We thus evaluated the affini-
ties of these complexes towards DNA by means of CD melting
assays, BLI, and SPR.
CD melting assays
Figure 2. Luminescence titrations of complexes 1–3 with DNA. Lumines-
cence titrations of complexes 1, 2, and 3 ((a), (b), and (c), respectively) were
carried out in 10 mm HEPES (pH 7.4), 35 mm NaCl, 50 mm KCl buffer by
adding increasing proportions of ds-DNA (GC-rich hairpin duplex
3’(GC)4TTTT(GC)45’, base pairs equivalents per Ru complex) or G-quadruplex
DNA (wtTel23, 3’TT(GGGATT)3GGG5’, G-quartet equivalents per Ru complex).
Solid lines were obtained by a modified McGhee–von Hippel fitting process
(see the Supporting Information) to evaluate the binding affinities. Excitation
at l=450 nm.
The ability of complexes 1–4 to interact with G-quadruplex
DNA was first investigated through CD experiments. CD analy-
ses were carried out in 10 mm Tris-HCl, 100 mm NaCl, or
100 mm KCl buffer. As anticipated, in buffer containing
100 mm NaCl, wtTel23 folded into an antiparallel topology
characterized by two positive peaks at 242 and 294 nm, re-
spectively, and a negative peak at 262 nm (Figure S33). Upon
addition of complexes 2–4, minor changes in the ellipticity of
wtTel23 were observed, suggesting that these complexes did
not induce major structural changes in the antiparallel confor-
mation of the G-quadruplex DNA (Figure S33). For complex 1,
we also observed the appearance of a shoulder at 270 nm, at-
tributable to a slight modification of the topology induced by
this complex (presence of hybrid II-type G4 folding). In buffer
containing 100 mm KCl, wtTel23 folded into a hybrid II-type G4
structure, characterized by a maximum at 290 nm and a
shoulder at 270 nm (Figure S34). Again, a minor change in the
ellipticity of wtTel23 was observed, thus revealing no major
structural changes upon binding. The duplex structure
(5’CGT3CGT5ACGA3CG3’ hairpin) was not affected as no change
in the CD spectrum was observed upon addition of complexes
1–4 (Figure S35).
enhancement of the luminescence upon the addition of in-
creasing amounts of wtTel23 or hairpin ODN for complexes 1
and 2. This behaviour is consistent with (i) protection of the
complex by the ODN, which decreases the rate of non-radia-
tive processes, (ii) the fact that no luminescence quenching
was observed in the presence of guanine moieties (see above).
In contrast, the luminescence of complex 3 was quenched
upon the addition of increasing amounts of DNA (Figure 2c),
in accordance with PET and the Stern–Volmer plot.
The above results from steady-state luminescence studies
with 1–3 suggested a strong interaction between DNA and
these complexes. We thus decided to further investigate the
interactions of the complexes with DNA in order to evaluate
whether they might display a specific affinity towards G-quad-
ruplex structures over double-stranded DNA.
Next, we performed CD melting assays to evaluate whether
complexes 1–4 exerted stabilizing or destabilizing effects on
the G-quadruplex and hairpin duplex DNA structures. CD melt-
ing curves were recorded in the absence or presence of each
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complex (at 5:1 complex/DNA ratio) in buffer containing
100 mm NaCl or 100 mm KCl (Table 3 and Figures S36 and S37)
in the case of wtTel23 and in buffer containing 100 mm NaCl
for hairpin duplex (Table 3 and Figure S38). The results of CD
Bio-layer interferometry studies
BLI is a label-free method for the measurement of affinity con-
stants. It allows the determination of kinetic parameters of an
interaction (such as SPR) and has been used to study biomo-
lecular interactions between large biomolecules, such as pro-
tein–membrane interactions.[56] We have hitherto employed an
SPR analysis method based on the use of a template-assem-
bled synthetic G-quadruplex (TASQ) that allows precise control
of G-quadruplex topology through the assembly of constrain-
ed structures on a template.[57–61] We have now adapted this
SPR method for BLI. Different G-quadruplex features were
used: intermolecular-like G-quadruplex motif A constrained in
a parallel G-quadruplex topology, intramolecular G-quadruplex
B (HTelo sequence in equilibrium between different topolo-
gies), human telomeric sequence (HTelo) C constrained in anti-
parallel topology, and hairpin DNA D (Figure 3). Each of the
systems A–C formed the desired G-quadruplex structure under
the conditions used for BLI analysis (10 mm HEPES pH 7.4,
35 mm NaCl, 50 mm KCl).[57,58]
Each evaluated complex showed KD values in the micromolar
range for G-quadruplex topologies A, B, and C (Table 4 and
Figures S39–S42). These values fall within the range of those
reported for related ruthenium(II) complexes interacting with
G-quadruplexes. It is noteworthy that the substitution of
carbon atoms by nitrogen atoms in the ligands (i.e., two phen
ligands in 1 replaced by two TAP ligands in 3; the CPIP ligand
in 1 replaced by the CPITAP ligand in 2) only weakly affects
the interaction with G-quadruplex DNA. More interestingly,
each ruthenium(II) complex showed a higher affinity for G-
quadruplex structures than for duplex DNA. Indeed, it was im-
possible to measure KD values for interaction with duplex
system D within the concentration range used in this study
(i.e., from 5 to 40 mm), which were presumably higher than
1 mm for each complex. The good selectivity was further con-
firmed by SPR analysis using systems B and D. We obtained KD
values for the G-quadruplex system B of the same order of
magnitude as from BLI analysis, whereas none of the com-
plexes showed an affinity with duplex DNA D within the con-
centration range used (Figure S43 and Table S3).
Table 3. Melting temperatures (Tm) of wtTel23 and duplex hairpin in the
absence or presence of ligands (5 equiv).[a]
Tm [8C] (Æ1)
Complex
wtTel23
hairpin
no complex
52.9 (61.9)
58.5 (76.0)
58.0 (64.0)
57.3 (67.0)
52.9 (61.9)
68.7
58.0
67.6
68.1
67.2
1
2
3
4
[a] WtTel23 sequence 3’TT(GGGATT)3GGG5’ was first annealed by heating at
958C for 5 min in Tris-HCl buffer (10 mm, pH 7.04) with 100 mm NaCl or
100 mm KCl (in brackets) and then allowed to cool to room temperature
overnight. Oligonucleotide concentration 2.5 mm. Hairpin sequence
5’CGT3CGT5ACGA3CG3’ was first annealed by heating at 958C for 5 min in
Tris-HCl buffer (10 mm, pH 7.04) with 100 mm NaCl and then allowed to
cool to room temperature overnight. The ellipticity was recorded at 290
and 252 nm for wtTel23 and duplex hairpin, respectively.
melting experiments clearly showed that complexes 1–3 in-
duced a slight stabilization of wtTel23, whereas none of the
complexes significantly affected the stability of duplex DNA
structures. The melting temperature assays thus confirmed the
ability of most of the investigated complexes to selectively in-
teract with G-quadruplex DNA over duplex DNA. It should be
mentioned that each complex is a racemic mixture of two
enantiomers and that preferential binding or a different bind-
ing mode of each enantiomer could arise. However, separation
of the respective enantiomers represents a new and challeng-
ing task. Thus, we are presently unable to comment on wheth-
er the changes observed by CD are due to different binding
modes of each enantiomer. Because this technique is not the
most appropriate for direct measurements of affinity constants,
we next performed BLI analysis, which allowed us to determine
the thermodynamic parameters for the interaction.
Figure 3. G-quadruplex systems A–C and duplex control D used for bio-layer interferometry studies: (A) parallel-stranded quadruplex (intermolecular-like G-
quadruplex), (B) intra quadruplex (intramolecular-like G-quadruplex), (C) antiparallel human telomeric sequence, and (D) duplex (hairpin).
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Table 4. Data for the interactions of complexes 1–4 with DNA structures
A–D from BLI analyses.
Complex
DNA
Constants
1
2
3
4
structure
A
B
C
D
kon (103 mÀ1 sÀ1
)
)
)
)
2.2Æ1.3
1.8Æ0.5
80Æ20
9.4Æ1.4
2.9Æ0.3
31Æ8
27Æ1.2
3.7Æ0.6
20Æ11
8.0Æ2.1
13Æ0.2
182Æ74
koff (10À1 sÀ1
KD (mm)[a]
)
kon (103 mÀ1 sÀ1
5.9Æ1.6
130Æ0.1 100Æ1.0 27Æ1.3
koff (10À1 sÀ1
KD (mm)[a]
)
0.37Æ0.8 1.5Æ0.1
1.5Æ1.4
2.0Æ0.5
4.6Æ3.0
22Æ10
6.0Æ4.0
22Æ1.0
1.6Æ0.4
8.0Æ2.0
n.d.[b]
1.0Æ0.5
62Æ1.2
1.8Æ0.1
3.0Æ0.5
n.d.[b]
n.d.[b]
n.d.[b]
kon (103 mÀ1 sÀ1
280Æ1.6 61Æ1.7
koff (10À1 sÀ1
KD (mm)[a]
)
2.4Æ0.8
1.0Æ1
n.d.[b]
9.4Æ1.6
10Æ8
n.d.[b]
n.d.[b]
n.d.[b]
kon (103 mÀ1 sÀ1
koff (10À1 sÀ1
KD (mm)[a]
)
n.d.[b]
n.d.[b]
n.d.[b]
n.d.[b]
[a] Equilibrium dissociation constants deduced from the kinetic rate con-
stants. [b] Due to very low binding of the different complexes with hair-
pin DNA, the kinetics of the interactions could not be determined (n.d.)
in the studied concentration range. This was confirmed by SPR analysis
(Figure S43). Running buffer: 10 mm HEPES pH 7.4, 35 mm NaCl, 50 mm
KCl.
Figure 4. Isoaffinity plot and kinetic characterization for G-quadruplex struc-
ture C. For PhenDC3, BRACO-19, MMQ1, and TMPyP4 (in red), the analyses
were performed by SPR;[61] for ruthenium(II) complexes (in green), data are
from BLI analysis. KD (parallel diagonal lines), kon (association kinetic constant,
y-axis), koff (dissociation kinetic constant, x-axis).
It was also noticed that the affinities of complexes 1–4 were
higher for G-quadruplex structures B and C, which contain TTA
loops, than for parallel-stranded quadruplex A. This is consis-
tent with interactions of the complexes with G-quadruplexes
through mixed p-stacking over the guanine tetrad and further
interactions with loops and grooves (see the Molecular Model-
ling section below). To obtain further information on the affini-
ty of complexes 1–4 for G-quadruplex DNA, the association
(kon) and dissociation (koff) constants of the interaction were de-
termined (Table 4), which again revealed the relatively minor
influence of the replacement of carbon by nitrogen atoms in
complexes 1–4. Indeed, for a given G-quadruplex structure, kon
and koff differ only slightly.
For the most biologically relevant G-quadruplex structure C,
kon and koff values were also compared with those of well-
known compounds that interact with G-quadruplex DNA (i.e.,
pyridostatine PDS, Phen-DC3, BRACO-19, MMQ1, and
TMPyP4).[61]
As illustrated in Figure 4, complexes 1–4 display association
and dissociation rates that are similar to those of BRACO,
MMQ1, and TMPyP4. In particular, the fact that our complexes
showed similar affinity to BRACO-19, which has demonstrated
anticancer activity through the stabilization of G-quadruplex at
the telomere,[62] prompted us to study their phototoxicity.
Figure 5. Molecular modelling of the interactions of complex 3 (D-isomer)
with G-quadruplex DNA. Docking calculations for interaction of complex 3
with G-quadruplex human telomeric DNA structure (PDB entry 1KF1): (A) in
p-stacked position over the guanine tetrad, (B) interaction with TTA loop.
tioning of 3 over the guanine tetrad, while the second in-
volved insertion of the complex into the TTA loop of the G-
quadruplex through the CPIP ligand. Initial calculations were
performed with the D-isomer of complex 3 to afford the two
best-ranked positions in Figure 5. However, similar docked po-
sitions were obtained with the other L-enantiomer (see Fig-
ure S44 in the Supporting Information).
Molecular modelling
To obtain further insights into the interactions with G-quadru-
plex DNA, molecular docking calculations were carried out.
The most interesting complex 3 (due to its photophysical
properties) was docked to the human telomeric DNA structure
(PDB entry 1KF1, wtTel23 in parallel conformation). From analy-
sis of the best-ranked docked positions, two binding modes
were obtained (Figure 5). The first involved a p-stacked posi-
Dynamic molecular mechanics simulations were then carried
out in order to assess the stabilities of these two docking posi-
tions. In both cases, the complex remained tightly bound in its
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relative position (either in interaction with the TTA loop or p-
stacked) for up to 20 ns, thus emphasizing the strong affinity
displayed by 3 towards G-quadruplex DNA. These results are
consistent with those of the BLI experiments. Indeed, the affini-
ty of complex 3 towards structure A (i.e., without a loop) is
lower than those towards structures with a loop (KD =20 mm
for A; KD ꢀ2 mm for B and C), confirming the importance of the
second docked position (i.e., loop interactions).
Cell penetration
By virtue of the ability of complex 1 to emit light in aqueous
media, its penetration into U2OS osteosarcoma cells could be
studied by confocal microscopy.[63] Cells were incubated with
20 mm complex 1 for 24 h. As shown in Figure 6, complex 1
showed efficient penetration into the cells, including in the
nuclei.
Figure 7. Cell viability studies. Percentage of viable U2OS cells after incuba-
tion with complexes 1–4 for 24 h in the dark followed by 30 min of irradia-
tion (orange bars) or not irradiated (blue bars). The tetrazolium salt-based
WST-1 assay was performed 24 h after irradiation. Values were normalized to
untreated and non-irradiated U2OS cells. Error bars indicate SD.
phototoxicity of complex 3 was confirmed by microscopic ob-
servation of the cells, as revealed by tetrazolium salt-based
metabolic assay, which revealed extensive cell death upon irra-
diation (dead cells can clearly be recognized by a change in
shape, see Figure S45).
A possible explanation for the phototoxicity of 1–3 is that
the internalized ruthenium complexes react with the biological
material through a type I photoreaction (i.e., photoelectron
transfer) or type II photoreaction (singlet oxygen photosensiti-
zation), both mechanisms being likely to induce DNA damage,
ultimately leading to cell death. On the contrary, the low
photo-cytotoxicity of 4 most likely originates from its short ex-
cited-state lifetime, resulting in a poor photo-damaging ability.
Conclusions
A series of new ruthenium(II) complexes has been designed to
target and photoreact with G-quadruplex DNA through incor-
poration of the CPIP ligand (or similar). As anticipated, the
photophysical properties of these complexes are consistent
with MLCT transitions and a metal-centred HOMO. Conse-
quently, these complexes are able to react with DNA through
type II photoreaction (i.e., formation of singlet oxygen) or
through photo-induced charge transfer (PET). All four designed
complexes 1–4 displayed a good affinity for G-quadruplex
DNA and selectivity over duplex DNA. Docking studies and
molecular dynamic simulations revealed that this affinity is due
to p-p stacking above the tetrad and interaction with the TTA
loop.
Figure 6. Cell penetration study. Fluorescence microscopy images of U2OS
cells after incubation with 20 mm of complex 1 for 24 h, DMEM buffer:
(A) RuII complex in green, (B) the nuclei in red, stained by Draq5; (C) bright-
field and (D) merged image. Scale bar 14 mm.
Photo-cytotoxicity
Preliminary photo-cytotoxicity studies were performed with
complexes 1–4 on U2OS osteosarcoma cells. Figure 7 depicts
the percentage of metabolically active cells after incubation
with 10 mm of complexes 1–4 for 24 h and subsequent irradia-
tion for 30 min (orange bars). Non-irradiated controls were also
performed (blue bars). Interestingly, non-irradiated cells dis-
played very low rates of mortality, whereas irradiation of the
cells led to a dramatic decrease in survival. Indeed, 100% mor-
tality was obtained at 10 mm for complexes 1 and 3, whereas
complex 2 was slightly less efficient, inducing 70% mortality.
As anticipated, complex 4 showed only very weak phototoxici-
ty in comparison with non-irradiated control cells. The strong
Strikingly, both complexes 1 and 3 elicited a dramatic
photo-cytotoxic effect, as 100% mortality was obtained upon
irradiation of U2OS osteosarcoma cells in their presence,
whereas very low mortality was observed in the dark at the
same drug concentration. Further studies are underway with
the aim of establishing whether this photo-cytotoxic effect is
mainly due to a type II photoreaction (i.e., singlet oxygen pho-
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argon. After cooling and addition of aqueous NH4PF6 solution, a
solid was formed. The latter was collected by filtration and washed
three times each with water, EtOH, and Et2O to afford the final
pure product as an orange powder (36 mg, 90%). 1H NMR
(500 MHz, CD3CN): d=9.05 (d, 1H, J=8.2 Hz), 8.89 (d, 1H, J=
8.4 Hz), 8.59 (dd, 4H, J=8.3 Hz, J=1.2 Hz), 8.28 (d, 2H, J=8.7 Hz),
8.25 (s, 4H), 8.07 (dd, 2H, J=10.5 Hz, J=4.8 Hz), 8.01 (d, 2H, J=
5.4 Hz), 7.96 (d, 2H, J=5.2 Hz), 7.67–7.59 ppm (m, 8H); HR-MS:
calcd for C43H27N8ClF6PRu (1À1PF6): 931.07595 Da; found
931.07688 Da.
tosensitization) or a type I photoreaction (i.e., photo-induced
charged transfer (PIET)). Further experiments are currently
being performed to investigate whether telomeric DNA
damage is induced in cells by complexes 1 and 3. Interestingly,
this photo-cytotoxicity should not involve the inhibition of te-
lomerase activity through the stabilization of G-quadruplex
DNA as U2OS osteosarcoma cells do not express the telomer-
ase enzyme. To the best of our knowledge, this would be the
first example of high photo-cytotoxicity based on the use of
metal complexes targeting telomeric DNA, through a mecha-
nism that does not involve the inhibition of telomerase. There-
fore, the photo-cytotoxicity of these two complexes will also
be comparatively evaluated towards both telomerase-express-
ing cancer cells and normal non-immortalized cells.
[Ru(phen)2CPITAP]·2PF6 (2): [Ru(phen)2Cl2] (20 mg, 0.037 mmol)
and CPITAP (15 mg, 0.044 mmol) were dissolved in ethylene glycol
(3 mL) and the solution was heated at 1208C for 20 h in the dark
under argon. After cooling and addition of aqueous NH4PF6 solu-
tion, a solid was formed. The latter was collected by filtration and
washed three times each with water, EtOH, and Et2O to afford the
crude product. Purification by preparative chromatography on SiO2
(CH3CN/H2O/NH4Cl(sat), 4:4:1, v/v/v) gave the final product as a red
Experimental Section
1
powder (18 mg, 64%). H NMR (500 MHz, CD3CN): d=8.86 (d, 2H,
J=2.9 Hz), 8.65 (d, 4H, J=8.3 Hz), 8.31 (d, 2H, J=8.8 Hz), 8.28 (s,
4H), 8.13 (d, 2H, J=5.0 Hz), 8.08 (d, 2H, J=2.9 Hz), 8.00 (d, 2H, J=
4.8 Hz), 7.69–7.62 ppm (m, 6H); HR-MS: calcd for C41H25N10ClRu
(2À2PF6): 394.05086 Da; found 394.05131 Da.
Material and methods
[Ru(phen)2Cl2],[64] [Ru(TAP)2Cl2],[42] 1,10-phenanthroline-5,6-dione,[65]
9,10-diamino-1,4,5,8-tetraazaphenanthrene,[65] and 2-(4-chlorophen-
yl)-1H-imidazo[4,5-f][1,10]phenanthroline (CPIP)[48] were synthesized
according to previously described literature protocols. The
oligonucleotides wtTel23 (3’TT(GGGATT)3GGG5’), GC-rich hairpin
duplex (3’(GC)4TTTT(GC)45’), and hairpin duplex sequence
5’CGT3CGT5ACGA3CG3’ were prepared by standard automated solid-
phase oligonucleotide synthesis on a 3400 DNA synthesizer. After
purification by RP-HPLC, they were thoroughly desalted by size-ex-
clusion chromatography (SEC). All solvents and reagents for the
synthesis were of reagent grade and were used without any fur-
ther purification. All solvents for the spectroscopic and electro-
chemical measurements were of spectroscopic grade. Water was
purified with a Millipore Milli-Q system. 1H and 13C NMR spectra
were measured from solutions in CDCl3 or CD3CN on a Bruker AC-
300 Avance II (300 MHz) or a Bruker AM-500 (500 MHz) spectrome-
ter at 208C. Chemical shifts (in ppm) were referenced to the residu-
al peak of the solvent as an internal standard. High-resolution
mass spectra (HRMS) were recorded on a Q-extractive Orbitrap
spectrometer from Thermo-Fisher, using reserpine as an internal
standard. Samples were ionized by electrospray ionization (ESI; ca-
pillary temperature 3208C, vaporizer temperature 3208C, sheath
gas flow rate 5 mLminÀ1).
[Ru(TAP)2CPIP]·2PF6 (3): [Ru(TAP)2Cl2] (20 mg, 0.037 mmol) and
CPIP (15 mg, 0.045 mmol) were dissolved in ethylene glycol (3 mL)
and the solution was heated at 1208C for 20 h in the dark under
argon. After cooling and addition of aqueous NH4PF6 solution, a
solid was formed. The latter was collected by filtration and washed
three times each with water, EtOH, and Et2O to afford the final
pure product as an orange powder (34 mg, 83%). 1H NMR
(500 MHz, CD3CN): d=9.01–8.94 (m, 6H), 8.62 (s, 4H), 8.27 (d, 2H,
J=8.6 Hz), 8.24 (d, 2H, J=2.8 Hz), 8.19 (d, 2H, J=2.7 Hz), 8.04 (d,
2H, J=4.3 Hz), 7.73 (dd, 2H, J=8.3 Hz, J=5.3 Hz), 7.66 ppm (d,
2H, J=8.6 Hz); HR-MS: calcd for C39H23N12ClF6PRu (3À1PF6):
941.053699 Da; found: 941.054467 Da.
[Ru(TAP)2CPITAP]·2PF6 (4): [Ru(TAP)2Cl2] (20 mg, 0.037 mmol) and
CPITAP (15 mg, 0.044 mmol) were dissolved in ethylene glycol
(3 mL) and the solution was heated at 1208C for 20 h in the dark
under argon. After cooling and addition of aqueous NH4PF6 solu-
tion, a solid was formed. The latter was collected by filtration and
washed three times each with water, EtOH, and Et2O to afford the
crude product. Purification by preparative chromatography on SiO2
(CH3CN/H2O/KNO3(sat), 7:2:1, v/v/v) gave the final product as a red
1
powder (10 mg, 25%). H NMR (500 MHz, CD3CN): d=9.01–8.98 (m,
4H), 8.97 (d, 2H, J=2.8 Hz), 8.64 (s, 4H), 8.32 (d, 2H, J=8.6 Hz),
8.29 (s, 2H), 8.24 (d, 2H, J=2.7 Hz), 8.15 (d, 2H, J=2.8 Hz),
7.66 ppm (d, 2H, J=8.5 Hz); HR-MS; calcd for C37H21N14ClRu
(4À2PF6): 396.04136 Da; found: 396.04171 Da.
Synthesis
2-(4-Chlorophenyl)-1H-imidazo[4,5-f]pyrazino[2,3-h]quinoxaline
(CPITAP): A solution of 9,10-diamino-1,4,5,8-tetraazaphenanthrene
(53 mg, 0.250 mmol) and 2-(4-chlorophenyl)-1H-imidazo[4,5-f][1,10]
phenanthroline (35 mg, 0.250 mmol) in EtOH (2.5 mL) was heated
under reflux for 24 h. AcOH (3 mL) was then added and the mix-
ture was heated at 110 8C for 60 h. After cooling, the AcOH was
evaporated under vacuum. The crude dark-green solid was then
purified by preparative chromatography on SiO2 (CHCl3/EtOH, 99:1)
Absorption and luminescence studies
UV/Vis absorption spectra were recorded on a Shimadzu UV-1700
spectrophotometer. The concentration of the complexes was
50 mm. Room temperature luminescence spectra were recorded on
a Varian Cary Eclipse instrument. Luminescence intensity at 77 K
was recorded on a FluoroLog 3 FL3-22 from Jobin Yvon equipped
with an 18 V 450 W short-arc xenon lamp and an R928P photomul-
tiplier, using an Oxford Instruments Optistat DN nitrogen cryostat
controlled by an Oxford Intelligent Temperature Controller
(ITC503S). Quantum yields were obtained using [Ru(bpy)3]2+ as a
reference.[51] Luminescence lifetime measurements were performed
after irradiation at l=400 nm obtained as the second harmonic of
a titanium:sapphire laser (picosecond Tsunami laser Spectra Physics
1
to afford pure CPITAP as a yellow powder (62 mg, 75%). H NMR
(300 MHz, CDCl3): d=9.13 (d, 2H, J=1.8 Hz), 9.06 (d, 2H, J=
2.0 Hz), 8.24 (d, 2H, J=8.6 Hz), 7.52 ppm (d, 2H, J=8.6 Hz);
13C NMR (75 MHz, CDCl3): d=151.71, 151.70, 145.73, 145.72, 143.79,
139.52, 137.6, 129.63, 129.62, 128.13 ppm; HR-MS: calcd for
C17H10N6Cl: 333.06500 Da; found 333.06489 Da.
[Ru(phen)2CPIP]·2PF6 (1): [Ru(phen)2Cl2] (20 mg, 0.037 mmol) and
CPIP (15 mg, 0.045 mmol) were dissolved in ethylene glycol (3 mL)
and the solution was heated at 1208C for 20 h in the dark under
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3950-M1BB+39868-03 pulse picker doubler) at a repetition rate of
80 kHz. A Fluotime 200 instrument from AMS Technologies was
used for the decay acquisition. It consists of a GaAs microchannel
plate photomultiplier tube (Hamamatsu model R3809U-50) fol-
lowed by a time-correlated single-photon counting system from Pi-
coquant (PicoHarp300). The ultimate time resolution of the system
is close to 30 ps. Luminescence decays were analysed with FLUO-
FIT software available from Picoquant.
erence sensors without DNA immobilization were used to subtract
the non-specific adsorption on the SA layer. The sensorgrams were
fitted using a heterogeneous model (see the sensorgrams in Fig-
ures S39–S42). The reported values are the means of representative
independent experiments, and the errors provided are standard
deviations from the mean. Each experiment was repeated at least
twice.
Computational studies
Electrochemical studies
Docking experiments were performed with complex 3, the geome-
try of which was first optimized at the B3LYP/6-31 g* level using
Gaussian 09 software. The AutoDock 4.0 software package was
then used on the crystal structure of parallel quadruplexes from
human telomeric DNA (PDB entry: 1KF1). A grid of 80”80”80
points with a spacing of 0.5 Š between these points was used;
non-polar hydrogen atoms were merged and Gasteiger–Hückel
charges were added on both the complex and the G-quadruplex.
The parameters for the Ru atom were set at r=2.96 Š, q= +2.0
and the van der Waals well depth was 0.056 kcalmolÀ1. The dock-
ing calculations involved a genetic algorithm search generating
100 docked structures. A default protocol was applied, with an ini-
tial population of 150 randomly placed individuals, a maximum
number of 2.5”105 energy evaluations, a maximum number of
2.7”104 generations, a mutation rate of 0.02, and a crossover rate
of 0.8. Results differing by less than 2 Š in positional root-mean-
square deviation (RMSD) were clustered together and represented
the result with the most favourable free binding energy. Regarding
the dynamic molecular mechanics simulation, the AMBER 12 soft-
ware package was chosen and applied to the two best-ranked po-
sitions obtained from the docking calculations. The complex was
broken down into its constituent ligands and their specific parame-
ters were generated with the ANTECHAMBER module and the
GAFF force field, whereas charges were calculated through an
RESP fitting of HF/6-31g*-level calculations. The docking structures
were solvated in a TIP3P water box, the dimensions of which were
set at least 10 Š larger than the solute in every direction. Sodium
cations were added until the global charge was neutral and long-
range electrostatic interactions were computed using the particle-
mesh Ewald method with a cut-off value of 10 Š. After minimiza-
tion and heating, an MD simulation was run at 300 K for 20 ns with
the time step set at 1 fs.
Cyclic voltammetry was carried out in a one-compartment cell,
using a glassy carbon disk working electrode (approximate area
0.03 cm2), a platinum wire counter electrode, and an Ag/AgCl refer-
ence electrode. The potential of the working electrode was con-
trolled by an Autolab PGSTAT 100 potentiostat through a PC inter-
face. Cyclic voltammograms were recorded at a sweep rate of
100 mVsÀ1 from solutions in dry acetonitrile (Sigma–Aldrich, HPLC
grade). The concentration of the complexes was 8”10À4 molLÀ1
,
with 0.1 molLÀ1 tetrabutylammonium perchlorate as supporting
electrolyte. Before each measurement, the samples were purged
with nitrogen. Redox potentials were determined by comparison
with ferrocene, added at the end of the measurement.
Luminescence titration experiments of complexes 1–3 with dGMP
were conducted with a Varian Cary Eclipse instrument. A solution
of dGMP (5 mm) was progressively added to a solution of the re-
spective complex (50 mm) in 50 mm Tris-HCl buffer, pH 7.4. Lumi-
nescence titration experiments with ODNs (GC-rich hairpin or
wtTel23 G-quadruplex DNA) were conducted by recording spectra
from solutions in 10 mm HEPES, 35 mm NaCl, 50 mm KCl (pH 7.4)
buffer on a Varian Cary Eclipse instrument for complexes 1 and 3,
and on a FluoroLog 3 FL3-22 from Jobin Yvon for complex 2. Titra-
tions were performed by starting from the highest DNA concentra-
tion (10 mm) and progressively decreasing it, whilst the concentra-
tion of the complex (5 mm) was kept constant. Fitting equations
are described in the Supporting Information.
CD experiments
Prior to CD analysis, oligonucleotides were annealed by heating at
958C for 5 min in buffered medium, then allowed to cool to room
temperature overnight. Spectra were recorded on a JASCO J-810
spectropolarimeter from solutions in 1 cm pathlength quartz cuv-
ettes at 58C increments from 258C to 908C over the wavelength
range from 220 to 330 nm. At each temperature, the spectrum was
an average of three scans with response time 0.5 s, data pitch
1 nm, bandwidth 4 nm, and scanning speed 200 nmminÀ1. For CD
melting experiments, the ellipticity was recorded at 290 and
252 nm for wtTel23 and duplex hairpin, respectively. Melting tem-
peratures were obtained through Boltzmann fitting with Origin
software. Each curve fit was only accepted with r>0.99.
Confocal laser scanning microscopy
U2OS cells were grown at 378C in a humidified atmosphere with
5% CO2 in DMEM medium (Westburg) containing 10% foetal
bovine serum (FBS) (Gibco) and 1% penicillin/streptomycin (West-
burg). 20000 cells were seeded onto a coated microscope slide
and incubated with 20 mm of complex 1 for 24 h in the dark. After
incubation, the medium containing the complex was removed,
and fresh medium was added to the cells. The cells were rinsed in
pre-warmed PBS, fixed in 4% paraformaldehyde (VWR) for 10 min,
and labelled with Draq5 (eBioscience) following the instructions of
the manufacturer. A confocal laser scanning microscopy system
(Zeiss LSM 710) was used to acquire the images, which were pro-
cessed with Zen software.
Bio-layer interferometry (BLI)
BLI sensors coated with streptavidin (SA sensors) were purchased
from Forte Bio (PALL). Prior to functionalization, they were im-
mersed for 10 min in buffer to dissolve the sucrose layer. They
were then dipped for 15 min in the requisite DNA solution (biotin-
ylated systems A–D) at 100 nm and rinsed in buffer solution
(10 mm HEPES pH 7.4, 35 mm NaCl, 50 mm KCl, and 0.5% v/v sur-
factant P20) for 10 min. The functionalized sensors were next
dipped in solutions of the respective ruthenium complexes at dif-
ferent concentrations (see the Supporting Information) for 2 min
interspersed by a rinsing step in the buffer solution for 4 min. Ref-
Photocytotoxicity experiments
U2OS cells were cultured for 24 h in DMEM (Westburg) containing
10% FBS (Gibco) and 1% penicillin/streptomycin (Westburg) in 96-
well plates to reach a density of 10000 cells/well. The supernatant
was then removed and fresh medium containing 10 mm of the
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requisite complex was added. After 24 h of incubation at 378C in
the dark, cells were rinsed twice with PBS to remove non-internal-
ized complex. They were then illuminated for 30 min with blue
LEDs (LED strip IP68 60 LEDmÀ1 from Prolumia, 405 nm at
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Acknowledgements
J.W., M.A., L.M., and B.E. gratefully acknowledge the Fonds Na-
tional pour la Recherche Scientifique (F.R.S.-FNRS), the Fonds
pour la Formation à la Recherche dans l’Industrie et dans l’Ag-
riculture (F.R.I.A.), the RØgion Wallonne, the UniversitØ catholi-
que de Louvain (UCLouvain), and the “Prix Pierre et Colette
Bauchau” for financial support. J.W. thanks the Erasmus pro-
gramme. This work was supported by the Agence National de
la Recherche (ANR-12-BSV8-0008-04 and ANR-16-CE11-0006-
01), Labex ARCANE (ANR-11-LABX-0003-01), and the RØgion
Auvergne-Rhône-Alpes. The NanoBio-ICMG platforms (FR 2607)
are acknowledged for their support. We also thank Dr. L.
Troian-Gautier for scientific advice concerning the synthesis of
CPITAP, Dr. L. Bonnat and Dr. T. Lavergne for providing biomo-
lecular systems A–D, and Prof. F. Loiseau for help with the
measurement of luminescence lifetimes. M.-C. Eloy is thanked
for help with the confocal microscopy experiments. We thank
L. Vriamont for the cover artwork.
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Keywords: DNA
photocytotoxicity · photo-electron transfer · ruthenium
complexes
structures
·
G-quadruplexes
·
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Manuscript received: September 18, 2018
Revised manuscript received: October 22, 2018
Accepted manuscript online: October 26, 2018
Version of record online: December 11, 2018
Chem. Eur. J. 2018, 24, 19216 –19227
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