DNA-binding and photocleavage studies of ruthenium(II) complexes containing asymmetric intercalative ligand

Article abstract of DOI:10.1016/j.saa.2011.11.014

A novel asymmetric ligand 2-(pyridine-2-yl)-1-H-imidazo[4,5-i]dibenzo[2,3- a:2′,3′-c]phenazine (pidbp) and its ruthenium complexes [Ru(L) ₂(pidbp)]²⁺ (L = bpy (2, 2′- bipyridine), phen (1, 10 - phenanthroline)), have been synthesized

Full text of DOI:10.1016/j.saa.2011.11.014

Spectrochimica Acta Part A 86 (2012) 554–561
Contents lists available at SciVerse ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
DNA-binding and photocleavage studies of ruthenium(II) complexes containing
asymmetric intercalative ligand
Xue-Wen Liua,b,∗, Yuan-Dao Chena,b, Lin Lia, Ji-Lin Lua,∗, Da-Shun Zhanga
a College of Chemistry and Chemical Engineering, Hunan University of Arts and Science, ChangDe 415000, PR China
b Key Lab of Environment-Friendly Chemistry and Application in Ministry of Education, Xiangtan University, Xiangtan 411105, PR China
a r t i c l e i n f o
a b s t r a c t
A novel asymmetric ligand 2-(pyridine-2-yl)-1-H-imidazo[4,5-i]dibenzo[2,3-a:2ꢀ,3ꢀ-c]phenazine (pidbp)
and its ruthenium complexes [Ru(L)2(pidbp)]2+ (L = bpy (2, 2ꢀ- bipyridine), phen (1, 10 – phenan-
throline)), have been synthesized and characterized by elemental analysis, ES-MS, 1H NMR. Various
methods support the conclusion that both Ru(II) complexes can intercalate into DNA base pairs. Complex
[Ru(bpy)2(pidbp)]2+ 4 exhibits its DNA “molecular light switch” properties. Furthermore, the two com-
plexes are efficient DNA-photocleavers under irradiation at 365 nm, and complex 5 exhibits a stronger
DNA-photocleavage efficiency than complex 4. The mechanism of DNA cleavage is an oxidative process
by generating singlet oxygen.
Article history:
Received 13 September 2011
Received in revised form 1 November 2011
Accepted 9 November 2011
Keywords:
Ru(II) complex
Asymmetric ligand
Light switch
DNA-binding
Photocleavage
© 2011 Elsevier B.V. All rights reserved.
[Ru(bpy)2(qdppz)]2+/[Ru(bpy)2(hqdppz)]2+ (qddpz = naphtho[2,3-
1. Introduction
a]dipyrido[3,2-h:2ꢀ,3ꢀ-f]phenazine-5,18-dione;
hqdppz = 5,18-dihydroxynaphtho[2,3-a]dipyrido[3,2-h:2ꢀ,3ꢀ-
f]phenazine) [19]; (iv) pH-induced molecular light switches, such
as [Ru(bpy)2(btppz)]2+ (btppz = btppz = benzo[h]tripyrido[3,2-
a:2ꢀ,3ꢀ-c:2ꢀꢀ,3ꢀꢀ-j]phenazine, which exhibits “on-off” emission
switch in water solutions with various pH values[20]. Among these
systems, DNA “light switches” have attracted particular attention,
owing to their possible applications such as detection of DNA base
mismatches [21], molecular-scale logic gates, DNA sensing, the
signaling of DNA protein binding [22–25] and luminescent probes
of DNA structure [16]. The requirements for these applications
are the DNA-binding and steady-state photophysical properties
of Ru(II) polypyridyl complexes. Recently, there has been a great
interest on the binding of ruthenium(II) polypyridyl complexes
with DNA, because it may provide important information for new
cancer therapeutic agents and potential probes of DNA structure
and conformation. In general, Ru(II) complexes can interact with
DNA through three non-covalent modes such as electrostatic bind-
ing, groove binding, or intercalation. Among these interactions,
intercalative binding mode is one of the most important DNA
binding modes, which is related to DNA “light switches” and the
antitumor activity of the complexes.
There has been tremendous interest recently in the design
of novel Ru(II) polypyridyl complexes due to their unique
photophysical properties and potential applications in stere-
oselective probes of nucleic acid structures, molecular “light
switches”, DNA-photocleavage reagents, catalysts for chem-
ical reactions, photodynamic therapy (PDT) agents and solar
energy utilizations[1–16]. Of these, research of molecular “light
switches” has received a lot of attentions, as one of the hot topics
of superamolecular chemistry. Up to now, Ru(II) polypyridyl
complex exhibits its “light switch” effect mainly through the
following four channels: (i) “off-on” light switches effect based
on DNA binding; such as [Ru(bpy)2(dppz)]2+ (L = bpy (2,2ꢀ-
bipyridine);
dppz = dipyrido[3,2-a:2ꢀ,3ꢀ-c]phenazine),
which
does not emit luminescence in aqueous solution but displays
strong photoluminescence in DNA solution after binding to
DNA in intercalation mode [9]; (ii) chemical cycling the DNA
light switch on and off based on DNA binding and modu-
lation by metal ions and EDTA, such as [Ru(bpy)2(tpphz)]2+
(tpphz = tetrapyrido[3,2-a:2ꢀ,3ꢀ-c:3ꢀꢀ,2ꢀꢀ-h:2ꢀꢀꢀ,3ꢀꢀꢀ-j]phenazine,
which exhibits this phenomenon through the successive addition
of Co2+ and EDTA after binding to DNA[17,18]; (iii) “electro-photo
switch” based on the redoxation of ligand, such as the redox couple
In addition, more recently, many ruthenium(II) complexes with
symmetric intercalative ligand have been synthesized and applied
to DNA-binding studies. However, the DNA-binding investigations
of such complexes containing asymmetric ligands have attracted
much less attention. In fact, these Ru(II) complexes with asym-
metric ligands also exhibit interesting properties upon binding to
∗
Corresponding authors. Tel.: +86 736 7186115; fax: +86 736 7186133.
E-mail addresses: liuxuewen050@sina.com (X.-W. Liu), lu jilin10@163.com
(J.-L. Lu).
1386-1425/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2011.11.014

X.-W. Liu et al. / Spectrochimica Acta Part A 86 (2012) 554–561
555
DNA[26–29]. A great number of reports have shown that varying
the shape of the intercalative ligand can create some interesting
difference in the space configuration and the electron density dis-
tribution of Ru(II) polypyridyl complexes, this will result in the
changes in the DNA-binding properties. Therefore, an extensive
study of some new complexes with asymmetric ligands is neces-
sary for further understanding the DNA-binding mechanism and
guiding the design of new Ru(II) complexes with an excellent bioac-
tivity.
The motivation of this research is stimulated by DNA
“light switch” effect of [Ru(bpy)2(dppz)]2+reported by Hartshorn
and Barton [10]. Ru(bpy)2(dppz)]2+ exhibits its DNA “light
switch” effect, because the intercalative ligand dppz possesses
phenazine ring, and DNA protected the phenazine nitrogen
atoms from interaction with water upon intercalation com-
pared to free in aqueous solutions [10]. In this work, we
report the synthesis and characterization of a novel asymmetric
ligand (pidbp = 2-(pyridine-2-yl)-1-H-imidazo[4,5-i]dibenzo[2,3-
a:2ꢀ,3ꢀ-c]phenazine) containing phenazine ring and its ruthenium
complexes [Ru(L)2(pidbp)]2+ (L = bpy (2,2ꢀ-bipyridine), phen (1, 10
– phenanthroline)). [Ru(bpy)2(pidbp)]2+ is demonstrated here to
act as a DNA “molecular light switch” with the luminescence
enhancement factor of 10.23 on binding to DNA. To the best of our
knowledge, it is the first example of Ru(II) complexes with asym-
metric ligand, as DNA “molecular light switch”. Furthermore, the
DNA-binding and DNA-photocleavage properties of the two novel
Ru(II) complexes were carefully studied.
Ruthenium–DNA solutions were allowed to incubate for 5 min
before the absorption spectra were recorded.
DNA viscosities were measured using an Ubbelohde viscometer
maintained at a constant temperature of 30.0 0.1 ◦C in a ther-
mostatic bath. The DNA samples for viscosity measurement were
prepared by sonication in order to minimize complexities arising
from DNA flexibility [37]. Every sample was measured at least three
times and an average flow time was calculated. The DNA viscosity
was calculated according to Ái = (ti − t0)/t0, whereÁi is the corre-
sponding values 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 were presented as (Á/Á0)1/3 vs. binding ratio
[38], where Á is the viscosity of DNA in the presence of complex
and Á0 is the viscosity of DNA alone.
The competitive binding experiments was conducted by adding
increasing amounts of Ru(II) complex directly into the samples con-
taining 5 ␮M ethidium bromide (EB) and 100 ␮M DNA in buffer
A. Emission spectra were recorded in the region 500–700 nm, and
samples were excited at 515 nm.
For the gel electrophoresis experiments, supercoiled pBR322
DNA (0.1 ␮g) was treated with Ru(II) complexes in buffer B (50 mM
Tris, 18 mM NaCl, pH = 7.8), and the solutions were incubated for
1 h in the dark, then irradiated at room temperature with an UV
lamp (365 nm, 10 W). The samples were analyzed by electrophore-
sis for 2 h at 75 V in TBE buffer C (89 mM Tris, 89 mM boric acid,
2 mM EDTA) containing 1% agarose gel. The gel was stained with
0.5 ␮g/ml ethidium bromide and then photographed under UV
light.
2. Experimental
2.3. Synthesis
All materials were commercially available and used without
further purification. Calf thymus DNA (CT-DNA) was obtained
from Sigma (St. Louis, MO, USA). Supercoiled pBR 322 DNA
was purchased from MBI Fermentas. Doubly distilled water was
used to prepare buffer. All DNA-binding experiments were car-
ried out in buffer A (50 mM NaCl, 5 mM Tris–HCl, pH = 7.2).
For DNA photocleavage experiments, samples were treated
in buffer B (50 mM Tris, 18 mM NaCl, pH = 7.8). Solutions of
2.3.1. 5,6-(p-Toluenesulfonamide)
-2-(pyridine-2-yl)-1H-benzimidazole(1)
A solution of methyl pyridine-2-carboximidate (ca. 0.4 mmol),
1,2-diamino-4,5-(p-toluenesulfamidobenzene) 0.178 g (0.4 mmol)
in 10 ml glacial acetic acid was refluxed under argon for 4 h. The
cooled solution was diluted with water and neutralized with con-
centrated aqueous ammonia. The khaki precipitate was collected
and dried under vacuum. 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+1] +
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 suf-
ficiently free of protein [30]. The concentration of DNA was
determined spectrophotometrically (ε260 = 6600 M−1 cm−1) [31].
The complexes cis-[RuCl2(bpy)2]·2H2O, cis-[RuCl2(phen)2]·2H2O
[32], methyl pyridine-2-carboximidate [33,34], 1,2-diamino-4,5-
(p-toluenesulfamidobenzene) [35,36], were prepared by the
literature methods.
2.3.2. 2-(Pyridine-2-yl)-5,6-diamino-1H-benzimidazole (2)
5,6-(p-Toluenesulfonamide)
-2-(2ꢀ-pyridineyl)-1H-
benzimidazole(1) 0.533 g (1.0 mmol) and 4 ml concentrated
sulfuric acid were heated at 100 ◦C for 4 h. 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. And the solution was extracted with dichloromethane
(3 ml × 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: m/z =226 [M+1]+.
2.1. Physical measurement
C, H, and N analyses were carried out with a Perkin–Elmer 240Q
elemental analyzer. Electrospray mass spectra were recorded on
a LQC system (Finngan MAT, USA) using CH3CN as mobile phase.
Fast atomic bombardment mass spectra (FAB-MS) were obtained
on a VG ZAB-HS spectrometer. 1H NMR spectra were recorded
on a Bruker ARX-500 spectrometer with (CD3)2SO for the com-
plexes at 500 MHz at room temperature. Absorption spectra were
recorded with a Shimadzu UV-2450 spectrophotometer and emis-
sion spectra on a Hitachi F-2500 spectrofluorophotometer at room
temperature.
2.3.3. 2-(Pyridine-2-yl)-1-H-imidazo[4,5-i]dibenzo[2,3-a:2ꢀ,3ꢀ-
c]phenazine(pidbp)
(3)
A mixture of 2-(pyridine-2-yl)-5,6-diamino-1H-benzimidazole
(2) 0.067 g (0.3 mmol) and phenanthrenequinone 0.062 g
(0.3 mmol) was refluxed for 2 hours in methanol (30 ml). The
cooled solution was poured into water. The resulting yellow
precipitate was filtered and washed with water, and then dried in
vacuo. Yield: 0.098 g, 82.1%. Anal (%): (Found: C, 78.42; H, 3.89; N,
2.2. DNA-binding and photocleavage experiments
The absorption titrations of Ru(II) complexes in buffer A were
performed by using a fixed ruthenium concentration (20 ␮M),
to which increments of the DNA stock solution were added.

556
X.-W. Liu et al. / Spectrochimica Acta Part A 86 (2012) 554–561
17.46%. Calcd for C26H15N5: C, 78.56; H, 3.81; N, 17.63%). ES-MS
2.0
1.5
1.0
0.5
0.0
1.0
0.8
0.6
0.4
0.2
0.0
(CH3CH2OH): m/z = 398.0 ([M+H]+).
(a)
2.3.4. [Ru(bpy)2(pidbp)](ClO4)2·H2O (4)
A mixture of pidbp 0.120 g (ca. 0.3 mmol), [Ru(bpy)2Cl2]·2H2O
(0.156 g, 0.3 mmol), and ethylene glycol (10 ml) was deoxygenated
with argon. The mixture was heated at 140 ◦C under argon for
6 h. When the solution was cooled to room temperature, 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 washed with small amounts
of water and diethyl ether, then dried under vacuum. The crude
product was purified by column chromatography on a neutral
alumina with acetonitrile–toluene (3:1, v/v) as an eluent. Yield:
0.209 g, 68.9%. Anal (%): (Found: C, 54.55; H, 3.16; N, 12.42%.
Calcd for C46H31N9O8RuCl2: C, 54.72; H, 3.09; N, 12.48%−). ES-MS
0
5
10
15
20
5
×
DNA 10 M
300
400
500
600
Wavelength / nm
(CH3CN): m/z = 810.1 ([M−2ClO4−−H]+), 405.5 ([M−2ClO4
] H
2+). 1
2.0
1.5
1.0
0.5
0.0
NMR (500 MHz, ppm, DMSO-d6): 9.28 (d, 1H, J = 7.5 Hz), 9.04 (d, 1H,
J = 8.0 Hz), 8.95 (d, 1H, J = 8.0 Hz), 8.85 (dd, 2H, J = 4.5 Hz, J = 7.5 Hz),
8.65 (d, 1H, J = 7.5 Hz), 8.51 (s, 1H), 8.46 (t, 1H, J = 8.0 Hz), 8.19 (t, 1H,
J = 8.0 Hz), 8.15 (t, 1H, J = 8.0 Hz), 8.10 (t, 1H, J = 7.5 Hz), 8.02 (t, 1H,
J = 8.0 Hz), 7.97 (t, 1H, J = 5.5 Hz), 7.92 (d, 1H, J = 5.0 Hz), 7.79 (m, 6H),
7.70 (d, 1H, J = 5.0 Hz), 7.58 (t, 1H, J = 6.5 Hz), 7.53 (m, 2H, J = 5.5 Hz),
7.48 (t, 1H, J = 6.5 Hz), 6.38 (s, 1H).
1.0
0.8
0.6
0.4
0.2
0.0
(b)
0
2
4
6
8
10 12 14
5
×
DNA 10 M
2.3.5. [Ru(phen)2(pidbp)](ClO4)2 (5)
This complex was synthesized by a similar procedure as that
described for complex 4, with [RuCl2(phen)2]·2H2O (0.170 mg,
0.3 mmol) in place of [Ru(bpy)2]Cl2·2H2O. Yield: 0.173 g, 54.6%.
Anal (%): (Found: C, 56.53; H, 3.08; N, 11.76%, Calc for
300
400
Wavelength / nm
500
600
C
50H31N9O8RuCl2: C, 56.77; H, 2.95; N, 11.92%). ES-MS (CH3CN):
m/z = 858.4 ([M−2ClO4−−H] +), 429.5 ([M−2ClO4
]
2+). 1H NMR
−
(500 MHz, ppm, DMSO-d6): 9.22 (d, 1H, J = 8.0 Hz), 9.00 (d, 1H,
J = 8.0 Hz), 8.95 (d, 1H, J = 7.5 Hz), 8.76 (m, 2H), 8.67 (d, 4H, J = 8.0 Hz),
8.46 (s, 2H), 8.41 (t, 3H, J = 9.0 Hz), 8.38 (s, 1H), 8.29 (d, 1H, J = 8.5 Hz),
8.21 (m, 2H), 8.13 (t, 1H, J = 7.5 Hz), 8.02 (t, 1H, J = 7.0 Hz), 7.94 (t, 1H,
J = 7.0 Hz), 7.87 (t, 1H, J = 6.5 Hz), 7.79 (m, 5H), 7.67 (d, 1H, J = 5.5 Hz),
7.38 (t, 1H, J = 6.5 Hz), 6.13 (s, 1H).
Fig. 1. Absorption spectra of complexes 4 (a) and 5 (b) in buffer A upon the addition
of CT-DNA, [Ru] = 20 ␮M, [DNA] = 0 − 200 ␮M. Arrow shows the absorbance chang-
ing upon the increase in DNA concentration. Inset: plots of (εa − εf)/(εb − εf) vs.
[DNA] for the titration of DNA to Ru(II) complexes.
the doubly charged species appeared as major peak. The measured
molecular weights were consistent with expected values.
Each of the above ClO4 salts was dissolved in the minimum
amount of acetone, and a saturated TBACl (Tetrabutylammonium
chloride) in acetone was added dropwise until precipitation was
complete. The water-soluble chloride salts were filtered off and
washed thoroughly with acetone, and then dried under vacuum
(yield ∼92% in each case).
The two Ru(II) complexes [Ru(bpy)2(pidbp)]2+
and
Ru(phen)2(pidbp)]2+ gave well-defined 1H NMR spectra (Fig. S1).
The proton chemical shifts were assigned via comparison with
those of similar Ru(II) complexes with asymmetric ligand [26–29].
The chemical shifts of all the protons in aromatic region were
presented in Section 2.
3. Results and discussion
3.2. Electronic absorption titration
3.1. Synthesis and characterization
The DNA binding properties of the complex is usually charac-
terized through electronic absorption titration. Complex bound to
DNA through intercalation usually results in different extents of
hypochromism and red shift (bathochromism), due to the interca-
lation mode involving a strong ␲–␲ stacking interaction between
aromatic chromophore and the base pairs of DNA. The extent of the
hypochromism in the visible 1MLCT band is commonly consistent
with the intercalative binding strength [10].
The synthetic pathway for the asymmetric ligand pidbp and its
two complexes is shown in Scheme 1. The precursor compound 1
was obtained on the basis of the method for imidazole ring prepara-
tion established by Schaefer and Peters [33,34,39,40]. Deprotection
of the tosyl groups was carried out in concentrated sulfuric acid to
give 2. Condensation of phenanthrenequinone with the precursor
diamine compound 2 in refluxing methanol gave the asymmetric
ligand 3 in good yields. The corresponding ruthenium(II) complexes
4 and 5 were prepared by direct reaction of ligand with the appro-
priate mol ratios of the precursor complexes in ethylene glycol. All
these complexes were purified for DNA-binding and photocleav-
age experiments, by chromatography on a neutral alumina column
using acetonitrile-toluene (3:1 and 2:1, respectively) as eluents,
and characterized by element analysis, ES-MS and 1H NMR. In the
ES-MS spectra for the two complexes 4 and 5, only the signals of
Fig.
1 shows that the absorption spectra of complexes
[Ru(bpy)2(pidbp)]2+ and Ru(phen)2(pidbp)]2+ at a constant com-
plex concentration (20 ␮M) in the absence and presence of
calf-thymus (CT) DNA. With increasing concentration of CT-
DNA, the UV–vis spectra of the two complexes showed a clear
hypochromism in absorption bands. For complex 4, the decreases in
the 1MLCT transitions reach as high as 28.7% at 436 nm at a ratio of
[DNA]/[Ru] = 10.1. For complex 5, upon addition of DNA, the MLCT
band at 438 nm exhibits hypochromism of about 32.6% at a ratio of
[M−2ClO4−−H]+ and [M−2ClO4
]
were observed. In both case,
−
2+

X.-W. Liu et al. / Spectrochimica Acta Part A 86 (2012) 554–561
557
NH2
NH2
NH Ts
NH Ts
O2N
O2N
NH Ts
fuming nitric acid
HAC
TsCl
NH Ts
NH Ts
NH
H
N
H2N
H2N
NH Ts
NH Ts
Na2S2O4
NaOH
N
OCH3
N
N
NH Ts
N
O
O
1
H
N
NH2
NH2
H
N
H2SO4
H2O
N
N
N
N
N
2+
2
3
H
N
N
N
N
N
Ru(bpy)2Cl2
Ru
N
N
N
N
2+
4
H
N
N
N
N
N
N
Ru
N
N
Ru(phen)2Cl2
N
5
Scheme 1. The synthetic routes of the ligand and its Ru(II) complexes [Ru(bpy)2(pidbp)]2+ 4 and Ru(phen)2((pidbp))]2+ 5.
(Kb = 5.1 × 106 M−1 [42]), but are much larger than that of
[DNA]/[Ru] = 6.7, respectively. No obvious red shift was observed
for both complexes. The DNA binding-induced hypochromism sug-
gest that both complexes bind to DNA with high affinity.
In order to evaluate quantitatively the DNA-binding affinities
of the complexes, the intrinsic binding constants K of these two
complexes to DNA were determined by monitoring the changes of
the 1MLCT absorbance at 436 nm for complex 4 and at 438 nm for
complex 5 using Eq. (1) [41].
Ru(II) complexes with asymmetric ligand, [Ru(bpy)2PYNI]2+
(PYNI = 2-(2ꢀ-pyridyl)naphthoimidazole) (3.81 × 104 M−1
)
[26],
[Ru(bpy)2PZNI]2+
(PZNI = 2-(pyrazin-2-yl)naphthoimidazole)
(3.42 × 104 M−1)[27], [Ru(phen)2PZNI]2+(5.86 × 104 M−1)[27], and
[Ru(dmb)2(pdta)]2+(2.37 × 105 M−1) [28]. A possible explanation
for these facts may be due to the different planarity of the inter-
calative ligand. From the results, we could deduce that the two
complexes bind to DNA with high affinities. In addition, complex
5 exhibits a stronger DNA-binding affinity than complex 4 due
to the different plane area and hydrophobicity of the ancillary
ligands. However, the DNA binding mode cannot be determined
exclusively using optical method, since surface aggregation leads
to similar results. Further investigation is needed to determine the
DNA-binding mode.
(b − (b2 − 2K2Ct[DNA]/s)1/2
2KCt
)
(εa − εf )
=
(1a)
(1b)
(εb − εf )
K[DNA]
b = 1 + KCt +
2s
where [DNA] is the concentration of DNA in M (nucleotide), the
apparent absorption coefficient εa, εb and εf correspond to the
extinction coefficient observed for the 1MLCT absorption band at a
given DNA concentration, the extinction coefficient of the complex
in the absence of DNA, and the extinction coefficient of the com-
plex fully bound to DNA. K is the equilibrium binding constant in
3.3. Viscosity properties
In order to further clarify the exact nature of both complexes
binding to DNA, DNA viscosity measurements were carried out
on CT-DNA by increasing the concentration of Ru(II) complexes.
The DNA viscosity measurement is an useful means of determin-
ing whether a complex intercalate into DNA, which is sensitive
to length change of DNA (i.e. viscosity and sedimentation) and
regarded as the least ambiguous and most critical tests for the
DNA-binding mode in the absence of crystallographic structural
data [43,44]. When a complex intercalate into DNA, the DNA helix
lengthens as base pairs are separated to accommodate the bound
M
−1, Ct is the total metal complex concentration, and s is the bind-
ing site size. The binding constants K were obtained by fitting the
absorption titration data using a non-linear least-square method.
The values of intrinsic binding constants
K
were
1.68 0.2 × 106 M−1
(s = 2.33)
and
3.09 0.1 × 106 M−1
(s = 1.71) for complex
4 and 5, respectively. The values are
smaller than that of those Ru(II) complex reported in the
literature, such as DNA intercalator [Ru(phen)2(dppz)]2+

558
X.-W. Liu et al. / Spectrochimica Acta Part A 86 (2012) 554–561
luminescence in Tris buffer at room temperature in the
1.25
1.20
1.15
1.10
1.05
1.00
absence of DNA, and upon the addition of DNA, the 10.23-fold
increase in emission intensity was observed. Obviously, complex
[Ru(bpy)2(pidbp)]2+ can act as DNA “molecular light switch”. Other
examples of DNA “molecular light switch” have been reported
previously, such as [Ru(bpy)2(dppz)]2+, [Ru(bpy)2(tpphz)]2+ and
[Ru(bpy)2(btppz)]2+ [9,17,18,20] et al. These complexes function
as “molecular light switches” in aqueous solution, exhibiting
negligible luminescence in the absence of DNA and strong lumi-
nescence upon addition of DNA. The luminescence enhancement
may be due to the protection of the phenazine nitrogens from
solvent. To the best of our knowledge, it is the first example
of Ru(II) complexes with asymmetric ligand, as DNA “molec-
ular light switch”. Although in most cases, the extent of the
luminescence enhancement of Ru(II) complexes upon binding to
DNA commonly (but not absolutely) parallels the intercalative
binding strength, here, [Ru(phen)2(pidbp)]2+ with higher DNA
affinities exhibits the lower luminescence enhancement compared
to [Ru(bpy)2(pidbp)]2+. A possible explanation of this fact is due
to the difference of the background luminescence between the two
complexes. This observation is similar to that of [Ru(phen)2dppz]2+
and [Ru(bpy)2dppz]2+. The luminescence enhancement was found
to be 3000 for [Ru(phen)2dppz]2+ with CT-DNA [46]. However, the
enhancement factor was > 104 for [Ru(bpy)2dppz]2+ upon binding
to DNA [10]. In addition, the titration results also indicate that two
complexes can bind to DNA with high affinities and be protected
by DNA efficiently, since the hydrophobic environment inside the
DNA helix reduces the accessibility of solvent water molecules to
the complex and the complex mobility is restricted at the binding
site, leading to the decrease of vibration modes of relaxation.
0.00
0.02
0.04
0.06
0.08
0.10
[Ru]/[DNA]
Fig. 2. Effects of the increase in amounts of EB (ꢀ), complex 4 (ꢁ), 5 (᭹) and
[Ru(bpy)3]2+ (ꢁ) on the relative viscosity of CT-DNA at 30 ( 0.1) ◦C, respectively.
The total concentration of DNA is 0.25 mM.
ligand, which result in the increase of DNA viscosity. In contrast, the
complex which interacts with DNA by a partial, non-classical inter-
calation could bend (or kink) the DNA helix, reducing its length and,
concomitantly, its viscosity [45]. In addition, electrostatic binding
mode has little effect on DNA viscosity.
Steady-state
emission
quenching
experiments
using
[Fe(CN)6]4− as quencher may provide further information about
the DNA-binding properties of the two complexes and DNA. As
shown in Fig. 4, in the absence of DNA, the emission of complex
5 were efficiently quenched by [Fe(CN)6]4−, complex 4 cannot be
studied by emission quenching experiment in absence of DNA due
to its negligible luminescence. However, in the presence of DNA,
the emission of the two complexes were difficult to be quenched
by [Fe(CN)6]4−. This may be explained by repulsion of the highly
negative [Fe(CN)6]4− from the DNA polyanion backbone which
hinders access of [Fe(CN)6]4− to the DNA-bound complexes [47].
The curvature reflects different extent of protection, a larger slope
for the Stern–Volmer curve parallels poorer protection and lower
binding. The results suggest that complex 5 binds to DNA more
strongly than complex 4, which is consistent with the results
observed by electronic absorption titration (Fig. 1).
The changes in DNA viscosity upon addition EB, complex 4, 5
and [Ru(bpy)3]2+ are shown in Fig. 2. As is well known, ethid-
ium bromide (EB) interacts with DNA through intercalative binding
mode, and can increase the relative DNA viscosity for lengthen-
ing of the DNA double helix; while complex [Ru(bpy)3]2+, which
bind to DNA in an electrostatic binding mode, has little effect on
DNA viscosity. On increasing the concentrations of Ru(II) com-
plexes 4 and 5, the relative viscosities of CT-DNA increase steadily,
similarly to the behavior of EB. The increased degree of viscosity,
which may depend on the DNA-binding mode and affinity, fol-
lows the order of EB > 5 > 4 > [Ru(bpy)3]2+. The results suggest that
these two complexes could interact with DNA through a classical
intercalative binding mode. Due to the more hydrophobic ability
of co-ligand phen, complex 5 can intercalate into DNA base pairs
deeper and thus show stronger DNA-binding affinity than complex
4. The experiment results are consistent with the above spectro-
scopic results.
The competitive binding experiments were carried out using a
molecular fluorophore ethidium bromide (EB) as a probe. The EB
competitive binding experiments is a well-established assay based
on the displacement of the intercalating drug EB from CT-DNA, and
may provide more information about the DNA-binding mode and
the DNA affinities of the complex. EB can emit strong fluorescence
in the presence of DNA due to its strong intercalation between the
adjacent DNA base pairs. If a complex could replace 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. Fluorescence quenching spectra of
DNA-bound EB by Ru(II) complexes are shown in Fig. 5. On the exci-
tation at 515 nm, both the two Ru(II) complexes in the absence and
presence of DNA and free EB emit negligible fluorescence, therefore
their emission has little influence on EB competitive binding exper-
iment. As shown in Fig. 5, additions of the complexes to EB-DNA
system resulted in sharp decreases in EB emission intensities. In
the plot of percentage of quenching fluorescence, (I0 − I)/I0 versus
[Ru]/[EB], we can see that 50% EB molecules were displaced from
adjacent DNA base pairs at a concentration ratio of [Ru]/[EB] = 4.26
3.4. Steady-state emission studies
Emission spectroscopy is one of the most common and sensitive
ways to investigate the interaction between complex and DNA. As
shown in Fig. 3, in the absence of DNA, complex 4 showes neg-
ligible luminescence in buffer A at ambient temperature; while
complex 5 can emit weak luminescence with maximum appear-
ing at 578 nm. Upon the addition of CT-DNA, an obvious increase
in emission intensity was observed for the two complexes, and a
red shift of 11 nm was also observed for complex 5. The emission
intensity increases steadily to ca. 10.23 times of the original for
complex 4, and 1.96 times for complex 5 (Fig. 3). From Fig. 3(a),
we can see that the complex [Ru(bpy)2(pidbp)]2+ emits negligible

X.-W. Liu et al. / Spectrochimica Acta Part A 86 (2012) 554–561
559
25
(b)
20
15
10
5
(a)
20
15
10
5
0
0
550
600
650
700
550
600
650
700
Wavelength / nm
Wavelength / nm
Fig. 3. Emission spectra of Ru(II) complexes (5 ␮M) 4 (a) and 5 (b) in Tris–HCl buffer at 298 K in the absence and the presence of CT-DNA. Arrow shows the intensity change
upon the increase in DNA concentration.
for complex 4, and 3.31 for complex 5. By taking the DNA binding
constant of 1.4 × 106 M−1for EB [48,49], the apparent DNA bind-
ing constants Kapp values of the two complexes were calculated
according to Eq. (2) [50].
ꢀ
ꢁ
200
150
100
50
[EB]50%
[Ru]50%
Kapp = KEB
(2)
(a)
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% fluo-
rescence, respectively. The values are 3.29 × 105 M−1 for complex
4, and 4.23 × 105 M−1 for complex 5, respectively, which is slightly
2.5
2.0
1.5
1.0
0
550
(b)
600
650
700
Wavelength / nm
200
150
100
50
0
550
600
650
700
Wavelength / nm
0.0
0.2
0.4
0.6
0.8
1.0
[Fe(CN)6]4- (mM)
Fig. 5. Fluorescence quenching spectra of EB bound to DNA by Ru(II) complexes
4 (a) and 5 (b), [DNA] = 100 ␮M, [EB] = 5 ␮M. Arrow shows the intensity change
upon increasing Ru(II) complexes concentration. Inset: plots of relative integrated
fluorescence intensity vs. [Ru]/[EB].
Fig. 4. Emission quenching curves of the complexes 4 + DNA (᭹), 5 (ꢀ), 5 + DNA (ꢁ)
with increasing concentration of [Fe(CN)6]4−. [Ru] = 5 ␮M, [DNA]/[Ru] = 40.

560
X.-W. Liu et al. / Spectrochimica Acta Part A 86 (2012) 554–561
(OH•) were not indeed in the DNA cleavage of the Ru(II) complexes
under irradiation, the mechanism of DNA cleavage is an oxidative
process by generating singlet oxygen. Similar cases are found in
other Ru(II) complexes [54,55].
Fig. 6. Photoactivated cleavage of pBR322 DNA in the presence of Ru(II) complexes
after 2 h irradiation at 365 nm. Lane 0, DNA alone; Lanes 1–4: complex 4 (a) and 5(b)
at 10, 20, 40 and 80 ␮M.
4. Conclusions
In this work, a novel asymmetric ligand 3 and its Ru(II) com-
plexes [Ru(bpy)2(pidbp)]2+ 4 and [Ru(phen)2(pidbp)]2+ 5 have
been synthesized and characterized as potential complexes for
DNA “light switch” and photocleavers. Various methods support
the conclusion that both Ru(II) complexes can bind to DNA in
an intercalative mode. Complex [Ru(bpy)2(pidbp)]2+ 4 exhibits it
DNA “molecular light switch” properties. Furthermore, the two
complexes are efficient DNA-photocleavers under irradiation at
365 nm, and complex 5 exhibits a stronger DNA-photocleavage effi-
ciency than complex 4. The mechanism experiments indicated that
the singlet oxygen may play an important role in the DNA photo-
cleavage of the two Ru(II) complexes.
Fig. 7. Agarose gel showing Cleavage of pBR322 DNA incubated with Ru(II) complex
4 (a), 5(b) and different inhibitors after 2 h irradiation at 365 nm, [Ru] = 80 ␮M. Lane
0:DNA alone, lane 1: DNA + Ru, lanes 2–6: DNA + Ru + 1 M DMSO, 100 mM mannitol,
1000 U ml−1 SOD, 25 mM NaN3, 1.2 mM histidine.
smaller than the Kb values derived from the absorption spectral
studies.
3.5. Photocleavage of pBR 322 DNA by Ru(II) complexes
Acknowledgements
Many Ru(II) complexes with polypyridyl ligands have been
shown to cleave DNA under irradiation. Most of them, commonly
known as “DNA photocleavers”, are activated by light, and generate
singlet oxygen, thus induce single-strand or double-strand cleav-
age of DNA [51]. Upon irradiation, the effective cleavage activity
is attributed to the well-behaved redox-active and photochemical
properties.
We are grateful to the supports of the Natural Science Foun-
dation of Hunan Province (07JJ3015), the Scientific Research
Foundation of Hunan Provincial Education Department and the
Open Project Program of Key Laboratory of Environmentally
Friendly Chemistry and Applications of Ministry of Education
(10HJYH05).
The abilities of the present complexes to cleavage DNA were
studied by gel electrophoresis using supercoiled pBR322 DNA in
TBE buffer (pH = 7.8). In general, 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 gener-
ate 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 [52].
Fig. 6 shows the results of the gel electrophoresis experiments
carried out with supercoiled pBR322 DNA cleavage induced by var-
ious concentrations of the Ru(II) complexes under irradiation at
365 nm. No obvious DNA cleavage was observed for controls in the
absence of the complex (Fig. 6: lane 0). The two complexes induced
efficient DNA cleavage under irradiation as evidenced by the con-
version of supercoiled to nicked circular form DNA. With increasing
concentration of the Ru(II) complex 4 (Fig. 6 (a)) and 5 (Fig. 6 (b)),
the amount of Form I of pBR322 DNA is decreased, whereas that of
Form II is increased. And the increase in the amounts of nicked DNA
was associated with the increase in the concentration of both com-
plexes. Notably, under the same experimental conditions, when the
concentration reached 80 ␮M, complex 5 can almost promote the
complete conversion of DNA from Forms I to II. The DNA cleav-
age results show that both complexes 4 and 5 can cleave DNA
upon irradiation and complex 5 exhibits a higher efficiency in DNA-
photocleavage than complex 4.
In order to determine the reactive species responsible for the
DNA photocleavage of the two Ru(II) complexes, the mechanism
experiments were performed in the presence of hydroxyl radical
(OH•) scavengers [30,31,38] (DMSO and mannitol), singlet oxygen
(1O2) scavengers [53] (NaN3 and histidine), and a superoxide anion
radical (O2•−) scavenger (SOD). As shown in Fig. 7, NaN3 and his-
tidine (lanes 5, 6) efficiently inhibited the DNA cleavage activity of
the two complexes, which suggest that singlet oxygen (1O2) is likely
to be the cleaving agent. In the presence of other scavengers DMSO,
mannitol or SOD, little inhibition was observed. These results indi-
cated that superoxide anion radical (O2•−) and hydroxyl radical
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.saa.2011.11.014.
References
[1] A. Pyle, J.K. Barton, in: S.J. Lippard (Ed.), Progress in Inorganic Chemistry, vol.
38, 1990, pp. 413–475, New York.
[2] A. Sigel, H. Sigel (Eds.), Metal Ions in Biological Systems, vol. 33, Marcel Dekker,
New York, 1996, pp. 177–252.
[3] L.N. Ji, X.H. Zou, J.G. Liu, Coord. Chem. Rev. 216–217 (2001) 513–536.
[4] B.M. Zeglis, V.C. Pierre, J.K. Barton, Chem. Commun. (2007) 4565–4579.
[5] J.C. Genereux, J.K. Barton, Chem. Rev. 110 (2010) 1642–1662.
[6] H.K. Liu, P.J. Sadler, Acc. Chem. Res. 44 (2011) 349–359.
[7] M.K. Nazeeruddin, C. Klein, P. Liska, M. Grätzel, M. Grätzel, Coord. Chem. Rev.
249 (2005) 1460–1467.
[8] S.S. Bhat, A.S. Kumbhar, P. Lönnecke, E. Hey-Hawkins, Inorg. Chem. 49 (2010)
4843–4853.
[9] C. Metcalfe, J.A. Thomas, Chem. Soc. Rev. 32 (2003) 215–224.
[10] R.M. Hartshorn, J.K. Barton, J. Am. Chem. Soc. 114 (1992) 5919–5925.
[11] S. Eyal, D.N. Hans, B.G. Harry, C.A. Fred, J. Am. Chem. Soc. 118 (1996) 1158–1163.
[12] K.A. O’Donoghue, J.M. Kelly, P.E. Kruger, Dalton Trans. (2004) 13–15.
[13] S. Shi, J. Liu, J. Li, K.C. Zheng, C.X.M. Huang, C.P. Tan, L.M. Chen, L.N. Ji, Dalton
Trans. (2005) 2038–2046.
[14] J.H. Zhou, J.H. Liu, Y.Y. Feng, S.H. Wei, X.T. Gu, X.S. Wang, B.W. Zhang, Bioorg.
Med. Chem. Lett. 15 (2005) 3067–3070.
[15] Y. Liu, A. Chouai, N.N. Degtyareva, S.A. Lutterman, K.R. Dunbar, C. Turro, J. Am.
Chem. Soc. 127 (2005) 10796–10797.
[16] A.T. Steven, K. Roni, S. Dietrich, Inorg. Chem. 38 (1999) 5196–5197.
[17] A. Ambroise, B.G. Maiya, Inorg. Chem. 39 (2000) 4256–4263.
[18] Y.M. Chen, Y.J. Liu, Q. Li, K.Z. Wang, J. Inorg. Biochem. 103 (2009) 1395–1404.
[19] B.M. Zeglis, J.K. Barton, J. Am. Chem. Soc. 128 (2006) 5654–5655.
[20] E. Ruba, J.R. Hart, J.K. Barton, Inorg. Chem. 43 (2004) 4570–4578.
[21] A.E. Friedman, J.C. Chambron, J.P. Sauvage, N.J. Turro, J.K. Barton, J. Am. Chem.
Soc. 112 (1990) 4960–4962.
[22] L.S. Ling, Z.H. He, G.W. Song, Y.E. Zeng, C. Wang, C.L. Bai, X.D. Chen, P. Shen,
Anal. Chim. Acta 436 (2001) 207–214.
[23] L.S. Ling, Z.K. He, F. Chen, Y.-E. Zeng, Talanta 59 (2003) 269–275.
[24] A.P. DeSilva, N.D. McClenaghan, Chem. Eur. J. 10 (2004) 574–586.
[25] Y. Jiang, X. Fang, C. Bai, Anal. Chem. 76 (2004) 5230–5235.
[26] Y.J. Liu, H. Chao, L.F. Tan, Y.X. Yuan, W. Wei, L.N. Ji, J. Inorg. Biochem. 99 (2005)
700–708.
[27] Y.J. Liu, H. Chao, J.H. Yao, L.F. Tan, Y.X. Yuan, L.N. Ji, Inorg. Chim. Acta 358 (2005)
1904–1910.
[28] X.L. Hong, H. Li, C.H. Peng, J. Mol. Struct. 990 (2011) 197–203.

X.-W. Liu et al. / Spectrochimica Acta Part A 86 (2012) 554–561
561
[29] X.H. Zou, B.H. Ye, H. Li, Q.L. Zhang, H. Chao, J.G. Liu, L.N. Ji, J. Biol. Inorg. Chem.
6 (2001) 143–150.
[43] S. Satyanarayana, J.C. Dabroniak, J.B. Chaires, Biochemistry 31 (1992)
9319–9324.
[30] J. Bolger, A. Gourdon, E. Ishow, J.P. Launay, Inorg. Chem. 35 (1996) 2937–
2944.
[44] S. Satyanaryana, J.C. Daborusak, J.B. Chaires, Biochemistry 32 (1993)
2573–2584.
[31] E. Ishow, A. Gourdon, J.P. Launay, Chem. Commun. (1998) 1909–1910.
[32] B.P. Sullivan, D.J. Salmon, T.J. Meyer, Inorg. Chem. 17 (1978) 3334–3341.
[33] F.C. Schaefer, G.A. Peters, J. Org. Chem. 26 (1961) 412–418.
[34] V.A. Milway, L. Zhao, T.S.M. Abedin, L.K. Thompson, Z.Q. Xu, Polyhedron 22
(2003) 1271–1279.
[45] J.K. Barton, J.M. Goldberg, C.V. Kumar, N.J. Turro, J. Am. Chem. Soc. 108 (1986)
2081–2088.
[46] C. Hiort, P. Lincoln, B. Norden, J. Am. Chem. Soc. 115 (1993) 3448–3454.
[47] C.V. Kumar, N.J. Turro, J.K. Barton, J. Am. Chem. Soc. 107 (1985) 5518–5523.
[48] M.J. Waring, J. Mol. Biol. 13 (1965) 269–282.
[35] G.W.H. Cheeseman, J. Chem. Soc. (1962) 1170–1176.
[36] F.E. Arnold, J. Polym. Sci. Part A 8 (1970) 2079–2089.
[37] J.B. Chaires, N. Dattagupta, D.M. Crothers, Biochemistry 21 (1982) 3933–3940.
[38] G. Cohen, H. Eisenberg, Biopolymers 8 (1969) 45–49.
[39] V.A. Milway, L. Zhao, T.S.M. Abedin, L.K. Thompson, Zh.Q. Xu, Polyhedron 22
(2003) 1271–1279.
[40] X.W. Liu, J.L. Lu, Y.D. Chen, L. Li, D.S. Zhang, Inorg. Chim. Acta 379 (2011) 1–6.
[41] J.D. McGhee, P.H. von Hippel, J. Mol. Biol. 86 (1974) 469–489.
[42] B.N. Rajesh, S.T. Emily, L.K. Shalawn, J.M. Catherine, Inorg. Chem. 37 (1998)
139–141.
[49] J.B. LePecq, C. Paoletti, J. Mol. Biol. 27 (1967) 87–106.
[50] D.L. Boger, B.E. Fink, S.R. Brunette, W.C. Tse, M.P. Hedrick, J. Am. Chem. Soc. 123
(2001) 5878–5891.
[51] H.Y. Mei, J.K. Barton, Proc. Natl. Acad. Sci. U.S.A. 85 (1988) 1339–1343.
[52] J.K. Barton, A.L. Raphael, J. Am. Chem. Soc. 106 (1984) 2466–2468.
[53] F.R. Nilsson, P.B. Merkel, D.R. Kearns, Photochem. Photobiol. 16 (1972) 117–124.
[54] H.J. Yu, S.M. Huang, L.Y. Li, H.N. Ji, H. Chao, Z.W. Mao, J.Z. Liu, L.N. Ji, J. Inorg.
Biochem. 103 (2009) 881–890.
[55] F. Gao, H. Chao, F. Zhou, Y.X. Yuan, B. Peng, L.N. Ji, J. Inorg. Biochem. 100 (2006)
1487–1494.