Synthesis and characterization of mononuclear copper(II) complex of tetradentate N2S2 donor set and the study of DNA and bovine serum albumin binding

Abstract

One mononuclear copper(II) complex, containing neutral tetradentate NSSN-type ligands, of formulation [Cu ^II(L ¹)Cl]ClO ₄ (1), was synthesized and isolated in pure form [where L ¹˭ 1,3-bis(3-pyridylmethylthio)propane]. Green-colored copper(II) complex was characterized by physicochemical, spectroscopic methods and conductivity measurement. These experimental data matched well with the proposed structure of the complex. Biological activity of the complex (1) toward calf thymus DNA and bovine serum albumin has been examined systematically and groove-binding behavior of the Copper(II) complex 1 with calf thymus DNA has been observed from the spectral study.

• Copper complex
• Nitrogen–sulfur ligand
• DNA binding

1. Introduction

Identifying molecules that intercalate into DNA helices have attracted considerable interest over the last few decades. The cleavage of nucleic acids may be considered as an enzymatic reaction which comprises various biological processes as well as the biotechnological manipulation of genetic material. The application of artificial DNA cleaving agents is manifold: biotechnology, structural studies of nucleic acids or development of new drug [1–6]. Compounds showing the property of effective binding as well as cleaving double-stranded DNA under physiological conditions are of importance since these could be used as diagnostic agents in medicinal and genomic research [4–12].

Serum albumins are the most important protein in plasma that functions in biological system to carry drugs as well as endogenous and exogenous substances [13]. Among the serum albumins, bovine serum albumin (BSA) has a wide range of physiological functions involving binding, transport and delivery of fatty acids, porphyrins, bilirubin, steroids, etc. The binding properties of BSA with various drugs have been fully investigated by many researchers [14–18], which are useful for understanding the reaction mechanism as well as for providing guidance for the application and design of new drugs [19]. BSA has been selected as our protein model due to its water-soluble nature which is important for interaction studies [20,21].

The potential role played by copper ions in the active sites of a large number of metalloproteins has stimulated efforts to design and characterize copper complexes as models for providing better understanding of biological systems and for assisting in the development of new homogeneous catalysts for selective oxidation. Copper(II) complexes are effective for interaction and cleavage agents of DNA and hydrolysis catalysts [22–24]. Particularly, the coordination chemistry and reactivity of copper complexes involving nitrogen–sulfur donor ligands has received considerable attention as models [25–29]. In particular, the CuN₂S₂ chromophore is present in blue copper proteins such as plastocyanine [30,31] and copper(II) chelates of N2S2 ligands have been found to have antineoplastic activities and to interact with biological systems [32–34].

The present work stems from our interest in designing and developing the chemistry of copper complexes that are capable of binding DNA. Herein, we report an account on synthesis, characterization of copper(II) complex having NS ligand moiety, (1,3-bis(3-pyridylmethylthio)-propane) and investigation of the mode of binding of the complex with DNA and BSA.

2. Experimental

2.1 Materials and physical measurement

All chemicals and reagents were obtained from commercial sources and used as received. Solvents were distilled using an appropriate drying agent [35,36].

The elemental (C, H, N) analyses were performed on a Perkin-Elmer model 2400 elemental analyzer. Electronic absorption spectra were recorded on a JASCO UV–VIS/NIR spectrophotometer model V-570. IR spectra (KBr discs, 4000–300 cm⁻¹) were recorded using a Perkin-Elmer FTIR model RX1 spectrometer. Fluorescence spectra were recorded on a Fluorometer Hitachi-2000. The room temperature magnetic susceptibility measurements were performed by using a vibrating sample magnetometer PAR 155 model. Electrochemical measurements were recorded on a computer-controlled EG&G PAR model 270 VERSTAT electrochemical instruments with tetrabutylammonium perchlorate (TBAP) as supporting electrolyte, a Pt-disk as working electrode and a Pt-wire as auxiliary electrode. All the measurements were made at 298 K with acetonitrile as solvent. Molar conductances () were measured in a systronics conductivity meter 304 model using ∼10⁻³ mol L⁻¹ solutions in appropriate organic solvents. Thermal analysis of the complex was carried out using Perkin-Elmer Diamond TG/DT analyzer.

2.2 DNA-binding experiments

Tris-(hydroxymethyl)aminomethane–HCl (Tris–HCl) buffer solution () was used in all the experiments involving calf thymus DNA (CT-DNA). The CT-DNA used in the experiments was sufficiently free from protein as the ratio of UV absorbance of the solutions of DNA in tris–HCl at 260 and 280 nm was almost ≈1.9 [37]. The concentration of DNA was determined with the help of the extinction coefficient ( L mol⁻¹ cm⁻¹) of DNA solution [38]. Stock solution of DNA was always stored at 4°C and used within 4 days. Stock solution of the copper(II) complex was prepared by dissolving the complex in DMSO and suitably diluted with tris–HCl buffer to the required concentration for all the experiments. Absorption spectral titration experiment was performed by keeping constant the concentration of the copper(II) complex and varying the CT-DNA concentration.

In the ethidium bromide (EB) fluorescence displacement experiment, 5 μL of the EB tris–HCl solution (1 mmol L⁻¹) was added to 1 mL of CT-DNA solution (at saturated binding levels) [39], and stored in the dark for 2 h. Then the solution of the copper(II) complex was titrated into the CT-DNA/EB mixture and diluted in tris–HCl buffer to 5 mL to get the solution with the appropriate copper(II) complex/CT-DNA mole ratio. Before measurements, the mixture was shaken up and incubated at room temperature for 30 min. The fluorescence spectra of EB bound to DNA were obtained at an emission wavelength of 610 nm ( nm) in the Fluorimeter (Hitachi-2000).

Figure 1. Ligand's structure and the procedures of the reactions.

2.2.1 Protein (BSA) binding experiments

Samples for spectroscopic measurements were prepared by dissolving BSA in water and administering the appropriate concentration of the Cu(II) complex. The samples were carefully degassed using pure nitrogen gas for 15 min. Quartz cells with high vacuum Teflon stopcocks were used for degassing.

2.3 Preparation of the ligand

The preparation of 1,2-bis(3-pyridylmethylthio)ethane (L¹) has been carried out following a common procedure with slight modification [40]. An ethanolic solution of 3-picolyl chloride, hydrochloride (1.64 g, 10.0 mmol) was added to 1,3-propandithiol (0.54 g, 5.0 mmol) in dry ethanol containing sodium ethoxide (0.46 g, 20.0 mmol) at low temperature (0–5°C). Then this mixture was stirred at room temperature for 0.5 h and then it was refluxed for 2 h. The mixture was cooled to room temperature, water was added and finally the ethanol was evaporated off using rotary evaporator. The product was extracted into dichloromethane and dried by using NaHSO₃. The product, 1,3-bis(3-pyridylmethylthio)propane (L¹), was obtained as a yellow oil by removing the dichloromethane. The other products were also obtained as liquids. Finally, the products were verified by ¹H NMR spectroscopy.

2.4 Preparation of Cu(II) complex 1

The copper(II) complex was synthesized following a common procedure as described below, using copper(II) perchlorate, hexahydrate and the organic compound in equimolar ratio. L¹ (292.0 mg, 1.0 mmol) was mixed with 1.0 mmol of copper(II) perchlorate hexahydrate, and the mixture was stirred for 4 h in methanol. Then sodium chloride (1.0 mmol) was added to the resulting mixture and stirred for another 1 h at room temperature. The green colored complex was precipitated out and the solid was filtered, washed with water, cold methanol and dried in vacuo.

Yield: 84–85 %.

[Cu(L¹)Cl]ClO₄(1): C₁₅H₁₆N₂CuCl₂O₄: Anal. Calcd: C, 40.45; H, 3.90; N, 6.41; Cu, 14.12. Found: C, 40.65; H, 3.80; N, 6.48; Cu, 14.19. IR (KBr cm⁻¹): , 1384; , 1091 and 626. Conductance Λo (ohm⁻¹ cm² mol⁻¹) in methanol: 145.

3. Result and discussion

The copper(II) complex was obtained in good yield from the reaction of copper(II) perchlorate with equimolar amounts of the respective organic moiety in the methanol medium. The organic moieties (L¹) (depicted in Scheme 1) act as tetradentate neutral ligands with four NSSN donor centers in this complex.

Microanalytical data (Table 1) establish the composition of the complex. Here, monomeric 1 complex is soluble in acetonitrile and DMF but sparingly soluble in methanol, ethanol and dichloromethane. At room temperature, the magnetic moment (μ) of complex 1 is 1.81 B.M., per copper atom. The conductivity measurement of complex 1 in acetonitrile shows the conductance values of 145 Λo mol⁻¹ cm⁻¹ at 300 K. These values suggest that complex 1 exists as 1: 1 electrolytes.

Table 1  Microanalytical^a and physicochemical data of complex 1.

	Elemental analyses^a (%)
					Magnetic moment^b	Conductance^c
Compd	C	H	N	Cu	(μ, B.M.)	(Λo, mho cm^2 mol^−1)
1	40.45	3.90	6.41	14.12	1.80	145
	(40.48)	(4.08)	(6.30)	(14.51)

^aCalculated values are in parenthesis.

^bAt room temperature.

^cIn MeOH.

3.1 Spectral studies

3.1.1 IR Spectra

Infrared spectral data of complex 1 represented in exhibit an intense band at approximately 1090 cm⁻¹ along with a weak band at 624 cm⁻¹, which have been assigned to and , respectively. The band is observed at 1465–1478 cm⁻¹ for the complex, in addition to the at 760 cm⁻¹. The IR spectrum of complex 1 is given in Figure 1.

Scheme 1. IR spectrum of complex 1.

3.1.2 UV–VIS spectra

The electronic absorption spectra (Figure 2) of complex 1 were recorded at room temperature using acetonitrile as solvent and the data have been tabulated in Table 2. The spectrum shown in Figure 2 exhibits a band around 360 nm assignable to the S(σ) → Cu(II) charge transfer (LMCT) transition [19] along with the transition at high-energy region corresponding to intramolecular and transitions [41–43]. A characteristic d‒d absorption band (above 640 nm) has also been observed in the electronic spectrum of this complex.

Table 2  UV–VIS spectral and electrochemical data^a.

		Electrochemical data^b E^o′, mV(, mV)
Compds	λnm (ϵ, dm^3 mol^−1 cm^−1)	[Epa, mV; Epc, mV]
1	210 (s, 12,227), 258 (s, 8,432), 362 (s, 3,971), 646 (b, 316)	−378 (182) [−287, −469]

^a1 in acetonitrile

^bScan rate of 100 mV s⁻¹.

Fig. 2. A UV–VIS spectrum of copper(II) complex 1 in acetonitrile.

3.2 Electrochemistry

Redox properties of complex 1 was examined by cyclic voltametry using a Pt-disk working electrode and a Pt-wire auxiliary electrode in dry MeOH and in presence of [n-Bu₄N]ClO₄ as supporting electrolyte. The potentials (Table 2 and Figure 3) are expressed with reference to Ag/AgCl electrodes. In solution, all compounds displayed a quasi-reversible voltammogram having in the range of −340 to −387 mV with Δ E=132–294 mV. Only one quasi-reversible Cu(II)/Cu(I) redox couple at negative potential was observed.

Fig. 3. CV diagram of copper complexes in acetonitrile for complex 1 using TBAP as supporting electrolyte at a scan rate of 100 mVs⁻¹ at room temperature.

3.3 DNA-binding studies

The interaction of copper(II) complex 1 with calf thymus DNA (CT-DNA) has been investigated by absorption and emission spectral studies in Figure 4.

Fig. 4. Electronic spectral of complex 1 through titration with CT-DNA in tris–HCl. The increase of DNA concentration is indicated by an arrow.

Electronic absorption spectroscopy is an effective method to examine the binding modes of metal complex with DNA. In general, binding of the metal complex to the DNA helix is testified by an increase of the CT band of copper(II) complex due to the involvement of strong interactions between an aromatic chromophore of complex and the base pairs of DNA [44].

In order to further illustrate the binding strength of the copper(II) complex with CT-DNA, the intrinsic binding constant K_b was determined from the spectral titration data using the following equation [45]: where [DNA] is the concentration of DNA, , and correspond to the extinction coefficient, respectively, for the free copper(II) complex, for each addition of DNA to the copper(II) complex and for the copper(II) complex in the fully bound form. A plot of [DNA]/() versus [DNA], gives K_b, the intrinsic binding constant as the ratio of slope to the intercept. From the [DNA]/() versus [DNA] plot (Figure 5), the binding constant K_b for the copper(II) complex 1 was estimated to be  M⁻¹ (R²=0.93964 for five points).

Fig. 5. Plot of [DNA]/(ϵ_a−ϵ_f) vs. [DNA] for the titration of CT-DNA with complex 1 in tris–HCl buffer; binding constant K_b=1×10⁵ M⁻¹ (R=0.95469 for five points).

Fluorescence intensity of EB bound to DNA at 610 nm ( nm) shows a decreasing trend with the increasing concentration of the copper(II) complex (Figure 6). Pink-colored spectrum indicates the maximum binding with copper(II) complex replacing EB. The quenching of EB bound to DNA by the copper(II) complex is in agreement with the linear Stern–Volmer equation [46]:

Fig. 6. Emission spectra of the CT-DNA–EB system in tris–HCl buffer based on the titration of complex. λ_ex=522 nm.

where I₀ and I represent the fluorescence intensities in the absence and presence of quencher, respectively, K_sv is a linear Stern–Volmer quenching constant and Q is the concentration of quencher. In the quenching plot (Figure 7) of I₀/I versus [complex], K_sv value is given by the ratio of the slope to intercept. The K_sv value for the copper(II) complex is (R²=0.96561 for five points), suggesting a strong affinity of the copper(II) complex to CT-DNA.

Fig. 7. Plot of I₀/I versus [complex] for the titration of CT-DNA–EB system with the complex using spectrofluorimeter; linear Stern–Volmer quenching constant (K_sv) for complex 1=1.125×10⁴; (R=0.99023 for five points).

3.4 Protein (BSA) binding experiments

3.4.1 Absorption characteristics of BSA–Cu(II) complex 1

The absorption spectra of BSA in the absence and presence of Cu(II) complex 1 at different concentrations were studied (Figure 8). From this study, we observed that upon increasing the concentration of the complex, the absorption of BSA increases regularly. It may be due to the adsorption of BSA on the surface of the complex. From these data, the apparent association constant (K_app) of the complex with BSA has been determined using the following equation [45]:

Fig. 8. Absorption spectrum of BSA in the presence of complex in the concentration range 0–6.34×10⁻⁵ M. Inset is the linear dependence of 1/A−A₀ on the reciprocal concentration of complex.

where A_obs is the observed absorbance of the solution containing different concentrations of the complex at 280 nm, A₀ and A_c are the absorbances of BSA and the complex at 280 nm, respectively, with a concentration of C₀, and K_app represents the apparent association constant. The enhancement of absorbance at 280 nm was due to adsorption of the surface complex, based on the linear relationship between versus reciprocal concentration of the complex with a slope equal to and an intercept equal to (Figure 8, inset). The value of the apparent association constant (K_app) determined from this plot is  M⁻¹.

3.4.2 Fluorescence quenching of BSA by complex 1

The effect of increasing the concentration of the complex on the fluorescence emission spectrum of BSA was studied and represented in Figure 9. With the addition of complex BSA, fluorescence emission is quenched. The fluorescence quenching is described by the Stern–Volmer relation [46]:

Fig. 9. Fluorescence quenching of BSA in the presence of various concentrations of complex 1, [complex]=0, 1, 2, 3, 4 and 5×6.35×10⁻⁶ M. Insert shows the Stern–Volmer plot.

where I₀ and I represent the fluorescence intensities of BSA in the absence and presence of quencher, respectively. K_SV is the linear Stern–Volmer quenching constant and [complex] the molar concentration of the quencher. A linear plot (inset, Figure 9) between I₀/I against [complex] was obtained and from the slope we calculated the K_SV as suggesting strong interaction between Cu(II) complex and BSA.

3.4.3 Cyclic voltammetric studies

The cyclic voltammetric technique has also been introduced to study the interaction between the metal complex and DNA which provides a useful complement to the previously used spectral studies [47]. Cyclic voltammograms of Cu(II) complex 1 in the absence and presence of CT-DNA exhibited significant shifts in the anodic peak potentials followed by a decrease in peak currents, indicating the interaction existing between the copper(II) complex and CT-DNA. The shift in the value of the formal potential (Δ E⁰) is used to calculate the ratio of equilibrium binding constants K₂₊/K₊ according to the following equation described by Bard and Carter [48]: where and are the formal potentials of the bound and free complex forms, respectively, and K₂₊ and K₊ are the corresponding binding constants for the binding of reduction and oxidation species to DNA, respectively. Ratio of equilibrium binding constants, K₂₊/K₊, is calculated to be 1.04 which indicates the same binding property with both states of copper species.

4. Conclusion

Here, tetradentate N2S2 neutral ligand (L¹) has been used to synthesize copper(II) complex having CuN₂S₂ chromophore. In addition to NaCl in the reaction mixture of copper perchlorate and organic moiety (L¹), penta-coordinated copper(II) complex, which is verified by the measurement of conductance and IR spectra, is formed where four coordinating zones are satisfied by four ligating zones of ligand molecule and fifth ligating site is occupied by one chloride ion. This penta-coordination of complex 1 was characterized by C,H,N analysis, UV–VIS, conductance and also IR spectrum. From TGA experiment, it is also concluded that there is no water molecule in their coordination zone. The interaction of the complex with calf thymus DNA and BSA has been investigated by using absorption, emission spectral and electrochemical study. The results indicate that the interaction mode of the complex with DNA may be the groove binding. For complex 1, it was observed from electrochemical technique that both the Cu(I) and Cu(II) forms interact with DNA almost to the same extent.

Funding

Financial support [UGC minor project PSW-175/11-12(ERO)] from the University grand commission (UGC), New Delhi, India, is gratefully acknowledged.

Authors are thankful to Dr Pabitra Chattopadhyay, Department of chemistry, The University of Burdwan, West Bengal, India for his constant encouragement and stimulating guidance.