Synthesis of a novel trinuclear palladium complex: the influence of an oxime chelate ligand on biological evaluation towards double-strand DNA, BSA protein and molecular modeling studies

Abstract

A trinuclear palladium complex with an aryl oxime ligand, [Pd₃(C₁₂H₈C=NO)₆], has been synthesized and structurally characterized by elemental analysis (C, H, N), IR, NMR resonance signals, and single crystal X-ray diffractometry. The crystal structure had a distorted square planar geometry. Binding interactions of the trinuclear Pd(II) complexes with calf thymus deoxyribonucleic acid (CT-DNA) and bovine serum albumin (BSA) were investigated by vivid spectroscopic techniques and molecular modeling studies. In vitro studies (UV-vis spectroscopy, competitive emission titration, circular dichroism (CD) and helix melting methods) show that the complex interacts with DNA via a groove mechanism binding mode. The intrinsic binding constants, K_b, of the complex with CT-DNA obtained from UV-vis absorption studies were 2 ± 0.02 × 10⁵ M⁻¹. Moreover, the addition of the complexes to CT-DNA (1 : 2) led to an increase in the melting temperature of DNA, up to 2.97 °C. Competitive studies with methylene blue (MB), as a fluorescence probe with trinuclear palladium, revealed that it cannot release MB molecules of the DNA-bound MB, suggesting there is no competition with MB. Furthermore, the microenvironment and secondary structure of BSA are changed in the presence of the trinuclear Pd(II) complex. Competitive binding using the site markers, Eosin and Ibuprofen, demonstrated that the complex binds to domain I (subdomain IIA) on BSA. Finally, molecular modeling studies were conducted to determine the binding sites of the DNA and BSA with the complex.

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1. Introduction

Over the past several decades, oxime chemistry has been a vital research field for inorganic and bioinorganic chemists because of the simple preparation and pharmacological applications of oximes.^1–4 Oxime derivatives are attractive synthetic goals because of their low toxicity to non-target organisms, antibacterial and antifungal properties,^5,6 and high index of antitumor activity^7,8 via intercalation. In addition, the biological relevance of oximes favors their use as ligands for potential metal based drugs. For instance, it has been reported that oxime complexes and other species bearing the oxime functional group can cause biological effects such as endothelium independent relaxation in blood vessels,^9,10 increased targeting of specific nuclear bases of DNA,¹¹ and oxidative DNA cleavage.¹² It is well known that deoxyribonucleic acid is an important genetic substance in organisms. Errors in gene expression can often cause diseases and play a secondary role in the outcome and severity of human diseases.¹³ DNA is generally the primary intracellular target of anticancer drugs. Therefore, the interaction between transition metal complexes and DNA can often cause DNA damage in cancer cells, blocking the division of cancer cells and resulting in cell death.^14,15 Transition metal complexes are known to bind to DNA via both covalent and/or non-covalent interactions. In the case of covalent binding, a labile ligand of the complex can be replaced by a nitrogen base of DNA such as guanine N7. The non-covalent DNA interactions include intercalative, electrostatic and groove (surface) binding of a metal complex outside of the DNA helix along major or minor grooves.^16,17 The toxicity and side effects of transition metal complexes such as cis-platin and other platinum metallo-drugs have shifted attention towards the synthesis of palladium metal complexes.¹⁸ Palladium complexes have been widely used as potential anticancer and anti-infective agents^19–21 because palladium(II) has a coordination mode and chemical properties similar to platinum(II). Some palladium complexes with aromatic N-containing ligands, e.g. derivatives of pyridine, quinoline, pyrazole and oxime, have shown very promising antitumor characteristics.²² Serum albumins, the most abundant proteins in blood plasma, often termed transport proteins, have many physiological functions. Many drugs and transition metal complexes are transported in the blood while bound to albumin. Therefore, investigations on the formation of complexes between proteins and drugs are important in order to find out the transport mechanism of a drug in the body.²³ In our earlier studies, we exhibited that amine and phosphorous ylide palladacyclic complexes have reasonable cytotoxic effects against some tumor cell lines and good DNA/BSA binding affinity.^24–26 In continuation of our research, we focused on the synthesis of a new class of trinuclear palladium oximes because of the pharmacological applications of oximes.²⁷ Since the chelating ligands minimize the high lability and fast hydrolysis of palladium complexes in biological environments, they have good clinical effects as cytotoxic agents on the treatment of various types of cancers. However, the application of chelating ligands in the synthesis of trinuclear palladium oximes was essential for improvement in their stability, which was further increased by generation of cyclopalladated compounds.²⁸ The present work stems from our continued interest in defining and evaluating the key DNA–BSA binding interactions of trinuclear palladium complexes and oxime based ligands and also our efforts to understand the structure activity relationships (SARs) of this new chemical compound. It would be interesting to probe whether the enhanced hydrophobicity and steric bulk of the 9-fluorenone oxime moiety would affect the DNA binding interaction and improve the effectiveness of these compounds as cytotoxic agents. Some hydrophobic molecules have been shown to migrate towards sites inside the grooves of DNA and release bound solvent molecules.²⁹ On the contrary, hydrophilic surfaces limit the cell uptake of the complexes due to the lipophilic character of the cell membrane.³⁰ Hence, we wish to describe here the synthesis and characterization of a new series of trinuclear palladium complexes and the pharmacological properties of the complexes for DNA/protein binding interactions. In addition, molecular docking experiments have been performed on the structures of BSA and DNA.

2. Experimental section

2.1. Materials

Reagent grade calf thymus DNA (CT-DNA), BSA and MB were obtained from Sigma Aldrich Chemical Company and used as received. Extra pure solvents used in the preparation of the compounds and for physical measurements were obtained from Merck Chemical Co. and used without further purification. DNA and BSA stock solutions were prepared using distilled water, a buffer containing 5 mM Tris–HCl [Tris(hydroxymethyl)-aminomethane] and 50 Mm NaCl, and was adjusted to pH 7.2 using hydrochloric acid. This was followed by exhaustive stirring at 4 °C for 3 days and keeping the solutions at 4 °C for no longer than a week. The stock solution of CT DNA gave a UV absorbance ratio at 260 and 280 nm (A260/A280) of 1.88, indicating that the DNA was sufficiently free of protein contamination.³¹ The DNA concentration per nucleotide was determined by the UV absorbance at 260 nm after a 1 : 20 dilution using ε = 6600 M⁻¹ cm⁻¹.³²

2.2. Instrumentation and physical measurements

Infrared (IR) spectra (400–4000 cm⁻¹) were recorded on a FT-IR JASCO 680 spectrophotometer using KBr pellets. The UV-vis spectra were recorded on a Varian Cary 100 UV-vis spectrophotometer using a 1 cm path length cell. Fluorescence spectra were recorded in solution on a Cary Eclipse fluorescence spectrophotometer. NMR spectra were recorded on a Bruker spectrometer at 400.13 MHz (¹H) and 100.61 MHz ¹³C-{¹H}. Elemental analyses were performed on a Leco, CHNS-932 apparatus. The T_m spectra were recorded on a Varian BioCary-100 UV-vis spectrophotometer using a 1 cm path length cell. Solutions were prepared by dissolving the complexes in water containing 5 mM Tris–HCl (pH 7.4) and 50 mM NaCl.

2.3. Preparation of ligand and compound

2.3.1. Synthesis of 9-fluorenone oxime ligand. 9-Fluorenone (0.540 g, 3 mmol) was dissolved in ethanol (5 mL). A solution of hydroxylamine hydrochloride (0.260 g, 3.75 mmol, H₂O, 0.5 mL) and anhydrous sodium hydroxide (0.12 g, 3 mmol) was then added to the resulting mixture. The reaction mixture was irradiated in the water bath of an ultrasonic cleaner at 25–35 °C for 2 h. Thereafter, the reaction mixture was cooled, poured into water (150 mL) and filtered. The solvent was evaporated to dryness under reduced pressure to give 9-fluorenone oxime in 82% yield as a yellow solid. Anal. calc. for C₁₃H₉NO: C 79.9; H 4.6; N 7.1%. Found. C 78.3; H 4.3; N 7.1%. IR (KBr pellet, cm⁻¹): ν(s, C=N) = 1606, ν(bs, OH) = 3250, ν(m, N–O) = 997, ν(m, C–H_aromtic) = 3075. ¹H NMR (400.13 MHz, CDCl₃, ppm): δ = 7.24–7.40 (m, 4H), 7.53 (d, 1H), 7.60 (d, 1H), 7.78 (d, 1H), 7.84 (d, 1H).

2.3.2. Synthesis of [Pd₃(C₁₂H₈C=NO)₆]. PdCl₂ (0.35 g, 2 mmol) and LiCl (0.17 g, 4 mmol) were dissolved in 20 mL of methanol. The resulting solution was heated to 50 °C and held at this temperature for 1 h. The color of the solution changed from orange to black. To a solution of Li₂PdCl₄ in MeOH, a methanolic solution (10 mL) of the corresponding 9-fluorenone oxime (0.48 g 2 mmol) and anhydrous sodium acetate (0.16 g, 2 mmol) was added. Then, the solution was stirred for 2–3 days at room temperature (Scheme 1). Following addition of water (10 mL), the corresponding trinuclear palladium complex precipitated and was filtered off and kept for crystallization. Slow evaporation of the filtrate over 15 days produced orange X-ray quality crystals. The crystals were filtered and washed with methanol. Yield: 64%. Anal. calc. for C₇₈H₄₈N₆O₆Pd₃: C 63.1; H 3.2; N 5.6%. Found. C 61.3; H 3.3; N 5.1%. IR (KBr pellet, cm⁻¹): ν(s, C=N) = 1602, ν(bs, OH) = 3370, ν(m, N–O) = 1064, ν(m, C–H_aromtic) = 3062. ¹H NMR (400.13 MHz, CDCl₃, ppm): δ = 10.87 (d, 6H₁), 7.83 (d, ), 7.72 (d, 6H₄), 7.45 (d, ), 7.64 (t, 6H₂), 7.55 (t, ), 7.16 (t, 6H₃), 6.73 (t, ). ¹³C-{1H} NMR (100.61 MHz, CDCl₃, ppm) 130.44, 128.09, 127.87, 123.75, 122.51, 120.22, 119.66.

Scheme 1 Synthesis of the trinuclear palladium complex.

2.4. Single crystal structure determination

X-ray diffraction experiments were carried out at 100 K using an Agilent Super Nova single crystal diffractometer (Mo K(α) radiation). Analytical numeric absorption corrections were made using a multi-faceted crystal model based on expressions derived by R. C. Clark & J. S. Reid.³³ The structures were solved by direct method using the SHELXS97 program and refined by the SHELXL (Sheldrick 2008) program. Hydrogen atoms were added in the calculated positions and were riding on their respective carbons during the refinement.

2.5. Molecular docking

Molecular docking is an important method for understanding the ligand receptor interactions. In this work, molecular docking was performed by an Autodock 4.2 package, which uses the free energy of binding as the basis for its empirical scoring function.^34,35 The polar hydrogens and Gasteiger charges were added to the structures by AutoDock Tools (ADT).³⁶ The Lamarckian genetic algorithm, one of the widely used stochastic search algorithms, which unites the properties of both the local (Solis and Wets algorithm)³⁷ and global search algorithms (genetic algorithm), was used. The crystal structure of BSA was obtained from the protein data bank (PDB ID: 4F5S) at a solution of 2.47 A. Also, the DNA sequence (CGCGAATTCGCG)2 was taken from the protein data bank (PDB ID: 1BNA) at a solution of 1.90 A. The coordination sphere of the trinuclear palladium complex was generated from its X-ray crystal structure as a CIF file. The CIF file was converted to the PDB format using the Mercury software (http://www.ccdc.cam.ac.uk/). The UCSF Chimera³⁸ package was used to produce molecular images and animations. LIGPLOT+,³⁹ a program for automatically plotting ligand–receptor interactions, was used to analyze the interactions between the DNA, BSA and trinuclear palladium complex.

3. Results and discussion

3.1. Synthesis and spectroscopic characterization

Palladium aryl oxime complex [Pd₃(C₁₂H₈C=NO)₆] was obtained in one step by stirring Li₂PdCl₄ with 9-fluorenone oxime and sodium acetate in MeOH for 2–3 days at room temperature (Scheme 1). The product obtained is orange, stable at room temperature, and soluble in chlorinated solvents such as CH₂Cl₂ and CHCl₃ and aprotic solvents like DMSO (dimethyl sulfoxide). The compound was characterized by IR spectroscopy, elemental analysis (C, H, N), resonance signals in the NMR, and single crystal X-ray crystallography.

The coordination mode of the ligand can be reliably followed by IR spectroscopy. The IR spectra of the complex showed typical bands at 3370, 3062 and 1064 cm⁻¹, assigned to ν (OH), ν (C–H aromatic) and ν (N–O), respectively. The band corresponding to the oxime complex in ν (N–O) of the free ligand (997 cm^−l) shifted to a higher wavenumber, and the ν (C=N) stretch at 1602 cm⁻¹ shifted to a lower wavenumber (compared to the free ligand) due to N-coordination of the oxime.⁴⁰ Formation of the Pd complex is further supported by detection of peaks in IR spectroscopy, NMR spectra ¹H, and ¹³C NMR spectral data. The data for the complex is incorporated in Fig. S1–S3 in the ESI.† The NMR spectrum of the complex was in good agreement with the proposed structure. In the ¹H NMR spectra, the signal due to the H₁ proton in the trinuclear complex appears at 10.8–10.6 ppm. This signal is clearly shifted strongly downfield with respect to the position in the oxime ligands (7.8 ppm) due to the anisotropic deshielding by oxime rings or C=NO group. However, the chemical shift does not change. Moreover, the ¹H NMR spectra display three doublets between 7.4 and 7.6 ppm and four triplets in the range of 6.7–7.7 ppm, signals attributed to the H₄, and H₂H₃ protons, respectively. The ¹³C{¹H} NMR spectra revealed the resonance for the C–NO carbon atom of the oxime group at δ = 130 ppm. As expected, six resonances are observed for the trinuclear complex ipso-carbon atom δ = 128.6 ppm, 123–119 for the heterocyclic ligands.

The electronic spectra of the Pd complex display bands around 236, 258 and 364 nm are due to intra-ligand (ILCT) transitions of (π–π*) and (n–π*) types and metal to ligand charge transfer (MLCT), respectively.

3.2. Molecular structure of the complex

The investigated complex was characterized in the solid phase by a single crystal X-ray diffraction study. Orange single crystals were obtained at room temperature by slow diffusion of n-hexane into a dichloromethane or chloroform solution of the complex. The molecular structure of the compound and packing diagrams are shown in (Fig. 1A and B). Relevant crystallographic data and structure refinement details are listed in Table S1 ESI.† Selected bond lengths and angles are listed in Table 1.

Fig. 1 (A) The molecular structure of the trinuclear palladium complex with its atom (50% probability level) labelling scheme. Hydrogen atoms have been omitted for clarity and (B) the packing diagram of the trinuclear complex formed by supramolecular interactions, each molecule of the complex is associated with three other units through π–π interactions, creating (011) sheets.

Table 1 Selected bond lengths (Å) and angles (°) for the Pd complex

Atoms	Bond lengths
Pd1–N1	2.017(6)
Pd1–N2	2.007(6)
Pd1–O6	2.016(5)
Pd1–O5	2.015(5)
Pd1–Pd2	2.924(7)
Pd1–Pd3	2.886(7)
O1–N1	1.342(7)

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Atoms	Bond angles
N2–Pd1–O5	88.3(2)
N2–Pd1–O6	177.9(2)
N1–Pd1–O6	90.2(2)
N1–Pd1–O5	171.2(2)
O6–Pd1–O5	90.92(19)
N1–Pd1–N2	90.3(2)

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The title complex crystallizes in the space group P2₁/n with Z = 4. Surprisingly, the deprotonated ligand 9-fluorenone oxime reacts with palladium chloride to form the oxime chelate complex. The palladium oxime complex forms trimeric units featuring three six membered ring skeletons (Pd–N–O–Pd–O–N) occupying the trans positions, which consist of alternating (distorted) square-planar palladium and oxime functional groups.

The trinuclear complex is held by Pd–N, Pd–O, N–O as well as Pd–Pd bonds. The Pd–N bond lengths are 2.008 (11) and 2.026 (12) Å, Pd–O bond lengths are 2.023 (10) and 2.027 (10) Å, N–O bond lengths are 1.338 (1) and 1.341 (1) Å and the Pd–Pd bond length is 2.894 (2) Å. The NO chelates are approximately symmetric, with the Pd–N–O angles ranging within 114.1(4)–112(4). The values are comparable to those observed in related complexes such as [Pd₃(ON=CPrⁱPh)₆] (av. Pd–N = 2.016 Å, Pd–O = 2.025 Å, N–O = 1.339 Å), which also feature bridging oxime groups.⁴¹ This structure is further stabilized by weak π⋯π [3.985 Å] interactions.

3.3. Interaction of the metal complex with bovine serum albumin

3.3.1. Absorption spectral studies. UV-vis absorption spectroscopy is a common method to explore the structural changes of proteins and investigate protein complex formation. The absorption spectrum of BSA shows two bands, a strong band in the range of 220–240 nm related to the absorption of the protein backbone (α-helix structure) and a weak one around 278 nm due to the absorption of aromatic amino acids (Trp, Tyr, and Phe).⁴² It is well known that the absorption maximum of BSA is highly sensitive to the surrounding microenvironment and displays a substantial spectral shift upon changes in the protein conformation.^43,44 Fig. 2 indicates that the BSA skeleton absorption intensity in the range of 220–240 nm decreases upon adding the Pd complex because of perturbation of the secondary structure of the protein.^45,46 Subtle changes in the maximum absorption at 278 nm were also seen, which indicate a perturbation of the α-helix induced by a specific interaction between the complexes and BSA.⁴⁷ The plot of 1/(A − A₀) versus 1/(complex concentration) is linear, and the binding constant (K) can be estimated from the ratio of the intercept to the slope (Fig. 2 inset), where A₀ is the initial absorbance of the free BSA at 278 nm and A is the recorded absorbance of BSA in the presence of different complex concentrations. The binding constants are estimated to be K_com BSA = 1.82 × 10⁴ M⁻¹. Therefore, the values of K_b show that BSA can be considered a good carrier for transfer of these complexes in vivo.^48,49

Fig. 2 UV absorption spectra of C(BSA) = 2 × 10⁻⁶ mol L⁻¹ in the absence and presence of complex(complex) = 0, 5.94 × 10⁻⁷, 1.17 × 10⁻⁶, 1.74 × 10⁻⁶, 2.30 × 10⁻⁶, 2.85 × 10⁻⁶, 3.39 × 10⁻⁶, 3.92 × 10⁻⁶, 4.44 × 10⁻⁶, 5.45 × 10⁻⁶, 6.42 × 10⁻⁶ mol L⁻¹ in 5 Mm Tris–HCl with 50 Mm NaCl.

3.3.2. Binding affinity and binding site number by fluorescence spectroscopy. An efficient approach for evaluating the interaction between metal complexes and BSA is the use of fluorescence spectroscopy. Generally, the fluorescence of a protein is caused by three intrinsic components, namely Trp-134, located on the surface of domain I, and Trp-213 located within the hydrophobic pocket of domain II, tyrosine, and phenylalanine residues. Fluorescence quenching can be due to a variety of molecular interactions including excited state reactions, molecular rearrangements, energy transfer, ground state complex formation and molecular collisions.⁵⁰ The fluorescence spectra have been recorded in the absence and presence of increasing concentrations of the Pd(II) complex. In the case of an interaction with the coordination compounds, the fluorescence intensity of the protein at around 345 nm decreases regularly as the concentration of the probe increases. Moreover, a red or blue shift of the emission maximum in the fluorescence spectrum of the albumin is indicative of an increase in the hydrophobicity of the microenvironment around the tryptophan residues;⁵¹ no change in the position of the emission maximum suggests no alteration in the local dielectric environment of BSA.⁵² It is evident from Fig. 3 that the fluorescence emission intensities of BSA at 345 nm show a remarkable decreasing trend with increasing concentration of the complex. This suggests a change in the conformation of BSA.⁵³

Fig. 3 Emission spectra of BSA upon the titration of the complex. C(BSA) = 2 × 10⁻⁶ mol L⁻¹, C(complex) = 0, 2.95 × 10⁻⁷, 6.76 × 10⁻⁷, 1.04 × 10⁻⁶, 1.39 × 10⁻⁶, 1.73 × 10⁻⁶, 2.06 × 10⁻⁶, 2.37 × 10⁻⁶, 2.83 × 10⁻⁶ mol L⁻¹. Arrow shows the change upon increasing the complex concentration. Inset: plots of F₀/F vs. [complex] for the titration of the complex to BSA.

Commonly, fluorescence quenching can be described by the Stern–Volmer eqn (1):⁵⁴

I₀/I = 1 + K_sv[Q] = 1 + K_qτ₀[Q] (1)

where I and I₀ are the fluorescence intensities of BSA with and without quencher (complex), respectively. K_q, K_SV, τ₀ and [Q] are the quenching rate constant of the biomolecule, the dynamic quenching constant, the average lifetime of the biomolecule without quencher and the concentration of quencher, respectively. In the present work, the value of K_sv for the Pd complex is 2.6 × 10⁵ M⁻¹ s⁻¹.

Since the fluorescence lifetime of the biopolymer is 10⁻⁸ s, the quenching rate constant, K_q, can be calculated using the following equation:

K_q = K_sv/τ₀

The K_q value for the Pd complex is 1.32 × 10¹³ M⁻¹ s⁻¹. However, the maximum scatter collision quenching constant, K_q, of various quenchers within the biopolymer is 2 × 10¹⁰ L mol⁻¹ s⁻¹.⁵⁵ Thus, the rate constant calculated by the protein quenching procedure is greater than the K_q from the scatter procedure. This indicates that a static quenching mechanism is in operation.⁵⁶

Therefore, the fluorescence quenching of BSA by the complex should be analyzed using the modified Stern–Volmer eqn (2):⁵⁷

where K_a is the association constant for the accessible fluorophores, f_a is the fraction of accessible fluorescence and ΔF is the difference in fluorescence intensity in the absence and presence of quencher at concentration [Q]. The dependence of I₀/ΔI on the reciprocal value of the quencher concentration, [Q]⁻¹, is linear, with a slope equal to the value of (f_aK_a)⁻¹. A quantitative estimate of the extent of the binding (K_a) is determined from the intercept to slope ratio of the modified Stern–Volmer equation. The K_a value for the BSA-Com system was computed as 4.3 × 10⁵ L mol⁻¹.

For the static quenching interaction, assuming that there are similar and independent binding sites in the macromolecule, the binding constant (K_b) and the number of binding sites (n) can be determined according to the method using the following eqn (3):⁵⁸

where I₀ and I are the same as those in eqn (3), K_b is the equilibrium constant for a site and n is the number of binding sites per BSA. The double logarithmic plot of log[I₀ − I/I] vs. log[com] is shown in Fig. 4. For the system complex and BSA, the values of K_b and n at 298 K were obtained (1.42 × 10⁵ L mol⁻¹, 1). Moreover, the linear correlation coefficient was calculated to be 0.996, which indicates that the assumptions underlying the derivation of eqn (3) were satisfied. The value of n is therefore, equivalent to 1. This indicates that there is a single class of binding sites for the complex on BSA.

Fig. 4 Scatchard plots of log[(F₀ − F)/F] vs. log[Q] for determination of the complex-BSA binding constant and the number of binding sites on BSA for the complex.

3.3.3. Site selective binding of the Pd complex on BSA. The crystal structure of BSA shows that BSA is a heart shaped helical monomer composed of three homologous domains named I, II, III, and each domain includes two sub-domains A and B to form a cylinder.⁵⁹ Eosin Y(EY) and Warfarin (WF), Ibuprofen (IB) and Flufenamic acid (FA) were used as site marker fluorescence probes for monitoring domains I and II of the serum albumin, respectively.^60,61 In this work, Eosin Y and Ibuprofen were used as domain I and II markers, respectively. In order to determine the Pd complex binding site on BSA, site marker competitive experiments are carried out using drugs (Eosin Y and Ibuprofen) known to specifically bind to a known domain or region on BSA. Information on the binding domain to which the complex binds can be obtained by monitoring the changes in the fluorescence of BSA after binding the complex in the presence of Eosin Y and Ibuprofen. As shown in Fig. 5A and B, the addition of a site marker (Eosin Y or Ibuprofen) into BSA decreased the fluorescence intensity compared to that without a site marker. To facilitate the comparison of the influence of Eosin Y and Ibuprofen on the binding of the complex to BSA, the binding constant in the presence of site markers was analyzed using the modified Stern–Volmer method (eqn (2)) (Fig. 5C and Table 2). The binding constant is surprisingly variable in the presence of Eosin Y, while a smaller influence is observed in the presence of Ibuprofen (somewhat lower than that with isolated BSA). The result indicates that the binding site of the complex is mainly located within domain I of BSA.

Fig. 5 Influence of selected site markers on the fluorescence of the complex bound to BSA (T = 298 K, λ_ex = 285 nm). (A) C(BSA) = C(Eosin Y) = 2 × 10⁻⁶ mol L⁻¹; (B) C(BSA) = C(Ibuprofen) = 2 × 10⁻⁶ mol L⁻¹; C(complex) 0, 2.95 × 10⁻⁷, 6.76 × 10⁻⁷, 1.04 × 10⁻⁶, 1.39 × 10⁻⁶, 1.73 × 10⁻⁶, 2.06 × 10⁻⁶, 2.37 × 10⁻⁶, 2.83 × 10⁻⁶ mol L⁻¹. (C) Modified Stern–Volmer plots for the complex-BSA system in the absence and presence of site markers (T = 298 K, pH 7.4). The inserts correspond to the molecular structures of the site markers.

Table 2 Estimated binding constants for site marker competitive experiments of the Pd complex-BSA system

Site marker	Ksv (L mol^−1)	Ka (L mol^−1)	R^a
Blank	2.64 × 10^5	4.39 × 10^5	0.992
Ibuprofen	1.60 × 10^5	2.76 × 10^4	0.998
Eosin Y	1.50 × 10^5	1.30 × 10^4	0.994

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3.3.4. Conformational changes of BSA induced by the Pd complex. Synchronous fluorescence spectra of BSA with different amounts of the Pd complex were recorded to explore the conformational change of BSA with various concentrations of the complex. The synchronous fluorescence spectroscopy of BSA can provide characteristic information on tyrosine or tryptophan residues when the wavelength interval (Δλ) between the excitation and emission wavelengths is 15 or 60 nm (Δλ = λ_emission − λ_excition).⁶² The synchronous fluorescence spectra of BSA upon addition of the Pd complex at Δλ = 15 and 60 nm are displayed in Fig. 6A and B. The fluorescence intensities of the protein decreased and a slight red shift is observed with increasing concentration of the complex. It is proposed that the interaction of the Pd complex with BSA influences the conformation of the tyrosine residues and the hydrophobicity around the tyrosine residues is strengthened,^61,63 thus resulting in the conformational changes of BSA.

Fig. 6 Synchronous fluorescence spectra of BSA in the presence of different concentrations of complex (λ = 15 nm (A) and λ = 60 nm (B)) at 298 K and pH 7.4. C(BSA) = 2 × 10⁻⁶ mol L⁻¹; C(complex) = 0, 2.95 × 10⁻⁷, 6.76 × 10⁻⁷, 1.04 × 10⁻⁶, 1.39 × 10⁻⁶, 1.73 × 10⁻⁶, 2.06 × 10⁻⁶, 2.37 × 10⁻⁶, 2.83 × 10⁻⁶, 3.05 × 10⁻⁶, 3.26 × 10⁻⁶, 3.47 × 10⁻⁶, 3.67 × 10⁻⁶ mol L⁻¹.

3.4. Nucleic acid binding studies

3.4.1. Electronic absorption spectroscopy. Electronic absorption spectroscopy is one of the most widely used methods to determine overall binding constants in DNA binding. Transition metal complexes can bind to nucleic acids via covalent or non-covalent interactions (electrostatic, groove binding (major and minor) and intercalation).⁶⁴ The electronic spectra of the Pd complex display bands around 236, 258 and 364 nm due to intra-ligand (ILCT) transitions of (π–π*) and (n–π*) types and metal to ligand charge transfer (MLCT), respectively. The absorption spectra of the Pd complex in the absence and presence of calf thymus DNA are shown in Fig. 7. Upon adding CT DNA to the Pd complex (R = [DNA]/[complex] = 0–2) in 2% DMSO/5 Mm Tris HCl/50 mM NaCl buffer solution, the ligand-based π → π* spectral band exhibits hypochromism (H% = 23.6) and only a small shift in the wavelength band position. The extent of hypochromism was calculated using the following equation:⁶⁵

H% = (A_Free − A_bounded)/A_Free × 100

Fig. 7 Electronic spectra of the complex in a buffer solution (5 mM Tris HCl/50 mM NaCl at pH 7.4) upon addition of CT-DNA. C(complex) = 1.5 × 10⁻⁵ mol L⁻¹, C(DNA) = 0–2.8 × 10⁻⁵ mol L⁻¹. Arrow shows the absorption intensities decrease upon increasing DNA concentration. Inset: plots of [DNA]/[ε_a − ε_f] vs. [DNA] for the titration of the complex with DNA.

The major decrease in the intensity at λ_max = 364 nm indicates that the interaction of the CT DNA with the complex takes place, resulting in the formation of a new complex with double helical CT DNA. In general, the absence of any shift in λ_abs max rules out the possibility of intercalative binding since this mode of complex binding with DNA usually causes a large shift in the absorption maxima, while groove binding results in an insignificant (or small) shift in the absorption spectra.⁶⁶ On the other hand, the trinuclear palladium complex is a very hydrophobic compound. Some hydrophobic molecules were shown to migrate towards sites inside the grooves of DNA and release bound solvent molecules.²⁹ This observation is ascribed to groove binding.

For the purpose of quantitatively evaluating the affinity of the complex with DNA, the intrinsic binding constant, K_b, was determined by monitoring the changes in absorbance at 364 nm according to eqn (4)⁶⁵

The absorption coefficients, ε_a, ε_f, and ε_b, correspond to A_obs/[DNA], the extinction coefficients for the free complex and the complex in the fully bound form, respectively. In particular, ε_f was determined by the calibration curve of the isolated Pd(II) complex in aqueous media following Beer's law. The slope and the intercept of the linear fit of the plot of [DNA]/[ε_a − ε_f] vs. [DNA] give 1/[ε_a − ε_f] and 1/K_b[ε_b − ε_f] (Fig. 7, inset). The intrinsic binding constant, K_b, (2 × 10⁵ M⁻¹) can be obtained from the ratio of the slope to the intercept.⁶⁷

From the values of the binding constant (K_b), the free energy (ΔG) of the compound-DNA complex was calculated using eqn (5):

ΔG = – RT ln K_b (5)

Binding constants are a measure of the compound-DNA complex stability while the free energy indicates the spontaneity/non-spontaneity of compound-DNA binding. Free energy of the Pd complex was evaluated as negative values (−7.20 kcal mol⁻¹), showing the spontaneity of the compound-DNA interaction.

3.4.2. Fluorescence studies: competitive interaction of the complex with MB–ds-DNA. No luminescence is observed for the Pd complex in any solvent or even in the presence of nucleic acid at ambient temperature. Therefore, competitive binding experiments using the Pd complex may afford further information for studying the binding of the complex to nucleic acids. Methylene blue (MB) is a planar cationic dye that is widely used as a sensitive fluorescence probe for native DNA. MB emits intense fluorescent light in the presence of DNA due to its strong intercalation between adjacent DNA base pairs.⁶⁸ The emission spectra of DNA bound MB in the presence of varying concentrations of the complex was monitored (Fig. 8). The experiment revealed that addition of the complex to the DNA bound MB does not cause the release of MB molecules, while the emission intensity decreases steadily. This implies that the two probes, the complex and the MB, bind with DNA independently and binding of one probe does not affect that of the other. Fig. 8 clearly reveals the decrease in the fluorescence intensity of the probe molecule (MB) ([DNA]/[MB] = 10) by adding the Pd(II) complex ([complex]/[DNA] = 2.5). Thus, the experiments confirmed the interaction between DNA and the Pd(II) complex is the groove interaction.

Fig. 8 The emission spectra of the DNA–MB system, in the presence of complex, C(DNA) = 5 × 10⁵ mol L⁻¹, C(complex) = 0–1 × 10⁻⁵ mol L⁻¹, C(MB) = 5 × 10⁻⁶ mol L⁻¹. The arrow shows the emission intensity changes upon increasing complex concentration. The inserts correspond to the molecular structures of methylene blue.

3.4.3. Helix melting experiment. Thermal denaturation studies of the B form of the DNA duplex provide a basis for modeling strand separation. The melting of DNA can be used to distinguish between molecules that bind via intercalation and groove mechanism. In the presence of metal-complex intercalators, T_m rises sharply until all the intercalating sites are saturated, after which the stabilization is due to electrostatic binding and T_m increases less steeply. The effect of the Pd complex and MB on the melting temperature (T_m) of calf thymus DNA in a buffer is shown in Fig. 9. In this experiment, the T_m of CT-DNA alone was 81.62 ± 0.2 °C. After the complex was added, ΔT_m increased by 2.97 °C. Common DNA metallo intercalators cause ΔT_m values of >10 °C,⁶⁹ and the organic intercalator EB (13 °C)⁷⁰ was higher than that of Pd complex. ΔT_m < 10 °C is indicative of weak DNA-binding abilities of the complex, which would most likely interact with DNA.⁷¹ This ΔT_m value is similar to those reported for typical groove binding, such as polynuclear complexes.^27,29 These results indicate that the binding strength between the complex and DNA was the groove bindings. It seems that binding to DNA should increase with the size of the complex, since the number of metal centers and consequently the hydrophobic contribution to groove binding increases.

Fig. 9 Plots of the changes of absorbance at 260 nm of CT-DNA (7.5 × 10⁻⁶ mol L⁻¹) upon heating in the absence and presence of the complex (37.5 × 10⁻⁶ mol L⁻¹) in 5 mM Tris HCl with 50 Mm NaCl.

3.4.4. Conformational change of the DNA double helix. CD spectroscopy is a powerful technique to monitor DNA conformational changes resulting from drug or metal complex binding. It also shows the potential to be a drug screening platform in the future. The CD spectrum of the CT DNA exhibits a positive band at 275 nm due to base stacking and a negative band at 245 nm due to the helicity of B DNA.⁷² The interaction of the Pd complex with DNA induces a change in the CD spectrum of B DNA (Fig. 10). Gradual addition of the complex to DNA causes a decrease in the CD spectra intensity in the positive as well as negative bands. The secondary structure of DNA is known to be perturbed by the intercalation of small molecules and, thus, increases the intensities of both bands, stabilizing the right handed B conformation of CT-DNA. Whereas, the simple groove binding intensities of both the negative and positive bands decrease significantly and show less or no perturbation on the base stacking and helicity bands. This suggests that the DNA binding of the complex induces certain conformational changes, such as the conversion from a more B-like to a more Z-like structure within the DNA molecule.⁷³ These changes are indicative of the groove binding mode.^74,75

Fig. 10 CD of CT-DNA (1 × 10⁻⁴ mol L⁻¹) in the absence and presence of the Pd(II) complex (5 × 10⁻⁵ mol L⁻¹) in 5 Mm Tris HCl with 50 Mm NaCl (pH = 7.4).

3.5. Molecular docking of the trinuclear palladium complex with DNA sequence d(CGCGAATTCGCG)₂

In order to obtain the binding site, blind docking was performed on the DNA duplex with a d(CGCGAATTCGCG)₂ sequence. The grid map was set to 28 × 28 × 26 ų along the x, y, and z axes with 1.0 Å grid spacing. The center of the grid map was set to 17.008, 22.868, and 8.944 Å. The conformations were ranked based on the lowest free binding energy. The results of the docking revealed that the Pd complex interactions with the DNA were major groove (Fig. 11). There are six hydrophobic interactions between the complex atoms and bases of DNA, viz., (i) between C51 and DG2; (ii) C42, C43 and DC3; (iii) C37, C38 and DG4; (iv) C8, C9 and DA17; (v) C19, C20 and DT19; and (vi) C20, C46, C47 and DT20. The binding free energy was found to be −6.54 kcal mol⁻¹. The binding free energy indicates a high binding affinity between the complex and DNA.

Fig. 11 (A) Molecular docking of the Pd complex with the major groove side of DNA by UCSF chimera, (B) two-dimensional interactions generated by LIGPLOT+.

3.6. Molecular docking of the trinuclear palladium complex with BSA

In the docking of the Pd complex with BSA, a grid map was set with dimensions of 48 × 36 × 44 ų with a grid point spacing of 1.0 Å. The center of the grid box was placed at points of 10.996, 28.52, and 83.093 Å. The conformations were ranked based on the lowest binding free energy. The molecular docking study of BSA showed that the Pd complex prefers the binding pocket of domain I. As shown in Fig. 12, there are five categories of hydrophobic contacts between the complex atoms and the amino acids of the binding site, viz., (i) between C22 and Asn44; (ii) C56, C61 and Asp129; (iii) C10, C11, C12, C28, C30, C31, C32, C33 and Lys131; (iv) C6, C8, C9 and Lys132; and (v) C6 and Val40. The binding free energy for the complex to BSA was found to be −6.81 kcal mol⁻¹. In addition, the docking study shows that the distance between the Trp134 residue and the complex is 4.33 Å. These results are in agreement with the fluorescence quenching of BSA emission in the presence of the Pd complex.

Fig. 12 (A) The Pd complex was docked in the binding pocket of BSA using UCSF chimera, (B) two-dimensional interactions generated by LIGPLOT+.

4. Conclusion

Recently oxime and its derivatives, which have shown to have favorable bioactivities, have attracted researchers in many areas, especially in the bioinorganic chemistry and pharmacological fields. This is because of their low toxicity to non-target organisms, antibacterial and antifungal properties and high index of antitumor activity. The present study offers a different mechanism for the synthesis and characterization of a novel trinuclear palladium(II) complex containing an oxime chelate ligand. The crystal structure of the complex [Pd₃(C₁₂H₈C=NO)₆] was determined by X-ray crystallography revealing a (distorted) square-planar geometry for Pd(II). DNA interaction activity of the synthetic trinuclear palladium complex was carried out, and the results were discussed. The UV-visible, fluorescence competitive, thermal denaturation and DNA solution circular dichroism (CD) measurements revealed the ability of the complex to bind to DNA and supported the fact that the complex binds to DNA via groove binding. The experimental results also show that the secondary structure of BSA molecules and the polarity of the microenvironment around the tyrosine and tryptophan residues change in the presence of the trinuclear palladium complex. The reactivity towards BSA revealed that the quenching of BSA fluorescence by the Pd complex was of the static type. The site marker competitive experiments indicate that the Pd complex binds specifically to the hydrophobic pocket of domain I (sub domain IIA) of BSA. The results of the molecular docking studies reiterate domain I as the favorable binding site of the BSA and groove binding of the DNA with the complex. Further studies on the trinuclear palladium complex in vivo and in vitro anticancer activities are currently in progress in our research group.

Acknowledgements

This study was funded and supported by Isfahan University of Technology and Tehran University of Medical Sciences (Grant number: 27496). We gratefully thank the Iran National Science Foundation (INSF) for financial support. Crystallography was provided by Institute of Physical Chemistry, Polish Academy of Sciences, Warsaw, Poland.

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Footnote

† Electronic supplementary information (ESI) available. CCDC 1423655 contains the supplementary crystallographic data for the complex. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c6ra08744b

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