CT-DNA/BSA protein binding and antioxidant studies of new binuclear Pd(II) complexes and their structural characterisation

Article abstract of DOI:10.1016/j.ica.2016.05.018

New palladium(II) binuclear complexes (1-4) of the type [Pd₂(Msal-Rtsc)₂(μ-dppm)] (R = H or CH₃ or C₂H₅ or C₆H₅) were prepared and characterised by using various spectro analytic

Full text of DOI:10.1016/j.ica.2016.05.018

Inorganica Chimica Acta 449 (2016) 107–118
Contents lists available at ScienceDirect
Inorganica Chimica Acta
journal homepage: www.elsevier.com/locate/ica
Research paper
CT-DNA/BSA protein binding and antioxidant studies of new binuclear
Pd(II) complexes and their structural characterisation
A. Shanmugapriya a, G. Kalaiarasi a, P. Kalaivani b, F. Dallemer c, R. Prabhakaran a,
⇑
a Department of Chemistry, Bharathiar University, Coimbatore 641 046, India
b Department of Chemistry, Nirmala College for Women, Bharathiar University, Coimbatore 641018, India
c Laboratoire Chimie Provence-CNRS, UMR7246, Université of Aix-Marseille, Campus Scientifique de Saint-Jérôme, Avenue Escadrille Normandie-Niemen, F-13397 Marseille Cedex 20,
France
a r t i c l e i n f o
a b s t r a c t
Article history:
New palladium(II) binuclear complexes (1–4) of the type [Pd2(Msal-Rtsc)2(l-dppm)] (R = H or CH3 or
Received 4 February 2016
Received in revised form 2 April 2016
Accepted 7 May 2016
C2H5 or C6H5) were prepared and characterised by using various spectro analytical techniques. The true
coordinating nature of the ligands in the complexes was confirmed by X-ray crystallographic studies.
From the X-ray crystallographic analysis, it is found that the ligands [H2L1–H2L4] were bound to metal
as tridentate ONS donors and phosphorus atom of bridged diphenylphosphinomethane satisfied the
fourth coordination site in both the metal units to form a binuclear complexes. The interaction mode
of the complexes (1–4) with calf-thymus DNA (CT-DNA) has been explored by absorption titrations
and ethidium bromide displacement study was used to confirm the mode of binding. Based on the results
obtained, electrostatic and intercalative binding modes have been proposed. Quenching the tryptophan
and tyrosine residues of Bovine Serum Albumin (BSA) by complexes were found as static. Anti-oxidant
properties of the complexes were tested against, 2,20-diphenyl-1-picrylhydrazyl radical (DPPH), superox-
ide anion radicals.
Available online 11 May 2016
Keywords:
Palladium(II) thiosemicarbazones
Spectroscopy
X-ray crystallography
CT-DNA/BSA protein binding
Anti-oxidant activity
Ó 2016 Elsevier B.V. All rights reserved.
1. Introduction
been used in the treatment of rapidly growing high grade prostate
cancer [36,37]. Palladium(II) complexes are most probably involves
The chemistry of thiosemicarbazone complexes of the transi-
tion metal ions has been receiving significant current attention,
largely because of their bioinorganic importance [1–7] and wide
range of medicinal applications owing to their potentially benefi-
cial biological and pharmaceutical activities such as antibacterial,
antimalarial, antiviral, antitumor, anti-proliferative, antifungal,
anti-inflammatory, anticonvulsant, antitubercular, antioxidant
effects and inhibition of tumour growth [8–25]. These activities
are due to their ability to form chelates with metals causing drastic
change in the biological properties. They are well known chelating
ligands coordinating to the metal ion and their coordination beha-
viour has been established elsewhere [26–32]. Apart from the bio-
logical properties, most of the thiosemicarbazone derivatives are
used as chemical intermediates, perfume bases, dyes, rubber accel-
erators and liquid crystals for electronics and ion sensing ability
[33,34]. Binuclear d8 complexes have considerable attention due
to their novel structural and solid state properties [35]. Among
the d8 complexes, palladium in its radioactive isotope 103Pd has
in the inhibition of ribonucleotide reductase, by converting ribonu-
cleotide to deoxyribonucleotide [38,39] and were screened against
the replication of wide-type herpes simplex virus (HSV-1) and
(HSV-2) strains [40]. Recently, phosphine chemistry has gained
attention due to the tremendous applications in organometallic
and coordination chemistry [41,42]. Bis(diphenylphosphino)
methane induces unusual cyclometalation of thiophene and phe-
nyl rings at the C2 carbon of thiosemicarbazone in Ru(II) com-
plexes [43]. Bis(diphenylphosphino)methane containing silver(I)
complexes were assayed for their antibacterial activity against
two gram positive bacterial strains (Bacillus subtilis ATCC 6633
and Staphylococcus aureus ATCC 6538) and two gram negative
bacterial strains (Pseudomonas aeruginosa ATCC 13525 and Escher-
ichia coli ATCC 35218) [44]. Bis(diphenylphosphino)methane com-
plexes of platinum were found to have strong anticancer activity
and weak denaturing effect against albumin proteins [45].
Cyclometalated organoplatinum(II) complexes containing bisphos-
phine shown to have in vitro and in vivo antitumor activities [46].
Diphenylphosphinomethane ruthenium(II) arene complexes found
to exhibit high chemoselectivity in the hydrogenation of aldehydes
[47]. The interaction of transition metal complexes with DNA is a
⇑
Corresponding author. Tel.: +91 422 2428319; fax: +91 422 2422387.
E-mail address: rpnchemist@gmail.com (R. Prabhakaran).
http://dx.doi.org/10.1016/j.ica.2016.05.018
0020-1693/Ó 2016 Elsevier B.V. All rights reserved.

108
A. Shanmugapriya et al. / Inorganica Chimica Acta 449 (2016) 107–118
vibrant area of research [48]. DNA regulates most aspects of cell
life and constitutes an important drug target. Designing the com-
plexes that can bind and react with selective nucleotide sequences
are of great importance in probing biological processes and in
developing therapeutic drugs. An advantage of using transition
metal complexes in such studies can be suited individual applica-
tions by conveniently varying their ligands and metal ions. A vari-
ety of palladium complexes are found to be effective in various
therapeutic applications [49,50]. In this article, we are reporting
CT-DNA/BSA (Bovine Serum Albumin)-protein binding and antiox-
idant studies of new binuclear palladium(II) complexes and their
structural characterisation.
been assigned to intra ligand transition taking place within the
ligands. The bands appeared around 298–301 nm have been
assigned to ligand to metal charge transfer (LMCT) transitions
and the bands at 341–406 nm have been assigned to metal to
ligand charge transfer (MLCT) transitions [56]. The 1H NMR spectra
of the ligands (H2L1-H2L4) and the corresponding complexes (1–4)
were recorded in DMSO and CDCl3 (Figs. S1–S8). In the spectra of
H2L1-H2L4,
a singlet which appeared in the range d 9.13–
10.00 ppm has been assigned to N(2)H proton [57]. However, in
the spectra of the new complexes (1–4), there was no resonance
attributable to N(2)H, indicating the coordination of thiolate sul-
phur in the anionic form after deprotonation at N(2) [58]. A sharp
singlet at d 11.34–11.76 ppm corresponding to the phenolic AOH
group in the free ligands was completely absent in all the new
complexes confirming the involvement of phenolic oxygen in coor-
dination. The aromatic protons corresponding to the coordinated
ligands and diphenylphosphinomethane were found as multiplet
at d 6.31–7.93 ppm, and a sharp singlet corresponding to the
AOCH3 group also appeared at d 3.60–3.70 ppm [59]. Two singlets
observed at d 8.36–8.50 and d 8.37–9.20 ppm were assigned to
azomethine and terminal ANH protons of the ligands [53]. How-
ever, a doublet was observed at d 7.95–8.20 ppm in the complexes,
to azomethine proton, may be due to the nuclear quadrupolar
effect of nitrogen atom [53]. A sharp singlet observed around d
7.45–8.17 ppm in 2 and 3 was assigned to the presence of terminal
ANH protons of the ligands; however in the complex 4, this reso-
nance was mixed with aromatic protons. Two broad singlets were
observed at d 7.84 and d 8.06 ppm corresponding to NH2 protons of
the H2L1 indicating the magnetic non-equivalence of the protons
which may be due to inter molecular hydrogen bonding [54]. How-
ever in the complex 1, this resonance appeared as a singlet at d
4.65 ppm and this up field shift may be due to the chelation which
increases the shielding effect. Further, a doublet observed at d
2.99 ppm in H2L2 was found at and d 2.71 ppm in the complex 2
indicating the presence of terminal CH3 protons. A multiplet
appeared at d 3.55–3.58 ppm and d 3.70–3.78 ppm in H2L3 and 3
was assigned to the methylene protons of ethyl group [59]. Fur-
ther, a triplet was also noted at d 1.13 ppm and d 1.11 ppm in
H2L3 and complex 3 corresponding to the presence of terminal
CH3 of ethyl group protons. In addition, a triplet was appeared
around d 4.79–4.82 ppm in the complexes indicating the presence
of methylene protons of coordinated 1,10-bis(diphenylphosphino)
methane [60].
2. Results and discussion
2.1. Synthesis of new palladium(II) complexes
The reactions of 3-methoxysalicylaldehyde-4(N)-substituted
thiosemicarbazones (H2L1-H2L4) with K2[PdCl4] and 1,10-bis
(diphenylphosphino)methane [dppm] in 1:1 methanol-dichloro-
methane resulted in the formation of new complexes (1–4)
(Scheme 1), the analytical data of which confirmed their stoi-
chiometry (1–4). The complexes are soluble in common organic
solvents such as dichloromethane, chloroform, ethanol, methanol,
dimethylformamide and dimethylsulphoxide.
2.2. Spectroscopic studies
The IR spectra of the ligands H2L1-H2L4 and the corresponding
complexes provided significant information about the metal ligand
bonding. A strong vibration observed at 1539–1593 cmꢀ1 in the
ligands corresponding to m(C@N) was shifted to 1593–1596 cmꢀ1
in the complexes indicating the coordination of azomethine nitro-
gen to palladium ion [29,51]. A broad band at 3297–3457 cmꢀ1 due
to the presence of AOH group in the free ligands completely disap-
peared in the IR spectra of the complexes (1–4) indicating the coor-
dination of phenolic oxygen to palladium after deprotonation. This
was further supported by the increase in the phenolic CAO stretch-
ing frequency from 1260–1275 cmꢀ1 to 1299–1313 cmꢀ1 [52]. A
sharp band observed at 772–788 cmꢀ1 corresponding to m(C@S) in
the ligands was absent in the spectra of the complexes and a
new band appeared at 730–734 cmꢀ1 corresponding to m(CAS)
indicating the coordination of thiolate sulphur atom after
enolisation and subsequent deprotonation [53,54]. In addition,
the characteristic stretching frequencies corresponding to the pres-
ence of bis(diphenylphosphino)methane were found in the region
1436–1437 cmꢀ1, 1096–1097 cmꢀ1 and 686–689 cmꢀ1 [55]. The
electronic spectra of palladium(II) complexes were recorded in
chloroform and they displayed three to four bands in the region
around 243–406 nm. The bands appeared at 243–244 nm have
2.3. X-ray crystallography
In order to confirm the exact structure of the complexes, X-ray
crystallographic studies were done for the new complexes (1–3).
The ORTEP diagram and the numbering scheme of the complexes
are given in Figs. 1–3. The crystallographic data, selected bond
N
N
HN
Pd
OCH3
S
PPh2
O
PPh2
OH
R
S
5h,
OCH3
R
+
K2[PdCl4]
+
N
N
H
N
H
[Methanol/CH2Cl2]
OCH3
PPh2
PPh2
O
S
R
Pd
N
N
H
N
R = H, CH3, C2H5,C6H5
R = H
(1)
R = CH3
R = C2H5
R = C6H5
(2)
(3)
(4)
Scheme 1. Preparation of new palladium(II) complexes.

A. Shanmugapriya et al. / Inorganica Chimica Acta 449 (2016) 107–118
109
distances 2.018(3) Å (1), 2.019(2) Å (2) and 2.022(3) Å (3), and
Pd1AS1 bond distances 2.245(1) Å (1), 2.2511(6) Å (2) and 2.231
(1) Å (3) were found as similar to the reported values [53,59,61].
The remaining binding site is occupied by the phosphorous atom
of 1,10-bis(diphenylphosphino)methane with Pd(1)AP(1) bond dis-
tances of 2.271(1) Å (1), 2.2775(8) Å (2) and 2.268(1) Å (3), respec-
tively with the [S1APd1AN1] bite angles of 84.3(1)°, 84.01(6)° and
84.5(1)° for 1,2 and 3 respectively. The trans angles [P1APd1AN1]
and [O1APd1AS1] were found as 172.4(1)°, 177.82(6)° and 176.3
(1)° for 1,2 and 3 respectively and 177.6(1)°, 171.67(6)° and
176.61(9)° for 1,2 and 3 respectively indicating the considerable
deviation from the ideal symmetry and significant distortion
around square planar palladium ion [61]. Two of these symmetri-
cal units are bridged through dppm ligand in the complexes 1–3.
In complex 1, there are two intermolecular hydrogen bonding
between hydrogen atoms of terminal nitrogen (N3) and with N2
and N5 hydrazinic nitrogen atoms, forming a 2D layer structure
(Fig. S9). The hydrogen bonding parameters are given in Table S1.
Complex 3 exhibited an intermolecular hydrogen bonding with
the oxygen atom of the water moiety which is present in the crys-
tal lattice (Fig. S10).
2.4. DNA binding studies
DNA binding is one of the main properties in pharmacology for
evaluating the anticancer property of any new compound, and
hence, the interaction between DNA and metal complexes is of
paramount importance in understanding the mechanism. Thus
the mode and tendency for binding of complexes 1–4 to CT DNA
were studied by electronic absorption and ethidium bromide (EB)
displacement experiments. The interaction of transition metal
complexes with DNA takes place via both covalent and/or non-
covalent interaction [62]. In the case of covalent binding, the labile
ligand of the complexes is replaced by a nitrogen base of DNA such
as guanine N7 while the non-covalent DNA interactions include
intercalative, electrostatic and groove binding of metal complexes
outside of a DNA helix.
Fig. 1. ORTEP diagram of [(Pd(Msal-tsc))2(l-dppm)] (1).
distances and bond angles are listed in Tables 1 and 2. Crystallo-
graphic analysis revealed that the complexes 1–3 crystallised in
monoclinic form with space group, P21/c. In the complexes (1–3),
each palladium atom is coordinated through phenolic oxygen, N1
hydrazinic nitrogen and thiolate sulphur atom by forming a six
member and five member rings. The Pd1AO1 bond distances
2.025(3) Å (1), 2.020(2) Å (2) and 2.038(3) Å (3) and PdAN1 bond
Fig. 2. ORTEP diagram of [(Pd(MSal-mtsc))2(l-dppm)] (2).

110
A. Shanmugapriya et al. / Inorganica Chimica Acta 449 (2016) 107–118
240 nm exhibited hyperchromism (A = 0.0224–0.1549) with red
shift of 4 nm was observed in the intra ligand band. The CT bands
at 314, 348 and 406 nm showed hyperchromism without any shift
in the absorption maxima and the binding of complex 2 to CT DNA
led to isosbestic spectral change with the isosbestic point at
261 nm. Complex 3 exhibited hyperchromism (IL) at 258 nm with-
out wavelength shift in the absorption maxima and complex 4
exhibited hyperchromism (IL) at 268 nm with a blue shift of
9 nm. The CT bands showed hypochromism at 345 nm (for 3)
and 352 nm (for 4) without undergoing any shift upon addition
of CT-DNA. The observed hyperchromic effect with red shift sug-
gested that complexes (1–4) bind to DNA by external contact, pos-
sibly due to electrostatic binding [63]. The intrinsic binding
constant Kb is a useful tool to monitor the magnitude of the binding
strength of compounds with CT-DNA (Table 3). It can be deter-
mined by monitoring the changes in the absorption in the IL band
at the corresponding kmax with increasing concentration of DNA
and is given by the ratio of the slope to the Y intercept in plots
of [DNA]/(Ɛa ꢀ Ɛf) versus [DNA] (Fig. 5). From the binding constant
values (Table 3), it is inferred that all the complexes bind with CT-
DNA efficiently. Among the four complexes, complex 4 binds more
strongly with CT-DNA compared to the remaining complexes and
the order of binding affinity is 1 < 2 < 3 < 4.
Fig. 3. ORTEP diagram of [(Pd(Msal-etsc))2(l-dppm)] (3).
2.4.1. Electronic absorption titration
Electronic absorption titration experiment was carried out to
study the DNA binding properties of the new Pd(II) complexes
(1–4). The absorption spectra of complexes (1–4) at a constant con-
2.4.2. Competitive studies with ethidium bromide
DNA binding study point out that the new palladium(II) com-
plexes effectively bind to DNA. Further, this has been confirmed
by ethidium bromide displacement experiments as done with the
reported procedure [59]. The fluorescence spectra of EB were mea-
sured using an excitation wavelength of 620 nm, and the emission
range was set between 550 and 750 nm. Addition of test com-
pounds to CT DNA pre-treated with EB ([DNA]/[EB] = 1) caused
reduction in the emission intensity (Fig. 6). The quenching extents
of the complexes were evaluated qualitatively by employing
Stern–Volmer Eq. (1). From the obtained values it is concluded that
EB was replaced by the compounds from the EB-DNA system (Fig. 7
and Table 4). Such a characteristic changes is often observed in
centration (10
CT-DNA (0–50
l
l
M) in the presence of different concentrations of
M) are given (Fig. 4). The absorption spectra of
complex 1 mainly consist of two resolved bands [intra ligand (IL)
and metal-to-ligand (MLCT) transitions] centred 246 nm (IL) and
347 nm (MLCT). While increasing the concentration DNA, a hyper-
chromism (A = 0.3759–0.5106) with red shift of 11 nm (up to 257)
was observed in the intra ligand band. A modest hypochromism
with negligible shift in the absorption maxima was found in the
MLCT band at 347 nm. An isosbestic spectral change with the isos-
bestic point at 294 nm was observed when complex 1 binds to CT
DNA. For complex 2, upon addition of DNA, the intra ligand band at
Table 1
Crystal data and structure refinement of new Pd(II) complexes.
[(Pd2(Msal-tsc))2(
l
-dppm)] (1)
[(Pd2(Msal-mtsc))2(
l
-ppm)] (2)
[(Pd2(Msal-etsc))2(l-dppm) ](3)
Empirical formula
Formula weight
T (K)
C43 H40 N6 O4 P2 Pd2 S2
1135.80
293
C45 H44 N6 O4 P2 Pd2 S2
1071.72
293
C47 H49 N6 O5 P2 Pd2 S2
1124.78
293
k (Å)
1.54184
1.54184
1.54184
Crystal system
Space group
Unit cell dimension
a (Å)
monoclinic
P21/c
monoclinic
P21/c
monoclinic
P21/c
14.1487(2)
11.1101(10)
10.44070(11)
b (Å)
14.19040(17)
22.39197(10)
15.83437(12)
c (Å)
32.5313(6)
22.6748(2)
31.1277(2)
a
(°)
90
90
90
b (°)
123.649(3)
128.7415
93.6697
c
(°)
90
90
90
V (Å3)
5437.14(16)
4399.86 (9)
5135.54(8)
Z
4
4
2
Density (Mg mꢀ3
)
1.388
6.980
2304
1.618
8.585
2168
1.455
7.405
2284
Absorption coefficient (mmꢀ1
F(000)
)
Crystal size (mm)
h range for data collection
Index range
Reflection collected
Completeness to theta
Refinement method
Data/restraints/parameters
Goodness of fit (GOF) on F2
0.16 ꢁ 0.12 ꢁ 0.06
0.16 ꢁ 0.11 ꢁ 0.07
0.02 ꢁ 0.12 ꢁ 0.08
4.42–73.67°
3.94–73.78°
3.98–73.78°
ꢀ17 6 h 6 17, ꢀ17 6 k 6 17, ꢀ39 6 l 6 39
21796
ꢀ13 6 h 6 13, ꢀ27 6 k 6 25, ꢀ28 6 l 6 27
288415
ꢀ12 6 h 6 12, ꢀ19 6 k 6 19, ꢀ38 6 l 6 38
27775
66.97°
66.97°
66.97°
Full-matrix least-square on F2
10748/6/568
Full-matrix least-square on F2
8793/0/554
Full-matrix least-square on F2
10235/0/596
1.093
1.036
1.110
Final R indices[I > 2
R indices (all data)
r
(I)]
R1 = 0.0558, wR2 = 0.1380
R1 = 0.0489, wR2 = 0.1484
R1 = 0.0306, wR2 = 0.0773
R1 = 0.0291, wR2 = 0.0793
R1 = 0.0487, wR2 = 0.1163
R1 = 0.0446, wR2 = 0.1204

A. Shanmugapriya et al. / Inorganica Chimica Acta 449 (2016) 107–118
111
Table 2
(where the compound concentration is the value at a 50% reduction
Selected bond lengths (Å) and angles (o) of new Pd(II) thiosemicarbazone complexes.
in the fluorescence intensity of EB, KEB (1.0 ꢁ 107 Mꢀ1) is the DNA
binding constant of EB, [EB] is the concentration of EB = 10 lM).
(1)
(2)
(3)
The calculated values of the quenching constant (Kq) binding con-
stant (Kapp) are listed in Table 4. Furthermore, the observed quench-
ing constants (Kq) and binding constants (Kapp) of the Pd(II)
complexes suggest that the interaction of all the complexes with
DNA should be of intercalation [65].
Bond lengths
Pd1AO1
Pd1AN1
Pd1AS1
Pd1AP1
Pd2AO3
Pd2AN4
Pd2AS2
Pd2AP2
2.025(3)
2.020(2)
2.038(3)
2.018(3)
2.245(1)
2.271(1)
2.016(6)
2.023(4)
2.246(2)
2.274(1)
2.019(2)
2.2511(6)
2.2775(8)
2.024(2)
2.024(2)
2.2464(7)
2.2692(7)
2.022(3)
2.231(1)
2.268(1)
2.015(4)
2.020(5)
2.254(1)
2.264(1)
2.5. Protein binding studies
Bond angles
O1APd1AN1
S1APd1AP1
N1APd1AS1
O1APd1AP1
N1APd1AP1
O1APd1AS1
O3APd2AN4
S2APd2AP2
N4APd2AS2
O3APd2AP2
N4APd2AP2
O3APd2AS2
The fluorescence analysis provide information on the binding
mechanism, binding mode, binding constant and binding sites of
small molecules to protein. The mechanism of quenching are usu-
ally classified by either dynamic quenching or static quenching.
Static quenching refers to fluorophore-quencher complex forma-
tion and the dynamic quenching refers to a process in which the
fluorophore and the quencher come into contact during the tran-
sient existence of the excited state.
93.2(1)
97.21(5)
84.3(1)
93.18(8)
96.31(3)
84.01(6)
86.79(6)
177.82(6)
171.67(6)
93.72(8)
99.19(3)
83.73(6)
83.37(6)
176.93(6)
177.44(6)
93.2(1)
94.65(4)
84.5(1)
87.51(9)
176.3(1)
176.61(9)
93.8(2)
98.13(4)
83.9(1)
84.0(1)
85.2(1)
172.4(1)
177.6(1)
92.6(2)
100.42(5)
84.5(1)
82.8(1)
173.9(1)
175.1(1)
176.3(1)
175.5(1)
2.5.1. UV absorption spectra of BSA
The binding of the complexes with BSA (Bovine Serum
Albumin) has been estimated from the concentration dependence
upon the change in the fluorescence intensity of protein after the
addition of complexes. Analysing the absorption spectra of the
BSA in the presence of complexes can give information about the
method of quenching [66]. The UV absorption spectra of BSA in
the absence and presence of four compounds showed that the
(Fig. 8) absorption intensity of BSA was enhanced while adding
intercalative DNA interaction [64]. Further, the apparent DNA
binding constant (Kapp
equation,
) were calculated using the following
KEB ½EBꢂ ¼ Kapp½complexꢂ
Fig. 4. Absorption titration of fixed concentration (10 lM) of complexes 1–4 with increasing concentrations (0–50 lM) of CT-DNA (TrisHCl buffer, pH 7).

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A. Shanmugapriya et al. / Inorganica Chimica Acta 449 (2016) 107–118
Table 3
Binding constant for interaction of complexes with CT-DNA.
System
Kb (Mꢀ1
)
CT-DNA + 1
CT-DNA + 2
CT-DNA + 3
CT-DNA + 4
3.30 ꢁ 104
1.89 ꢁ 105
4.83 ꢁ 105
8.71 ꢁ 105
Fig. 7. Plot of [Q] vs. I0/I.
Table 4
Quenching constant and Binding constant for interaction of complexes with DNA.
System
Kq (ꢁ103 Mꢀ1
)
Kapp (ꢁ106 Mꢀ1
)
Complex 1
Complex 2
Complex 3
Complex 4
4.68
3.53
4.45
4.10
2.383
2.011
2.241
2.341
Fig. 5. Plot of [DNA] vs. [DNA]/(Ɛa ꢀ Ɛf).
Fig. 6. Fluorescence quenching curves of ethidium bromide bound to DNA 1–4. [DNA] = 10 lM, [EB] = 10 lM and [compound] = 0–100 lM.

A. Shanmugapriya et al. / Inorganica Chimica Acta 449 (2016) 107–118
113
where K is the binding constant of quencher with BSA, n is the num-
ber of binding sites, F0 and F are the fluorescence intensity in the
absence and the presence of the quencher. The value of K can be
determined from the slope of the plot log[F0 ꢀ F/F] versus log[Q]
(Fig. 11). The calculated value of the binding constant (K) and the
number of binding sites (n) are listed in Table 5. Complex 4 has a
higher magnitude of binding than other complexes. This confirms
the effect of substitution on binding with BSA.
2.5.3. Synchronous fluorescence spectroscopic studies of BSA
Synchronous fluorescence spectral study was used to obtain
information about the molecular environment in the vicinity of
the fluorophore moieties of BSA [70]. Synchronous fluorescence
spectra show tyrosine residue of BSA only at wavelength interval
D
k of 15 nm whereas tryptophan residues of BSA at
The concentration of complexes (0–100 M) added to BSA
(10 M) is increased, a decrease in the fluorescence intensity with
Dk of 60 nm.
l
l
blue shift in the tryptophan emission maximum is observed for all
the complexes (S11). In contrast, the emission intensity of tyrosine
residue increases without any change in the wavelength of emis-
sion. These observations indicate that the test compounds did
not affect the microenvironment of tyrosine residue during the
binding process significantly but the tryptophan microenviron-
ment to a larger extent.
Fig. 8. UV absorption spectra of BSA (10 lM) in the presence of complexes (10 lM).
the complexes with a blue shift of 3 nm. The changes in the absorp-
tion spectra of fluoropore + quencher complex indicate the interac-
tion of complexes with BSA. It is well known that dynamic
quenching only affects the excited state of fluorophore and does
not change the absorption spectrum. However, the formation of
non-fluorescence ground state complex induced the change in
absorption spectrum of fluorophore. Thus, possible quenching
mechanism of BSA by complexes was found as static quenching
[67].
2.6. Antioxidant activity
Since the palladium complexes exhibited good DNA and protein
binding affinity, it was considered worthwhile to study the antiox-
idant properties of these compounds. Free radicals play an impor-
tant role in the inflammatory process. The compounds with
possible antioxidant properties could play a crucial role against
inflammation and lead to potentially effective drugs. Antioxidants
that exhibit radical scavenging activity are receiving increased
attention since they present interesting anticancer, anti-ageing
and anti-inflammatory activities. Therefore, compounds with
antioxidant properties may offer protection against rheumatoid
arthritis and inflammation. The radical scavenging activities of
our compound along with standards, such as ascorbic acid and
butylated hydroxyl toluene (BHT) in a cell free system, have been
examined with reference to DPPH (2-20-diphenyl-1-picrylhy-
drazide radical (DPPHꢀ) and hydroxyl radical (OHꢀ). The DPPH_scav-
enging ability of the Pd(II) complex was slightly low (Fig. 12), the
ability was excellent for the other radicals when compared to the
standard and their IC50 values are listed in Table 6.
2.5.2. Fluorescence quenching studies of BSA
In order to get more insight into the binding affinity of the com-
plexes with BSA, emission titrations were done with BSA and
increasing concentrations of test complexes. Among the three flu-
orophores, namely, tryptophan, tyrosine, and phenylalanine in
BSA, the intrinsic fluorescence is mainly due to tryptophan alone,
because phenylalanine has a very low quantum yield and the fluo-
rescence of tyrosine is almost quenched when it becomes ionised
or near to an amino group, a carbonyl group, or a tryptophan resi-
due [68]. Changes in the emission spectra of tryptophan are com-
mon in response to protein conformational transitions, subunit
associations, substrate binding, or denaturation. Hence, the inter-
action of BSA with compounds was studied by fluorescence mea-
surement at room temperature and the binding constants of the
compounds were calculated. On increasing the concentration of
complexes, a progressive decrease in the fluorescence intensity
was observed, accompanied with a blue shift (Fig. 9). The observed
blue shift may be due to the binding of compounds with the active
sites of BSA [69]. The quenching effect indicates the interaction of
BSA with new Pd(II) complexes. The fluorescence quenching data
have been analysed by Stern–Volmer equation.
The reducing power of the complexes increases with increase in
the concentration of complexes (20–100
bance values 0.289, 0.288, 0.283 and 0.267 nm was shown at
100 g/ml concentration of the complexes 1, 2, 3 and 4 respec-
lg/ml), and the absor-
l
tively are shown (Fig. 13). The complex 1 has higher activity when
compared to the other complexes. The superoxide anion radical
activity of the complexes increased with increasing the concentra-
tion of the complexes (Fig. 14). It is seen from the results that com-
plexes 1–4 possess significant antioxidant activity. The total
antioxidant activities of new palladium complexes were also
assessed by phosphor molybdenum method [71] and the values
are listed in Table 7. Among the four complexes, complex 4 showed
the best activity.
I0=I ¼ 1 þ Ksv½Qꢂ
ð1Þ
where I0 and I are the fluorescence intensities of the fluorophore in
the absence and presence of quencher, Ksv is the quenching constant
and [Q] is the quencher concentration. A plot of I0/I against the con-
centration of 1–4 resulted in a linear plot (Fig. 10) and the Ksv value
is obtained from the slope. The observed linearity in the plots indi-
cated the ability of the complexes to quench the emission intensity
of BSA.
3. Conclusion
When small molecule bind to the active site of BSA, the equilib-
rium binding constant and the number of binding sites can be anal-
ysed by using the Scatchard Eq. (2)
Four new binuclear palladium(II) complexes have been synthe-
sized and characterised by various spectro and analytical tech-
nique. The structure of the complexes 1,2, and 3 were confirmed
by X-ray crystallographic studies and form the studies it is found
log½F0 ꢀ F=Fꢂ ¼ log K þ n log½Qꢂ
ð2Þ

114
A. Shanmugapriya et al. / Inorganica Chimica Acta 449 (2016) 107–118
Fig. 9. Fluorescence quenching of BSA (1 ꢁ 106 M; kexi = 276; kemi = 346 nm) in the absence and presence of various concentration of complexes (0–100
lM).
Fig. 10. Plot of [Q] vs. I0/I.
Fig. 11. Plot of log [Q] vs. log (F0 ꢀ F/F).
that the ligand coordinated with metal as ONS tridentate bibasic
donor. From the results of DNA binding studies, it is concluded that
the complexes exhibited significant binding affinity by showing
electrostatic binding mode. Whereas, the complexes quench the
fluorescence of BSA by forming quencher + fluorophore complex
by static quenching mechanism and thus made significant confor-
mational change to the protein structure. The complexes exhibited
significant anti oxidant activity by showing some degree of radical
scavenging ability with the tested radicals.
4. Experimental
4.1. Synthesis and characterisation
1,10-bis(diphenylphosphino)methane was purchased from
Sigma Aldrich Ltd, PdCl2 was purchased from Thomas Baker and
CT-DNA, EB and BSA were obtained from Himedia. The ligands
[H2L1-H2L4] and palladium complexes K2[PdCl4] were synthesized
according to the standard literature procedures [72–74]. All the

A. Shanmugapriya et al. / Inorganica Chimica Acta 449 (2016) 107–118
115
Table 6
The radical scavenging activity of the compounds.
Compounds
IC50 values (lM)
DPPH_
O2ꢀ
Standard
39.26
39.70
63.26
44.68
40.23
43.44
27.74
42.26
35.34
34.15
1
2
3
4
Table 7
Estimation of total antioxidant capacity of new Pd(II) complexes.
Sample
lg Ascorbic acid equivalents/ml
1
2
3
4
52
25
70
89
Fig. 12. DPPH scavenging activity of the new Pd(II) complexes (1–4).
reagents used were analar grade, were purified and dried according
to the standard procedure [75]. Infrared spectra were measured as
KBr pellets on a JASCO FT-IR 4100 instrument between 400 and
4000 cmꢀ1. The electronic spectra of the complexes have been
recorded in chloroform using a JASCO V-630 spectrophotometer
in the 200–800 nm range. Emission spectra were recorded by using
a
JASCO FP 6600 spectrofluorometer.1H NMR spectra were
recorded in CDCl3 and DMSO at room temperature with Bruker
400 MHz instrument, chemical shift relative to tetramethylsilane.
Melting points were measured in a Labindia apparatus. Single crys-
tal data collection and correction for the new Pd(II) complexes 1–3
were done at 293 K with CCD kappa diffractometer using graphite
mono chromated Mo Ka (k = 1.54184 Å) radiation [76]. The struc-
tural solution were done by using SHELXTL-97 [77] and refined by full
matrix least square on F2 using SHELXTL-97 [78].
Fig. 13. Hydroxyl scavenging activity of the new Pd(II) complexes (1–4).
4.2. Preparation of new palladium(II) complexes
4.2.1. Preparation of [Pd2(Msal-tsc)2(l-dppm)] (1)
0.081 g of 3-methoxysalicyaldehyde thiosemicarbazone
[H2-Msal-tsc] (0.3063 mmol) was dissolved in dichloromethane
(30 cm3) and added to K2[PdCl4] (0.100 g, 0.3063 mmol) in hot
methanol (30 cm3). The mixture was refluxed for 10 min. To this
0.058 g of 1,10-bis(diphenylphosphino)methane (0.1531 mmol)
was added. After 5 h refluxing, the reaction mixture was allowed
to stand for 3 days at room temperature. A reddish brown solid
formed was filtrated, washed with petroleum ether (60–80 °C)
and recrystallized from toluene and methanol to yield orange red
crystals. Yield: 59%, M.p. 146 °C. FT-IR (cmꢀ1) in KBr: 1596 (
1299 ( CAO), 734 (
CAS), 1437, 1096, 689 cmꢀ1 (for PPh2(CH2)
mC@N),
m
m
PPh2); UV–Vis (CHCl3), kmax (nm): 243 nm (intra-ligand transition);
298 nm (LMCT); 342, 401 nm (MLCT); 1H NMR (CDCl3, ppm): d 4.60
(s, 2H, NH2), d 7.95 (d (J = 12.8 Hz), 1H, CH = N), d 3.64 (s, 3H,
OCH3), d 6.31–7.85 (m, aromatic), d 4.82 (t, 2H, CH2).
The very similar method was followed to synthesize other
complexes.
Fig. 14. Superoxide scavenging activity of new Pd(II) complexes (1–4).
4.2.2. Preparation of [Pd2(Msal-mtsc)2(l-dppm)] (2)
Table 5
The complex 2 was prepared by the procedure as used for 1,
Quenching constant (Ksv), binding constant (K) and number of binding sites (n) for
interaction of complexes (1–4) with BSA.
with
3-methoxysalicylaldehyde-4(N)-methylthiosemicarbazone
[H2-Msal-mtsc] (0.085 g, 0.3063 mmol), K2[PdCl4] (0.100 g,
0.3063 mmol) and 1,10-bis(diphenylphosphino)methane (0.058 g,
0.1531 mmol). Yield: 60%, M.p.206 °C. FT-IR (cmꢀ1) in KBr: 1593
System
Kq (Mꢀ1
)
Kb (Mꢀ1
)
n
BSA + 1
BSA + 2
BSA + 3
BSA + 4
5.40 ꢁ 103
2.42 ꢁ 102
1.83 ꢁ 102
2.79 ꢁ 102
1.719 ꢁ 103
3.470 ꢁ 104
3.908 ꢁ 104
1.115 ꢁ 105
0.876
1.050
1.094
1.169
(mC@N), 1311 (mCAO), 732 (m
CAS), 1437, 1097, 689 cmꢀ1 (for
PPh2(CH2)PPh2); UV–Vis (CHCl3), kmax (nm): 244 nm (intra-ligand
transition); 301 nm (LMCT); 342, 397 nm (MLCT); 1H NMR

116
A. Shanmugapriya et al. / Inorganica Chimica Acta 449 (2016) 107–118
constant and [Q] is the concentration ratio of the complex. The Ksv
values have been obtained as a slope from the plot of I0/I versus [Q].
(DMSO-d6, ppm): d 7.45 (s, 1H, NHCH3), d 8.07 (d (J = 14 Hz), 1H,
CH@N), d 3.60 (s, 3H, OCH3), d 6.42–7.85 (m, aromatic), d 2.71 (d
(J = 4.4 Hz), 3H, CH3), d 4.82 (t, 2H, CH2).
4.4. Bovine Serum Albumin (BSA) binding study [66]
4.2.3. Preparation of [Pd2(Msal-etsc)2(
l-dppm)] (3)
The protein binding study was performed by tryptophan fluo-
rescence quenching experiments using Bovine Serum Albumin
(BSA, 10 lM) as the substrate in phosphate buffer (pH 7). Quench-
ing of the emission intensity of tryptophan residues of BSA at
346 nm (excitation wavelength at 276 nm) was monitored using
compound as quenchers with increasing compound concentration.
Emission spectra were recorded on a JASCO FP-6600 spectrofluo-
rometer. A 3 ml solution containing appropriate concentration of
BSA (1 ꢁ 10ꢀ6 M), was titrated with successive additions of the
complex. For synchronous fluorescence spectra of BSA with various
The complex 3 was prepared by the procedure as used for 1,
with
3-methoxysalicylaldehyde-4(N)-ethylthiosemicarbazone
[H2-Msal-etsc] (0.090 g, 0.3063 mmol), K2[PdCl4] (0.100 g,
0.3063 mmol) and 1,10-bis(diphenylphosphino)methane (0.058 g,
0.1531 mmol). Yield: 52%, M.p. 164 °C. FT-IR (cmꢀ1) in KBr: 1592
(mC@N), 1310 (mCAO), 733 (m
CAS), 1437, 1097, 686 cmꢀ1 (for
PPh2(CH2)PPh2); UV–Vis (CHCl3), kmax (nm): 243 nm (intra-ligand
transition); 300 nm (LMCT); 343, 400 nm (MLCT); 1H NMR
(DMSO-d6, ppm): d 8.17 (s, 1H, NHC2H5), d 8.05 (d (J = 13.6 Hz),
1H, CH@N), d 3.60 (s, 3H, OCH3), d 6.42–7.72 (m, aromatic), d
3.70–3.78 (m, 2H, CH2), d 1.11 (t, 3H, CH3), d 4.79 (t, 2H, CH2).
concentration of complexes (0–100
to 500 nm when k = 60 nm and from 290 to 500 nm when
k = 15 nm. The excitation and emission slit widths were 5 and
lM) were obtained from 300
D
D
4.2.4. Preparation of [Pd2(Msal-ptsc)2(
l-dppm)] (4)
6 nm, respectively. Fluorescence and synchronous measurements
were performed by using a 1 cm quartz cell on JASCO FP-6600
spectrofluorometer.
The complex 4 was prepared by the procedure as used for 1,
with
3-methoxysalicylaldehyde-4(N)-phenylthiosemicarbazone
[H2-Msal-ptsc] (0.104 g, 0.3063 mmol), K2[PdCl4] (0.100 g,
0.3063 mmol) and 1,10-bis(diphenylphosphino)methane (0.058 g,
0.1531 mmol). Yield: 57%, M.p. 189 °C. FT-IR (cmꢀ1) in KBr: 1593
4.5. Evaluation of antioxidant activity
(mC@N), 1313 (mCAO), 732 (m
CAS), 1436, 1097, 687 cmꢀ1 (for
4.5.1. DPPH (1,1-diphenyl-2-picryl-hydrazyl) radical scavenging assay
The potential antioxidant activity of the new complexes (1–4)
was evaluated by 2,20-diphenyl-1-picrylhydrazyl (DPPH) radical
scavenging assay was determined by the szabo method [81]. DPPH
free radicals are used for rapid analysis of antioxidants. While scav-
enging the free radicals, the antioxidants donate hydrogen and
form a stable DPPH molecule. Different concentration of the com-
PPh2(CH2)PPh2); UV–Vis (CHCl3), kmax (nm): 243 nm (intra-ligand
transition); 341, 406 nm (MLCT); 1H NMR (CDCl3, ppm): d 8.20
(d (J = 13.2 Hz), 1H, CH@N), d 3.70 (s, 3H, OCH3), d 6.42–7.93 (m,
aromatic), d 4.82 (t, 2H, CH2).
4.3. DNA binding study
plexes (20–100 lg/ml) were prepared and subjected to antioxidant
CT-DNA solution of various concentrations (0–50
in a trisHCl (pH 7) were added to the palladium complexes 1–4
(10 M dissolved in a DMSO–H2O mixture). Absorption spectra
were recorded after equilibrium at 20 °C for 10 min. The intrinsic
binding constant Kb was determined using the Stern–Volmer Eq.
(3) [79,80].
lM) dissolved
tests. To 1 ml of each of the extracts, 5 ml of 0.1 mM methanol
solution of DPPH was added, vortexes, followed by incubation at
27 °C for 20 min. The radical scavenging capacity was measured
every 10 min using a spectrophotometer (ELICO) by monitoring
the decrease in the absorbance at 517 nm.
l
4.5.2. Reductive ability
ð½DNAꢂ=½ea
ꢀ
ef ꢂÞ ¼ ½DNAꢂ=½eb
ꢀ
ef ꢂ þ 1=Kb½eb
ꢀ
ef
ꢂ
ð3Þ
The reducing power of the compounds has been investigated
using the Oyaizu method [82]. 1 ml of sample solution at different
concentrations was mixed with 2.5 ml of phosphate buffer
(0.2 mol/l, pH 6.6) and 2.5 ml of potassium ferricyanide (1%). The
mixture was incubated at 50 °C for 20 min 2.5 ml of trichloroacetic
acid (TCA, 10%) was added to the mixture and centrifuged at 3000 g
for 10 min. The supernatant (5 ml) was mixed with 1 ml of ferric
chloride (0.1%) and the absorbance was measured at 700 nm in a
Spectrophotometer. Increased absorbance of the reaction mixture
indicated increased reducing power.
The absorption coefficients Ɛa, Ɛf, and Ɛb correspond to Aobsd
/
[DNA], the extinction coefficient for the free complexes and the
extinction coefficient for the complexes in the fully bound form,
respectively. The slope and intercept of the linear fit of the plot
of [DNA]/[Ɛa ꢀ Ɛf] versus [DNA] give 1/[Ɛa ꢀ Ɛf] and 1/Kb[Ɛb ꢀ Ɛf],
respectively. The intrinsic binding constant Kb can be obtained
from the ratio of the slope to the intercept. It can be determined
by monitoring the changes in the absorbance in the intra ligand
band at the corresponding kmax with increasing concentration of
DNA and is given by the ratio of slope to the Y intercept in plots
of [DNA]/[Ɛa ꢀ Ɛf] versus [DNA].
4.5.3. Superoxide anion scavenging activity
The superoxide anion radical scavenging activity of new Pd(II)
complexes were done as per the Liu method [83]. Superoxide rad-
icals were generated in PMS-NADH systems by oxidation of NADH
and assayed by the reduction of nitrobluetetrazolium (NBT). 3 ml
of sample solutions at different concentrations were mixed with
4.3.1. Competitive binding with ethidiumbromide [48]
In order to know the mode of attachment of CT-DNA to the
complexes fluorescence quenching experiments of EB-DNA were
carried out by adding 10
l
L portion of 10
lM palladium(II) com-
plexes every time to the sample containing 10
l
M EB, 10
lM
1 ml of NBT (156
was initiated by adding 0.1 ml of phenazinemethosulphate (PMS)
solution (60 M) to the mixture. The reaction was incubated at
lM) and 1 ml of NADH (468 lM). The reaction
DNA and Tris buffer (pH 7). Before measurements, the system
was shaken and incubated at room temperature for ꢃ5 min. The
emission was recorded at 530–750 nm. On the basis of the classical
Stern–Volmer equation, the quenching constant has been analysed
by following equation.
l
25 °C for 5 min, and the absorbance at 560 nm was measured
against a blank. Decreased absorbance of the reaction mixture indi-
cates increased superoxide anion scavenging activity.
I0=I ¼ Ksv½Qꢂ þ 1
ð4Þ
4.5.4. Estimation of Total antioxidant capacity [71]
where I0 and I represent the emission intensities in the absence and
presence of the complexes, respectively. Ksv is the quenching
Total antioxidant was determined by the phosphomolybdenum
method followed by samples and standard (1 ml) was mixed with

A. Shanmugapriya et al. / Inorganica Chimica Acta 449 (2016) 107–118
117
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Acknowledgment
Department of Science and Technology (DST-SERB)(SR/FT/CS-
32-/2011 dated 04.05.2012) and Council of Science and Industrial
Research (CSIR) (01(2553)/12/EMR-II dated 03.04.2012) New
Delhi, India are greatly acknowledged for their financial support.
The author A.S. gratefully acknowledges the University Grants
Commission (UGC), New Delhi for UGC-BSR Fellowship, New Delhi,
India [Grand No. G2/14308/17750/UGC BSR/Selection Comt./2014
dt.15.09.2014]. The author G.K. gratefully acknowledges the
Department of Science and Technology (DST) New Delhi for
INSPIRE Fellowship, New Delhi, India [IF140225 dt.23.01.2014].
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(c) Y.W. Jung, S.J. Lippard, Chem. Rev. 107 (2007) 1387.
CCDC 1445118, 1445119 and 1445120 contains the supplemen-
tary crystallographic data. These data can be obtained free of
charge from The Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data_request/cif. Supplementary data associ-
ated with this article can be found, in the online version, at http://
dx.doi.org/10.1016/j.ica.2016.05.018.
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