Palladium(II) and platinum(II) complexes of deprotonated N,N′-bis(2-p-yridinecarboxamide)-1, 2-benzene: Synthesis, structural characterization and binding interactions with DNA and BSA

Article abstract of DOI:10.1016/j.ica.2013.04.033

Two neutral complexes [ML] (where M = Pd (1a) and Pt (1b), L = bis(pyridine-2-carboxamide)benzene dianion) have been synthesized and characterized by physico-chemical and spectroscopic tools along with the detailed structural analysis by single crystal X-

Full text of DOI:10.1016/j.ica.2013.04.033

Accepted Manuscript
Palladium(II) and platinum(II) complexes of deprotonated N,N’-bis(2-pyridi‐
necarboxamide)-1,2-benzene: synthesis, structural characterization and binding
interactions with DNA and BSA
Titas Mukherjee, Buddhadeb Sen, Ennio Zangrando, Geeta Hundal, Basab
Chattopadhyay, Pabitra Chattopadhyay
PII:
S0020-1693(13)00223-5
http://dx.doi.org/10.1016/j.ica.2013.04.033
ICA 15438
DOI:
Reference:
To appear in:
Inorganica Chimica Acta
Received Date:
Revised Date:
Accepted Date:
18 December 2012
20 March 2013
23 April 2013
Please cite this article as: T. Mukherjee, B. Sen, E. Zangrando, G. Hundal, B. Chattopadhyay, P. Chattopadhyay,
Palladium(II) and platinum(II) complexes of deprotonated N,N’-bis(2-pyridinecarboxamide)-1,2-benzene:
synthesis, structural characterization and binding interactions with DNA and BSA, Inorganica Chimica Acta (2013),
doi: http://dx.doi.org/10.1016/j.ica.2013.04.033
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Palladium(II) and platinum(II) complexes of deprotonated
N,N'-bis(2-pyridinecarboxamide)-1,2-benzene: synthesis, structural
characterization and binding interactions with DNA and BSA
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Titas Mukherjee , Buddhadeb Sen , Ennio Zangrando , Geeta Hundal , Basab Chattopadhyay ,
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and Pabitra Chattopadhyay *
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Department of Chemistry, Burdwan University, Golapbag, Burdwan-713104, India
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Dipartimento di Scienze Chimiche e Farmaceutiche, Via Licio Giorgieri 1, 34127 Trieste, Italy
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Department of Chemistry, Guru Nanak Dev University, Amritsar-143005, India
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Department of Solid State Physics, Indian Association for the Cultivation of Science, Jadavpur,
Kolkata 700032, India
Abstract
Two neutral complexes [ML] (where M = Pd (1a) and Pt (1b), L = bis(pyridine-2-
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carboxamide)benzene dianion) have been synthesized and characterized by physico-chemical
and spectroscopic tools along with the detailed structural analysis by single crystal X-ray
crystallography and theoretical (DFT) study. In solid state the compounds are isomorphous and
isostructural showing the formation of [ML] .3H O trimeric species. Electrochemical study of 1a
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showed a quasi-reversible reductive response at E1/2 = –1.148 V assignable to the Pd(II)/Pd(I)
couple, while a metal centered irreversible oxidative peak centred at +0.977 V was observed in
the voltammogram of 1b. The interaction of the complexes with CT-DNA has been investigated
using spectroscopic tools and viscosity measurement. In each case the association constant (Kb)
was deduced from the absorption spectral study and the number of binding sites (n) and the
binding constant (K) were calculated from relevant fluorescence quenching data, suggesting a
non-covalent interaction between the metal complex and DNA, which could be assigned to an
intercalative binding. In addition, the interaction of 1a and 1b was ventured with bovine serum
albumin (BSA) with the help of absorption and fluorescence spectroscopy measurements.
Through these techniques, the apparent association constant (K ) and the binding constant (K)
app
could be calculated for each complex.
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Keywords: Palladium(II) complex; platinum(II) complex; crystal structure; DNA and BSA
binding study.
*Corresponding author: E-mail: pabitracc@yahoo.com
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1. Introduction
The last few decades have witnessed a remarkable interest in pyridine carboxamide
complexes in various fields of biological relevance like asymmetric catalysis, [1,2] dendrimer [3]
and molecular receptor synthesis, [4] and also in the synthesis of compounds with possible
antitumour properties [5]. The carboxamide [–C(O)NH–] group of the primary structure of
proteins represents an important ligand construction unit in coordination chemistry, since its
chelating rigid nature imparts a unique balance of stability versus reactivity, and has allowed for
impressive developments in a variety of catalytic transformations.
Deprotonated amide groups readily coordinate metal ions through the amide-N and/or -O
atom [6] forming a stable delocalized electronic system. The anticancer properties of cisplatin
and palladium(II) complexes stem from the ability of the cis-MCl fragment to bind to DNA
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bases. However, cisplatin also interacts with non-cancer cells, mainly binding molecules
containing -SH groups, resulting in nephrotoxicity [7]. This has aroused interest in the design of
novel palladium(II) [8] and platinum(II) complexes with better efficacy and lower toxicity.
Serum albumins are the major soluble protein constituents of the circulatory system and serve as
a depot- and as a transfer protein along with several physiological functions. BSA has been one
of the most extensively studied of these proteins, particularly because of its structural homology
with human serum albumin [9].
In the present work we have turned our attention to explore the amide functionality of the
'
N,N -bis(2-pyridinecarboxamide)-1,2-benzene ligand (H L) towards palladium(II) and
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platinum(II) ion. We report herein two novel Pd(II) (1a) and Pt(II) (1b) complexes with the
tetradentate ligand having two deprotonated amide-N and two pyridinic-N donors. These species
were structural characterized by X-ray diffraction and also by means of different physico-
chemical, spectroscopic and computational studies. In addition, interaction of complexes 1a and
1b with CT-DNA and also with bovine serum albumin (BSA) have been studied. In order to
establish the association mode of these small molecules to DNA, the binding (K ) and the
b
quenching constant (K ) of the complexes with the double-helix has been determined from UV-
q
Vis study and fluorescence displacement experiments using ethidium bromide as spectral probe.
Beside this, the strong binding activity of complexes 1a and 1b with BSA was examined using
absorption-fluorescence spectroscopy, further supported by viscosity measurements.
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2. Experimental
2.1. Materials and Instrumentation
All chemicals and reagents were obtained from commercial sources and used as received,
unless otherwise stated. 2-Pyridinecarboxylic acid, 1,2-diaminobenzene and triphenyl phosphite
were purchased from Aldrich and K PtCl and PdCl procured from Across, were used as such.
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The solvents used were purified following the standard procedures, all other chemicals used were
of analytical reagent grade.
The elemental (C, H, N) analyses were performed on a Perkin Elmer model 2400 elemental
analyzer. Electronic absorption spectra and IR spectra were obtained on a JASCO UV–Vis/NIR
spectrophotometer model V-570 and on a Perkin-Elmer FTIR model RX1 spectrometer (KBr
-1
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discs, 4000-300 cm ), respectively. H NMR spectra were recorded on a Bruker AC300
spectrometer using TMS as an internal standard in DMSO-d solvent. Electrospray ionization
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(ESI) mass spectra of complexes 1a and 1b were recorded with a QtofMicro Instrument (Waters,
YA263). The fluorescence spectra complex bound to DNA were obtained at an excitation
wavelength of 522 nm in the Fluorimeter (Hitachi-2000). Viscosity experiments were conducted
on an Ostwald’s viscometer, immersed in a thermostated water-bath maintained at 25 °C. Redox
potentials were measured in CHI620D potentiometer in DMF using TBAP as supporting
electrolyte at room temperature. Electrochemical setup was a three-electrode cell with glassy
carbon, Ag/AgCl and a platinum wire as a working, reference and counter electrode,
respectively. Molar conductances ( ) were measured in a systronics conductivity meter 304
M
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model in dimethylformamide at complex concentration of ~10 mol L .
2.2. Preparation of the ligand
The ligand H L was synthesized following reported method [10] with slight modification.
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Pyridinic solution (10 ml) of 2-pyridinecarboxylic acid (1.23 g, 10 mmol) and 1,2-
diaminobenzene (0.54 g, 5 mmol) was mixed under stirring condition followed by the dropwise
addition of triphenyl phosphite (3.1 g, 10 mmol) at 80 °C for 4 hrs. and settled overnight. A pale
brown crystalline solid resulted, was washed with methanol to give long white needles. Yield;
96%. Anal. Calc. for C H N O : C, 67.91; H, 4.43; N, 17.60. Found: C, 68.16; H, 3.94; N,
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1762.18. Anal. Calc.: IR (KBr, cm ): ν
1677; νNH, 3317.9. ESI-MS (m/z): parent 318.11 (100
C=O,
+
% abundance); [M+H ] 319.12 (19.7 % abundance).
2.3. Preparation of the complexes
2.3.1. Synthesis of Pd(L) (1a)
To a solution of H L (0.636 g, 0.50 mmol) in dry DMF (10 mL) was added NaH (0.0237 g,
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1.00 mmol) and the resulting suspension was stirred for 30 min. To the resulting light yellow
solution, PdCl (0.089 g, 0.50 mmol) dissolved in DMF was added in portions with continuous
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stirring for a period of 3 h. The yellow precipitate resulted was filtered and washed with diethyl
ether and vacuum dried. The residue was further dissolved in DMF (6.0 mL), filtered, and the
volume of the filtrate was reduced to 3.0 mL. Yellow crystals, suitable for X-ray diffraction,
were obtained by slow evaporation.
(1a).H O: C H N PdO : Anal. Calc.: C, 49.05; H, 3.20; N, 12.71. Found: C, 48.26; H,
2
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3.94; N, 12.18. IR (KBr,cm ): ν
1630; Pd-N, 415. ESI-MS (m/z): [M+Na ], 463.01 (16 %
C=O,
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abundance); [M+H ] 442.01 (96.2 % abundance). Conductivity (Λo, ohm cm mol ) in DMF:
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51. UV-Vis in DMF, λ nm (ε) (ε, dm mol cm ): 281 (8,264), 305 (6,960). Yield: 65-68%.
2.3.2. Synthesis of Pt(L) (1b)
To a solution of H L (0.636 g, 0.50 mmol) in dry DMF (10.0 mL), NaH (0.0237 g, 1.00
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mmol) was added and the resulting suspension was stirred for 30 min. To the resulting light
yellow solution, aqueous K PtCl (0.2075 g, 0.50 mmol) was added in portions with vigorous
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stirring under nitrogen atmosphere and stirring has continued for further 10 h. The resulting
orange-red solution was allowed to evaporate slowly, obtaining orange micro crystals suitable
for X-ray diffraction studies.
(1b).H O: C H N PtO : Anal.: C, 40.83; H, 2.66; N, 10.58 Found: C, 40.67; H, 2.64; N,
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10.54. IR (KBr, cm ): ν
1637; Pt-N, 435. ESI-MS (m/z): [M+Na ], 554.22 (20 %
C=O,
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abundance); [M+H ], 532.22 (81 % abundance). Conductivity (Λo, ohm cm mol ) in DMF:
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48. UV-Vis in DMF, λ nm (ε) (ε, dm mol cm ): 278 (5,142), 313 (5,564). Yield: 50-52 %.
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2.4. X-ray data collection and structural determination
Data collections of compounds 1a and 1b were carried at room temperature on a Bruker
Smart Apex diffractometer equipped with CCD. (λ = 0.71073 Å). Cell refinement, indexing and
scaling of the data sets were done by using programs Bruker Smart Apex and Bruker Saint
packages. [11] The structures were solved by direct methods and subsequent Fourier analyses
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and refined by the full-matrix least-squares method based on F with all observed reflections
[12]. Hydrogen atoms were placed at calculated positions, those of lattice water molecules of
compound 1b were located on the ΔFourier map and analogously for molecule Ow1 of 1a. All
the calculations were performed using the WinGX System, Ver 1.80.05. [13] Crystal data and
details of refinements are given in Table 1.
2.5. DNA binding studies of palladium(II) and platinum(II) complexes
The binding experiments with calf thymus DNA for complexes 1a and 1b were monitored
following the same procedure previously reported by us [14] with UV-Vis and fluorescence
spectroscopic tools and also by viscosity and cyclic voltammetry measurements.
All the experiments involving the interaction of the complexes with CT-DNA were carried
out in MilliQ water containing Tris–HCl buffer (pH 8.04). The solution of CT-DNA in the buffer
gave a ratio of UV absorbance of ca. 1.8 –1.9:1 at 260 and 280 nm, indicating that the CT-DNA
was sufficiently free of protein [15]. Stock solution of DNA was always stored at 4 ºC in the dark
and used within four days. The CT-DNA concentration per nucleotide was determined
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spectrophotometrically by employing an extinction coefficient of 6600 M cm at 260 nm [16].
The complexes were dissolved in a solvent mixture of 1% DMSO and 99% tris–HCl buffer at
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1.0×10 M concentration. Absorption spectral titration experiment was performed by keeping
constant the concentration of the complex (10 µM) and varying the CT-DNA concentration.
While measuring the absorption spectra, an equal amount of CT-DNA was added to both the
complex solutions and the reference solution to take into account the absorbance of DNA itself.
In the emission quenching experiment, ethidium bromide (EB) was used as a common
fluorescent probe for the DNA in order to examine the mode and process of metal complex
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binding to the double-helix [17]. A 5.0 µL of the EB tris–HCl buffer solution (1.0 mmol L ) was
added to 1.0 mL of DNA solution (at saturated binding levels) [18], stored in the dark for 2 h.
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Then the solution of each of the Pd(II) and Pt(II) complexes was titrated into the DNA/EB
mixture and diluted in tris–HCl buffer to 5.0 mL to get the solution with the appropriate
complex/ CT-DNA mole ratio. After the incubation at room temperature for 30 min, the
fluorescence spectra of EB bound to DNA were recorded (λ = 522 nm) in the Hitachi 4500
ex
Fluorimeter. All measurements were performed at ambient temperature.
The binding interaction of the metal complexes with DNA was studied by the well known
method employing the Ostwald’s viscometer. The CT-DNA solution (5 µM) was titrated with
Pt(II) and Pd(II) complexes (0.5–3.5 µM), following the change of the viscosity in each case.
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Data are presented as (η / η0) versus the ratio of the concentration of the compound and CT-
DNA, where η is the viscosity of CT-DNA in presence of the compound and η is the viscosity
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of CT-DNA alone. Viscosity values were calculated from the observed flow time of CT-DNA-
containing solution corrected from the flow time of buffer alone (t ), η = t-t [19].
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2.6. Protein (BSA) binding experiments of palladium(II) and platinum(II) complexes
The binding study with bovine serum albumin (BSA) for complexes 1a and 1b were done
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dissolving the BSA in MilliQ water (1.0×10 M ) and the stock solutions of each of the
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complexes were prepared in DMSO-H O (1:99 v/v) mixture at 1.0×10 M concentration. Both
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the absorption and fluorescence quenching experiments (λ = 280 nm) were performed by
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gradually increasing the complex concentration, keeping fixed the concentration of BSA. All the
experimental sets were carefully degassed purging pure nitrogen gas for 5 min.
2.7. Computational details
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The DFT calculations for the isolated complexes 1a and 1b were performed using Dmol
code [20] in the framework of a generalized-gradient approximation (GGA) [21]. The geometry
of the molecules were fully optimized using the hybrid exchange-correlation functional BLYP
[22] and a double numeric plus polarization (DNP) basis set (Supplementary material, Figs. s1-
s3). The electronic structures were also calculated at the same level. No constraints on bond
lengths, bond angles or dihedral angles were applied in the calculations, and all atoms were free
to optimize. Convergence was assumed to be reached when the total energy change between two
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consecutive self-consistent field (SCF) cycles was less than 1x10 a.u.
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3. Results and Discussion
3.1. Synthesis and characterization
The organic ligand L was synthesized by the reaction of 1, 2-diaminobenzene with 2-
pyridinecarboxylic acid stirring at 80-85 °C in pyridine medium, and it has been characterized by
1
IR and H NMR spectral analyses. The palladium(II) and platinum(II) complexes 1a and 1b were
obtained in good yield from the reaction of palladium(II) chloride and potassium tetrachloro
platinate(II), respectively with the tetradentate ligand L in 1:1 molar ratio in the DMF medium
with prolonged stirring at room temperature (viz Scheme 1).
The monomeric complexes 1a and 1b are soluble in DMF and DMSO but insoluble in
methanol and ethanol. The conductivity measurement of complexes in DMF showed the
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-1
conductance values in the range of 42-53 Λ mol cm at 300 K. These values suggest that the
o
complexes exist as non-electrolytes in solution [23].
3.2. Structural description of 1a and 1b
The X-ray structural analysis show that complexes 1a and 1b are isomorphous and
isostructural showing the formation of [ML] .3H O aggregates (M = Pd and Pt, respectively L =
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bis(pyridine-2-carboxamide)benzene dianion. The independent crystallographic unit comprises
of one and half neutral metal complex, being one located on a two-fold axis passing through the
metal and bisecting the Namide-M-Namide bond angle, as shown in Figs. 1 and 2 for the Pt
derivative 1b. The crystals contain also some disordered water lattice molecules.
As expected, in 1a and 1b the metal ion is chelated by the tetradentate dianionic ligand L
in a square planar coordination geometry and due to the nature of the ligand all the atoms in each
complex are coplanar. The bond lengths reported in Table 2 indicate that the values relative to
the pyridine N donors are significantly longer by ca. 0.1 Å than those of the amide nitrogen
atoms, either in the Pd and Pt complexes, and the Pd-N bond values are in agreement with those
reported in analogous complexes [24]. On the other hand the Pd-N bond lengths appear slightly
longer with respect to the correspondent Pt-N values in agreement with the metal ionic radius.
It is worth noting of the supramolecular arrangement observed in these compounds, being
the complexes arranged as trimeric entities where complex Pt2 (or Pd2) of C2 symmetry (Figs. 1
and s4) is stacking in between a pair of two symmetry related Pt1 (or Pd1) units (Figs. 3 and s5).
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Within this trimer the Pt metal ions in 1b are separated by 3.2897(1) Å, in an almost collinear
arrangement forming a Pt(1)-Pt(2)-Pt(1) angle of 178.64(1)°. The correspondent figures in 1a are
close comparable being of 3.2903(4) Å and 179.33(1)°, respectively. This packing feature is not
unusual for square planar complexes having aromatic ligands and the aggregation of complexes
are stabilized by a combination of π-π stacking interactions between the pyridine and phenyl
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rings of the ligands and d -d metallophilic contacts. [25-27]. A structural indication of the latter
interaction between the metals is the slight displacement from the N donor set plane of atom
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M(1) by ca. 0.03 Å towards M(2) (M = Pt or Pd) inside each trimer. A rotation of ca. 140° is
requested to complex Pt1 in order to be superimposed to Pt2 that occupies the center (Fig. 3),
and similarly for the Pd complex.
Since the number of water molecules and crystal packing are close comparable for 1a and
1b, we limit here the description for the former compound. The lattice water molecules reside
sideways to the trimer entities and are connected through H-bonds. In fact O2w, which is located
on a two-fold axis, is weakly bound to O1w (O…O = 3.16 Å), which in turn connects the
carbonyl oxygens O(1) and O(3) of symmetry related complexes (O….O = 2.80, 3.01 Å,
respectively) forming a 2D packing arrangement parallel to the crystallographic ab plane.
3.3. Spectral properties
3.3.1. Electronic absorption spectral studies
The electronic absorption spectra of complexes 1a and 1b were recorded at room
temperature using DMF as solvent. The spectra exhibit a sharp band around 280 nm assignable
to the intramolecular π  π* transition. Another relative high intensity band around 310 nm is
due to a charge transfer from amide ligand core to metal, i.e. LMCT. The low energy tail of the
charge transfer band that appears in the visible region of the spectrum is responsible for the
yellow and orange-yellow color of the solution containing 1a and 1b, respectively. Here, it is
observed that the transition for the palladium(II) complex shifted to lower energy compared to
that of the platinum(II) derivative, and this study is in accordance with the theoretical calculation
of the energy of HOMO and LUMO for 1a and 1b, being LUMO for complex 1a lower lying
compared to 1b (Fig. s3).
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3.3.2. Electrochemistry
The electrochemical properties for 1a and 1b have been studied by cyclic voltammetry (CV)
at a platinum working electrode in dimethylformamide (0.1 M TBAP as supporting electrolyte)
at room temperature. The cyclic voltammograms of 1a and 1b are displayed in Figs. 4 and 5,
respectively. The CV scan of 1a revealed a one-electron quasi-reversible reductive response at
E1/2 = –1.148 V (E = –1.245 V and E = –1.051 V; E = 194 mV) assignable to Pd(II)/Pd(I)
pc
pa
–
couple. On the contrary, platinum(II) in 1b is irreversibly oxidized to platinum(IV) by 2e
stoichiometry [28, 29] centred at +0.977 V. From the theoretical calculation of HOMO and
LUMO energy of both complexes, it may be derived that the comparatively lower energy of
LUMO of 1a may be responsible for the reduction of Pd(II)/Pd(I) and accordingly the higher
energy of HOMO of 1b for the irreversible oxidation of Pt(II) to Pt(IV) (Fig. s3).
3.4. Binding experiments with calf thymus-DNA
The mode of interaction of the complexes 1a and 1b with calf thymus DNA (CT-DNA) has
been investigated using absorption and emission spectroscopic tools as well as by viscosity and
cyclic voltammetry measurements.
3.4.1. Absorption spectroscopy
Electronic absorption spectroscopy is used as a distinctive characterization tool for
examining the binding mode of metal complexes with DNA [18, 30]. In intercalative binding
mode, the * orbital of the intercalated ligand can couple with the  orbital of the base pairs, thus
decreasing the * transition energy and resulting in bathochromism. On the other hand, the
coupling  orbital was partially filled by electrons, thus decreasing the transition probabilities
and concomitantly resulting in hypochromism [31]. The absorption spectra of the free metal
complexes and of their adducts with CT-DNA (at a constant concentration of the compounds) are
given in Figs. 6 and s6 for complexes 1a and 1b, respectively. The extent of hyperchromism in
the absorption band is generally consistent with the strength of intercalative interaction [28-31].
As the concentration of CT-DNA is increased, it was found that the Pd and Pt complexes at 270
nm and 268 nm exhibit hyperchromicity of 3.5/19.76% and 3.7/24.02%, respectively. This
feature might be ascribed to the fact that both of the co-complexes could uncoil the helix
structure of DNA and made more bases embedding in DNA exposed [32-34]. In order to
2
2
2
2
2
2
2
2
2
63
64
65
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67
68
69
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71
9

2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
3
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
00
establish the binding strength of the metal complexes with CT-DNA, the apparent association
constant K was determined from the spectral titration data using the following equation [35]
b
1/Δ = 1/ (ΔK D) + 1/Δ
ap
b
where, Δ =  - , Δ=  - , D = [DNA], and   and  are respectively the apparent, bound
ap
a
f
b
f
a,
b
f
–
and free extinctions coefficient of each of the compound in respective cases. K , expressed as M
b
1
, is derived from the slope of the graph obtained by plotting the [DNA]/( - ) vs [DNA] (Fig.
a
f
4
–1
7). The K values for complexes for 1a and 1b were estimated to be 0.36 × 10 M (R = 0.99999,
b
4
–1
n = 5 points) and 0.93× 10 M (R = 0.99885, n = 5 points), respectively.
In order to corroborate the binding mode of intercalation of the Pd(II) and Pt(II) complexes
with CT-DNA, we employed ethidium bromide (EB) that, interacting with DNA, represents a
characteristic indicator of intercalation. The maximal absorption of EB at 479 nm decreased and
shifted to 499 nm in presence of DNA (Fig. s7), typically indicating insertion between the base
pairs [36]. The absorption spectra of the mixture solution of EB, palladium(II) complex 1a and
DNA and similarly for platinum(II) complex 1b are reported as supplementary (Figs. s7(a) and
s7(b), respectively). The observed behavior could be indicative of: (1) being EB strongly bound
to complex 1a (or 1b), the result is a decrease amount of EB intercalated into DNA; or (2) there
exists a competition between the palladium(II) (or platinum(II)) complex and EB towards DNA
intercalation, so releasing some free EB from DNA–EB complex. However, here the former
account could be irrelevant because of the appearance of a new absorption band.
3.4.2. Fluorescence quenching analysis
The binding propensity of palladium and platinum complexes to CT-DNA has been
analyzed by the steady-state emission quenching experiments using the emission intensity of
ethidium bromide (EB). It is well known that EB can intercalate nonspecifically with DNA,
causing a strong fluorescence. Other compounds competing with EB to intercalation in DNA will
induce displacement of bound EB and a decrease in the fluorescence intensity. This
fluorescence-based competition can provide indirect evidence for the DNA-binding mode. The
fluorescence intensity of the EB/DNA system (with excitation wavelength of 522 nm) is reduced
by the increasing concentration of the complexes (Figs. 8 and s8), and caused by EB migration
1
0

3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
from a hydrophobic to an aqueous environment [37]. The quenching of EB bound to DNA by 1a
and 1b is in agreement with the linear Stern–Volmer equation [38]:
I /I = 1+ K [Q]
0
q
where I and I represent the fluorescence intensities in the absence and presence of quencher,
0
respectively. K is a linear Stern–Volmer quenching constant, Q is the concentration of the
q
quencher. In the quenching plot (insets of Figs. 8 and s8) of I /I vs. [complex], Kq is given by
0
4
4
the ratio of the slope to the intercept. The K values are 0. 14  10 and 0.44 10 for complexes
q
1a and 1b respectively implies that both complexes can insert between DNA base pairs and that
the platinum(II) complex can bind to DNA more strongly than the palladium(II) complex which
is consistent with the absorption data.
The titration data obtained from the fluorescence experiment can be helpful also to
calculate the number of binding sites and the apparent binding constant. In the following
equation [39]:
log[(I - I)/I] = logK + n log[Q]
0
K and n represent the binding constant and number of binding sites of palladium complex to CT-
DNA, respectively. The number of binding sites n, determined from the intercept of log[(I – I)/I]
0
vs log[Q] (Fig. 9), are 1.07 and 1.18 for 1a and 1b, respectively, indicating the existence of about
a single binding site in DNA and a weaker association for the complexes. The K values were
4
4
calculated to be 0.32  10 and 0.77  10 for 1a and 1b, respectively, with a trend similar to the
apparent association constant values of the complexes.
3.4.3. Viscosity measurements
Since optical photophysical probes generally provide necessary, but insufficient clues to
further clarify the interactions between the complex and DNA, viscosity measurements were
carried out. Hydrodynamic measurements, sensitive to length change (i.e. viscosity and
sedimentation), are regarded as the least ambiguous and the most critical tests of binding in
solution in the absence of crystallographic structural data. A classical intercalation model
demands that the DNA helix lengthens as base pairs are separated in order to accommodate the
binding ligand, leading to an increase in DNA viscosity. In contrast, a partial, non-classical
intercalation of compound could bend (or kink) the DNA helix, reducing its effective length and,
1
1

3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
concomitantly, its viscosity [19]. The results obtained in these viscosity measurement studies
suggest that both the compounds 1a and 1b can intercalate between adjacent DNA base pairs,
causing an extension of the helix with a concomitant increase of the DNA viscosity. The effects
of both compounds on the viscosity of DNA are shown in Fig. 10.
3.5. Binding experiments with bovine serum albumin (BSA)
3.5.1. Absorption spectral characterization
The binding mode of complexes 1a and 1b with BSA were examined by electronic
absorption titration with BSA. The absorption spectra of the free metal complexes and of their
adducts with BSA are given in Figs. 11 and s9 for complex 1a and 1b, respectively. The spectra
indicate a significant increase in the absorbance of BSA by increasing the concentration of the
complex and are indicative of the fact that BSA adsorbs strongly the complex on its surface [40].
From these titration data the apparent association constant (K ) of the complexes with BSA has
app
been determined using the following equation [31]:
1/(Aobs – A ) = 1/(A – A ) + 1/ K (A – A ) [comp]
0
c
0
app
c
0
where, Aobs is the observed absorbance of the solution containing different concentrations of the
complex, A and Ac are the absorbance of BSA and of the complex at 280 nm, respectively. The
0
enhancement of the absorbance at 280 nm was attributable to the complex absorption at BSA
surface. Based on the linear relationship between 1/(Aobs - A ) vs the reciprocal concentration of
0
the complex with a slope of 1/K (A - A ) and an intercept equal to 1/(A - A ) (Fig. 12), the
app
c
0
c
0
5
-1
5
value of Kapp was determined to be 1.262× 10 M (R = 0.99967, n = 5 points) and 1.402× 10
-1
M (R = 0.99991, n = 5 points), for 1a and 1b, respectively.
3.4.2. Fluorescence quenching analysis
In the fluorescence quenching experiment, the fluorescence emission spectrum of BSA was
studied increasing the concentration of the quencher (Figs. 13 and s10). The fluorescence
quenching is described by the Stern–Volmer relation, [40] similarly as described above for CT-
DNA binding experiments. From the slope of the regression line in the derived plot of I /I vs
0
[complex] (insets of Figs. 13 and s10) the K values for the complexes were determined to be
q
1
2

4
4
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
4.29 10 for 1a (R = 0.99825 for five points) and 4.62  10 for 1b (R = 0.99948 for five
points), indicating a strong affinity of both of the complexes to BSA.
4. Conclusions
Two novel square planar palladium(II) and platinum(II) complexes 1a and 1b of
'
deprotonated tetradentate ligand N,N -bis(2-pyridinecarboxamide)-1,2-benzene have been
synthesized and characterized using various spectroscopic measurements. The X-ray structural
characterization revealed that the palladiumd(II) and platinum(II) derivatives are isomorphous
and are packed to form a trimeric motif with complexes connected by π-π interactions between
the aromatic rings of the ligands and metallophilic bonding. The complexes have been found to
interact with CT-DNA through an intercalative mode, which was investigated by absorption,
fluorescence and viscosity measurement tools. The quenching rate constant, binding constant and
number of binding sites were calculated according to the relevant fluorescence data. The binding
constants indicate that the DNA-binding affinity, as well as the binding trend with BSA,
increases from Pd(II) to Pt(II), in accordance with the relevant viscosity measurement study. The
information obtained from the present work is indicative of the development of potential probes
of DNA structure in future applications.
5. Supporting material
Crystallographic data for the structural analyses have been deposited with the Cambridge
Crystallographic Data Centre, CCDC Nos. 856198 and 873080 for compounds 1a and 1b,
respectively. Copies of this information are available on request at free of charge from CCDC,
12 Union Road, Cambridge, CB21EZ, UK (fax : +44-1223-336-033; e-mail: deposit@ccdc.ac.uk
or http://www.ccdc.cam.ac.uk). MO diagrams from DFT calculations (Figs. s1-s3); ORTEP
drawings of the independent units of 1a (Fig.s s4, s5) and the spectral data of the binding
interactions of complex 1b with DNA and BSA (Fig.s s6-s10). Supplementary data associated
with this article can be found, in the online version, at http:// XXXXXXX.
1
3

3
3
3
3
3
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3
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3
3
3
3
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4
4
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4
4
4
4
88
89
90
91
92
93
94
95
96
97
98
99
00
01
02
03
04
05
06
07
08
Acknowledgements
Financial support from Council of Scientific and Industrial Research (CSIR), New Delhi,
India is gratefully acknowledged. E. Zangrando thanks MIUR-Rome, PRIN 2007HMTJWP_002
for financial support.
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1
5

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48
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55
Figures’ legend
Fig.1. ORTEP drawing (35% ellipsoid probability) of complex B of 1b located on a
crystallographic two-fold axis. (The same label scheme applies also to 1a; primed atoms at -
x, y, -z+1/2).
Fig. 2. ORTEP drawing (35% ellipsoid probability) of complex A of 1b. (The same label scheme
applies also to 1a).
Fig. 3. Complex trimers in the crystal packing of 1b connected through H-bonds occurring
among carbonyl groups and lattice water molecules. A similar packing is observed in 1a.
4
4
56
57
Fig.4. Cyclic voltammogram (scan rate 100 mV/s) of 1a in DMF solution of 0.1 M TBAP, using
platinum working electrode.
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
Fig.5. Cyclic voltammogram (scan rate 100 mV/s) of 1b in DMF solution containing 0.1 M
TBAP, using platinum working electrode
Fig.6. Electronic spectral titration of complex 1a with CT-DNA at 271 nm in Tris-HCl buffer;
-5
-6
-6
6
-6
[1a] = 2.62×10 ; [DNA]: (a) 0.0, (b) 1.25×10 , (c) 2.5×10 , (d) 3.75×10 , (e) 5.0×10 , (f)
-6
-1
6.25×10 mol L . The arrow denotes the gradual increase of DNA concentration.
Fig. 7. Comparative plot of [DNA]/( - ) vs [DNA] for the absorption titration of CT-DNA with
a
f
4
-1
complexes 1a and 1b in Tris-HCl buffer; association constant K : 0.36 × 10 M (R =
b
4
-1
0.99999, n = 5 points) for 1a; 0.93× 10 M (R = 0.99885, n = 5 points) for 1b.
Fig.8. Emission spectra of the CT-DNA-EB system in Tris-HCl buffer upon titration with
-5
-5
-5
complex 1a. λ = 522 nm; [EB] = 9.6×10 ; [DNA] = 1.25×10 ; [1a]: (a) 0.0, (b) 1.31×10 ,
ex
-5
-5
-5
-5
-1
(c) 2.62×10 , (d) 3.93×10 , (e) 5.24×10 , (f) 6.55×10 mol L . The arrow denotes the
gradual increase of complex concentration. Inset shows the plot of I /I vs. [1a]; K = 0.16
0
q
4
10 (R = 0.99876, n = 5 points)
Fig.9. Comparative plot of log[I -I/I] vs. log[complex] for the titration of CT-DNA-EB system
0
with complexes 1a and 1b in tris-HCl buffer medium.
Fig.10. Effect of increasing amounts of Pd(II) and Pt(II) complexes on the relative viscosity of
CT-DNA at 25 °C.
Fig.11. Absorption titration spectra of BSA in presence of complex 1a. Concentration range of
-6
-1
complex is 0-6.25× 10 M
1
6

_
4
4
4
4
4
4
77
78
79
80
81
82
Fig. 12. The linear dependence of 1/A A on the reciprocal concentration of complexes 1a and
0
1b.
Fig.13. Fluorescence quenching titration of BSA varying the concentrations of complex 1a,
-6
[complex] = 0, 1, 2, 3, 4 and 5 × 6.35 ×10 M. Inset shows the Stern–Volmer plot.
Scheme 1. Synthetic routes of the complexes
1
7

4
4
4
83
84
85
Table 1. Crystallographic data for compounds 1a and 1b.
1
a.1.5H20
1b.1.5H20
Empirical Formula
C H N O Pd
C H N O Pt
54 42 12 9 3
5
4
42 12
9
3
Fw
1322.20
1588.27
Crystal system
Space group
a, Å
Monoclinic
C 2/c
14.5777(5)
14.5231(17)
20.2671(17)
16.4470(17)
97.365(3)
b, Å
20.2335(6)
16.4016(5)
97.237(2)
4799.2(3)
c, Å
β, deg
3
V, Å
4801.1(9)
Z
4
–
3
Dcalcd, g cm
1.829
2.198
–
1
μ (Mo-Kα) mm
F(000)
1.188
8.799
2640
3024
θ range, deg
1.73 - 28.22
26979
1.73 - 31.00
36287
no. of reflns collcd
no. of indep reflns
Rint
5836
0.0513
3951
357
7530
0.0325
5593
362
no. of reflns (I>2σ(I))
no. of refined params
2
goodness-of-fit (F )
1.025
1.010
[
a]
R1, wR2 (I >2σ(I))
0.0344, 0.0767
0.0623, 0.0890
0.0231, 0.0487
0.0428, 0.0554
R indices (all data)
4
4
86
87
[
a]
2
2
2
2 2 ½
R1 = Σ Fo–Fc / Σ Fo, wR2 = [Σ w (Fo – Fc ) / Σw (Fo ) ]
4
4
88
89
1
8

4
90
Table 2. Coordination bond lengths (Å) and angles (°) for compounds 1a and 1b.
1
a, M= Pd
1b, M= Pt
M(1)-N(1)
M(1)-N(4)
M(1)-N(2)
M(1)-N(3)
M(2)-N(5)
M(2)-N(6)
M(1)-M(2)
2.054(3)
2.055(3)
1.939(3)
1.951(3)
2.053(3)
1.946(3)
3.2903(4)
2.040(3)
2.044(2)
1.953(2)
1.944(3)
2.043(3)
1.958(2)
3.2897(1)
N(1)-M(1)-N(2)
N(1)-M(1)-N(3)
N(1)-M(1)-N(4)
N(2)-M(1)-N(3)
N(2)-M(1)-N(4)
N(3)-M(1)-N(4)
N(5)-M(2)-N(6)
N(5)-M(2)-N(6')
N(5)-M(2)-N(5')
N(6)-M(2)-N(6')
M(1)-M(2)-M(1')
81.33(10)
165.28(11)
112.51(10)
84.22(11)
166.13(11)
81.91(11)
81.85(11)
165.73(11)
112.41(15)
83.90(15)
179.33(1)
81.28(10)
165.79(10)
112.64(10)
84.81(10)
166.01(10)
81.22(10)
81.37(11)
165.90(10)
112.72(14)
84.55(15)
178.64(1)
4
91
Primed atoms at -x, y, -z+1/2.
4
4
4
4
92
93
94
95
1
9

4
4
96
97
4
4
5
5
5
98
99
00
01
02
5
5
5
03
04
05
Fig. 1. ORTEP drawing (35% ellipsoid probability) of complex B of 1b located on a
crystallographic two-fold axis. (The same label scheme applies also to 1a; primed atoms at
-x, y, -z+1/2).
5
5
5
5
5
5
5
06
07
08
09
10
11
12
5
5
13
14
Fig. 2. ORTEP drawing (35% ellipsoid probability) of complex A of 1b. (The same label scheme
applies also to 1a).
5
5
5
15
16
17
2
0

5
5
5
5
5
5
5
5
5
18
19
20
21
22
23
24
25
26
5
5
27
28
Fig. 3. Complex trimers in the crystal packing of 1b connected through H-bonds occurring
among carbonyl groups and lattice water molecules. A similar packing is observed in 1a.
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
Fig.4. Cyclic voltammogram (scan rate 100 mV/s) of 1a in DMF solution of 0.1 M TBAP, using
platinum working electrode
2
1

5
5
5
5
5
44
45
46
47
48
5
5
5
5
5
5
5
5
49
50
51
52
53
54
55
56
5
5
5
57
58
59
Fig.5. Cyclic voltammogram (scan rate 100 mV/s) of 1b in DMF solution containing 0.1 M
TBAP, using platinum working electrode
5
5
5
5
5
5
5
5
60
61
62
63
64
65
66
67
2
2

5
5
5
5
5
5
5
68
69
70
71
72
73
74
0
0
0
0
.12
.10
.08
.06
f
a
0.04
0
.02
.00
0
300
400
500
600
700
800
Wavelength(nm)
5
5
5
75
76
77
Fig.6. Electronic spectral titration of complex 1a with CT-DNA in Tris-HCl buffer; [1a] =
-5
-6
-6
6
-6
2.62×10 ; [DNA]: (a) 0.0, (b) 1.25×10 , (c) 2.5×10 , (d) 3.75×10 , (e) 5.0×10 , (f)
-6
-1
6.25×10 mol L . The arrow denotes the gradual increase of DNA concentration.
5
5
5
5
5
5
5
78
79
80
81
82
83
84
4
.0
1
b
3.5
3
.0
.5
1a
2
2.0
1
.5
.0
1
1
2
3
4
5
6
6
[DNA]X10
5
5
5
85
86
87
Fig. 7. Comparative plot of [DNA]/( - ) vs [DNA] for the absorption titration of CT-DNA with
a
f
4
-1
complexes 1a and 1b in Tris-HCl buffer. Association constant K : 0.36 × 10 M (R =
b
4
-1
0.99999, n = 5 points) for 1a; 0.93× 10 M (R = 0.99885, n = 5 points) for 1b.
5
5
88
89
2
3

5
5
5
5
5
5
90
91
92
93
94
95
1.12
1.10
1
1
1
350
200
050
a
f
1
.08
.06
1
1.04
.02
1.00
1
9
00
750
00
450
1
2
3
4
5
5
6
7
[Complex]X10
6
3
00
50
0
1
575
600
625
650
675
700
725
750
775
Wavelength(nm)
5
5
5
5
6
96
97
98
99
00
Fig.8. Emission spectra of the CT-DNA-EB system in Tris-HCl buffer upon titration with
-5
-5
-
complex 1a. λ = 522 nm; [EB] = 9.6×10 , [DNA] = 1.25×10 ; [1a]: (a) 0.0, (b) 1.31×10
ex
5
-5
-5
-5
-5
-1
, (c) 2.62×10 , (d) 3.93×10 , (e) 5.24×10 , (f) 6.55×10 mol L . The arrow denotes the
gradual increase of complex concentration. Inset shows the plot of I /I vs. [1a]; K = 0.16
0
q
4
10 (R = 0.99876, n = 5 points).
6
6
6
6
6
6
6
6
01
02
03
04
05
06
07
08
-1.0
-1.2
-1.4
-1.6
-1.8
-2.0
1
b
1a
-
4.9 -4.8 -4.7 -4.6 -4.5 -4.4 -4.3 -4.2 -4.1
log[Complex]
6
6
09
10
Fig. 9. Comparative plot of log[I -I/I] vs. log[complex] for the titration of CT-DNA-EB system
0
with complexes 1a and 1b in tris-HCl buffer medium.
6
11
2
4

6
6
6
6
6
6
6
6
6
12
13
14
15
16
17
18
19
20
0
0
0
0
0
0
.60
.55
.50
.45
.40
.35
1b
1a
0.30
0.1
0.2
0.3
0.4
0.5
0.6
0.7
[
M]/DNA
6
6
21
22
Fig. 10. Effect of increasing amounts of Pd(II) and Pt(II) complexes on the relative viscosity of
CT-DNA at 25 °C.
6
6
6
6
6
6
23
24
25
26
27
28
1
1
0
0
0
0
0
.2
.0
.8
.6
.4
.2
.0
f
a
6
6
6
6
6
6
29
30
31
32
33
34
2
80
350
420
490
560
630
Wave length (nm)
Fig. 11. Absorption titration spectra of BSA in presence of complex 1a. Concentration range of
-6
-1
the complex is 0-6.25× 10 M .
2
5

6
6
6
6
35
36
37
38
1
1
1
1
6
4
2
0
8
6
4
2
1
b
1
a
6
6
6
6
39
40
41
42
2
4
6
8
5
10
6
6
6
6
43
44
45
46
(
1 / [Complex]) x 10
_
Fig. 12. The linear dependence of 1/A A on the reciprocal concentration of 1a and 1b.
0
6
6
6
6
6
6
6
6
47
48
49
50
51
52
53
54
1
1
1
1
1
1
1
1
.35
.30
.25
.20
.15
.10
.05
.00
a
f
2
2
1
1
500
000
500
000
1
2
3
4
5
6
7
6
[
Complex]x10
5
00
0
300
320
340
360
380
400
Wavelength(nm)
6
6
55
56
Fig. 13. Fluorescence quenching titration of BSA varying the concentration of 1a, [complex] = 0,
-6
1, 2, 3, 4 and 5 × 6.35 ×10 M. Inset shows the Stern–Volmer plot.
6
57
2
6

6
6
6
6
6
6
6
6
58
59
60
61
62
63
64
65
O
O
N
N
N
N
PdCl2
Pd
DMF
Stir, 3 h
O
O
NH HN
1a
N
N
K2PtCl4
H L
O
O
2
N
N
DMF-H O, N
Stir, 12 h
2
2
Pt
N
N
1b
6
6
66
67
Scheme 1. Synthetic routes of the complexes
2
7

Highlights





Two isomorphous and isostructural complexes of general formulation [ML].H O
2
M= Pd (1a) and Pt (1b), L = bis(pyridine-2-carboxamide)benzene dianion)
Synthesis, structural characterisation and redox behavior
Spectroscopic study on the interaction of both 1a and 1b with CT-DNA and BSA
Both complexes have strong noncovalent intercalative binding ability with CT-DNA

Graphical Abstract
PdCl2
DMF
Stir, 3 h
O
O
NH
N
HN
N
K2PtCl4
DMF-H O, N2
2
Stir, 12 h
1
b
for 1a Pt = Pd