Synthesis, characterization and DNA binding studies of two Ru(II) complexes containing guanidinium ligands

Article abstract of DOI:10.1016/j.poly.2016.02.038

Two new Ru(II) complexes containing guanidinium groups have been synthesized, characterized and analyzed according to their interactions with different G-quadruplexes and duplex DNA. A FRET assay and a competitive FRET assay showed that both complexes pro

Full text of DOI:10.1016/j.poly.2016.02.038

Accepted Manuscript
Synthesis, characterization and DNA binding studies of two Ru(II) complexes
containing guanidinium ligands
Wen-Xiu Chen, Xing-Dong Song, Jia-Xi Chen, Xuan-Hao Zhao, Jie-Hua Xing,
Jun-Rui Ren, Tie Wu, Jing Sun
PII:
S0277-5387(16)00132-7
DOI:
http://dx.doi.org/10.1016/j.poly.2016.02.038
POLY 11854
Reference:
Polyhedron
To appear in:
Received Date:
Accepted Date:
23 December 2015
16 February 2016
Please cite this article as: W-X. Chen, X-D. Song, J-X. Chen, X-H. Zhao, J-H. Xing, J-R. Ren, T. Wu, J. Sun,
Synthesis, characterization and DNA binding studies of two Ru(II) complexes containing guanidinium ligands,
Polyhedron (2016), doi: http://dx.doi.org/10.1016/j.poly.2016.02.038
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Synthesis, characterization and DNA binding studies of two Ru(II)
complexes containing guanidinium ligands
Wen-Xiu Chen†a, Xing-Dong Song†a, Jia-Xi Chen*a, Xuan-Hao Zhaoa, Jie-Hua Xinga, Jun-Rui Rena
Tie Wua, Jing Sun ab*
a School of Pharmacy, Guangdong Medical University, Dongguan, 523808, China
bGuangdong Key Laboratory for Research and Development of Natural Drugs, Guangdong Medical
University, Zhanjiang, 524023, China
———
† Contributed equally to this work.
* Corresponding author. Tel.: +86-769-2289-6322; E-mail address: sunjing03@foxmail.com (Jing Sun);
cppcc@qq.com (Jia-Xi Chen).
1

Abstract
Two new Ru(II) complexes containing guanidinium groups have been synthesized, characterized and
analyzed according to their interactions with different G-quadruplexes and duplex DNA. A FRET
assay and a competitive FRET assay showed that both complexes promote the formation and
stabilization of the human telomeric (h-telo) G-quadruplex and exhibit higher selectivity compared to
promoters (such as c-myc, c-kit and bcl2) or duplex DNA. After binding to G-quadruplex, the two
complexes have shown different DNA affinity and fluorescence enhancement. CD analyses further
indicate that the two complexes display the ability to induce and stabilize the formation of antiparallel
G-quadruplex structures in K+, Na+ or ion-free buffers. The binding stoichiometry with h-telo was of
the order of three ruthenium complexes per quadruplex.
Keywords: Synthesis; Ru(II) complex; G-quadruplex DNA; Guanidinium; H-telo
2

1. Introduction
It is well known that nucleic acid sequences rich in guanosine (G) can form a special class of
DNA structures known as G-quadruplexes (G4). These structures are comprised of a stack of G-
tetrads, which are four guanines connected to each other through Hoogsteen hydrogen bonds to
form a square planar structure [1-3]. Higher-ordered DNA structures are found in human telomeres
(h-telo) and the promoter regions of many oncogenes, such as c-myc, c-kit and bcl2, and are
guanine-rich. Telomerase is an enzyme present in over 85-90% of tumor cells, but it is not found in
most normal cells [4,5]. This ribonucleoprotein increases the length of telomeres by adding
polynucleotides to the ends, causing normal cells to divide uncontrollably and thus become tumor
cells. Results show that the telomerase activity or oncogene expression, which are closely related to
apoptosis, cellular proliferation and tumorigenesis [6,7], could be directly inhibited by the
formation and stabilization of G-quadruplex structures. Moreover, the variety of the G-quadruplex
DNA (G4-DNA) topological structures is different from duplex DNA, which further emphasizes
that telomerase is an ideal probe for tumor diagnosis and a target for antitumor research [8,9]. It is
crucial to find new compounds that are able to selectively interfere with the h-telo expression by the
formation/stabilization of specific structures. Previous studies reported that G-quadruplex binders
are organic compounds, and a number of metal complexes interact effectively with this DNA
secondary structure [10-14]. The relative ease of the synthesis and the ability to vary the ligands and
interchange the metal center increase the advantages of using metal complexes over their organic
counterparts.
Successful quadruplex DNA binders should not only interact strongly with their target but also
exhibit high selectivity for quadruplex DNA versus duplex DNA. The guanidinium group, the
positively charged residues of arginine, plays a key role in many biological activities, such as
molecular recognition and catalysis [15,16]. Meanwhile, the added positive charge increases the
3

non-specific binding of the metal-peptide conjugate due to electrostatic association with the
negatively charged DNA backbone [17-19]. In this paper, we report the synthesis and
characterization of G-quadruplex DNA binding of two new ruthenium(II) complexes (Scheme 1),
[Ru(bpy)2L1]3- (complex 1) and [Ru(bpy)2L2]3+ (complex 2) using 2,2-bipyridine (bpy), 1-(4-(1H-
imidazo[4,5-f][1,10]phenanthrolin-2-yl)phenyl)guanidine hydrochloride (L1) and 1-(3-(1H-
imidazo[4,5-f][1,10]phenanthrolin-2-yl)phenyl)guanidine hydrochloride (L2). Both structures
proved to exhibit higher selectivity than promoters and duplex DNA. The obtained results will
hopefully be of value for the future synthesis of Ru(II) complexes as potent telomerase inhibitors
and also set a foundation for the rational design of new cancer chemotherapy drugs.
2. Materials and methods
2.1. Materials
DNA oligomers of 5′-AG3[T2AG3]3-3′ DNA (h-telo), the fluorescent labelled oligonucleotide
employed FRET probes: h-telo (5′-FAM-AG3[T2AG3]3-TAMRA-3′, FAM = 6-carboxyfluorescein,
TAMRA = 6-carboxy-tetramethylrhod amine), three promoter sequences c-myc (5′-FAM-
[TG4AGGGTGGGGAGGGTGGGGAAGG]-TAMRA-3′);
[CGGGCGGGCGCGAGGGAGGGG]-TAMRA-3′)
c-kit
bcl2
(5′-FAM-
(5′-FAM-
;
[AGGGGCGGGCGCGGGAGGAAGGGGGCGGGAGCGGGGCTG]-TAMRA-3′) and a duplex
DNA sequence F10T (5′-FAM-[TATAGCTATA-HEG-TATAGCTATA]-TAMRA-3′ (the HEG linker
is [(-CH2-CH2-O-)6]) were obtained from the Sangon Biotechnology Company. CT-DNA (calf
thymus DNA) was purchased from the Sigma Company. Other reagents and solvents were
commercially available. Four buffers were used in this work. Buffer A: 60 mM potassium
cacodylate, pH 7.4; Buffer B: 100 mM KCl, 10 mM Tris, pH 7.4; Buffer C: 100 mM NaCl, 10 mM
Tris, pH 7.4; Buffer D: 10 mM Tris, pH 7.4.
2.2. Physical measurements
4

1
Microanalysis (C, H and N) was carried out with a Perkin-Elmer 240C elemental analyzer. H
NMR spectra were recorded on a Varian Mercury-plus 300 NMR spectrometer with (CD3)2SO and
CD3CN as solvents and SiMe4 as an internal standard at 300 MHz at room temperature.
Electrospray mass spectra (ESI-MS) were performed on an LQC system (Finngan MAT, USA).
UV-Vis spectra were recorded on a Varian Cary 300 spectrophotometer. Emission spectra were
recorded on a Perkin-Elmer Lambda-850 spectrophotometer and an Ls55 spectrofluorophotometer.
2.3. Preparation of the ligands and complexes
1,10-Phenanthroline-5,6-dione [20], 4-(1H-imidazo[4,5-f][1,10]phenanthrolin-2-yl)aniline, 3-
(1H-imidazo[4,5-f][1,10]phenanthrolin-2-yl)aniline [21] and cis-[Ru(bpy)2Cl2]·2H2O [22] were
synthesized according to the methods reported in the referred literature.
2.3.1
Synthesis
of
1-(4-(1H-imidazo[4,5-f][1,10]phenanthrolin-2-yl)phenyl)guanidine
hydrochloride (L1·HCl)
A mixture of 4-(1H-imidazo[4,5-f][1,10]phenanthrolin-2-yl)aniline (0.4 g, 1.3 mmol) was
completely dissolved in methanol (12 mL) and hydrochloric acid (1.5 mL) with stirring for 30 min.
Next 50% cyanamide (4 mL) was added and refluxed for 20 h with the pH < 3. The solution was
cooled to room temperature. After filtration, the yellow compound obtained was washed with cool
ethanol and dried in a vacuum. Yield: 0.32 g (58.2%). Anal. Calcd for C20H16ClN7·2H2O (425.87):
C, 56.41; H, 4.73; N, 23.02. Found: C, 56.43; H, 4.72; N, 23.04%. ESI-MS, m/z: 354.2 [M+H+]+
1
(100), 707.0 [2M+H+]+ (10). H NMR ((CD3)2SO) δ (ppm): 10.38 (s, H,); 9.17 (dd, 2H); 8.57 (d,
2H); 8.20 (dd, 2H); 7.78 (s, 4H); 7.46 (t, 3H); 7.30 (s, H); 7.17 (s, 1H).
2.3.2
Synthesis
of
1-(3-(1H-imidazo[4,5-f][1,10]phenanthrolin-2-yl)phenyl)guanidine
hydrochloride (L2·HCl)
A mixture of 3-(1H-imidazo[4,5-f][1,10]phenanthrolin-2-yl)aniline (0.4 g, 1.3 mmol) was
completely dissolved in methanol (12 mL) and hydrochloric acid (1.5 mL) with stirring for 30 min.
5

Next 50% cyanamide (4 mL) was added and refluxed for 48 h with the pH < 3. Upon cooling, a pink
precipitate was obtained by dropwise addition of saturated aqueous Na2CO3 solution. The product
was washed with H2O until the pH was 7.0. The solid was then dissolved in 5 mL hydrochloric acid
(3 M) and refluxed for 30 min; a pink compound was obtained after the solution was evaporated.
Yield: 0.36 g (65.5%). Anal. Calcd for C20H16ClN7·2H2O (425.87): C, 56.41; H, 4.73; N, 23.02.
Found: C, 56.40; H, 4.75; N, 23.05%. ESI-MS, m/z: 354.5 [M+H+]+ (100), 176.0 [M+2H+]2+ (20).
1H NMR (CD3CN) δ (ppm): 10.24 (s, H,); 9.63 (d, 2H); 9.19 (d, 2H); 8.45 (d, H); 8.34 (s, H); 8.27
(dd, 2H); 7.68 (t, 5H); 7.41 (d, 1H).
2.3.3 [Ru(bpy)2L1](HPF6)(PF6)2 (1)
Cis-Ru(bpy)2Cl2·2H2O 0.14 g (0.26 mmol) and L1 0.11 g (0.26 mmol) were added to 20 mL
ethanol-water (9:1, v/v). The mixture was refluxed for 2 h under the protection of argon. Upon
cooling, a red complex was obtained after the addition of ammonium hexafluorophosphate. The
crude product was purified by reversed-phase HPLC. Yield: 0.14 g (43.3%). Anal. Calcd for
C40H32F18N11P3Ru·2H2O (1238.75): C, 38.78; H, 2.93; N, 12.44. Found: C, 38.76; H, 2.90; N,
− 2+
− 2+
− 3+
12.47%. ESI-MS, m/z: 456 [M-2PF6 ] (12), 383 [M-HPF6-2PF6 ] (31), 256 [M-3PF6 ] (100).
1H NMR (CD3CN) δ (ppm): 9.12 (d, 2H); 8.62 (dd, 4H); 8.50 (d, 2H); 8.20 (t, 2H); 8.14 (d, 2H);
8.12 (d, 2H); 7.96 (d, 2H); 7.91 (dd, 3H); 7.70 (d, 2H); 7.62 (d, 2H); 7.57 (t, 3H); 7.34 (t, 3H); 6.47
(s, 3H). UV-Vis ((λ (nm), ε (M-1 cm-1)) (H2O): 457 (12100), 283 (78300).
2.3.4 [Ru(bpy)2L2](HPF6)(PF6)2 (2)
This complex was obtained in an identical manner to that described for complex 1. L2 (0.11 g,
0.26 mmol) was used in place of L1. Yield: 0.15 g (47.3%). Anal. Calcd for
C40H32F18N11P3Ru·2H2O (1220.73): C, 39.36; H, 2.81; N, 12.62. Found: C, 39.35; H, 2.84; N,
− +
12.60%. ESI-MS, m/z:1057 [M-PF6 ] (2), 456 [M-2PF6 ] (28), 383 [M-HPF6-2PF6 ] (63), 256
− 2+
− 2+
− 3+
1
[M-3PF6 ] (100). H NMR (CD3CN) δ (ppm): 9.12 (d, 2H); 8.62 (dd, 5H); 8.40 (d, 1H); 8.32 (s,
6

1H); 8.22 (t, 3H); 8.14 (d, 2H); 8.12 (d, 2H); 7.97 (d, 3H); 7.91 (dd, 3H); 7.85 (d, 1H); 7.72 (d, 2H);
7.58 (m, 4H); 7.33 (t, 3H). UV-Vis ((λ (nm), ε (M-1 cm-1)) (H2O): 457 (13900), 282 (87400).
2.4 FRET assay
A FRET melting point assay was used to investigate the ability of the Ru(II) complexes to
stabilize the different G-quadruplexes. Fluorescence melting curves were determined by a Roche
Light Cycler II real-time PCR machine. The fluorescent labelled DNA was monitored alone and in
the presence of the complexes in buffer A. The total reaction volume was 20 µL with 200 nM of the
labelled oligonucleotide and different concentrations of the complexes (0, 1 and 2 µM).
Measurements were made on a RT-PCR with excitation at 470 nm and detection at 530 nm.
Fluorescence readings were taken at intervals of 1 °C from 37-99 °C with a constant temperature
being maintained for 30 s prior to each reading to ensure the sample had reached equilibrium. Tm is
the mid-point of a melting curve at which the complex is 50% dissociated.
The competition FRET-melting assay was similar to the FRET melting point assay, except
different concentrations of duplex CT-DNA were added. This experiment was carried out to explore
the selectivity of the Ru(II) complexes between h-telo DNA and duplex DNA.
2.5 Absorption and emission spectra
Absorption and emission titrations were carried out using 3 mL solutions of complexes 1 and 2
(10 µM, in buffer B) at room temperature, to which increments of the h-telo DNA stock solution
were added. The Ru(II)-DNA solutions were allowed to incubate for 5 min before the spectra were
recorded. The intrinsic binding constant Kb of the Ru(II) complex to DNA was calculated based on
equations (1a) and (1b), which was applied to absorption titration data for non-cooperative
metallointercalators binding to DNA [23-26], where [DNA] is the DNA concentration in base pairs.
εa, εf, and εb correspond to the extinction coefficient (Aabs/[M]) observed for the MLCT absorption
band at a given DNA concentration, the extinction coefficients for the free Ru(II) complex and
7

Ru(II) complex in the fully bound form, respectively. Kb is the equilibrium binding constant (M-1);
Ct is the total metal complex concentration and s is the binding site size.
2
(εa − εf)/(εb − εf) = (b − (b2 − 2Kb Ct[DNA]/s)1/2/2KbCt
(1a)
b = 1 + KbCt + Kb [DNA]/2s
2.6 Circular dichroism studies
(1b)
Circular dichroism (CD) studies were used to observe the effect of the complexes on the structure
of the h-telo DNA; these were performed on a JASCO J-810 spectropolarimeter at room
temperature. Spectral data were collected from 200 to 350 nm with a scanning speed of 500 nm
min-1, scanning three times for each CD spectrum. The oligomers were re-suspended in buffers B, C
and D. CD spectra were baseline-corrected for signal contributions due to the buffers. The CD
titration was then performed at a constant DNA concentration (3 µM) with various concentrations of
the complexes. All solutions were mixed thoroughly and allowed to equilibrate for 5 min before
data collection.
2.7 Continuous Variation Analysis
The binding stoichiometries were obtained for the two complexes and h-telo according to the
method of various proportions [27,28]. The mole fractions of the Ru(II) complex and DNA were
varied from 0 to 1 in 0.1 increments in Buffer B, while the sum of the concentrations was kept at 10
µM. Each solution was equilibrated at 4 °C for 12 h with no light. The fluorescence intensities of
these mixtures were measured at room temperature using an excitation wavelength of 458 nm for
the two complexes. The Fmax (fluorescence) was collected in the range 500-750 nm. The resulting
curves show a break point at the molar fraction according to the binding stoichiometry of the
complex.
3. Results and discussion
The synthetic route for the complexes is summarized in Scheme1. Each synthetic step involved
8

here is straight forward and provides a moderate yield of the desired product in the pure form.
These products were characterized by elemental analysis, 1H NMR, ESI-MS and UV-Vis
spectroscopy. The absorption spectra of the two Ru(II) complexes in water are characterized by a
metal to ligand charge transfer (MLCT) in the visible region, at 457 nm, and an intense ligand-
centered transition (LC) in the UV region, around 282 nm, typical of polypyridyl ruthenium(II)
complexes [28,29]. The visible bands at 457 nm are attributed to the overlap of Ru(dπ) → bpy(π*)
and Ru(dπ) →L1 or L2 (π*) transitions. The ultraviolet bands around 282 nm for the two complexes
can be attributed to (bpy) π→π* transitions for [Ru(bpy)2L]3+ [29].
(Scheme 1.)
FRET studies were used to investigate the thermodynamic stability of the two complexes to h-
telo, promoter G-quadruplex DNA sequences (c-myc, c-kit and bcl2) and a duplex DNA sequence
F10T. The ∆Tm values represent the increase in melting temperature between the initial DNA and
DNA after addition of the Ru(II) complexes and representing the ability of the Ru(II) complexes to
stabilize the different DNA. Reliable FRET melting curves (Figure 1) and ∆Tm values (Table 1)
were obtained [30]. As shown in Table 1, the two complexes exhibit selectivity to h-telo G-
quadruplex DNA at different concentrations. Both complexes had ∆Tm values > 23 °C with h-telo
G-quadruplex; in contrast, the two complexes had ∆Tm values < 9 °C with c-myc, ∆Tm values < 18
°C with c-kit, ∆Tm values < 8 °C with bcl2 and ∆Tm values < 11 °C with duplex DNA at 2 uM. At
the same time, the two Ru(II) complexes displayed high ∆Tm values with h-telo G-quadruplex of
22.5 and 19.2 °C ([Ru]/[DNA] 5/1) for 1 and 2 respectively, which were higher than that of the
=
complex [Ru(bpy)2(dppz)]2+ (4.7 °C, [Ru]/[DNA] 1/1) [25], but smaller than that of the complex
=
[Ru(bpy)2(ptpn)]2+ (26 °C, [Ru]/[DNA] 5/1) [11]. Although most of the ∆Tm values observed for
=
complex 2 were smaller than those for complex 1 in K+ buffer, the results suggest that the two Ru(II)
complexes are good h-telo G-quadruplex stabilizers.
9

(Figure 1.)
(Table 1.)
In addition to the high stabilization ability, the two Ru(II) complexes also exhibited high
selectivity for quadruplex DNA over duplex DNA. The DNA competition FRET-melting assay was
carried out to show the ∆Tm change for 2.0 µM of the Ru(II) complexes with 200 nM h-telo by
adding varying concentrations of the double-stranded CT-DNA in K+ buffer [31]. Figure 2 shows
the melting point change with 2.0 µM Ru(II) complexes by adding CT-DNA in buffer A. As shown
in Table 2, the ∆Tm values do not change significantly, even though the CT-DNA is present at a
concentration 50 times that of h-telo, showing duplex DNA has a negligible effect on the binding of
ligands to G-quadruplex DNA. The above results indicate that both the Ru(II) complexes not only
have a high stabilization ability, they also have better selectivity for h-telo DNA.
(Figure 2.)
(Table 2.)
Absorption titration spectra were performed to determine the binding affinity of the complexes
with h-telo. The electronic spectral traces of the complexes titrated with DNA are shown in
Figure 3. The data of the UV absorption titration with DNA for complexes 1 and 2 are listed in
Table 3. Upon addition of h-telo, the MLCT transition bands of the two complexes exhibit different
degrees of hypochromism and red shifting changes. There is a significant difference in the DNA-
binding intrinsic constants. The intrinsic binding constants, Kb, of the two complexes 1 and 2 in K+
buffer were respectively (1.37 0.09) × 106 and (1.08 0.80) × 105 M-1, in which complex 1 is much
greater and has a stronger DNA-binding affinity than complex 2. The results obtained by absorption
spectra titrations overall are consistent with those obtained by the FRET assay. The two complexes
have the same ancillary ligand (bpy). The difference in DNA affinity ability mostly originates from
the difference between the intercalative ligands, which can be explained by the lower steric
10

hindrance of L1 in complex 1 than that of L2 in complex 2. In general, a larger steric hindrance in
the intercalative ligand will reduce the interaction of the complexes with DNA.
(Figure 3.)
(Table 3.)
Luminescence titration measurements were used to further clarify the nature of the interaction
between the complexes and h-telo DNA. Figure 4 displays the results of the luminescence titration
for the two complexes with h-telo. Upon excitation using a wavelength of 458 nm, both complexes
exhibit luminescence in buffer B with a maximum wavelength at about 600 nm. The luminescence
intensity of the complexes increases with an increase in the DNA concentration and reaches a
maximum at a ratio of [DNA]/[Ru] ≈ 13:1, at which there is a 2.44 and 2.19-fold increase in the
fluorescence intensity for complexes 1 and 2, respectively. The binding constants of the two
complexes interacting with DNA from the emission spectra were obtained using the luminescence
titration method [32]. The binding data obtained from the emission spectra were fitted using the
McGhee-von Hippel equation [33] to acquire the binding parameters. The intrinsic binding
constants Kb of 1.44( 0.23) × 106 M-1 for complex 1 and 9.84( 0.98)× 105 M-1 for complex 2 were
determined. Although the binding constants obtained from fluorescence spectra with the McGhee–
von Hippel method are different from those obtained from absorption spectra, both sets of binding
constants show that complex 1 binds to DNA more avidly than complex 2.
(Figure 4.)
CD spectroscopy was employed to characterize the solution formation of h-telo G-quadruplex
induced by Ru(II) complex. It has been reported that h-telo G-quadruplexes generally consist of a
mixed conformation (parallel/antiparallel) in K+ solution [34-36]. The CD spectrum showed a large
positive band around 290 nm, a small positive band at 250 nm and a negative band at 235 nm. In
the presence of Na+ ions, the CD spectrum had a 295 nm positive band and a 265 nm negative band,
11

which may be characteristic of an antiparallel G-quartet structure [37,38]. The CD spectrum of the
h-telo sequence indicated the coexistence of a single strand and two types of quadruplex structures,
parallel and antiparallel G-quadruplexes, in the absence of metal cations [34,39,40].
As shown in Figure 5(a), upon addition of the two complexes, dramatic changes in the CD
spectra were observed. The maximum at 290 nm was gradually suppressed, and two positive bands
at 281 and 295 nm increased. At the same time, the positive band at 248 nm was gradually
suppressed and a major negative band at 260 nm started to appear. It seemed that in the
presence of the complexes, the mixed parallel G-quadruplex decreased, forming an
antiparallel G-quadruplex instead, and the CD signal at 281 nm may be induced through the
strong absorbance of the two complexes at about 285 nm (Figure 3) [41]. In Na+ solution, the
intensity of the bands at 260 nm increased on addition of the two Ru(II) complexes, while a minor
shoulder positive band at about 280 nm was observed, and the intensity of the band centered at 295
nm increased significantly [Figure 5(b)]. These changes suggest that the two Ru(II) complexes can
further stabilize the antiparallel G-quadruplex structure. In the ion-free system, the CD spectrum of
h-telo consists of a small negative band at 240 nm, a major positive band at 255 nm, and a rather
broad and small positive signal around 295 nm. Addition of the complexes increased the CD
intensity of the band at 295 nm and decreased the band at 255 nm; the negative band at 260 nm
began to appear and gradually increased [Figure 5(c)]. This is typical for the antiparallel G-
quadruplex structure as described above. All of the results imply that the two complexes can induce
h-telo into an antiparallel G-quadruplex structure and stabilize it even at high metal strength.
(Figure 5.)
A luminescence-based Job plot was used to determine the stoichiometry interactions between the
complexes and h-telo DNA. From the intersection points obtained in Figure 6, binding
stoichiometries for complexes 1 and 2 in K+ buffer were obtained. The Ru(II) complexes
12

demonstrated binding stoichiometries of the order of three ruthenium complexes per G-quadruplex.
(Figure 6.)
Small molecules can potentially bind to a quadruplex by externally stacking below the quartets,
intercalating between the quartets, or non-specifically binding to some random location on the DNA
strand [41,42]. The most important interaction between G-quartets and the octahedral Ru(II)
complexes in solution is thought to partially stack on or intercalate the G-tetrads [43]. Given the CD
results, the structure of the two complexes and the positive charge of the metal ion, the main mode
of interaction is thought to occur through the intercalative mode. Meanwhile, the guanidinium
groups with the cationic charge probably give rise to an additional interaction with the negatively
charged phosphate backbones of DNA through hydrogen-bonding and electrostatic interactions
[19,44]. Detailed investigations on the binding modes and the biological activity are in progress.
4. Conclusion
In summary, two new Ru(II) complexes, [Ru(bpy)2L]3+, containing guanidinium ligands have
been synthesized and characterized. Both complexes show good selectivity for h-telo G-quadruplex
compared to promoters and duplex DNA. The results of absorption and emission titrations indicate
that complex 1 has a greater DNA affinity than complex 2. The 3:1 stoichiometry suggests that
three complexes molecules interact with one molecule of G-quadruplex DNA. Given the CD spectra,
we propose that the Ru(II) complexes most likely interact with h-telo DNA through the intercalative
mode.
Appendix A. Supplementary data
1
Nine figures (Figure S1-9) for ESI-MS spectra of L1, L2, 1 and 2, H NMR spectra of L1, L2, 1
and 2, and UV-Vis spectra of 1 and 2, respectively.
Acknowledgments
13

We are grateful to the National Natural Science Foundation of China (21101034), the Yong
Teachers Training Plan of Guangdong Province (Yq2013086), the Science and Technology Plan of
Dongguan City (2011108102046) and the innovative items for undergraduates of Guangdong
Province (201510571038).
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16

Table 1. The effect of complexes 1 and 2 on the Tm of h-telo, c-myc, c-kit, bcl2 and duplex DNA
F10T determined from FRET ([DNA] = 200 nM)
h-telo
c-myc
c-kit
bcl2
F10T
Complex
∆Tm(°C)
17.80
14.00
16.39
9.00
1 (2 uM)
1 (1 uM)
2 (2 uM)
2 (1 uM)
25.74
22.54
23.20
19.20
8.80
5.20
6.94
6.14
2.60
0.26
7.40
5.20
9.60
4.60
10.50
8.26
17

Table 2. The effect of complexes 1 and 2 on the Tm of h-telo DNA ([DNA] = 200 nM)
h-telo
h-telo+2 uM Ru(II)
h-telo+2 uM Ru(II)
+5 uM CT-DNA
h-telo+2 uM Ru(II)
+10 uM CT-DNA
Complex
Tm (°C )
∆Tm(°C )
51.34
27.03
23.40
27.43
29.00
23.80
1
2
51.34
23.63
18

Table 3. Absorption spectra (λmax/nm) and DNA-binding constants Kb (M-1) of complexes 1 and 2
Complex
H/(%) Kb/ M-1
s
λ
max(free)
λmax(bound) ∆λ/nm
457
285
458
284
460
289
463
287
3
4
5
3
32.63 1.37 0.9(×106) 0.24 0.04
1
40.48
18.19 1.08 0.8(×105) 0.025 0.02
19.16
2
19

Scheme and Figure Captions
Scheme 1. Synthetic routes for the preparation of the ligands L1 and L2, and the complexes 1 and 2.
Figure 1. FRET-melting curves for experiments carried out in buffer A (60 mM potassium
cacodylate, pH 7.4) with h-telo, c-myc, c-kit, bcl2 and duplex DNA F10T separately with the two
complexes 1 and 2; [DNA] = 200 nM.
Figure 2. FRET competition experiments of the complexes 1 and 2 at 2 µM concentration showing
the h-telo DNA (200 nM) melting temperature with different concentration of CT-DNA in buffer A
(60 mM potassium cacodylate, pH 7.4).
Figure 3. Absorption spectra of complexes 1 and 2 in buffer B (100 mM KCl, 10 mM Tris, pH 7.4)
with increasing amounts of h-telo DNA; [Ru] = 10 µM, [DNA] = 0-14 µM from top to bottom.
Arrows refer to the change in absorbance upon increasing the DNA concentration. Inset: plot of (εa-
εf)/(εb-εf) vs. [DNA] and the non-linear fit for the titration of DNA to Ru(II) complexes.
Figure 4. Emission spectra of complexes 1 and 2 in buffer B (100 mM KCl, 10 mM Tris, pH 7.4)
with increasing amounts of h-telo DNA; [Ru] = 10 µM, [DNA] = 0-14 µM. Arrow refers to the
emission intensity changes upon increasing DNA concentrations.
Figure 5. CD spectra of G-quadruplex induced by complexes 1 and 2 with 3 µmol L-1 h-telo at room
temperature. (a) 1 and 2 in a buffer of 100 mM KCl, 10 mM Tris, pH 7.4; (b) 1 and 2 in a buffer of
100 mM NaCl, 10 mM Tris, pH 7.4; (c) 1 and 2 in a buffer of 100 mM NaCl, 10 mM Tris, pH 7.4.
Arrows indicate the increasing amounts of complexes. r = 0 ~ 6 (r = [Ru]/ [DNA])
■) and ●) with a final G
Figure 6. Job plot using luminescence data for complexes 1 ( 2 (
-quadruplex
at 10.0 µM in buffer solution of 100 mM KCl, 10 mM Tris, pH 7.4.
20

Scheme 1
21

1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.8
0.6
0.4
0.2
0.0
c-myc
c-myc+2µM 1
c-myc+1µM 1
c-myc+2µM 2
c-myc+1µM 2
h-telo
h-telo+2µM 1
h-telo+1µM 1
h-telo+2µM 2
h-telo+1µM 2
40
50
60
Temperature (°C)
70
80
90
100
40
50
60
70
80
90
100
Temperature (°C)
1.0
1.0
0.8
0.6
0.4
0.2
0.0
0.8
0.6
0.4
0.2
0.0
c-kit
bcl2
c-kit+2µM 1
bcl2+2µM 1
bcl2+1µM 1
bcl2+2µM 2
bcl2+1µM 2
c-kit+1µM 1
c-kit+2µM 2
c-kit+1µM 2
40
50
60
70
80
90
100
40
50
60
70
80
90
100
Temperature (°C)
Temperature (°C)
1.0
0.8
0.6
0.4
0.2
0.0
F10T
F10T+2µM 1
F10T+1µM 1
F10T+2µM 2
F10T+1µM 2
40
50
60
70
80
90
100
Temperature (°C)
Figure 1.
22

1.2
1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.8
0.6
0.4
0.2
0.0
2
1
h-telo
h-telo
h-telo+2µM 1
h-telo+2µM 2
h-telo+2µM 1+5µM CT-DNA
h-telo+2µM 1+10µM CT-DNA
h-telo+2µM 2+5µM CT-DNA
h-telo+2µM 2+10µM CT-DNA
40
50
60
70
80
90
100
40
50
60
70
80
90
100
Temperature (°C)
Temperature (°C)
Figure 2.
23

1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.8
0.6
0.4
0.2
0.0
1
0
2
4
[DNA]*100000
6
8
10 12 14
300
400
Wavelength (nm)
500
600
1.0
0.8
0.6
0.4
0.2
1.0
0.8
0.6
0.4
0.2
0.0
2
0.0
0
2
4
[DNA]*100000
6
8
10 12 14
300
400
500
600
Wavelength/nm
Figure 3.
24

2.6
2.4
2.2
2.0
1.8
1.6
1.4
1.2
1.0
600
400
200
0
1
0
2
4
6
[DNA]/[Ru]
8
10
12
14
500
600
Wavelength (nm)
700
2.2
2.0
1.8
1.6
1.4
1.2
1.0
400
300
200
100
0
2
0
2
4
6
[DNA]/[Ru]
8
10 12 14
500
600
700
Wavelength (nm)
Figure 4.
25

16
12
8
0
1
2
3
4
5
6
15
10
5
0
1
2
3
4
5
6
a-2
a-1
4
0
0
250
300
350
250
300
350
Wavelength(nm)
-5
Wavelength(nm)
-4
0
1
2
3
4
5
6
15
10
5
0
1
2
3
4
5
6
10
5
b-2
b-1
0
250
300
350
0
Wavelength(nm)
250
300
350
-5
-5
Wavelength(nm)
-10
-15
-10
-15
15
15
10
5
0
1
2
3
4
5
6
0
1
2
3
4
5
6
10
5
c-2
c-1
0
250
300
350
0
-5
Wavelength(nm)
250
300
350
Wavelength(nm)
-5
-10
Figure 5.
26

150
120
90
60
30
0
1
2
0.0
0.2
Mole fraction of complex
0.4
0.6
0.8
1.0
Figure 6.
27

1.0
0.8
0.6
0.4
0.2
0.0
h-telo
h-telo+2µM 1
h-telo+1µM 1
h-telo+2µM 2
h-telo+1µM 2
40
50
60
70
80
90
100
Temperature (°C)
Synthesis, characterization and DNA binding studies of two Ru(II) complexes
containing guanidinium ligands
Wen-Xiu Chen, Xing-Dong Song, Jia-Xi Chen, Xuan-Hao Zhao, Jie-hua Xing, Jun-Rui Ren,
Tie Wu, Jing Sun
Two new Ru(II) complexes, [Ru(bpy)2L]3+ (1 and 2), containing guanidinium ligands have been
synthesized and characterized. Both complexes show good selectivity for h-telo G-quadruplex DNA
compared to promoters and duplex DNA.
28