A mononuclear complex of Zn(II), [Zn(DIP)2 (DMP)] (NO3)2·2H2O in which DIP is 4,7-diphenyl-1,10-phenanthroline and DMP is 4,4′-dimethyl-2,2′-bipyridine has been prepared and characterized by 1HNMR spectroscopy, FT-IR, UV-Vis and elemental analysis techniques. DNA-binding properties of the complex were studied using UV-vis spectra, circular dichroism (CD) spectra, fluorescence, cyclic voltammetry (CV), and viscosity measurements. The results indicate that this zinc(II) complex can intercalate into the stacked base pairs of DNA and compete with the strong intercalator ethidium bromide for the intercalative binding sites.
In recent years, many researches [
All chemicals such as Zn(NO3)2·6H2O, 4,7-diphenyl-1,10-phenanthroline (DIP), and 4,7-dimethyl-1,10-phenanthroline (DMP) were purchased from Merck, and Tris-HCl highly polymerized calf thymus DNA (CT-DNA) were purchased from Sigma Co. Experiments were carried out in Tris-HCl buffer at pH = 7.0. A solution of calf thymus DNA gave a ratio of UV absorbance at 260 and 280 nm more than 1.8, indicating that DNA was sufficiently free from protein [
The complex [Zn(DIP)2(DMP)](NO3)2·2H2O (Figure
The structure of [Zn(DIP)2(DMP)](NO3)2·H2O complex.
1HNMR spectra were recorded using a Bruker Avance DPX200 MHz (4.7 Tesla) spectrometer with CDCl3 as the solvent. The elemental analysis was performed using a Heraeus CHN elemental analyzer. Absorbance spectra were recorded using an HP spectrophotometer (Agilent 8453) equipped with a thermostated bath (Huber Polystat cc1). Absorption titration experiments were conducted by keeping the concentration of complex constant (2.9 × 10−5 M) while varying the DNA concentration from 0 to 1.9 × 10−4 M (
CD measurements were recorded on a JASCO (J-810) spectropolarimeter, keeping the concentration of DNA constant (5.9 × 10−5 M) while varying the complex concentration (
Viscosity measurements were made using a viscosimeter (SCHOT AVS 450) maintained at
All fluorescence measurements were carried out with a JASCO spectrofluorimeter (FP6200) by keeping concentration of complex constant while varying the DNA concentration from 0 to 36.9 × 10−5 (
The cyclic voltammetric, linear sweep voltammetry, and differentials pulse voltammetry (DPV) measurements were performed using an AUTOLAB model (PG STAT C), with a three-electrode system: a 0.10 cm diameter Glassy carbon (GC) disc as working electrode, an Ag/AgCl electrode as reference electrode, and a Pt wire as counter electrode. Electrochemical experiments were carried out in a 25 mL voltammetric cell at room temperature. All potentials are referred to the Ag/AgCl reference. Their surfaces were freshly polished with 0.05 mm alumina prior to each experiment and were rinsed using double-distilled water between each polishing step. The supporting electrolyte was 0.01 M of Tris-HCl buffer solution (pH = 7.4) which was prepared with double-distilled water. Before experiments, the solution was deaerated via purging with pure nitrogen gas for 1 min, and during measurements a stream of nitrogen was passed over the solution. The current-potential curves and experimental data were recorded on software GPES [
The complex conforms to the formula [Zn(DIP)2(DMP)](NO3)2·2H2O determined on the basis of elemental analysis. The IR spectrum of the complex was characterized by the appearance of a band at 486 cm−1 due to the
The UV-Vis spectra of the free ligands (DIP and DMP) and the zinc complex.
Absorption spectroscopy is one of the most useful techniques to study the binding of any drug to DNA. The extent of hypochromism generally indicates the intercalative binding strength [
Absorption spectra of Zn(II) complex (2.9 × 10−5 M) in the absence and presence of increasing amounts of CT-DNA:
Structurally, intercalation to DNA may not be one of the binding modes, since the tris(bidentate) ligand strands wrap around the zinc center, possessing pseudo-threefold rotation axis that passes through the metal ions. Therefore, the complete intercalation of the ligands between a set of adjacent base pairs is sterically impossible, but some partial intercalation can be envisioned [
The intrinsic binding constant,
As the zinc complex is luminescent in the absence of DNA, it does show appreciable increase in emission upon addition of CT-DNA (Figure
Dynamic enhancement, bimolecular enhancement and formation constants of the complex at different temperatures.
Temperature (K) |
|
|
|
|
---|---|---|---|---|
298 | 0.343 |
|
0.496 | 2.87 |
308 | 1.083 |
|
0.484 | 1.97 |
318 | 1.07 |
|
0.517 | 1.34 |
Circular dichroism spectra of CT-DNA (5.9 × 10−5 M) in Tris buffer (10 mM), in the presence of increasing amounts of the complex.
By considering the equivalency of the bimolecular quenching and enhancement constants, it can be seen that the latter is greater than the largest possible value (1.0 × 1010 M−1s−1) in aqueous medium. Thus, the fluorescence enhancement is not initiated by a dynamic process; it is suggested that a static process involves complex formation in the ground state [
The binding constant (
To have a better understanding of thermodynamics of the reaction between the complex and DNA, it is useful to determine the contributions of enthalpy and entropy of the reaction. Therefore, the evaluation of formation constant for the complex-DNA at three different temperatures (298, 308, 318 K) allows determining the thermodynamic parameters such as enthalpy (
The analogous [Ru(phen)3]2+ [
To establish in more detail whether binding of the complexes brings about any significant conformational change of the DNA double helix, CD spectra of CT-DNA were recorded at increasing complex/CT-DNA ratios. The observed CD spectrum of natural calf thymus DNA consists of a positive band at 275 nm (UV:
The viscosity measurements of CT-DNA are regarded as the least ambiguous and the most critical tests of a binding model in solution in the absence of crystallographic structural data. A classical intercalation model demands that the DNA helix lengthens as base pairs are separated to accommodate the bound ligand, leading to the increase of DNA viscosity. In contrast, a partial, nonclassical intercalation of ligand could bend (or kink) the DNA helix, reducing its length and, concomitantly, its viscosity. In addition, complexes that bind exclusively in the DNA grooves by partial and/or nonclassical intercalation, under the same conditions, typically cause less pronounced (positive or negative) or no change in DNA solution viscosity [
Effect of increasing amounts of Zn(II) complex on the viscosity of CT-DNA.
Recently, the electrochemical techniques extensively were used as a simple and rapid method to study DNA interaction with different compounds. The electrochemical behaviour of zinc is well known and was strongly influenced by the electrode material. A well-defined and sensitive peak was observed from the solutions of the complex with a GC electrode rather than the Pt one. Therefore a GC electrode was used in this investigation. When CT-DNA is added to a solution of complex both the anodic and cathodic peak current heights of the complex decreased in the same manner of increasing additions of DNA (Figure
Cyclic voltammetry for the Zn(II) complex in the presence of different concentrations of CT-DNA.
In summary, we have synthesized a new tris-chelate complex of Zn(II), [Zn(DIP)2(DMP)](NO3)2·2H2O, which exhibits high binding affinity to CT-DNA. Different instrumental methods were used to finding the interaction mechanism. The following results supported the fact that the [Zn(DIP)2(DMP)](NO3)2·2H2O complex can bound to CT-DNA by the mode of partial intercalation and electrostatic binding. In absorption spectrum, the absorption intensity of the complex increased (hyperchromism) evidently after the addition of DNA, which indicated the interactions between DNA and the complex. The intrinsic binding constant ( Fluorescence studies showed appreciable increase in the complex emission upon addition of DNA. The positive slope in van’t Hoff plot indicated that the reaction of the Zn(II) complex and DNA was enthalpy favored ( The changes in the CT-CD spectra of DNA in the presence of increasing amounts of the complex show stabilization of the right-handed B form of CT-DNA. The positive shift in the CV peak potentials of the complex is indicative of intercalative binding mode of the complex with DNA. Increase of the relative viscosity of CT-DNA in the presence of the complex showed that the intercalative binding must be predominant.
Financial support from the Razi University Research center is gratefully acknowledged.