Flexible RuII Schiff Base Complexes: G-Quadruplex DNA Binding and Photo-Induced Cancer Cell Death

Article abstract of DOI:10.1002/chem.202001409

A series of new Ru^II Schiff base complexes built on the salphen moiety has been prepared. This includes four flexible monometallic Ru^II compounds and six rigid bimetallic analogues that contain Ni^II, Pd^II or Pt^II cations into the salphen complexation site. Steady state luminescence titrations illustrated the capacity of the compounds to photoprobe G-quadruplex (G4) DNA. Moreover, the vast array of the Schiff base structural changes allowed to extensively assess the influence of the ligand surface, flexibility and charge on the interaction of the compounds with G4 DNA. This was achieved thanks to circular dichroism melting assays and bio-layer interferometry studies that pointed up high affinities along with good selectivities of Ru^II Schiff base complexes for G4 DNA. In cellulo studies were carried out with the most promising compounds. Cellular uptake with location of the compounds in the nucleus as well as in the nucleolus was observed. Cell viability experiments were performed with U2OS osteosarcoma cells in the dark and under light irradiation which allowed the measurements of IC₅₀ values and photoindexes. They showed the substantial role played by light irradiation in the activity of the drugs in addition to the low cytotoxicity of the molecules in the dark. Altogether, the reported results emphasize the promising properties of Ru^II Schiff base complexes as a new class of candidates for developing potential G4 DNA targeting diagnostic or therapeutic compounds.

Full text of DOI:10.1002/chem.202001409

Accepted Article
Accepted Article
Title: Flexible RuII Schiff base complexes: G-quadruplex DNA binding
and photo-induced cancer cell death
Authors: Martin Gillard, Justin Weynand, Hugues Bonnet, Frédérique
Loiseau, Anabelle Decottignies, Jérôme Dejeu, Eric Defrancq,
and Benjamin Elias
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of the final Version of Record (VoR). This work is currently citable by
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To be cited as: Chem. Eur. J. 10.1002/chem.202001409
Link to VoR: https://doi.org/10.1002/chem.202001409
01/2020

10.1002/chem.202001409
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Flexible RuII Schiff base complexes: G-quadruplex DNA binding
and photo-induced cancer cell death
Martin Gillard,[a] Justin Weynand,[a,b] Hugues Bonnet,[b] Frédérique Loiseau,[b] Anabelle Decottignies,[c]
Jérôme Dejeu*,[b] Eric Defrancq*[b] and Benjamin Elias*[a]
Abstract:
constitute a major step forward in the survival of cancer patients.
However, this class of drugs turned out to be highly cytotoxic,
often resulting in heavy side effects due to the weak selectivity of
the compounds towards cancer cells.[2] During the last decades,
the pursuit for more selective anticancer agents has emulated the
development of new drugs able to recognize non-canonical DNA
substructures that are found in cancer cells such as mismatches[3-
7] and G-quadruplexes (G4s).[8-9] G4s, in which DNA assembles in
the stacking of at least two guanine quartets, constitute a more
and more studied target as they are well differentiated from duplex
DNA.[10-12] Furthermore, G4 DNA was reported to play a key role
in the development of cancer being involved in the immortalisation
process by virtue of their abundance in telomeric regions.[13]
Indeed, reactivation of the telomerase protein in most cancer cells
promotes the telomere extension in turn favoring the proliferation
of cancer.[14] Therefore, G4 DNA appeared to be in higher
prevalence in cancer cells, which has opened a new field for more
targeted therapies.[15-17]
Plenty of small molecules have already been described for their
tight binding of G4s in vitro; with some compounds being at
advanced stages of clinical trials.[8-9] Among the designed
compounds, organic molecules based either on the acridine,
phenanthroline or more recently quinazoline frameworks showed
good selectivities for G4;[18-22] while in terms of metal complexes,
many metalloporphyrins and metal-salphens also revealed to
possess high affinities and selectivities towards G4.[23-29] In
addition, mono and dinuclear ruthenium (II) complexes carrying a
polyazaaromatic and rigid planar extended ligand were developed
and were shown to bind tightly to the G4 scaffold.[30-36] The high
affinity of these compounds mainly arises from efficient π-
stacking interactions with the G4 external quartet and additional
interaction with the G4 grooves also appeared to play a prominent
role in the interaction of some complexes.[37-39] More recently,
studies focused on the importance of designing more flexible
ligands for recognizing G4 over duplex DNA thanks to favored
groove interactions.[40-41]
A series of new RuII Schiff base complexes built on the salphen moiety
has been prepared. This includes four flexibile monometallic
RuII compounds and six rigid bimetallic analogues that contain NiII,
PdII or PtII cations into the salphen complexation site. Steady state
luminescence titrations illustrated the capacity of the compounds to
photoprobe G-quadruplex DNA. Moreover, the vast array of the Schiff
base structural changes allowed to extensively assess the influence
of the ligand surface, flexibility and charge on the interaction of the
compounds with G-quadruplex DNA. This was achieved thanks to
circular dichroism melting assays and bio-layer interferometry studies
that pointed up high affinities along with good selectivities of
RuII Schiff base complexes for G4 DNA. In cellulo studies were
carried out with the most promising compounds. Cellular uptake with
location of the compounds in the nucleus as well as in the nucleolus
was observed. Cell viability experiments were performed with U2OS
osteosarcoma cells in the dark and under light irradiation which
allowed the measurements of IC50’s and photoindexes. They showed
the substantial role played by light irradiation in the activity of the
drugs in addition to the low cytotoxicity of the molecules in the dark.
Altogether, the reported results emphasize the promising properties
of RuII Schiff base complexes as a new class of candidates for
developing potential G4 DNA targeting diagnostic or therapeutic
compounds.
Introduction
DNA has been one of the privileged cellular target in cancer
research since the understanding of the nitrogen mustard gas
mode of action reported in 1946.[1] Chemotherapeutics targeting
DNA have proven to be extremely effective drugs and to
[a]
M. Gillard, Dr. J. Weynand and Prof. B. Elias
Université catholique de Louvain (UCLouvain)
Institut de la Matière Condensée et des Nanosciences (IMCN),
Molecular Chemistry, Materials and Catalysis (MOST)
Place Louis Pasteur 1, bte L4.01.02, 1348 Louvain-la-Neuve
(Belgium)
In the present paper, we were interested in studying new kinds of
RuII complexes that do not contain a rigid polyazaaromatic
interacting ligand but a more flexible interacting ligand with a view
of enhancing the selectivity for G4 over duplex DNA. RuII
compounds carrying Schiff base type ligand 1-4 were chosen for
their high structural versatility as well as for their interacting part
flexibility (Figure 1). Indeed, in addition to be electronically
delocalized ligands, the studied Schiff bases contain multiple
single bonds that allow free rotational movements, being at the
origin of the flexibility of the whole structure. Furthermore, the
salphen moiety presents in complexes 1-3 was selected as it
offers the possibility to chelate a second metal cation into the
E-mail: Benjamin.Elias@uclouvain.be
Dr. J. Weynand, Dr. J. Dejeu, Prof. E. Defrancq
Université Grenoble Alpes (UGA)
Département de Chimie Moléculaire, UMR CNRS 5250
CS 40700 - 38058 Grenoble (France).
E-mail: eric.defrancq@univ-grenoble-alpes.fr
jerome.dejeu@univ-grenoble-alpes.fr
[b]
[c]
Prof. A. Decottignies
Université catholique de Louvain (UCLouvain)
de Duve Institute
Avenue Hippocrate 75 , 1200 Brussels (Belgium)
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salphen site. This chelation prevents the free rotations occurring Results and Discussion
in the non-metallated Schiff base subunit in turn leading to the
stiffening of the interacting part. The presence of the d8 cations
NiII, PdII and PtII in compounds 5-10 leads to planar and rigid RuII
bimetallic complexes containing the metal-salphen scaffold as
interacting fragment, metal-salphens being well-known G4 DNA
binders as already mentioned. Overall, the reported complexes 1-
10 will allow to study the influence of the interacting ligand
flexibility on the G4 recognition. In addition, the compelling
perspective of combining the rich and tuneable photochemistry of
RuII compounds with the high G4 DNA affinity and selectivity of
metal-salphens is of great interest. Indeed, in addition to ensure
good solubility in biological conditions, RuII complexes display
suitable cellular uptake properties and the ability to induce cellular
damages under light irradiation, which emphasizes the relevance
of this class of compounds in the search for more targeted
therapies.[42-45] In here, we describe the study of the photophysics
of the RuII Schiff base complexes 1-10 by UV-vis absorption, light
emission spectroscopy and excited state lifetime measurements.
Their ability to selectively bind to G4 over duplex DNA was fully
studied by steady state luminescence titrations, circular dichroism
studies and bio-layer interferometry analysis. Eventually, cellular
uptake and localization of the compounds were determined by
confocal microscopy along with their ability to induce cell death
under light irradiation (IC50 measurements). This constitutes the
first study of this type of RuII complexes in the context of G4
recognition and cellular photo-cytotoxicity assays.
Synthesis of RuII Schiff base and bimetallic d8-analogs
The synthesis of four Schiff base type ligands was achieved by
the condensation of 5,6-diamino-1,10-phenanthroline with two
equivalents of the appropriate aldehyde (i.e. salicylaldehyde, 2-
hydroxy-4-(2-(piperidin-1-yl)ethoxy)
benzaldehyde,
7-
hydroxycoumarin-8-carbaldehyde and 8-quinolinecarbaldehyde)
in the presence of triethylamine in refluxing ethanol (yield: 38-44%,
see supporting information). The choice of the piperidine
substituted salicylaldehyde was motivated by the works reported
by Vilar and co-workers on metal-salphen compounds indicating
increased affinities towards G4 thanks to the presence of the
charged piperidine groups.[25-26] Aiming at extending the ligand
surface, the coumarin and quinoline based aldehydes were
chosen for their ease of preparation. Indeed, salicylaldehyde and
8-quinolinecarbaldehyde were commercially available; while 7-
hydroxycoumarin-8-carbaldehyde was prepared via the Duff
formylation of 7-hydroxycoumarin. 2-hydroxy-4-(2-(piperidin-1-
yl)ethoxy) benzaldehyde was prepared via the alkylation of 2,4-
dihydroxybenzaldehyde by 1-(2-chloroethyl)piperidine (see
supporting information). The structure of the ligands was
confirmed by 1H-NMR spectroscopy and HRMS analysis (see
supporting information). The corresponding RuII complexes were
synthesized by the direct chelation of the ligands onto a
[Ru(phen)2Cl2] precursor in refluxing ethanol (yield: 62-81%, see
supporting information). Complexes 1-4 were isolated as orange
powders and characterized by 1H-NMR spectroscopy and HRMS
analysis (see supporting information). 1H-NMR spectroscopy
shows unambiguously the symmetry of the compounds, which
induces the equivalence of (i) the protons of the ligands and (ii) of
the phenanthroline moieties.
Bimetallic analogues of complexes 1-3 containing a d8 transition
metal cation (NiII, PdII or PtII) into the salphen complexation site
were synthesized to study the influence of the rigidity of the
extended ligand on the G4 recognition capability of the
compounds. Another synthesis strategy was followed for these
structures by modifying the conditions described by Onozawa-
Komatsuzaki et al. for similar compounds.[46] The key reaction
consists in a one-step assembly of a [Ru(phen)25,6-diamino-1,10-
phenanthroline]2+ precursor prepared by reported method[47] with
2 eq. of the appropriate aldehyde and one equivalent of the d8
metal cation acetate salt (see supporting information). The
reactions were performed in DMSO at 100 °C in the presence of
triethylamine over the course of three hours. They led to the
formation of the corresponding bimetallic complexes 5-10 as dark
red powders with proper yields (28-64%) (Figure 1). This synthetic
pathway proved to be very convenient as it allowed the
preparation of a vast array of analogs by simply changing the
nature of the aldehyde or/and of the metal cation in the one pot
reaction. While RuII-NiII, RuII-CuII and RuII-ZnII complexes were
already reported by similar methods,[46] those are to the best of
our knowledge the first described syntheses of RuII-PdII and RuII-
PtII salphen complexes.
Figure 1. Structure of the RuII Schiff base complexes 1-10.
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Electrochemical study
to the apparition of a larger band at ca. 360 nm compared to the
salicylic analogs (Figure 2 A and see supporting information).
To study the intrinsic photophysical properties of compounds 1-
10, electrochemical analyses were first implemented by cyclic
voltammetry in dry deoxygenated CH3CN (Table 1). The oxidation
stage revealed very similar results for all the complexes showing
a one electron oxidation wave within the investigated window,
which is close to that of the Ru2+/Ru3+ oxidation of [Ru(phen)3]2+
(1.32 V vs. Ag/AgCl). Concerning the reduction, three reversible
one electron waves were detected at very similar potentials for all
complexes, which were found notably easier to reduce than
[Ru(phen)3]2+. The first reduction wave (-0.86 to -0.91 V vs.
Ag/AgCl) is ascribed to the reduction of the imine fragments as
already proposed for similar complexes.[48] The two other
reduction waves are attributed to the subsequent reductions of
the two phenanthroline ancillary ligands. As expected, the
complexation of a metal cation (NiII, PdII or PtII) into the salphen
site led to more positive potentials of the first reduction (∆V ≈
0.07V). Altogether, systems 1-10 exhibit very similar
electrochemical properties with data indicating that their HOMO is
located onto the RuII centre and that their LUMO is located onto
the Schiff base unit of the extended ligand.
Table 1. Oxidation (E1/2 ox) and reduction (E1/2 red) potentials of complexes 1-10.
Complex
E1/2ox[a][V][b]
E1/2 red [a] [V][b]
-1.47
[Ru(phen)3]2+
[Ru(phen)2salicyl]2+
1.32
1.36
1.36
1.34
1.33
1.37
1.36
1.36
1.36
1.34
1.35
-1.30
-0.91
-0.90
-0.88
-0.86
-0.84
-0.84
-0.81
-0.86
-0.87
-0.86
-1.68
-1.64
-1.66
-1.63
-1.65
-1.68
-1.69
-1.63
-1.62
-1.61
-1.61
1
-1.45
[Ru(phen)2salicylpip]2+
[Ru(phen)2coumarin]2+
[Ru(phen)2quinoline]2+
[Ru(phen)2Ni-salicyl]2+
2
3
4
5
-1.47
Figure 2. Absorption spectra in acetonitrile under air of A) complexes 1-4 and
B) complexes 5, 8 and 9.
-1.43
-1.44
Table 2. Absorption data in CH3CN for complexes 1-10.
-1.45
Complex
Absorption λmax [nm] (ε [104 L.mol−1.cm−1])[a]
[Ru(phen)2Ni-salicylpip]2+
[Ru(phen)2Ni-coumarin]2+
6
7
-1.40
[Ru(phen)3]2+
265, 418 (sh), 447 (1.84)
-1.42
[Ru(phen)2salicyl]2+
1
265, 288 (sh), 313 (sh), 338 (sh), 458 (1.04)
265, 296 (sh), 333 (2.71), 461 (1.28)
265, 363 (1.95), 428(1.44), 457 (1.69)
265, 281 (sh), 341 (sh), 433 (sh), 460 (1.71)
265, 320, 428 (1.94), 454 (1.74), 580 (0.46)
265, 294, 427 (1.90), 454 (1.71), 580 (0.44)
265, 294, 348 (3.77), 422 (2.18), 481 (1.76)
265, 329, 389 (2.24), 478 (1.96), 514 (20.1)
265, 414 (1.94), 452 (1.79), 540 (0.50)
265, 412 (1.91), 452 (1.77), 540 (0.49)
[Ru(phen)2Pd-salicyl]2+
[Ru(phen)2Pt-salicyl]2+
[Ru(phen)2Pt-salicylpip]2+ 10
8
-1.45
[Ru(phen)2salicylpip]2+
[Ru(phen)2coumarin]2+
[Ru(phen)2quinoline]2+
[Ru(phen)2Ni-salicyl]2+
2
3
4
5
9
-1.45
-1.44
[a] Measured in dry acetonitrile. [b] Potentials are given vs. Ag/AgCl.
[Ru(phen)2Ni-salicylpip]2+
[Ru(phen)2Ni-coumarin]2+
6
7
Light absorption
Light absorption data of the complexes were recorded at ambient
temperature in acetonitrile under air (Table 2) and in water (data
not shown). The spectra of complexes 1-4 display typical shapes
and molar extinction coefficients of polyazaaromatic RuII
complexes. Indeed, in the visible part of the spectra (at ca. 460
nm), we notice the occurrence of absorption bands (ε ≈ 15000
L.mol-1cm-1) assigned to MLCT transitions. Strong absorption
bands resulting from LC transitions centred on the phenanthroline
ligands are also observed in the UV-region (250 nm). Noticeably,
the presence of the coumarin moiety in complexes 3 and 7 leads
[Ru(phen)2Pd-salicyl]2+
[Ru(phen)2Pt-salicyl]2+
[Ru(phen)2Pt-salicylpip]2+ 10
8
9
[a] Measurements were performed with 1 x 10−5 mol L−1 solutions of the
complex at room temperature. Extinction coefficients are reported in brackets.
sh = shoulder.
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Besides, the addition of a d8 cation into the salphen site
substantially increased the absorption intensity in the visible
(Figure 2 B). This is attributed to the overlay of the MCLT
transitions of RuII and d8 salphen moieties and to an increased
conjugation occurring between the d8 salphen and the
phenanthroline parts of the complexes as already proposed in the
literature.[46, 49-50] Interestingly, the fact that RuII-NiII analogs 5-7
display the most bathochromic absorption among the bimetallic
lifetimes (4-44 ns) and low emission quantum yields (i.e. <10-5).
This is in agreement with the few similar bimetallic RuII-NiII
complexes reported in the literature and is attributed to an
electron transfer through the metal-salphen fragment in turn
leading to luminescence quenching.[46, 52] The luminescence of
the compounds being crucial to perform biological assays, it was
of high importance to obtain luminescent bimetallic analogs. This
encouraged us to synthesize a series of RuII-PtII analogs.
analogs 5-10 is in accordance with their higher reduction potential. Interestingly, the RuII-PtII compounds 9 and 10 showed much
This can be correlated with the fact that the NiII cation is more
electron withdrawing than PdII and PtII cations that possess larger
ionic radii and higher electronic density.
stronger luminescence than the RuII-NiII and RuII-PdII analogs. As
already proposed for the monometallic PtII salphen complexes,
the involved transition likely consists in a triplet metal-ligand to
ligand charge transfer (MLLCT) occurring between the PtII
phenoxide moiety and the imine fragments (Pt(d)/O(p)→
π∗(N^N)).[49-50] This assumption is supported by the fact that
Light emission
Light emission properties of complexes 1-10 were investigated in
acetonitrile and in water. Strong luminescence of complexes 1-4
was observed with wavelengths, quantum yields and excited state
lifetimes similar to those of [Ru(phen)3]2+ (Table 3). These typical
complexes
9 and 10 possess very similar photophysical
properties to those of PtII salphen complexes with (i) very close
emission wavelength maxima (λ = 641-644 nm in acetonitrile) and
(ii) the same characteristic large hypsochromic shift of the
characteristics indicate
a
3MLCT-type emitting state for
complexes 1-4 as already reported for [Ru(phen)3]2+.[51] In addition, emission maxima from acetonitrile to water (∆λ ≈ 25 nm). This
complexes 1-4 also displayed longer excited state lifetimes in
water than in acetonitrile as observed for [Ru(phen)3]2+ which can
be linked to a difference of the capability of the complex to reach
increase of the luminescence energy in a more polar solvent
suggests that RuII-PtII complexes are less polar at the excited
state than at the ground state.
3
the MC non-emissive state.[51] The bimetallic RuII-NiII and RuII-
PdII compounds 5-8 were very poorly luminescent with short
Table 3. Emission data in CH3CN and H2O at 298 K under ambient air for complexes 1-10.
[c,d]
Emission
Φem
τ[c] [ns]
kr [103 s-1]
CH3CN
λmax[a,b][nm]
Complex
CH3CN
H2O
CH3CN
H2O
CH3CN
H2O
H2O
[Ru(phen)3]2+
604
599
599
599
598
680
678
682
670
644
641
606
600
600
601
600
n.d.
n.d.
n.d.
n.d.
619
618
0.028
0.018
0.022
0.019
0.032
0.072
0.075
0.081
0.065
0.092
460
688
687
576
670
7
920
1222
1210
862
1080
n.d.
n.d.
n.d.
n.d.
460
458
60.9
26.2
32.0
33.0
47.8
n.d.
75.0
61.4
66.9
75.4
85.2
n.d.
[Ru(phen)2salicyl]2+
1
[Ru(phen)2salicylpip]2+
[Ru(phen)2coumarin]2+
[Ru(phen)2quinoline]2+
[Ru(phen)2Ni-salicyl]2+
2
3
4
5
[e]
[e]
–
–
[e]
[e]
[Ru(phen)2Ni-salicylpip]2+
[Ru(phen)2Ni-coumarin]2+
6
7
–
–
5
n.d.
n.d.
[e]
[e]
–
–
4
n.d.
n.d.
[e]
[e]
[Ru(phen)2Pd-salicyl]2+
[Ru(phen)2Pt-salicyl]2+
[Ru(phen)2Pt-salicylpip]2+ 10
8
–
–
44
n.d.
n.d.
9
0.0041
0.0037
0.0068
0.0066
380
352
14.8
19.6
8.04
8.95
[a] Measurements were made with solutions 5 × 10−6 mol L−1 in complex under air. [b] λexc = 450 nm. [c] Measurements were made with 5
× 10−6 mol L−1 solutions of the complex under nitrogen. [d] Measurements relative to [Ru(bpy)3]2+ in nitrogen purged aqueous solution (Φem
= 0.063) and in nitrogen purged acetonitrile (Φem = 0.094); errors are estimated as 5%.[53] [e] The luminescence was observed with Φem
10-5. n.d. stands for not determined.
<
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DNA interaction studies
Binding constant are obtained using a McGhee–von Hippel type equation; the
To investigate the ability of the compounds to bind tightly and
selectively to the G-quadruplex topologies (G4s), their interaction
with both duplex and G4 DNA was studied via steady state
luminescence titrations, circular dichroism and bio-layer
interferometry analyses.
binding site corresponds to base pair or G-quartet (best fit). Errors estimated to
5%.
The excited state of RuII-PtII Schiff base complexes 9 and 10
showed to be rather influenced by the presence of G4 compared
to duplex DNA. Indeed, a notable hypsochromic shift of the
emission maximum occurs while interacting with G4 DNA (∆λ =
18 nm for G4 compared to 6 nm for duplex DNA) due to the more
hydrophobic environment afforded by the nucleic bases.
Luminescence titrations
The ability of the luminescent complexes 1-4 and 9-10 to
photoprobe duplex and G4 DNA was first studied by steady state
luminescence
titrations
using
a
20-mer
hairpin
(5’CGT3CGT5ACGA3CG3’) (HPATGC) as duplex model and the
human telomeric wtTel23 sequence (3’TT(GGGATT)3GGG5’) as
G4 model. All the studied RuII Schiff base complexes displayed
increased luminescence in the presence of small concentrations
of duplex and G4 DNA which had not been reported for these
types of systems. This behavior indicates strong binding towards
these structures along with efficient protection of the excited state
from non-radiative deexcitation sources such as collisions with
solvent or oxygen photosensitization when interacting with
oligonucleotides. This can be related to a “light switch” type effect
as reported for the detection of duplex DNA in the case of
[Ru(bpy)2dppz]2+.[54] Complexes 1 and 3 appeared to be the most
promising compounds according to luminescence titrations as
they exhibit a stronger enhancement of their luminescence in the
presence of G4 compared to duplex DNA (Figure 3), probably
arising from a different protection of the complex from non-
radiative deexcitation sources. Moreover, the fact that less
equivalents of complexes 1 and 3 are required to reach the light
emission maximum intensity in the case of G4 oligonucleotides
may indicate a stronger binding of these complexes towards G4s.
A McGhee-von Hippel type equation (see supporting information)
was implemented to fit the titration curves. This allowed to assess
the dissocation constant (KD) for the luminescent compounds 1-4
and 9-10 towards double stranded hairpin and G4 DNA. As
depicted in Table 4, most complexes display affinities towards G4
in the micromolar range or lower. Concerning the most promising
candidates in terms of selectivity, compound 3 possess the best
affinity for G4 DNA (KD = 1.9 µM) compared to complex 1 (KD = 15
µM). Moreover, complexes 1 and 3 displayed much lower
affinities towards the hairpin shape oligonucleotides (101 and 61
µM respectively) revealing good selectivities for G4 over duplex
DNA structures.
Figure 3. Relative luminescence intensity (I/I0) of 1 and 3 as a function of the
relative oligonucleotide concentration ([site]/[Ru]). With I0, the luminescence of
the complex in the absence of DNA (λexc = 450 nm; λem = 599 nm). One site
corresponds to base pair or G-quartet. The fitted curves are obtained using a
modified McGhee–von Hippel equation (see supporting information) and drawn
as dashed or solid curves for hairpin and G4 DNA respectively.
Circular dichroism
CD melting assays were implemented to further investigate the
impact of the complexes 1-10 binding on the stability of the duplex
and G4 DNA structures. CD analyses were performed using the
wtTel23 sequence as G4 model and the hairpin HPATGC as duplex
model. The experiments were carried out in 10 mM Tris-HCl and
100 mM NaCl buffer to ensure that the G4 structure was folded
into a single antiparallel conformation and not into hybrid
conformations as it is the case with the use of K+ (see supporting
information).[55] It should be noted that, although well appropriate
for our measurements, these saline conditions are not biologically
relevant conversely to the conditions used for BLI analysis and
luminescence titrations experiments. The proper folding of
wtTel23 into the antiparallel G4 topology was confirmed by the
presence of two positive peaks at 242 and 294 nm in the 100 mM
NaCl buffer (see supporting information).[56-57] The folding of
HPATGC was characterised by a negative peak at 252 nm. CD
melting assays were carried out to study the impact of the
presence of the complex on the stability of the G-quadruplex and
the hairpin DNA structures. CD melting curves were recorded in
the presence (5 equivalents) and in the absence of the complex
in 100 mM NaCl containing buffer for wtTel 23 and for the duplex
hairpin on the temperature range 20-95°C (Table 5). No CD signal
could be observed above 310 nm, which might be explained by a
too low sensitivity of our equipment (it should be noted that the
intensity of MLCT transitions is quite low compared to the DNA
absorption in the concentration ratio used). The so measured
melting temperatures show a strong stabilization of the G4
Table 4. Equilibrium dissociation constants (KD) for the interaction of
complexes 1-4 and 9-10 with duplex hairpin and G4 DNA measured from steady
state luminescence titrations.
HPATGC
KD (µM)
101
1.9
G4 wtTel23
KD (µM)
15
Compound
I/I0 max
2.26
I/I0 max
4.12
[Ru(phen)2salicyl]2+
1
[Ru(phen)2salicylpip]2+
[Ru(phen)2coumarin]2+
[Ru(phen)2quinoline]2+
[Ru(phen)2Pt-salicyl]2+
2
3
4
9
2.41
1.6
3.21
61
2.42
1.9
5.22
7
2.51
4.2
4.71
5.2
1.40
1.4
2.22
[Ru(phen)2Pt-salicylpip]2+ 10
2.4
1.44
0.88
2.34
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Table 5. Melting temperatures (Tm) for the wtTel23 G4 and the HPATCG hairpin oligonucleotides in the absence or in the presence of 5 eq of the complexes.[a]
Oligonucleotide
No complex
1
2
3
4
5
6
7
8
9
10
wtTel23
Tm [°C][b]
50.2
0
59.2
9
60.1
9.9
61.3
7.1
60.5
10.3
56.3
2.1
58.8
8.6
59.6
5.4
57.1
6.9
59.2
5.0
57.9
7.7
62.6
8.4
55.3
5.1
58.3
4.1
56.8
6.6
60.0
5.8
56.7
6.5
60.9
6.7
58.8
8.6
∆ Tm [°C][c]
Tm [°C]b]
HPATGC
54.2
0
54.6
0.4
63.0
8.8
∆ Tm [°C][c]
[a] Oligonucleotides were first annealed by heating at 95 °C for 5 min in Tris-HCl buffer (10 mM, pH 7.4) with 100 mM NaCl and then allowed to cool to room
temperature overnight. Oligonucleotide concentrations were kept at 2.5 µM. The ellipticity was recorded at 294 and 252 nm for wtTel23 and duplex hairpin,
respectively. [b] Error in Tm is estimated to 0.5 °C. [c] Error on ∆ Tm is estimated to 1 °C.
structure by the complexes with ∆Tm ranging from 5.1 to 10.3 °C.
Interestingly, the stabilisation of G4s was found less effective with
bimetallic compounds compared to monometallic ones. Indeed
∆Tm from 9 to 10.3 °C were observed for complexes 1-3 whereas
for bimetallic complexes 5-10 slightly lower ∆Tm were obtained
(from 5.1 to 8.6 °C). The effect of the piperidine arms was also
noticed as a slight increase of ∆Tm was observed from 1 to 2, from
5 to 6 and from 9 to 10, respectively. Remarkably, complexes 1
and 3 displayed much weaker stabilizing effect on duplex DNA
than on G4 DNA which is in accordance with the differences
observed by luminescence titrations. However, it should be
noticed that circular dichroism analyses as well as steady state
luminescence titrations are not the most appropriate methods for
the direct measurements of affinity constants with G-quadruplex
DNA.[58] Therefore, bio-layer interferometry was used to
determine the thermodynamic parameters of the interaction.
between small molecules and different DNA secondary structures
such as mismatches and G-quadruplexes.[7, 34, 59-61] In this study,
BLI analysis was performed for complexes 1-10 using wtTel23
and a GC rich hairpin duplex (3’(GC)4TTTT(GC)45’) (HPGC
)
sequences which are both 3’-biotinylated for attachment on the
BLI sensors.
Affinities of complexes 1-10 for G4s cover two orders of
magnitude of concentrations standing from 0.24 µM for the
strongest affinity of complex 6 to 21 µM for the lowest affinity of
complex 1 which shows the substantial impact of the Schiff base
part structural changes on the interaction strength (Table 6 and
see supporting information). Concerning complex 1, a lower
interferometry signal was observed for the interaction with G4
compared to the other studied compounds (see supporting
information). This is probably due to a different interaction type
(π-stacking, loop or groove interaction) that can bring a fluctuant
variation of the layer thickness. It could also arise either from a
difference of optical properties (in particular difference in
refractive index) or a lower accessibility of compound 1 to the
entire G4 DNA immobilized. The addition of positively charged
piperidine chains on the salphen moiety increased significantly
the affinity for G4 and appears when comparing the G4 affinity of
complex 1 versus 2 (21 µM and 1 µM, respectively), complex 5
versus 6 (11 µM and 0.24 µM, respectively) and to a lesser extent
complex 9 versus 10 (0.84 µM and 0.63 µM, respectively). This
can be explained by favourable interactions with the G4 grooves
as previously reported for metal salphen complexes.[26-29] These
results were in accordance with CD melting experiments.
Bio-layer interferometry
Bio-layer interferometry analysis (BLI) is an optical technique that
has been recently implemented to study biomolecular interactions
Table 6. Equilibrium dissociation constants (KD) for the interaction of
complexes 1-10 with G4 and duplex DNA.
HP
wtTel 23
KD (µM)[a]
Complex
KD (µM)[a]
n.d.[b]
[Ru(phen)2salicyl]2+
1
21 ± 2
[Ru(phen)2salicylpip]2+
[Ru(phen)2coumarin]2+
[Ru(phen)2quinoline]2+
[Ru(phen)2Ni-salicyl]2+
2
3
4
5
1.0 ± 0.2
48 ± 11.7
3.9 ± 0.8
16 ± 7
1.0 ± 0.02
2.5 ± 0.9
1.5 ± 0.1
11± 0.7
Replacing the salicyl moiety by the coumarin or quinoline moieties
in complexes 3 and 4, respectively, allowed an enhancement of
the affinity for G4. This observation can be justified by a higher π-
stacking interactions through the aromatic coumarin and quinoline
part relative to the salicyl one. It was noted that the stiffening of
the systems via the addition of NiII, PdII and PtII in the salphen
moiety led to improved affinities for G4s. Indeed, bi-metallic
complexes 5, 8 and 9 showed an affinity of 11, 6.5 and 0.84 µM,
respectively whereas the parent complex 1 has an affinity of 21
µM. The same trend was observed for complexes 6 and 10
(affinity for G4s of 0.24 µM and 0.63 µM, respectively) in
comparison with the parent complex 2 (1.0 µM). Interestingly, the
affinity for G4 also seems to follow the trend of the cation size with
the PtII version 9 showing the highest affinity towards G4 (840 nM).
Overall, the majority of the studied RuII complexes 1-10 show
affinities towards G4 with the same order of magnitudes
compared to other RuII complexes that have been already
published in the literature.[33-34, 36, 62] It should be noted that for
[Ru(phen)2Ni-salicylpip]2+
[Ru(phen)2Ni-coumarin]2+
6
7
0.37 ± 0.03
14 ± 6
0.24 ± 0.02
3.8 ± 0.9
6.5 ± 0.4
0.84 ± 0.2
0.63 ± 0.04
[Ru(phen)2Pd-salicyl]2+
[Ru(phen)2Pt-salicyl]2+
[Ru(phen)2Pt-salicylpip]2+ 10
8
18 ± 1
9
1.0 ± 0.1
1.4 ± 0.1
[a] Equilibrium dissociation constants were deduced from the kinetic rate
constants. [b] Due to the very low binding of the complex with hairpin DNA,
the kinetics of the interaction could not be determined (n.d.) in the studied
concentration range.
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complexes 6 and 10 a higher affinity similar to the well-known
BRACO-19 G4 ligands was observed.[63]
in cellulo. Figure 5 unambiguously shows a better co-localization
of compound 2 with the nucleus marker DRAQ5 compared to
compound 1. Also, complex 2 appeared to accumulate in the
nucleoli, which display high density of nucleic acids (Figure 5; E).
A significant selectivity of complexes 1 and 3 for G4 relative to
duplex DNA was observed. Despite an interesting increase of
affinity for G4, a selectivity decrease was observed for the bi-
metallic analogues. It was also noticed that although the presence
of piperidine arms on the salphen fragment led to remarkable
affinities towards G4 a loss of the selectivity occurred. This is
likely due to increased non-specific electrostatic interactions
caused by the protonation of the piperidine fragments at pH 7.4.
Finally, it is also interesting to compare the kinetic constants of
the interaction between the complexes 1-10 for G4 and duplex
DNA to better understand the observed affinity differences (Figure
4). We observed that the dissociation rates span on a larger range
(from 8,11.10-2 s-1 to 2,2.10-3 s-1) than the association rates (from
1,6.104 M-1.s-1 to 1,8.105 M-1.s-1). We could notice that the better
affinities towards G4 relative to duplex DNA of complexes 1, 3, 7
and 8 was due to a slower dissociation with G4. Second, the non-
selective complexes 6, 9, 10 display the lowest dissociation
constants both for duplex and G4 DNA. Whereas complexes 2, 4
and 6 possess the highest association rate.
Figure 5. Cell penetration study. Confocal microscopy pictures of U2OS cancer
cells after incubation with 20 µM of complex 1 (A-C) or 2 (D-F) in DMEM for 1,5
hour. Nucleus was stained in red by DRAQ5 (A and D); RuII complexes appear
in yellow (B and E). C and F are merged images (orange). Scale bar 10 µm.
Similar observations were made for complex 10 (see supporting
information). This increased targeting of complex 2 towards the
genetic material is likely due to the additional positive charge
brought by the piperidine arms that are protonated in biological
conditions. The RuII-PtII compounds 9 and 10 both displayed
great compartmentalisation within the nucleus, especially
compared to 1. This might be explained by the higher affinities
towards oligonucleotides of the mentioned PtII compounds .
Cellular photo-cytotoxicity
In view of the ability of compounds 1-3, 9 and 10 to
penetrate cells and to locate in the nucleus, their ability to kill cells
under light irradiation was studied using the same U2OS cancer
cell line. The half maximal concentration of each complex required
to kill U2OS cells (IC50) was measured in the dark and under light
irradiation by testing a range of concentrations. Cells were
incubated with drugs for one hour, before light irradiation for 30
minutes. Control cells were not irradiated. Cells were then
incubated in culture medium for 24 hours before their metabolic
activity was measured to quantify cell viability. Whereas toxicity
was very low in the dark for all tested compounds, cell viability
decreased dramatically under light irradiation (405 nm; 15.7 Wm-
2) for complexes 1-3, 9 and 10, displaying IC50 values in the
micromolar range or lower (Table 7). Interestingly, incorporation
of piperidine arms to our systems such as in complexes 2 and 10
led to increased photo-cytotoxicities, which might be related to
their increased ability to target the genetic material as suggested
by the cell penetration assays. However, both compounds also
showed higher toxicity in the dark (Table 7). Presence of PtII into
the salphen site of compounds 9 and 10 led to very similar IC50
values compared to their respective non d8 metallated analogs,
complexes 1 and 2 respectively.
Figure 4. Two-dimensional isoaffinity kinetic plot of rate constants measured by
BLI. The dashed diagonals depict the equilibrium binding constants and are
shown to help with the visualization of the affinity distribution. Complexes 1-10
are plotted in the presence of duplex DNA hairpin (HP) and G4 DNA (G4).
In cellulo studies
Cell penetration
Due to their promising luminescence properties, the ability
of complexes 1-3, 9 and 10 to penetrate cells and to target the
genetic material was studied by confocal microscopy using U2OS
osteosarcoma cell line as model. The selected compounds
allowed to study the impact of the coumarin and the piperidine
moieties and of the chelated PtII onto the cell localization of the
drugs. Complexes 1-3, 9 and 10 were found to penetrate cells with
differentiated
intracellular
localization
(see
supporting
information). At the studied concentration, compounds 1, 3 and 9
localize both outside and inside the nucleus, with complexes 3
and 9 being in higher prevalence within the nucleus. Noticeably,
the presence of piperidine fragments in complexes 2 and 10 led
to enhanced ability of the complexes to target the genetic material
In conclusion, all tested compounds display high photoindex,
highlighting the prominence of light excitation for the activation of
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the drugs as well as their remarkably weak cytotoxicity in the dark
(Table 7).
studies were conducted using the U2OS osteosarcoma cell line
to assess the ability of the molecules to penetrate the cell and to
induce mortality under light irradiation. Confocal microscopy
showed that all the studied complexes 1-3, 9 and 10 penetrate the
cell and mainly localize to the nucleus. Cell viability experiments
conducted either in the dark or under light irradiation revealed
suitable IC50’s of the compounds (from 0.326 to 2.71 µM) along
with high photoindex, showing the strong impact of light excitation
in the activity of the drugs as well as the remarkably weak toxicity
of the compounds in the dark. However, the link between the
targeting of the drugs to specific DNA secondary structures and
the observed cytotoxicity still needs to be established and will be
a challenging project for the future. In any case, the RuII Schiff
base scaffold proves to be promising in terms of theranostic
applications according to luminescence, interactions and in
cellulo studies.
Table 7. IC50 values of the complexes determined from dose-dependent growth
inhibitory curves vs. U2OS cancer cell line under irradiation and in the dark.
IC50 value[a] [µM]
Complex
Light
PI[b]
(Dark)
[Ru(phen)2salicyl]2+
1
> 47
2.1 ± 0.6
(>100)
[Ru(phen)2salicylpip]2+
[Ru(phen)2coumarin]2+
[Ru(phen)2Pt-salicyl]2+
2
3
0.33 ± 0.06
(47 ± 7)
142
> 71
> 37
45
1.4 ± 0.2
(>100)
9
2.7 ± 0.6
(>100)
[Ru(phen)2Pt-salicylpip]2+ 10
0.73 ± 0.20
(33 ± 7)
Experimental Section
[a] IC50 values are means ± standard errors calculated from 3 independent
experiments (4 wells per condition). [b] PI = photoindex = IC50 dark/IC50 light
Materials and instrumentation
5,6-diamino-1,10-phenanthroline,
[Ru(phen)2Cl2]
and
Conclusions
[Ru(phen)25,6-diamino-1,10-phenanthroline]2+ were synthesized
according to previously described literature protocols.[47] The
oligonucleotides wtTel23 (3’TT(GGGATT)3GGG5’), mixed AT-GC
hairpin duplex sequence HPATGC (3’CGT3CGT5ACGA3CG5’) and
GC-rich hairpin duplex HP (3’(GC)4TTTT(GC)45’) 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-exclusion chromatography (SEC). All
solvents and reagents for the synthesis were of reagent grade and
were used without any further purification. All solvents for the
spectroscopic and electrochemical 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, d6-DMSO, CD3OD or CD3CN on a Bruker AC300
Avance II (300 MHz) or a Bruker AM-500 (500 MHz) spectrometer
at 20 °C. Chemical shifts (in ppm) were referenced to the residual
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;
capillary temperature 320 °C, vaporizer temperature 320 °C,
sheath gas flow rate 5 mL min-1).
In this paper, we reported on the synthesis of ten new RuII Schiff
base complexes 1-10. The structures of these compounds were
inspired by the metal-salphen and the polyazaaromatic RuII
complexes developed for G4 recognition and are distinct from the
RuII compounds usually described in the field. We prepared
bimetallic RuII-PtII salphen based complexes 9 and 10 that both
showed strong luminescence in addition to photophysical
properties that indicate an excited state centred on the PtII salphen
moiety. The luminescence of complexes 1-4, 9 and 10 increases
significantly in the presence of DNA with complexes 1 and 3
showing well differentiated behavior in the presence of G4 relative
to duplex DNA. Interestingly complexes 9 and 10 also revealed
luminescence wavelength maxima that are well different between
duplex and G4 DNA. The interaction of the complexes with duplex
and G4 DNA was studied thanks to melting assays using circular
dichroism. The presence of the d8 cation in the salphen
complexation site led to weaker stabilization of G4. More
generally, the results obtained could confirm the observations
made in luminescence titrations with notably complexes 1 and 3
leading to stronger stabilizing effect on G4 than on duplex DNA.
Finally, bio-layer interferometry was implemented to better
characterize the kinetics and the strength of the interaction of the
complexes with the two type of DNA secondary structures. This
analysis show that the compounds interact with dissociation
constants in the micromolar range or lower. Interestingly, it was
noticed that the presence of the d8 cation into the salphen site of
complexes 5-10 led to higher affinity for G4 DNA than their
monometallic analogs 1-4. Moreover, the monometallic
complexes 1 and 3 proved to be selective for G4 DNA compared
to duplex DNA. All combined with the stronger stabilizing effect
induced by complexes 1-4 on G4 recorded by CD experiments,
these results could be explained by the fact that complexes that
possess a more flexible interacting part give rise to a different
mode of interaction with G4 DNA. Also, the presence of piperidine
arms in complexes 6 and 10 led to stronger interactions with both
duplex and G4 DNA, probably due to the additional charge
brought by the piperidine that are protonated at pH 7.4. In cellulo
Synthesis procedures and characterization
Preparation of [Ru(phen)2salicyl]2+ 1.
The dichloro precursor [Ru(phen)2Cl2] (20 mg, 0.038 mmol) and
the salicylsalphen ligand (16 mg, 0.038 mmol) were mixed in a
solution of absolute ethanol. The reaction medium was then
stirred at 80 °C until the precursor was consumed as monitored
by TLC. Afterwards, ethanol was evaporated and addition of small
portions of NH4PF6 yielded to the formation of a precipitate. After
centrifugation, the crude solid was washed several times with
water and was then dried under reduced pressure. The residue
was finally purified by flash chromatography on silica gel
(CH3CN/H2O/ KNO3sat 10:1:1/4) to provide the title compound 1
as an orange solid (35 mg, 0.030 mmol, 80%). Rf 0.31
(CH3CN/H2O/ KNO3sat 10:1:1/4). The counter-anion exchange
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from PF6 to Cl was performed by adding small portions of NBu4Cl
to a solution of the complex in acetone. This led to the
precipitation of the complex which was then washed three times
with acetone to remove any exceeding salts. 1H NMR (CD3CN,
500 MHz) δ 9.03 (broad m, 4H), 8.62 – 8.60 (m, 2H), 8.60 – 8.58
(m, 2H), 8.25 (s, 4H), 8.12 – 8.06 (m, 4H), 8.02 (dd, J = 5.2, 1.2
Hz, 2H), 7.99 (dd, J = 5.3, 1.2 Hz, 2H), 7.68 (dd, J = 8.3, 5.3 Hz,
2H), 7.63 (d, J = 5.2 Hz, 2H), 7.61 (d, J = 5.2 Hz, 2H), 7.51 – 7.44
(m, 2H), 7.17 – 7.06 (m, 4H). HRMS-ESI calculated for
[C43H28N8O96Ru]2+ : m/z 384.07255, found: m/z 384.07322.
under reduced pressure. Addition of water and small portions of
NH4PF6 led to the formation of a precipitate that was centrifuged.
The crude product was purified by flash chromatography on silica
(acetonitrile/water/KNO3
with a ratio 10:1:1/2) to give
sat
compound 5 as a dark-red solid (16.4 mg, 0.0170 mmol, 64%).
-
The counter-anion exchange from PF6 to Cl- was performed by
adding small portions of NBu4Cl to a solution of the complex in
acetone. Rf 0.32 (CH3CN/H2O/ KNO3sat 10:1:1/2); 1H NMR
(CD3CN, 500 MHz) δ 8.76 (d, J = 8.5 Hz, 2H), 8.63 (d, J = 8.2 Hz,
2H), 8.61 (d, J = 8.4 Hz, 2H), 8.58 (s, 2H), 8.27 (s, 4H), 8.10 (d, J
= 4.7 Hz, 2H), 8.03 (d, J = 4.9 Hz, 2H), 8.00 (d, J = 4.7 Hz, 2H),
7.68 (dd, J = 8.2, 5.2 Hz, 2H), 7.63 (dd, J = 8.2, 5.3 Hz, 2H), 7.59
(dd, J = 8.6, 5.2 Hz, 2H), 7.55 (d, J = 7.5 Hz, 2H), 7.40 (m, 2H),
6.92 (d, J = 8.9 Hz, 2H), 6.77-6.72 (m, 2H). HRMS-ESI calculated
for [C50H32N8O2NiRu]2+ : m/z 468.05193, found: m/z 468.05215.
Preparation of [Ru(phen)2Ni-salicylpip]2+ 6. Using the same
procedure as for 5 with [Ru(phen)2diamino-phenanthroline]2+ (20
mg, 0.021 mmol) and nickel(II) acetate tetrahydrate (6 mg, 0.025
mmol) in DMSO (3 mL) with the addition of 2-hydroxy-4-(2-
(piperidin-1-yl)ethoxy)benzaldehyde (16 mg , 0.063 mmol) gave
the crude product that was purified by flash chromatography on
silica (acetonitrile/water/KNO3 sat with a ratio 10:1:3/4) to yield
compound 6 as a dark-red solid (15.1 mg, 0.0102 mmol, 49%). Rf
0.31 (CH3CN/H2O/ KNO3sat 10:1:3/4);1H NMR (CD3CN, 500 MHz)
δ 8.73 (d, J = 8.7 Hz, 2H), 8.66 (d, J = 8.2 Hz, 2H), 8.63 (dd, J =
8.3, 1.1 Hz, 2H), 8.45 (s, 2H), 8.30 (s, 4H), 8.12 (d, J = 4.8 Hz,
2H), 8.03 (dd, J = 8.9, 3.4 Hz, 4H), 7.71 (dd, J = 8.3, 5.3 Hz, 2H),
7.65 (dd, J = 8.3, 5.3 Hz, 2H), 7.59 (dd, J = 8.4, 5.3 Hz, 2H), 7.51
(d, J = 9.0 Hz, 2H), 6.48 (d, J = 9.0 Hz, 2H), 6.41 (broad m, 2H),
4.36 (broad m, 4H), 3.59 (broad m, 4H), 3.46 (broad m, J = 17.1
Hz, 4H), 3.00 (broad s, 4H), 1.30 (broad m, 4H). HRMS-ESI
calculated for [C64H58N10O4NiRu]2+ : m/z 592.15305, found: m/z
592.15453.
Preparation of [Ru(phen)2salicylpip]2+ 2. Using the same
procedure as for 1 with [Ru(phen)2Cl2] (20 mg, 0.038 mmol) and
salicylsalphen piperidine ligand (21 mg, 0.038 mmol) gave the
crude product that was purified by flash chromatography on silica
(CH3CN/H2O/ KNO3sat 10:1:1/4) to provide the title compound 2
as an orange solid (35 mg, 0.029 mmol, 72%). Rf 0.32
(CH3CN/H2O/ KNO3sat 10:1:1/2); 1H NMR (CD3CN, 500 MHz) δ δ
9.12 – 9.03 (m, 4H), 8.62 (m, 4H), 8.28 (s, 4H), 8.17 (d, J = 8.8
Hz, 2H), 8.10 (d, J = 5.2 Hz, 2H), 8.04 (dd, J = 5.2, 1.2 Hz, 2H),
8.00 (d, J = 5.2 Hz, 2H), 7.71 – 7.67 (m, 2H), 7.65 (m, 4H), 6.79
(d, J = 8.8 Hz, 2H), 6.73 (m, 2H), 4.43 (t, 2H), 3.48 (broad s, 4H),
1.30 (broad s, 4H). HRMS-ESI calculated for [C64H60N10O496Ru 2+
m/z 564.19320, found: m/z 564.19332.
:
Preparation of [Ru(phen)2coumarin]2+ 3. Using the same
procedure as for 1 with [Ru(phen)2Cl2] (20 mg, 0.038 mmol) and
coumarinsalphen ligand (21 mg, 0.038 mmol) gave the crude
product that was purified by flash chromatography on silica
(CH3CN/H2O/ KNO3sat 10:1:1/4) to provide the title compound 3
as an orange solid (38 mg, 0.029 mmol, 77%). Rf 0.27
1
(CH3CN/H2O/ KNO3sat 10:1:1/4); H NMR (CD3CN, 500 MHz) δ
8.62-8.58 (m, 4H), 8.26 (s, 4H), 8.08 (d, J = 5.3, 2H), 8.02 (m, 8H),
7.97 (d, J = 9.6 Hz, 2H), 7.71 (d, J = 8.7 Hz), 7.63 (d, J = 4.8 Hz,
2H, m, 2H), 7.60 (d, J = 5.2 Hz, 2H), 7.14 (d, J = 8.7 Hz, 2H), 6.38
(d, J = 9.6 Hz, 2H). HRMS-ESI calculated for [C52H34N8O696Ru]2+
m/z 505.08332, found: m/z 505.08356.
:
Preparation of [Ru(phen)2Ni-coumarin]2+ 7. Using the same
procedure as for 5 with [Ru(phen)2diamino-phenanthroline]2+ (20
mg, 0.021 mmol) and nickel(II) acetate tetrahydrate (6 mg, 0.025
mmol) in DMSO (3 mL) with the addition of 7-hydroxycoumarin-8-
carbaldehyde (20 mg, 0.105 mmol) gave the crude product that
Preparation of [Ru(phen)2quinoline]2+ 4. Using the same
procedure as for 1 with [Ru(phen)2Cl2] (20 mg, 0.038 mmol) and
phenDK ligand (19 mg, 0.038 mmol) gave the crude product that
was purified by flash chromatography on silica (CH3CN/H2O/
KNO3sat 10:1:1/4) to provide the title compound 4 as an orange
solid (38 mg, 0.030 mmol, 80%). Rf 0.29 (CH3CN/H2O/ KNO3sat
10:1:1/4); 1H NMR (CD3CN, 500 MHz) δ 9.28 (dd, J = 4.2, 1.8 Hz,
2H), 9.18 (dd, J = 7.4, 1.4 Hz, 2H), 9.12 (d, J = 7.7 Hz, 2H), 8.61
– 8.59 (m, 4H), 8.55 (dd, J = 8.4, 1.8 Hz, 2H), 8.26 (s, 4H), 8.17
(dd, J = 8.2, 1.3 Hz, 2H), 8.11 (broad, 2H), 8.03 (dd, J = 5.2, 1.1
Hz, 2H), 7.98 (dd, J = 5.2, 1.0 Hz, 2H), 7.90 – 7.85 (m, 2H), 7.75
(dd, J = 8.3, 4.3 Hz, 2H), 7.69 (d, J = 5.2 Hz, 2H), 7.68 (d, J = 5.2
Hz, 2H), 7.64 (d, J = 5.3 Hz, 2H), 7.62 (d, J = 5.2 Hz, 2H). HRMS-
ESI calculated for [C56H36N1096Ru]2+ : m/z 472.10947, found: m/z
472.10969.
was
purified
by
flash
chromatography
on
silica
(acetonitrile/water/KNO3
with ratio 10:1:1/2) to yield
a
sat
compound 7 as a dark-red solid (16.7 mg, 0.0123 mmol, 59%). Rf
0.30 (CH3CN/H2O/ KNO3sat 10:1:1/2);1H NMR (CD3CN, 500 MHz)
δ 9.93 – 9.81 (broad s, 2H), 8.84 (d, J = 8.5 Hz, 2H), 8.68 – 8.62
(m, 4H), 8.29 (s, 4H), 8.21 (d, J = 4.9 Hz, 2H), 8.10 (d, J = 5.2 Hz,
2H), 8.05 (d, J = 5.0 Hz, 2H), 7.76 – 7.70 (m, 4H), 7.69 – 7.64 (m,
4H), 7.43 (d, J = 8.8 Hz, 2H), 6.47 (d, J = 9.0 Hz, 2H), 6.04 (d, J
= 9.4 Hz, 2H). HRMS-ESI calculated for [C52H32N8O6NiRu]2+ : m/z
536.04186, found: m/z 536.04198.
Preparation of [Ru(phen)2Pd-salicyl]2+ 8. Using the same
procedure as for 5 with [Ru(phen)2diamino-phenanthroline]2+ (20
mg, 0.021 mmol) and palladium(II) acetate (8 mg, 0.025 mmol) in
DMSO (3 mL) with the addition of salicylaldehyde (22 µL, 0.21
mmol) gave the crude product that was purified by flash
chromatography on silica (acetonitrile/water/KNO3 sat with a ratio
10:1:1/4) to yield compound 8 as a dark-red solid (9.9 mg, 0.0077
mmol, 37%). Rf 0.38 (CH3CN/H2O/ KNO3sat 10:1:1/4);1H NMR
(CD3CN, 500 MHz) δ 8.85 (d, J = 8.6 Hz, 2H), 8.75 (s, 2H), 8.66
– 8.61 (m, 4H), 8.28 (s, 4H), 8.17 (dd, J = 5.2, 1.0 Hz, 2H), 8.08
(dd, J = 5.2, 0.7 Hz, 2H), 8.03 (dd, J = 5.2, 1.2 Hz, 2H), 7.70 (dd,
J = 8.3, 5.3 Hz, 2H), 7.66 – 7.61 (m, 4H), 7.59 (dd, J = 8.1, 1.5 Hz,
Preparation of [Ru(phen)2Ni-salicyl]2+ 5. According to a
modified procedure of Onozawa-Komatsuzaki, to a refluxing
solution of [Ru(phen)2diamino-phenanthroline]2+ (20 mg, 0.021
mmol) and nickel(II) acetate tetrahydrate (6 mg, 0.025 mmol) in
deoxygenated DMSO were added the salicylaldehyde (22 µL,
0.21 mmol) and triethylamine (50 µL, 0.68 mmol) .The reaction
mixture was then stirred at reflux during 3 hours in the dark under
argon while the solution turned from dark orange to dark red. After
cooling down to room temperature, the volatiles were removed
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10.1002/chem.202001409
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2H), 7.45 – 7.40 (m, 2H), 6.92 (d, J = 8.7 Hz, 2H), 6.76 – 6.71 (m,
xenon lamp and an R928P photomultiplier, using an Oxford
Instruments Optistat DN nitrogen cryostat controlled by an Oxford
Intelligent Temperature Controller (ITC503S). Quantum yields
2H). HRMS-ESI calculated for [C50H32N8O2PdRu]2+
491.53700, found: m/z 491.53731.
: m/z
Preparation of [Ru(phen)2Pt-salicyl]2+ 9. First, Pt(OAc)2 was
prepared in situ by stirring K2PtCl4 (14 mg, 0.034 mmol) and
NaOAc (5 mg, 0.056 mmol) in DMSO (3 mL) at 100 °C over the
course of 15 minutes. Then, using the same procedure as for 5
[Ru(phen)2diamino-phenanthroline]2+ (20 mg, 0.021 mmol) and
salicylaldehyde (22 µL, 0.21 mmol) were added which gave the
crude product that was purified by flash chromatography on silica
were obtained using [Ru(bpy)3]2+ as
a
reference.[53]
Luminescence lifetime measurements were performed after
irradiation at λ=450 nm obtained as the second harmonic of a
titanium: sapphire laser (picosecond Tsunami laser Spectra 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) followed by a time-correlated single-photon
counting system from Picoquant (PicoHarp300). The ultimate
time resolution of the system is close to 4 ps. Luminescence
titration experiments with ODNs (GC-rich hairpin or wtTel23 G-
quadruplex DNA) were conducted by recording spectra from
solutions in 10 mM HEPES, 50 mM NaCl, 100 mM KCl (pH 7.4)
buffer on a Varian Cary Eclipse instrument. Titrations were
performed by starting from the highest DNA concentration and
progressively decreasing it, whilst the concentration of the
complex (5 µm) was kept constant.
(acetonitrile/water/KNO3
with a ratio 10:1:1/4) to yield
sat
compound 9 as a dark-red solid (11 mg, 0.0081 mmol, 36%). Rf
0.34 (CH3CN/H2O/ KNO3sat 10:1:1/4); 1H NMR (CD3CN, 500 MHz)
δ 9.36 (s, 2H), 8.97 (d, J = 8.7 Hz, 2H), 8.86 (d, J = 8.5 Hz, 2H),
8.61 (d, J = 8.3 Hz, 2H), 8.58 (d, J = 8.3 Hz, 2H), 8.25 (s, 4H),
8.14 (dd, J = 5.3, 1.1 Hz, 2H), 8.10 (dd, J = 8.3, 1.1 Hz), 7.87 (dd,
J = 8.1, 1.5 Hz, 2H), 7.70 – 7.50 (m, 8H), 7.42 (dd, J = 8.8, 5.1 Hz,
2H), 7.38 (d, J = 8.5 Hz, 2H), 6.98 – 6.92 (m, 2H). HRMS-ESI
calculated for [C50H32N8O2PtRu]2+ : m/z 532.07165, found: m/z
532.07345.
Preparation of [Ru(phen)2Pt-salicylpip]2+ 10. First, Pt(OAc)2
was prepared in situ by stirring K2PtCl4 (14 mg, 0.034 mmol) and
NaOAc (5 mg, 0.056 mmol) in DMSO (3 mL) at 100 °C over the
course of 15 minutes. Then, using the same procedure as for 5
[Ru(phen)2di-amino-phenanthroline]2+ (20 mg, 0.021 mmol), 2-
hydroxy-4-(2-(piperidin-1-yl)ethoxy)benzaldehyde (16 mg , 0.063
mmol) which gave the crude product that was purified by flash
chromatography on silica (acetonitrile/water/KNO3 sat with a ratio
10:1:3/4) to yield the title compound 10 as a dark-red solid (9.4
mg, 0.0058 mmol, 28%). Rf 0.31 (CH3CN/H2O/ KNO3sat 10:1:3/4);
1H NMR (CD3CN, 500 MHz) δ 8.70 (d, J = 8.7 Hz, 2H), 8.63 (d, J
= 8.2 Hz, 4H), 8.60 (dd, J = 8.3, 1.1 Hz, 2H), 8.42 (s, 2H), 8.26 (s,
4H), 8.09 (d, J = 4.8 Hz, 2H), 8.00 (dd, J = 8.9, 3.4 Hz, 4H), 7.68
(dd, J = 8.3, 5.3 Hz, 2H), 7.62 (dd, J = 8.3, 5.3 Hz, 2H), 7.56 (dd,
J = 8.4, 5.3 Hz, 2H), 7.48 (d, J = 9.0 Hz, 2H), 6.45 (d, J = 9.0 Hz,
2H), 6.38 (s, 2H), 4.33 (broad s, 4H) 3.61 (broad s, 4H), 3.49
(broad s, J = 17.1 Hz, 4H), 3.03 (broad s, 4H), 1.33 (broad s, 2H).
CD experiments
Prior to CD analysis, oligonucleotides were annealed by heating
at 95 °C 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 path length
quartz cuvettes at 5 °C increments from 25 °C to 95 °C
(temperature ramp: 1 °C/min) over the wavelength range from
230 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 nm min-1. For CD
melting experiments, the ellipticity was recorded at 292 and 252
nm for wtTel23 and duplex hairpin, respectively. Measurements
were performed in Tris-HCl buffer (10 mM, pH 7.4) with 100 mM
NaCl and oligonucleotide concentrations were fixed at 2.5 µM.
Melting temperatures were obtained through Boltzmann fitting
with Origin software. Each curve fit was only accepted with
HRMS-ESI calculated for [C64H58N10O4190Pt96Ru]2+ : m/z 659.6667, r2>0.99.
found: m/z 659.6683.
Bio-layer interferometry
Electrochemical studies
Bio-layer interferometry experiments were performed using
sensors coated with streptavidin (SA sensors) purchased from
Forte Bio (PALL). Prior to use, they were immersed for 10 minutes
in a buffer before functionalization to dissolve the sucrose layer.
Then the sensors were dipped for 15 minutes in DNA containing
solutions (biotinylated hairpin oligonucleotides) at 100 nM and
rinsed in the buffer solution (10 mM HEPES, 50 mM NaCl, 100
mM KCl (pH 7.4), 0.5% v/v surfactant P20) for 10 minutes. The
functionalized sensors were next dipped in the ruthenium
complex containing solution at different concentrations (see
supporting information) for 2 minutes interspersed by a rinsing
step in the buffer solution for 4 minutes. Reference sensors
without DNA immobilization were used to subtract the non-
specific adsorption on the SA layer. The sensorgrams were fitted
using a 1 : 1 interaction model. 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 two times using a concentration
range from 0.5 μM to 10 μM.
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
reference electrode. The potential of the working electrode was
monitored by an Autolab PGSTAT 100 potentiostat through a PC
interface. Cyclic voltammograms were recorded at a sweep rate
of 100 mVs-1 from solutions in dry acetonitrile (Sigma–Aldrich,
HPLC grade). The concentration of the complexes was 8x10-4 mol
L-1, with 0.1 mol L-1 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.
Photophysical measurements
UV/Vis absorption spectra were recorded on a Shimadzu UV-
1700 spectrophotometer. 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
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10.1002/chem.202001409
Chemistry - A European Journal
FULL PAPER
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U2OS cells were grown at 37 °C in a humidified atmosphere with
5% CO2 in DMEM medium (Westburg) containing 10% foetal
bovine serum (FBS) (Gibco) and 1% penicillin/streptomycin
(Westburg). 20000 cells were seeded onto a coated microscope
slide and incubated with 20 μM of complex for 1,5 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)
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Photo-cytotoxicity 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
the complex was added. After 1.5 h of incubation at 37 °C in the
dark, cells were rinsed twice with 1x PBS to remove non-
internalized complex. They were then illuminated for 30 min with
blue LEDs (LED strip IP68 60 LEDm-1 from Prolumia, 405 nm at
15.7 Wm-2). The distance between the light source and the culture
plate was 10 cm. Cultures were rinsed with 1x PBS before
illumination still in 1x PBS to avoid absorption by red phenol-
containing culture medium. Dark controls were protected from
illumination with aluminium foil. Illuminated and control cultures
were directly put back in the incubator after addition of fresh
medium and incubated for an additional 24 hours. Cell viability
was then measured (1 day post-irradiation) using 10 µL/well of
WST-1 reagent (Roche) following the manufacturer’s instructions.
The ratio of the optical density at λ = 450 nm under each set of
conditions to that of control cells (non-transfected and non-
irradiated, 100% viability) was used to determine the relative
viability. Experiments were performed as biological triplicate with
4 measurements each time.
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[22] J. Jamroskovic, M. Doimo, K. Chand, I. Obi, R. Kumar, K. Brännström,
M. Hedenström, R. Nath Das, A. Akhunzianov, M. Deiana, K. Kasho, S.
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M. G., M.D., M.A. and B. E. gratefully acknowledge the Université
catholique de Louvain (UCLouvain), the Fonds National pour la
Recherche Scientifique (F.R.S.-F.N.R.S.) and the Prix Pierre et
Colette Bauchau for financial support. We also thank Simon De
Kreijger and Robin Bevernaegie for their scientific support. This
work was partially supported by the“Agence National de la
Recherche”, Labex ARCANE (ANR-16-CE11-0006-01), CBH-
EUR-GS (ANR-17-EURE-0003), and the rꢀgion Auvergne-
Rhꢁne-Alpes. The NanoBio-ICMG platforms (FR 2607) are
acknowledged for their support.
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Keywords: G-quadruplex DNA • Ruthenium complexes • Schiff
base • Photo-cytotoxicity • Bio-layer interferometry
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10.1002/chem.202001409
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Ruthenium(II) Schiff base complexes are relevant compounds
for G-quadruplex DNA binding and display interesting photo-
cytotoxicity properties.
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