DNA and BSA-Binding Studies of Dinuclear Palladium(II) Complexes with 1,5-Naphtiridine Bridging Ligands

Chemicals and reagents ethylenediamine (en), 1,3-propylenediamine (1,3-pd), 1,5-naphthyridine (1,5-nphe), deoxyribonucleic acid isolated from calf thymus (CT-DNA), bovine serum albumin (BSA), ethidium bromide (EtBr), 0.01 M phosphate buffer and K₂[PdCl₄] were purchased from Sigma-Aldrich Chemical Co. Bidistilled water was used to prepare a solution of these reagents. Other chemicals used in this work were commercial products of analytical purity and were purchased from a domestic manufacturer.

All pH measurements were performed at 25 °C. For this purpose, a pH meter Mettler Toledo Seven Copmact S220-U was used, which was calibrated in relation to Mettler Toledo buffer solutions for pH = 4.0 and pH = 7.0. The measured pH values were not corrected for the deuterium effect.

Elemental microanalysis for C, H, and N parameters were performed in the Microanalytical Department of the Institute of Chemistry, Faculty of Chemistry, University of Belgrade.

D₂O as the solvent and TSP (trimethyl-silylpropane-3-sulfonate) as the reference standard were used to record the ¹H and ¹³C NMR spectrum. The spectra were recorded on a Varian Gemini 200 MHz spectrometer. All reactions were performed in 5 mm diameter NMR cuvettes.

Electronic absorption spectra were recorded on a Shimadzu UV-Vis spectrophotometer. Concentrations of dinuclear palladium(II) complexes were 5 · 10⁻⁵ mol/dm³. The electron absorption spectra were recorded in the wavelength range 200–500 nm at 25 °C and the experimental results were processed using the computer program Microsoft Office Excel 2003.

Infra-red (IR) spectra were recorded on a Perkin-Elmer Spectrum One FT-IR spectrometer using the KBr technique, in the wavelength range of 450 – 4000 cm⁻¹.

Fluorescence measurements were performed on an RF-1501 PC spectrophotometer (Shimadzu, Japan).

Mononuclear palladium(II) complexes [Pd(en)Cl₂] and [Pd(1,3-pd)Cl₂] (en and 1,3-pd, are bidentate coordinated diamine ligands ethylenediamine and 1,3-propylenediamine, respectively) were synthesized according to a modified procedure previously described in the literature (29, 30, 31).

0.1632 g (5.00 · 10⁻⁴ mol) of K₂[PdCl₄] was dissolved in 25 cm³ of water and the resulting brown solution was transferred to a 100 cm³ double-necked flask supplied with reflux condenser and dropper. The pH value of the solution was adjusted to about 2–3 by adding a solution of HCl with a concentration of 0.1 M. Obtained solution was heated on a water bath, with slow instilment of an equimolar amount (5.00 · 10⁻⁴ mol) of ethylenediamine or 1,3-propylenediamine. Instilment is performed slowly for one hour with constant heating, stirring and occasional control of pH values (pH 2-3). During the reaction, the color of the solution changes from brown to light yellow. After refluxing for 5 h, the reaction mixture was left at room temperature overnight. The complexes crystallized from aqueous solution at room temperature. The obtained crystals were filtered off, washed with ethanol and air dried. The yield was about 90%.

Dinuclear palladium(II) complexes, [{Pd(en)Cl}₂(μ-1,5-nphe)](NO₃)₂ (Pd1) and [{Pd(1,3-pd)Cl}₂(μ-1,5-nphe)](NO₃)₂ (Pd2), were obtained from the corresponding mononuclear complexes according to a modified procedure previously described in the literature (32, 33, 34).

In 5 cm³ of dimethylformamide 0.0572 g (3.37 · 10⁻⁴ mol) of AgNO₃ was dissolved and a suspension of the mononuclear [Pd(en)Cl₂] or [Pd(1,3-pd)Cl₂] complex (3.43 · 10⁻⁴ mol) in 10 cm³ of dimethylformamide was added. The reaction mixture was leaved overnight with stirring at room temperature and in the dark. Precipitated AgCl was removed by filtration and in the pale yellow solution of palladium(II) complex, [Pd(en)(dmf)Cl]⁺ or [Pd(1,3-pd)(dmf)Cl]⁺, solution of 1,5-naphthyridine (1,5-nphe) in dimethylformamide (molar ratio 2 : 1) was edded dropwise. The reaction mixture was stirred at room temperature for about 4 h in the dark. The volume of the solution was reduced by evaporation of dimethylformamide on a rotary vacuum evaporator.

After the addition of about 20 cm³ of dichloromethane, a light yellow precipitate of the complex [{Pd(en)Cl}₂(μ-1,5-nphe)](NO₃)₂ and [{Pd(1,3-pd)Cl}₂(μ-1,5-nphe)](NO₃)₂ was obtained. The resulting precipitate of dinuclear palladium(II) complexes was separated by filtration, washed with methanol, then ether and air dried. The yield of dinuclear palladium(II) complexes was 65% for Pd1 and 50% for Pd2.

Anal. Calcd. for Pd1 (C₁₂H₂₂N₈Cl₂O₆Pd₂: FW = 658,11): C, 21.90%; H, 3.37%; N, 17.03%. Found: C, 22.11%; H, 3.26%; N, 17.29%. ¹H NMR (200 MHz, D₂O, δ, ppm): 8.14 (m, 2H, H3, H7), 9.58 (m, 2H, H4, H8), 10.21 (d, 2H, H2, H6). ¹³C NMR (50 MHz, D₂O, δ, ppm): 130 (C3, C7), 143 (C4a, C8a), 147 (C4, C8), 160 (C2, C6). IR (KBr, ν, cm⁻¹): ~3226, 3100 (N-H stretch); 1582, 1509 (C=C/C=N 1,5-naphthyridine group stretch); 1373, 1356, 1336 (ν_as(NO₃)). UV-vis (H₂O, λ_max, nm): 303 (ɛ = 7.8 · 10³ M⁻¹cm⁻¹), 314 (ɛ = 8.0 · 10³ M⁻¹cm⁻¹).

Anal. Calcd. for Pd2 (C₁₄H₂₆N₈Cl₂O₆Pd₂: FW = 686,15): C, 24.51%; H, 3.82%; N, 16.33%. Found: C, 24.28%; H, 3.55%; N, 16.53%. ¹H NMR (200 MHz, D₂O, δ, ppm): 8.23 (m, 2H, H3, H7), 9.55 (d, 2H, H4, H8), 10.17 (d, 2H, H2, H6). ¹³C NMR (50 MHz, D₂O, δ, ppm): 131 (C3, C7), 143 (C4a, C8a), 146 (C4, C8), 160 (C2, C6). IR (KBr, ν, cm⁻¹): ~3241, 3135 (N-H stretch); 1659, 1600, 1589, 1510 (C=C/C=N 1,5-naphthyridine group stretch); 1367, 1295 (ν_as(NO₃)). UV-vis (H₂O, λ_max, nm): 303 (ɛ = 7.5 · 10³ M⁻¹cm⁻¹), 313 (ɛ = 7.2 · 10³ M⁻¹cm⁻¹).

Emission fluorescence spectroscopy were used to examine interactions of dinuclear palladium(II) complexes [{Pd(en)Cl}₂(μ-1,5-nphe)](NO₃)₂ and [{Pd(1,3-pd)Cl}₂(μ-1,5-nphe)](NO₃)₂ with bovine serum albumin (BSA). Emission spectra were recorded in the wavelength range 300–500 nm, with extinction at 295 nm (32). The binding effect of the tested albumin complexes was observed based on the reduction of the albumin emission intensity (1.6 · 10⁻⁶ M in 0.01 M PBS) to 352 nm after the addition of palladium(II) complexes (0–4.0) · 10⁻⁵ M. All emission spectra were recorded under the same experimental conditions.

The Stern-Volmer constant (K_sv) was determined based on the equation: I₀/I = 1 + K_sv[Q] = 1 + k_qτ₀[Q], where I₀ is the initial fluorescence intensity of tryptophan in BSA, and I is the fluorescence intensity of tryptophan in BSA after addition of palladium(II) complex in protein solution, k_q is the fluorescence quenching constant, τ₀ is the average time for fluorescence of albumin in the absence of the complex and [Q] is the concentration of the complex. The binding constant (K_a) as well as the number of binding sites (n) were obtained on the basis of Scatchard's equation: log(I₀–I)/I = logK_a + n · log[Q]. The results are presented graphically as the dependence of log(I₀–I)/I from log [Q], the value of K_a is aquired from the intersection of the obtained line with the y axis, and the number of connecting points (n) from the slope of the line.

Dinuclear palladium(II) complexes, [{Pd(en)Cl}₂(μ-1,5-nphe)](NO₃)₂ (Pd1) and [{Pd(1,3-pd)Cl}₂(μ-1,5-nphe)](NO₃)₂ (Pd2), were obtained from the corresponding mononuclear complexes according to a modified procedure previously described in the literature (32, 33, 34).

As shown in Figure 1, the synthesized dinuclear palladium(II) complexes have the same bridging 1,5-naphthyridine ligand and different bidentate coordinated diamine ligands. ¹H and ¹³C NMR spectra of 1,5-naphthyridine, ethylediamine, 1,3-propylenediamine as well as Pd1 and Pd2 complexes were recorded in D₂O as solvent. NMR data for ligands and dinuclear palladium(II) complexes is given in Table 1, while ¹H and ¹³C NMR spectra of Pd1 and Pd2 complexes in Figure 2.

Figure 2.

¹H and ¹³C NMR spectra of [{Pd(en)Cl}₂(μ-1,5-nphe)](NO₃)₂ (Pd1) and [{Pd(1,3-pd)Cl}₂(μ-1,5-nphe)](NO₃)₂ (Pd2) complexes.

After coordination of diamine ligand to the palladium(II) ion, the signal shifts downfield (Fig. 2 and Table 1). The shifts of the signals, which correspond to the 1,5-nphe protons after coordination for the palladium(II) ion, are a consequence of the delocalization of the charge transmitted through the pyridine rings (20,32,42). The multiplet at 2.76 ppm in the ¹H NMR spectrum of the Pd1 complex, correspond to methylene protons of bidentate coordinated ethylenediamine. This signal was shifted by Δδ = 0.12 ppm downfield in regard to free ethylenediamine. In the aliphatic part of the ¹H NMR spectrum of the Pd2 complex, two multiplets appear at 1.85 and 2.60 ppm due to the CH₂ protons at position 2 and the CH₂ protons at position 1 and 3 (Fig. 1, Table 1). Signals derived from these protons after coordination were shifted downfield (Δδ = 0.25 ppm for methylene protons at position 2 and Δδ = 0.19 ppm for same protons at positions 1 and 3) in regards to the uncoordinated 1,3-pd.

In the ¹³C NMR spectra of the Pd1 and Pd2 complexes, in the aromatic region, four signals from the carbon atoms of the 1,5-naphthyridine ligand (C2 and C6, C4 and C8, C4a and C8a, C3 and C7) appears. The position of these signals differs significantly from those of the uncoordinated 1,5-nphe (Table 1). After coordination of 1,5-nphe for Pd(II) the signals for the carbon atoms in the heterocyclic aromatic ring move downfield. The signal at 50 ppm originating from from the carbon atoms of bidentate-coordinated ethylediamine in Pd1, are shifted by Δδ = 5 ppm in regarde to the uncoordinated en ligand. Also, in the spectrum of the Pd2 complex, the signals corresponding to the C atoms of the coordinated 1,3-pd ligand are shifted by Δδ = 1–5 ppm downfield relative to the signals of the uncoordinated ligand.

The UV-Vis spectra of the Pd1, Pd2 complexes and free 1,5-nphe ligand are given in Figure 3. The first absorption maximum is located in the wavelength range of 296 to 310 nm, while the second maximum is in the range of 310 to 320 nm. All absorption maxima of the synthesized complexes have a batochromic shift after the coordination of 1,5-naphthyridine, due to π….π electron transitions in the heterocyclic 1,5-nphe ligand.

Figure 3.

UV-Vis spectra of dinuclear [{Pd(en)Cl}₂ (μ-1,5-nphe)]²⁺ and [{Pd(1,3-pd)Cl}₂(μ-1,5-nphe)]²⁺ complexes and free 1,5-nphe ligand.

IR spectra of Pd1 and Pd2 complexes, recorded in the wavelength range 4000 - 450 cm-1, show bands corresponding to coordinated 1,5-nphe ligand, as well as bands of bidentate coordinated ethylenediamine (en) and 1,3-propylenediamine (1,3-pd) (Fig. 4).

Figure 4.

IR spectra of Pd1 and Pd2 complexes, KBr, 4000-450 cm⁻¹.

In the IR spectra of dinuclear palladium(II) complexes, two very strong bands in the range of 3280 - 3098 cm⁻¹ were observed, which correspond to asymmetric and symmetric vibrations of the coordinated amino group of ethylenediamine and 1,3-propylenediamine. The medium-strength band in the region of 1500–1650 cm⁻¹ correspond to the C = C and C = N vibrations of the aromatic 1,5-naphthyridine ring. A strong and wide band at ~1364 - 1384 cm⁻¹ indicates the existence of NO_3⁻ ions in the outer coordination sphere of dinuclear palladium(II) complexes.

Serum albumin, as the most common blood plasma protein, is one of the most studied proteins (23,25). It plays an important role in the transport of metal ions and their complexes through the blood system to cells and tissues. Bovine serum albumin (BSA) is the most studied serum albumin due to its structural similarity to human serum albumin (HSA). Namely, human serum albumin contains one tryptophan at the Trp-214 position, while BSA contains two tryptophan residues, Trp-134 and Trp-214. The bovine serum albumin (BSA) solution shows intense fluorescent emission at λ_{em, max} = 352 nm, and after excitation at 295 nm (47). After the addition of the Pd1 or Pd2 complex to the BSA solution, the fluorescence intensity at λ = 352 nm decrease (Fig. 7) based on it can be concluded that the tested complexes interact with this biomolecule. The decrease in fluorescence intensity can be attributed to changes in protein structure, which occur due to changes in the tryptophan environment in BSA, because of protein binding to complexes (48).

Figure 7.

BSA emission spectra in the presence of [{Pd(en)Cl}₂(μ-1,5-nphe)]²⁺ and [{Pd(1,3-pd)Cl}₂(μ-1,5-nphe)]²⁺ complexes. [BSA] = 1.60 · 10⁻⁶ M, [Pd(II) complex] = (0 – 4.00) · 10⁻⁵ M; λ_ex = 295 nm. Inserted graph: Dependence of Io/I from concentration [Q].

The values of the dynamic Stern-Volmer constant (K_sv) and the fluorescence quenching constant (k_q) for complex interactions with BSA were determined using the Stern-Volmer equation. The values of the constants K_sv and k_q for the interaction of the studied Pd1 and Pd2 complexes with BSA are given in Table 4. In addition, Table 4 shows the values of binding constants (K_a) as well as the number of albumin binding sites (n) for Pd(II) complexes, which were obtained on the basis of Scatchard's equation (Experimental part 2.6). The obtained results are graphically presented as the dependence of log(I₀–I)/I from log[Q], the value of K_a is obtained from the intersection of the line with the y axis, and the number of connecting points (n) from the slope of the line.

Based on the linearity in Stern-Volmer graphs, it can be concluded that both complexes lead to the quenching of tryptophan emission in the BSA molecule. K_sv values for Pd1 and Pd2 complex (Table 4) indicate a very similar binding affinity of BSA for the tested complexes. The values of n for both complexes are 1.8, which indicates that there are approximately two possible sites in the protein suitable for complex binding. The quenching constants (k_q) are greater than 10¹⁰ and indicate a static emission quenching mechanism (49). K_a value leads to conclusion that the Pd1 and Pd2 complexes can bind to BSA and thus be transported to the cell and hydrolyze the bond with BSA at the appropriate site in the cell.