Binding of oxindole-Schiff base copper(II) complexes to DNA and its modulation by the ligand

Journal of Inorganic Biochemistry 105 (2011) 1692–1703

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Binding of oxindole-Schiff base copper(II) complexes to DNA and its modulation by the ligand Vivian Chagas da Silveira a, 1, Henri Benezra a, Juliana Silva Luz b, Raphaela Castro Georg b, Carla Columbano Oliveira b, Ana Maria da Costa Ferreira a,⁎ a b

Departamento de Química Fundamental, Instituto de Química, Universidade de São Paulo, Av. Prof. Lineu Prestes, 748, São Paulo 05508-900, Brazil Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, Av. Prof. Lineu Prestes, 748, São Paulo 05508-900, Brazil

a r t i c l e

i n f o

Article history: Received 19 May 2011 Received in revised form 7 September 2011 Accepted 8 September 2011 Available online 6 October 2011 Keywords: Copper(II) complexes Oxindole-Schiff bases DNA binding EPR spectroscopy Oxidative cleavage

a b s t r a c t Previous studies on copper(II) complexes with oxindole-Schiff base ligands have shown their potential antitumor activity towards different cells, inducing apoptosis through a preferential attack to DNA and/or mitochondria. Herein, we better characterize the interactions between some of these copper(II) complexes and DNA. Investigations on its binding ability to DNA were carried out by fluorescence measurements in competitive experiments with ethidium bromide, using plasmidial or calf-thymus DNA. These results indicated an efficient binding process similar to that observed with copper(II)-phenanthroline species, [Cu(o-phen)2] 2+, with binding constants in the range 3 to 9 × 10 2 M− 1. DNA cleavage experiments in the presence and absence of distamycin, a recognized binder of DNA, indicated that this binding probably occurs at major or minor groove, leading to double-strand DNA cleavage, and being modulated by the imine ligand. Corroborating these data, discrete changes in EPR spectra of the studied complexes were observed in the presence of DNA, while more remarkable changes were observed in the presence of nucleotides (AMP, GMP, CMP or UMP). Additional evidence for preferential coordination of the copper centers to the bases guanine or cytosine was obtained from titrations of these complexes with each nucleotide, monitored by absorption spectral changes. Therefore, the obtained data point out to their action as groove binders to DNA bases, rather than as intercalators or covalent cross-linkers. Further investigations by SDS PAGE using 32P-ATP or 32P-oligonucleotides attested that no hydrolysis of phosphate linkage in DNA or RNA occurs, in the presence of such complexes, confirming their main oxidative mechanism of action. © 2011 Elsevier Inc. All rights reserved.

1. Introduction Three classical DNA-binding agents have been described, the intercalators which are usually planar chromophoric compounds that can be inserted between adjacent DNA base pairs [1], the covalent crosslinkers, and the groove binders that interact to available binding sites at major or minor grooves [2,3]. Particularly, a wide variety of small ligands, including distamycin A and derivatives, has been discovered or synthesized, that fits structural features for interactions at the minor groove [4,5]. On the other hand, there is a wide range of metal complexes whose interactions with DNA have been investigated, trying to recognize its specific or target binding sites. The modes of recognition of DNA structural features by transition metal complexes are based on intercalation, electrostatic binding, hydrogen bonding, and groove interaction [6,7]. It is well established that the anticancer agent cisplatin covalently binds to susceptible

⁎ Corresponding author. Tel.: + 55 11 3091 2151; fax: + 55 11 3815 5579. E-mail address: [email protected] (A.M.C. Ferreira). 1 Present address: Universidade Federal do Espírito Santo, Campus São Mateus, ES, Brazil. 0162-0134/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.jinorgbio.2011.09.016

sites in the major groove, notably at N7 or O6 of guanine, and prefers guanine sites surrounded in sequences with negative electrostatic potential, such as short runs of guanines [8]. In contrast, binding of some ruthenium(II) complexes to DNA was described as coordination to guanine N7, and hydrophobic interactions between the arene ligands and the nucleic acid, including intercalation, depending on the ligand [9]. The binding of metal ions or metal complexes causes DNA damage [10,11], with single or double-strand cleavage by different types of nuclease mechanism, including photoinduced, oxidative or hydrolytic cleavage [12,13]. Non-redox active metal ions such as magnesium(II) or zinc(II) preferentially promote hydrolytic cleavage [14], whereas redox active metal ions such as iron or manganese are usually assumed to operate via an oxidative mechanism. Copper compounds seem to have access to both pathways, with mononuclear complexes favoring oxidative mechanisms and dinuclear species usually promoting hydrolysis of phosphate bonds [15,16]. In the case of imine–copper complexes, earlier investigations on the chemical nuclease activity of the bis(1,10-phenanthroline)copper(I) complex, [Cu(o-phen)2] +, mediated by H2O2, indicated that this complex induces oxidative strand scission mediated by free radicals [17,18]. The active oxo-species

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formed attacks the deoxyribose sugar proton of the nucleotide which is in the vicinity of the metal complex in the minor groove, initiating a series of oxidative reactions mediated by free radicals that leads to DNA strand scission [19]. We have been studying some oxindole-Schiff base copper(II) complexes with different structural features that exhibited potential antitumor activity, and remarkable ability to bind to DNA, promoting double-strand cleavage, upon H2O2 activation [20,21]. Further, these complexes were able to catalyze the D-2-deoxy-ribose oxidation in a process modulated by the ligand. The main purpose of this work is to recognize the preferential binding modes of oxidole-Schiff base copper(II) complexes to DNA and to verify their possible mechanisms of action. Experiments by diverse techniques (fluorescence, gelelectrophoresis with labeled 5′- 32P-oligonucleotides, EPR and CD spectroscopies) were carried out with such complexes (shown in Scheme 1), using plasmidial and/or calf-thymus DNA and the respective nucleotides NMP, in order to investigate their possible interactions with nucleic acids, and its implication on subsequent damage. Further experiments with labeled 32P-ATP or 32P-oligonucleotides helped on elucidating their mechanism of action as potential antitumor agents. 2. Experimental 2.1. Materials All the copper(II) complexes have been prepared according to already described methods, using P.A. grade reagents from SigmaAldrich or Merck Chemical Co., and characterized by different techniques, as previously reported [20,22]. Briefly, the corresponding imine ligands were obtained by condensation reaction of the appropriate carbonyl and amine precursor compounds, under strict control of pH, followed by metallation with perchlorate copper(II), usually in ethanol or methanol aqueous solutions. Calf-thymus DNA, as sodium salt, and distamycin were purchased from Sigma-Aldrich, and the nucleotides from Sigma or Acros. The plasmid pBluescript II, obtained from Stratagene, was purified using Qiagen plasmid purification kit (Qiagen). The oligonucleotide probes were labeled using T4 polynucleotide kinase (Biolab). Deionized water, from a Barnstead D 4700 apparatus, was used in the preparation of all solutions.

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in a DNA solution (1 μM) was monitored by addition of increasing amounts of an EB solution, in the range 0.4 to 4 μM (see Fig. S1, in Appendix A, Supplementary Information). A saturation concentration of 2 μM or 0.79 μg/mL EB was then determined. Afterwards, titrations of a saturated solution of DNA with 2 μM EB were performed with the different copper(II) complexes 1–3 solutions, ranging from 0.6 to 60 μM, in 50 mM phosphate buffer (pH 7.4). The corresponding spectra were recorded at room temperature, 10 min after each complex addition. Analogous experiments with the [Cu(o-phen)2] 2+ complex were performed for comparison, since it was the first copper(II) complex showing efficient nuclease activity [24]. For complex [Cu(enim)H2O]2+ 2, analogous experiments with calf-thymus DNA (CT-DNA) were also performed. Purity of the final CT-DNA preparation was checked by monitoring its absorption spectrum, giving a ratio of A260/A280 N 1.8, which was indicative of a DNA free from protein [25], and DNA concentration per nucleotide was determined by spectrophotometric analysis (ε = 6600 M− 1 cm− 1 at 260 nm). In this case, the CT-DNA concentration was 3.2 μM, and the EB emission spectra were recorded varying its concentration in the range 10 to127 μM, in the absence or presence of DNA. The saturation point in this case was achieved at 51 μM. The binding constant (K na) of each copper complex to DNA and the number of copper species interacting with DNA were obtained according to reference [26]. The Stern–Volmer dynamic quenching constant KSV was calculated according to the classical Stern–Volmer equation [27]: F0 ¼ 1 þ KSV ½D F

ð1Þ

where F0 and F are the fluorescence intensities of DNA–EB in the absence and in the presence of copper complexes, respectively, [D] is the copper complex concentration, ranging from 0.6 to 60 μM, and KSV is the Stern–Volmer quenching constant, equal to the product of bimolecular quenching constant and the fluorescence lifetime in the absence of the quencher. The reaction of copper complexes (D) and DNA modified by EB (N) can be expressed as N þ nD⇄NDn :

ð2Þ

Therefore, the binding constant Kna is determined by the expression:

2.2. Methods 2.2.1. Competitive DNA binding experiments in the presence of ethidium bromide All the fluorescence measurements were performed on a SPEXFluorolog 2 spectrophotometer, using a quartz cuvette of 1 cm path length. The samples were excited at 492 nm and the emitted fluorescence recorded around 620 nm. All experiments were carried out in phosphate buffer (50 mM, pH 7.4) containing 0.1 M NaCl, in order to avoid ethidium bromide (EB) binding to secondary sites of DNA [23]. The usual experimental plasmidial DNA concentration was 0.001 mg/mL. Previously, the saturation of the fluorescent intensity

½ND  n Ka ¼ h in hn i Nf Df

ð3Þ

where Kna is the equilibrium constant for the reaction (2), [Df] is the free concentration of copper complex and [Nf] is free concentration of DNA–EB complex. Based on Eq. (3), the following relationship is found [26]: log

  F0 −F F −F n ¼ n log Ka þ n log ½Dt –n½Nt  0 F F0

Scheme 1. Structures of the oxindole-Schiff base copper(II) complexes studied.

ð4Þ

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where [Nt] and [Dt] are the total concentration of DNA–EB and CuLcomplex, respectively. From the corresponding curve of log(F0 − F)/F versus log([Dt] − n[Nt](F0 − F)/F0), obtained using the Matlab Simulink software (from MathWorks), the n value and K na were calculated, fitting a linear regression.

[Form III], as well as the supercoiled plasmid DNA [Form I] were quantified by using Image J program [30]. The ability of distamycin to protect DNA molecules against copper(II) complexes reactivity was measured by calculating the ratio of open circle DNA species in relation to the supercoiled DNA species, [Form III]/[Form I].

2.2.2. Circular dichroism studies Circular dichroism spectra were registered in a JASCO J-720 spectropolarimeter, using a quartz cuvette of 0.1 cm path length, at room temperature, in the range 200–300 nm. The initial experimental DNA concentration was 800 μM, and the spectra were registered in the absence or in the presence of 10 to 120 μM of each complex studied.

2.2.4. Assay of phosphate hydrolysis by the copper complexes DNA or RNA oligomers were labeled with α 32P-ATP using T4 polynucleotide kinase. 1 pmol samples of these oligonucleotides were added to different copper(II) complexes 1–3 (25 and 50 μM), in 50 mM phosphate buffer (pH 7.4), and 120 μM H2O2. The resulting 200 μL reaction mixtures were then incubated at 37 °C, for 60 min. Reaction products were analyzed on 8% non-denaturing (RNA) or denaturing (DNA) polyacrylamide gels by electrophoresis.

2.2.3. DNA double strand cleavage in the presence of distamycin The cleavage of plasmidial DNA was monitored using agarose gel electrophoresis. Reaction mixtures (20 μL total volume) containing 200 ng of supercoiled DNA (Form I), 50 mM phosphate buffer (pH 7.4), in the presence of 120 μM of H2O2, and copper(II) complexes 1–3 (25 and 50 μM), were incubated at 37 °C, for 30 or 60 min [28]. In the inhibition reactions, distamycin was added to the supercoiled DNA and the incubation carried out for 15 min, at 37 °C, prior to the addition of each complex and H2O2 [29]. After incubation, a quench buffer solution (4 μL) was added, and the final solution was submitted to electrophoresis on an 1% agarose gel, in 1× TAE buffer (40 mM Tris–acetate, 1 mM EDTA), at 100 V. The resultant species from DNA cleavage by [CuL] complexes, ss-cleavage [Form II] and ds-cleavage

Fluorescence Intensity (u.a)

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2.2.5. EPR measurements Possible interactions between DNA or nucleotides and the studied complexes were monitored by EPR spectra, recorded in a Bruker EMX instrument, working at X-band (9.65 GHz frequency, 20 mW power, 100 kHz modulation frequency). DPPH (α, α′–diphenyl-βpicrylhydrazyl) was used as magnetic field calibrator (g = 2.0036), and measurements were carried out with frozen aqueous solutions of the different complexes and DNA, in Wilmad quartz tubes. Usually, 15 G modulation amplitude, 7.96 × 10 3 or 3.56 × 10 4 receiver gain, and 2 or 4 scans were standard conditions for the spectra recording, at 77 K. CT-DNA and respective nucleotides (AMP, GMP, CMP or

without CuL

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0 550

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Fig. 1. Fluorescence emission spectra of EB bound to plasmidial DNA, in the absence and in the presence of the copper(II) complexes. [EB] = 2 μM, [DNA] = 1 μM, [CuL] = 0.6 to 60 μM. The arrow indicates fluorescence intensity decrease with increasing addition of the complexes.

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2+

[Cu(isaepy) 2] [Cu(isaenim)]

100

-0.8

2+

[Cu(enim)H 2O]

2+

2+

log[(Fo-F)/Fo]

Relative Intensity

[Cu(o -phen)2]

96

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UMP) were used in these experiments and the corresponding spectra were registered at DNA:[CuL] ratio 1:1 or NMP:[CuL] ratios 1:1 and 3:1, for a constant copper complex concentration (300 μM for DNA, and 600 μM for nucleotides) in phosphate buffer 50 mM, pH = 7.4. The reaction mixtures were incubated for 12 h, at room temperature, before recording the spectra, and aliquots were diluted 1:1 with ethylene glycol, required to form a glass adequate for these measurements. The corresponding spectroscopic parameters were calculated after simulation using WinEPR program from Bruker. 2.2.6. Analysis of nucleotides coordination to the copper complexes by UV/Vis spectroscopy Interactions between the different copper(II) complexes and the deoxy-nucleotides monophosphate (NMP = AMP, GMP, CMP or UMP) were also monitored in a UV-1650PC Shimadzu spectrophotometer, using quartz cuvettes of 0.1 cm path length, at room temperature, in the range 220–450 nm. Spectrophotometric titrations were carried out using 2.5 mL solution (1.8 × 10 − 5 M) of each copper complex, [Cu(isaepy)2] 2+ 1, [Cu(enim)] 2+ 2 or [Cu(isaenim)] 2+ 3, and successive addition of 10 μL aliquots of a 2.0 × 10 − 3 M solution of each NMP, up to a final volume of 3.0 mL. After each addition, a corresponding spectrum was recorded. The experimental conditions included 50 mM phosphate buffer (pH 7.4) containing 0.1 M sodium perchlorate, to keep the ionic strength constant. Data were analyzed by the Benesi–Hildebrand method [31], in order to determine the apparent formation constant (Kapp) of ternary species, [CuL(NMP)].

-5.0

-4.5

-4.0

Fig. 3. Graphs of log(F0 − F)/F versus log([Dt]− n[Nt](F0 − F)/F0), based on Eq. (4), for the different copper(II) complexes. (A) [Cu(isaepy)2]2+ 1, (B) [Cu(enim)H2O]2+ 2, (C) [Cu (isaenim)]2+ 3, and (D) [Cu(o-phen)2]2+. [EB]= 2 μM, [DNA]= 1 μM, [CuL]=0.6 to 60 μM.

Therefore, these copper complexes act as uncoupler agents, depleting significantly the adenosine triphosphate (ATP) concentration [32]. Herein, some further experiments were performed by fluorescence, CD and EPR spectroscopies, with the aim of comparing the binding ability of the studied copper(II) complexes to DNA and nucleotides

15

10

CD intensity, mdeg

Fig. 2. Effect on the fluorescence intensity of EB bound to plasmidial DNA upon addition of the copper(II) complexes.

-5.5

log{Dt-n[Nt](Fo-F)/Fo}

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-10

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300

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3. Results and discussion

Table 1 Binding constants (Kna) and corresponding binding sites number for Schiff base-copper (II) complexes in DNA, determined by linear regression plots, in competitive experiments with EB inserted in plasmidial DNA. Compounds

Binding sites number (n) 2+

[Cu(isaepy)2] 1 [Cu(enim)H2O]2+ 2 [Cu(isaenim)]2+ 3 [Cu(o-phen)2]2+

0.68 0.46 0.73 0.60

Kna, 102 M− 1 3.64 4.13 8.85 7.55

CD intensity, mdeg

10

The studied copper(II) complexes have already shown good proapoptotic properties, with preferential attack to DNA and/or mitochondria, indicating their potentiality as antitumor agent [21,22]. In more recent studies, it was also observed that damage to mitochondria occurs with loss of mitochondrial transmembrane potential.

5 2+

0

[Cu(isaepy) 2] : DNA DNA 800 μM 1: 20 1:10 1:5 1:2 1:1

-5 -10 -15 240

260

280

300

Wavelength, nm Fig. 4. CD spectra of CT-DNA (800 μM) in phosphate buffer 50 mM/NaCl 0.1 M, in the absence and presence of the copper(II) complexes, [Cu(enim)H2O]2+ 2 and [Cu (isaepy)2]2+ 1, at varied concentrations.

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with that of known DNA binder, as well as of verifying if these complexes show preferential sites for interaction with DNA. 3.1. Competitive binding experiments in the presence of ethidium bromide Fluorescence is a very useful method to verify the binding mode of complexes to DNA [33,34]. Ethidium bromide (EB) is frequently used as a sensitive probe, since its fluorescence intensity greatly enhanced in the presence of DNA, due to its strong intercalation between adjacent base pairs [35,36]. First, an optimized ratio of DNA to EB was determined for this assay, by fixing a 1 μM DNA concentration and varying the EB concentration. As expected, an increase in fluorescence signal was observed with increasing EB concentrations, attaining a saturation value at 2 μM EB (data in Fig. S1, Supplementary Information at Appendix A). Control experiments showed no emission for solutions containing only DNA, or the studied complexes. Subsequently, different amounts of each copper complex were added to this saturated EB–DNA solution. The corresponding emission spectra, in the absence and in the presence of increasing amounts of each copper complex, are given in Fig. 1. All the complexes were able to reduce the fluorescence intensity, indicating that they are interacting with DNA, probably competing with EB for the same binding sites [37], or interacting at different sites, according to the equilibria bellow: CuL þ DNA−EB⇄DNA−CuL þ EB K or CuL þ DNA−EB⇄EB−DNA−CuL K′: In Fig. 2 the corresponding curves of fluorescence intensity decay versus copper complex concentration are displayed, in comparison to that for complex [Cu(o-phen)2] 2+. The binding constant and the corresponding number of binding sites were then calculated using the fluorescence intensities before and after complex addition, according to S. Bi et al. [26], based on linear regression fit of Eq. (4), as specified in Experimental section. These results, shown in Table 1 and Fig. 3, demonstrated that all the complexes were efficient on binding to DNA, in competitive experiments with EB. Values of binding sites number (n) were obtained in the range 0.5–0.7, consistent with the value verified for [Cu(o-phen)2] 2+ (n = 0.60), and indicating interaction of one complex molecule per EB bound to DNA. By using plasmidial DNA, which is more coiled than calf-thymus DNA, smaller values for Ka were obtained (10 2 M − 1, instead of 10 4 usually verified with CT-DNA). Further, the DNA-binding affinity Ka in the presence of EB was verified to be dependent on the ligand. Complex [Cu(isaenim) H2O] 2+ 3 having a more tetragonal geometry, shows the highest saturation binding constant in this series, with a comparable binding ability to that of [Cu(o-phen)2] 2+ complex, a well known DNA binder. On the contrary, the [Cu(isaepy)2] 2+ 1 complex, with a more distorted structure, exhibits the lower Ka value in the series. 3.2. CD spectra studies CD spectra is a useful technique in diagnosing changes in DNA morphology during drug–DNA interactions, since CD signals are quite sensitive to the mode of DNA interactions with small molecules [38]. In the case of CT-DNA interacting with metal complexes, the characteristic CD spectra consist of two bands: a positive one at 275 nm due to the base stacking between the compounds and DNA bases, and a negative band at 245 nm due to the right-handed helicity B form of DNA [39,40]. Observed changes in those CD signals of DNA are usually assigned to corresponding changes in its structure. The simple groove binding or electrostatic interaction between small

molecules and DNA causes less or no perturbation on the base stacking and helicity bands, whereas a classical intercalation enhances both CD bands, stabilizing the CT-DNA form B conformation, as observed for intercalative ligands [41]. Complexes [Cu(isaepy)2] 2+ 1 and [Cu(isaenim)] 2+ 3 exhibited different binding constant values, determined in fluorescence experiments in the presence of EB. However, after the complex addition to CT-DNA it was only verified small perturbations in negative and positive bands of CD spectra for both complexes, as shown in Fig. 4. Chen and co-workers [42] observed an increase in both positive and negative bands after incubating ruthenium(II) complexes with dipyridoquinoxaline and 1-methylimidazole ligands with DNA, attributed to a typical intercalative mode, involving π–π* stacking and stabilization of the right-handed form of CT-DNA. For our complexes, on the contrary, the results indicated a probable non-intercalative mode of binding, suggesting instead a groove binding nature. It is known that the complex [Cu(o-phen)2] 2+ is first reduced in solutions where thiols or superoxide ions are electron donors, and the resulting reduced complex binds reversibly to the minor groove of DNA. Afterwards, it suffers a one-electron oxidation by hydrogen peroxide, and generates a copper-oxo species that is directly responsible for the DNA cleavage. The coordination of the complex may be directed or occur more easily to the hydrogen at C1 of the deoxy-ribose moiety. In B-DNA, this H (C1) is located at the floor of the minor groove [17,43]. Metal complexes containing extended aromatic heterocyclic ligands have been shown to bind DNA non-covalently through intercalation at the major groove, where the heterocyclic ligand is inserted partially between the base pairs so as to maximize stacking interactions. This was observed for an imine–rhodium(III) complex, where the 9,10-phenanthrenequinone diimine ligand leads to classical intercalation, favoring a 5′-GC-3′ base sequence [41]. Complex [Cu (isaepy)2] 2+ 1 contains extended aromatic ligands, showing a higher molecular volume and a more pronounced tetrahedral distortion than the other complexes studied, and therefore this complex probably interacts at the major groove of DNA, involving stacking. On the contrary, complexes [Cu(enim)] 2+ 2 and [Cu(isaenim)] 2+ 3 probably show intercalation similar to that of [Cu(o-phen)2] 2+, where the oxidative attack is initiated at hydrogen of C1 at the deoxy-ribose moiety, located at the minor groove. It was previously verified that these later complexes 2 and 3 are efficient on causing deoxy-ribose oxidative damage [22]. 3.3. DNA damage in the presence of distamycin The oxidative cleavage of DNA in the presence of the copper complexes studied and hydrogen peroxide has been already investigated, monitoring its extent by agarose gel electrophoresis assays [21]. In order to verify the mechanistic aspects of such reactions, the effect of the minor groove binder distamycin on the DNA strand cleavage was verified (Figs. 5–7). Distamycin is known to bind side-by-side to the minor groove of duplex DNA, but it can also interact with quadruplex DNA arrangements [44], and the structural characteristics of interactions verified in both cases are analogous [45]. The assays were performed by binding distamycin to plasmidial DNA before the treatment with the copper complexes, and analyzing its possible inhibition effect on DNA cleavage. In the case of [Cu(isaepy)2] 2+ 1, no inhibition of its DNA cleavage ability was observed (Fig. 5A, lanes 5–8). Evaluation of the relative ss- and ds-breaks accumulation (Forms II and III) by densitometry measurements complemented these results (Fig. 5B). From these values it seems clear that this complex binds preferentially at the DNA major groove. In contrast, distamycin showed to protect DNA cleavage caused by complexes [Cu(enim)] 2+ 2 or [Cu(isaenim)] 2+ 3, even when higher concentrations of the copper complex (50 and 100 μM, respectively) were used (Figs. 6 and 7). In the case of 3, its DNA cleavage ability

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Fig. 5. A) Agarose gel electrophoresis of pBluescript II KS(+/−) plasmid DNA, incubated with different concentrations of complex [Cu(isaepy)2]2+ 1, for 30 or 60 min in the presence of hydrogen peroxide (120 μM) with (+) or without (−) distamycin addition (300 μM). Controls: MW, marker; DNA, DNA length Standard; H2O2, DNA + H2O2; [Cu(H20)4]2+, [Cu(H20)4]2+, DNA + [Cu(H20)4]2+ (50 μM) (tinc = 60 min) + H2O2; different times (30 or 60 min) and [CuL] (25 or 50 μM) + DNA + H2O2. B) Form II/Form I DNA ratio as determined by densitometry of gel electrophoresis bands in part (A). 1, 25 μM, tinc = 30 min; 2, 50 μM, tinc = 30 min; 3, 25 μM, (tinc = 60 min); 4, 50 μM, tinc = 60 min.

was inhibited by distamycin at lower concentrations, in both incubation periods (Fig. 7, lanes 5 and 7). However, at higher concentrations, this protection effect decreased, indicating that this complex initially prefers the minor groove binding, but it can also be bonded to the major groove. Altogether, these results show that binding of those complexes to DNA is very dependent of the imine ligand, and a prerequisite for their nuclease activity. 3.4. Investigation on the mechanism of nucleic acids cleavage by copper (II) complexes In order to verify if the copper(II) complexes studied are also capable to catalyze the hydrolytic cleavage of the phosphate groups in nucleic acids, they were incubated with DNA or RNA oligomers, labeled at phosphorus 5′-end. No significant hydrolysis was observed in DNA as well as in RNA oligomers with all those complexes even at higher concentrations, as shown in Fig. 8A and B, respectively. Only very weak lines were observed in the gel, in addition to those of the added oligomers. Therefore, those complexes act mostly by

an oxidative mechanism. In the hydrolytic mechanism, they should be effective on generating nucleophile species at neutral pH, capable of causing significant phosphate hydrolysis or cleavage [46]. 3.5. Interactions of copper complexes with nucleotides The two most important and available binding sites for covalent and/or electrostatic interaction between metal ions and DNA corresponds to the N electron donor groups in the bases, mainly guanine, and the O in anionic phosphate groups. To verify such interactions between the copper(II) complexes studied and DNA or its nucleotides, UV/Vis and EPR spectroscopies were used. In Fig. 9, the EPR spectra of the copper complexes in the presence of each nucleotide, at stoichiometric ratio 1:1, were compared to those of the free complexes. After addition of CMP or GMP to the aqua complex [Cu(H2O)4] 2+, used as control, a new signal was observed, with parameters A// = 147 × 10 − 4 cm − 1 and g// = 2.333, calculated after simulation of the corresponding spectra, indicating a probable interaction of the copper ion with these nucleotides. No remarkable spectral changes

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Fig. 6. A) Agarose gel electrophoresis of pBluescript II KS(+/−) plasmid DNA, incubated with different concentrations of complex [Cu(enim)H2O]2+ 2, for 30 or 60 min in the presence of hydrogen peroxide (120 μM) with (+) or without (−) distamycin addition(300 μM). Controls: MW, marker; DNA, DNA length Standard; H2O2, DNA + H2O2; [Cu(H20)4]2+, [Cu(H20)4]2+, DNA + [Cu(H20)4]2+ (50 μM) (tinc = 60 min) + H2O2; different times (30 or 60 min) and [CuL] (25 or 50 μM) + DNA + H2O2. B) Form II/Form I DNA ratio as determined by densitometry of gel electrophoresis bands in part (A). 1, 25 μM, tinc = 30 min; 2, 50 μM, tinc = 30 min; 3, 25 μM, tinc = 60 min; 4, 50 μM, tinc = 60 min.

were observed in analogous experiments with UMP or AMP. The corresponding spectroscopic parameters, g⊥, g// and A//, were also determined for all the studied complexes after simulation of the corresponding spectra, and are given in Table 2. The same parameters were verified when using excess of nucleotides (3:1 or 5:1) in analogous experiments. Different studies in the literature indicated that strand breakage often occurs near guanine residues, suggesting that the copper ions bind to DNA preferentially at these sites [47,48]. Complex [Cu (enim)H2O] 2+ 2 interacts efficiently with CMP showing the same signal, and the same parameters observed when this nucleotide was added to the aqua complex. This complex 2 has a labile aqua ligand in its coordination sphere, which can be easily replaced by a nitrogen base. Also, this complex is smaller than the others, not exhibiting an oxindole moiety. Complexes [Cu(isaepy)2] 2+ 1 and [Cu(isaenim)] 2+ 3 probably interacted preferentially with GMP and CMP, respectively.

Despite the EPR parameters for these complexes in the presence of such nucleotides are almost the same obtained with the compounds alone, the addition of GMP or CMP caused substantial changes in their UV/Vis spectra (shown in Fig. 10), probably indicating that the bases guanine or cytosine, respectively, can occupy the fifth coordination site around the copper ion. The analogous EPR spectra, observed with each of these complexes incubated with CT-DNA for 12 h, are shown in Fig. S3 (at Appendix A, Supplementary Information). However, no significant changes in such spectra were observed, suggesting that those interactions occur mostly into the grooves of DNA (major or minor grooves), without noteworthy modifications in the copper ion environmental geometry. Complementary data by UV/Vis spectroscopy were also obtained, in order to clarify these possible interactions between the copper species and the nucleotides. The coordination of the different nucleotides

V.C. da Silveira et al. / Journal of Inorganic Biochemistry 105 (2011) 1692–1703

1699

Fig. 7. A) Agarose gel electrophoresis of pBluescript II KS(+/−) plasmid DNA, incubated with different concentrations of complex [Cu(isaenim)]2+ 3, during 30 or 60 min in the presence of hydrogen peroxide (120 μM) with (+) or without (−) distamycin addition (300 μM). Controls: MW, marker; DNA, DNA length Standard; H2O2, DNA + H2O2; [Cu (H20)4]2+, [Cu(H20)4]2+, DNA + [Cu(H20)4]2+ (50 μM) (tinc = 60 min) + H2O2; different times (30 or 60 min) and [CuL] (25 or 50 μM) + DNA + H2O2. B) Form II/Form I DNA ratio as determined by densitometry of gel electrophoresis bands in part (A). 1, 25 μM, tinc = 30 min; 2, 50 μM, tinc = 30 min; 3, 25 μM, tinc = 60 min; 4, 50 μM, tinc = 60 min.

to the copper complexes forming a ternary species can be described by the following equilibrium: ½CuL þ NMP⇄½CuLðNMPÞ

ð5Þ

with an apparent formation constant, defined as Kapp = [CuL (NMP)]/[CuL][NMP]. In order to determine the corresponding Kapp values for the copper (II) complexes studied, UV/Vis titration curves were recorded after addition of increasing amounts of each nucleotide to the complex solutions. Those curves, shown in Fig. 10, were analyzed both qualitative and quantitatively, monitoring the changes in the spectra profile [49]. The main equation used for data analysis in these experiments was: 2

CC CN =ΔA þ ΔA=Δε ¼ 1=ΔεKapp þ ðCC þ CN Þ1=Δε

ð6Þ

where CC is the total copper concentration, added as [CuL], and CN is the total nucleotide concentration in the solution, respectively. During the experiment, the changes in absorbance and molar absorptivity can be expressed as: ΔA ¼ Aexp −lðεC CC þ εN CN Þ ¼ lðΔεÞCCN þ l εN CN :

ð7Þ

Or, if (l εN CN) is negligible at the wavelength where [CuL(NMP)] is monitored, ΔA ¼ lðΔεÞCCN ; and Δε ¼ ðεCN −εC −εN Þ: The factor l corresponds to the optical length (1 cm); εC, εN and εCN are the corresponding molar absorptivity of the complex [CuL], the nucleotide (NMP) and the resulting species [CuL(NMP)]. (See

1700

V.C. da Silveira et al. / Journal of Inorganic Biochemistry 105 (2011) 1692–1703

A

Oligo DNA

further addition of nucleotides. At the beginning of the titration (linear step), [NMP] is low, and a stoichiometric ratio 1:1 [CuL]:NMP is considered, according to Benesi–Hildebrand method [31]. With further addition of nucleotide, different species can be formed, with different stoichiometries, as shown in Fig. 10. The nucleotides are preferentially coordinated to the copper center through the electron lone-pair of a nitrogen atom in the purine or pyrimidine rings of the nucleobases, forming ternary species, since all the copper complexes studied exhibits a fourth or fifth coordination site available for this interaction. As can be observed in Table 4, all the copper(II) complexes tested were capable to form stable ternary species with the nucleotides. In general, higher values for Kapp were obtained with complex [Cu (enim)] 2+ 2. For complex [Cu(isaepy)2] 2+ 1 the interaction with GMP seems preferential, as indicated by the higher stability constant, while for [Cu(isaenim)] 2+ 3 the interaction with CMP was favored. On the contrary, in case of complex [Cu(enim)H2O] 2+ 2, the corresponding stability constants determined for the nucleotides GMP, AMP and CMP were high and very similar. This can be explained by the larger accessibility of the copper ion, since this complex exhibits a tridentate imine ligand with less steric hindrance, and a fourth labile coordination site.

4. Conclusions

B

Oligo RNA

Fig. 8. A) Denaturing polyacrylamide gel 8% of DNA oligomers labeled at phosphorus 5′- end after incubation with the complexes. Lane 1: DNA oligomer labeled; Lane 2: DNA + H2O2 (120 μM); Lanes 3 and 4: H2O2 (120 μM) + [Cu(H2O)4]2+, 25 or 50 μM; Lanes 5 and 6: H2O2 (120 μM) + [Cu(isaepy)2]2+, 25 or 50 μM; Lanes 7 and 8: H2O2 (120 μM) + [Cu(enim)H2O]2+, 25 or 50 μM; Lanes 9 and 10: H2O2 (120 μM) + [Cu(isaenim)]2+, 25 or 50 μM. B) Non denaturing polyacrylamide gel 8% of RNA oligomers labeled at phosphorus 5′- end after incubation with the complexes. Lane 1: RNA oligomer labeled; Lane 2: RNA + H2O2 (120 μM); Lanes 3 and 4: [Cu(H2O)4]2+, 25 or 50 μM, + H2O2 (120 μM); Lanes 5 and 6: [Cu(isaepy)2]2+, 25 or 50 μM, + H2O2 (120 μM); Lanes 7 and 8: [Cu(enim)]2+, 25 or 50 μM, + H2O2 (120 μM); Lanes 9 and 10: [Cu(isaenim)]2+, 25 or 50 μM, + H2O2 (120 μM).

derivation of this Eq. (6) at Appendix A, Supplementary Information.) Maximum wavelength and corresponding molar absorptivity coefficients for the oxindole-Schiff base copper(II) complexes and each nucleotide (NMP) are shown in Table 3. Since the term ΔA/Δε 2 is very small, it was neglected in the Benesi–Hildebrand equation. The curve profile without this term ΔA/Δε 2 was very similar to that predicted with the addition of this term. The measurements were focused at 250–270 nm, because in this range the more significant spectral changes were observed. Plots of CC CN/ΔA vs. (CC +CN) exhibited initially a linear dependence up to (CC +CN)≈1, followed by a change in the angular coefficient with

The binding behaviors of oxindole-Schiff base copper(II) complexes toward CT-DNA or plasmidial DNA were investigated by fluorescence, absorption spectroscopy, CD and EPR measurements. In competitive experiments with EB, all the complexes were able to displace this compound from plasmidial DNA, although the extension of substitution was dependent on the imine ligand. The corresponding DNA-binding constants were comparable to that determined for complex [Cu(o-phen)2]2+, in the range 102 M− 1. The highest value (K=8.85×102 M− 1) was verified for [Cu(isaenim)H2O]2+ 3 exhibiting a more tetragonally-distorted geometry, similarly to [Cu(o-phen)2]2+ complex. Titrations of the complexes with each DNA nucleotide (AMP, GMP, CMP or UMP), monitored spectrophotometrically, indicated some preferential binding to one of the DNA bases, depending on the imine ligand. Complex [Cu(isaepy)2] 2+ 1 binds preferentially to guanine, while complex [Cu(isaenim)] 2+ 3 prefers cytosine. For the small complex [Cu(enim)H2O] 2+ 2, binding to either guanine, cytosine or adenine seems to lead to species equally thermodynamically stable. The corresponding determined equilibrium constants, Kapp, are in the range 1–2 × 10 5 M − 1. EPR measurements after addition of each nucleotide to the copper complexes corroborated these results, with a more remarkable change in the spectroscopic parameters in the case of complex [Cu(enim)H2O] 2+ 2 with CMP. In analogous EPR experiments with CT-DNA, less significant changes in the spectra were observed, as expected if only the fourth or fifth coordination site of copper is occupied by the bases, and the metal ion coordination spheres in the complexes are not considerably disturbed. As a consequence of this binding to nucleic bases, the copper(II) complexes cause oxidative damage to DNA, as previously reported [20]. Further experiments carried out in the presence of distamycin, a known minor groove binder, indicated that for complex [Cu (isaepy)2] 2+ 1 no changes in its DNA cleavage ability were observed. On the contrary, for complexes [Cu(enim)H2O] 2+ 2, and [Cu(isaenim)] 2+ 3, some inhibition of cleavage was observed, due to protection by distamycin. These data indicated that complex 1 binds preferentially to major grooves, as expected for a bulky species, and the other two bind first to the minor groove, competing with distamycin for the same sites. Therefore, the obtained data point out to

Fig. 9. EPR spectrum of Schiff base-copper(II) complexes and copper-aqua complex solutions after incubation with DNA nucleotides, in phosphate buffer 50 mM, pH 7.4, at 37°C. Spectra registered at 77 K, in frozen solutions containing ethyleneglycol (50%). For determination of corresponding parameters, all experimental spectra were simulated, as illustrated for the aqua-complex in the figure.

V.C. da Silveira et al. / Journal of Inorganic Biochemistry 105 (2011) 1692–1703

2+

A

[Cu(H2O)4]

B C D

+ CMP

+ AMP + GMP

E

2400

1701

+ UMP

2800

3200

3600

Magnetic Field, G 2+

A A B

[Cu(isaepy)2]

[Cu(enim)]

B

2+

+ AMP

C

C

+ AMP

D

+ CMP

D

E

+ GMP

E

+ CMP + GMP

+ UMP

2400

2800

3200

+ UMP

2400

3600

2800

3200

Magnetic Field, G

Magnetic Field, G A 2+

[Cu(isaenim)]

B C

+ AMP

D E

+ CMP + GMP + UMP

2400

2800

3200

3600

Magnetic Field, G 2+

A spectra [Cu(H2O)4] + CMP 2+

B simulated spectra, [Cu(H2O)4] signal C simulated spectra, [CuL(CMP)] signal

A B C D E F 2+

D spectra [Cu(H2O)4] + GMP 2+

E simulated spectra, [Cu(H2O)4] signal F simulated spectra, [CuL(GMP)] signal 2600

2800

3000

Magnetic Field, G

3200

3400

3600

1702

V.C. da Silveira et al. / Journal of Inorganic Biochemistry 105 (2011) 1692–1703

Table 2 EPR parameters determined for frozen solutions of Schiff base-copper(II) complexes (600 μM), after 30 min incubation with nucleotides (NMP, 600 μM), in phosphate buffer 50 mM, pH 7.4, at 77 K.

A 2.5 3.0 2.5

[Cu(H2O)4]2+

2.416 2.414 2.414 2.416 2.414

A//(G)

g//

147 145

2.333 2.360

g⊥ 2.085 2.085 2.085 2.048 2.085

2.0 1.5

-9

121 121 121 121 121

2

2

g//

10 (CCCN/Δ A)

1

Absorbance

1

A//(G)

[Cu(H2O)4]2+ AMP CMP GMP UMP

2.0

1.5

1.0 0.5 0.0

1.0

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

-4

10 (CC + CN)

1

g⊥

180 180 180 180 180

2.249 2.249 2.249 2.249 2.249

2.067 2.067 2.067 2.067 2.067

[Cu(isaepy)2]2+ 1 AMP CMP GMP UMP 1

1

180 180 180 180 180

2.243 2.243 2.243 2.243 2.243

198 198 198 198 198

2.202 2.202 2.202 2.202 2.202

A//(G)

[Cu(enim)H2O] AMP CMP GMP UMP

2+

2

[Cu(isaenim)]2+ 3 AMP CMP GMP UMP

g//

g//

2

A//(G)

145

2

g//

2.363

g⊥

0.5

0.0 240

320

360

400

B

2.063 2.063 2.063 2.063 2.063 2.075 2.075 2.075 2.075 2.075

280

λ ,nm

0

2.0

-2

1.6

Absorbance

A//(G)

-9 10 (CCCN/Δ A)

1

1.2

-4

-6

-8

0.8

0,0

0,4

0,8

1,2

1,6

2,0

-4

10 (CC + CN)

0.4

their action as groove binders to DNA bases, rather than as intercalators or covalent cross-linkers. Additional experiments with DNA or RNA labeled at phosphorus 5′-end attested that these copper complexes are not capable, or only in negligible extension, of catalyzing the hydrolytic cleavage of the phosphate groups in nucleic acids. Those data are consistent with previous results, and confirm that their mechanism of action is mostly oxidative.

0.0 250

300

350

400

450

λ ,nm

C 3.0 2,5

Abbreviations

10-9(CCCN/ΔA)

right-handed helicity DNA circular dichroism calf thymus DNA ethidium bromide imine ligand prepared from 4(5)-imidazolecarboxaldehyde and ethylenediamine NMP nucleotide monophosphates (AMP, CMP, GMP or UMP) ds-cleavage double strand cleavage isaenim asymmetric imine ligand obtained from isatin, ethylenediamine and 4(5)-imidazolecarboxaldehyde isaepy ximine ligand prepared from isatin and 2-(2-aminoethyl) pyridine phen 1,10-phenanthroline or o- phenanthroline ss-cleavage single strand cleavage tinc time of incubation

2,0

Absorbance

B-DNA CD CT-DNA EB enim

2.5 2.0 1.5

1,5

1,0 0,5 0,0

1.0

0,0

0,5

1,0

1,5

2,0

2,5

3,0

-4

10 (CC + CN )

0.5 0.0 240

280

320

360

400

440

λ ,nm Fig. 10. Electronic spectra recorded after increasing addition of a nucleotide to each copper(II) complex solution: A) GMP added to [Cu(isaepy)2]2+; B) AMP added to [Cu (enim)]2+; C) CMP added to [Cu(isaenim)]2+. Insets: plots of (CC CN/ΔA) vs. (CC + CN).

(INCT Redox Processes in Biomedicine — Redoxoma) for financial support. We also thank Dr. Marcos Brown Gonçalves for the pictogram.

Acknowledgments

Appendix A. Supplementary data

The authors are grateful to FAPESP (Projetos Temáticos No. 05/605968 to A.M.D.C.F.; and 05/56493-9 to C.C.O.) and to CNPq/FAPESP/MCT

Saturation curve of the fluorescence intensity obtained with the addition of EB to DNA; Insets of Fig. 10, with complementary results

V.C. da Silveira et al. / Journal of Inorganic Biochemistry 105 (2011) 1692–1703 Table 3 Maximum wavelength and corresponding molar absorptivity coefficients for oxindoleSchiff base copper(II) complexes and nucleotides (NMP). Compound

λmax (nm)

ε (mol− 1 L cm− 1)

AMP GMP CMP UMP [Cu(isaepy)2]2+ [Cu(enim)H2O]2+ [Cu(isaenim)]2+

259 253 272 262 247 264 268

1.5 × 104 4.6 × 103 5.2 × 103 4.9 × 103 5.6 × 103 1.1 × 104 1.9 × 104

Table 4 Equilibrium constants Kapp (105 M− 1) and corresponding CD/CP ratios (in parentheses) for nucleotide–copper complexes species, [CuL(NMP)], evaluated from fitting experimental data, according to the Benesi–Hildebrand method. NMP/CuL

[Cu(isaepy)2]2+ 1

[Cu(enim)H2O]2+ 2

[Cu(isaenim)]2+ 3

AMP GMP CMP UMP

0.45 1.06 0.60 0.96

1.53 1.69 1.40 1.00

0.78 0.64 1.95 0.92

(0.55) (0.24) (0.40) (0.19)

(0.24) (0.55) (0.24) (0.18)

(0.55) (0.75) (0.34) (0.24)

for determination of formation constants (Kapp) relative to species [CuL(NMP)]; EPR spectra of oxindole-Schiff base copper(II) complexes solutions after 12 h incubation with CT-DNA; and derivation of Eq. (6) for (CC CN/ΔA) vs. (CC + CN) were included as supplementary material. Supplementary data to this article can be found online at doi:10.1016/j.jinorgbio.2011.09.016. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14]

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