2+: A colorimetric molecular "light switch" and powerful stabilizer for G-quadruplex DNA

Article abstract of DOI:10.1039/c3dt32640c

A new ruthenium complex, [Ru(bpy)₂dppz-idzo]²⁺ (bpy = 2,2′-bipyridine, dppz-idzo = dipyrido-[3,2-a:2′,3′-c] phenazine-imidazolone), was synthesized and characterized. The luminescent titrations showed that the Ru-complex exhibited an outstanding "light switch" effect with an emission enhancement factor of about 300 in the presence of G-quadruplex DNA in a K⁺ solution. This remarkable "light switch" behavior can even be observed by the naked eye under irradiation with UV light. To get an insight into the "light switch" mechanism, quantum-chemical calculations were performed based on the DFT/TDDFT/PCM method at the B3LYP/6-31G* level. Furthermore, the CD titrations and thermal melting experiments indicated that [Ru(bpy) ₂dppz-idzo]²⁺ could not only induce the formation of an antiparallel G-quadruplex structure in the absence of monocations, but also has the ability to stabilize the G-quadruplex architecture, implying potential applications in anticancer therapeutics. Both the "light switch" effect and the structure stabilization ability of [Ru(bpy)₂dppz-idzo] ²⁺ were found to be superior to the well-known DNA molecular "light switch" [Ru(bpy)₂dppz]²⁺. Finally, a "sandwich-like" binding model was proposed on the basis of molecular docking simulations.

Full text of DOI:10.1039/c3dt32640c

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2
+
[
Ru(bpy) dppz-idzo] : a colorimetric molecular “light
2
switch” and powerful stabilizer for G-quadruplex DNA†
Cite this: Dalton Trans., 2013, 42, 5661
Jun-Liang Yao, Xing Gao, Wenliang Sun, Shuo Shi* and Tian-Ming Yao*
2
+
2
A new ruthenium complex, [Ru(bpy) dppz-idzo] (bpy = 2,2’-bipyridine, dppz-idzo = dipyrido-[3,2-
a:2’,3’-c] phenazine-imidazolone), was synthesized and characterized. The luminescent titrations showed
that the Ru-complex exhibited an outstanding “light switch” effect with an emission enhancement factor
+
of about 300 in the presence of G-quadruplex DNA in a K solution. This remarkable “light switch”
behavior can even be observed by the naked eye under irradiation with UV light. To get an insight into
the “light switch” mechanism, quantum-chemical calculations were performed based on the
DFT/TDDFT/PCM method at the B3LYP/6-31G* level. Furthermore, the CD titrations and thermal melting
2+
2
experiments indicated that [Ru(bpy) dppz-idzo] could not only induce the formation of an antiparallel
G-quadruplex structure in the absence of monocations, but also has the ability to stabilize the
Received 5th November 2012,
Accepted 22nd January 2013
G-quadruplex architecture, implying potential applications in anticancer therapeutics. Both the “light
2
+
switch” effect and the structure stabilization ability of [Ru(bpy)
to the well-known DNA molecular “light switch” [Ru(bpy)
2
dppz-idzo] were found to be superior
DOI: 10.1039/c3dt32640c
2+
2
dppz] . Finally, a “sandwich-like” binding
www.rsc.org/dalton
model was proposed on the basis of molecular docking simulations.
from illegitimate recombination, degradation, and end-to-end
Introduction
1
3,14
fusion.
In humans, telomeres consist of a repetition of the
Among a variety of non-canonical DNA structures, the G-quad- double-stranded DNA sequence (5′-TTAGGG)/(5′-CCCTAA), with
ruplex architectures have attracted intense attention as pro- the G-rich 3′ region extending beyond the duplex to form a
1
5,16
spective targets for the chemical intervention of biological single stranded overhang, called the G-overhang.
These
1
–3
functions.
structures containing planar G-quartets stabilized by Hoogs- present on all chromosomal ends.
G-quadruplexes are four-stranded nucleic acid 3′ overhangs are relatively long (50–210 bases in length) and
1
5,17
4
–6
teen hydrogen bondings.
The structures and stability of
G-quadruplexes are quite sensitive to monocations. For instance,
+
Na induces antiparallel quadruplex folding in the telomere
+
sequence d[AGGG(TTAGGG) ] (22AG), while K induces a
3
hybrid-type mixed parallel/antiparallel G-quadruplex structure
7
–9
(
Fig. 1). The folding pathways of these specific G-quadruplex
1
0
conformations have also been reported recently.
The first glimpse of a new era for DNA-targeted therapeutics
came through the realization that telomeres can form G-quad-
1
1,12
ruplexes.
Telomeres, a specialized functional nucleopro-
tein structure located at the ends of eukaryotic chromosomes,
play an important role in structural chromosome integrity.
Telomeres function to cap and protect chromosome termini
Department of Chemistry, Tongji University, Shanghai, China.
E-mail: shishuo@tongji.edu.cn, tmyao@tongji.edu.cn; Fax: +86-21-6598-2287;
Tel: +86-21-6598-3292
†
Electronic supplementary information (ESI) available: absorption titrations and
some detailed results of the computational calculations. See DOI:
0.1039/c3dt32640c
Fig. 1 Structures of intramolecular G-quadruplexes formed from human telo-
+
+
meric DNA sequences in K (a) and Na (b) solutions. The potassium and
sodium ions are shown as lavender and orange spheres, respectively.
1
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Because of the repetition of guanines, the G-overhang is interactions with G-quartets. Also, some of the electropositive
prone to form four-stranded polymorphic G-quadruplex struc- metals can, in principle, be positioned at the center of the
tures. G-quadruplex conformations may be capable of both guanine quartet, increasing the electrostatic stabilization by
shortening the telomere and directly causing telomere uncap- substituting the cationic charge of the potassium or sodium
4
4
ping, leading to the inhibition of the catalytic lengthening that would normally occupy this site.
activity of telomerase by disrupting the interaction between Among all the metal complexes, Ru(II) complexes have
1
8
the enzyme and its substrate. It has been shown that telo- attracted considerable attention not only because of their
merase is over-expressed in approximately 85% of cancer cells DNA-binding properties, but also, more importantly, their
1
9,20
2+
and plays an essential role in their immortalization.
“light switch” effects for DNA. In particular, [Ru(bpy)
2
dppz]
2
+
Numerous observations, notably that inhibiting telomerase and [Ru(phen)
2
dppz] (bpy = 2,2′-bipyridine, phen = 1,10-phen-
activity and/or interfering with the telomere capping function anthroline and dppz = dipyrido-[3,2-a:2′,3′-c] phenazine),
limits tumor cell growth, have given rise to the proposal that known as the most famous DNA “light switch” complexes,
4
5–47
telomeres and telomerase are potential targets for cancer have been studied a lot during the last decade.
Many
2
1–23
chemotherapy.
developing small molecules that bind and stabilize telomeric elucidate the DNA “light switch” mechanism.
G-quadruplex structures.
Hence, there is great current interest in experimental and theoretical methods have been carried out to
4
5,47–50
A detailed
The biological and therapeutic description of the “light switch” behavior remains elusive. One
significance of telomeric G-quadruplexes is well appreciated leading theory is that the “light switch” behavior results from
2
4–26
3
and continues to be an active field for drug discovery.
the presence of two MLCT states involving the dppz ligand: in
3
In addition, the abundance of G-rich repeats was also aprotic solvents, the lowest MLCT state is a bright state (BS)
found to be linked with nuclease hypersensitive regions within associated with the bipyridine (bpy) fragment and thus lumi-
DNA promoter regions of the human genome, suggesting that nescence was observed, but in protic environments, the hydro-
guanine-rich duplex DNA sequences within promoter regions gen bonding with the phenazine (phz) nitrogens lowered the
2
7,28
are involved in modulating gene transcription.
Although energy of the dark state (DS), localized largely on the phz
these sequences are usually paired with complimentary DNA portion, to below that of the BS and quenched the lumines-
strands, during transcription and replication duplex unwind- cence by a decay process from the DS via nonradiative
5
1
ing would expose the single G-rich strand and allow intra- vibrational relaxation back to the ground state.
29,30
molecular folding.
The therapeutic potential of gene promoter
Many Ru(II) complexes have been reported to intercalate
G-quadruplexes has sparked great interest in the design of between the duplex DNA base pairs and stabilize the ds-
3
1–33
52–54
molecules that can act as G-quadruplex stabilizers.
In DNA.
Recently, our laboratory found that Ru(II) complexes
terms of therapeutic targets, the gene promoter regions, with can also serve as a prominent molecular “light switch” for
5
5
their various sequences, may provide specific scaffolds that are G-quadruplexes. Furthermore, we have described the first
1
1
ideal for designing selective ligands. Moreover, recent inves- example of a new G-quadruplex DNA “light switch” complex,
tigations found that transcribed single-stranded G-rich non- which can be repeatedly cycled on and off through the
5
6
coding RNA sequences, particularly within mRNA, could addition of external agents. Nevertheless, most of the com-
readily form more stable G-quadruplexes in vitro than plexes have a low fluorescence enhancement with G-quadru-
3
4–36
DNA.
Further characterization of these RNA quadruplexes plex DNA and can’t be observed with the naked eye. Some of
could provide the basis for a rational approach towards transla- them exhibit residual emission in a DNA-free water solution or
tional control of gene expression by employing small-molecule have a weak stabilization ability with the G-quadruplex
ligands. In comparison with DNA, RNAs are inherently single- structure.
stranded, distributed more widely in cells and can readily fold
into very stable G-quadruplex topologies, indicating that G-rich (bpy) dppz-idzo] (dppz-idzo = dppz-10,11-imidazolone), as a
In the present work, we report a new Ru(II) complex, [Ru-
2
+
2
RNAs may prove to be a class of more attractive therapeutic molecular “light switch” for G-quadruplex DNA (22AG,
3
7
targets.
3
d[AGGG(TTAGGG) ]). The molecular structure is shown in
It has been reported that a number of metal complexes can Scheme 1. We introduced an imidazolone group to the main
also interact strongly and selectively with quadruplex DNA, ligand (dppz) of the Ru-complex and dramatically enhanced
apart from the purely organic heteroaromatic compounds the emission and selectivity to detect G-quadruplex DNA even
3
8–43
reported previously.
pounds, metal complexes have a very broad range of structural plex structure by [Ru(bpy)
and electronic properties that can be successfully exploited terized in comparison with [Ru(bpy)
In comparison with organic com- with naked eye. Moreover, the stabilization of the G-quadru-
2
+
2
dppz-idzo] (complex 1) was charac-
2
+
2
dppz] (complex 2). The
+
when designing quadruplex DNA binders. The metal center G-quadruplex structure in a K solution, which is considered
can play a major structural role in organizing ligands in to be biologically more relevant due to the higher intracellular
specific geometries and relative orientations for optimal concentration of K , is extensively investigated in this article.
+
quadruplex interaction. Moreover, the electron density on co- We hope that our research findings will be helpful for the
ordinated aromatic ligands can be reduced by the electron- understanding of G-quadruplex DNA recognition by Ru(II) com-
withdrawing properties of metal centers and consequently, plexes as well as laying the foundation for the rational design
electron-poor systems are expected to exhibit stronger π of new anticancer drug candidates.
5
662 | Dalton Trans., 2013, 42, 5661–5672
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2+
Scheme 1 Chemical structures of [Ru(bpy)
2
dppz-idzo]
(1) and [Ru-
2+
2
(bpy) dppz] (2).
Experimental section
Materials
1
5
,10-Phenanthroline-5,6-dione,
,6-diaminobenzimidazolone and cis-Ru(bpy) Cl ·2H O were
5,6-dinitrobenzimidazolone,
2
2
7–60
2
5
synthesized according to the literature methods.
The other
2
+
2
Scheme 2 Synthetic route to [Ru(bpy) dppz-idzo] .
chemicals were obtained from commercial sources and used
without further purification. The synthetic route to [Ru(bpy) -
2
2
+
dppz-idzo] is shown in Scheme 2 and the synthetic details
are given below. The DNA oligomer 22AG (5′-AGGGTTAGGG-
TTAGGGTTAGGG-3′) and 22CT (5′-CCCT AACCCTAACCCTAA-
CCCT-3) were purchased from Sangon (Shanghai, China). Calf
thymus DNA (CT-DNA) was obtained from Sigma. The concen-
trations of these oligomer samples were determined by
measuring the absorbance at 260 nm. Single-strand extinction
coefficients were calculated from mononucleotide data using a
nearest-neighbour approximation. The formation of an intra-
molecular G-quadruplex was performed according to the fol-
lowing procedures: the oligonucleotide samples dissolved in a
added. The precipitate was filtered and washed with successive
batches of water and 95% ethanol. The residue was dried
under vacuum to give 4.7 g of off-white benzimidazolone-2 in
a yield of 70%.
5
,6-Dinitrobenzimidazolone-2
3.35 g, 25 mmol) was dissolved in 13 mL of 98% sulfuric acid.
The colorless solution was cooled to 0–5 °C in an ice bath and
mL of 90% fuming nitric acid in 13 mL of 98% sulfuric acid
(3). Benzimidazolone-2
(
4
was added dropwise to the cooled, stirred solution. The reac-
tion temperature was not allowed to go above 5 °C during the
addition. After that, the cold solution was rapidly poured onto
+
K buffer were heated to 90 °C for 5 minutes, gently cooled to
room temperature and then incubated at 4 °C overnight.
Buffer A: 10 mM tris-HCl, 100 mM KCl, pH 7.0; Buffer B:
150 g of ice. The yellow precipitate was collected via filtration
and washed thoroughly with cold water. After drying under
vacuum, 4.2 g of yellow 5,6-dinitrobenzimidazolone-2 was
obtained in a yield of 82%.
1
0 mM KH PO –K HPO , 100 mM KCl, pH 7.0.
2
4
2
4
5
,6-Diaminobenzimidazolone-2 (4). 10 mL of hydrazine
Synthesis of [Ru(bpy)
2
6 2
dppz-idzo]·(PF )
hydrate (85%) was added dropwise to a mixture of 2.24 g
Benzimidazolone-2 (2). A mixture of 5.4 g (0.05 mol) of (10 mmol) of 3 and 0.3 g of palladium on activated carbon
o-phenylenediamine, 3.4 g (0.057 mol) of urea and 25 mL of (10% Pd/C) in 150 mL of refluxing water–methanol (1 : 2, v/v)
glycol were stirred for 1 h at 130–140 °C and then heated at a within 5 min. The reaction mixture was refluxed for a further
maximum temperature of 170 °C for 7 h. The solution was 4 h and then filtered while hot. After concentrating under
cooled down to 40–50 °C and 5 mL of 95% ethanol was added vacuum, the off-white 5,6-diaminobenzimidazolone-2 was pre-
with stirring for 10 min and then 20 mL of distilled water was cipitated from the filtrate (1.2 g, yield 75%).
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Dipyrido[3,2-a:2′,3′-c]phenazine-10,11-imidazolone-2 (dppz- The concentration of the bound compound was calculated
6
1
idzo) (5). 0.27
g
of 1,10-phenanthroline-5,6-dione (1) using eqn (1)
(
0
1.28 mmol) was dissolved in 30 mL of ethanol with stirring.
.21 g of 5,6-diaminobenzimidazolone-2 (1.28 mmol) was
0
max
0
Cb ¼ Ct½ðF ꢀ F Þ=ðF
ꢀ F Þꢁ
ð1Þ
added to this solution and a yellow precipitate formed
immediately. The reaction mixture was then refluxed for a
further 15 min and the yellow solid was collected by filtration.
After drying thoroughly, 0.4 g of dppz-idzo (5) was obtained in
a yield of 90%.
where C
t
is the total compound concentration, F is the
observed fluorescence emission intensity at a given DNA con-
centration, F is the intensity in the absence of DNA and F
is the fluorescence of the totally bound compound. The
affinity constant of the Ru-complex with G-quadruplex DNA
was calculated using the Scatchard equation (eqn (2)).
0
max
2 6 2 2 2
[Ru(bpy) dppz-idzo]·(PF ) (6). 0.208 g of cis-[Ru(bpy) Cl ]·
2
H O (0.4 mmol) and 0.21 g of dppz-idzo (0.60 mmol) were
2
r=Cf ¼ ꢀrKa þ nKa
where r is the binding ratio C /[DNA]
ð2Þ
mixed in 40 ml of glycol–water (7 : 1, v/v). The mixture was
refluxed for 6 h. Upon cooling, the reaction mixture was
diluted with 40 mL of water and filtered to remove solid impu-
b
t
and C
f
is the free
complex concentration, n is the number of binding sites per
DNA molecule and K is the affinity constant. The lumines-
cence quantum yields were calculated with reference to [Ru-
4 6
rities. Then, 2 g of NH PF was added to the filtrate and the
a
crude product precipitate was dried and collected. The
complex was then purified by alumina chromatography using
MeCN–methanol (5 : 1, v/v) as the eluent and further recrystal-
lized from acetone–diethyl ether (1 : 5, v/v). Yield: 240 mg,
5
2+
62,63
(
bpy)3] (φstd = 0.028)
in aerated water at room tempera-
ture using eqn (3), where φ and φstd are the quantum yields,
A and Astd are the absorbance at the excitation wavelength and
I and Istd are the integrated emission intensities for the
unknown and standard samples, respectively.
1
6%. H NMR [(CD ) SO]: δ 11.71 (2H, s), 9.61 (2H, d), 8.88
3
2
(
7
4H, dd), 8.22 (4H, m), 8.14 (2H, t), 8.01 (2H, dd), 7.84 (2H, d),
.76 (4H, d), 7.60 (2H, t), 7.38 (2H, t). Calc. for
C H F N OP Ru: C, 44.97; H, 2.52; N, 13.45. Found: C,
φ ¼ φstdðAstd=AÞðI=IstdÞ
ð3Þ
3
9
26 12 10
2
4
26
4.95; H, 2.53; N, 13.43. MALDI-MS: calcd For C39H N10ORu
+
+
7
51.76 [M] ; found 751.1 [M] .
Colorimetric experiments
General procedures
In the colorimetric experiments, the final concentrations of
the DNA and Ru-complexes were adjusted to 2.5 and 5.0 μM,
respectively. The total volume of every sample was 2 mL. All
the samples were photographed under irradiation with UV
light (Vilber Lourmat, Bio-Print, VL) at 365 nm without any
incubation.
All synthetic reactions were performed under an argon atmos-
phere. Elemental analyses (C, H, and N) were performed on a
Perkin-Elmer 240C elemental analyzer. The H NMR spectra of
1
Ru(bpy) dppz-idzo in (CD ) SO were collected on a Bruker
2
3 2
ARX-400 NMR spectrometer. Matrix assisted laser desorption
ionization mass spectra (MALDI-MS) were measured on an Ion
Spec HiResMALDI spectrometer. The UV-visible absorption
spectra were recorded on a Perkin-Elmer Lambda Bio 40 spec-
trometer. UV-visible, colorimetric and emission spectrophoto-
metric measurements were carried out in buffer A. The CD
measurements were performed in buffer B. Deionized water
was used to prepare all the Ru(II) complex aqueous solutions.
Circular dichroism measurements
Circular dichroism (CD) titrations were performed on a Jasco
J-810 spectropolarimeter at room temperature using a quartz
cell with a 1 cm path length. The DNA (22AG) samples were
dissolved in two different solutions in this study: (a) 10 mM
tris-HCl, pH 7.0; (b) 100 mM KCl in a 10 mM PBS buffer, pH
7
.0. The process of the CD titrations was similar to that of
Fluorescence titrations
fluorescence titrations. The DNA samples, at a concentration
Fluorescence spectra were measured on a Hitachi F-7000 fluor- of 2.5 μM, were dissolved in corresponding solutions and
escence spectrophotometer at room temperature. The exci- placed in a quartz cuvette (2000 μL of the sample). During the
2
+
tation wavelength was set at 460 nm and the emission titrations, an aliquot (5 μL) of
2
a [Ru(bpy) dppz-idzo]
spectrum was collected from 500 to 800 nm. The excitation (200 μM) solution was added to the cuvette each time and
and emission slits were both set at 10 nm. Luminescence titra- mixed thoroughly. After a further equilibrium of 5 min, the CD
tions were performed as following: 2000 μL of 2.5 μM [Ru- spectra were recorded. The titration processes were repeated
2
+
(
2
bpy) (dppz-idzo)] in a 1.0 cm path length quartz cuvette was until there was almost no change in the spectra, indicating
loaded into the fluorimeter sample block. After 5 min to allow binding saturation had been achieved. For each sample, at
the sample to equilibrate, the first spectrum was recorded and least four spectrum scans were accumulated over the wave-
then 5 μL of a 100 μM DNA solution was added to the sample length range of 225–350 nm at a scanning rate of 100 nm
−
1
cell followed by thorough mixing. After 5 min, the spectrum min . During the measurement, the instrument was flushed
was taken again. The titration processes were repeated until continuously with dry nitrogen gas. The scan of the buffer
there was no apparent change in the spectra for at least four alone (baseline) was subtracted from the average scan for each
cycles, indicating the achievement of the binding saturation. sample.
5
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Thermal DNA denaturation experiments
dispersion/repulsion, hydrogen bonding, electrostatics and
desolvation. The LGA in AutoDock 4.2 was applied to search
for the conformational and orientational space of the Ru(II)-
complex while keeping the G-quadruplex structure rigid. Auto-
Grid performed precalculated atomic affinity grid maps for
each atom type in the ligand plus an electrostatics map and a
separate desolvation map present in the substrate molecule.
Then, during the AutoDock calculation, the energetics of a par-
ticular ligand configuration was evaluated using the values
from the grids. Finally, the Ru(II)–DNA docked complex was
selected according to the criteria of interacting energies
matched with geometric quality. The output from AutoDock
was imported into Accelrys Discovery Studio 2.5 Client for
The melting temperatures of the G-quadruplex DNA were
measured by employing a Jasco J-810 spectropolarimeter
equipped with a Peltier temperature-control programmer
(±0.1) °C. DNA samples (2.5 μM) dissolved in buffer B were
measured using a 1 cm path quartz cell in the absence and
presence of 5 μM Ru-complexes, respectively. Melting plots
were collected by a CD signal as a function of temperature.
Characteristic signals of the quadruplex DNA were measured at
6
4
2
95 nm. The temperature was increased from 40 to 90 °C at
−
1
the rate of 1 °C min
.
Theoretical calculations
7
9
further rendering.
2
+
[
Ru(bpy) dppz-idzo] is made of one Ru(II) atom, one main
2
ligand (dppz-idzo) and two ancillary ligands (bpy), as shown in
Scheme 1. There are 77 atoms involved in the complex, which
Results and discussion
Synthesis of the complex
has C symmetry. The geometric and energy optimizations for
1
2
+
the ground states of [Ru(bpy) dppz-idzo] were performed
2
2
+
with the Gaussian 03 program based on the density functional
2
The synthetic procedure for the complex [Ru(bpy) dppz-idzo]
6
5
theory (DFT) method. The subsequent frequency analysis
shows that the structure is a local minima on the potential
energy surface. Vertical singlet transition energies of the Ru(II)
complex were also obtained using time-dependent DFT
includes mainly four steps: (1) the 5,6-dinitrobenzimidazo-
lone-2 was synthesized through cyclization and nitration
reactions, as reported by Zehui Yang, (2) the 5,6-diaminoben-
zimidazolone-2 was synthesized through catalysis hydrogen-
ation by Pd/C, (3) a moderate condensation reaction of
,6-diaminobenzimidazolone-2 with 1,10-phenanthroline-5,6-
dione rapidly produced the main ligand dppz-idzo, (4) the
final product was synthesized through the coordination
between the main ligand dppz-idzo and the precursor complex
5
8
6
6–68
5
9
(TDDFT).
All the calculations were performed by employ-
ing Becke’s three parameter hybrid functional with the Lee–
5
69–71
6
7
Yang–Parr correlation functional (B3LYP) method.
The
DFT calculations with the B3LYP method have been shown to
be very effective for bio-molecular interactions, such as DNA–
7
2
small molecule interactions. Thus, we have adopted this
method for our investigations. The LANL2DZ basis set was
used to treat the Ru atom, whereas the 6-31G* basis set was
[
2 2 2
Ru(bpy) Cl ]·2H O. After further purification, the complex was
obtained with good purity and a relatively high yield. The
structure was characterized by H NMR, MALDI-MS and
1
7
3,74
used to treat all the other atoms (C, N, O, H).
To clarify the
elemental analysis.
nature of the excited state, orbital analysis of the complexes
were also performed.
Fluorescent and colorimetric studies
2
+
The fluorescence behaviour of [Ru(bpy)
2
dppz-idzo] (1) and
Molecular docking
2+
[
Ru(bpy) dppz] (2) with the G-quadruplex DNA structure were
2
2
+
The electronic structure of [Ru(bpy) dppz-idzo] was opti- investigated. The effects of successive additions of 22AG DNA
2
mized using the DFT-B3LYP method with the 6-31G* basis set on the emission spectra of the Ru-complexes are depicted in
for the C, N, O, H atoms and LanL2DZ for the Ru atom with Fig. 2. It was clear that in the absence of the DNA, both the
the G03 quantum chemistry program-package. The hybrid-type complexes were almost nonemissive. Upon successive
+
mixed parallel/antiparallel G-quadruplex structure in a K solu- additions of the DNA, the luminescence of complex 1 and 2
tion was obtained from the protein data bank (PDB ID: 2HY9) rose sharply by about 300- and 80-fold enhancements in the
as an initial model to study the interaction between [Ru- emission intensity (I/I ≈ 300 and 80, φ = 0.067 and 0.020) at
0
2
+
(
bpy)
2
(dppz-idzo)]
and the 22AG telomeric DNA. Before [DNA]/[Ru] = 1.0 ([DNA] = [Ru] = 2.5 μM), respectively, behaving
docking, necessary modifications were carried out according like DNA molecular “light switches”. These emission changes
5
6
to a literature report. Two adenines located at the end of the of the Ru(II) complexes induced by the addition of the DNA
initial model were removed and slight modifications to clearly indicate that they are bound to the G-quadruplex struc-
increase the separation between loop base pairs and the ture. Based on the emission enhancement, the intrinsic
7
5
G-quartet were performed, as reported in the literature. The binding constant (K ) of each complex was obtained according
b
molecular docking was performed with the AutoDock 4.2 to the Scatchard equation. The values of the binding constant
7
6–78
6
6
Lamarckian Genetic Algorithm (LGA).
molecular docking process, a DNA molecule was enclosed in a respectively.
0 Å cubic grid created by the AutoGrid algorithm (a subpro- The binding stoichiometry was determined by utilizing the
In the automated were about 4.8 × 10 and 2.3 × 10 for complex 1 and 2,
6
gram of AutoDock) with 0.375 Å spacing and the default par- method of continuous variation analysis (Job plot). The total
ameters (supplied with the program package) were used for concentrations of DNA and Ru-complexes were held constant,
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Fig. 3 Job plots constructed from mixing the Ru-complexes and 22AG
+
together in variable ratios but a constant total concentration (2.5 μM) in a K
buffer (10 mM tris-HCl, 100 mM KCl, pH 7.0). The normalized peak values of the
2
emission are plotted versus the fraction of the Ru-complex ([Ru(bpy) dppz-
2
+
2+
idzo] ( ) and [Ru(bpy)
2
dppz] (◆)).
2
Fig. 2 Changes in the emission spectra (λex = 460 nm) of 2.5 μM [Ru(bpy) -
2+
2+
dppz-idzo] (a) and [Ru(bpy)
2AG DNA (0–3.0 μM) in 10 mM tris-HCl, 100 mM KCl, pH 7.0. Insets: plots of
I vs. [DNA]/[Ru] and the best fit for the titrations of Ru(II) complexes with
2AG DNA.
2
dppz] (b) with increasing concentrations of
2
Fig. 4 Images of the “light switch” behavior of Ru(II) complexes (5 μM) for
DNA (2.5 μM) under UV light at 365 nm in 10 mM tris-HCl, 100 mM KCl, pH 7.0.
2
while the relative molar ratios changed for each sample, from
0
6
: 1 (all DNA) to 1 : 0 (all complex). The emission data at assay can reach upto 6 nM at a signal-to-noise ratio of 3
05 nm and 615 nm were used to generate Job plots for (S/N = 3). In addition, the selectivity of the title complex
complex 1 and 2, respectively. As shown in Fig. 3, it is clear towards the G-quadruplex DNA structure was also studied
that two molecules of complex 1 bind to each molecule of the using fluorescence measurements and the results are shown
G-quadruplex, whereas only one molecule of complex 2 binds in Fig. 5. We can see that our complex displayed specificity
+
to a molecule of the G-quadruplex.
towards the G-quadruplex in a K solution, indicating a
UV-visible titrations were also carried out to investigate the potential application in the visual detection of a hybrid-type
+
interaction between the complexes and 22AG in a K solution G-quadruplex structure. The fluorescence titration of the title
(
see Fig. S1 and S2, ESI†). Both complexes exhibited apparent complex with ds-DNA (calf thymus DNA, CT-DNA) was also
hypochromism in the absorption band. Based on the absorp- investigated (Fig. S3, ESI†) and the luminescence selectivity
tion results, the binding constants of 1 and 2 towards hybrid- results are shown in Fig. S4 in the ESI.† It is obvious that the
6
type G-quadruplex DNA were calculated to be 3.17 × 10 and title complex showed a good fluorescence selectivity towards
6
1
.69 × 10 , respectively. These values were comparable to those quadruplex over duplex DNA when both of them were at the
obtained from fluorescence titrations.
same concentration ([G4] = [CT] = [Ru] = 2.5 μM), while a
The strong enhancement of the intrinsic fluorescence of modest selectivity for quadruplex over duplex was observed
2
+
[
Ru(bpy)
2
dppz-idzo] when bound to DNA provides a possi- when the amount of ds-DNA was much larger than that of the
bility for the visual detection of the G-quadruplex structure. As quadruplex DNA ([CT] = 12.5 μM > [G4] = 2.5 μM). Further-
shown in Fig. 4, this dramatic “light switch” effect can easily more, a competition dialysis assay was also conducted to inves-
be observed by the naked eye under UV light. The results show tigate the quadruplex selectivity in the binding affinity
2
+
80,81
that [Ru(bpy)
lar “light switch” for 22AG DNA in a K solution. The limit of (bpy)
detection (LOD) for the G-quadruplex with this “switch on” quadruplex over duplex DNA at the same DNA concentration,
2
dppz-idzo] is a prominent colorimetric molecu- according to the literature.
As shown in Fig. S5, ESI† [Ru-
+
2+
2
dppz-idzo] showed good binding selectivity towards
5
666 | Dalton Trans., 2013, 42, 5661–5672
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2
+
2
The electronic absorption spectra of [Ru(bpy) dppz-idzo]
in neutral water in the vertical singlet excited states were also
calculated by TD-DFT at the B3LYP//LanL2DZ/6-31G* level. The
transition results of the six calculated lowest-lying excited
singlet states are shown in Table 1. The transition contri-
butions show that all of the five excited states in this table are
1
1
MLCT in character. The lowest-energy MLCT state of the
complex was calculated to be at 2.70 eV (459.33 nm). Based on
the calculated oscillator strength, the measured low-energy
2
+
absorption band of [Ru(bpy)
2
dppz-idzo]
at 450 nm is
assigned to the ES1 (459 nm), ES2 (452 nm) and ES3 (452 nm)
states, which are mainly composed of the HOMO → LUMO/
LOMO + 1/LOMO + 3 transitions. The calculated absorption
band is very close to the experimental data (Fig. 7). According
2+
Fig. 5 Specific luminescent selectivity of [Ru(bpy)
2
dppz-idzo] towards hybrid to the orbital distributions, these transitions originate mainly
G-quadruplex. The concentration of the Ru complex and DNAs was 2.5 μM.
Buffer of each sample: (a) 10 mM tris-HCl, 100 mM KCl, pH 7.0; (b) 10 mM tris-
HCl, 100 mM NaCl, pH 7.0; (c) 10 mM tris-HCl, 100 mM NaCl, pH 5.5; (d) 10 mM
tris-HCl, 50 mM NaCl, 1 mM EDTA, pH 7.5.
from dRu orbits to π*dppz-idzo, with a small contribution from
dRu orbits to π*bpy. In addition, the ES4 (430 nm), ES5
(
428 nm) and ES6 (424 nm) states, characterized by relatively
higher energy and oscillator strength, are responsible for the
experimentally observed stronger absorption band at 400 nm.
1
These MLCT states are mainly assigned to the HOMO −
while still displaying moderate selectivity towards G-quadru-
plex DNA even in the presence of a vast amount of ds-DNA.
1/HOMO − 2 → LUMO/LUMO + 1/LUMO + 3 transitions.
It has been reported that charge transfer from a central Ru(II)
atom to the distal phz part of the dppz ligand gives rise to a
nonemissive state, whereas the emissive state arises from the
Computational studies
MLCT transition from Ru(II) to the proximal bpy of the dppz
To further investigate the mechanism of the light-switch
effect, DFT and time-dependent DFT (TD-DFT) calculations
were performed for [Ru(bpy)
lated coordination dihedral angles are shown in the ESI
45,48,49,51,82
ligand or the ancillary ligands.
In a water solution,
hydrogen bonding between the phz nitrogen and the solvent
would lead to the decay of the excited state via nonradiative
vibrational relaxation back to the ground state and quench the
luminescence. Therefore, the key factor that determines the
BS/DS property of an excited state is whether the excited state
2
+
2
dppz-idzo] . The selected calcu-
(Table S1†). We can see that the imidazolone group is coplanar
2
+
2
with dppz in [Ru(bpy) dppz-idzo] , with the dihedral angles
close to 180.00°. The distributions of the frontier molecular
orbitals obtained by the DFT calculations are shown in Fig. 6.
The highest occupied molecular orbital (HOMO), the HOMO −
has the phz nitrogen atomic orbital’s character or not. For [Ru-
2+
(
2
bpy) dppz-idzo] , the electron density of the LUMO + 2 is
localized on the proximal bpy portion of the dppz-idzo and all
the other LUMOs (LUMO, LUMO + 1 and LUMO + 3) contain
phz nitrogen atomic orbitals. Thus, the transitions from Ru(II)
to LUMO + 2 would result in an emissive MLCT excited state,
whereas the MLCT excited states involving the LUMO,
LUMO + 1 and LUMO + 3 should be nonemissive or weakly
emissive. In addition, it has been proposed that the MLCT
states possessing >75% of the nonemissive transitions should
be assigned to DSs, as BSs contained only emissive transitions,
1
and HOMO − 2 are primarily characterized by the d orbitals
of the Ru atom with a small distribution on the ligands. The
HOMO − 3 resides on both the ruthenium center and the
main ligand (dppz-idzo). Its lowest unoccupied molecular
orbital (LUMO) is distributed over the whole dppz-idzo ligand
(
π*dppz-idzo). The LUMO + 1 and LUMO + 2 are mainly located
on the ancillary ligands (π*bpy), while the LUMO + 3 receives a
dominant contribution from the proximal bpy portion of
dppz-idzo.
82
and all the others should be labelled mixed-states (MS). On
the basis of the transition contributions and the coefficients of
the singlet excited states shown in Table 1, we can conclude
that all the listed excited states belong to DSs. This should be
responsible for the nonemissive state of the Ru(II) complex in
water without DNA. DFT/TD-DFT calculations of the free Ru-
complex molecule without water as a solvent were also per-
formed and the results are shown in the ESI (Fig. S6,
Table S2†). We can see that the first three excited states (ES1,
ES2 and ES3) are BSs, while ES4 and ES5 are MSs and only ES6
is DS. This is the explanation for the fluorescence enhance-
2
+
ment of [Ru(bpy)
ruplex DNA.
2
dppz-idzo] under the protection of G-quad-
2
Fig. 6 Contour plots of some selected frontier molecular orbitals of [Ru(bpy) -
2+
dppz-idzo]
.
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2
Table 1 TD-DFT calculated energies, oscillator strengths, transition contributions, and coefficients of the six lowest-energy excited singlet states of [Ru(bpy) dppz-
2
+
idzo] at the B3LYP//Lanl2dz/6-31G* level
Excited state
ES1
λ
abs/nm (eV)
Oscillator strength
0.0000
Transition contribution
Coefficient
459.33(2.70)
HOMO → LUMO
0.5111(52.24%)
0.1834(6.73%)
−0.4486(40.25%)
0.6974(97.28%)
0.6231(77.65%)
0.3171(20.11%)
0.1136(2.58%)
0.5950(70.81%)
0.3201(20.49%)
−0.1239(3.07%)
0.4737(44.87%)
0.1119(2.50%)
−0.4349(37.83%)
−0.2185(9.55%)
0.1618(5.23%)
0.1220(2.98%)
0.2887(16.67%)
0.4269(36.45%)
0.2905(16.88%)
−0.1843(6.79%)
0.2455(12.05%)
HOMO → LUMO + 1
HOMO → LUMO + 3
HOMO → LUMO + 3
HOMO → LUMO + 1
HOMO → LUMO + 3
HOMO − 3 → LUMO + 1
HOMO − 1 → LUMO
HOMO − 1 → LUMO + 1
HOMO − 1 → LUMO + 3
HOMO − 2 → LUMO
HOMO − 2 → LUMO + 1
HOMO − 2 → LUMO + 3
HOMO − 1 → LUMO + 2
HOMO − 3 → LUMO + 2
HOMO − 2 → LUMO
HOMO − 2 → LUMO + 3
HOMO − 1 → LUMO + 1
HOMO → LUMO + 2
HOMO → LUMO
ES2
ES3
452.26(2.74)
452.24(2.74)
0.0000
0.0007
ES4
ES5
ES6
430.51(2.88)
428.28(2.89)
424.00(2.92)
0.2430
0.0026
0.0020
HOMO → LUMO + 3
2
+
Fig. 7 Experimental electronic absorption spectra of [Ru(bpy)
10 μM) at room temperature in pure water.
2
dppz-idzo]
(
CD measurements
Circular dichroism (CD) spectroscopy, which is extremely
useful in conformational studies, is widely used to study the
8
3
conformations of G-quadruplexes. It has been reported that
the CD spectra of 22AG without any metal cations exhibits a
major positive band at 257 nm, a negative band centered at
2
35 nm and a minor negative band at 280 nm, which probably
8
4,85
corresponds to the random coil structure.
Upon the titra-
tion of 22AG with increasing amounts of the title complex in a
free cation aqueous solution, a dramatic change in the CD
spectrum was observed (Fig. 8a). With the successive addition
2
+
Fig. 8 CD titrations of 22AG (2.5 μM) by adding [Ru(bpy)
2
dppz-idzo] (a) in a
+
tris buffer without metal ions (10 mM tris-HCl, pH 7.0) and (b) in a K buffer
2
+
2
of [Ru(bpy) dppz-idzo] , the major positive band at 257 nm
(
2 4 2 4
10 mM KH PO –K HPO , 100 mM KCl, pH 7.0) at room temperature. The con-
gradually decreased and shifted to 245 nm, the negative band centrations of the Ru-complex range from 0 (solid black) to 5 μM (solid red).
at 235 nm disappeared, the minor negative band at 280 nm
slightly increased and moved to 265 nm and the small positive
band at 295 nm clearly increased. These changes in the CD As the concentration of the title complex increased to 5 μM,
signals indicate a significant conformational conversion. the CD spectrum of this new DNA conformation was almost
5
668 | Dalton Trans., 2013, 42, 5661–5672
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2
+
2
Fig. 10 The calculated model of [Ru(bpy) dppz-idzo] binding to the mixed
hybrid-type G-quadruplex DNA. (a) The G-quartets are displayed in a ball and
stick mode; (b) the G-quadruplex is rendered with a solvent surface and colored
by interpolated charge.
Fig. 9 Normalized melting curves of the G-quadruplex DNA (2.5 μM)
+
measured by circular dichroism in a K buffer (10 mM KH
2
PO
4
–K
2
HPO
4
, 100 mM
KCl, pH 7.0) in the absence of the Ru(II) complex (■), in the presence of 5 μM
2+
2+
2+
[Ru(bpy)
2
dppz] ( ) and 5 μM [Ru(bpy)
2
dppz-idzo]
(
), respectively.
[Ru(bpy) dppz] complex according to the significant increase
2
in the melting temperature of the G-quadruplex.
Molecular docking
identical to that of the antiparallel G-quadruplex structure
described in previous researches, which was characterized by a
major positive peak at 295 nm with a smaller negative peak at
Finally, molecular docking simulations were carried out to get
an insight into the interaction between [Ru(bpy) dppz-idzo]
2
2+
and the G-quadruplex. The structure of the title complex was
optimized on the basis of the DFT-B3LYP method using Gaus-
sian03. The mixed hybrid-type G-quadruplex was obtained
from the protein data bank (PDB entry: 2HY9) as a template
for the docking studies. Two extra adenines were removed
from each end of the 26-mer mixed hybrid-type structure
8
,86
2
65 nm and a positive peak at 245 nm.
This indicates that
the title complex is capable of promoting the formation of the
human telomeric intramolecular G-quadruplex structure inde-
pendently without any other metal ions.
The structures and stability of the G-quadruplex DNA are
largely influenced by the presence of monocations, especially
75
before docking, according to the literature. The results of the
+
+
Na and K . Due to the higher intracellular concentration of
Job plot (Fig. 3) indicate that every G-quadruplex molecule
probably binds with two molecules of the title complex. As
shown in Fig. 10, the docking study confirms that each intra-
+
+
K , the G-quadruplex in a K solution was considered to be bio-
logically more relevant and introduced to investigate the inter-
action with the title Ru(II) complex. As shown in Fig. 8b, in the
absence of the Ru(II) complex, 22AG adopted a typical hybrid-
molecular G-quadruplex molecule binds to two molecules of
2+
[
2
Ru(bpy) (dppz-idzo)] . It has been suggested that “End
+
type G-quadruplex structure in a K buffer, exhibiting a distinct
pasting” was a possible model of the interaction between
many metal complexes and G-quadruplexes. This means the
CD spectrum containing a strong positive peak at 295 nm with
a shoulder peak around 268 nm and a smaller negative peak at
binders commonly stack on the surface of either the 5′- or 3′-
8
,84
2
35 nm.
After successive additions of [Ru(bpy)
2
dppz-
31,38,56,87
terminal G-quartet plane.
(
Our results reveal that [Ru-
2+
idzo] , no apparent changes were observed in the CD spec-
trum, indicating that the interaction between the title complex
and the DNA hardly changed the G-quadruplex structure in a
2+
bpy) (dppz-idzo)] probably bound to the mixed hybrid-type
2
G-quadruplex structure at both termini (5′ and 3′ terminus)
with potential π–π stacking interactions. This strong inter-
action and quite different binding model might result from
the introduction of an imidazolone group to the main ligand
of the Ru-complex, which effectively extended the square π-aro-
matic surface. This “sandwich-like” end-stacking of the title
Ru(II) complex onto the G-quartets surfaces explained why its
+
K solution.
Thermal melting experiments were also performed, employ-
ing the circular dichroism to demonstrate the stabilization of
hybrid G-quadruplex DNA by the Ru(II) complexes. The nor-
+
malized thermal denaturation profiles of 22AG in a K buffer
are depicted in Fig. 9. It should be noted that T1/2 is used
instead of Tm to describe the transition temperature as G-quad-
quadruplex stabilization ability was much stronger than that
2+
of the classic [Ru(bpy) dppz] complex.
2
6
4
ruplex DNA–ligand interactions are not reversible. As shown
+
in Fig. 9, the T1/2 of 2.5 μM 22AG in a K solution increased
from 60.7 to 72.2 °C in the presence of 5 μM [Ru(bpy) dppz-
2
Conclusions
2+
2
idzo] and increased to 65.8 °C in the presence of [Ru(bpy) -
2
+
dppz] . The enhancements in T1/2 of the G-quadruplex DNA Our previous investigations of the interactions between Ru(II)
indicated that both Ru complexes stabilized the G-quadruplex complexes and G-rich DNA showed that many of the complexes
+
structure in a K buffer. Obviously the title complex exhibited can serve as a prominent “light switch” for G-quadruplex struc-
5
5,56,85
a much more powerful stabilization ability than the classic tures, especially for the mixed hybrid-type quadruplex.
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However, most of the complexes exhibit a relatively low
quantum yield with G-quadruplex DNA and can hardly be
observed with the naked eye. Herein, a novel Ru(II) complex,
3 S. Neidle and G. Parkinson, Nat. Rev. Drug Discovery, 2002,
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+
2
[Ru(bpy) dppz-idzo] , has been synthesized and characterized.
The fluorescence titrations showed that the title compound
acted as an excellent molecular “light switch” for hybrid-type
G-quadruplex DNA (φ = 0.067), which was superior to the well-
2
+
known DNA molecular “light switch” [Ru(bpy) dppz] (φ =
2
0
.020). The title complex bonds to G-quadruplex DNA with a
6
−1
binding constant of 4.8 × 10
M
in a 10 mM tris-HCl,
1
00 mM KCl (pH = 7.0) buffer solution, as evidenced by UV-
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8,89
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2
+
2
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15 V. L. Makarov, Y. Hirose and J. P. Langmore, Cell, 1997, 88,
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+
2
CD titrations have revealed that [Ru(bpy) dppz-idzo] can
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+
+
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2
+
ation ability of [Ru(bpy)
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2
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3
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This work was supported by the National Natural Science
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