Synthesis, DNA-binding, and photocleavage studies of ruthenium(II) complexes with an asymmetric ligand

Article abstract of DOI:10.1080/00958972.2011.639364

An asymmetric ligand (pdpiq=2-(pyridine-2-yl)-6,7-diphenyl-1-H-imidazo[4,5- g]quinoxaline) and its ruthenium complexes with [Ru(L)2pdpiq]²⁺ (L=bpy (2,20-bipyridine) or phen (1,10-phenanthroline)) have been synthesized and characterized by eleme

Full text of DOI:10.1080/00958972.2011.639364

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Synthesis, DNA-binding, and
photocleavage studies of ruthenium(II)
complexes with an asymmetric ligand
Xue-Wen Liu a b , Lin Li a , Ji-Lin Lu a , Yuan-Dao Chen a b & Da-
Shun Zhang a
a College of Chemistry and Chemical Engineering, Hunan
University of Arts and Science , ChangDe 415000 , P.R. China
b Key Lab of Environment-Friendly Chemistry and Application in
Ministry of Education, Xiangtan University , Xiangtan 411105 , P.R.
China
Published online: 02 Dec 2011.
To cite this article: Xue-Wen Liu , Lin Li , Ji-Lin Lu , Yuan-Dao Chen & Da-Shun Zhang (2011)
Synthesis, DNA-binding, and photocleavage studies of ruthenium(II) complexes with an asymmetric
ligand, Journal of Coordination Chemistry, 64:24, 4344-4356, DOI: 10.1080/00958972.2011.639364
To link to this article: http://dx.doi.org/10.1080/00958972.2011.639364
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Journal of Coordination Chemistry
Vol. 64, No. 24, 20 December 2011, 4344–4356
Synthesis, DNA-binding, and photocleavage studies of
ruthenium(II) complexes with an asymmetric ligand
XUE-WEN LIU*yz, LIN LIy, JI-LIN LU*y, YUAN-DAO CHENyz and
DA-SHUN ZHANGy
yCollege of Chemistry and Chemical Engineering, Hunan University of Arts and Science,
ChangDe 415000, P.R. China
zKey Lab of Environment-Friendly Chemistry and Application in Ministry of Education,
Xiangtan University, Xiangtan 411105, P.R. China
(Received 1 September 2011; in final form 31 October 2011)
An asymmetric ligand (pdpiq ¼ 2-(pyridine-2-yl)-6,7-diphenyl-1-H-imidazo[4,5-g]quinoxaline)
and its ruthenium complexes with [Ru(L)2pdpiq]2þ (L ¼ bpy (2,20-bipyridine) or phen
(1,10-phenanthroline)) have been synthesized and characterized by elemental analysis,
ES-MS, and 1H NMR. The DNA-binding behaviors of these complexes were studied by
spectroscopic methods and viscosity measurements. The results indicate that the complexes can
intercalate into DNA base pairs. When irradiated at 365 nm, the two complexes promote the
cleavage of plasmid pBR322DNA. The mechanism of DNA cleavage is an oxidative process by
generating singlet oxygen.
Keywords: Ru(II) complex; Asymmetric ligand; DNA-binding; Photocleavage
1. Introduction
There has been considerable interest in studies on the interactions of transition metal
complexes with nucleic acid, ruthenium(II) polypyridyl complexes in particular. These
Ru(II) complexes have attracted considerable attention due to their rich photophysical
properties and potential applications in biology, such as the design and development of
non-radioactive probes of nucleic acid structure, new therapeutic reagents, synthetic
restriction enzymes, versatile catalysts for divergent organic reactions, DNA foot
printing agents, and possible DNA cleaving agents [1–10]. In general, Ru(II)
polypyridyl complexes interact with DNA through electrostatic binding, groove
binding, or intercalation. Among these interactions, intercalation is the most important
because many potential applications such as antitumor activity and molecular ‘‘light
switch’’ [11–16] require that the complexes bind to DNA in an intercalative mode.
[Ru(bpy)2(dppz)]2þ (bpy ¼ 2,20-bipyridine; dppz ¼ dipyrido[3,2-a:20,30-c]phenazine) is
the most extensively investigated ruthenium(II) complex as a molecular ‘‘light switch’’
for DNA, because the complex shows negligible luminescence in aqueous solution at
*Corresponding authors. Email: liuxuewen050@sina.com; lu_jilin10@163.com
Journal of Coordination Chemistry
ISSN 0095-8972 print/ISSN 1029-0389 online ß 2011 Taylor & Francis
http://dx.doi.org/10.1080/00958972.2011.639364

Ruthenium(II) with an asymmetric ligand
4345
ambient temperature but displays strong luminescence after binding to DNA by
intercalation [15]. Clarification of the trends in DNA-binding of Ru(II) polypyridyl
complexes will facilitate understanding and control of interactions between the
complexes and DNA, and thus mechanisms of DNA mutation and damage, as well
as the design of new clinic anti-cancer drugs and complexes with biochemical
activity [1–5].
In order to further understand the DNA-binding mechanism of Ru(II) complexes
and obtain Ru(II) complexes with excellent bioactivity, many polypyridyl Ru(II)
complexes were designed and studied for DNA-binding behavior, DNA cleavage, and
spectral properties [17–28]. A number of reports have shown that varying the shape of
the ligand can create different space configuration and electron density distribution of
Ru(II) polypyridyl complexes, resulting in changes in DNA-binding properties. Studies
of such changes will help to more clearly understand the DNA-binding mechanism of
Ru(II) polypyridyl complexes. Therefore, further design and studies on different ligands
are necessary to evaluate and understand the factors that determine DNA-binding
mode. Designs of ligands for modulating DNA affinities of Ru(II) complexes are
mainly classified into two categories: (i) structural change on the intercalative ligand,
including symmetric (e.g., dppz, tpphz) [14–22] and asymmetric ones (e.g., dppt, ptdb,
PZNI) (dppt ¼ 3-(1,10-phenanthrolin-2-yl)-5,6-diphenyl-as-triazine; ptdb ¼ 3-(pyridine-
2-yl)-5,6-diphenyl-as-triazine;
PZNI ¼ 2-(pyrazin-2-yl)naphthoimidazole)
[23–25];
(ii) changing the ancillary ligands [28–31]. Attention has primarily focused on
symmetric aromatic ligands such as 1,10-phenanthroline and its derivatives [14–22];
the influence of asymmetric ligands on DNA-binding properties of Ru(II) complexes
has attracted much less attention. Some of these complexes with asymmetric ligands
exhibit interesting properties upon binding to DNA [23–25].
In order to obtain more insight into DNA-binding and photocleavage properties of
ruthenium(II) complexes, herein, we report the synthesis and characterization of an
asymmetric
ligand
(pdpiq ¼ 2-(pyridine-2-yl)-6,7-diphenyl-1-H-imidazo
[4,5-g]quinoxaline) and its ruthenium complexes, [Ru(L)2pdpiq]2þ (L ¼ bpy or phen).
DNA-binding and DNA-photocleavage properties of these Ru(II) complexes were
carefully studied. We hope the results are of value in further understanding
DNA-binding mechanisms.
2. Experimental
2.1. Materials
cis-[Ru(bpy)2Cl2] ꢀ 2H2O, cis-[Ru(phen)2Cl2] ꢀ 2H2O [32], methyl pyridine-2-carboximi-
date [33, 34], and 1,2-diamino-4,5-(p-toluenesulfamidobenzene) [35, 36] were prepared
by literature methods. All DNA-binding experiments were carried out in buffer A
(50 mmol Lꢁ1 NaCl, 5 mmol Lꢁ1 Tris–HCl, pH ¼ 7.2). For DNA photocleavage
experiments, samples were treated in buffer B (50 mmol Lꢁ1 Tris, 18 mmol Lꢁ1 NaCl,
pH ¼ 7.8). Calf thymus DNA (CT-DNA) was obtained from Sigma (St. Louis, MO,
USA). Supercoiled pBR 322 DNA was purchased from MBI Fermentas. Solutions of
CT-DNA in buffer A gave a ratio of UV-Vis absorbance of 1.8–1.9 : 1 at 260 and
280 nm, indicating that the DNA was sufficiently free of protein [37]. The concentration

4346
X.-W. Liu et al.
of DNA was determined spectrophotometrically ("260 ¼ 6600 (mol L
)
ꢁ1 ꢁ1 cmꢁ1) [38].
Other materials were commercially available and used without purification.
2.2. Physical measurement
C, H, and N analyses were carried out with a Perkin-Elmer 240Q elemental analyzer.
1H NMR spectra were recorded on a Bruker ARX-500 spectrometer with (CD3)2SO as
solvent at room temperature. Electrospray mass spectra were recorded on an LQC
system (Finnigan MAT, USA) using CH3CN as mobile phase. Fast atomic bombard-
ment mass spectra (FAB-MS) were obtained on a VG ZAB-HS spectrometer.
Absorption spectra were recorded with a Shimadzu UV-2450 spectrophotometer and
emission spectra on a Hitachi F-2500 spectroFuorophotometer at room temperature.
2.3. DNA-binding experiments
2.3.1. Electronic absorption titration. Absorption spectral titrations of Ru(II) com-
plexes in buffer A were carried out at room temperature to determine the DNA-binding
affinities. Ruthenium-DNA solutions were allowed to incubate for 5 min before the
absorption spectra were recorded. The intrinsic binding constants K of these two
complexes to DNA were obtained by monitoring the changes of the 1MLCT
absorbance according to equation (1) [39],
1=2
ð"a ꢁ "fÞ=ð"b ꢁ "fÞ ¼ ðb ꢁ ðb2 ꢁ 2K2Ct½DNAꢂ=sÞ Þ=2KCt,
ð1aÞ
ð1bÞ
b ¼ 1 þ KCt þ K½DNAꢂ=2s,
1
where "a is the extinction coefficient observed for the MLCT absorption band at a
given DNA concentration, "f is the extinction coefficient of the complex in the absence
of DNA, "b is the extinction coefficient of the complex fully bound to DNA. K is the
equilibrium binding constant in (mol L )
ꢁ1 ꢁ1, Ct is the total metal complex concentra-
tion, [DNA] is the concentration of DNA in mol Lꢁ1 (nucleotide), and s is the binding
site size. The experimental absorption titration data were fitted to obtain the binding
constants with a non-linear least-squares method.
2.3.2. Competitive binding experiment. The competitive binding experiments were
conducted by adding increasing amounts of Ru(II) complex directly into the samples
containing 5 mmol Lꢁ1 ethidium bromide (EB) and 100 mmol Lꢁ1 DNA in buffer A.
Emission spectra were recorded in the region 500–700 nm and samples were excited at
340 nm. To further illustrate the DNA-binding strength of the two Ru(II) complexes,
a competitive binding model was applied to calculate the apparent binding constants of
Ru(II) complexes from EB competitive experiments using equation (2) [40],
Kapp ¼ KEBð½EBꢂ50%=½Ruꢂ50%Þ,
ð2Þ
where Kapp is the apparent DNA-binding constant of the Ru(II) complex, KEB is the
DNA-binding constant of EB, and [EB]50% and [Ru]50% are the EB and Ru(II) complex
concentrations at 50% fluorescence, respectively.

Ruthenium(II) with an asymmetric ligand
4347
2.4. Viscosity studies
DNA viscosities were measured using an Ubbelohde viscometer maintained at
30.0 ꢃ 0.1ꢄC in a thermostatic bath. DNA samples for viscosity measurement were
prepared by sonication in order to minimize complexities arising from DNA flexibility.
Every sample was measured at least three times and an average flow time was
calculated. The DNA viscosity was calculated according to ꢀi ¼ (ti ꢁ t0)/t0, where ꢀi is
the corresponding value of DNA viscosity, ti is the flow time of the solutions in the
presence or absence of the complex, and t0 is the flow time of buffer alone. Data are
presented as (ꢀ/ꢀ0)1/3 versus binding ratio [41], where ꢀ is the viscosity of DNA in the
presence of complex and ꢀ0 is the viscosity of DNA alone.
2.5. DNA photocleavage experiment
The DNA photocleavages by Ru(II) complexes were examined by gel electrophoresis
experiments. Supercoiled pBR322 DNA (0.1 mg) was treated with Ru(II) complex in
buffer B (50 mmol Lꢁ1 Tris, 18 mmol Lꢁ1 NaCl, pH ¼ 7.8), and the solutions were
incubated for 1 h in the dark, then irradiated at room temperature with a UV lamp
(365 nm, 10 W). The samples were analyzed by electrophoresis for 2 h at 75 V on a 1%
agarose gel in Tris-borate-EDTA (TBE) buffer C (89 mmol Lꢁ1 Tris-boric acid,
2 mmol Lꢁ1 EDTA, pH ¼ 8.3). The gel was stained with 1 mg mLꢁ1 EB and then
photographed under UV light.
2.6. Synthesis
2.6.1. 5,6-(p-toluenesulfonamide)-2-(pyridine-2-yl)-1H-benzimidazole (1). A solution of
methyl pyridine-2-carboximidate (ca 0.4 mmol) in CH3OH was added to a solution of
1,2-diamino-4,5-(p-toluenesulfamidobenzene) 0.178 g (0.4 mmol) in 10 mL glacial acetic
acid and the mixture was refluxed under argon for 4 h. The cooled solution was diluted
with water and neutralized with concentrated aqueous ammonia. The khaki precipitate
was collected and dried in vacuo. Yield: 0.195 g, 91.3%. Anal. (%): (Found: C, 58.37;
H, 4.23; N, 12.92. Calcd for C26H23N5O4S2: C, 58.52; H, 4.35; N, 13.13). FAB-MS:
534 [M þ 1]þ.
2.6.2. 2-(pyridine-2-yl)-5,6-diamino-1H-benzimidazole
(2). 5,6-(p-toluenesulfona-
mide)-2(pyridine-2-yl)-1H-benzimidazole (1) 0.533 g (1.0 mmol) and 4 mL concentrated
sulfuric acid were stirred for 24 h at room temperature. The dark violet solution was
then added dropwise to ice water. Treatment of the resulting solution with a saturated
Na2CO3 solution gave a clear green solution; the solution was extracted with
dichloromethane (3 ꢅ 100 mL). The combined extracts were dried over MgSO4.
Removal of the solvent at reduced pressure gave the product as a yellow solid. Yield:
0.089 g, 39.6%. Anal. (%): (Found: C, 63.11; H, 4.99; N, 30.82. Calcd for C12H11N5:
C, 63.99; H, 4.92; N, 31.09). FAB-MS: 226 [M þ 1]þ.
2.6.3. 2-(pyridine-2-yl)-6,7-diphenyl-1-H-imidazo [4,5-g]quinoxaline (pdpiq) (3). A mix-
ture of 2-(20-pyridineyl)-5,6-diamino-1H-benzimidazole (2) 0.067 g (0.3 mmol) and

4348
X.-W. Liu et al.
benzil was refluxed for 2 h in methanol (30 mL). After cooling to room temperature, the
solution was poured into water, and the resulting yellow precipitate was collected and
dried in vacuo. Yield: 0.094 g, 78.3%. Anal. (%): (Found: C, 77.87; H, 4.35; N, 17.36.
Calcd for C26H17N5: C, 78.18; H, 4.29; N, 17.53). ES-MS (CH3CH2OH): m/z ¼ 400.0
([M þ 1]þ).
2.6.4. [Ru(bpy)2pdpiq](ClO4)2 (3a). A mixture of pdpiq 0.120 g (ca 0.3 mmol),
[Ru(bpy)2Cl2] ꢀ 2H2O (0.156 g, 0.3 mmol), and ethylene glycol (10 mL) was refluxed
under argon for 4 h. Upon cooling, the resulting clear red solution was diluted with
water (ca 60 mL), then treated with a saturated aqueous solution of NaClO4. The
orange precipitate was collected and purified by column chromatography on neutral
alumina with acetonitrile-toluene (2 : 1, v/v) as eluent. Yield: 0.179 g, 64.5%. Anal. (%):
(Found: C, 54.38; H, 3.33; N, 12.32. Calcd for C46H33N9O8RuCl2: C, 54.59; H, 3.29; N,
12.46). ES-MS (CH3CN): m/z ¼ 811.3 ([M ꢁ 2ClOꢁ4 ꢁH]þ), 406.5 ([M ꢁ 2ClO4ꢁ]2þ).
1H NMR (500 MHz, ppm, DMSO-d6): 8.85 (dd, 3H, J ¼ 8.0 Hz), 8.72 (d, 1H,
J ¼ 8.5 Hz), 8.58 (d, 1H, J ¼ 7.5 Hz), 8.28 (s, 1H), 8.26 (7, 1H, J ¼ 8.0 Hz), 8.16 (t, 1H,
J ¼ 8.0 Hz), 8.12 (t, 1H, J ¼ 7.0 Hz), 8.09 (t, 1H, J ¼ 7.5 Hz), 8.05 (d, 1H, J ¼ 5.5 Hz),
8.02 (t, 1H, J ¼ 8.0 Hz), 7.94 (d, 1H, J ¼ 5.5 Hz), 7.90 (d, 1H, J ¼ 5.5 Hz), 7.66 (m, 3H),
7.56 (t, 1H, J ¼ 6.5 Hz), 7.52 (t, 1H, J ¼ 5.5 Hz), 7.47 (t, 1H, J ¼ 7.0 Hz), 7.45 (t, 1H,
J ¼ 6.5 Hz), 7.40 (d, 2H, J ¼ 6.5 Hz), 7.34 (brs, 8H), 6.20 (s, 1H).
2.6.5. [Ru(phen)2pdpiq](ClO4)2 (3b). This red complex was obtained by a similar
procedure as described for complex 3a, with the only difference being that
[Ru(phen)2]Cl2 ꢀ 2H2O (0.170 mg, 0.3 mmol) was used instead of [Ru(bpy)2]Cl2 ꢀ 2H2O.
Yield: 0.180 g, 61.4%. Anal. (%): (Found: C, 56.41; H, 3.22; N, 11.81, Calcd for
C50H33N9O8RuCl2: C, 56.65; H, 3.14; N, 11.90). ES-MS (CH3CN): m/z ¼ 860.2
1
([M ꢁ 2ClOꢁ4 ꢁH]þ), 430.5 ([M ꢁ 2ClO4ꢁ]2þ). H NMR (500 MHz, ppm, DMSO-d6):
8.82 (d, 1H, J ¼ 8.0 Hz), 8.75 (d, 1H, J ¼ 8.0 Hz), 8.72 (d, 1H, J ¼ 8.0 Hz), 8.69 (d, 1H,
J ¼ 8.0 Hz), 8.60 (d, 1H, J ¼ 7.5 Hz), 8.41 (d, 1H, J ¼ 5.0 Hz), 8.38 (m, 6H), 8.14 (d, 1H,
J ¼ 5.0 Hz), 8.09 (t, 2H, J ¼ 8.0 Hz), 7.93 (t, 1H, J ¼ 8.0 Hz), 7.85 (t, 2H, J ¼ 5.5 Hz,
J ¼ 8.5 Hz), 7.74 (t, 1H, J ¼ 5.5 Hz), 7.62 (m, 1H), 7.31 (brm, 11H), 5.92 (s, 1H).
Each of the above ClO4 salts was dissolved in the minimum amount of acetone and a
saturated solution of tetrabutylammonium chloride (TBACl) in acetone solution was
added dropwise until precipitation was complete. The water-soluble chloride salts were
filtered off, washedthoroughly with acetone, and vacuum dried (yield ꢆ91% in each case).
3. Results and discussion
3.1. Synthesis and characterization
The outline of the synthesis of the ligand and its complexes is presented in scheme 1.
Compound 1 was obtained on the basis of the method for imidazole ring preparation
established by Schaefer and Peters [33, 34, 42]. The diamino compound 2 was prepared
by detosylation of the corresponding tosylated 1. The synthesis of pdpiq 3 was
performed by the condensation of benzil with the precursor diamine 2.

Ruthenium(II) with an asymmetric ligand
4349
NH
H
NH Ts
NH Ts
N
H2N
H2N
NH Ts
NH Ts
N
OCH3
O
N
1
N
O
H
NH2
NH2
H
N
N
H2SO4
H2O
N
N
N
N
N
N
3
2
2+
H
N
N
N
N
N
Ru(bpy)2Cl2
Ru
N
N
N
N
2+
3a
H
N
N
N
N
N
N
Ru
N
N
Ru(phen)2Cl2
N
3b
Scheme 1. The synthetic routes of the ligand, [Ru(bpy)2(pdpiq)]2þ and Ru(phen)2(pdpiq)]2þ
.
The corresponding ruthenium(II) complexes 3a and 3b were prepared by direct reaction
of ligand with the appropriate mol ratios of the precursor complexes in ethylene glycol.
All these complexes were purified by column chromatography and characterized by
elemental analysis, ES-MS, and 1H NMR. In the ES-MS spectra for the two complexes
3a and 3b, only the signals of [M ꢁ 2ClOꢁ4 ꢁH]þ and [M ꢁ 2ClOꢁ4 ]2þ were observed.
The measured molecular weights were consistent with expected values.
The two Ru(II) complexes [Ru(bpy)2pdpiq]2þ and Ru(phen)2pdpiq]2þ gave
1
well-defined H NMR spectra (figure S1). The proton chemical shifts were assigned
via comparison with those of similar Ru(II) complexes with asymmetric ligand
[23–25, 43–47]. The chemical shifts of all the protons in aromatic region are presented in
section 2.
3.2. Electronic absorption titration
Small molecules bound to DNA by intercalation are associated with hypochromism
and red shift (bathochromism), because of the strong ꢁ–ꢁ stacking interaction between

4350
X.-W. Liu et al.
(a)
1.5
1.0
0.5
1.0
0.8
0.6
0.4
0.2
0.0
0
5
10 15 20 25 30 35 40
DNA ×10 molL
5
–1
0.0
2.0
300
400
500
600
Wavelength (nm)
(b)
1.0
0.8
0.6
0.4
0.2
0.0
1.5
1.0
0.5
0.0
0
2
4
6
8 10 12 14 16
5
–1
DNA ×10 molL
300
400
500
600
Wavelength (nm)
Figure 1. Absorption spectra of 3a (a) and 3b (b) in buffer
A upon the addition of CT-
DNA, [Ru] ¼ 20 mmol Lꢁ1, [DNA] ¼ 0ꢁ360 mmol Lꢁ1. Arrow shows the absorbance changing upon the
increase in DNA concentration. Inset: plots of ("aꢁ"f)/("bꢁ"f) vs. [DNA] for the titration of DNA to Ru(II)
complexes.
aromatic chromophore and the base pairs of DNA. The extent of the hypochromism in
the visible 1MLCT band depends on the strength of intercalative interaction [15].
Figure 1 shows absorption spectra of 3a and 3b in the absence and presence of
CT-DNA (at a constant concentration of complexes, [Ru] ¼ 20 mmol Lꢁ1).
Absorption spectra of the two complexes in buffer A exhibited three well-resolved
absorptions from 200 to 650 nm. The lowest energy bands at 478 nm for 3a and 472 nm
for 3b are attributed to the metal-to-ligand charge transfer (MLCT) transition, the
bands at 400 nm for 3a and 402 nm for 3b are assigned to ꢁ–ꢁ* transitions, and the

Ruthenium(II) with an asymmetric ligand
4351
bands at 289 nm for 3a and 264 nm for 3b are assigned to bpy-centered ꢁ–ꢁ* transitions
and phen-centered ꢁ–ꢁ* transitions in comparison with [Ru(bpy)3]2þ and
[Ru(phen)3]2þ, respectively. Increasing the amount of DNA for 3a decreases the
1MLCT transitions as much as 17.6% at 478 nm at a ratio of [DNA]/[Ru] ¼ 18.2. For
3b, upon addition of DNA, the MLCT band at 472 nm exhibits hypochromism of about
21.4% at a ratio of [DNA]/[Ru] ¼ 7.4.
In order to compare quantitatively the DNA-binding affinities of the complexes, the
intrinsic binding constants K of these two complexes to DNA were determined by
1
monitoring the changes of the MLCT absorbance at 478 nm for 3a and at 472 nm for
3b according to equation (1) [39]. The intrinsic binding constants K of 3a and 3b were
)
2.8 ꢃ 0.1 ꢅ 105 (mol L
)
ꢁ1 –1 (s ¼ 3.79, figure 1a) and 7.6 ꢃ 0.5 ꢅ 105 (mol L
ꢁ1 ꢁ1 (s ¼ 1.93,
figure 1b), respectively. Complex 3b exhibits a stronger DNA-binding affinity than 3a
due to the different plane area and hydrophobicity of the ancillary ligands. These values
are compared to that of Ru(II) complexes with the asymmetric ligand,
[Ru(dmb)2(pdta)]2þ (2.37 ꢅ 105 (mol Lꢁ1 ꢁ1
)
)
[47], [Ru(bpy)2PZNI]2þ (3.42 ꢅ
104 (mol L
)
ꢁ1 ꢁ1) [23], and [Ru(phen)2PZNI]2þ (5.86 ꢅ 104 (mol L
)
ꢁ1 ꢁ1) [23], but smaller
than that of the classical intercalator, [Ru(phen)2dppz]2þ (5.1 ꢅ 106 (mol L
)
ꢁ1 ꢁ1) [48].
From the results, we deduce that the two complexes bind to DNA with high affinities.
Further studies are needed to elucidate the DNA-binding mode of the complexes.
3.3. Competitive binding experiments
The two Ru(II) complexes do not emit distinct fluorescence in buffer A even in the
presence of DNA. Therefore, the DNA-binding properties of these complexes and
DNA cannot be directly studied in emission spectra. The competitive binding
experiments were carried out using a molecular fluorophore EB as a probe. EB emits
strong fluorescence in the presence of DNA due to its strong intercalation between
adjacent DNA base pairs. If a complex replaces EB from DNA-bound EB, the
fluorescence of the solutions would be efficiently quenched as free EB shows no
apparent emission intensity in buffer A because of solvent quenching. Figure 2 shows
fluorescence quenching spectra of DNA-bound EB by Ru(II) complexes. Upon
addition of Ru(II) complexes, sharp decreases in EB emission intensities were observed,
indicating that the two Ru(II) complexes could displace EB from DNA. The
competitive binding experiment results suggest that the two Ru(II) complexes interact
with DNA through intercalation. In the plot of percentage of quenching fluorescence,
(I0 ꢁ I)/I0 versus [Ru]/[EB], we see that 50% EB molecules were displaced from adjacent
DNA base pairs at a concentration ratio of [Ru]/[EB] ¼ 5.34 for 3a and 2.52 for 3b, as
shown in figure 2. By taking the DNA-binding constant of 1.4 ꢅ 106 (mol L
)
ꢁ1 ꢁ1 for EB
[49, 50], the apparent DNA-binding constants Kapp values of the two complexes were
) for 3a and
for 3b, respectively, in agreement with the Kb values derived
calculated according to equation (2) [39] as 2.62 ꢅ 105 (mol Lꢁ1 ꢁ1
5.56 ꢅ 105 (mol Lꢁ1 ꢁ1
)
from the absorption spectral studies.
3.4. Viscosity properties
The DNA viscosity measurement is useful to determine whether a complex intercalates
into DNA, which is sensitive to length change of DNA (i.e., viscosity and

4352
X.-W. Liu et al.
(a)
1.0
0.8
0.6
0.4
0.2
0.0
300
250
200
150
100
50
0
5
10 15 20
[Ru]/[EB]
0
500
550
600
Wavelength (nm)
650
(b)
300
250
200
150
100
50
1.0
0.8
0.6
0.4
0.2
0.0
0
4
8
12 16
[Ru]/[EB]
0
500
550
600
650
Wavelength (nm)
Figure 2. Fluorescence quenching spectra of EB bound to DNA by 3a (a) and 3b (b),
[DNA] ¼ 100 mmol Lꢁ1, [EB] ¼ 5 mmol Lꢁ1. Arrow shows the intensity change upon increasing Ru(II)
complex concentration. Inset: plots of relative integrated fluorescence intensity vs. [Ru]/[EB].
sedimentation) and regarded as the least ambiguous and most critical tests for a
classical intercalation model in solution in the absence of crystallographic structural
data [42, 43]. When a complex intercalates into DNA, the DNA helix lengthens as base
pairs are separated to accommodate the bound ligand, which results in increase of DNA
viscosity. In contrast, a partial, non-classical intercalation of ligand could bend
(or kink) the DNA helix, reducing its length and, concomitantly, its viscosity [51, 52];
electrostatic binding has little effect on DNA viscosity.

Ruthenium(II) with an asymmetric ligand
4353
1.25
1.20
1.15
1.10
1.05
1.00
0.00 0.02 0.04 0.06 0.08 0.10
[Ru]/[DNA]
Figure 3. Effects of the increase in amounts of EB (h), 3a (4), 3b (m), and [Ru(bpy)3]2þ (g) on the relative
viscosity of CT-DNA at 30 (ꢃ0.1)ꢄC, respectively. The total concentration of DNA is 0.25 mmol Lꢁ1
.
Figure 3 shows the changes in DNA viscosity upon addition of EB, 3a, 3b, and
[Ru(bpy)3]2þ. EB, as a typical intercalator, increases the relative DNA viscosity;
[Ru(bpy)3]2þ, which bind to DNA in an electrostatic binding mode, has little effect on
DNA viscosity. On increasing amounts of 3a and 3b, the relative viscosities of
CT-DNA increases steadily, but smaller than those of DNA bound with EB. The
increased viscosity, which may depend on the DNA-binding mode and affinity,
follows the order EB 4 3b 4 3a 4 [Ru(bpy)3]2þ. In a study by Chao et al. [24] on
DNA-binding of Ru(II) complexes containing asymmetric tridentate ligand similar to
our ligand, the two phenyl rings in the asymmetric dppt are rotated away from the
1,2,4-triazine ring with large dihedral angles, causing severe steric constraints when
the complex interacts with DNA, resulting in a partial, non-classical intercalation.
However, some similar examples of Ru(II) complexes with the bidentate ligand
containing two rotated phenyl rings, [Ru(bpy)2(dptatp)]2þ [53] and [Ru(bpy)2(ptdb)]2þ
[25] (dptatp ¼ 2,3-diphenyl-1,4,8,9-tetraazatriphenylene), have been reported by Zheng
et al., suggesting that [Ru(bpy)2(dptatp)]2þ and [Ru(bpy)2(ptdb)]2þ interact with DNA
through intercalation despite steric constraints caused by the two phenyl rings. The
different results may be due to the difference between the structure of the complexes
and ligands. Considering these reported results and our experimental results
(absorption titration, competitive binding and viscosity experiments), we deduce
that 3a and 3b intercalate between the base pairs of DNA, consistent with the
spectroscopic results.
In addition, due to the more hydrophobic ability of phen, 3b can intercalate deeper
into DNA base pairs and thus show stronger DNA-binding affinity than 3a.

4354
X.-W. Liu et al.
Figure 4. Photoactivated cleavage of pBR322 DNA in the presence of Ru(II) complexes after 2 h irradiation
at 365 nm. Lane 0, DNA alone; Lanes 1ꢁ4: 3a (a) and 3b (b) at 10, 20, 40, and 80 mmol Lꢁ1
.
Figure 5. Agarose gel showing cleavage of pBR322 DNA incubated with 3a (a), 3b (b) and different
inhibitors after 2 h irradiation at 365 nm, [Ru] ¼ 80 mmol Lꢁ1. Lane 0: DNA alone, lane 1: DNA þ Ru, lanes
2–6: DNA þ Ru þ 1 mol Lꢁ1 DMSO, 100 mmol Lꢁ1 mannitol, 1000 U mLꢁ1 SOD, 25 mmol Lꢁ1 NaN3,
1.2 mmol Lꢁ1 histidine.
3.5. Photocleavage of pBR 322 DNA by Ru(II) complexes
There has been interest in transition metal complexes which cleave nucleic acids. Ru(II)
complexes with polypyridyl ligands have well-behaved redox-active and rich photo-
chemical properties, making them good candidates for DNA photocleavers. Upon
irradiation, most generate singlet oxygen, thus inducing single-strand or double-strand
cleavage of DNA [54].
The photocleavage reactions of the present complexes on supercoiled pBR322 DNA
were studied by gel electrophoresis in TBE buffer (pH ¼ 8.3). When circular plasmid
DNA is subjected to gel electrophoresis, relatively fast migration will be observed for
the intact supercoil form (Form I). If scission occurs on one strand (nicked circulars),
the supercoil will relax to generate a slower moving nicked circular form (Form II).
If both strands are cleaved, a linear form (Form III) that migrates between Forms I and
II will be generated [55].
Figure 4 shows the photoactivated cleavage of pBR322DNA in the presence of
different concentrations of 3a and 3b upon irradiation at 365 nm. No obvious DNA
cleavage was observed for controls in the absence of complex (lane 0). On increasing the
concentration of complexes, the amount of Form I of pBR322 DNA is decreased and
that of Form II (nicked circular DNA) is increased. Both complexes can induce
single-strand scissions in supercoiled DNA. However, under the same experimental
conditions, when the concentration reached 80 mmol Lꢁ1, 3b promotes the conversion of
DNA from Form I to Form II. These results show that 3a and 3b can cleave DNA upon
irradiation and 3a exhibits a higher efficiency in DNA-photocleavage than 3b.
In order to determine the reactive species responsible for the DNA photocleavage of
the two Ru(II) complexes, mechanism experiments were performed in the presence of
.
hydroxyl radical (OH ) scavengers [41, 43, 56] (DMSO and mannitol), singlet oxygen
(1O2) scavengers [57] (NaN3 and histidine), and a superoxide anion radical (O.2ꢁ
)
scavenger (SOD). As shown in figure 5, NaN3 and histidine inhibited the DNA cleavage
activity of the two complexes, suggesting that singlet oxygen (1O2) is the cleaving agent.
In the presence of other scavengers DMSO, mannitol or SOD, little inhibition

Ruthenium(II) with an asymmetric ligand
4355
was observed. These results indicated that superoxide anion radical (O.2ꢁ) and hydroxyl
.
radical (OH ) were not involved in DNA cleavage by 3a and 3b under irradiation and
the mechanism of DNA cleavage is an oxidative process by generating singlet oxygen.
Similar cases are found in other Ru(II) complexes [58, 59].
4. Conclusion
An asymmetric ligand 3 and its Ru(II) complexes [Ru(bpy)2pdpiq)]2þ (3a) and
[Ru(phen)2pdpiq]2þ (3b) have been synthesized and characterized. The DNA-binding
and photocleavage properties of the two complexes have been investigated by
absorption spectroscopy, competitive binding, viscosity, and agarose gel electrophore-
sis. Both complexes bind to DNA in an intercalative mode. Also, the two complexes are
efficient DNA-photocleavers upon irradiation at 365 nm and 3a exhibits stronger
DNA-photocleavage efficiency than 3b. The mechanism experiments indicate that
singlet oxygen may play an important role in the DNA photocleavage of the two Ru(II)
complexes.
Acknowledgments
We are grateful to the Natural Science Foundation of Hunan Province (07JJ3015), the
Doctoral Scientific Research Foundation of Hunan University of Arts and Science, and
the Open Project Program of Key Laboratory of Environmentally Friendly Chemistry,
and Applications of Ministry of Education (10HJYH05) for the support.
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