DNA Interaction Studies of Cobalt (II) Mixed-Ligand Complexes Containing Dimethyl-1,10-Phenanthroline and Dipyrido[3,2-a:2′,3′-c]Phenazine: The Role of Methyl Substitutions on the Mode of Binding

Abstract

Two cobalt (II) complexes containing a dipyrido[3,2-a:2′,3′-c]phenazine (dppz) base with the general formulation [Co(dppz)(dmp)₂]Cl₂, where dmp is 4,7-dimethyl-1,10-phenanthroline ligand (4,7-dmp) (1) and 2,9-dimethyl-1,10-phenanthroline ligand (2,9-dmp) (2) were synthesized and characterized. Binding interactions of these complexes with calf thymus DNA were investigated by emission, absorption, circular dichroism, and viscosity studies, and the effects of the positions of methyl substitutions in phenanthroline coligands were investigated. The DNA binding constants obtained from the absorption spectral titrations decrease in the order of 1 > 2, which is consistent with the trend in apparent emission enhancement of the complexes on binding to calf thymus DNA. These observations were supported by circular dichroism spectroscopy and viscosity measurements and reveal that DNA binding affinity of the complexes depends on the position of methyl groups on the phenanthroline ligands.

Introduction

I n the last decades, the synthesis of transition metal polypyridyl complexes that interact with DNA has attracted wide interest (Gielen and Tiekink, 2005; Maheswari et al., 2006a; Arockiasamy et al., 2009). These complexes can bind to DNA in noncovalent modes such as electrostatic, intercalative, and groove binding (Cowan, 2001; Ji et al., 2001). Many useful applications of these complexes require that the complexes bind to DNA through an intercalative mode with the ligand intercalating into the adjacent base pairs of DNA. Varying the substituted groups or their positions in the intercalative ligand or ancillary ligands (coligands) can create differences in the space configuration and the electron density distribution of transition metal complexes. These will result in some differences in spectral properties and the DNA-binding behaviors of the complexes and will be helpful in understanding the binding mechanism of transition metal complexes to DNA (Ortmans et al., 1998; Metcalfe and Thomas, 2003; Chao and Ji, 2005). Such studies mainly focus on the interaction of Ru(II) complexes with DNA, and the reports on the other metal complexes are relatively limited (Cheng et al., 1999; Zhang et al., 2001, 2002; Sastri et al., 2003; Wang et al., 2004, 2005; Arounaguiri et al., 1996; Srinivasan et al., 2005; Wu et al., 2005). Among these complexes, the Ru(II)-dppz (dppz: dipyrido[3,2-a:2′,3′-c]phenazine) complexes with bidentate ancillary ligands, for example, bpy (bipyridine), phen (1,10-phenanthroline), dmb (4,4'-dimethyl-2,2'-bipyridine), dmp (2,9-dimethyl-1,10-phenanthroline [2,9-dmp]), have been reported to have interesting properties (Liu et al., 2001; Biver et al., 2007). Currently, efforts are focused on modifying the main ligand, dppz (Moucheron et al., 1997; Ambroise and Maiya, 2000; Komatsuzaki et al., 2000; Liu et al., 2005; Gao et al., 2006, 2007; Lawrence et al., 2006). In addition, the effects of some of the bidentate coligands mentioned above are also being investigated.

Cobalt complexes have gained importance due to their application as potential hypoxia-activated prodrugs (Ware et al., 2000; Blower et al., 2001; Bonnitcha et al., 2006). Our studies focus on cobalt complexes, which have the same interesting characteristics and DNA cleaving properties as ruthenium complexes, but have not received as much attention as the ruthenium(II) systems (Arounaguiri and Maiya, 1996; Jin and Yang, 1997).

In this article, we describe synthesis of the mixed-ligand coordination complexes of cobalt (II) containing dppz (Fig. 1) and dimethyl-phenanthroline (4,7-dimethyl-1,10-phenanthroline [4,7-dmp] and 2,9-dmp), then extend our studies to complex-DNA binding using a variety of physical methods. The effects of the positions of methyl substitutions in phenanthroline coligands are investigated. The DNA interaction mechanism of this type of cobalt (II) mixed-ligand complexes is not well-studied. The results should be valuable in understanding the mode of the binding of the complexes to DNA and in laying the foundation for rational design of DNA structure probes and antitumor drugs.

FIG. 1.

Schematic representation and ¹H NMR labeling of 1,10-phenanthroline-5,6-dione, dppz, [Co(4,7-dmp)₂(dppz)]Cl₂ (1) and [Co(2,9-dmp)₂(dppz)]Cl₂ (2) complexes.

Materials and Methods

Materials

2,9-dmp, o-phenylenediamine, 4,7-dmp, dichloromethane, nitric acid, 1,10-phenanthroline (Phen), absolute ethanol, cobalt (II) chloride, diethyl ether, dichloromethane, butanol, methanol, aceton, and Tris-HCl were purchased from Merck. Doubly distilled deionized water was used throughout. Highly polymerized calf thymus DNA (CT-DNA) and Tris-HCl buffer were purchased from Sigma.

Experiments were carried out in Tris buffer at pH 7.2. Solutions of CT-DNA gave an UV absorbance ratio (260 over 280 nm) of >1.8, indicating that the DNA was sufficiently free of protein. Stock solution of CT-DNA was prepared by dissolving ∼1–2 mg of CT-DNA fibers in 2 mL Tris HCl (10 mM), shaken slowly, and stored for 24 h at 4°C. The concentration of DNA solution was expressed in monomer units, which was determined by spectrophotometry at 260 nm using an extinction coefficient (ɛ_p) of 6600 M⁻¹ cm⁻¹ value, and the solution was used within 4 days.

Synthesis of cobalt (II) complexes

Synthesis of 1, 10-phenanthroline-5, 6-dione

A mixture of 1,10-phenanthroline (1.00 g, 5.04 mmol), potassium bromide (5.94 g, 50.0 mmol), concentrated sulfuric acid (20 mL), and concentrated nitric acid(10 mL) was stirred for 2 h at 80°C–85°C and cooled to room temperature. The solution was poured into 400 mL of water, neutralized with sodium hydroxide, and then extracted with dichloromethane. Removal of dichloromethane gave 1,10-phenanthroline-5,6-dione as yellow needles. ¹H NMR (ppm, CDCl₃): 9.12 (¹H), 8.51 (³H), 7.55 (²H); UV-Vis:λ_max: 304, 260, 232; IR data (Cm⁻¹): 1684, 1559, 1458; Yield: 0.91 g (86%).

Synthesis of dppz

A mixture of phen-5,6-dione (0.50 g, 2.40 mmol) and o-phenylenediamine (0.50 g, 4.60 mmol) was refluxed in ethanol for 1 h. Brown solid precipitated after cooling to room temperature was collected by filtration and successively washed with ethanol and acetone. ¹H NMR (ppm, CDCl₃): 7.8(²H), 7.95(³H), 8.36(⁵H), 9.27(⁴H), 9.65(¹H); Yield: 1.32 g (69%).

Synthesis of [Co(4,7-dmp)₂(dppz)]Cl₂. 2H₂O (1)

A mixture of CoCl₂ · 6H₂O (238 mg, 1 mmol) and 4,7-dmp (416 mg, 2 mmol) was dissolved in 50 mL 1-butanol and stirred at reflux temperature for 1 h. The brown precipitate, [Co(4,7-dmp)₂Cl₂], was collected and washed with 1-butanol and ether. This product was dissolved in water-methanol (20 mL, 1:2 V/V) and dppz (282 mg, 1 mmol) was added. The solution was stirred under reflux. After 3 h, the reaction mixture was cooled to room temperature and filtered. The precipitate was washed with ice water and methanol then dried under vacuum. Yield: 65%. ¹H NMR (ppm, DMSO): 11.19 (¹ H); 10.07(⁴H); 9.66(⁵H); 8.89 (³H); 8.6(²H); 2.71 (Me groups) (Fig. 1). Analysis calculated (Anal. Calc.) for C₄₆H₃₄Cl₂N₈Co · 2H₂O: C, 63.93; H, 3.97; N, 13.02; found: C, 64.6; H, 4.3; N, 13.7%. FT-IR data (cm⁻¹): 1530 (ring), 1515 (C = C), 1429 (CCH), 847 (Phen), 723 (Phen), 1342 (Me), 411 (Co-N); Molar conductance, (Ω⁻¹cm²mol⁻¹) in DMF: 148 (1:2 electrolyte). (Geay, 1971); μ_eff = 2.05 BM.

Synthesis of [Co(2,9-dmp)₂(dppz)]Cl₂ · 2H₂O (2)

This complex was obtained by a similar procedure as 1, but 2,9-dmp was used (416 mg, 2 mmol). Yield: 63%. ¹H NMR (ppm, DMSO): 10.9 (¹H); 9.76 (⁴H); 9.40 (⁵H); 8.49 (³H); 8.12(²H); 2.87 (Me groups). Anal. Calc. for C₄₆H₃₄Cl₂N₈Co · 2H₂O: C, 63.93; H, 3.97; N, 13.02; found: C, 63.7; H, 3.7; N, 14%. FT-IR data (cm⁻¹): 1530 (ring), 1515 (C = C), 1429 (CCH), 847 (Phen), 723 (Phen), 1342 (Me), 411 (Co-N); Molar conductance, (Ω⁻¹cm²mol⁻¹) in DMF: 130 (1:2 electrolyte) (Geay, 1971); μ_eff = 1.95 BM.

Instrumentation

¹H NMR 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. The molar conductance of complexes was measured in DMF at room temperature on an ELICO (CM 82T) conductivity bridge. Magnetic susceptibility measurements were made at room temperature using AGFM and VSM methods (Meghnatis Kavir Kashan Co.).Absorbance spectra were recorded using an HP spectrophotometer (Agilent 8453) equipped with a thermostated bath (Huber polysat cc1). Absorption titration experiments were conducted by keeping the concentration of complexes constant (5 × 10⁻⁵ M) while varying the DNA concentration from 0 to 1 × 10⁻⁴ M (r _i = [DNA]/[complex] = 0.0, 0.1, 0.2, 0.4, 0.6, 0.8, 1. Absorbance values were recorded after each successive addition of DNA solution, followed by an equilibration period.

All fluorescence measurements were carried out with a JASCO spectrofluorometer (FP6200) by keeping the concentration of complex constant (5 × 10⁻⁵ M) while varying the DNA concentration from 0 to 40.5 × 10⁻⁵ M (r _i = 0.0, 0.5, 1, 2, 3, 4) r_i = [DNA]/[complex] at three different temperatures (279, 293, 310 K).Circular dichroism (CD) measurements were recorded on a JASCO (J-810) spectropolarimeter by keeping the concentration of DNA constant (5 × 10⁻⁵ M) while varying the complex concentration from 0 to 1 × 10⁻⁵ M (r _i = [complex]/[DNA] = 0, 0.02, 0.05, 0.1, 0.15, 0.2).

Viscosity measurements were made using a viscosimeter (SCHOT AVS 450) that was maintained at 25°C ± 0.5°C using a constant temperature bath. The DNA concentration was fixed at 5 × 10⁻⁵ M, and flow time was measured with a digital stopwatch. The mean values of three replicated measurements were used to evaluate the viscosity η of the samples. The values for relative specific viscosity (η/η_o)^1/3, where η_o and η are the specific viscosity contributions of DNA in the absence (η_o) and in the presence of the Co(II) complexes (η), were plotted against r_i (r_i = [complex]/[DNA]) (Bloomfield et al., 1974).

Results

Synthesis and characterization

The ligand dppz was prepared by condensation of 1,10-phenanthroline-5, 6-dione, and o-phenylenediamine in ethanol. dppz is soluble in ethanol, and its UV-Vis spectrum is shown in Figure 2. It is worth noting that the UV-Vis spectrum of the ligand shows characteristic peaks at 249 and 270 nm, a shoulder at 290 nm, and a multiple in the range 325–400 nm, the latter interpreted as a π-π^* transition (Terenzi et al., 2009). The electronic absorption spectra of [Co(4,7-dmp)₂(dppz)]²⁺ and [Co(2,9-dmp)₂(dppz)]²⁺ are well characterized by two intense ligand centered transitions in the UV region. The bands at 325–400 nm are characteristic of the interligand (IL) π-π* transition of the dppz ligand (Fig. 2). The low energy bands at 447 nm are assigned as Co(dπ) dppz (π*) metal-to-ligand charge transfer transition.

FIG. 2.

Electronic spectra of dppz ligand, [Co(4,7-dmp)₂(dppz)]Cl₂ (1) and [Co(2,9-dmp)₂(dppz)]Cl₂ (2) complexes.

The FT-IR spectra of two complexes show bands in the 411 cm⁻¹ ascribable to the Co-N stretching.

The observed magnetic moments of Co(II) complexes are 2.05 and 1.95 for complex 1 and 2, respectively. Magnetic susceptibility measurements of the titled complexes are generally diagnostic of the coordination geometry about the metal ions and reveal that the complexes have low spin (s = 1/2) systems, because in low spin octahedral cobalt (II) complexes, the magnetic moments are close to 1.8–2.0 BM (Faus et al., 1993; Constable et al., 2003).

In the ¹H NMR spectra of the two cobalt (II) complexes, the peaks due to the protons of 4,7-dmp, 2,9-dmp, and dppz were shifted in comparison with the corresponding free ligands, suggesting complexation. Molar conductance (Ω⁻¹ cm²mol⁻¹) in DMF showed 1:2 electrolytes (Geay, 1971).

Multispectroscopic methods

The absorption spectra of the complexes in the presence of increasing amounts of CT-DNA (R = [DNA]/[complex]) were measured (Fig. 3). As the DNA concentration is increased, pronounced hypochromism is observed for both complexes. An isobestic point at 385 nm is observed for complex 2. The hypochromism (32.7%) and a red shift (4 nm) observed for the 378 nm band in complex 1 and the hypochromism (23.6%) and a red shift (5 nm) observed for the 378 nm band in complex 2 typical for a stacking interaction of the dppz ligand with the DNA base pairs. To compare the binding strengths of the complexes quantitatively, the intrinsic binding constant k_b was determined by using the equation 1 (Kashanian et al., 2007) \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland,xspace}\usepackage{amsmath,amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6} \begin{document} \begin{align*} \frac { \varepsilon_a - \varepsilon_f } { \varepsilon_b - \varepsilon_f } &= \frac { b - \left( b^2 - \frac { 2k_{b}^{2}C_t [ DNA ] } { s } \right) ^ { 1 / 2 } } { 2k_{b}C_{t} } \\ b &= 1 + k_b C_t + \frac { k_b [ DNA ] } { 2s } \tag { 1 } \end{align*} \end{document}

FIG. 3.

Electronic absorption spectra for the titration of 5.0 × 10⁻⁵ M of complexes [Co(4,7-dmp)₂(dppz)]Cl₂ (1) and [Co(2,9-dmp)₂(dppz)]Cl₂ (2) with calf thymus DNA (CT-DNA) (r _i = 0.00, 0.1, 0.2, 0.4, 0.6, 0.8, and 1).

where ɛ _a is the extinction coefficient observed for the metal-to-ligand charge transfer absorption band at a given DNA concentration, ɛ _f is the extinction coefficient of the complex free in solution, ɛ _b the extinction coefficient of the complex when fully bound to DNA (it is assumed when further addition of DNA does not change the absorbance, all complex is bound and ɛ _b can be calculated from Beer's Law), K_b the equilibrium binding constant, C_t the total complex concentration in nucleotide, and s the binding site size. The plots of (ɛ_a–ɛ_f)/(ɛ_b–ɛ_f) versus [DNA] have been shown in Figure 4. (K_b = 4.8 ± 0.1 × 10⁵ M⁻¹ (s = 1.04) for complex 1 and K_b = 3.6 ± 0.2 × 10⁵ M⁻¹ (s = 1.06) for complex 2, respectively).

FIG. 4.

Plots of (ɛ_a-ɛ_f)/(ɛ_b-ɛ_f) versus [DNA] for the titration of DNA with the complexes [Co(4,7-dmp)₂(dppz)]Cl₂ (1) and [Co(2,9-dmp)₂(dppz)]Cl₂ (2).

For both complexes, 1 and 2, the viscosity of DNA increases with increased concentration of the added complexes; however, the increase in viscosity is less than that for known intercalators such as EtBr. (Fig. 5) (Li et al., 2006). The plots of relative specific viscosities (η/η₀, where η, η₀ are the specific viscosities of DNA in the presence and absence of complexes, respectively) versus 1/R (1/R = [DNA]/[complex]) are shown in Figure 5.

FIG. 5.

Effect of increasing amounts of [Co(4,7-dmp)₂(dppz)]Cl₂ (1) (▪) and [Co(2,9-dmp)₂(dppz)]Cl₂ (2) (▴) complexes on the viscosity of CT-DNA (5 × 10⁻⁵ M) in 10 mM Tris–HCl buffer (r _i = 0.1, 0.3, 0.6, and 0.9).

The two Co(II) complexes can emit luminescence in Tris-HCl buffer with a maximum wavelength of about 558 nm, excited at about 450 nm. The results of emission titration for the two complexes with CT-DNA are shown in Figure 6. On the addition of CT-DNA, the emission intensities at about 558 nm of two complexes increase by about 1.5 and 1.26 times for complexes [Co(4,7-dmp)₂(dppz)] and [Co(2,9-dmp)₂(dppz)], respectively. The dynamic enhancement constants of cobalt complexes in different temperatures were calculated (Table 1). Stern-Volmer plots are shown in Fig. 7. The linear equations for log (F-F₀)/F versus log [DNA] at different temperature values are shown in Table 2.

FIG. 6.

Emission spectra of the complexes [Co(4,7-dmp)₂(dppz)]Cl₂ (1) and [Co(2,9-dmp)₂(dppz)]Cl₂ (2) in Tris–HCl buffer in the absence and presence of CT-DNA. r _i = [DNA]/[Co] = 0.0, 0.5, 1, 2, 3, 4.

FIG. 7.

Stern–Volmer plots for the observed fluorescence enhancement of complexes [Co(4,7-dmp)₂(dppz)]Cl₂ (1) and [Co(2,9-dmp)₂(dppz)]Cl₂ (2) after addition of CT-DNA at different temperatures.

Table 1.

Dynamic Enhancement and Bimolecular Enhancement Constants for Interactions Between Co(II) Complexes and Calf Thymus-DNA at Different Temperatures

[Co(4,7-dmp)₂(dppz)]Cl₂ ( 1 )
Temperature (K)	Linear equation	K_D	K_B
279	Y = 1.01–6715X	6715	6.71 × 10¹¹
293	Y = 0.97–9451.2X	9451.2	9.45 × 10¹¹
310	Y = 0.96–14711X	14711	1.47 × 10¹²

[Co(2,9-dmp)₂(dppz)]Cl₂ ( 2 )
Temperature (K)	Linear equation	K_D	K_B
279	Y = 1.01–4999.3X	4999.3	4.99 × 10¹¹
293	Y = 0.99–8027.1X	8027.1	8.02 × 10¹¹
310	Y = 0.96–11261X	11261	1.12 × 10¹²

Table 2.

Linear Equations of log (F-F₀)/F Versus log [DNA] and K_F Values of Co(II) Complexes with DNA at Different Temperatures

[Co(4,7-dmp)₂(dppz)]Cl₂ ( 1 )
Temperature (K)	Linear equation	R²	K_f
279	Y = 1.31X + 5.27	0.957	1.8 × 10⁵
293	Y = 1.02X + 3.97	0.988	9.3 × 10³
310	Y = 0.7X + 2.54	0.999	3.46 × 10²

[Co(2,9-dmp)₂(dppz)]Cl₂ ( 2 )
Temperature (K)	Linear equation	R²	K_f
279	Y = 1.28X + 4.95	0.975	8.9 × 10⁴
293	Y = 1.03X + 3.91	0.94	8.1 × 10³
310	Y = 0.68X + 2.61	0.97	4.07 × 10²

In Figure 8, the changes in the CD spectra of CT-DNA in the presence of increasing concentrations of the two complexes are shown. After addition of incremental amounts of the complexes, the intensity of positive bands diminish and new negative bands at about 292 nm and positive bands at about 260 nm develop for complexes 1 and 2, respectively. The molar ellipticity of the negative bands at 240 nm also decreases with increasing concentrations of the complexes.

FIG. 8.

Circular dichroism spectra of DNA (5.0 × 10⁻⁵) in 10 mM Tris–HCl buffer, in the presence of increasing amounts of [Co(4,7-dmp)₂(dppz)]Cl₂ (1) and [Co(2,9-dmp)₂(dppz)]Cl₂ (2) complexes (r _i = [complex]/[DNA] = 0.0, 0.02, 0.05, 0.1, 0.15, and 0.2).

Discussion

Electronic absorption spectral studies

The absorption spectra are the most common means to examine the interaction between metal complex and DNA. In general, the absorption spectra of metal complexes bound to DNA through intercalation exhibit significant hypochromism and red shift due to the strong π-π stacking interaction between the aromatic chromophore ligand, metal complex and base pairs of DNA. The absorption spectra of the complexes 1 and 2 in the visible region were characterized by maximum wavelengths (λ_max) of 362 and 378 nm, respectively. The band at 378 nm is characteristic of the IL π-π* transition of dppz ligand.

The results in Figure 3 are consistent with preferential intercalation of the dppz ligand into the base pairs of the DNA helix when the dppz ligand of these complexes intercalate into the base pairs of DNA; its π* orbital is coupled with the π orbital of the DNA base pairs to give rise to the decrease in the π-π* transition energies. As a result, the λ_max of the IL transition of the dppz ligand is shifted to the longer wavelength (red shift). The extent of hypochromicity in complex 2 is lower than complex 1 and shows that the interaction of complex 2 is weaker than 1.

The intrinsic binding constants (K_b) are 4.8 ± 0.1 × 10⁵ M⁻¹ (s = 1.14) for complex 1 and 3.6 ± 0.2 × 10⁵ M⁻¹ (s = 1.06) for complex 2, respectively. Introduction of 2,9-dimethyl groups on the phen rings (complex 2) decreases the DNA binding affinity by sterically hindering the insertion of dppz ligand into the DNA base stack, whereas 4,7-dimethyl groups (complex 1) increase the DNA-binding affinity by exhibiting favorable hydrophobic interaction of the methyl groups on the surface of DNA major groove (Maheswari et al., 2006b). The values of binding site sizes we obtained (s > 1), which have been observed for other intercalators (Yu et al., 2009), support the hypothesis that the complexes can bind to DNA via intercalative mode of binding. Further, the higher binding site size of the 4,7-dmp complex is expected for its bulkiness with methyl groups of the ligand occupying more space within the interior of the DNA surface. Similar results have been previously observed (Maheswari et al., 2006b).

Viscosity studies

Viscosity measurements on solutions of CT-DNA incubated with the cobalt (II) complexes were made to explore DNA binding mode. The results in Figure 5 indicate that the increase in relative viscosity, expected to correlate with the compounds' DNA-intercalating propensities, followed the order complex 1 (4,7-dmp) > complex 2 (2,9-dmp). These results suggest that complexes 1 and 2 both bind to DNA through intercalation, the difference in binding strength is probably caused by the different ancillary ligands. The Me groups of 2,9-dmp ligands are expected to give rise to much more steric hindrances than those in the 4,7-dmp ligands. Therefore, complex 1 is probably more deeply intercalated and more tightly bound to adjacent DNA base pairs than complex 2.

Fluorescence spectroscopic studies

The two Co(II) complexes can emit luminescence in Tris-HCl buffer with a maximum wavelength of about 558 nm, excited at about 450 nm. The results of emission titration for the two complexes with CT-DNA are shown in Figure 6. Afteraddition of CT-DNA, the emission intensities at about 558 nm of two complexes increase by about 1.5 and 1.26 times for complexes [Co(4,7-dmp)₂(dppz)] and [Co(2,9-dmp)₂(dppz)], respectively. This implies that these complexes can interact with CT-DNA and be protected by DNA efficiently, as the hydrophobic environment inside the DNA helix reduces the accessibility of solvent water molecules to the complexes and the complexes' mobilities are restricted at the binding sites, leading to decrease of the vibrational modes of relaxation.(Liu et al., 2009; Sun et al., 2010).

Analogous to the quenching constant in a quenching process, an enhancement constant may be obtained from the following equilibrium: \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland,xspace}\usepackage{amsmath,amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6} \begin{document} \begin{align*} F + E \leftrightarrow F - E \tag{2} \end{align*} \end{document}

where F, E, and F–E are the fluorophore, enhancer, and Co(II) complex, respectively. Analogous to a static quenching process (Li et al., 2008) for fluorescence enhancement, the dependence of the intensity on enhancer concentration can be easily derived from the association constant for complex formation (Li et al., 2008). Equation 3 gives the enhancement constant. \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland,xspace}\usepackage{amsmath,amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6} \begin{document} \begin{align*} \frac { F_0 } { F } = 1 - K_E [ E ] \tag { 3 } \end{align*} \end{document}

If a dynamic process is part of the enhancing mechanism, the above equation can be written as follows (Shahabadi and Fatahi, 2010): \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland,xspace}\usepackage{amsmath,amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6} \begin{document} \begin{align*} \frac { F_0 } { F } = 1 - K_D [ E ] = 1 - K_B \tau_0 [ E ] \tag { 4 } \end{align*} \end{document}

where K_D is the dynamic enhancement constant (analogous to a dynamic quenching constant), K_B is the bimolecular enhancement constant (analogous to a bimolecular quenching constant), and τ₀ is the lifetime of the fluorophore in the absence of an enhancer. The dynamic enhancement constants of cobalt complexes at different temperatures were calculated using Equation 4 (Fig. 7 and Table 1). Since fluorescence lifetimes are typically near 10⁻⁸ s, the bimolecular enhancement constant (K_B) was calculated from K_D = K_Bτ₀ (Table 1).

If the bimolecular quenching and enhancement constants are equivalent, the latter constant (K_B) would be greater than the largest possible value (1 × 10¹⁰ M⁻¹ s⁻¹) (Li et al., 2008) in aqueous solutions. Thus, the fluorescence enhancement is not initiated by a dynamic process, but is instead due to a static process involving ground state complex formation. The possibility for ground state complex formation is supported by observed changes in the absorption spectra during the titration of the Co(II) complex with CT-DNA, as shown in Figure 3. Since dynamic quenching only affects the excited state, no changes are expected in the ground state. Alternatively, a static process only involves complex formation in the ground state (Li et al., 2008).

Equilibrium binding titration

Fluorescence titration data were used to determine the binding constant (K_f) and binding stoichiometry (n) for the complex formed between the Co(II) complex and CT-DNA. Figure 6 shows the fluorescence spectra of Co(II) complexes in the presence of different concentrations of CT-DNA. The data show that the fluorescence intensity at 454 nm increases in the presence of CT-DNA. This change in fluorescence intensity at 454 nm was used to estimate K_f and n for the binding of Co(II) complexes to CT-DNA from the following equation (Song et al., 2005): \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland,xspace}\usepackage{amsmath,amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6} \begin{document} \begin{align*} \log ( { \rm F}_0 - { \rm F} ) / { \rm F} = \log { \rm K}_{ \rm f} + { \rm n} \log [ { \rm DNA} ] \tag{5} \end{align*} \end{document}

Here, F₀ and F are the fluorescence intensities of the fluorophore in the absence and presence of different concentrations of CT-DNA, respectively. In the case of enhanced emission intensity, that is, F₀ < F, Equation 5 becomes \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland,xspace}\usepackage{amsmath,amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6} \begin{document} \begin{align*} \log ( { \rm F} - { \rm F}_0 ) / { \rm F} = \log { \rm K}_{ \rm f} + { \rm n} \log [ { \rm DNA} ] \tag{6} \end{align*} \end{document}

The linear equations for log (F-F₀)/F versus log [DNA] at different temperature values are shown in Table 2. The values of K_f clearly underscore the remarkably high affinity of the Co(II) complexes for DNA.

The size of the binding site makes it possible to distinguish between intercalating and nonintercalating binding agents (Krishna et al., 1998). Molecules with large binding site sizes are indicative of nonintercalative binding mechanisms, which require correspondingly lower concentrations to saturate the sites. Wilson and coworkers (1989) reported that the binding of 4′,6-diamino-2-phenylindole to polyGC conforms to an intercalative mechanism following the neighbor exclusion model with K_f = 1.2 × 10⁵ M⁻¹ and n = 2 (approximately). Our results appear to follow a similar trend. Therefore, the calculated binding site size is again indicative of intercalative binding of Co(II) complexes to CT-DNA.

Circular dichroic spectral studies

Circular dichroic spectra provide information about the chirality of spectroscopically active species in solution. They are particularly sensitive to the degenerate/nondegenerate exciton coupling expected to arise when chromophores closely located in space strongly interact (Holmlin et al., 1996). Thus, rac-metal complexes give a zero CD but show induced circular dichroic (ICD) signals on enantiopreferential binding to DNA providing further confirmation of their DNA binding (Hirot et al., 1990; Delaney et al., 2002). Thus, the CD spectral technique has been used to study the enantiopreferential DNA binding of rac-metal complexes. In addition, the technique is useful in diagnosing changes in DNA morphology during drug-DNA interaction, as CD signals are quite sensitive to the mode of DNA interactions of small molecules (Maheswari et al., 2006b).

CT-DNA in the B conformation shows two conservative CD bands, a positive band at 276 nm (due to base stacking) and a negative band at 240 nm (due to helicity) in the UV region. The changes in the CD spectra of CT-DNA in the presence of increasing concentrations of the two complexes are depicted in Figure 8. This is typical of exciton coupled ICD arising due to enantiopreferential binding of the Δ-enantiomer of the rac-complex and/or ligand–ligand interactions among DNA bound/unbound complexes. When the concentration of the complex is systematically increased from 1/R = 0 to 1 (=[Ru complex]/[DNA]), keeping the DNA concentration constant, both the positive and negative components of the ICD increase in intensity. The transitions complete at r _i values of 0.2 and 0.15 for complex 1 and 2, respectively. (Fig. 8). The complexes begin to stack on the DNA acting as chiral nanotemplates and result in large exciton CD signals. A similar induced CD has been observed (Lincoln et al., 1997) when equimolar amounts of Δ-[Rh(phi)₂(bpy)]²⁺ (RU) and Δ-[Ru(phen)₂(dppz)]²⁺ (RH) are mixed with polyAT. The observed CD intensities are higher than the sum of those for the two complexes bound separately to polyAT, which is due to the adjacent binding of the complexes leading to RU–RH interaction through exciton coupling.

Conclusion

Two novel Co(II) complexes [Co(4,7-dmp)₂(dppz)]Cl₂ (1) and [Co(2,9-dmp)₂(dppz)]Cl₂ (2) have been synthesized and characterized. The results indicate that both complexes bind to DNA in an intercalation mode. Further, ancillary ligands, 4,7-dmp and 2,9-dmp, have an important effect on the DNA-binding of the complexes and lead to the order of DNA-binding affinity of the complexes being (1) > (2). The Me groups in 2,9-dmp ligands are expected to give rise to much more steric hindrances than those in 4,7-dmp ligands. Therefore, complex 1 is probably more deeply intercalated and more tightly bound to adjacent DNA base pairs than complex 2. Interestingly, the Δ–enantiomers of rac-complexes show the potential to bind preferentially to right-handed B-DNA.

Footnotes

Acknowledgments

Financial support from Razi University Research center is gratefully acknowledged.

Disclosure Statement

No competing financial interests exist.

References

Ambroise

, Maiya

B.G.

2000. Ruthenium(II) complexes of 6,7-dicyanodipyridoquinoxaline: synthesis, luminescence studies, and DNA interaction. Inorg Chem, 39:4264–4272.

Arockiasamy

D.L.

, Radhika

, Parthasarathi

, Nair

B.U.

2009. Synthesis and DNA-binding studies of two ruthenium(II) complexes of an intercalating ligand. Eur J Med Chem, 44:2044–2051.

Arounaguiri

, Maiya

B.G.

1996. Dipyridophenazine complexes of cobalt(III) and nickel(II): DNA-binding and photocleavage studies. Inorg Chem, 35:4267–4270.

Biver

, Cavazza

, Secco

, Venturini

2007. The two modes of binding of Ru(phen)₂dppz²⁺ to DNA: thermodynamic evidence and kinetic studies. J Inorg Biochem, 101:461–469.

Bloomfield

V.A.

, Crothers

D.M.

, Tioco

1974. Physical Chemistry of Nucleic Acids. Harper and Row: New York, 432–434.

Blower

P.J.

, Dilworth

J.R.

, Maurer

R.I.

, Mullen

G.D.

, Reynolds

C.A.

, Zheng

2001. Towards new transition metal-based hypoxic selective agents for therapy and imaging. J Inorg Biochem, 85:15–22.

Bonnitcha

P.D.

, Hall

M.D.

, Underwood

C.K.

, Foran

G.J.

, Zhang

, Beale

P.J.

, Hambley

T.W.

2006. XANES investigation of the Co oxidation state in solution and in cancer cells treated with Co(III) complexes. J Inorg Biochem, 100:963–971.

Chao

, Ji

L.N.

2005. DNA interactions with ruthenium(II) polypyridine complexes containing asymmetric ligands. Bioinorg Chem Appl, 3:15–28.

Cheng

C.C.

, Kuo

Y.N.

, Chuang

K.S.

, Luo

C.F.

, Wang

W.J.

1999. A new Co(II) complex as a bulge-specific probe for DNA. Angew Chem Int Ed, 38:1255–1257.

10.

Constable

, Hart

C.P.

, Housecroft

C.E.

2003. Cobalt (II) and iron(II) bis(2,2′:6,2′′-terpyridine)complexes functionalized with alkynes and cobalt carbonyl clusters. Appl Organomet Chem, 17:383–387.

11.

Cowan

J.A.

2001. Chemical nucleases. Curr Opin Chem Biol, 5:634–642.

12.

Delaney

, Pascaly

, Bhattaracharya

, Han

, Barton

J.K.

2002. Oxidative damage by ruthenium complexes containing the dipyridophenazine ligand or its derivatives: a focus on intercalation. Inorg Chem, 41:1966–1974.

13.

Faus

, Julve

, Lloret

, Munoz

M.C.

1993. Bis(dimethylviolurateo)(phenanthroline)cobalt(II), a low spin octahedral cobalt (II) complex. Crystal structure of [Co(dmvi)₂phen].2CHCl₃ . Inorg Chem, 32:2013–2017.

14.

Gao

, Chao

, Zhou

, Xu

L.C.

, Zheng

K.C.

, Ji

L.N.

2007. Synthesis, characterization, and DNA-binding properties of the chiral ruthenium(II)complexes Δ- and Λ-[Ru(bpy)₂ (dmppd)]²⁺ (dmppd = 10,12- dimethylpteridino[6,7-f] [1,10]phenanthroline-11,13(10H,12H)-dione; bpy = 2,2′-bipyridine) Helv Chim Acta, 90:36–51.

15.

Gao

, Chao

, Zhou

, Yuan

Y.X.

, Peng

, Ji

L.N.

2006. DNA interactions of a functionalized ruthenium(II) mixed-polypyridyl complex [Ru(bpy)₂ppd]²⁺ J Inorg Biochem, 100:1487–1494.

16.

Geay

W.J.

1971. The use of conductivity measurements in organic solvents for the characterization of coordination compounds. Coord Chem Rev, 7:81–122.

17.

Gielen

Tiekink

E.R.T.

2005. Metallotherapeutic Drugs and Metal-Based Diagnostic Agents: The Use of Metals in Medicine. John Wiley & Sons, Hoboken: NJ, 441–461.

18.

Hirot

, Norden

, Rodger

1990. Enantiopreferential DNA binding of [ruthenium(II)(1,10-phenanthroline)3]2+ studied with linear and circular dichroism. J Am Chem Soc, 112:1971–1982.

19.

Holmlin

R.E.

, Stemp

E.D.A.

, Barton

J.K.

1996. Os(phen)₂dppz²⁺ in photoinduced DNA-mediated electron transfer reactions. J Am Chem Soc, 118:5236–5244.

20.

L.N.

, Zou

X.H.

, Liu

J.G.

2001. Shape- and enantioselective interaction of Ru(II)/Co(III) polypyridyl complexes with DNA. Coord Chem Rev, 216–217:513–536.

21.

Jin

, Yang

1997. Synthesis and DNA binding studies of Co^III mixed-ligand complex containing dipyrido [3,2-a: 2′,3′-c]phenazine and phen. Polyhedron, 16:3395–3398.

22.

Kashanian

, Gholivand

M.B.

, Ahmadi

, Taravati

, Hosseinzadeh Golkar

2007. DNA interaction with Al–N,N′-bis(salicylidene) 2,2′-phenylendiamine complex. Spectrochem Acta A, 67:472–478.

23.

Komatsuzaki

, Katoh

, Himeda

, Sugihara

, Arakawa

, Kasuga

2000. Structure and photochemical properties of ruthenium complexes having dimethyl-substituted DPPZ or TPPHZ as a ligand. J Chem Soc Dalton Trans, 3053–3054.

24.

Krishna

A.G.

, Kumar

D.V.

, Khan

B.M.

, Rawal

S.K.

, Ganesh

K.N.

1998. Taxol–DNA interactions: fluorescence and CD studies of DNA groove binding properties of taxol. Biochim Biophys Acta, 1381:104–112.

25.

Lawrence

, Vaidyanathan

V.G.

, Nair

B.U.

2006. Synthesis, characterization and DNA binding studies of two mixed ligand complexes of ruthenium(II) J Inorg Biochem, 100:1244–1251.

26.

F.H.

, Zhao

G.H.

, Wu

H.X.

, Lin

, Wu

X.X.

, Zhu

S.R.

, Lin

H.K.

2006. Synthesis, characterization and biological activity of lanthanum (III) complexes containing 2-methylene–1,10-phenanthroline units bridged by aliphatic diamines. J Inorg Biochem, 100:36–43.

27.

T.R.

, Yang

Z.Y.

, Wang

B.D.

, Qin

D.D.

2008. Synthesis, characterization, antioxidant activity and DNA-binding studies of two rare earth (III) complexes with naringenin-2-hydroxy benzoyl hydrazone ligand. Eur J Med Chem, 43:1688–1695.

28.

Lincoln

, Tuite

, Norden

. 1997. Short-circuiting the molecular wire; cooperative binding of Δ-[Ru(phen)₂ dppz]²⁺ and Δ-[Ru (phi)₂ bpy]³⁺ to DNA. J. Am. Chen. Soc., 119:1454–1455.

29.

Liu

J.G.

, Zhang

Q.L.

, Shi

X.F.

, Ji

L.N.

2001. Interaction of [Ru(dmp)₂(dppz)]²⁺ and [Ru(dmb)₂(dppz)]²⁺ with DNA: effects of the ancillary ligands on the DNA-binding behaviors. Inorg Chem, 40:5045–5050.

30.

Liu

X.W.

, Li

, Zheng

K.C.

, Chao

, Ji

L.N.

2005. Synthesis, characterization, DNA-binding and photocleavage of complexes [Ru(phen)₂(6-OH-dppz)]²⁺ and [Ru(phen)₂(6-NO₂-dppz)]²⁺ J Inorg Biochem, 99:2372–2380.

31.

Liu

Z.C.

, Wang

B.D.

, Yang

Z.Y.

, Li

, Qin

D.D.

, Li

T.R.

2009. Synthesis, crystal structure, DNA interaction and antioxidant activities of two novel water-soluble Cu(²⁺) complexes derivated from 2-oxo-quinoline-3-carbaldehyde Schiff-bases. Eur J Med Chem, 44:4477–4484.

32.

Maheswari

P.U.

, Rajendiran

, Palaniandavar

, Parthasarati

, Sabramanian

2006a. Synthesis, characterization and DNA-binding properties of rac-[Ru(5,6-dmp)₂(dppz)]²⁺–Enantiopreferential DNA binding and co-ligand promoted exciton coupling. J Inorg Biochem, 100:3–17.

33.

Maheswari

P.U.

, Rajendiran

, Palaniandavar

, Thomas

, Kulkarini

G.U.

2006b. Mixed ligand ruthenium(II) complexes of 5,6-dimethyl-1,10-phenanthroline: the role of ligand hydrophobicity on DNA binding of the complexes. Inorg Chim Acta, 359:4601–4612.

34.

Metcalfe

, Thomas

J.A.

2003. Kinetically inert transition metal complexes that reversibly bind to DNA. Chem Soc Rev, 32:215–224.

35.

Moucheron

, Mesmaeker

A.K.D.

, Kelly

J.M.

1997. Deoxyribonucleic acid (DNA) J Photochem Photobiol B, 40:91–106.

36.

Ortmans

, Moucheron

, Mesmaeker

A.K.D.

1998. Ru(II) polypyridine complexes with a high oxidation power. Comparison between their photoelectrochemistry with transparent SnO₂ and their photochemistry with desoxyribonucleic acids. Coord Chem Rev, 168:233–271.

37.

Sastri

C.V.

, Eswaramoorthy

, Giribabu

, Maiya

B.G.

2003. DNA interactions of new mixed-ligand complexes of cobalt(III) and nickel(II) that incorporate modified phenanthroline ligands. J Inorg Biochem, 94:138–145.

38.

Shahabadi

, Fatahi

2010. Multispectroscopic DNA-binding studies of a tris-chelate nickel(II) complex containing 4,7-diphenyl 1,10-phenanthroline ligands. J Mol Struct, 970:90–95.

39.

Song

, Yan

, He

2005. Studies on interaction of norfloxacin, Cu²⁺, and DNA by spectral methods. J Fluorescence, 15:673–678.

40.

Srinivasan

, Annaraj

, Athappan

P.R.

2005. Spectral and redox studies on mixed ligand complexes of cobalt(III) phenanthroline/bipyridyl and benzoylhydrazones, their DNA binding and antimicrobial activity. J Inorg Biochem, 99:876–882.

41.

Sun

, Wang

Y.C.

, Qian

, Chu

, Liang

S.M.

, Chao

, Ji

L.N.

2010. Synthesis, characterization and DNA-binding studies of chiral ruthenium(II) complexes with 2-(5-nitrofuran-2-yl)-1H-imidazo[4,5-f][1,10]phenanthroline. J Mol Struct, 963:153–159.

42.

Terenzi

, Barone

, Silvestri

, Ciuliani

A.M.

, Ruggirello

, Liveri

V.T.

2009. The interaction of native calf thymus DNA with Fe^III-dipyrido[3,2-a:2′,3′-c]phenazine. J Inorg Biochem, 103:1–9.

43.

Wang

X.L.

, Chao

, Hong

X.L.

, Liu

Y.J.

, Ji

L.N.

2005. Bis(2,2′-bipyridine)cobalt(III) complexes containing asymmetric ligands: synthesis, DNA-binding and photocleavage studies. Trans Metal Chem, 30:305–311.

44.

Wang

X.L.

, Chao

, Li

, Hong

X.L.

, Liu

Y.J.

, Tan

L.F.

, Ji

L.N.

2004. DNA interactions of cobalt(III) mixed-polypyridyl complexes containing asymmetric ligands. J Inorg Biochem, 98:1143–1150.

45.

Ware

D.C.

, Brothers

P.J.

, Clark

G.R.

, Denny

W.A.

, Palmer

B.D.

, Wilson

W.R.

2000. Synthesis, structures and hypoxia-selective cytotoxicity of cobalt(III) complexes containing tridentate amine and nitrogen mustard ligands. Dalton Trans, 925–932.

46.

Wilson

W.D.

, Tanious

F.A.

, Barton

H.J.

, Jones

R.L.

, Strekowski

, Boykin

D.W.

1989. Binding of 4′,6-diamidino-2-phenylindole (DAPI) to GC and mixed sequences in DNA: intercalation of a classical groove binding molecule. J Am Chem Soc, 111:5008–5010.

47.

Y.B.

, Chen

H.L.

, Yang

, Xiong

Z.H.

2005. Racemic D,L-[Co(phen)₂dpq]³⁺–DNA interactions: investigation into the basis for minor-groove binding and recognition. J Inorg Biochem, 99:1126–1134.

48.

H.J.

, Huang

S.M.

, Li

L.Y.

, Jia

H.N.

, Chao

, Mao

Z.W.

, Liu

J.Z.

, Ji

L.N.

2009. Synthesis, DNA-binding and photocleavage studies of ruthenium complexes [Ru(bpy)₂(mitatp)]²⁺ and [Ru(bpy)₂(nitatp)]²⁺ J Inorg Biochem, 103:881–890.

49.

Zhang

Q.L.

, Liu

J.G.

, Chao

, Xue

G.Q.

, Ji

L.N.

2001. DNA-binding and photocleavage studies of cobalt(III) polypyridyl complexes: [Co(phen) IP]³⁺ and [Co(phen) PIP]³⁺ J Inorg Biochem, 83:49–55.

50.

Zhang

Q.L.

, Liu

J.G.

, Liu

J.Z.

, Li

, Yang

, Xu

, Chao

, Ji

L.N.

2002. Effect of intramolecular hydrogen-bond on the DNA-binding and photocleavage properties of polypyridyl cobalt(III) complexes. Inorg Chim Acta, 339:34–40.

DNA Interaction Studies of Cobalt (II) Mixed-Ligand Complexes Containing Dimethyl-1,10-Phenanthroline and Dipyrido[3,2-a:2′,3′-c]Phenazine: The Role of Methyl Substitutions on the Mode of Binding

Abstract

Introduction

Materials and Methods

Materials

Synthesis of cobalt (II) complexes

Synthesis of 1, 10-phenanthroline-5, 6-dione

Synthesis of dppz

Synthesis of [Co(4,7-dmp)2(dppz)]Cl2. 2H2O (1)

Synthesis of [Co(2,9-dmp)2(dppz)]Cl2 · 2H2O (2)

Instrumentation

Results

Synthesis and characterization

Multispectroscopic methods

Discussion

Electronic absorption spectral studies

Viscosity studies

Fluorescence spectroscopic studies

Equilibrium binding titration

Circular dichroic spectral studies

Conclusion

Footnotes

Acknowledgments

Disclosure Statement

References

Synthesis of [Co(4,7-dmp)₂(dppz)]Cl₂. 2H₂O (1)

Synthesis of [Co(2,9-dmp)₂(dppz)]Cl₂ · 2H₂O (2)