Journal of the Iranian Chemical Society

, Volume 10, Issue 5, pp 1001–1011

Novel 2,2′-bipyridine palladium(II) complexes with glycine derivatives: synthesis, characterization, cytotoxic assays and DNA-binding studies

Authors

    • Nanotechnology and Advanced Materials DepartmentMaterials and Energy Research Center
  • Hassan Mansouri-Torshizi
    • Department of ChemistryUniversity of Sistan and Baluchestan
  • Adele Divsalar
    • Department of Biological SciencesTarbiat Moallem University
  • Ali Akbar Saboury
    • Institute of Biochemistry and BiophysicsUniversity of Tehran
Original Paper

DOI: 10.1007/s13738-013-0237-1

Cite this article as:
Saeidifar, M., Mansouri-Torshizi, H., Divsalar, A. et al. J IRAN CHEM SOC (2013) 10: 1001. doi:10.1007/s13738-013-0237-1
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Abstract

The two water-soluble designed palladium(II) complexes, [Pd(bpy)(pip-Ac)]NO3 and [Pd(bpy)(mor-Ac)]NO3, (where bpy is 2,2′-bipyridine, pip-Ac is 1-piperidineacetato and mor-Ac is 4-morpholineacetato) have been synthesized and characterized by elemental analyses, molar conductivity measurements and spectroscopic methods (FT-IR, 1H NMR, UV–Vis). The complexes have been tested for in vitro cytotoxic activity against human breast cancer cell line, T47D. The binding of these complexes with DNA has been investigated by absorption spectroscopy, fluorescence titration spectra, EB displacement and gel chromatography. The results suggest that the complexes can bind to DNA cooperatively through a static mechanism at low concentrations (~0.57 μM). The thermodynamic parameters indicated that the van der Waals and hydrogen binding might play a major role in the interaction of these complexes with DNA.

Keywords

DNA-bindingPd(II) complex1-piperidineacetate4-morpholinoacetateCytotoxicity

Introduction

cis-Diamminedichloroplatinum(II) (cisplatin) received FDA approval in 1979 for use as an anticancer drug. Cisplatin is widely used and is especially effective against testicular, head and neck, non-small-cell lung, and cervical cancers [1, 2]. It is still one of the most effective agents in cancer chemotherapy in the clinic, although its high activity is accompanied by severe side effects and development of resistance [36]. There is strong evidence indicating that the biological target for cisplatin action is DNA [7], and that the covalent binding of cisplatin to cellular DNA mediates the cytotoxicity of this anticancer agent [8]. The interaction of cisplatin with DNA results in covalent cisplatin–DNA adducts that can inhibit DNA replication [7, 9]. Therefore, to overcome intrinsic and acquired resistance to cisplatin, a rational approach would be to prepare platinum complexes whose mechanisms of interaction with DNA are different from that of cisplatin and its analogs [10, 11]. Because palladium chemistry is similar to that of platinum, it was speculated that palladium complexes might also exhibit antitumor activities with fewer side effects [12]. In this context, development of palladium anticancer drugs has been promising and their design has been based mainly on the structure–activity relationship used for platinum anticancer drugs, as well as good models for the analogous Pt(II) complexes in solution [13]. As a result, some palladium complexes with aromatic N-containing ligands, e.g., derivatives of pyridine, quinoline, pyrazole, 1,10-phenanthroline and 2,2′-bipyridine, have shown very promising antitumor characteristics [1417].

As a continuation to our study on this class of palladium(II) compounds which might be of interest as antitumor agents, in the present work we report the synthesis, characterization as well as spectroscopic properties and gel chromatography of two palladium(II) complexes of 2,2′-bipyridine and glycine derivatives. The in vitro antitumor activity of complexes was investigated against human breast cancer cell line, T47D. We also described the DNA-binding, thermodynamic parameters and evaluation of binding modes of the complexes. These parameters may throw light on the interaction mechanisms of these types of complexes with DNA of the cells and possible side effects of these agents. It is also speculated that this mechanism may be different from that reported for cisplatin.

Experimental

Reagents and instruments

Palladium(II) chloride anhydrous, sodium chloride, sodium hydroxide and potassium bromide were obtained from Fluka (Switzerland). Piperidine, morpholine, bromoacetic acid were purchased from Aldrich (England). 2,2′-bipyridine, Sephadex G-25 and Tris–HCl [tris(hydroxymethyl) amino methane hydrochloride] were obtained from Merck (Germany). Calf thymus DNA and ethidium bromide (EB) were obtained from Sigma Chemical Co. (USA) and used as received. [Pd(bpy)Cl2] were prepared by the procedures described in the literature [18]. All reagents and solvents are of analytical reagent grade and double distilled water is used all along. Solutions of DNA in 20 mM NaCl, 20 mM Tris–HCl (pH 7.0) gave a ratio of UV–Vis absorbance of 1.8–1.9:1 at 260 and 280 nm, indicating that the DNA was sufficiently free of protein [19]. The DNA concentration per nucleotide was determined by absorption spectroscopy using the molar absorption coefficient (6,600 M−1 cm−1) at 260 nm [20]. The stock solutions of Pd(II) complexes (5 mM for [Pd(bpy)(pip-Ac)]NO3 and [Pd(bpy)(mor-Ac)]NO3 complexes) were made in Tris–HCl buffer by gentle stirring and heating at 308 K. The stock solution of DNA (4 mg/mL) at 278 K was stirred until homogenous and used after not more than 4 days. The cell line for the cytotoxicity studies was obtained from the Cell Bank of Pasteur Institute in Tehran (IRAN).

Electronic absorption spectra of the title ligands and metal complexes were measured on a JAS.CO UV/Vis-7850 recording spectrophotometer. Infrared spectra of the ligands and metal complexes were recorded on a JAS.CO-460 Plus FT-IR spectrophotometer in the range of 4,000–400 cm−1 in KBr pellets. Microchemical analysis of carbon, hydrogen and nitrogen for the ligands and complexes was carried out on a Herause CHNO-RAPID elemental analyzer. 1H NMR spectra were recorded on a Brucker DRX-500 Avance spectrometer at 500 MHz in DMSO-d6 using tetramethylsilane as internal reference. Fluorescence measurements were carried out on a Hitachi spectrofluorimeter model MPF-4. Melting point were measured on a Unimelt capillary melting point apparatus and reported uncorrected. Conductivity measurements of the above palladium complexes were carried out on a Systronics conductivity bridge 305, using a conductivity cell of cell constant 1.0. All spectrometric measurements were performed in a quartz cuvette with a 1-cm path length.

Synthesis of ligands and metal complexes

Synthesis of 1-piperidineacetate sodium salt (pip-AcNa)

This ligand was synthesized by the following procedure, in which bromoacetic acid (13.9 g, 100 mmol) and sodium hydroxide (4.0 g, 100 mmol) were added to 30 mL ethanol and stirred in an ice bath. 100 mmol sodium hydroxide (4.0 g) was added to above contents after 4 min. Piperidine (9.9 mL, 100 mmol) diluted in 10 mL ethanol was added dropwise while vigorous stirring. The white solution so obtained was stirred for another 1 h at 0 °C and at room temperature overnight. It was then heated under reflux with stirring at 60 °C for 3 h and then evaporated at 35–40 °C to complete dryness (Scheme 1 (I)). The solid was washed with acetone. Recrystallization was carried out by stirring the crude product in 40 mL methanol–acetonitrile (1:3 v/v) mixture and filtering the undissolved particles out. Diffusion of ether into the filtrate gave white needle-like crystals. The crystals was isolated by filtration, washed with 15 mL acetone and dried at 40 °C. The yield was 13.53 g (82 %); m.p. 320–322 °C. Anal. Calc. for C7H12NO2Na (165 g/mol): C, 50.91; H, 7.27; N, 8.48 %. Found: C, 50.87; H, 7.25; N, 8.53 %. FT-IR (KBr ν/cm−1): 1,362, ν(N–C); 1,606, ν(COO) and 2,945, ν(C–H)aliphatic. 1H NMR (500 MHz, DMSO-d6, ppm, s = singlet, sb = singlet broad, d = doublet and m = multiplet) [21]: 1.31 (d, 2H, H–d), 1.43 (m, 4H, H-c,c’), 2.32 (sb, 4H, H-b,b’) and 2.57 (s, 2H, H-a), (Scheme 3 (I)).
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Scheme 1

Syntheses of ligands I and II

Synthesis of 4-morpholineacetate sodium salt (mor-AcNa)

This ligand was synthesized by the method as described to that of pip-AcNa except that morpholine was replaced by piperidine (8.7 mL, 100 mmol) (Scheme 1 (II)). The yield was 14.53 g (87 %); m.p. 305–307 °C. Anal. Calc. for C6H6NO2Na (147 g/mol): C, 43.11; H, 5.99; N, 8.38 %. Found: C, 43.15; H, 5.95; N, 8.43 %. FT-IR (KBr ν/cm−1): 1,338, ν(N–C); 1,605, ν(COO) and 2,832, ν(C–H)aliphatic. 1H NMR (500 MHz, DMSO-d6, ppm, s = singlet and t = triplet) [21]: 2.38 (s, 4H, H-c,c’), 2.53 (t, 4H, H-b,b’) and 2.63 (s, 2H, H-a), (Scheme 3 (II)).

Synthesis of 2,2′-bipyridine 1-piperidineacetatopalladium(II) nitrate [Pd(bpy)(pip-Ac)]NO3

[Pd(bpy)(H2O)2](NO3)2 was prepared following the previous literature procedure [22], to the clear yellow filtrate containing above aqua complex (1.5 mmol), a solution of 1.5 mmol (0.247 g) 1-piperidineacetate sodium salt in 10 mL water was slowly added with stirring under dark. The clear yellowish orange solution so obtained was evaporated at 35–40 °C to complete dryness and the solid was washed three times with acetone. The solid residue was then dissolved in 30 mL of methanol–acetonitrile (1:1 v/v) mixture at 30–35 °C and filtrated. Diffusion of ether into the filtrate gave yellow powder after 2 days. The powder was isolated by filtration, washed with 15 mL acetone and dried at 40 °C (Scheme 2 (1)). Yield: 0.456 g (65 %) and decomposes at 337.2–337.7 °C. Anal. Calc. for C17H20N4O5Pd (466): C, 43.78; H, 4.29; N, 12.02 %. Found: C, 43.76; H, 4.26; N, 12.08 %. Λm (5 × 10−4 M, distilled water): 131.4 Ω−1 mol−1 cm2. FT-IR (KBr ν/cm−1): 1,323, ν(N–C); 1,686, ν(COO); 2,936, ν(C–H)aliphatic and 1,383, ν(NO3). UV–Vis (λ, nm): 309 (ε = 1.51), 245 (ε = 1.90) and 209 (ε = 4.58). 1H NMR (500 MHz, DMSO-d6, ppm, s = singlet, sb = singlet broad, d = doublet, t = triplet, m = multiplet) [23]:1.42 (sb, 2H, H–d), 1.72 (d, 2H, H-c), 1.92 (s, 2H, H-c’), 3.45 (d, 4H, H-b,b’), 4.06 (s, 2H, H-a), 7.84 (t, 1H, H-2), 7.90 (t, 1H, H-2′), 8.43 (m, 3H, H-4,4′,3), 8.63 (t, 2H, H-1,3′), 8.69 (d, 1H, H-1′) (Scheme 3 (1)) [24].
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Scheme 2

Syntheses of complexes 1 and 2

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Scheme 3

Proposed structures and proton NMR numbering schemes of pip-AcNa (I), mor-AcNa (II) ligands and [Pd(bpy)(pip-Ac)]NO3 (1) and [Pd(bpy)(mor-Ac)]NO3 (2) complexes

Synthesis of 2,2′-bipyridine 4-morpholineacetatopalladium(II) nitrate [Pd(bpy)(mor-Ac)]NO3

This complex was prepared by a similar method to that of [Pd(bpy)(pip-Ac)]NO3 except that pip-AcNa was replaced by mor-AcNa (Scheme 2 (2)). Yield: 0.312 g (67 %) and decomposes at 345.1–345.6 °C. Anal. Calcd. for C16H18N4O6Pd (468): C, 41.02; H, 3.85; N, 11.96 %. Found: C, 41.07; H, 3.80; N, 11.98 %. Λm (5 × 10−4 M, distilled water): 107.2 Ω−1 mol−1 cm2. FT-IR (KBr ν/cm−1): 1,321, ν(N–C); 1,670, ν(COO); 2,817, ν(C–H)aliphatic and 1,384, ν(NO3). UV–Vis (λ, nm): 309 (ε = 2.20), 245 (ε = 2.85) and 209 (ε = 6.98). 1H NMR (500 MHz, DMSO-d6, ppm, s = singlet, sb = singlet broad, m = multiplet) [23]: 3.54 (s, 4H, H-c,c’), 3.81 (sb, 4H, H-b,b’), 4.23 (s, 2H, H-a), 7.85 (s, 1H, H-2), 7.95 (s, 1H, H-2′), 8.44 (m, 3H, H-4,4′,3), 8.64 (m, 3H, H-3′,1,1′) (Scheme 3 (2)) [24].

In vitro antitumor experiments

Cell proliferation was evaluated using a system based on the tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT] that is reduced by living cells to yield a soluble formazan product that can be assayed colorimetrically [2527]. The MTT assay is dependent on the cleavage and conversion of the soluble yellowish MTT to the insoluble purple formazan by active mitochondrial dehydrogenase of living cells. The T47D human breast cancer cell line was maintained in a RPMI medium supplemented with 10 % heat-inactivated fetal calf serum and 2 mM l-glutamine, streptomycin and penicillin (5 μg/ml), at 310 K under a 5 % CO2/95 % air atmosphere. In this experiment, the clear stock solution (2 mM, in deionized water) was sterilized by filtering through sterilizing membrane (0.1 nm) and then varying concentrations of the sterilized complex (0–250 μM) were added to harvested cells. Harvested cells were seeded into 96-well plates (2 × 104 cell/mL) with varying concentrations of the sterilized complex and incubated for 48 h. At the end of the 4-h incubation period, 25 μL of MTT solution (5 mg/mL in PBS) was added to each well containing fresh culture media [28]. The insoluble formazan produced was then dissolved in solution containing 10 % SDS and 50 % DMF (under dark condition for 2 h at 310 K), and optical density (OD) was read against the reagent blank with a multi-well scanning spectrophotometer (ELISA reader, Model Expert 96, Asys Hitchech, Austria) at a wavelength of 570 nm. Absorbance is read as a function of concentration of converted dye. The OD value of study groups was divided by the OD value of untreated control and presented as a percentage of the control (as 100 %). Results were analyzed for statistical significance using a two-tailed Student’s t test. Changes were considered significant at p < 0.05. Experiments were carried out in triplicates and the mean values reported here.

Spectroscopic studies on DNA interaction

The DNA-metal complex solutions were incubated at 300 and 310 K, separately. Then, the spectrophotometric readings at λmax of the palladium complexes (314 nm) where DNA has no absorption were measured. Using trial and error, the incubation time for solutions of DNA-metal complexes at 300 and 310 K were found to be 5 h. No further changes were observed in the absorption readings after longer incubation, indicating that the reaction had gone completion. All experiments were repeated multiple times to obtain consistent results.

DNA-binding experiments and determination of thermodynamic parameters

In this experiment, the sample cell was filled with 1.8 mL DNA (~0.20 mM). However, the reference cell is filled with 1.8 mL Tris–HCl buffer only. Both cells were set separately at a constant temperature of 300 or 310 K and then 25 μL stock solution of Pd(II) complexes were added to each cell. After 3 min, the absorption was recorded at 260 nm for DNA and at 640 nm to eliminate the interference of turbidity. Addition of metal complexes to both cells was continued until no further changes in the absorption readings were observed [29, 30].

Electronic spectral studies

Electronic absorption titration experiments were performed with fixed concentrations of each metal complex (100 μM), while gradually increasing the concentration of DNA (25.8–43 μM and 28–43 μM for [Pd(bpy)(pip-Ac)]NO3 and [Pd(bpy)(mor-Ac)]NO3 complexes, respectively) in a total volume of 2 mL for obtaining the maximum ΔAAmax), i.e., change in the absorbance when all binding sites on DNA were occupied by the metal complexes. To affirm quantitatively the affinity of the complexes bound to DNA, the binding parameters of metal complexes to DNA were obtained. In this experiment, a fixed amount of DNA (21.4 μM) was titrated with varying concentrations of each of Pd(II) complexes (50–100 and 50–70 μM for [Pd(bpy)(pip-Ac)]NO3 and [Pd(bpy)(mor-Ac)]NO3 complexes, respectively) in a total volume of 2 mL. Each sample solution was scanned at 314 nm for two complexes.

Fluorescence spectral studies

Quantitatively, the affinity of the compounds bound to DNA was compared by the luminescence titration method. In this experiment, a series of solutions with various concentrations of each of the two complexes (0.45–0.87 mM for [Pd(bpy)(pip-Ac)]NO3 and 0.59–0.98 mM for [Pd(bpy)(mor-Ac)]NO3) were added to a solution of DNA (60 μM) and ethidium bromide (2 μM). The samples were excited at 471 nm and the emission spectra were observed between 540 and 700 nm at different temperatures of 300, 310, and 320 K. The fluorescence intensities of the Pd(II) complexes at the highest denaturant concentrations at 471 nm excitation wavelength have been checked, and the emission intensities of these compounds were negligible.

Further studies to characterize the mode of binding of Pd(II) complexes to DNA were carried out by Scatchard analysis [31]. In this experiment, the wavelengths of excitation and emission were set at 540 and 700 nm, respectively, with 0.5 nm slit widths. The Pd(II) complexes were incubated with DNA at 300 K for 5 h. The appropriate amount of EB was added before an additional incubation at room temperature (300 K) for 5 h before the final fluorescence spectral measurement. Saturation curves of fluorescence intensity for [Pd(bpy)(pip/mor-Ac)]+-DNA systems at different rf values (7.50, 10.67 and 12.50 for [Pd(bpy)(pip-Ac)]NO3 complex and 10, 12.5, and 15 for [Pd(bpy)(mor-Ac)]NO3 complex) were obtained in the presence of increased concentrations of EB (2, 4, …, 20 μM).

Gel filtration

Each of the above-mentioned Pd(II) complexes (187.5 μM) was incubated with calf thymus DNA (42.8 μM) for 5 h at 301 K in Tris–HCl buffer (pH 7.0). It was then passed through a Sephadex G-25 column equilibrated with the same buffer. The elution of the column fraction of 2.0 mL was monitored at 314 and 260 nm for DNA-Pd(II) complexes.

Results and discussion

Syntheses and characterization

Two new glycine derivative ligands have been prepared by the reaction between piperidine and morpholine with bromoacetic acid in presence of sodium hydroxide (Scheme 1). Sodium salts of these two bidentate ligands have been reacted with [Pd(bpy)(H2O)2](NO3)2 and thus two new mixed-ligand complexes of formula [Pd(bpy)(pip-Ac)]NO3 and [Pd(bpy)(mor-Ac)]NO3 (where bpy is 2,2′-bipyridine, pip-Ac is 1-piperidineacetato and mor-Ac is morpholineacetato) were prepared (Scheme 2). These two ligands and their corresponding palladium(II) complexes have been characterized by elemental analysis, molar conductivity measurements and spectroscopic methods (FT-IR, 1H NMR, UV–Vis). The characterization data are collected in the experimental section. These data support the formulation of the ligands and metal complexes proposed in Scheme 3. [Pd(bpy)(pip-Ac)]NO3 and [Pd(bpy)(mor-Ac)]NO3 have been found to show growth inhibition of human breast cancer cell line, T47D and the interaction of them with calf thymus DNA has been studied using absorption and fluorescence techniques.

Cytotoxicity results

The effect of prepared complexes on human breast cancer cell line, T47D, was evaluated by MTT assay. In this study, various concentrations of Pd(II) complexes ranging from 0 to 25 μM of stock solution (2 mM) were used in the culture medium of the tumor cell line for 48 h. The 50 % cytotoxic concentration (Ic50) value of the [Pd(bpy)(pip-Ac)]NO3 and the [Pd(bpy)(mor-Ac)]NO3 complexes are 0.42 and 0.48 mM, respectively. As shown in Fig. 1 for [Pd(bpy)(pip-Ac)]NO3 complex and the inset of [Pd(bpy)(mor-Ac)]NO3 complex, the number of growing cells was significantly reduced after 48 h in the presence of various concentrations of the palladium complexes. These results suggest that our newly designed Pd(II) complexes may be potential antitumor agents.
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Fig. 1

The growth suppression activity of the [Pd(bpy)(pip-Ac)]NO3 complex and the inset for [Pd(bpy)(mor-Ac)]NO3 complex on T47D cell line

DNA-binding studies

DNA interaction data and evaluation of thermodynamic parameters

Electronic absorption spectroscopy is one of the most useful techniques for DNA-binding studies of metal complexes [30, 32]. The result of UV–Vis spectrum analysis of the interaction of DNA with Pd(II) complexes at two temperatures of 300 and 310 K is shown in Fig. 2 and are summarized in Table 1. It is noticeable that, absorbance of DNA at 260 nm should increase in presence of increasing amount of each metal complex. However, the opposite trend was observed for the two complexes. The decrease in the absorbance at 260 nm with increasing amount of Pd(II) complexes added to DNA may be due to: (1) potential interaction between DNA and the metal complex that can cause the double helix of DNA to become more straight leading to stacking. This stacking may cause conformational changes leading to some degree of denaturation, or (2) each strand after denaturation is associated with a more stacked structure and (3) metal complex slips into the helix and masks the hydrophobic bases leading to a decrease in absorbance. These profiles have shown that the concentration of metal complexes in the midpoint of transition, [L]1/2, is comparable to [L]1/2 values of binding of analogous complexes [Pd/Pt(bpy)(mor-dtc)]NO3 [33] and is lower than values of crocin, crocetin, and dimethylcrocetin (DMC) [34] with DNA. The important observation of this work is the low values of [L]1/2 for these complexes [3537] i.e., both complexes can interact with DNA at low concentrations (~0.57 μM). Thus, if these complexes are to be used as antitumor agents, low doses of these compounds may be administered, resulting in fewer side effects. The improving effect of temperature, leading to the decrease in [L]1/2 indicates that the increase in temperature lowers the stability of DNA against denaturation caused by these complexes [22].
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Fig. 2

The changes of absorbance of DNA at λmax = 260 nm due to increasing the total concentration, [L]t, of [Pd(bpy)(pip-Ac)]NO3 and the inset for [Pd(bpy)(mor-Ac)]NO3, at constant temperatures of 300 and 310 K

Table 1

Thermodynamic parameters of DNA denaturation by palladium(II) complexes

Compound

Temperature (K)

[L]1/2 (mM)

m (kJ/mol)(mmol/L)−1

\( \Updelta G_{{({\text{H}}_{ 2} {\text{O}})}}^{ \circ } \) (kJ/mol)

\( \Updelta H_{{({\text{H}}_{ 2} {\text{O}})}}^{ \circ } \) (kJ/mol)

[Pd(bpy)(pip-Ac)]NO3

300

0.57

45.83

28.49

97.30

310

0.55

46.19

26.18

[Pd(bpy)(mor-Ac)]NO3

300

0.66

45.57

30.07

59.99

310

0.65

43.18

29.06

Furthermore, some thermodynamic parameters found in the process of DNA interaction are discussed here. Using the DNA interaction plots given in Fig. 2 and the Pace method [38, 39], the values of K, i.e., unfolding equilibrium constant and ∆G°, unfolding free energy of DNA at two temperatures of 300 and 310 K in the presence of [Pd(bpy)(pip-Ac)]NO3 and [Pd(bpy)(mor-Ac)]NO3 have been calculated. The Pace method assumes the two-state mechanism, including equilibrium between native and denatured forms, and calculates the unfolding free energy of DNA i.e., (∆G°) using Eqs. 1 and 2:
$$ K = \left( {A_{\text{N}} - A_{\text{obs}} } \right)/\left( {A_{\text{obs}} - A_{\text{D}} } \right) $$
(1)
$$ \Updelta G^{ \circ } = - RT\;\ln \,K $$
(2)
Aobs is the absorbance readings in the transition region, AN and AD are absorbance readings of the natural and denatured conformations of DNA, respectively. The values of ΔG˚ are plotted against the concentrations of each metal complex in the transition region at 300 and 310 K (Fig. 3). The equation for the straight lines can be written as follows [40]:
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Fig. 3

The molar Gibbs free energies of unfolding (∆G° vs [L]t) of DNA in the presence of [Pd(bpy)(pip-Ac)]NO3 and the inset for [Pd(bpy)(mor-Ac)]NO3

$$ \Updelta G^{ \circ } = \Updelta G^{ \circ }_{{({\text{H}}_{ 2} {\text{O}})}} - m[{\text{complex}}] $$
(3)

Here the values of \( \Updelta G_{{({\text{H}}_{ 2} {\text{O}})}}^{ \circ } \) for each curve are measured from the intercept on ordinate of the plots and it is conformational stability of DNA in the absence of metal complex. Higher values of ∆G° indicate larger changes in the conformational stability of DNA. However, the values of ∆G° (Table 1) are decreased by increasing the temperature for both complexes. This is expected because in general, the decrease in \( \Updelta G_{{({\text{H}}_{ 2} {\text{O}})}}^{ \circ } \) value is the main reason for the decrease in DNA stability [44]. The slope of each curve in the same plots, m, is a measure of the metal complex ability to interact DNA with metal complexes and the results are summarized in Table 1. The values of m for the [Pd(bpy)(pip-Ac)]NO3 complex are higher than that of the [Pd(bpy)(mor-Ac)]NO3 complex, which indicates the greater ability of [Pd(bpy)(pip-Ac)]NO3 complex to interact DNA. This interpretation is confirmed by higher m-value and lower [L]1/2 (Table 1) for [Pd(bpy)(pip-Ac)]NO3 in comparison to [Pd(bpy)(mor-Ac)]NO3 [39, 44].

Another important thermodynamic parameter found is the molar enthalpy of DNA interaction in absence of metal complexes i.e., \( \Updelta H_{{({\text{H}}_{ 2} {\text{O}})}}^{ \circ } \). To calculate this, the molar enthalpy of DNA interaction in the presence of metal complexes, ∆H°conformation or ∆H°denaturation, in the range of the two temperatures using Gibbs–Helmholtz equation was used [41]. Upon plotting the values of these enthalpies versus the concentrations of metal complexes, straight lines were obtained as shown in Fig. 4 for [Pd(bpy)(pip-Ac)]NO3 complex and the inset of [Pd(bpy)(mor-Ac)]NO3 complex. Intrapolation of these lines (intercept on ordinate i.e., absence of metal complex) give the values of \( \Updelta H_{{({\text{H}}_{ 2} {\text{O}})}}^{ \circ } \) (Table 1). These plots show that the changes in the enthalpies in the presence of two complexes in the range of 300 and 310 K are descending. These observations indicate that an increase in the concentration of these complexes, decreases the stability of DNA. In addition, it can be concluded that the interaction of Pd(II) complexes with DNA is exothermic [42].
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Fig. 4

The molar enthalpies of DNA denaturation in the interaction with [Pd(bpy)(pip-Ac)]NO3 and the inset for [Pd(bpy)(mor-Ac)]NO3 complexes in the range of 300–310 K

Generally, thermodynamic processes in the absence of Pd(II) complexes were shown that DNA was stable and nature because \( \Updelta G_{{({\text{H}}_{ 2} {\text{O}})}}^{ \circ } \) and \( \Updelta H_{{({\text{H}}_{ 2} {\text{O}})}}^{ \circ } \) were positive. However, the process of Gibbs free energy and enthalpy with increasing concentration of two complexes is negative. Thus, the binding of Pd(II) complexes to DNA is a spontaneous reaction and in presence of these complexes enthalpy is favorable.

Electronic absorption titration and determination of binding parameters

The absorption spectral titration experiment was performed by maintaining a constant concentration of the complexes against varying concentrations of DNA. In this experiment, the change in absorbance, ∆A, was calculated by subtracting the absorbance reading of mixed solutions of each metal complex with various concentrations of DNA, from absorbance reading of free metal complex. The values of ∆Amax, change in absorbance when all binding sites on DNA were occupied by metal complex, are given in Table 2. These values were used to calculate the concentration of metal complexes bound to DNA, [L]b, and the concentration of free metal complexes, [L]f and v, ratio of the concentration of bound metal complexes to the total concentration of DNA in the next experiment, that is, titration of fixed amount of DNA with varying amounts of each metal complex. Using these data (v, [L]f), Scatchard plots were constructed for the interaction of each metal complexes at 300 and 310 K [43]. The Scatchard plots are shown in Fig. 5 for [Pd(bpy)(pip-Ac)]NO3 complex and the inset for [Pd(bpy)(mor-Ac)]NO3 complex. These plots are curvilinear concave upwards, suggesting cooperative binding [29]. To obtain the binding parameters, the above experimental data (v and [L]f) were substituted in Eq. (4), i. e., Hill equation [44].
$$ v = g\left( {K\left[ L \right]_{\text{f}} } \right)^{n} /\left( {1 + \left( {K\left[ L \right]_{\text{f}} } \right)^{n} } \right) $$
(4)
Table 2

Values of ΔAmax and binding parameters in the Hill equation for interaction between Pd(II)complexes and DNA in 20 mmol/L Tris–HCl buffer and pH 7.0

Compound

Temperature (K)

ΔAmax

g

K × 103 (M)−1

n

Error

[Pd(bpy)(pip-Ac)]NO3

300

0.193

4

6.35 ± 0.2

1.86

7.5 × 10−3

310

0.224

7.19 ± 0.3

2.03

5.5 × 10−4

[Pd(bpy)(mor-Ac)]NO3

300

0.082

4

11.72 ± 0.1

6.57

0.015

310

0.060

11.43 ± 0.2

6.55

0.017

https://static-content.springer.com/image/art%3A10.1007%2Fs13738-013-0237-1/MediaObjects/13738_2013_237_Fig5_HTML.gif
Fig. 5

Scatchard plots for binding of [Pd(bpy)(pip-Ac)]NO3 and the inset for [Pd(bpy)(mor-Ac)]NO3 to DNA

Our equations contain the following unknown parameters n, K and g where n is Hill coefficient, g is the number of binding sites per 1,000 nucleotides of DNA and K is apparent binding constant. Using Eureka software, the theoretical values of these parameters were deduced (Table 2). The values of n for two complexes will be more than 1, which means that the systems are cooperative, thus, the binding at one site increases the affinity of binding at the other sites [45]. The apparent binding constants of two complexes were obtained and are comparable to those of [Cr(phen)3]3+ i.e., 3.5 × 103 M−1 and [Ni(DIP)3](NO3)2 i.e., 4.34 × 104 M−1 [46, 47]. The maximum errors between experimental and theoretical values of v are low as shown in Table 2. Knowing the experimental (dots) and theoretical (lines) values of v in the Scatchard plots and super impossibility of the two, the values of v were plotted versus the values of ln[L]f. Finding the area under the above plots of binding isotherms and using Wyman-Jons equation [48], we can calculate the Kapp and ∆G° at 300 and 310 K for each particular v and also ∆H°. Plots of the values of ∆H° versus the values of [L]f are shown in Fig. 6 for [Pd(bpy)(pip-Ac)]NO3 complex and the inset of [Pd(bpy)(mor-Ac)]NO3 complex at 300 K. Deflections are observed in both plots which may be due to binding of metal complexes to macromolecules or might indicate macromolecule denaturation. Similar observations can be seen in the literature where Pd(II) and Pt(II) complexes have interacted with DNA [22, 29, 49].
https://static-content.springer.com/image/art%3A10.1007%2Fs13738-013-0237-1/MediaObjects/13738_2013_237_Fig6_HTML.gif
Fig. 6

Molar enthalpies of binding in the interaction between DNA and [Pd(bpy)(pip-Ac)]NO3 and the inset for [Pd(bpy)(mor-Ac)]NO3 versus free concentrations of complexes at pH 7.0 and 300 K

Fluorescence spectroscopic studies

No fluorescence was observed for the above palladium(II) complexes in aqueous solution or in the presence of calf thymus DNA. So the binding of palladium(II) complexes and DNA cannot be directly presented in the emission spectra. Hence, competitive ethidium bromide (EB) binding studies were undertaken to determine the extent of binding of palladium(II) complexes to DNA. Figure 7 showed the emission spectra of DNA-EB system in the absence and presence of the Pd(II) complexes. Upon addition of Pd(II) complexes, a remarkable decrease in fluorescence of DNA-EB system was observed which indicates that the complexes bind to DNA [50, 51]. This behavior can be analyzed through the stern–volmer equation:
https://static-content.springer.com/image/art%3A10.1007%2Fs13738-013-0237-1/MediaObjects/13738_2013_237_Fig7_HTML.gif
Fig. 7

Fluorescence emission spectra of interacted EB-DNA in the absence (1) and presence of different concentrations of [Pd(bpy)(pip-Ac)]NO3 and the inset for [Pd(bpy)(mor-Ac)]NO3: 0.11–1.37 and 0.11–0.83 mM, respectively

$$ F_{0} /F = 1 + K_{\text{SV}} \left[ L \right] $$
(5)
F0 and F are the fluorescence intensities of DNA in the absence and in the presence of Pd(II) complexes, respectively. KSV is the Stern–Volmer dynamic quenching constant and [L] is the total concentration of quencher (Pd(II) complexes). The plots of F0/F on the quencher concentration ([L]) are shown in Fig. 8 and KSV values of Pd(II) complexes is simulated in Table 3.
https://static-content.springer.com/image/art%3A10.1007%2Fs13738-013-0237-1/MediaObjects/13738_2013_237_Fig8_HTML.gif
Fig. 8

Stern-Volmer curve for quenching of DNA by [Pd(bpy)(pip-Ac)]NO3 (filled diamond) and [Pd(bpy)(mor-Ac)]NO3 (filled triangle) complexes in 20 mM NaCl solution, 20 Mm Tris–HCl at pH 7.0 and 320 K

Table 3

Binding parameters for interaction of palladium complexes on DNA

Compound

KSV × 103 (M)−1

Kb × 103 (M)−1

n

\( \Updelta G^{ \circ } \)(kJ/mol)

\( \Updelta H^{ \circ } \)(kJ/mol)

\( \Updelta S^{^\circ } \)(kJ/mol K)

300

310

320

[Pd(bpy)(pip-Ac)]NO3

0.33 ± 0.2

1.05 ± 0.2

3.40

−2.85

−0.49

−0.13

−45.34

−1.45

[Pd(bpy)(mor-Ac)]NO3

1.62 ± 0.2

9.12 ± 0.2

3.45

−6.97

−5.94

−5.88

−25.00

−0.06

For static quenching, the following equation was employed to obtain various binding parameters. The binding constant (K) and number of binding sites (n) were calculated according to the equation [52, 53]:
$$ \log \, \left( {F_{0} - F} \right)/F = \log K_{\text{b}} + n \, \log \left[ L \right] $$
(6)
F0 and F are the florescence intensity without and with the Pd(II) complexes, respectively [58]. A plot of log[(F0F)/F] versus log[L] gave a straight line using least square analysis whose slope was equal to n (binding site number) and the intercept on Y-axis to logKb (Kb = binding constant) (Fig. 9; Table 3). The number of binding sites for these complexes is in good agreement with the above absorption spectral results. As shown in Table 3, Kb is higher than KSV. Thus, this suggests that DNA-binding with above complexes is a static quenching process [54].
https://static-content.springer.com/image/art%3A10.1007%2Fs13738-013-0237-1/MediaObjects/13738_2013_237_Fig9_HTML.gif
Fig. 9

The best linear plot of log(F0/F)/F versus log[L] to the Eq. (6) for [Pd(bpy)(pip-Ac)]NO3 (filled diamond) and [Pd(bpy)(mor-Ac)]NO3 (filled triangle) complexes at 320 K

Considering the dependence of binding constant on temperature, a thermodynamic process was considered to be responsible for the formations of a complex. Therefore, the thermodynamic parameters dependent on temperatures were analyzed to further characterize the interaction forces between Pd(II) complexes and DNA. The interaction forces between a small molecule and macromolecule mainly include hydrogen bonds, van der Waals force, electrostatic force and hydrophobic interaction force. The thermodynamic parameters of binding reaction are the main evidence to determine the binding force. If the enthalpy change (ΔH°) does not vary significantly over the temperature range studied, then its value and that of entropy change (ΔS°) can be determined from the van’t Hoff equation:
$$ \ln K = - \Updelta H^{ \circ } /RT + \Updelta S^{ \circ } /R $$
(7)
K and R are binding constant and gas constant, respectively. The temperatures used were 300, 310, and 320 K. The enthalpy change (ΔH°) and entropy change (ΔS°) were obtained from the slope and intercept of the linear van’t Hoff plot based on logK versus 1/T (Fig. 10). The free energy change (ΔG°) is estimated from the Eq. 2.
https://static-content.springer.com/image/art%3A10.1007%2Fs13738-013-0237-1/MediaObjects/13738_2013_237_Fig10_HTML.gif
Fig. 10

Van’t Hoff plot for the binding of Pd(II) complexes with DNA

The values of ΔH°, ΔS°, and ΔG° are listed in Table 3. From Table 3, it can be seen that the negative value of ΔG˚ revealed the interaction process is spontaneous reaction. It can also be seen in Table 3 that the formations of the Pd(II)-DNA complexes are exothermic reactions accompanied by negative enthalpy (ΔH°) and negative entropy (ΔS°), which indicates that ΔG° values were mainly due to the contribution of ΔH° but not ΔS°. Therefore, the thermodynamic parameters for the interaction of Pd(II) complexes and DNA can be explained on the basis of van der Waals and hydrogen binding [55].

Further studies to characterize the mode of binding of [Pd(bpy)(pip-Ac)]NO3 and [Pd(bpy)(mor-Ac)]NO3 to DNA were carried out [56, 57]. The number of EB molecules intercalated to DNA in presence of different concentrations of the Pd(II) complexes was calculated using Scatchard analysis [58]. The fluorescence Scatchard plots obtained for binding of EB to DNA in absence (♦) and presence (◊, Δ, ○) of various concentrations of Pd(II) complexes were shown in Fig. 11. Consequently, it might be concluded that the Pd(II) complexes noncompetitively inhibit the EB binding to DNA (type-D behavior) [48] where number of binding site, (intercept on the abscissa) and the slope of the graphs that is Kapp (apparent association constant) decrease with increasing the concentration of the complexes. The values of K and n are given in Table 4. Therefore, it can be concluded that van der Waals and hydrogen bonding interaction might play a major role in the interactions of Pd(II) complexes with DNA and the complexes do not intercalate in DNA [59].
https://static-content.springer.com/image/art%3A10.1007%2Fs13738-013-0237-1/MediaObjects/13738_2013_237_Fig11_HTML.gif
Fig. 11

Competition between [Pd(bpy)(pip-Ac)]NO3 and the inset for [Pd(bpy)(mor-Ac)]NO3 with ethidium bromide for the binding sites of DNA (Scatchard plot). Scatchards plot was obtained with calf thymus DNA alone (60 μM) (filled diamond), 0.46 mM (open diamond), 0.64 mM (open triangle) and 0.8 mΜ (open circle) for [Pd(bpy)(pip-Ac)]NO3 complex and 0.6 mM (open diamond), 0.75 mM (open triangle) and 0.9 mΜ (open circle) for [Pd(bpy)(mor-Ac)]NO3 complex. Solutions were in 20 mM NaCl, 20 mM Tris–HCl (pH 7.0). Experiments were performed at room temperature

Table 4

Binding parameters for the effect of palladium complexes on the fluorescence of EB in the presence of DNA

Compound

rf

K × 104 (M)−1

n

[Pd(bpy)(pip/mor-Ac)]NO3

0.00

5.31 ± 0.1

0.039

[Pd(bpy)(pip-Ac)]NO3

7.5

7.27 ± 0.2

0.028

10.67

9.18 ± 0.2

0.019

12.5

12.46 ± 0.1

0.012

[Pd(bpy)(mor-Ac)]NO3

10

6.49 ± 0.4

0.029

12.5

8.57 ± 0.2

0.021

15

12.27 ± 0.3

0.014

Binding modes

The solution of each interacted DNA–metal complex was passed through a Sephadex G-25 column equilibrated with Tris–HCl buffer. Elution was done with buffer and each fraction of the column was monitored spectrophotometrically at 314 and 260 nm for Pd(II)-DNA systems. Gel chromatograms are obtained by plotting the absorbance readings at the two wavelengths versus column fractions in the same plot. These results are given in Fig. 12 for [Pd(bpy)(pip-Ac)]NO3 complex and the inset of [Pd(bpy)(mor-Ac)]NO3 complex. These plots show that the peak obtained for the two wavelengths are resolved and suggests that DNA has separated from the metal complexes. This implies that the binding between DNA and metal complexes mainly involved hydrogen binding. However, other non-covalent interactions such as van der Waals interaction seem to play less important roles [60]. This result further supports our data obtained by fluorescence method.
https://static-content.springer.com/image/art%3A10.1007%2Fs13738-013-0237-1/MediaObjects/13738_2013_237_Fig12_HTML.gif
Fig. 12

Gel chromatograms of [Pd(bpy)(pip-Ac)]NO3 and the inset for [Pd(bpy)(mor-Ac)]NO3 obtained on Sephadex G-25 column

Conclusion

In order to investigate the influence of some ligands in palladium complexes on DNA-binding properties and cytotoxic activities, we selected 1-piperidine- and 4-morpholinoacetate as bidendente ligand to design and synthesize two new water-soluble palladium(II) complexes, [Pd(bpy)(pip-Ac)]NO3 and [Pd(bpy)(mor-Ac)]NO3. They show cytotoxicity against the T47D human breast cancer cell line. The absorption and fluorescence spectra studies on the interaction of the palladium(II) complexes with DNA show that the complexes interact with DNA cooperatively and at low concentrations. The results of fluorescence titration revealed that the DNA was quenched by Pd(II) complexes through the static quenching procedure. Thermodynamic parameters, enthalpy change (ΔH°), and entropy change (ΔS°), were calculated according to relevant fluorescent data and Van’t Hoff equation.

In addition, gel chromatography demonstrated that the binding between DNA and metal complexes mainly involve weak bonding. We also demonstrated several binding and thermodynamic parameters. The results suggest that both complexes interact with DNA possibly through van der Waals and hydrogen binding and they lay a foundation for the rational design of novel agents for probing and targeting nucleic acids.

Acknowledgments

We are grateful for the financial support of the Materials and Energy Research Center, the University of Sistan and Baluchestan and of the University of Tehran.

Copyright information

© Iranian Chemical Society 2013