Synthesis, characterization, catalase functions and DNA cleavage studies of new homo and heteronuclear Schiff base copper(II) complexes

Polyhedron 28 (2009) 3967–3974

Contents lists available at ScienceDirect

Polyhedron journal homepage: www.elsevier.com/locate/poly

Synthesis, characterization, catalase functions and DNA cleavage studies of new homo and heteronuclear Schiff base copper(II) complexes Bulent Dede, Ismail Ozmen, Fatma Karipcin * Süleyman Demirel University, Sciences and Arts Faculty, Department of Chemistry, Isparta, Turkey

a r t i c l e

i n f o

Article history: Received 22 August 2009 Accepted 15 September 2009 Available online 30 September 2009 Keywords: Copper(II) complexes Polynuclear complexes Liquid–liquid extraction Catalase function Chemical nuclease DNA cleavage

a b s t r a c t A new tetradentate diimine–dioxime ligand containing a donor set of N4, and its homo-, heterodinuclear and homotrinuclear copper(II) complexes were prepared and characterized on the basis of their elemental analysis, FT-IR, 1H and 13C NMR spectra, molar conductivity and magnetic moment measurements. The extraction ability of N,N0 0 -bis[1-biphenyl-2-hydroxyimino-1-ethylidene]-diethylenetriamine was also evaluated in chloroform by using several transition metal picrates such as Mn(II), Co(II), Ni(II), Cu(II), Zn(II), Pb(II), Cd(II) and Hg(II). It has been seen that the ligand shows strong binding ability toward the copper(II) ion. Moreover, the catalytic activities of the complexes for the disproportionation of hydrogen peroxide were investigated in the presence of imidazole. The synthesized complexes display efficiency in the disproportion reactions of hydrogen peroxide, producing water and dioxygen in catalase-like activity. The interaction between these complexes and DNA has also been investigated by agarose gel electrophoresis. We found that the homo- and heterodinuclear copper complexes can cleave supercoiled pBR322 DNA to nicked and linear forms. The dinuclear complexes including phenanthroline (2–4), with H2O2 as a co-oxidant, exhibited the strongest cleaving activity. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction A broad variety of Schiff Base metal complexes can be utilized for the design of molecular ferromagnets, as catalysts for many organic reactions, models of reaction centers of metalloenzymes, optical materials, luminescence materials, DNA binding and cleavage reagents etc. [1–7]. Transition metal complexes with tunable coordination environments and versatile spectral and electrochemical properties offer a great scope of design for species that are suitable for catecholase, DNA binding and cleavage activities [8]. Hence, the synthesis of symmetrical and unsymmetrical binuclear Cu(II) complexes has gained more attention in recent years. Among the variety of methodologies applied to synthesize polynuclear coordination compounds, the use of mononuclear complexes as ligands, which have free coordination sites that can bind the second metal of the same or a different kind, is very useful and successful. This work focuses on the synthesis of homo- and heteronuclear and symmetrical and unsymmetrical Cu(II) complexes with a new Schiff base ligand containing oxime groups and 1,10-phenanthroline. Some kinds of metal complexes that interact with DNA could induce breakage of DNA strands by appropriate methods. In the case of cancer genes, after the DNA strands are cleaved, the DNA * Corresponding author. Tel.: +90 246 2114112; fax: +90 246 2371106. E-mail address: [email protected] (F. Karipcin). 0277-5387/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.poly.2009.09.020

double strands break, and the replication ability of the cancer gene is destroyed [9]. Cupric ion has been shown to bind to the DNA bases adenine, guanine and cytosine at the N(7) of purines and N(1) of pyrimidines [10]. Many copper-coordinated complexes exhibit nuclease activity [11–13]. The copper(II) complex of 1,10phenanthroline effectively exhibits nuclease-like activity [14–18]. In this study, mimic superoxide dismutase (SOD)-like activity for the disproportionation of hydrogen peroxide and the interaction with plasmid DNA (pBR322 DNA) employing gel electrophoresis of the compounds are also investigated. Transition metal ions such as Pb(II), Cd(II) and Hg(II) are recognized as highly toxic, which makes their presence in environmental waters or soils undesirable. Such metals can accumulate in the environment and produce toxic effects in plants and animals even at very low concentrations. However, it is not useful to describe elements as ‘‘toxic” or ‘‘non-toxic”. Even so-called ‘‘toxic compounds” can usually be tolerated in low doses, and may exhibit therapeutic effects within narrow concentration ranges, and biologically-essential elements such as Co(II), Ni(II), Cu(II), Zn(II) etc. can become toxic at high doses. Therefore, separation of these trace metals is vital due to potential health and ecological hazards [19]. The process of solvent extraction is one of the most versatile procedures used for the removal, separation and concentration of metallic species, broadening its applications in the recycling of resources in the field of metallurgy and waste water treatment as demand increases for the development of new

3968

B. Dede et al. / Polyhedron 28 (2009) 3967–3974

approaches to resolve the various problems presented. For this purpose, many oxime and Schiff base derivatives have been synthesized and their extraction properties investigated by solvent extraction [20–25]. We have investigated the extraction properties of the Schiff base ligand including oxime groups by solid–liquid extraction.

2. Experimental 2.1. Materials and measurements 4-Acetylbiphenyl [26] and 4-isonitrosoacetylbiphenyl [27,28] were prepared using literature methods. pBR322 DNA was purchased from Fermantas. All other chemicals and solvents were of analytical grade and used as received, without any further purification. The 1H and 13C NMR spectra of the free ligand were recorded on a Bruker AVANCE 300 MHz spectrometer, using TMS as an internal standard and CDCl3 as a solvent. The IR spectra of the free ligand and its metal complexes were measured using a Schimadzu IRPrestige-21 FT-IR spectrophotometer within the range 4000–400 cm1, using KBr discs. Elemental analyses (C, H, N) were performed using a LECO 932 CHNS analyzer and metal contents were estimated on a Perkin Elmer Optima 5300 DV ICP-OES spectrometer. The spectrophotometric measurements were carried out with a Perkin Elmer k 20 UV–Vis spectrometer. Molar conductances in DMF (103 M solution) were measured at room temperature on an Optic Ivymen System conductivity meter. Melting point determinations were performed with a digital melting point instrument from Electrothermal model IA 9100. Magnetic susceptibilities were determined on a Sherwood Scientific Magnetic Susceptibility Balance (Model MX1) at room temperature. All statistical analysis was performed using Statistica software release 6.0. Analysis of variance was performed and means were separated using Fisher’s protected least significant differences (LSD) test at P 6 0.05. 2.2. Synthesis 2.2.1. Preparation of the Schiff base ligand [H2L] To a solution of 4-isonitrosoacetylbiphenyl (30 mmol) in warm ethanol (10 mL), diethylenetriamine (1.547 g, 15 mmol) was added under constant stirring. The mixture was stirred at ambient temperature for 1 h. On addition of distilled water, a yellow colored ligand [H2L] was precipitated. The solid was separated by filtration, washed with distilled water, ethanol and diethyl ether, and dried in air. Yellow compound; yield: 71%; m.p.: 80 °C. Anal. Calc. for C32H31N5O2: C, 74.25; H, 6.04; N, 13.53. Found: C, 74.42; H, 6.30; N, 13.38%; 1H NMR (CDCl3, ppm): 8.24 (s, 2H, O–H), 7.32–7.91 (m, 18H, Ar–H), 7.96 (s, 2H, –CH–), 7.20 (s, 1H, N–H), 2.35 (t, 4H, –CH2–), 1.28 (t, 4H, –CH2–); 13C NMR (CDCl3, ppm): 169.53 (C2), 142.20 (C1), 121.35–139.74 (C3–C14), 57.32 (C15), 42.48 (C16); FT-IR (KBr, cm1): 3361 m (N–H), 3224 b (O–H), 1668 m (C@N(imine)), 1612 s (C@N(oxime)), 1430 s (N–O), 1476 m (C–N) (b, broad; s, strong; m, medium; w, weak). 2.2.2. Preparation of the mononuclear Cu(II) complex) Caution: All the perchlorate salts reported here are potentially explosive and therefore should be handled with care. To a suspension of the ligand (1 mmol) in Me2CO (25 mL), a solution of Cu(ClO4)26H2O (370 mg, 1 mmol) in Me2CO (10 mL) was added, and the mixture was refluxed for 1 h. A crude oily product was obtained on evaporating the resulting solution and the mononuclear copper(II) complex was used without further purification.

2.2.3. Preparation of the binuclear Cu(II) complex (2) The binuclear Cu(II) complex was prepared from a general synthetic procedure in which to a stirred suspension of the mononuclear complex (1 mmol) in methanol (25 mL), a methanolic solution of Et3N (101 mg, 1 mmol) was added, and the mixture was stirred for 30 min. Then methanolic solutions (10 mL) of Cu(ClO4)26H2O (370 mg, 1 mmol) and 1,10-phenanthroline monohydrate (397 mg, 2 mmol) were successively added to the above solution and refluxed for 3 h. The resulting solids were filtered off, washed with distilled water, methanol and diethyl ether, and dried over P4O10. Green complex; yield: 46%; m.p.: 218 °C. Anal. Calc. for C56H47N9O11Cu2Cl2: C, 55.13; H, 3.88; N, 10.33; Cu, 10.42. Found: C, 55.21; H, 3.67; N, 10.47; Cu, 10.51%; KM (DMF solution, X–1 cm2 mol–1): 176; leff = 2.20 B.M.; FT-IR (KBr, cm1): 3531 b (O–H), 3345 m (N–H), 1652 m (C@N(imine)), 1585 s (C@N(oxime)), 1421 s (N–O), 1536 m (C–N), 1097 s, 1183 w, 626 w (ClO4), 512 w (M–O), 421 (M–N) (b, broad; s, strong; m, medium; w, weak). 2.2.4. Preparation of the binuclear Cu(II)–Mn(II) (3) and Cu(II)–Co(II) (4) complexes The mononuclear copper complex (1 mmol) was mixed with Et3N (101 mg, 1 mmol) in methanol (20 mL) and stirred for 0.5 h. A solution of Mn(OAc)24H2O (268 mg, 1 mmol) or Co(OAc)24H2O (249 mg, 1 mmol) in methanol (10 mL) and 1,10-phenanthroline monohydrate (397 mg, 2 mmol) in methanol (10 mL) were successively added to the resulting solution. A stoichiometric amount of NaClO4 (123 mg, 1 mmol) was then added to the resulting mixture, which was refluxed for 3 h. The product was filtered off, washed with distilled water, methanol and diethylether, and dried over P4O10. For Cu(II)–Mn(II), green complex; yield: 53%; m.p.: 226 °C. Anal. Calc. for C56H47N9O11CuMnCl2: C, 55.52; H, 3.91; N, 10.41; Cu, 5.25; Mn, 4.54. Found: C, 55.41; H, 3.96; N, 10.32; Cu, 5.21; Mn, 4.48%; KM (DMF solution, X–1 cm2 mol–1): 183; leff = 3.62 B.M.; FT-IR (KBr, cm1): 3527 b (O–H), 3321 m (N–H), 1645 m (C@N(imine)), 1587 s (C@N(oxime)), 1398 s (N–O), 1516 m (C–N), 1099 s, 1180 w, 626 w (ClO4), 513 w (M–O), 418 (M–N) (b, broad; s, strong; m, medium; w, weak). For Cu(II)–Co(II), Brown complex; yield: 68%; m.p.: 230 °C. Anal. Calc. for C56H47N9O11CuCoCl2: C, 55.34; H, 3.90; N, 10.37; Cu, 5.23; Co, 4.85. Found: C, 55.63; H, 3.78; N, 10.51; Cu, 5.28; Co, 4.74%; KM (DMF solution, X–1 cm2 mol–1): 169; leff = 2.81 B.M.; FT-IR (KBr, cm1): 3510 b (O–H), 3324 m (N–H), 1649 m (C@N(imine)), 1593 s (C@N(oxime)), 1398 m (N–O), 1544 m (C–N), 1099 s, 1180 w, 626 w (ClO4), 513 w (M–O), 422 (M–N) (b, broad; s, strong; m, medium; w, weak). 2.2.5. Preparation of the trinuclear Cu(II) complex (5) To a stirred suspension of the mononuclear complex (2 mmol) in Me2CO (25 mL), a solution of Cu(ClO4)2.6H2O (370 mg, 1 mmol) in Me2CO (25 mL) was added and the mixture was refluxed for 2 h. The resulting solution was filtered while hot and concentrated slowly. As the solution cooled, a powder product precipitated. The resulting solid was filtered off, washed with diethyl ether, and dried over P4O10. Green complex; yield: 64%; m.p.: 228 °C. Anal. Calc. for C64H62N10O14Cu3Cl2: C, 52.77; H, 4.29; N, 9.61; Cu, 13.09. Found: C, 52.84; H, 4.17; N, 9.56; Cu, 13.17%; KM (DMF solution, X–1 cm2 mol–1): 172; leff = 2.57 B.M.; FT-IR (KBr, cm1): 3564 b (O–H), 3332 w (N–H), 1642 m (C@N(imine)), 1579 s (C@N(oxime)), 1400 m (N–O), 1480 m (C–N), 1096 s, 1182 w, 626 w (ClO4), 510 w (M–O), 426 (M–N) (b, broad; s, strong; m, medium; w, weak). 2.3. Studies on catalase function 2.3.1. Studies on catalase-like function (method I) Volumetric measurements of evolved dioxygen during the reactions of the copper(II) complexes 2, 3, 4 and 5 with H2O2 were car-

B. Dede et al. / Polyhedron 28 (2009) 3967–3974

ried out as follows: a 50 mL three-necked round-bottom flask containing a solution of the complexes (0.005 mmol solid sample) in DMF (10 mL) was placed in a water bath (25 °C). One of the necks was connected to a burette and the others were stoppered by a rubber septum. While the solution was stirring, H2O2 (1.33 mmol, 0.150 mL) was injected into it through the rubber septum using a microsyringe. Volumes of evolved dioxygen were measured at 1 min time intervals by volumometry. In the cases where imidazole (50 mg) was added, this was introduced into the reaction vessel before the addition of H2O2 (in the absence of the imidazole, the complexes were either inactive or very weak catalysts for this reaction). 2.3.2. Preparation of the haemolysate and the effects of the complexes on catalase activity (method II) Blood from different five humans collected in EDTA (20– 25 years old healthy male) was centrifuged (15 min, 2500g). Preparation of the haemolysate was done as described by Ninfali et al. [29]. The CAT activity was determined as follows. The haemolysate (20 lL) was added to a tube containing 1000 lL of sodium phosphate buffer saline (PBS, 0.01 M, pH 7.4), and 1000 lL of 9.7 M H2O2 (dissolved in sodium phosphate buffer) and 20 lL complex solution was added to start the reaction. The evolved dioxygen (mL) was recorded following the reduction of hydrogen peroxide during 3 min by volumometry. 2.4. Cleavage of pBR322 DNA For the agarose gel electrophoresis experiments, 0.5 lg/lL supercoiled pBR322 DNA (0.5 ll) was treated with 1 lL of 1 mM the tested ligand and its complexes in DMF and 2 lL of 0.1 M Tris–HCl (pH 8.0) buffer in the absence and presence of 2 lL of 5.0 mM hydrogen peroxide as a co-oxidant reagent. After incubation at 37 °C for 2 h, 1 lL of loading buffer (0.25% bromophenol blue, 0.25% xylene cyanol, 30% glycerol in H2O) was added to each tube and the mixed solution was loaded on 1% agarose gel. The electrophoresis was carried out for 1.5 h at 100 V in TBE buffer (89 mM Tris–borate, pH 8.3, 2.5 mmol L1 EDTA). Gels were stained with ethidium bromide (1 mg mL1) for 10 min prior to being photographed under UV light. The efficiency of the DNA cleavage was measured by determining the ability of the complex to form linked circular (LC) or nicked circular (NC) DNA from its supercoiled (SC) form by quantitatively estimating the intensities of the bands using the Biolab UVItec Gel Documentation System. The fraction of each form of DNA was calculated by dividing the intensity of each band by the total intensities of all the bands in the lane. 2.5. Solvent extraction Picrate extraction experiments were performed following Pedersen’s procedure [30]. The extraction properties of the oxime ligand H2L (1) were investigated under liquid–liquid phase and neutral conditions using selected metal picrates (Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, Hg2+, Pb2+) as substrates and measuring by UV– Vis spectroscopy the amounts of metal picrate in the aqueous phase before and after treatment with the compounds. About 10 mL of 2  105 M aqueous picrate solution and 10 mL of 1  103 M solution of ligand in CHCl3 were vigorously agitated in a stoppered plastic tube with a mechanical shaker for 2 min, then magnetically stirred at 25 °C for 1 h, and finally left standing for an additional 30 min. The concentration of the picrate ion remaining in the aqueous phase was then determined spectrophotometrically. Blank experiments showed that no picrate extraction occurred in the absence of ligand. Metal picrates were prepared by successive addition of a 1  102 M metal nitrate solution to

3969

2  105 M aqueous picric acid solution, shaking at 25 °C for 1 h. These metal picrates (Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, Hg2+, Pb2+) were measured by UV–Vis spectroscopy using the maximum wavelength 352 nm. For each combination of host and metal picrate, the picrate extraction was conducted on three different samples and the average value of percent picrate extracted, with a standard deviation, was calculated. In the absence of host, a blank experiment, no metal ion picrate extraction was detected. The extractability was calculated by using the equation below:

Extractabilityð%Þ ¼ ½ðA0  AÞ=A0   100 where A0 is the absorbance in the absence of ligand. A denotes the absorbance in the aqueous phase after extraction. 3. Results and discussion 1-(Biphenyl)-2-hydroxyimino-1-ethanone was prepared according to the reported procedures [4,26–28]. Condensation of the 1-(biphenyl)-2-hydroxyimino-1-ethanone with diethylenetriamine (DETA) gives the corresponding imine (H2L) (Scheme 1), which was identified by its IR, elemental and thermal analysis, 1 H and 13C NMR spectra, where replacement of the carbonyl by the imine group results in: (i) a lowering of the energy of the m(C@O) stretch in the IR spectrum and (ii) a shift to higher field of the CH@N proton signal in the 1H NMR spectrum. The mono- (Fig. 1), homodi- (Fig. 2), homotri- (Fig. 3) and heterodinuclear Cu(II)–Mn(II) and Cu(II)–Co(II) complexes (Fig. 2) with the tetradentate diimine–dioxime ligand were prepared by treating a solution of the ligand in acetone or methanol and one equivalent of Et3N with copper(II), manganese(II) or cobalt(II) salts. All the metal(II) chelates were stable at room temperature, intensely colored, insoluble in water and infusible at high temperature. All the compounds are stable in solution. The structures of the ligand and its complexes have been verified by elemental analysis, FT-IR, 1 H and 13C NMR spectra, molar conductivity and magnetic moment measurements. Various attempts to obtain single crystals of the complexes have so far been unsuccessful. The melting points, yields, colors, magnetic susceptibility, molar conductivity values and elemental analysis of complexes and the ligand are given in the Section 2. The metal:ligand:phen ratio was found to be 2:1:2 for the dinuclear complexes and 3:2:0 for the trinuclear complexes, according to the elemental analysis results. 3.1. 1H and

13

C NMR spectroscopy

In order to identify structure of the diimine–dioxime ligand, the H and 13C NMR spectra were recorded in CDCl3. The chemical shifts, expressed in ppm downfield from tetramethylsilane, are given in the Section 2. The 1H NMR spectrum of the ligand (H2L) shows a D2O exchangeable peak at 8.24 ppm as a singlet, which is characteristic of the (@N–OH) group’s proton in the molecule. The peaks observed in the range 7.32–7.91 ppm are assignable to the protons of benzene units as multiplet peaks. The chemical shift observed at 7.96 ppm is assigned to the protons of the azomethine group (CH@N) as a singlet. The 1H NMR resonances with the expected integrated intensities were observed as a singlet at 7.20 (1H), as triplets at 2.35 (4H) and 1.28 (4H) ppm for the N–H and methylene group protons of the diethylenetriamine, respectively. The 1H NMR spectral data of the ligand are supported by the 13C NMR spectrum. The chemical shifts for the carbon atoms of the aromatic rings, which were numbered C(3) to C(14), were recorded between 121.35 and 139.74 ppm. The signal for the carbon atom C(2) of the azomethine group is observed at 169.53 ppm, which 1

3970

B. Dede et al. / Polyhedron 28 (2009) 3967–3974

O

+

H3C

O

AlCl 3

Cl

CH3 OH

9

8

10

5 7

11

6

12

1

4 3

13

N

RONO/Na

2

N

14

15 16

O

DETA

HN

N OH N N

OH

Scheme 1. Synthetic route for the synthesis of H2L (1).

OH2

OH2 N

H N

N NH

O

Cu N

H N

(ClO4)2

O

NH

M

Cu N

N

N O

N

(ClO4)2

N O

N N

Fig. 1. Proposed structure of the mononuclear copper(II) complex of H2L.

also confirms the structure of the ligand. The chemical shift which belongs to the C(1) atom bonded to the oxime group was obtained at 142.20 ppm. In the 13C NMR spectrum of the ligand H2L, the signals at 57.32 and 42.48 ppm are attributed to C(15) and C(16), respectively. All the protons and carbons were found to be in their expected regions and are in good agreement with values previously reported [31–34]. The results strongly suggest that the proposed dimine–dioxime ligand has been formed. Because of the paramagnetic nature of the complexes, their 1H and 13C NMR spectra could not be obtained. 3.2. IR spectroscopy The significant IR absorption bands of the prepared ligand and its homo-, heterodinuclear and homotrinuclear copper(II) com-

Fig. 2. Proposed structure of the homodinuclear Cu(II) (2), heterodinuclear Cu(II)– Mn(II) (3) and Cu(II)–Co(II) (4) complexes of H2L (1).

plexes are given in the Section 2. The characteristic absorption bands of the ligand are shifted on complex formation and new characteristic vibrational bands of the complexes appear. The IR spectrum of the ligand did not display a characteristic band for a C@O group after the condensation reaction of 1-(biphenyl)-2-hydroxyimino-1-ethanone with diethylenetriamine. This result indicates that the formation of Schiff base was completed. The medium band located at 1668 cm1 is assigned to the m(C@N) stretching vibration of the azomethine group of the ligand. This band is shifted by 16–26 cm1 to lower wavenumber after complexation. These shifts to lower wavenumbers support the participation of the azomethine group of this ligand in binding to the copper(II) ion [35]. This can be explained by the donation of electrons from the nitrogen to the empty d-orbitals of the metal atoms.

3971

B. Dede et al. / Polyhedron 28 (2009) 3967–3974

OH2

N O

N NH

N

O N Cu

Cu N

H2O

N O

NH

Cu O N

(ClO4)2

N

spin-only magnetic moment of an S = 1/2 (1.73 BM), Cu(II) d9 system. The low magnetic moment values of the homodi- and trinuclear copper(II) complexes are indicative of antiferromagnetic exchange [42]. Room temperature effective magnetic moment values for the heterodinuclear Cu(II–)Mn(II) and Cu(II)–Co(II) complexes are 3.62 and 2.81 BM, respectively and deviated slightly from the spin-only values. The observed magnetic moment values of complexes 2, 3, 4 and 5 are less than the expected values. This may be due to antiferromagnetic interactions persisting in the complexes, where neighboring magnetic dipoles tend to align in the opposite direction, resulting in a zero moment or a decrease in the magnetic moments. 3.5. Catalase activity

In the FT-IR spectrum of the ligand, a broad band at 3224 cm1 was assigned to the O–H stretching vibration of the oxime group. The deprotonation of the oxime group is indicated by the absence of a band in the metal complexes at 3224 cm1. A strong band at 1430 cm1 in the spectrum of the free ligand is attributed to m(N–O). According to the IR spectra of the complexes, the shifting of the N–O stretching frequency to a lower frequency by about 9–32 cm1 indicates the formation of coordination bonds between the metal and oxygen atom of the oxime group [25]. The coordination mode of the ligand is further supported by new frequencies occurring in the 510–513 and 418–426 cm1 ranges, which have been assigned tentatively to m(M–O) and m(M–N), respectively [36,37]. All of the perchlorate salts show a weak band near 1180–1183 cm1 and a strong band at 1096– 1099 cm1 (antisymmeric stretch), and a weak band at 626 cm1 (antisymmetric band), indicative of uncoordinated perchlorate anions [38–40]. In addition, broad peaks appearing between 3510 and 3567 cm–1 in the spectra of the metal complexes indicate that H2O is coordinated to the Cu(II) ions. Therefore, from the IR spectra, it is concluded that the H2L ligand behaves as a tetradentate ligand coordinated to the metal ions via the azomethine and oxime groups. 3.3. Molar conductance The chelates were dissolved in DMF and the molar conductivities of their 103 M solutions at 25 °C were measured. The molar conductance values of the complexes are given in the Section 2. It is concluded from the results that the chelates are found to have molar conductance values of 169–183 X1 cm2 mol1, indicating that these chelates are 1:2 electrolytes in these solvents [41]. 3.4. Magnetic moment studies Magnetic susceptibility measurements of the complexes were carried out in the solid state at room temperature and provide information regarding their structures. These measurements in the solid state show that the homo-, heterodinuclear and homotrinuclear Cu(II) complexes are paramagnetic at ambient temperature. The observed magnetic moment values for the homodi- and trinuclear Cu(II) complexes are 2.20 and 2.57 BM, respectively. These values for complexes 2 and 5 are not consistent with the expected

4

3

2

5

18

Evolved Oxygen (mL)

Fig. 3. Proposed structure of the homotrinuclear Cu(II) (5) complex of H2L (1).

3.5.1. In vitro catalase-like activity (method I) All the complexes showed activity for the catalytic decomposition of H2O2 in the presence of added imidazole. Among them, the heterodinuclear Cu(II)–Co(II) complex (4) is the most effective. Experiments were repeated several times to ensure consistency of the results. The catalytic activity of the complexes 2, 3, 4 and 5 towards H2O2 was investigated in N,N-dimethylformamide. The time course of the O2 evolution is shown in Fig. 4. The H2O2 disproportionation efficiency of the complexes in the presence imidazole follows the order: 4 > 2 > 5 > 3. In the absence of heterocyclic base, the complex decomposes hydrogen peroxide slowly, but the decomposition of H2O2 is enhanced in the presence of a heterocyclic base such as 1-methylimidazole (1-MeimH), imidazole (imH) or pyridine (py) because of their strong p-donating ability. On the other hand heterocyclic bases themselves cause only a very slight disproportionation of the peroxide. The evolution profile in Fig. 4 shows the involvement of a fast catalytic process occurring at the initial stage followed by a short slow period process to finish the reaction. It was suggested that these bases may be essential in the catalysis disproportionation of H2O2 by manganese catalase since they are known to be present in the vicinity of the active site of catalase and other manganoenzymes [43]. Furthermore the presence of the bidentate chelating nitrogen donor ligand phenanthroline in the coordination sphere of the metal significantly enhances the ability of the manganese to disproportionate H2O2 and the phenanthroline (phen) and 2,20 -bipyridine (bipy) species were found to be the more aggressive peroxide disproportionation catalysts [44,45]. As a result of the catalase-like activity studies, the present the copper(II) complexes 4 and 2 have a high disproportionation efficiency when compared to the other synthesized complexes.

16 14 12 10 8 6 4 2 0 0

2

4

6

8

10

12

time (min) Fig. 4. Time courses of dioxygen evolution in the disproportionation of H2O2 by the complexes 2, 3, 4 and 5 in DMF. [Complex] = 0.005 mmol, [H2O2] = 1.33 mmol, 298 K.

3972

B. Dede et al. / Polyhedron 28 (2009) 3967–3974

3.5.2. Effects of the complexes on catalase activity (method II) The effects of the complexes 2, 3, 4 and 5 on the catalase activity of haemolysate to disproportionate H2O2 into H2O and O2 were examined in N,N-dimethylformamide at room temperature. The catalytic activity of haemolysate was investigated in the presence and absence of the complexes. Fig. 5 shows that the catalytic activity of haemolysate toward H2O2 increases with added complex. The catalytic activity of haemolysate-copper(II) complexes mixture is similar to the in vitro catalase-like activity and the efficiency of the complexes follows the decreasing order 4 > 2 > 5 > 3. 3.6. DNA cleavage activity The cleavage of the supercoiled form of pBR322 DNA with the ligand (1), its homodinuclear copper(II) (2), heterodinuclear copper(II)–manganese(II) (3), heterodinuclear copper(II)–cobalt(II) (4) and homotrinuclear copper(II) (5) complexes was studied in the absence or presence of H2O2 as a co-oxidant. DNA cleavage was analyzed by monitoring the conversion of supercoiled DNA (form I) to nicked circular DNA (form II) and linear DNA (form III) under aerobic conditions. When circular plasmid DNA is subjected to electrophoresis, a relatively fast migration will be observed for the intact supercoil form (form I). If scission occurs on one strand (nicking), the supercoil will relax to generate a slower moving open circular form (form II). If both strands are cleaved, a linear form (form III) that migrates between form I and form II will be generated [7,46]. The results of the gel electrophoresis separations of plasmid pBR322 DNA by the ligand (1) and its complexes (2–5) in the absence or presence of H2O2 are depicted in the Fig. 6. Control experiments were applied using only DNA and DNA + H2O2. As shown in Fig. 6, incubation of the pBR322 DNA at 37 °C for 2 h with 1 lg of the compounds cause the conversion of form I to form II. The supercoiled (form I) DNA was cleaved to form II and form III in the absence of H2O2 with the ligand (lane 3) and all the complexes except complex 2 (lanes 5–7). For complex 2, the

Fig. 5. Time courses of dioxygen evolution in the disproportionation of H2O2 by a haemolysate – complex 2, 3, 4 and 5 mixture in DMF. [Haemolysate] = 40 lL, [H2O2] = 9.7 M, [complex] = 1 mM, [PBS] = 0.01 M, pH 7.4, 298 K.

cleavage is found to be much more efficient, it cleaved the supercoiled pBR322 DNA into form II, form III and a much smaller fragment, and the circular supercoiled DNA (form I) band disappeared completely (lane 4). The cleavage efficiency after incubation for 2 h in the absence of H2O2, follows the order: 2 > 4 > 3 > 5 > 1. The cleavage percentages are listed in Table 1. These results indicate that although the examined complexes induces very similar conformational changes on supercoiled DNA with conversion of the supercoiled form to the nicked form then the linear form in a sequential manner, complexes 3 and 5 are less effective than complexes 2 and 4. On the other hand, pBR322 DNA treated with ligand 1 showed less changes in the form levels compared with the complexes. Namely, the ligand 1 alone is less effective. The different DNA cleavage efficiencies of the ligand and the complexes may be due to the different binding affinity of the complexes to DNA [7,47,48]. The degradation of pBR322 DNA is also dependent on co-oxidant used. The cleavage mechanism of pBR322 DNA induced by compounds 1–5 was investigated (Fig. 6) and clarified in the presence of H2O2 as a co-oxidant (lanes 8–12). In the ligand, the intensity of the circular supercoiled DNA (form I) band was found to decrease, while that of the nicked (form II) and linear DNA (form III) bands increase (lane 8) in the presence of H2O2. The dinuclear complexes including phenanthroline (2–4) with H2O2 exhibited the strongest cleaving activity. They cleaved the supercoiled pBR322 DNA into much smaller fragments (lanes 9–11). The activity of complex 5, however, was weaker than the dinuclear complexes. It could not cleave all the linear DNA into smaller fragments. These observations suggest that the complex mediated cleavage reaction proceeds via an oxidative pathway mechanism and implies that the singlet oxygen plays a role in the cleavage chemistry. In the presence of H2O2 all the Cu(II) complexes (2–5) are remarkably at degrading pBR322 DNA by an oxidative (O2dependent pathway) cleavage mechanism using the singlet oxygen as the reactive species [7,49]. These results are similar to those observed for some Cu(II) and Co(II) complexes as chemical nucleases [50–52]. In addition, the chemical environment around the central metal ions and their geometric structures may also effect the nucleolytic efficiency of the complexes [14,53]. Therefore, the difference in the DNA cleavage activities of the Schiff base complexes may be attributed to their proximity to the DNA on binding since the phenanthroline units present in 2, 3 and 4 may provide much more effective binding than 5, which has no such structural units. This may also imply that the binding of 2, 3 or 4 to DNA makes the metal ions more approachable to the DNA backbone than for 5. Therefore, the difference in the cleavage behavior of 5 is consistent with a distinct oxidative cleavage pathway. Therefore, these observations suggest that the coordination environment of the central metal ions in the complexes not only governs the DNA binding but also determines the nucleolytic action [14]. Furthermore, the interaction with DNA may be related to the extraction ability of the ligand for the same chemical environment complexes (2–4). The

Fig. 6. Gel electrophoresis diagram showing the cleavage data of pBR322 plasmid DNA (0.1 lg) by the ligand and its complexes in DMF – Tris buffer medium (pH 8.0) in air after incubation at 37 °C for 2 h. Lane 1, untreated pBR322 plasmid DNA; lane 2, pBR322 plasmid DNA + H2O2; lanes 3–7, pBR322 plasmid DNA + the compounds; lanes 8–12, pBR322 plasmid DNA + the compounds + H2O2 (the compounds = 1, 2, 3, 4, 5, respectively).

3973

B. Dede et al. / Polyhedron 28 (2009) 3967–3974 Table 1 DNA cleavage data of pBR322 plasmid DNA (0.1 lg) by 1–5. Lane no.

Reaction conditions

Ink. time (h)

Form I %SC

Form II %NC

Form III %LC

Form IV % SF

1 2 3 4 5 6 7 8 9 10 11 12

DNA DNA + H2O2 DNA + 1 DNA + 2 DNA + 3 DNA + 4 DNA + 5 DNA + 1 + H2O2 DNA + 2 + H2O2 DNA + 3 + H2O2 DNA + 4 + H2O2 DNA + 5 + H2O2

2 2 2 2 2 2 2 2 2 2 2 2

73.8 66.6 64.2 ND 61.8 61.2 64.3 63.8 ND ND ND ND

15.3 16.3 25.8 19.6 24.5 20.8 22.2 11.3 ND ND ND ND

10.9 17.1 10.0 65.8 13.7 18.0 13.6 24.9 ND ND ND 45.1

ND ND ND 14.6 ND ND ND ND ND ND ND 54.9

SC, NC, LC, SF are supercoiled, nicked circular and linked circular, smaller fragment forms of DNA, respectively. ND: not detected.

1

80

Extractability (%)

70 60 50 40 30 20 10 0 Mn(II) Co(II) Ni(II) Cu(II) Zn(II) Pb(II) Cd(II) Hg(II) Fig. 7. Extraction percentages of the metal picrates with the ligand H2L (1). H2O/ CHCl3 = 10/10 (v/v): [picric acid] = 2  105 M, [ligand] = 1  103 M, [metal nitrate] = 1  102 M, 298 K, 1 h contact time.

1

Extractability (%)

80 70 60 50 40 30

estimated by solvent extraction of selected transition metal picrates (Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, Hg2+, Pb2+) from the aqueous to an organic phase under neutral conditions. After complete phase separation, the equilibrium concentration of picrate in the aqueous phase was determined spectrophotometrically. The data are expressed as percentages of the cation extracted (E%) by the ligand and are given in Fig. 7. The affinities of metals for ligand 1 in decreasing order is Cu(II) > Hg(II) > Pb(II) > Co(II) > Ni(II) > Cd(II) > Zn(II) > Mn(II). It is clear that ligand 1 shows a high extraction for Cu(II) ion compared to the other metal ions. This is an expected result because of the interaction of the soft donor atom – soft metal cation. Due to the presence of the oxime group in the ligand, it acts as a soft base and binds selectively to soft and border line acids. The effectiveness of ligand 1 in transferring Cu(II) ions indicates that the oxime groups (–C@N–OH) appear to be operative and play an important role at the water–chloroform interphase, since the metal ions could possibly be interacting with these soft ligating sites [54]. These results are in accord with our recent findings [25,55]. The extremely high binding capacity of ligand 1 towards Cu(II) is a desired feature of a chelating compound designed for the removal and recovery of heavy metal ions from environmental and industrial waters. The results for the dependence of copper ion uptake on contact time for ligand 1 are presented in Fig. 8. These results indicate fast rates of equilibration; the rate of metal ion uptake increases in the first 60 min and reaches a steady state after this period. Therefore, an equilibrium time of 60 min was adopted for subsequent extraction studies to ensure complete extraction.

20 10

4. Conclusions

0 0

20

40

60

80

100

120

time (min) Fig. 8. Effect of equilibrium time on the extraction of metal picrates with the ligand H2L (1). H2O/CHCl3 = 10/10 (v/v): [picric acid] = 2  105 M, [ligand] = 1  103 M, [copper(II) nitrate] = 1  102 M, 298 K, 1 h contact time.

affinities of the metals for the ligand in decreasing order is Cu(II) > Co(II) > Mn(II). The DNA cleavage efficiency of complexes 2–4 also follows the same order. 3.7. Extraction ability of the ligand H2L (1) Oximes, an important class of chelating agents, have found numerous applications as highly selective reagents for the separation and determination of a number of metal ions. The metal binding property of the ligand containing a diimine–dioxime unit was

A new diimine–dioxime ligand (1) and its homo-, heterodinuclear and homotrinuclear copper(II) complexes are reported and characterized by using different spectroscopic techniques. From the elemental analyses data, the complexes were proposed to have the general formulae [Cu(L)(H2O)M(phen)2](ClO4)2 [where M = Cu(II), Mn(II), Co(II)] and [Cu3(L)2(H2O)2](ClO4)2 for the dinuclear and trinuclear complexes, respectively. The molar conductance data reveal that all the metal chelates are 1:2 electrolytes. It is concluded that H2L is coordinated to the metal ions in a tetradentate manner by the diimine and dioxime groups. The ligand complexing properties were studied by liquid–liquid extraction of selected transition metals (Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, Hg2+, Pb2+). It has been observed that the ligand 1 behaves as a good extractant and complexing agent for Cu(II). In addition we have tested the catalytic activity of the complexes toward the disproportionation of hydrogen peroxide. The catalytic results indicated that the complexes have good catalase activity and may be suitable and functional as a model for the pseudocatalase enzyme.

3974

B. Dede et al. / Polyhedron 28 (2009) 3967–3974

The DNA cleavage results showed that the homo- and heterodinuclear copper complexes can effectively cleave supercoiled DNA to form nicked or linear DNA by performing single strand and double strand scissions under aerobic conditions. The complexes cleaved the supercoiled pBR322 DNA into much smaller fragments in the presence of hydrogen peroxide as a co-oxidant. References [1] S. Di Bella, I. Fragala, Synth. Mater. 115 (2000) 191. [2] M. Zhao, C. Zhong, C. Stern, A.G.M. Barrett, B.M. Hoffman, J. Am. Chem. Soc. 127 (2005) 9769. [3] H.C. Wu, P. Thanasekaran, C.H. Tsai, J.Y. Wu, S.M. Huang, Y.S. Wen, K.L. Lu, Inorg. Chem. 45 (2006) 295. [4] F. Karipcin, B. Dede, Y. Caglar, D. Hür, S. Ilican, M. Caglar, Y. Sahin, Opt. Commun. 272 (2007) 131. [5] N.A. Rey, A. Neves, A. Bortoluzzi, C.T. Pich, H. Terenzi, Inorg. Chem. 46 (2007) 348. [6] C. Maxim, T.D. Pasatoiu, V. Ch Kravtsov, S. Shova, C.A. Muryn, R.E.P. Winpenny, F. Tuna, M. Andruh, Inorg. Chim. Acta 361 (2008) 3903. [7] S. Anbu, M. Kandaswamy, P. Suthakaran, V. Murugan, B. Varghese, J. Inorg. Biochem. 103 (2009) 401. [8] M. Roy, B. Pathak, A.K. Patra, E.D. Jemmis, M. Nethaji, A.R. Chakravarthy, Inorg. Chem. 46 (26) (2007) 11122. [9] N. Raman, J.D. Raja, A. Sakthivel, J. Chem. Sci. 119 (2007) 303. [10] R.B. Martin, Y.H. Marian, in: H. Sigel (Ed.), Metal ions in Biological Systems, Marcel Dekker Inc., New York, 1987. pp. 57–124. [11] R.R. Joshi, K.N. Ganesh, Biochem. Biophys. Res. Commun. 182 (1992) 588. [12] S. Ramakrishnan, S.M. Palaniandavar, J. Chem. Soc., Dalton Trans. 29 (2008) 3866. [13] F. Arjmand, M. Aziz, Eur. J. Med. Chem. 44 (2009) 834. [14] N. Saglam, A. Colak, K. Serbest, S. Dülger, S. Güner, S. Karaböcek, A.O. Beldüz, BioMetals 15 (2002) 357. [15] M.E. Katsarou, E.K. Efthimiadou, G. Psomas, A. Karaliota, D. Vourloumis, J. Med. Chem. 51 (2008) 470. [16] J.L. García-Giménez, M. González-Álvarez, M. Liu-González, B. Macías, J. Borrás, G. Alzuet, J. Inorg. Biochem. 103 (2009) 923. [17] R.S. Kumar, S. Arunachalam, Eur. J. Med. Chem. 44 (2009) 1878. [18] J. Hernández-Gil, L. Perelló, R. Ortiz, G. Alzuet, M. González-Álvarez, M. LiuGonzález, Polyhedron 28 (2009) 138. [19] M. Ak, D. Taban, H. Deligöz, J. Hazard. Mater. 154 (2008) 51. [20] S. Abe, T. Sone, K. Fujii, M. Endo, Anal. Chim. Acta 274 (1993) 141. [21] N. Hirayama, J. Taga, S. Oshima, T. Honjo, Anal. Chim. Acta 466 (2002) 295. [22] J.H.P. Tyman, S.J.A. Iddenten, J. Chem. Technol. Biotechnol. 80 (2005) 1319.

[23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55]

R. Gup, E. Girizoglu, Spectrochim. Acta, Part A 65 (2006) 719. U. Ocak, H. Alp, P. Gokce, M. Ocak, Sep. Sci. Technol. 41 (2006) 391. B. Dede, F. Karipcin, M. Cengiz, J. Hazard. Chem. 163 (2009) 1148. L.M. Long, H.R. Henze, J. Am. Chem. Soc. 63 (1944) 1939. J.V. Burakevich, A.M. Lore, G.P. Volpp, J. Org. Chem. 36 (1971) 1. I. Karatas, H.I. Ucan, Synth. React. Inorg. Met. Org. Chem. 28 (1998) 383. P. Ninfali, T. Orsenigo, L. Barociani, S. Rapa, Prep. Biochem. 20 (1990) 297. C.J. Pedersen, Fed. Am. Soc. Exp. Biol. 27 (1968) 1305. S.Y. Uçan, B. Mercimek, Synth. React. Inorg. Met. Org. Chem. 35 (2005) 197. M.J. Prushan, A.W. Addison, R.J. Butcher, L.K. Thompson, Inorg. Chim. Acta 358 (2005) 3449. D. Steinborn, M. Rausch, C. Bruhn, J. Organomet. Chem. 561 (1998) 191. K. Serbest, S. Karabocek, I. Degirmencioglu, S. Guner, F. Kormali, Transition Met. Chem. 26 (2001) 375. I. Yılmaz, Transition Met. Chem. 33 (2008) 259. J.R. Ferraro, Low Frequency Vibrations of Inorganic and Coordination Compounds, Plenum Press, New York, 1971. M.A. David, Metal-Ligand and Related Vibrations, Adward Arnold Ltd., London, 1967. B.J. Hathaway, A.E. Underhill, J. Chem. Soc. (1961) 3091. K. Nakamoto, Infrared and Raman Spectra of Inorganic and Coordination Compounds, Wiley, New York, 1986. M.R. Rosenthall, J. Michael, J. Chem. Educ. 50 (1973) 331. W.J. Geary, Coord. Chem. Rev. 7 (1971) 81. F. Akagi, Y. Michihiro, Y. Nakao, K. Matsumoto, T. Sato, W. Mori, Inorg. Chim. Acta 357 (2004) 684. E.J. Larson, V.L. Pecoraro, in: V.L. Pecoraro (Ed.), Manganese Redox Enzymes, Wiley-VCH, New York, 1992. M. McCann, M.T. Casey, M. Devereux, M. Curran, V. McKee, Polyhedron 16 (1997) 2741. M. McCann, M.T. Casey, M. Devereux, M. Curran, G. Ferguson, Polyhedron 16 (1997) 2547. Q.L. Zhang, J.G. Liu, H. Chao, G.Q. Xue, L.N. Ji, J. Inorg. Biochem. 83 (2001) 49. Q.L. Zhang, J.G. Liu, J.Z. Liu, H. Li, Y. Yang, H. Xu, H. Chao, L.N. Ji, J. Inorg. Chim. Acta 339 (2002) 34. X.-L. Wang, H. Chao, H. Li, X.-L. Hong, Y.-J. Liu, L.-F. Tan, L.-N. Ji, J. Inorg. Biochem. 98 (2004) 1143. W. Qian, F. Gu, L. Gao, S. Feng, D. Yan, D. Liao, P. Cheng, J. Chem. Soc., Dalton Trans. (2007) 1060. D.-Y. Kong, Y.-Y. Xie, Polyhedron 19 (2000) 1527. L.-P. Lu, M.-L. Zhu, P. Yang, J. Inorg. Biochem. 95 (2003) 31. M.S.S. Babu, K.H. Reddy, P.G. Krishna, Polyhedron 26 (2007) 572. C. Liu, M. Wang, T. Zhang, H. Sun, Coord. Chem. Rev. 248 (2004) 147. _ Pekacar, M.A. Özler, M. Ersöz, Sep. Sci. Technol. 34 (1999) 3297. H. Deligöz, A.I. B. Dede, F. Karipcin, M. Cengiz, J. Chem. Sci. 121 (2009) 163.