Interaction of a ruthenium(II)-chalcone complex with double stranded DNA: Spectroscopic, molecular docking and nuclease properties

Article abstract of DOI:10.1016/j.jphotochem.2011.04.005

The interaction of a well characterized new complex 1 cis,fac-[RuCl(dmso-S) ₃(L)] LH = 1-(2-hydroxyphenyl)-3-(4-chlorophenyl) propenone with Calf-thymus DNA (CT-DNA) is monitored using UV-vis titration (K_b = 3.8 × 10⁷) and

Full text of DOI:10.1016/j.jphotochem.2011.04.005

Journal of Photochemistry and Photobiology A: Chemistry 220 (2011) 145–152
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Journal of Photochemistry and Photobiology A:
Chemistry
journal homepage: www.elsevier.com/locate/jphotochem
Interaction of a ruthenium(II)–chalcone complex with double stranded DNA:
Spectroscopic, molecular docking and nuclease properties
Ruchi Gaura, Rais Ahmad Khanb, Sartaj Tabassumb, Priyanka Shahc,
Mohammad I. Siddiqic, Lallan Mishraa,∗
a Department of Chemistry, Faculty of Science, Banaras Hindu University, Varanasi-221005, India
b Department of Chemistry, Aligarh Muslim University, Aligarh 202002, India
c Molecular and Structural Biology Division, Central Drug Research Institute, Lucknow-226001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
The interaction of
a well characterized new complex 1 cis,fac-[RuCl(dmso-S)3(L)] LH = 1-(2-
Received 30 December 2010
Received in revised form 23 March 2011
Accepted 2 April 2011
hydroxyphenyl)-3-(4-chlorophenyl) propenone with Calf-thymus DNA (CT-DNA) is monitored using
UV–vis titration (Kb = 3.8 × 107) and ethidium bromide displacement studies (Ksv = 3.2). The molecular
docking of the complex with DNA sequence d(ACCGACGTCGGT)2 reveals that complex is stabilized by
additional electrostatic and hydrogen bonding interaction with DNA besides probable displacement of a
labile DMSO by the N7 of guanine. The coordination of guanine is further supported by the isolation and
characterization of its adduct with the complex 1.gua (gua = guanine). The nuclease property of complex
1 in the absence and presence of different activators and trappers demonstrates that complex efficiently
cleaves supercoiled pBR322 plasmid DNA and binds through major groove of the DNA.
© 2011 Elsevier B.V. All rights reserved.
Available online 14 April 2011
Keywords:
Ru(II) complex
X-ray crystallography
DNA binding
Molecular docking
Gel electrophoresis
1. Introduction
considered important components in the construction of overall
geometry of the metal complexes. The chalcones are also exploited
The interaction of transition metal complexes with DNA is found
interesting owing to their probable applications in cancer therapy,
molecular biology [1,2] and photodynamic therapy (PDT) [3]. In
this context, after cisplatin and carboplatin, ruthenium complexes
are explored extensively for their antitumor properties [4]. A pre-
liminary understanding of such properties has been explored by
targeting DNA molecule [5]. The ruthenium metal ions appended
with ligand like dimethyl sulphoxide has been exploited due to
labile nature of dmso molecule providing its easy substitution
with biomolecules. Thus, such ligand frame work is considered
as important building block in the construction of metal based
active drug [6]. The Ru(II) centre bearing labile ligands could form
monofunctional adducts with DNA. However, among complexes
containing ꢀ-bonded ligands, metal arenes form hydrophobic and
van der Waals contacts with nucleobases [7]. Thus, a combina-
tion of suitable metal as well as design of ligand is considered
important prerequisite for the construction of a highly efficient
drug. Chalcones, being an important class of ligands due to their
novel structure as well diverse bioactivities [8,9] like anticancer,
anti-inflammatory, antimalarial, antifungal and antiviral agents are
as a precursor for the synthesis of flavones, potential antioxidant.
Thus, a chalcone is considered ‘privileged structure’ [10]. Based on
these precedence, initially a well known ruthenium(II) complex
cis,fac-[RuCl2(dmso-S)3(dmso-O)] was allowed to react with 1-(2-
hydroxyphenyl)-3-(4-chlorophenyl)-propenone(LH). The resulting
composition cis,fac-[RuCl(dmso-S)3(L)] has been characterized
using single X-ray diffraction as well as spectroscopic techniques.
This complex was then allowed to interact with DNA and its mode
of binding with DNA and nuclease property in the presence of com-
plex has been investigated.
2. Experimental
2.1. Materials and methods
Starting materials were purchased from Sigma–Aldrich and
used without further purification. Infrared, UV–vis and lumines-
cence spectra were recorded on VARIAN 3100 FTIR, JASCO V-630
spectrometer, and Perkin Elmer LS-45 spectrophotometer respec-
tively. However, 1H NMR and 13C NMR spectra were recorded on
JEOL AL 300 MHz spectrometer using TMS as internal reference. Ele-
mental analysis and mass spectral measurements were carried out
using a Carbo-Erba elemental analyzer 1108 and JEOL SX-102 mass
spectrometer respectively. Cleavage experiments were performed
∗
Corresponding author. Tel.: +91 542 670 2449; fax: +91 542 236 8174.
E-mail address: lmishrabhu@yahoo.co.in (L. Mishra).
1010-6030/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.jphotochem.2011.04.005

146
R. Gaur et al. / Journal of Photochemistry and Photobiology A: Chemistry 220 (2011) 145–152
with the help of electrophoresis supported by Genei power supply
with a potential range of 50–500 V, visualized and photographed by
Vilber-INFINITY gel documentation system. The starting material
cis,fac-[RuCl2(dmso-S)3(dmso-O)] was prepared from RuCl3·3H2O
using reported procedure [11].
r is the ratio of the total concentration of complex to that of DNA.
The value of Ksv is given by the ratio of slope to intercept in a plot
of Io/I vs. [Complex]/[DNA].
2.3.3. Molecular docking study
MGL tools 1.5.4 with AutoGrid4 and AutoDock4 [19,20] were
used to set up and perform blind docking calculations between the
complex and DNA sequence. DNA sequence d(ACCGACGTCGGT)2
obtained from the Protein Data Bank (PDB id: 423D) at a resolution
2.2. X-ray crystallographic studies
The crystal data were collected on Oxford diffraction XCALIBUR-
˚
S
CCD area detector diffractometer using Mo K˛ radiation
of 1.60 A was used for the docking studies. Due to unavailability
˚
(ꢁ = 0.71073 A) at 120 K and was solved by direct method and
refined by full matrix least squares SHELXL-97 [12]. Drawings were
carried out using MERCURY [13] and special computations were
carried out with PLATON [14].
of standard parameters for Ru (II) in autodock4 parameter file,
Zn (II) parameters were used instead [21]. X-ray crystallographic
coordinates were used for the complex for the docking study.
Receptor (DNA) and ligand (complex) files were prepared using
AutoDock Tools. First of all the heteroatoms including water
molecules were deleted and polar hydrogen atoms and Kollman
charges were added to receptor molecule then rotatable bonds in
ligands were assigned. All other bonds were allowed to rotate. The
DNA was enclosed in a box with number of grid points in x × y × z
2.3. Binding studies of complex
2.3.1. Absorption titration
The concentration of DNA was determined by UV–vis
˚
absorbance
using
the
molar
absorption
coefficient
directions, 106 × 100 × 76 and a grid spacing of 0.375 A. Lamarckian
(6600 M−1 cm−1
)
at 260 nm. The absorption ratio at 260 nm
genetic algorithms, as implemented in AutoDock, were employed
to perform docking calculations. All other parameters were default
settings. For each of the docking cases, the lowest energy docked
conformation, according to the Autodock scoring function, was
selected as the binding mode.
and 280 nm of CT DNA solutions was found as 1.9:1. It showed
that DNA is sufficiently free from protein [15]. The absorption
spectral titrations were performed using a fixed concentration of
complex (100 ␮M) in Na-phosphate buffer (pH 7.2) containing
DMSO (0.01%) while the concentrations of CT-DNA were varied
within with 10–100 ␮M. From the absorption data, the intrinsic
association constant with DNA was determined from a plot of
[guest]/(εa − εf) vs. [Host] using [16] Eq. (1)
2.3.4. DNA cleavage
Cleavage experiments of supercoiled pBR322 DNA (300 ng) by
complex (20–100 ␮M) in buffer (5 mM Tris–HCl/50 mM NaCl at pH
7.2), were carried out and the reaction was followed by agarose
gel electrophoresis. The samples were incubated for 1 h at 37 ◦C.
A loading buffer containing 25% bromophenol blue, 0.25% xylene
cyanol, 30% glycerol was added and electrophoresis was carried
out at 60 V for 1 h in Tris–HCl buffer using 1% agarose gel contain-
ing 1.0 ␮g/mL ethidium bromide. The reaction was also monitored
upon addition of various radical inhibitors and/or activators such
as dimethylsulphoxide (DMSO), sodium azide (NaN3), superox-
ide dismutase (SOD), mercaptopropionic acid (MPA), glutathione
(GSH), Hydrogen peroxide (H2O2), sodium ascorbate (Asc), groove
binders; methyl green (MG) and 4ꢀ,6-diamidino-2-phenylindole
(DAPI). The inhibition reactions were carried out by adding the
reagent prior to the addition of the complex. The standard protocols
were followed for these experiments. The samples were incubated
for 45 min at 37 ◦C. The gel was visualized by photographing the
fluorescence of intercalated ethidium bromide under a UV illumi-
nator. The cleavage efficiency was measured by determining the
ability of the complex to convert the supercoiled DNA (SC or Form
I) to nicked circular form (NC or Form II) and linear form (LC or Form
III).
[guest]
(εa − εf )
[guest]
(εb − εf ) + [Kb(εb − εf )]−1
=
(1)
where [guest] refers to the concentration of DNA (in base pair). The
absorption coefficients εa, corresponds to Aobsd/[complex], εb and
εf are the molar absorptivities of the fully bound and free form of
the complex respectively. Plot of [DNA]/ꢂε vs. [DNA] (Scatchard
plot) according to Eq. (1) gives the binding association constant Kb,
are determined from the ratio of slope to the intercept.
2.3.2. Competitive binding with ethidium bromide
The relative binding of the complex to CT DNA was moni-
tored by fluorescence spectral method using ethidium bromide
(EB) bound to CT DNA solution in a Na phosphate buffer solu-
tion (pH 7.2). The solution of EB has been used as a spectral
probe as it shows no apparent emission intensity in the buffer
solution because of solvent quenching. However, enhanced emis-
sion intensity was observed when it intercalates to DNA [17]. The
complex was allowed to interact with DNA-EB solution, the emis-
sion from the resulting solution was decreased. This decrease in
emission-intensity could be considered in view of the replace-
ment of EB (DNA bound) by the complex which consequently
quenches the over-all emission-intensity. Competitive DNA bind-
ing studies of the complex with that of the ethidium bromide
are carried out using recording of ethidium bromide fluorescence
quenching after successive addition of 0–0.25 ␮M of complex to
10 ␮M DNA solutions containing 10 ␮M ethidium bromide in Na-
phosphate buffer (pH 7.2). For this experiment, sample was excited
at ꢁex 510 nm and emission spectra are recorded between 550 and
700 nm. Stern–Volmer quenching constant [18] was then calcu-
lated using the given equation
2.4. Synthesis of (LH)
1-(2-hydroxyphenyl)-3-(4-chlorophenyl)propenone
The ligand 1-(2-hydroxyphenyl)-3-(4-chlorophenyl)propenone
(LH) was synthesized using literature procedure [22] by the con-
densation of 2ꢀ-hydroxy acetophenone and ethanolic solution of
4-chlorobenzaldehyde in basic medium (50% NaOH). The ligand
was further characterized by its spectral data and melting point
(154–156 ◦C).
2.5. Synthesis and characterization of complex 1
Io
= 1 + Ksvr
(2)
A hot methanolic solution (20 cm3) of cis,fac-[RuCl2(dmso-
S)3(dmso-O)] (0.484 g, 1 mmol) was added dropwise to a solution
of LH (0.258 g, 1 mmol) dissolved in methanol (25 cm3) contain-
ing equimolar NEt3 while stirring. The reaction mixture was
I
where Io and I are the fluorescence intensities of the solution con-
taining EB and DNA in the absence and presence of the complex
respectively, Ksv is a linear Stern–Volmer quenching constant and

R. Gaur et al. / Journal of Photochemistry and Photobiology A: Chemistry 220 (2011) 145–152
147
then stirred further for 12 h at room temperature. A clear solu-
tion thus obtained was reduced to half of its volume under
reduced pressure. The block shaped red crystals suitable for X-
ray analysis were isolated from the solution in a refrigerator
after 24 h. The crystals were filtered and washed with diethyl
ether and then dried in vacuo. The crystals were partially solu-
ble in water, hot ethanol and hot methanol but highly soluble
in dichloromethane, acetonitrile, dimethylsulfoxide, chloroform,
tetrahydrofuran and dimethylformamide. Yield: 0.300 g (45%). Ele-
mantal analysis: For C22H32Cl2O6S3Ru Found: C, 39.85; H, 4.36
Cal.: C, 40.00; H, 4.88; ESI-MS: m/z: 627[M−H]+, IR (KBr pellet,
cm−1): 3015(m) ꢃ(C–H), 1620(s) ꢃ(C O), 1106(s) ꢃ(S O), 426(m)
ꢃ(Ru–S). 1H NMR (300 MHz, CDCl3, ı ppm): 3.09–3.55 (m, 18H;
dmso), 2.14 (s, 3H; CH3), 7.74, 6.89(d, 2H; HC CH), 6.53–7.76(m,
8H; phenyl). 13C NMR (75 MHz, CDCl3): 188.501 (–C O); 171.672
(PhC–O); 132.120, 133.422–(CH CH); 115.307, 121.595, 122.469,
124.801, 125.642, 129.326, 136.735, 141.961 (Phenyl), 42.855,
44.132, 44.940, 46.003, 47.437 (DMSO). UV–vis (dmso, 10−4 M):
However, OH proton observed at ı 12.2 ppm in the spectrum of free
LH disappered in the spectrum of the complex. The peaks displayed
at (ı ppm.) 188.50, 171.67, 129.33 and 132.12, 115.30–141.96
in its 13C NMR spectrum of complex were assigned to coordi-
nated >C O–, phenyl (–C–C–O–), CH CH and phenyl carbon atoms
respectively. The methyl carbon atoms of dimethylsulfoxide groups
were displayed at ı 42.86–47.44 ppm.
Additionally, ESI-MS of the complex (S1) in methanol displayed
an intense peak at m/z as 627 which corresponds to molecular
ion [M−H]+. Thus, ESI-MS together with NMR spectra recorded in
solution suggested that complex is stable in solution.
The absorption spectrum of complex recorded in
dichloromethane displayed band at ꢁmax 498 nm. This band
is considered to arise from dꢀ(RuII) → ꢀ*(L) metal-to-ligand
charge-transfer (MLCT) transitions [23]. However, bands observed
at ꢁmax 262 nm, 337 nm are red shifted as compared to bands
observed in the spectrum of the LH (ꢁmax 250 nm and 322 nm).
These bands are attributed to intraligand and ꢀ–ꢀ* type transitions
respectively.
ꢁmax (dmso)/nm (εmax
M ) 262(5000), 336 (14190),
−1 cm−1
498sh (2550). Crystallographic data: C22H32Cl2O6RuS3, fw = 660.63,
The crystals of the complex belong to rectangular shape
monoclinic crystal system with space group C2/c and exhibit
distorted-octahedral geometry around the low spin d6 Ru(II) ion
Fig. 1(a). It consists of a deprotonated chalcone (O, O) in bis-
chelating mode (ŋ2), three S-coordinated dmso and one Cl. The
chelating angle was found as 87.32◦. A molecule of methanol
was also detected in the molecular structure of the complex. The
bond length data for Ru–O, Ru–S as well as Ru–Cl were com-
parable with Ru(II)-chloride–DMSO complexes. The two oxygen
˚ ˚
C 2/c, a = 32.06(4) A, b = 8.2776(17) A,
red block, monocl◦inic,
◦
◦
3
˚
˚
c = 25.34(4) A, ˛ = 90 , ˇ = 126.69(2) , ꢄ = 90 , V = 5392(11) A , Z = 8,
3
˚
Dc = 1.628 Mg/m , MoK\˛ radiation (ꢁ = 0.71073 A), 20557 reflec-
tions, 4736 with Full-matrix, least squares on F2 used for refinement
(R = 0.0326, wR2 = 0.0662, GoF = 0.893, hydrogens calculated).
2.6. Synthesis of 1.gua
˚
atoms of the chalcone are bonded to Ru(II) ion at 2.061(4) A
A solution of guanine (0.75 g, 0.5 mmol) in methanol (10 cm3)
containing few drops of aqueous HCl solution was added to a
methanolic solution (10 cm3) of complex 1 (0.330 g, 0.5 mmol)
while stirring. It was refluxed for 3 h, the solution was then kept
in a refrigerator. After 12 h, a yellow crystalline solid obtained, was
filtered and washed with methanol. However, the solid adduct thus
obtained was found unsuitable for its X-ray crystallographic study.
It was found insoluble in water, dichloromethane, acetonitrile,
chloroform, tetrahydrofuran, hot ethanol, hot methanol but it was
found highly soluble in dimethylsulfoxide and dimethylformamide.
Yield 0.200 g (56%) Anal. Calc. for C24H27Cl2N5O5S2Ru, Found (%):
C, 41.7; H, 3.8; N, 9.8. Cal.: C, 41.1; H, 3.8; N, 9.9. FAB-MS: m/z: 700
[M]+, 664[M−Cl]+, 587[M−Cl-dmso]+, 508 [M−Cl-2dmso]+. IR (KBr
pellet, cm−1): 3327(m) ꢃ(NH), 3037(m) ꢃ(C–H), 1718(s), 1604(s)
ꢃ(C O), 1668(s) (NH2), 1099(s) ꢃ(S O), 420(m) ꢃ(Ru–S). 1H NMR
(300 MHz DMSO, ı ppm): 3.09–3.55 (s, 12H; dmso), 12.430 (s, NH),
11.178 (s, NH), 6.76 (s, NH2), 8.55(s, CH guanine), 8.23, 7.79(s,
2H; HC CH), 6.98–8.085 (m, 8H; Ar). UV–vis (dmso, 10−4 M): ꢁmax
˚
and 2.060(2) A for Ru(1)–O(1) and Ru(1)–O(2) bonds respectively,
slightly shorter than the corresponding values. However, Ru–S and
˚
˚
Ru–Cl bond distances varied from 2.244(10) A to 2.274(4) A and
˚
2.424(3) A respectively and found longer than the reported values
[23].
A PLATON analysis showed the formation of one conventional
hydrogen bond and twelve non-conventional hydrogen bonds
(S2). The non conventional bonds are formed through a DMSO
molecule as H-donor (CH3) while its O atom together with a
Cl atom acted as H-acceptors. The crystal packing of the com-
plex displayed independent inter-chain (O–H O) formation. The
inter-chain hydrogen bond was formed between the O–H group
of the coordinated methanol molecule and uncoordinated oxy-
˚
gen of DMSO at a distance of 2.768(7) A with O–H–O angle of
173◦ as depicted in Fig. 1(b). Molecular structure of complex
also displayed the presence of extensive intra- and intermolec-
ular C–H–Cl interaction. The packing diagram of complex along
crystallographic axis c forms butter fly structure. It clearly shows
formation of a double helical arrangement supported by co-
crystallized methanol molecules. The Cl atom play important role
in the formation of this network through CH· · ·Cl interaction. The
packing diagram of complex along crystallographic axis c forms
supramolecular network (butter fly structure) as depicted in Fig. 2.
It clearly shows the formation of a double helical arrangement
supported by co-crystallized methanol molecules. The Cl atom
play important role in the formation of this network through
CH· · ·Cl interaction. The Kitaigorodskii packing index [24] of 70.4%
with no grid points indicated compact packing in its crystal lat-
tice.
(dmso)/nm (εmax
M
−1 cm−1) 418 (43104), 501sh (4824).
3. Results and discussion
3.1. Structural characterization
The complex 1 was found air stable and non electrolyte in
dimethylsulfoxide (10−3 M) solution. The complex displayed IR
peaks at 1620, 1107 and 424 cm−1, assigned to coordinated ꢃ(C O),
ꢃ(S O) and ꢃ(Ru–S) vibrations respectively. It showed that ꢃ(C O)
vibration of free ligand (1639 cm−1) shifted to lower wavenumber
owing to its coordination with Ru(II) metal centre. Additionally
ꢃ(OH) vibration of the free ligand observed at 3047 cm−1 was
not observed in the spectra of its complex. It showed that (OH)
group of LH was deprotonated during coordination with the metal
ion.
To understand the binding mode of the complex with
DNA base on preliminary level, a complex of the composition
Ru(L)(dmso)2guaCl (1.gua) was isolated and characterized. Its
molecular composition was further supported by its mass at m/z
700 assigned to [M]+ (S3). Thus, the composition of the complex
showed that one DMSO coordinated in complex 1 is substituted
by one guanine molecule and the resulting complex 1.gua was
found air stable and soluble in dimethyl sulphoxide and dimethyl-
These assignments are further supported by 1H NMR spectrum
of the complex which showed peaks (ı ppm) at 3.09–3.55 (m, 18H;
dmso), 7.74, 6.89 (d, 2H; HC CH) and 6.53–7.76(m, 8H; phenyl).

148
R. Gaur et al. / Journal of Photochemistry and Photobiology A: Chemistry 220 (2011) 145–152
Fig. 1. (a) Molecular structure of complex with 50% probability ellipsoid (H atoms omitted for clarity). (Selected bond length and bond angles:
˚
˚
˚
˚
˚
˚
˚
˚
Ru(1)–O(2) = 2.060 A, Ru(1)–O(1) = 2.061 A, Ru(1)–S(1) = 2.244 A, Ru(1)–S(3) = 2.271 A, Ru(1)–S(2) = 2.274 A, Ru(1)–Cl(1) = 2.424 A, Cl(2)–C(13) = 1.734 A, S(1)–O(3) = 1.487 A,
S(1)–C(16) = 1.757 A, O(1)–C(1) = 1.305 A, O(2)–C(7) = 1.282 A, O(2)–Ru(1)–O(1) = 87.32◦, O(2)–Ru(1)–S(1) = 172.85◦, S(1)–Ru(1)–S(3) = 92.91◦, O(1)–Ru(1)–Cl(1) = 87.71◦,
S(3)–Ru(1)–Cl(1) = 173.83◦, O(3)–S(1)–C(16) = 106.56◦, C(16)–S(1)–C(17) = 98.2◦, C(16)–S(1)–Ru(1) = 110.38◦, O(4)–S(2)–Ru(1) = 121.11◦, O(1)–C(1)–C(6) = 126.1◦) and (b) Two
molecules of complex linked to a methanol (CH3OH) through C–H· · ·O interaction.
˚
˚
˚
to its closer proximity to the metal centre [26]. In the 1H NMR spec-
trum of 1.gua, H8 proton appeared at ı = 8.55 ppm which is upfield
as compared to H8 proton of free guanine observed at ı = 8.94 ppm.
Thus, it is considered that one guanine molecule is coordinated to
Ru(II) centre via its N7 atom.
formamide. Its IR spectrum displayed peaks at 1668, 1604, 1099,
1718 and 3327 cm−1 which were assigned to coordinated ı(NH2),
ꢃ(C O) (due to chalcone), ꢃ(S O) ꢃ(C O) (due to guanine) and
ꢃ(NH2) vibrations. The ı (NH2), ꢃ(C O) and ꢃ(NH2) vibrations of
free guanine were shifted to lower energy in the spectrum of 1.gua.
It could be considered owing to its coordination with the metal ion
in view of earlier report [25].
In general, coordination of nucleobase, nucleoside and
nucleotide to ruthenium ion displays H8 proton of the correspond-
ing nucleobases to upfield and considerably more broadened owing
The UV–vis spectrum of 1.gua recorded in DMSO (10−4 M)
showed intraligand ꢀ–ꢀ* transitions at ꢁmax 418 nm. This band was
red shifted as compared to intraligand transition observed in the
spectrum of complex 1 (ꢁmax 337 nm). The other band observed at
ꢁmax 502 nm is assigned to MLCT transition.
Fig. 2. The packing diagram of complex (butter fly structure) along crystallographic axis c.

R. Gaur et al. / Journal of Photochemistry and Photobiology A: Chemistry 220 (2011) 145–152
149
Fig. 3. (a) Absorption spectra of [Complex] = 100 ␮M in the absence and presence of increasing concentration of DNA = 10–100 ␮M. Arrow shows the absorbance changes
upon increasing DNA concentrations (inset: plot of [DNA]/(εa − εf) vs. [DNA].) and (b) fluorescence quenching pattern of ethidium bromide bound to DNA by complex.
[EB] = 10 ␮M, [DNA] = 10 ␮M, [Complex] = 0–0.67 ␮M. Arrow shows the intensity changes upon increasing complex concentrations. (Inset: Plot of Io/I vs. [complex]/[DNA]).
3.2. CT-DNA interaction with complex using UV–Vis spectroscopy
ble quenching of its emission by the solvent [32]. However, intense
emission is observed when EB strongly intercalates with the adja-
cent DNA base pairs. But a decrease in emission intensity results
from the displacement of EB by a quencher molecule. The extent
of emission quenching could be used to determine the extent of
binding between the metal complex with DNA. The emission spec-
tra of EB-DNA system in the absence and presence of complex are
displayed in Fig. 3(b). The quenching plot between Io/I vs. [Com-
plex]/[DNA] is in good agreement with the linear Stern–Volmer
equation and Stern–Volmer quenching constant (Ksv) is calculated
as 3.2.
Electronic absorption spectroscopy is an effective method to
examine the binding mode of DNA with metal complexes [27].
It has been reported earlier [27] that intercalation of a metal
complex with DNA involves ꢀ* orbital of the intercalated lig-
and which may couple with the ꢀ orbital of the DNA base pairs,
concomitantly lowers the ꢀ–ꢀ* transition energy hence displays
a bathochromic shift in the transition. On the other hand, the
coupling of a ꢀ orbital with partially filled electrons decreases
the transition probabilities hence results hypochromic shift [28].
Thus, such observation prompted us to investigate the binding
of newly prepared complex with DNA using spectroscopic titra-
tions of a solution of the complex with CT-DNA. On increasing
the CT-DNA concentration, a hypochromic shifts in both chal-
cone centered band and MLCT band were observed. The complex
can bind with double-stranded DNA in various binding modes
on the basis of its structure. However, hypochromic effect could
be attributed to the stacking interaction between the aromatic
rings of the ligand framework and DNA base pairs as well. The
hypochromism and bathochromic shifts may commonly vary in
consistence with the strength of intercalative interaction of the
complex with DNA helix as well as overall conformation of the
DNA. The intrinsic binding constant (Kb) was calculated by plotting
the changes in the absorbance of the complex upon incremental
addition of increasing concentration of DNA as shown in Fig. 3(a).
The significant magnitude of binding constant (Kb = 3.8 × 107)
obtained from the ratio of slope to the intercept from the plots
of [DNA]/(εa − εf) vs. [DNA] showed many folds stronger bind-
ing with DNA as compared to the earlier reported values for
ruthenium–porphyrin complex [29] as well as ruthenium–ammine
complexes [30,31].
3.4. Molecular docking of the complex with DNA sequence
d(ACCGACGTCGGT)2
After satisfactory spectroscopic measurement of DNA binding
study of the complex, molecular docking study was performed
to understand the preferred orientation of sterically acceptable
complex using chalcone ligand with the DNA sequence. Accord-
ing to this docking experiment, complex reasonably bind with
DNA sequence d(ACCGACGTCGGT)2. The minimum energy docked
structure obtained (Fig. 4) suggested the best possible conforma-
tion of the ligand interaction mainly through phenyl ring inside
the DNA major groove. It has been observed that the complex is
stabilized by electrostatic hydrogen bonding with DNA bases, par-
ticularly involving N6 of adenine from chain A and N7 of guanine
from the chain B, in addition to van der Waal’s and stacking ꢀ–ꢀ
bond interactions between electron deficient chalcone ring system
and purine–pyrimidine bases. Dimethyl sulfoxide (dmso) coordi-
nated to Ru(II) ion, participates in hydrogen bonding whereas the
side chains attached to Ru(II) complex enabled the molecules to
stay in the groove with the help of van der Waals forces. The bind-
ing energy values presented in Table 1a suggested that van der
Waal interactions are dominant over electrostatic interactions. The
hydrogen bonding interactions involving the energy-minimized
Docked poses of d(ACCGACGTCGGT)2 with complex and is shown
in Table 1b.
3.3. Competitive binding of complex with ethidium bromide using
flouresence spectroscopy
The ability of a complex to change the fluorescence intensity of
ethidium bromide (EB) in its EB-DNA adduct has been reported as a
standard intercalating agent of DNA and it is a reliable tool to mea-
sure the affinity of the complex for DNA, irrespective of the binding
modes. Therefore, a solution of EB has been used as a spectral
probe since it does not emit in the buffer solution due to proba-
Thus, molecular docking study together with spectroscopic
studies indicated that complex 1 interacts with the DNA through
both covalent and non-covalent interactions which perhaps owe to
its stronger bonding with DNA.

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R. Gaur et al. / Journal of Photochemistry and Photobiology A: Chemistry 220 (2011) 145–152
Table 1a
Molecular docking parameters of complex 1.
Binding energy
Kcal/mole
Inter-mol energy
Kcal/mole
Vdw hb desolv
energy Kcal/mole
Electrostatic
energy Kcal/mole
Total internal
energy Kcal/mole
Torsional energy
Kcal/mole
−8.24
−10.62
−10.42
−0.2
−1.23
2.39
Table 1b
Hydrogen bonding interactions involving the energy-minimized docked poses of
d(ACCGACGTCGGT)2 with complex 1.
Acceptor group (Y-H)
Donor
group Z
Distance
(A)
˚
H(N6)(A5)(DNA-chain A)
H(N7)(G19)(DNA-chain B)
O5(complex)
O4(complex)
2.096
1.963
3.5. DNA cleavage activity
3.5.1. DNA cleavage in presence of complex
After exploring strong binding of the complex with CT DNA both
experimentally and theoretically, the nuclease activity of the com-
plex was analyzed by monitoring the unwinding of supercoiled
pBR322 plasmid DNA (Form I) to nicked DNA (Form II) or Linear
circular (Form III). The amounts of strand scission were assessed
by agarose gel electrophoresis. A concentration-dependent DNA
cleavage experiment was performed, by mixing pBR322 DNA with
different concentrations (20–100 ␮M in acetonitrile) of ruthenium
complex in buffer (5 mM Tris–HCl/50 mM NaCl at pH 7.2) and the
mixture was then incubated at 310 K for 30 min. The cleavage pat-
terns as a consequence of increasing concentration of the complex
showed conversion of supercoiled (Form I) into the nicked circu-
lar form (Form II) without conversion to linear circular form (Form
III) and are depicted in Fig. 5(a). Thus, it indicated that complex
is involved in single strand DNA cleavage. The complex converts
more than 90% of SC form into NC form at a concentration of
100 ␮M.
Fig. 4. Molecular docking of the complex 1 with DNA.
3.5.2. DNA cleavage in presence of minor and major groove
binding agents
The nuclease efficiency of complexes is usually dependent on
activators. Therefore, to understand the cleavage mechanism, a
lower concentration (40 ␮M) of the complex was allowed to stay
in the presence of some activators like H2O2, 3-mercaptopropionic
acid (MPA) and glutathione (GSH). The degrees of binding of com-
plex to DNA in presence of H2O2, ASC, MPA, GSH were found as
74%, 43%, 40%, and 30%, respectively. As displayed in Fig. 5(b), the
nuclease activity of complex was significantly enhanced in the pres-
ence of activators and their activating efficacy follows the order of
H2O2 > ASC > MPA > GSH.
The potential interacting site of the complex with supercoiled
plasmid pBR322 DNA was performed by recognition elements,
minor groove binding agent 4ꢀ,6-diamidino-2-phenylindole (DAPI)
and major groove binding agent methyl green (MG) [33]. The super-
coiled DNA was treated separately with DAPI and MG prior to the
addition of the complex. The degree of binding of the complex with
pBR322 DNA in presence of DAPI and MG was found as 38% and 66%
respectively. Thus, present complex binds DNA through its major
groove, Fig. 6(a) which supports the result obtained from molecular
docking study.
Fig. 5. (a) Agarose gel electrophoresis showing cleavage patterns of the for pBR322 plasmid DNA (300 ng) by the complex. Lane 1, DNA; Lane 2, 20 ␮M complex + DNA; Lane
3, 40 ␮M complex + DNA; Lane 4, 60 ␮M complex + DNA; Lane 5, 80 ␮M complex + DNA; Lane 6, 100 ␮M complex + DNA and (b) agarose gel electrophoresis pattern for the
cleavage pattern of pBR322 plasmid DNA (300 ng) by lower concentration (40 ␮M) of the complex in presence of different activating agents. Lane 1, DNA control; Lane 2,
DNA + complex + GSH (0.4 mM); Lane 3, DNA + complex + Asc (0.4 mM); Lane 4, DNA + complex + MPA (0.4 mM); Lane 5, DNA + complex + H2O2 (0.4 mM).

R. Gaur et al. / Journal of Photochemistry and Photobiology A: Chemistry 220 (2011) 145–152
151
Fig. 6. (a) Agarose gel electrophoresis pattern for the cleavage pattern of pBR322 plasmid DNA (300 ng) by the complex (40 ␮M). Lane 1, DNA control; Lane 2,
DNA + complex + DAPI (8 ␮M); Lane 3, DNA + complex + methyl green (2.5 ␮L of a 0.01 mg/mL solution) and (b) agarose gel electrophoresis pattern showing cleavage
of pBR322 plasmid DNA (300 ng) by the complex (80 ␮M). Lane 1, DNA control; lane 2, DNA + complex + D2O (70%); Lane 3, DNA + complex + NaN3 (0.4 mM); Lane 8,
DNA + complex + superoxide dismutase (15 Units), respectively.
The experiments were further carried out in presence of sev-
eral reactive oxygen trappers in view of the report that interaction
between a metal complex and dioxygen or redox reagents is
believed to be a major cause of DNA damage. Thus, to investi-
gate this mechanistic pathway of the DNA cleavage, the complex
was allowed to interact with DNA separately with tert-butyl
alcohol and DMSO as a hydroxyl radical scavenger under iden-
tical conditions. But cleavage was not significant as compared
to DNA cleavage without them. Therefore, it was considered
that diffusible (•OH) hydroxyl radicals are not responsible for
DNA cleavage (S4). However, with other oxygen trappers like
sodium azide as singlet oxygen scavenger, the cleavage was sig-
nificantly exhibited, revealing the involvement of 1O2 in the DNA
cleavage. The involvement of 1O2 was further supported by the
significant enhancement of the cleavage activity in D2O, where
lifetime of 1O2 is notably longer than that in water [34]. In
D2O, a linear form also appears that indicated increase in the
cleavage. However, presence of superoxide dismutase (SOD), a
facile superoxide anion radical (O2•−) quencher, cleavage was
observed similar to that observed in presence of complex only. It
revealed that superoxide anion is not active specie in the cleav-
age of pBR322 DNA as depicted in Fig. 6(b). Hence, the cleavage
pattern thus observed supported the oxidative cleavage path-
way.
1, selected parameters for weak interactions in complex,
FAB mass of 1.gua, and nuclease activity in presence of
trappers.
Acknowledgements
Authors are grateful to authorities of DBT and UGC, New Delhi
India for their financial supports.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.jphotochem.2011.04.005.
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