Rich spectroscopic and molecular dynamic studies on the interaction of cytotoxic Pt(II) and Pd(II) complexes of glycine derivatives with calf thymus DNA

Article abstract of DOI:10.1080/07391102.2015.1015056

Some amino acid derivatives, such as R-glycine, have been synthesized together with their full spectroscopic characterization. The sodium salts of these bidentate amino acid ligands have been interacted with [M(bpy)(H₂O)₂](NO₃)₂ giving the corresponding some new complexes with formula [M(bpy)(R-gly)]NO₃ (where M is Pt(II) or Pd(II), bpy is 2,2′-bipyridine and R-gly is butyl-, hexyl-and octyl-glycine). Due to less solubility of octyl derivatives, the biological activities of butyl and hexyl derivatives have been tested against chronic myelogenous leukemia cell line, K562. The interaction of these complexes with highly polymerized calf thymus DNA has been extensively studied by means of electronic absorption, fluorescence and other measurements. The experimental results suggest that these complexes positive cooperatively bind to DNA presumably via groove binding. Molecular dynamic results show that the DNA structure is largely maintained its native structure in hexylglycine derivative-water mixtures and at lower temperatures. The simulation data indicates that the more destabilizing effect of butylglycine is induced by preferential accumulation of these molecules around the DNA and due to their more negative free energy of binding via groove binding.

Full text of DOI:10.1080/07391102.2015.1015056

This article was downloaded by: [West Virginia University]
On: 10 March 2015, At: 12:10
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer
House, 37-41 Mortimer Street, London W1T 3JH, UK
Journal of Biomolecular Structure and Dynamics
Publication details, including instructions for authors and subscription information:
http://www.tandfonline.com/loi/tbsd20
Rich Spectroscopic and Molecular Dynamic Studies
on the Interaction of Cytotoxic Pt(II) and Pd(II)
Complexes of Glycine derivatives with Calf Thymus
DNA
a
b
c
Mahboube Eslami Moghadam , Maryam Saidifar , Adeleh Divsalar , Hassan Mansouri-
d
e
a
f
Torshizi , Ali-Akbar Saboury , Hossein Farhangian & Maryam Ghadamgahi
a
Chemistry & Chemical Engineering Research Center of Iran, Tehran, Iran
b
Nanotechnology and Advanced Materials Department, Materials and Energy Research
Center, Karaj, Iran
c
Department of Biological Sciences, Kharazmi (Tarbiat Moallem) University, Tehran, Iran
Click for updates
d
Department of Chemistry, University of Sistan & Baluchestan, Zahedan, Iran
e
Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran
f
School of Chemistry, Damghan University, Damghan, Iran
Accepted author version posted online: 03 Mar 2015.
To cite this article: Mahboube Eslami Moghadam, Maryam Saidifar, Adeleh Divsalar, Hassan Mansouri-Torshizi, Ali-Akbar
Saboury, Hossein Farhangian & Maryam Ghadamgahi (2015): Rich Spectroscopic and Molecular Dynamic Studies on the
Interaction of Cytotoxic Pt(II) and Pd(II) Complexes of Glycine derivatives with Calf Thymus DNA, Journal of Biomolecular
Structure and Dynamics, DOI: 10.1080/07391102.2015.1015056
To link to this article: http://dx.doi.org/10.1080/07391102.2015.1015056
Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a service
to authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting,
typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication
of the Version of Record (VoR). During production and pre-press, errors may be discovered which could
affect the content, and all legal disclaimers that apply to the journal relate to this version also.
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained
in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no
representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of
the Content. Any opinions and views expressed in this publication are the opinions and views of the authors,
and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied
upon and should be independently verified with primary sources of information. Taylor and Francis shall
not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other
liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or
arising out of the use of the Content.
This article may be used for research, teaching, and private study purposes. Any substantial or systematic
reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any
form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://
www.tandfonline.com/page/terms-and-conditions

Publisher: Taylor & Francis
Journal: Journal of Biomolecular Structure and Dynamics
DOI: http://dx.doi.org/10.1080/07391102.2015.1015056
Rich Spectroscopic and Molecular Dynamic Studies on the Interaction of Cytotoxic
Pt(II) and Pd(II) Complexes of Glycine derivatives with Calf Thymus DNA
1
,*
2
3
Mahboube Eslami Moghadam , Maryam Saidifar , Adeleh Divsalar , Hassan
4
5
1
Mansouri-Torshizi , Ali-Akbar Saboury , Hossein Farhangian and Maryam
6
Ghadamgahi
1
2
Chemistry & Chemical Engineering Research Center of Iran, Tehran, Iran
Nanotechnology and Advanced Materials Department, Materials and Energy Research
Center, Karaj, Iran
3
Department of Biological Sciences, Kharazmi (Tarbiat Moallem) University, Tehran, Iran
4
Department of Chemistry, University of Sistan & Baluchestan, Zahedan, Iran
5
Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran
6
School of Chemistry, Damghan University, Damghan, Iran
*
Corresponding author. Tel.: +98 21 44580772; fax: +98 21 44580753; e-mail:
eslami_moghadam@ccerci.ac.ir & eslami_moghadam@yahoo.com

Rich Spectroscopic and Molecular Dynamic Studies on the Interaction of Cytotoxic
Pt(II) and Pd(II) Complexes of Glycine derivatives with Calf Thymus DNA
Abstract
Some amino acid derivatives, such as R-glycine, have been synthesized together with
their full spectroscopic characterization. The sodium salts of these bidentate amino acid
ligands have been interacted with [M(bpy)(H O) ](NO ) giving the corresponding
2
2
3 2
some new complexes with formula [M(bpy)(R-gly)]NO (where M is Pt(II) or Pd(II),
3
bpy is 2,2'-bipyridine and R-gly is butyl-, hexyl- and octyl-glycine). Due to less
solubility of octyl derivatives, the biological activities of butyl and hexyl derivatives
have been tested against chronic myelogenous leukemia cell line, K562. The interaction
of these complexes with highly polymerized calf thymus DNA has been extensively
studied by means of electronic absorption, fluorescence and other measurements. The
experimental results suggest that these complexes positive cooperatively bind to DNA
presumably via groove binding. Molecular dynamic results show that the DNA structure
is largely maintained its native structure in hexylglycine derivative–water mixtures and
at lower temperatures. The simulation data indicates that the more destabilizing effect of
butylglycine is induced by preferential accumulation of these molecules around the
DNA and due to their more negative free energy of binding via groove binding.
Keywords: Platinum and palladium complexes; Cytotoxicity; Amino acid derivative;
Calf thymus DNA; Molecular dynamics simulation.
List of abbreviations
protein data bank (PDB)
Particle-Mesh Ewald (PME)
molecular dynamic (MD)
root mean square deviation (RMSD)
gyration radius (Rg)

hydrogen bonds (H-B)
radial distribution function (RDF)
Introduction
Design and synthesis of new cisplatin analogs, as potential anticancer drugs, have
been renewed by the utilization of bidentate ligands in the development and the
treatment of cancer (1). Cisplatin has been used in the treatment of testicular cancers,
and solid malignancies such as bone, neck and bladder cancers (2). Due to the dose
limiting toxic side-effects of cisplatin, much current research work is aimed at the
discovery of platinum complexes which are not cross-resistant with cisplatin, have
fewer toxic side-effects, are active against a broad range of types of cancer and can be
administered orally (3-7). In response to these needs, one approach is using a variety of
amino acids as ligands.
Several water soluble platinum and palladium complexes with bidentate essential
amino acids have been reported (8-14). These complexes have shown anticancer
activity comparable to or greater than cisplatin. Also suggested that the palladium
complexes may be useful for the treatment of tumors of the gastrointestinal region
where cisplatin fails (2).
In addition, the substitution of chloride ligands of cisplatin or its palladium analogs
with bidentate ligands such as amino acid or amino acid derivatives that are found in
biological systems may decrease their toxic side effects or increase the concentration of
drug inside the cell and thus improve the antitumor activities of these agents (15). It has
been reported that many active complexes could react with DNA and inhibit its
synthesis (16,17). Recently, several platinum complexes of diamine chelating ligands
bearing amino-acid type substituents have been reported (18). They have been
interacted with a mononucleotide and DNA. Their results suggested that the presence of
different amino acids derivatives in these complexes can modulate their solubility and
cytotoxic activity. We have earlier reported the synthesis, characterization and protein
binding studies of a Pd(II) complex bearing amino acid derivative (19). The present
study deals with synthesis and characterization of some novel mixed-ligand platinum(II)
and palladium(II) complexes of 2,2'-bipyridine and amino acid derivatives (see scheme
1) including in vitro anti-tumor and detail calf thymus DNA binding studies. Computer
simulations can examine these interactions and describe important features for DNA–

ligand recognition (20). MD simulation has been applied to evaluate the binding of
newly mentioned complexes to DNA. The LIE algorithm has been applied to calculate
and compare the binding free energies of DNA-ligand systems (21).
Experimental
Materials and Methods
Octylamine, hexylamine, butylamine, sodium chloride, sodium bicarbonate, sodium
hydroxide, hydrochloric acid, sulfuric acid, potassium tetrachloroplatinate, 2,2'-
bipyridine, highly polymerized calf thymus DNA sodium salt and Tris-HCl buffer were
purchased from Merck (Germany). Palladium(II) chloride anhydrous was obtained from
Fluka (Switzerland). Bromo acetic acid, carbon disulfide and ethidium bromide were
obtained from Aldrich (England). [Pt(bpy)Cl ] and [Pd(bpy)Cl ] were prepared
2
2
according to the literature procedure (22). Solvents were purified prior to use by the
standard procedures. Other chemicals used were of analytical reagent or higher purity
grade.
The melting points of the compounds were determined on a Unimelt capillary
-1
melting point apparatus and reported as they were. Infrared spectra (4000-400 cm )
were determined in KBr disks on a JASCO-460 plus FT-IR spectrophotometer. UV-Vis
1
spectra were recorded on a JASCO UV/VIS-7850 recording spectrophotometer. H NMR
spectra were measured on a Brucker DRX-500 Avance spectrometer at 500 MHz, using
TMS as the internal reference in DMSO-d . The fluorescence spectra were carried out
6
on a Hitachi MPF-4 spectrofluorimeter. Conductivity measurements of the above
platinum and palladium complexes were carried out on a Systronics Conductivity
Bridge 305, using a conductivity cell of cell constant 1.0 and doubly distilled water was
used as solvent. Carbon, hydrogen and nitrogen in the ligand and the complexes were
analyzed on a Herause CHNO-RAPID elemental analyzer.
Synthesis of Ligands and Metal Complexes
Synthesis of Butylglycine, Hexylglycine and Octylglycine
To a solution of bromoethylacetate (6 mL, 53 mmol) in benzene (10 mL), a solution
of Alkyl amine (106 mmol) in benzene (40 mL) was added dropwise with constant
stirring (19). The reaction mixture was refluxed for two hours and then cooled. The

amine hydrobromide was filtered off, washed with small amount of benzene and
discarded. From the combined filtrate, all the benzene was removed by rotaevaporator
until a white fume was observed. 2.12 g sodium hydroxide (53 mmol) in 8 ml distilled
water was added, refluxed for 30 min. and cooled. The solution was transferred to a
separator funnel and washed with diethyl ether (two times, each time 20 mL) to remove
undesired organic materials. The aqueous layer was acidified with HCl to pH=2 and
°
slowly evaporated at 35 C until crystals appeared and left aside for 24 h.
Recrystallization was carried out by dissolving the filtered crystals is minimum amount
of distilled water, acidifying to pH=2, and slow evaporation. The pure crystals so
obtained was filtered and dried in a desiccator under vacuum over KOH pellets. This
reaction proceeds in accord with Scheme 2.
Butylglycine (CH -(CH ) -NH-CH -COOH.HCl), Yield: 80% with a melting point of
3
2 3
2
°
1
99-201 C. Analytical calculated for C H NO Cl (167.5): C, 42.98; H, 8.36; N, 8.36.
6 14 2
−
1
Analytical found: C, 42.95; H, 8.33; N, 8.4%; IR (cm , solid): 3415(w, O-H), 2942(w,
1
N-H), 2813(w, C-H), 1766 (s, C=O), 1208(s, C-O). H NMR (300 MHz, DMSO-d , δ in
6
ppm, J in MHz): 0.85 (t, 3H, 18.75), 1.28 (m, 2H), 1.6 (m, 2H), 2.86 (m, 2H), 3.78 (s,
2H), 9.3 (sb, NH and OH).
Hexylglycine (CH -(CH ) -NH-CH -COOH.HCl), Yield: 69% with a melting point of
3
2 5
2
°
2
06-208 C. Analytical calculated for C H NO Cl (195.5): C, 49.10; H, 9.20; N, 7.16.
8 18 2
−
1
Analytical found: C, 49.08; H, 9.18; N, 7.15%; IR (cm , solid): 3412(w, O-H), 2946(w,
1
N-H), 2829(w, C-H), 1752(s, C=O), 1215(s, C-O); H NMR (300 MHz, DMSO-d , δ in
6
ppm, J in MHz): 0.875 (t, 3H, 12.11), 1.338 (m, 6H), 1,57 (m, 2H), 2.81 (m, 2H), 3.85
(s, 2H), 9.35 (sb, NH and OH).
Octylglycine (CH -(CH ) -NH-CH -COOH.HCl), Yield: 81% with a melting point of
3
2 7
2
°
2
16-217 C. Analytical calculated for C H NO Cl (225.5): C, 52.6; H, 9.7; N, 6.0.
10 22 2
−
1
Analytical found: C, 52.6; H, 9.6; N, 6/0%; IR (cm , solid): 3400 (w, O-H), 2950 (w,
1
N-H), 2900 (w, C-H), 1740 (s, C=O), 1220 (s, C-O); H NMR (300 MHz, DMSO-d , δ
6
in ppm): 0.86 (t, 3H, 6.193), 1.25(m, 10H), 1.64(m, 2H), 2.87(t, 2H, 7.80), 3.8 (s, 2H),
9.3 (sb, NH and OH).
Synthesis of [Pt/Pd(bpy)(Butylgly)]NO complexes
3
1
mmol of [Pt(bpy)Cl ] or [Pd(bpy)Cl ] was suspended in 120 mL of doubly distilled
2 2
water. To this solution, 0.34 mg of AgNO (2 mmol) in 20 mL water was added slowly
3

°
with continuous stirring. The reaction mixture was heated for 7 h at 75 C and then for
15 h at room temperature under dark with constant stirring. The AgCl precipitate was
°
removed by filtering through Whatman filter paper No.42. This filtrate at 50 C was
mixed with butyl-glycine hydrochloride (0.17 g, 1 mmol) and NaHCO (0.17 g, 2
3
mmol) dissolved in 10 mL distilled water. The reaction mixture was further stirred and
°
concentrated at 50 C for 45 min. to 60 mL and then cooled. The trace amount of
turbidity formed was filtered and the clear yellow filtrate was further concentrated to
about 5 mL at 35 ºC (very fine needle crystals of the complex not suitable for X-Ray
crystallography were obtained by slow evaporation of this solution). The crystals
formed was filtered and washed with little amount of chilled double distilled water and
°
then dried in an oven at 45 C.
°
[
Pt(bpy)(Butylgly)]NO : Yield: 48% and decomposes at 205-207 C. Analytical
3
calculated for C H N O Pt (544): C, 35.36; H, 3.68; N, 10.31%. Analytical found: C,
1
6
20
4
5
-
1
-1
-4
3
3
3
5.4; H, 3.65; N, 10.33%; UV Band maxima in nm ( ε in liter mole cm ×10 ):
M
−
1
63(0.37), 321(0.85), 310(0.73), 283(1.36), 207(3.23); IR (cm , solid): 3435 (w, N-H),
-
1
048 (w), 2903 (w), 1680 (s, C=O), 1607 and 1471 (s, C=C), 1384 (s, NO ); H NMR
3
(
300 MHz, DMSO-d6, δ in ppm, J in MHz): 0.8 (t, 3H, 12.5), 1.34(m, 2H), 1.75,1.95(m,
H, 143.7), 2.89,2.98(m, 2H, 68.75), 3.47,4.96(m, 2H, 468.7), 8.45(s, NH), Aromatic
protons: 7.85(d, 2H), 8.49(t, 2H), 8.64(m, 2H); Molar conductance of 0.06 mM aqueous
2
2
-1
-1
solution: 120 cm ohm mol .
°
[
Pd(bpy)(Butylgly)]NO : Yield: 41% and decomposes at 197.7-198.9 C. Analytical
3
calculated for C H N O Pd (455): C, 42.29; H, 4.41; N, 12.33%. Analytical calculated
1
6
20
4
5
-1
-1
-
for: C, 42.34; H, 4.44; N, 12.30%; UV Band maxima in nm ( ε in liter mole cm ×10
M
4
−1
)
: 309 (0.98), 239 (1.22), 207 (4.02); IR (cm , solid): 3434 (w, N-H), 3080 (w), 2923
-
1
(
w), 1673 (s, C=O), 1602 and 1466 (s, C=C), 1384 (s, NO ); H NMR (300 MHz,
3
DMSO-d6, δ in ppm, J in MHz): 0.87 (t, 3H, 12.5), 1.37 (m, 2H), 1.82,2(m, 2H, 187.5),
2.83, 2.92(m, 2H, 62.5), 3.26,4.15(m, 2H, 718.7), 6.71(s, NH), Aromatic protons:
7.87(d, 2H), 8.65(m, 4H), 9.04(m, 2H); Molar conductance of 0.06 mM aqueous
2
-1
-1
solution: 136 cm ohm mol .
°
[
Pt(bpy)(hexylgly)]NO : Yield: 45% and decomposes at 187.3-188.6 C; Analytical
3
calculated for C H N O Pt (572): C, 37.83; H, 4.20; N, 9.81%. Analytical calculated
1
8
24
4
5
-1
-1
-
for: C, 37.86; H, 4.25; N, 9.88%; UV Band maxima in nm ( ε in liter mole cm ×10
M
4
−1
)
: 341(0.25), 318(1.29), 306(1.07), 247(1.97), 202(2.49); IR (cm , solid): 3435 (w, N-

1
H), 3113 (w), 2929 (w), 1677 (s, C=O), 1602 and 1470 (s, C=C), 1384 (s, NO ); H
3
NMR (300 MHz, DMSO-d6, δ in ppm, J in MHz): 0.8 (t, 3H, 12.5), 1.22 (m, 4H), 1.31
(m, 2H), 1.75,1.95 (m, 2H, 200), 2.9, 2.98(m, 2H, 50), 3.47,3.95(m, 2H, 375), 7.95 (s,
NH), Aromatic protons: 7.87(m, 2H), 8.47(m, 2H), 8.7(m, 2H), 8.9(d, 2H); Molar
2
-1
-1
conductance of 0.04 mM aqueous solution: 124.2 cm ohm mol .
[
Pd(bpy)(hexylgly)]NO and [Pd(bpy)(octylgly)]NO were synthesized by a method
3
3
similar to one described previously (19).
Pt(bpy)(ocylgly)]NO : Yield: 41% and decomposes at 186.6-188.4 C; Analytical
°
[
3
calculated for C H N O Pt (600): C, 40.07; H, 4.67; N, 9.35%. Analytical calculated
2
0
28
4
5
-1
-1
-
for: C, 40.12; H, 4.69; N, 9.30%; UV Band maxima in nm ( ε in liter mole cm ×10
M
4
−1
)
: 366(0.38), 321(0.72), 309(0.67), 285(1.17), 207(2.73); IR (cm , solid): 3435 (w, N-
1
H), 3118 (w), 2924 (w), 1677 (s, C=O), 1609 and 1467 (s, C=C), 1394 (s, NO ); H
3
NMR (300 MHz, DMSO-d6, δ in ppm, J in MHz): 0.79 (t, 3H, 12.5), 1.95 (m, 8H), 1.32
(m, 2H), 1.74,1.93 (m, 2H, 200), 2.91, 2.98(m, 2H, 50), 3.53,3.98(m, 2H, 500), 7.67 (s,
NH), Aromatic protons: 7.88(m, 2H), 8.49(m, 2H), 8.67(m, 2H), 8.78(d, 2H); Molar
2
-1
-1
conductance of 0.05 mM aqueous solution: 123 cm ohm mol .
Cell culture
Human tumor cell line K562 (which was obtained from National Cell Bank of Iran
(
NCBI)-Pasteur Institute of Iran), was selected for study of various Pd(II) complexes
cytotoxicity. Cells were grown in the RPMI medium (RPMI, Sigma). The medium was
supplemented with L-glutamine (2 mM), streptomycin and penicillin (5 μg/mL) and
°
1
0% heat-inactivated fetal calf serum, at 37 C under a 5% CO / 95% air atmosphere.
2
In vitro cytotoxicity study
The in vitro cytotoxicity was studied using the MTT assay. The cleavage and the
conversion of the soluble yellowish MTT to the insoluble purple formazan by active
mitochondrial dehydrogenase of living cells had been used to develop an assay system
alternative to other assays for measurement of cell proliferation. Harvested cells were
5
seeded into a 24-well plate (2×10 cell/ml) with different amounts of free Pd(II)
complexes (0-240 µM) for 24 h. Four hours to the end of the incubations, 50 µL of
MTT solution (5 mg/mL in PBS) was added to each well containing fresh and cultured
medium. At the end of the incubation, the insoluble formazan produced was dissolved

in a solution containing 1 ml of isopropanol, 4% HCl 1 N (left for 24 h at room
temperature in dark conditions). Finally, the optical density (OD) was read against a
reagent blank with multi well scanning spectrophotometer (ELISA reader, Asys
Hitchech, Austria) at a wavelength of 570 nm. The cell viability was calculated using
the following equation:
Cell viability (%) = (Atreated/Acontrol) × 100
where Atreated and Acontrol are the absorbance of the treated and untreated cells,
respectively. The 50% cytotoxic concentration (Cc ) was measured as the
5
0
concentration of Pd(II) complexes at which 50% of the cells were viable compared with
that of the control (23).
Biochemical Studies
All experiments involving the interaction of the complexes with ct-DNA were carried
-1
out in Tris-HCl buffer of pH 7.4 medium containing 10 mmolL sodium chloride
-1
(
24,25). The stock solutions of Pt(II) and Pd(II) complexes (5 mmolL ) were made in
°
this medium by gentle stirring and heating at 35 C, while that of DNA (4 mg/mL) at 4
°
C until homogenous. The metal complex solutions, with and without DNA were
°
°
incubated at 27 C and 37 C, separately. Then, the spectrophotometric readings at λmax,
21, 309, 318 and 314 nm, for [Pt(bpy)(But-gly)]NO , [Pd(bpy)(But-gly)]NO ,
3
3
3
[
Pt(bpy)(Hex-gly)]NO and [Pd(bpy)(Hex-gly)]NO , respectively, where DNA has no
3
3
absorption were measured. Using trial and error method, the incubation time for
°
°
solutions of DNA-metal complexes at 27 C and 37 C found to be 2.5 h. No further
changes were observed in the absorbance reading after longer incubation. The
concentration of DNA was determined spectrophotometrically using a molar
-1
-1
absorptivity of 6600 Lmol cm (258 nm) (26). Different techniques to probe the
changes on DNA structure induced by complexes have been used:
Electronic absorption titration
Electronic absorption spectroscopy is universally employed to determine the binding
parameters (n, K, g) of metal complexes with DNA as reported earlier (11, 27). Where n
is the Hill coefficient, g is the number of binding sites per 1000 nucleotides of DNA and
K is apparent binding constant. Also, the other thermodynamic binding parameters:
molar Gibbs free energy of binding (ΔG °), molar enthalpy of binding (ΔH °) and molar
b
b
entropy of binding (ΔS °) were determined according to reported method (14). All
b

°
°
measurements were performed separately at 27 C and 37 C and repeated three times
for each complex.
Denaturation of DNA with Pt(II) and Pd(II) complexes
The application of UV absorption method to the study of denaturation of DNA with
Pt(II) or Pd(II) complexes were similar to that reported earlier (14). In these studies, the
concentration of each metal complex at midpoint of transition, [L] , was determined.
1
/2
Also, thermodynamic parameters such as: ΔG°(H2O), conformational stability of DNA in
the absence of metal complex; ΔH°(H2O), the heat needed for DNA denaturation in the
absence of metal complex; ΔS°(H2O), the entropy of DNA denaturation by metal
complex as well as m, measure of the metal complex ability to denature DNA were
found out using Pace method (14,28,29). All measurements were performed in 0.01
-1
°
°
molL Tris-HCl buffer (pH 7.4) at 27 C and 37 C, separately.
Modes of binding
The modes of binding of some platinum (II) and palladium (II) complexes with ct-
DNA were done by the gel filtration, ethanol precipitation, electronic absorption and
fluorescence experiments as reported earlier (11,30). The modes of binding between
DNA and the above synthesized complexes were investigated by gel filtration
experiments. G-25 Sephadex can be used for separation of double-stranded DNA
fragments. The exclusion limits for 10 base pairs. For preparation of the column
chromatograph, G-25 sephadex is placed in the usage buffer and allowed to swell for at
least 3 h at 20 ˚C. The slurry mixture is poured into the column chromatograph in
continuous motion to 20 ml void volume of the gel filtration column. Then, the column
was filled to the top with buffer.
Fluorescence studies
Ethidium bromide (EB), one of the most sensitive fluorescence probes having a planar
-1
structure binds DNA by intercalative mode (31,32). At first, DNA (60 μmolL ) was
-1
added to 2 μmolL aqueous ethidium bromide solution (3.7 μL DNA + 16 μL EB +
4
4
30.3 μl buffer) and maximum quantum yield for ethidium bromide was achieved at
71 nm, so we selected this wavelength as excitation radiation for all of the samples at
different temperatures (27 °C and 37 °C) in the range of 540-700 nm. The widths of the
excitation and the emission slit were set at 5.0 nm. To this solution (containing EB and
-
DNA) different concentrations of the Pt(II) or Pd(II) complex (0.4, 0.5 and 0.6 mmolL

1
)
were added. The fluorescence intensities of the Pd(II) and Pt(II) complexes at the
highest denaturant concentration at 471 nm excitation wavelength have been checked
and the emissions intensities of these compounds were very small and negligible.
Molecular dynamic simulation
The structures of butyl- and hexyl-glycine derivatives were drawn using HyperChem 7
software, pre-optimized with Molecular Mechanics Force Field (MM+), and their final
geometries were obtained with the semi-empirical AM1 method. Their force field
parameters were generated using PRODRG2 server (33). The starting conformation for
the simulations of the DNA was obtained from the protein data bank (PDB) structure
453D (34). The DNA was solvated with a mixture of five butyl- or hexyl-glycine
derivatives and TIPP4 water and placed in a cubic large enough boxes to contain the
DNA and 1 nm of solvent on all sides. Molecular dynamics simulation was performed
using GROMACS software version 3. 3. 1 (35). AMBER 01 force field was used to
model atomic interactions (36). In all simulations, temperature was kept close to the
°
°
intended values (27 C and 37 C) by using the Nose-Hoover algorithm (37). Pressure
was also kept constant at 1 bar by applying Parrinello-Rahman algorithm (37). Leap-
Frog integration algorithm was used to solve the motion equation by a time step of two
femtoseconds. All bonds, including hydrogen bonds, were constrained by using LINCS
algorithm (37). Electrostatic interactions between charged groups within 1.5 nm were
calculated explicitly, while long-range electrostatic interactions were calculated using
the Particle-Mesh Ewald (PME) method (38). Lenard-Jones and van der Waals’s
interactions were calculated with a 1.5 nm range cutoff. Grid algorithm was used to
search neighbors (39). Table I lists a summary of the simulations. All the simulations
were equilibrated by 1 ns with position restraints on the DNA and complex to relax
solvent molecules. After equilibration, the molecular dynamic was run for 20 ns.
Production runs of all the systems were obtained three times to check repeatability and
analyses were averaged from all three independent trajectories. To estimate the affinity
of complex in binding to DNA, relative binding free energy is calculated for each
complex using semi empirical linear interaction energy (LIE) methodology introduced
vdw
vdw
el
el
by Aqvist et al (ΔGbinding=α (<Vl-s >bound-<Vl-s >free) + β (<Vl-s >bound-<Vl-s > )+γ)
free
(40).
el
bound
vdw
Where V
and V
are the electrostatic and van der Waals interaction energies
bound
between the ligand and the solvated DNA from a molecular dynamics (MD) trajectory

el
free
vdw
with ligand bound to DNA and V
and V
are the electrostatic and van der
free
Waals interaction energies between the ligand and the water from another MD
trajectory with the ligand in water. The angle bracket indicates Boltzmann average and
the subscripts l and s refer to the complex and the surround, respectively. For several
ligand-DNA systems, a set of values of α=0.5 and β=0.16 and γ=0 was found to give
good results (40). In order to obtain free energy of binding using this method, system
No. 5-8 of Table I were established and computations were only tested for palladium
complexes. The Lennard-Jones parameters for the palladium were taken from reference
(
41) and inserted manually and compared with parameters calculated from Gaussian
software obtained with density functional theory (DFT) B3LYP method and 6-
11++G(d,p) basis set (42).
3
Results and discussion
Cytotoxicity evaluation
The in vitro anti-tumor properties of Pt(II) and Pd(II) complexes with butyl- and hexyl-
glycine were carried out with human tumor cell line K562 (23). In this experiment, the
cell growth was measured after incubation of cells in the presence of compounds (from
-1
°
0
to 250 μmolL ) to be tested at 37 C for 24 h. In Figures 1 the cell growth (in %)
-1
versus concentration μmolL ) of above complexes is represented. The 50% cytotoxic
-1
-1
concentration (Cc ) of each compound was determined >96 μmolL and 30 μmolL
5
0
-1
-1
for butylglycine-Pd(II) and -Pt(II) complexes and >96 μmolL and 80 μmolL for
hexylglycine-Pt(II) and -Pd(II) complexes respectively.
Moreover, the Cc value of cisplatin under the same experimental conditions was
5
0
-1
determined to be 154 μmolL , which is higher than the four prepared complexes.
However, the Cc values of these complexes are slightly higher than that of our
5
0
analogous Palladium(II) amino acid derivative complexes reported earlier (23).
DNA Binding Studies
Evaluation of binding parameters
In metal complex-DNA titration experiments, the amount of free and bound complex
was determined as follow:
-1
The isothermal titration of each metal complex (25 µL of 1 mmolL stock) with
different concentrations of DNA (50-100 µL for butyl system and 150-300 µL of stock

for hexyl system) in total volume of 2 mL was performed by applying electronic
absorption spectroscopy at 27 °C and 37 °C, separately. The change in absorbance when
all binding sites on DNA were occupied by metal complex, ΔAmax, was determined
from the plot of 1/[ΔA] Vs 1/[DNA], where ΔA=ADNA+Acom-ADNA-com) and shown in
Table II. These values were used to calculate the concentration of metal complex bound
to DNA, [L] =ΔA.[L] /ΔA , and the concentration of free metal complex, [L] =[L] -
b
t
max
f
t
[
[
L] and ν=[L] /[DNA] , ratio of the concentration of bound metal complex to total
b
b
t
DNA] in the next experiment, that is, titration of fixed amount of DNA (50 mL of
stock) with varying amount of each metal complex (50-100 µL for butyl system and
-
1
1
50-300 µL of 1 mmolL stock for hexyl system) in total volume of 2 mL at 27 °C and
°
3
7 C, separately.
Using these data (ν and [L] ), the Scatchard plots were constructed for the
f
°
°
interaction of each metal complex at the two temperatures 27 C and 37 C. The
Scatchard plots are shown in Figures 2A for [Pt(bpy)(but-gly)]NO and the insert for
3
[
[
Pd(bpy)(but-gly)]NO and 2B for [Pt(bpy)(Hex-gly)]NO and the insert for
3 3
Pd(bpy)(Hex-gly)]NO . These Scatchard plots are almost curvilinear concave
3
downwards, suggesting positive cooperative binding (for determining binding
parameters with minimum error, two final experimental points in curve at high ν ratio
were eliminated) (27). To obtain the binding parameters, the above experimental data (ν
n
n
and [L] ) were substituted in Hill equation, [ν =g(K[L] ) /(1+(K[L] ) )], to get a series of
f
f
f
equation with unknown parameters n, K and g. Using Eureka software, the theoretical
values of these parameters could be deduced. The results are tabulated in Table II which
are comparable with those of 2,2'-bipyridine-platinum and -palladium complexes of
dithiocarbamate as reported earlier (43-46).
The maximum errors between experimental and theoretical values of ν are also
shown in Table II which is quite low. The K, apparent binding constant and n, the Hill
coefficient in the interaction of hexyl-glycine–Pt or –Pd complexes with DNA is higher
than that of butyl-glycine-Pt or –Pd complexes with DNA (see Table II). Moreover,
these data indicate that the cooperativity of [Pd(bpy)(Hex-gly)]NO is more than
3
5
[
Pt(bpy)(Hex-gly)]NO . This may be because palladium complexes are about 10 times
3
more labile than their platinum analogs (44). Moreover, finding the area under the
sigmoidal binding isotherm curves (the values of ν were plotted versus the values of Ln

[
L] ) and using Wyman-Jons equation (27), the K , ΔG° and ΔH° at 27 °C and 37 °C
f
app
b
b
for each compound with DNA could be calculated. Plots of the values of ΔH ° versus
b
the values of [L] are shown in Figures 3 for Pt complexes and the insert for Pd
f
complexes at 27 °C. Deflections are observed in all plots. These deflections indicate
that at particular [L] , there is a sudden change in enthalpy of binding which may be due
f
to binding of metal complex to DNA or DNA denaturation. Similar observations can be
seen in the literature where Pd(II) complexes interact with proteins (27,14).
Thermodynamic parameters in denaturation studies
The maximum unfolding of DNA by interaction with above platinum and palladium
complexes occurs when all binding sites are occupied. In this experiment, the sample
-1
cell was filled with 1.8 mL Tris-HCl buffer containing ~0.07 molL DNA for Pd(II)
-1
complexes and ~0.09 molL DNA for Pt(II) complexes where absorption of these
solutions are ~0.5 and ~0.7, respectively. However, reference cell was filled with 1.8
mL Tris-HCl buffer only. Both cells were set separately at constant temperature of 27
°
°
C or 37 C and then 15 μL of Pt(II) or Pd(II) complex from stock solutions was added
to each cell. After 3 min., the absorption was recorded at 258 nm for DNA and at 640
nm to eliminate the interference of turbidity. Addition of metal complex to both cells
was continued until no further changes in the absorption readings were observed. The
profiles of denaturation of DNA by Pt- and Pd-butyl-glycine and Pt and Pd-hexyl-
°
glycine at two temperatures of 27 °C and 37 C are shown in Figures 4A and 4B,
respectively. The concentration of metal complexes in the midpoint of transition, [L] ,
1
/2
°
°
for Pt(II) and Pd(II) complexes at 27 C and 37 C are shown in Table III. The important
observation of this work is the low values of [L]1/2 for these complexes (26,31,47) i.e.
all the complexes (in particular [Pd(bpy)(Hex-gly)]NO ) can denature DNA at low
3
concentrations (~180-610 µM). Thus, if these complexes will be used as anti-tumor
agents, low doses will be needed, which may have fewer side effects.
Another significant observation of this work is the type of denaturation plots shown in
figure 4. The inserts of fig.4A and 4B show ascending sigmoidal curves for both Pd(II)
complexes. These results indicate the ability of both Pd(II) complexes to separate the
two strands of the parent DNA resulting exposure of purine and pyrimidine bases to
UV-light and hence absorption increases. However, the analogous Pt(II) complexes (fig
4A and 4B) exhibit descending sigmoidal denaturation curves which is probably
because the winding the parent douplex DNA resulted in its precipitation. Thus the

difference in the interaction behavior of these analogous complexes may be due to the
kinetics of central metal ions in the structure of them. Incidentally, palladium(II)
5
6
complexes react about 10 -10 times faster than platinum(II) complexes giving reaction
rate that are too fast and may causes winding and hence precipitating the DNA.
Furthermore, some thermodynamic parameters found in the process of DNA
denaturation are discussed here: The standard Gibbs free energy of DNA denaturation,
°
ΔG (the work needed for DNA denaturation) was calculated as a function of complex
concentration by assuming two-state mechanism and using the equations K=[AN-
°
A ]/[A -A ] and ΔG =-RTlnK, where A is the observed absorbance used to follow
obs
obs
D
obs
unfolding in the transition region, A and A are the values of absorbance to the native
N
D
and denatured conformations of DNA, respectively. The free energy of unfolding is
calculated based on the data in the Fig. 4, which varies linearly with complex
°
°
concentration in the transition region (28). A straight line with formula of ΔG =ΔG H2O-
°
m[complex] is obtained when the values of ΔG are plotted versus the concentrations of
°
°
each metal complex in the transition region at 27 C and 37 C. These plots are shown in
Figures 5 for Pt(II) and the insert for Pd(II) systems. The m, slope of these plots (a
measure of the metal complex ability to denature DNA) and the intercept on ordinate,
ΔG°(H2O), (conformational stability of DNA in the absence of metal complex) are
summarized in Table III. Similar values have been observed for DNA denaturation with
°
dithiocarbamate (43-45). Also, the values of ΔG (see Table III) are decreased by
increasing the temperatures for all complexes. This is as expected because in general,
most of the macromolecules are less stable at higher temperature. The values of m for
Pd(II)-hexyl-glycine complex are more than that of other three systems which indicate
the higher ability of [Pd(bpy)(Hex-gly)]NO ) to denature DNA.
3
Another important thermodynamic parameter found is the molar enthalpy of DNA
denaturation in absence of metal complexes i.e. ΔH°(H2O). For this, we calculated the
°
molar enthalpy of DNA denaturation in presence of each metal complex, ΔH
conformation
°
or ΔH denaturation, in the range of the two temperature using Gibbs-Helmholtz equation
°
°
(
°
(
ΔH =(ΔG /T -ΔG /T )/(1/T -1/T )) (48). On plotting the values of these
T1)
1
(T2)
2
1
2
enthalpies versus the concentrations of each metal complex, straight lines will be
obtained which are shown in Figures 6. Intrapolation of these lines (intercept on
ordinate i.e. absence of metal complex) give the values of ΔH°(H2O) (see Table III).

Figures 6 shows that in the range of 27 °C to 37 °C the changes in the enthalpies in
the presence of Pt(II)- and Pd(II)-hexyl-glycine complexes and Pt(II)-butyl-glycine are
descending while that of Pd(II)-butyl-glycine is ascending. These observations indicate
that on increasing the concentration of three first complexes, the stability of DNA is
decreased while in the case of [Pd(bpy)(but-gly)]NO the opposite trend was observed
3
which may be due to low tendency of interaction of Pd(II)-butylglycine than other
°
complexes with DNA. In addition, the entropy (ΔS (H2O)) of DNA unfolding by Pt(II)
°
°
°
and Pd(II) complexes have been calculated using equation ∆G (H2O)=∆H (H2O)-T∆S
(H2O)
and the data are given in Table III. These data show that the metal-DNA complex is
more disordered than that of native DNA, because ΔS° is positive and ΔG° is negative
and finally complex binding to DNA is a spontaneous process (see Table III). These
data again show that ability of the hexyl-glycine systems in the denaturation of DNA is
more than that of the butyl-glycine systems.
Evaluation of binding modes
In this evaluation, at first, a solution of DNA was passed through a Sephadex G-25
column and each eluted fraction of 2 ml was monitored spectrophotometrically at 258
nm to detect the DNA coming out and the absorption readings were plotted versus
eluted fractions to construct chromatogram. Numbers of peaks in the chromatogram
indicate the number of DNA fractions. When DNA alone is passed through column, a
single peak is observed Figures 7A and 8A. Then, the solution of each interacted DNA-
metal complex, 350 µL of stock solution complex and 50 µL DNA of stock solutions in
-
1
-1
2
.5 mL buffer (0.7 molL complex and 0.175 molL DNA) was passed through a
Sephadex G-25 column equilibrated with the same buffer. Elution was done with buffer
and each fraction of the column was monitored spectrophotometrically at λmax of each
complex (see Table II) and 258 nm for all systems for detecting DNA. The gel
chromatograms obtained from these experiments are given in Figures 7 for Pt(II)- and
Pd(II)-butyl-glycine complexes and Figures 8 for Pt(II)- and Pd(II)-hexyl-glycine
complexes. These results show that the two peaks obtained at two wavelengths were not
clearly resolved which indicate that metal complexes have not separated from DNA and
their binding with DNA is strong enough that not readily break (43-46). Moreover, all
four complexes break the DNA into two fractions, one smaller (left) and the other larger
(right) and complexes interact with both factions because, in both peaks of

chromatograms, absorptions due to DNA (at 258 nm) and λmax of complexes (see Table
II) were observed.
Fluorescence titration studies
The fluorescence of ethidium bromide (EB) increases after intercalating in DNA. If the
complex intercalates into DNA, it leads to a decrease in the binding sites of DNA
available for EB-DNA system (31,32,49). The changes of fluorescence emission of
intercalated EB in DNA with increasing concentrations of Pd(II) and Pt(II) complexes
°
at 27 C are shown in figures 9. These figures show a slowly reduction of the ethidium
intensity by adding the different concentrations of each complex. (Similar observations
°
were made at 37 C). These results suggest that the above metal complexes presumably
bind to DNA and provide conformation changes in it. As a result of these
conformational changes some of EB molecules could not fit in the intercalation sites
properly and thus fluorescence intensity of DNA intercalated EB is quenched.
Further, studies to characterize the mode of binding of Pd(II) complexes to ct-DNA
were supported using fluorescence Scatchard analysis (50). Saturation curves of the
fluorescence intensity for a series of DNA-Pt(II)/Pd(II) complexes, at increasing
-1
concentrations of each complex (0.3, 0.4 and 0.5 mmolL ) are obtained by adding
-1
increasing concentrations of EB (2,4,.. 20 µmolL ). The binding isotherms for the
interaction of metal complexes are represented as fluorescence Scatchard plots (r/[L]f
against r) and are given in Figures 10. In r/[L] =nK -rK formula, r is the moles of EB
f
a
a
bound per mole of DNA, [L] is the molar concentration of the free EB, n is the number
f
of binding sites per nucleotide and K is the apparent binding constant. The complexes
a
show non-competitive inhibition of EB binding (Type-D behavior), in which the slope
that is K with no change in the presence of increasing amounts of metal complexes,
a
with decreases in the intercept on the abscissa that is n (51). This implies that all
complexes are bound to DNA via hydrogen and groove binding but not intercalation.
Molecular Dynamics Simulation
We first analyzed the root mean square deviation of DNA in the butyl and hexyl
derivatives at two temperatures. In Figure 11A, the Root mean square deviation
(RMSD) of systems was calculated at two temperatures. As found, DNA square
deviation is more at higher temperatures and in butyl complex. In this work the RMSD
rises almost continuously over the first 4 ns. In butyl complex and at 310 K, the RMSD

overcomes 3.5 nm and the most structure deviation is observed. Figure 11B shows
averaged solvent accessible surface area of DNA in 20 ns time interval in the presence
of butyl and hexyl derivatives at 27 °C and 37 °C. This figure shows more increase of
surface in the presence of butyl at 37 °C. It is obvious that DNA structure is unfolding
and average surface area of DNA in system with butyl at 37 °C is increased more.
Figure 11C shows the averaged gyration radius (R ) of DNA in 20 ns time interval and
g
it confirms gyration radius of DNA increase at higher temperatures and in the presence
of butyl derivative. This result is in good accordance with increase of surface area of
DNA. This shows that the DNA structure has been unfolded and denatured more in
butyl system at 37 °C.
Figure 12A and 12B show the average intermolecular hydrogen bonds (H-B) and
hydrogen bonds between the DNA and solvent over the course of the MD simulations.
The total number of hydrogen bonds between DNA and water and intermolecular
hydrogen bond decrease with temperature and in butyl derivative. These data suggest
that the butyl molecules attach themselves to the DNA better than hexyl derivative at
higher temperatures, and the solvent-DNA hydrogen bonds are replaced by DNA-butyl
hydrogen bonds. Thus, the interaction between the DNA and butyl molecules are
thought to stabilize the denatured state and lead to DNA denaturation and as a result of
denaturation, intermolecular hydrogen bond decreases. We have calculated atomic
radial distribution functions (RDF) to compare the strength of direct interactions
between DNA and complex. The radial distribution function of DNA-complex
presented in Figure 12C confirms more attach of butyl derivative to DNA. The strength
of RDF profile (denoted by g(r)) at higher temperatures and in the presence of butyl is
higher compared to the g(r) magnitude in hexyl. Thus, butyl interacts more strongly
with the DNA than water, suggesting that accumulation of butyl around the DNA
causes poor salvation of the DNA and unfolding of structure. The present work suggests
that the number of hydrogen bonds between DNA and with water decreases with time
but this decrease is more prominent at higher temperatures and in butyl comparing to
hexyl derivative.
Results of g(r) of complex-solvent (Figure 13A) shows that RDF decreases by
temperature in the presence of butyl derivative. It reveals that butyl is excluded from
solvent surface. In order to explain this phenomenon, we have used gradual hydration
and repulse concepts at lower temperatures and in the presence of hexyl derivative.

Results show that solvent molecules are increased in hydration layer around DNA and
this is due to hexyl molecules that go away from DNA surface. We can conclude from
g(r) diagrams that a thin hydration layer is formed on DNA surface in lower
temperatures in the presence of hexyl derivative. The g(r) of solvent around DNA
versus distance is shown in Figure 13B. At high temperatures, butyl derivative exclude
solvent away from DNA more and this result is in good agreement with hydrogen bond
results. As a consequence, both complexes cause solvent exclusion from DNA and this
effect is higher for butyl due to its less steric hindrance and hydrophobic property.
°
Time-coursed snapshots of the butyl system at 27 C depicted in Figure 14 indicate
interaction of butyl with the DNA. During the simulation, the butyl came near the DNA
and it began to bind the DNA via groove binding at the time of 20 ns which is indicated
by an orange circle.
A number of papers have shown LIE as a method with fast and reliable estimates of
binding free energies (52,53). Table IV presents the difference in average interaction
energies (free energies) of complex and DNA. When the ligand locates far from DNA,
the Gibbs free energy is less negative and the value rapidly decreases after the ligand
interacting with DNA or after binding to DNA. This result is in good accordance with
those obtained from previous studies. According to previous studies, negative free
energy confirms that the ligand prefers to bind to the DNA. The most negative value of
free energy is at higher temperatures and in the presence of butyl derivative. Equilibrium
of system has been monitored by quantities such as temperature and pressure of the systems
presented in Table V. The steady state in these parameters confirms equilibrium in NPT
ensemble indicating that all systems can be used for subsequent data sampling. Also, bond
lengths and force constant (Table VI) were obtained from quassian software, then
needed parameters for palladium were calculated from them.
Conclusion
In summery, cytotoxic Data show that the Cc values of Pt(II) and Pd(II) complexes are
5
0
lower than cisplatin which indicating of the more cytotoxicity and anti-cancer activity
of these compounds. Maybe the presence of glycinato moiety with hydrophobic tail in
the structure of metal complexes has great influences on the growth suppression activity
of these complexes on K562 cells. Also, the experimental binding results taken together
indicate that all complexes cooperatively interacted with DNA at low concentration in

the mode of hydrogen or van der Waals binding and presumably groove binding.
Molecular dynamics simulation results show that hydrogen bond interactions between
butyl molecules and the DNA, solvent accessible surface area of DNA and the radius of
gyration of DNA vary with temperature and complex. The direct relationship between
these parameters and hydrophobicity of complex is the dominant mechanism by which
the DNA is unfolded at higher temperatures. The butyl molecules interact and bind
strongly to the DNA via groove binding and have the most negative value of binding
free energy. This strong binding and interaction confirmed by RDF data leads to the
unfolding of DNA. Finally we find that with the decreasing of hydrophobicity or steric
hindrance of complex, the complexes tend to have groove binding interaction and
eventually they can bind on the DNA surface stably. Trend and results of structural
parameters of simulation was compatible with experimental results.
Acknowledgments
Financial assistance from the Research Council of Chemistry & Chemical Engineering
Research Center of Iran and of University of Sistan and Baluchestan and University of
Tehran are gratefully acknowledged.
References and notes
1
.
H. Sigel, Metal Ions in Biological Systems, Marcel Dekker, New York, vol.11,
pp. 1-196 (1980).
2
.
.
A. S. Abu-Surrah, M. Kettunen, Curr. Med. Chem. 13, 1337-1357 (2006).
V. Alverdi, L. Giovagnini, C. Marzano, R. Seraglia, F. Beltio, S. Sintran, R.
3
Graziani, D. Fregona, J. Inorg. Biochem. 98, 1117-1128 (2004).
N. J. Farrell, In Catalysis by Metal Complexes, B. R. James, R.Ugo (Eds.),
vol.11, Kluwer Academic Publishers, Dordrecht, The Netherlands (1989).
4
.
5
.
N. Manav, A. K. Mishra, N. K. Kaushik, Spectrochim. Acta A. 60, 3087-3092
(2004).
6
.
Y. E. Kwon, K. J. Whang, Y. J. Park, K. H. Kim. Bioorg. Med .Chem. 11, 1669-
76 (2003).
7
. C. Marzano, A. Trevisan, L. Giovagnini, D. Fregona, Toxicol.Vitro. 16, 413-9
(2002).

8
.
A.S. Abu-Surrah, H. H. Al-Sa’doni, M. Y. Abdalla, Cancer Therapy, 6, 1-10
(2008).
9
1
1
1
.
M. P. M. Marques, ISRN Spectroscopy, 2013, 1-29 (2013).
0.
1.
2.
V. X. Jin, J. D. Ranford, Inorg. Chim. Acta, 304, 38-44 (2000).
A. I. Anzellotti, M. Sabat, N. Farrell, Inorg. Chem. 45(4) 1638-45 (2006).
A. A. Saboury, A. A. Shamsaei, A. A. Mosavi Movahedi, H. Mansouri-Torshizi,
J. Chin. Chem. Soc. 46, 917-922 (1999).
3. E. Agostinelli, M. P. M. Marques, R. Calheiros, F. P. S. C. Gil, G. Tempera, N.
Viceconte, V. Battaglia, S. Grancara, A. Toninello, Amino Acids, 38(2) 393-403 (2010).
4. H. Mansouri-Torshizi, M. Islami-Moghaddam, A. A. Saboury, Acta Bioch.
Bioph. Sin. 35, 886-890 (2003).
5. Mylonas, S.; Valavanidis, A.; Voukouvalidis, V.; Inorg. Chim. Acta. 55, 125-
28 (1981).
6. J. K. Barton, S. J. Lipard, Heavy Metal interactions with nucleic acids. T. G.
Spiro (Ed.) Metal Ions in Biology. Vol. 1, New York, USA: Wiley- Interscience, 31-114
1980).
7.
8.
1
1
1
1
1
(
1
R. N. Bose, S. K. Ghosh, S. Moghaddas, J. Inorg. Biochem. 65, 199-205 (1997).
1
S. Moradell, J. Lorenzo, A. Rovira, S. V. Zutphen, F. X. Aviles, V. Moreno, R.
de Llorens, M. A. Martinez, J. Reedijk, A. Llobet, J. Inorg. Biochem. 98, 1933-46
2004).
9.
Biomol. Struct. Dyn. 25, 173-82 (2007).
(
1
A. Divsalar, A. A. Saboury, H. Mansouri-Torshizi, A. A. Mosavi-Movahedi, J.
2
0.
2004).
S. A. Shaikh, S. R. Ahmed, B. Jayaram, Arch. Biochem. Biophys. 429, 81-99
(
2
2
2
1.
2.
3.
B. O. Brandsdal, J. Aqvist, A. O. Smalas, Protein Sci. 10, 1584-95 (2001).
F. A. Palocsay, J. V. Rund, Inorg. Chem. 8, 524-528 (1969).
A. Divsalar, A. A. Saboury, R. Yousefi, A. A. Moosavi-Movahedi, H.
Mansouri-Torshizi, Int. J. Biol. Macro. 40, 381-386 (2007).
4. M. Eslami Moghadam, M. Saidifar, F. Rostami-Charati, D. Ajloo, M.
Ghadamgahi, Comb. Chem. High T. Scr. 17, 781-789 (2014).
5. M. Saeidifar, H. Mansouri-Torshizi, Y. Palizdar, M. Eslami-Moghadam, A.
Divsalar, A. A. Saboury, Acta Chim. Slov. 61, 126–136 (2014).
6. Z. H. Xu, F. J. Chen, P. X. Xi, X. H. Liu, Z. Z. Zeng, J. Photochem. Photobiol
A: Chem. 196, 77-83 (2008).
2
2
2

2
2
2
7.
8.
9.
A. A. Saboury, J. Iran. Chem. Soc. 3, 1-21 (2006).
R. F. Greene, C. N. Pace, J. Biol. Chem. 249, 5388-5393 (1974).
S. Z. Bathaie, A. Bolhasani, R. Hoshyar, B. Ranjbar, F. Sabouni, A. A.
Moosavi-Movahedi, DNA and Cell Biol. 26, 533-540 (2007).
3
3
0.
1.
R. Mital, N. Jain, T. S. Srivastava, Inorg. Chim. Acta. 166, 135-140 (1989).
L. Peres-Flores, A. J. Ruiz-Chica, J. G. Delcros, F. M. Sanches, F. J. Ramirez,
Spectrochim. Acta. Part A. 69, 1089-1096 (2008).
2. A. G. Krishna, D. V. Kumar, B. M. Khan, S. K. Rawal, K. N. Ganesh, Biochim.
Biophys. Acta. 1381, 104-112 (1998).
3
3
3
3
3
3
3
3
4
4
4
3.
4.
5.
6.
7.
8.
9.
0.
1.
2.
A. W. Schuttelkopf, D. M. F. van Aalten, Acta Crystallogr. 60, 1355-63 (2004).
http://www.rcsb.org.
D. Van Der Spoel, J. Comput. Chem. 26, 1701-1718 (2005).
M. G. Wolf, G. Groenhof, J. comput. Chem. 33, 2225-32 (2012).
G. Bussi, D. Donadio, M. Parrinello, J. Chem. Phys. 126, 14101-14107 (2007).
D. Fincham, Mol. Sim, 8, 165-178 (1992).
H. Gao, Nano. Lett. 3(4) 471-473 (2003).
J. Åqvist, C. Medina, J. E. Samuelsson, Protein Eng. 7, 385-91 (1994).
S. Erkoc, Ann. Rev. Comput. Phys. IX., 1, 1-103 (2001).
S. Jalili, A. Jaberi, M. G. Mahjani, M. Jafarian, Molecular Physics: An
International Journal at the Interface between Chemistry and Physics. 110, 361-368
2012).
3.
Med. Chem. 16, 9616-9625 (2008).
4. M. Islami-Moghaddam, H. Mansouri-Torshizi, A. Divsalar, A. A. Saboury, J.
Iran. Chem. Soc. 6(3) 552-569 (2009).
5. H. Mansouri-Torshizi, M. I-Moghaddam, A. Divsalar, A. A. Saboury, J. Biomol.
Struct. Dyn. 26(5) 575-586 (2009).
6. H. Mansouri-Torshizi, M. Eslami Moghaddam, A. Divsalar, A. A. Saboury,
Acta Chim. Slov. 58(4) 811-822 (2011).
7. P. X. Xi, Z. H. Xu, X. H. Liu, F. J. Cheng, Z. Z. Zeng, Spectrochim. Acta. Part
A: Mol. Biomol. Spectrosc. 71, 523-8 (2008).
8. G. M. Barrow, In Physical Chemistry, chap. 7, 5th ed.; M.C. Graw-Hill, New
York (1988).
9. J. L. Butour, J. P. Macquet, Eur. J. Biochem. 78, 455-463 (1977).
(
4
H. Mansouri-Torshizi, M. I-Moghaddam, A. Divsalar, A. A. Saboury, Bioorg.
4
4
4
4
4
4

5
5
0.
1.
G. Scatchard, N. Y. Ann, Acad. Sci. 51, 660-672 (1949).
M. Howe-Grant, K. C. Wu, W. R. Bauer, S. J. Lippard, Biochemistry, 15, 4339-
4346 (1976).
5
2.
3.
R. A. Nome, J. Braz. Chem. Soc. 21, 2189-2204 (2010).
5
J. J. Tan, R. Kong, C. X. Wang, W. Z. Chen, J. Mol. Struct. (THEOCHEM),
6
82, 9-15 (2004).

Table I Summary of studied systems
system No. water T ( C)
o
No
1
DNA-[Pd(bpy)(Buylgly)]NO
DNA-[Pd(bpy)(Butylgly)]NO
DNA-[Pd(bpy)(hexylgly)]NO
DNA-[Pd(bpy)(hexylgly)]NO
[Pd(bpy)(Butylgly)]NO
3
3
3
3
3
3
3
3
3968
3968
3910
3910
4248
4248
3967
3967
27
37
27
37
27
37
27
37
2
3
4
5
6
[Pd(bpy)(Butylgly)]NO
7
[Pd(bpy)(hexylgly)]NO
8
[Pd(bpy)(hexylgly)]NO
Table II Values of Amax and binding parameters in the Hill equation for
interaction between Pt(II) and Pd(II) complexes and DNA in 10 mmol/L Tris-
HCl buffer and pH 7.4
a
b
c
d
-1
e
f
Compound
Temperature

max(nm)
321
Amax
g
K(Lmol )
n
error
º
[
[
Pt(bpy)(But-gly)]NO
3
27 C
0.138
0.062
0.097
0.051
9
0.009
0.015
0.017
0.006
3.42
4.83
4.39
2.65
0.006
0.032
0.066
0.059
º
3
7 C
9
9
9
º
Pd(bpy)(But-gly)]NO
3
3
27 C
306
º
3
7 C
º
[
[
Pt(bpy)(Hex-gly)]NO
27 C
318
314
0.045
0.019
0.022
0.017
7
0.021
0.029
0.048
0.109
5.64
5.24
2.17
3.24
0.08
0.08
0.06
0.07
º
3
7 C
7
º
Pd(bpy)(Hex-gly)]NO
3
27 C
7
7
º
3
7 C
a
b
c
d
e
f
wavelengths monitored to detect complexes. In this region DNA has no absorption.
change in the absorbance when all the binding sites on DNA were occupied by each of the metal complexes.
the number of binding sites per 1000 nucleotides.
the apparent binding constant.
the Hill coefficient (as a criterion of cooperativity).
maximum error between theoretical and experimental values of .

Table III Thermodynamic parameters of DNA denaturation by platinum
II) and palladium (II) complexes
(
a
b
c

d

e

Compound
Temperature
[L]1/2
m

G(
H
S
H2O)
(H2O)
(H2O)
º
2
7 C
0.29
74.2
21.6
[
Pt(bpy)(But-gly)]NO
3
_____________________________________
117.2
56.5
0.32
0.1
º
3
7 C
º
0.25
0.41
73.6
63.5
18.3
26.1
2
7 C
[
Pd(bpy)(But-gly)]NO
3
_________________________________
º
3
2
7 C
0.35
0.55
71.7
55
25.1
º
7 C
33.64
[
[
Pt(bpy)( Hex-gly)]NO
3
___________________________________
223.4
177.5
0.63
0.52
º
3
7 C
º
0.61
0.24
48
27.28
20.7
2
7 C
104
Pd(bpy)( Hex-gly)]NO
3
__________________________________
º
3
7 C
0.18
61
15.45
a
The concentration of ligand in the midpoint of transition (mol/L)
b
c
d
e
-1
measure of the metal complex ability to destabilize DNA )kJ/mol))mmol/L)
conformational stability of DNA in the absence of metal complex (kJ/mol)
the entropy of DNA denaturation by metal complex (kJ/mol)
the heat needed for DNA denaturation in the absence of metal complex (kJ/molK)

Table IV Free energy values of systems obtained from LIE method.
-1
-1
Bounded(kJ.mol )
free(kJ.mol )
Free Energy
No
-
1
Electrostatic
VDW
-4495
Electrostatic
VDW
-4396
(kJ.mol )
-38.84
1
2
3
4
42.67
49.85
-27.08
-75.29
-26.40
-24.49
-68.92
-36.39
-5914
-5762
-4299
-4198
-64.05
-9.810
-20.74
-4332
-4227
Table V. Temperature and pressure value of studied systems during simulation (No column is based on Table I numbering)
No
1
Temperature(˚C)
27.15
Pressure (bar)
1.03
2
37.68
0.92
3
27.71
0.90
4
37.07
1.17
Table VI. Bond lengths and force constant of Pd with its neigbouring atoms according to scheme 1.
-
1
Atom number
Force constant(mDyn.A )
Bond length (A)
a
Pd- N
1.92
1.94
2.17
1.19
1.93
1.88
1.93
1.96
Pd-Oa
Pd- N
b
Pd- N
a
Force constant and bond length of Pd-N of cyclohexene ring.
Force constant and bond length of Pd-N of cyclopentane ring.
b

N
N
NO3
M
a
R
NH
O
C
c
H
2
O
M= Pt(II) or Pd(II)
R= CH –(CH
n= 3, 5, 7
3
2 n
) –
Scheme 1. Proposed structures of [M(bpy)(R-gly)]NO
3
(where M is Pt(II) or Pd(II), R-
gly is butyl-, hexyl- or octyl-glycine and bpy is 2,2' bipyridine.

Scheme 2. General preparation route of the ligands R-Gly (R = butyl, hexyl or octyl).

120
(A)
100
80
60
40
20
0
0
30
60
90
120
-1
[
complex] (mol L )
(
B)
1
00
8
6
4
2
0
0
0
0
0
0
30
60
90
120
-1
[
Complex] (mol L )
Figure 1. The growth suppression activity A) of the Pd(II)- (◊) and Pt(II)-butylglycine
complex() and B) of the Pd(II)- (◊) and Pt(II)-hexyl-glycine complexes() on K562
cell line was incubated with varying concentrations of the complexes for 24 h.

30
35
30
25
20
15
10
5
0
25
20
15
10
5
0
A
°
S e2 r 7i es C1
°
S 3e r7i es C3
0
1
2

Serie °s 1
27 C
Seri e° s3
37 C
0
0.2
0.4
0.6
0.8
1
n
3
00
°
S e3 r7i es C1
27
1
40
20
00
250
200
°
Series3
C
B
1
50
00
1
1
1
50
0
0
0.5
1
1.5
2
n
80
60
40
20
0
Series1°
7 C
Seri e2 s7 3 C
3
°
0
0.5
1
1.5
2
n
Figure 2. Scatchard plots for binding of A) Pt(II)-butyl-gly and B) Pt(II)-hexyl-gly with
DNA. The inserts are Scatchard plots for binding of their Pd(II) analogs with DNA.