Synthesis and Reactivity in Inorganic, Metal-Organic, and Nano-Metal Chemistry, 42:944–950, 2012 C Taylor & Francis Group, LLC Copyright ISSN: 1553-3174 print / 1553-3182 online DOI: 10.1080/15533174.2011.652274
Synthesis, Characterization, Electrochemical Behavior, and Biological Studies of 1-p-Diphenylmethane-2-hydroxyimino2-(arylamino)-1-ethanone Copper(II) and Ruthenium(III) Complexes Fatma Karipcin,1 Sabriye Percin-Ozkorucuklu,2 Ismail Ozmen,2 and Gulsen Baskale-Akdogan2 1
Department of Chemistry, Sciences and Arts Faculty, Nevs¸ehir University, Nevs¸ehir, Turkey Department of Chemistry, Sciences and Arts Faculty, S¨uleyman Demirel University, Isparta, Turkey
2
New copper and ruthenium mononuclear complexes of the type [ML2 (H2 O)X] [X = H2 O for M = Cu(II) and X = Cl for M = Ru(III)] have been prepared from 1-p-diphenylmethane-2-hydroxyimino-2-(4-chloroanilino)-1-ethanone (HL1) and 1-p-diphenylmethane-2-hydroxyimino-2-(4-toluidino)-1-ethanone (HL2). The complexes were characterized by elemental analyses, magnetic susceptibility, molar conductance, IR, thermal analysis, and cyclic voltammetry. Stoichiometric and spectral results of the metal complexes indicated that the metal:ligand ratios in the complexes were found to be 1:2 and the ligands behave as a bidentate ligand forming neutral metal chelates through the carbonyl and oxime oxygen. The electrochemical behavior of the ligands and their complexes were obtained by cyclic voltammetry. The interaction between these complexes with DNA has also been investigated by agarose gel electrophoresis. The copper(II) complexes (3 and 4) with H2 O2 as a co-oxidant exhibited strongest cleaving activity. Moreover, catalytic activities of the complexes for the disproportionation of hydrogen peroxide were also investigated in the presence of imidazole. Keywords
catalase-like, copper(II) complexes, DNA cleavage, electrochemistry, oxime, ruthenium(III) complexes
INTRODUCTION Oxime ligands and their complexes have received considerable attention because of their great potential in a number of applications in industry, medicine, detection, and determination of various metal ions. Apart from their relevance in various application fields, coordination chemistry of the oxime ligands
Received 13 June 2011; accepted 1 November 2011. This work was financially supported by the Scientific and Technical Research Council of Turkey (TUBITAK), project no. TBAGHD/223 (106T723) (Ankara, Turkey) and the Research Fund of S¨uleyman Demirel University, project no. 1270-YL-04 (Isparta, Turkey). Address correspondence to Fatma Karipcin, Department of Chemistry, Sciences and Arts Faculty, Nevs¸ehir University, Nevs¸ehir, Turkey. E-mail:
[email protected]
is also interesting with regard to their variable binding modes. The oximes usually display two modes of binding to metal ions, through the imine nitrogen and through the oxime oxygen. Therefore, oximes and their coordination compounds have been studied extensively.[1–8] Ruthenium is well known for its ability to cover a wide range of oxidation states (–2 to +8) and to adopt various coordination geometries. The chemistry of ruthenium has been receiving considerable current interest, because of the fascinating photochemical, photophysical, and redox properties exhibited by complexes of this metal. These complexes can be used in molecular photoelectronic devices, as well as electron and energy carriers, photosensitizers in catalytic systems, and luminescent probes in biochemistry. As properties are dependent mostly on the coordination environment around the metal center, complexation of ruthenium by ligands of selected types is of significant importance. Because of such a broad range of applications, there is a continuous interest to synthesize new complexes of ruthenium with different types of ligands.[9–16] Copper(II) complexes are also known to play a significant role either in naturally occurring biological systems such as transport, storage and activation of molecular oxygen or as pharmacological agents.[17–19] Copper is an essential micronutrient for feeding and a cofactor of several enzymes involved in oxidative metabolism: β-hydroxylases, quercetinase, ceruloplasmine, cytochromoxidase, monoaminoxidase, superoxydismutase, and ascorbic acid oxidase and tyrosinase. The catalytic role of these enzymes is the result of two processes: (a) the reduction of the Cu2+ cation to Cu+ and (b) the fixation of the molecular oxygen.[20,21] Therefore, structural and electronic properties of the copper(II) complexes that mimic biological systems are of interest. We have previously reported on investigations of the metal complexes of the ligand 1-p-diphenylmethane-2-hydroxyimino2-(naphthylamino)-1-ethanone.[22] The complexes have been characterized by various physicochemical methods. In the present paper we describe the synthesis, characterization, and
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CU(II), RU(III) OF A NEW SCHIFF BASE
thermal studies of new mononuclear Ru(III) and Cu(II) complexes with the ligands derived from the condensation of 1-pdiphenylmethane-2-hydroxyimino-2-chloro-1-ethanon with 4toluidine and 4-chloroaniline. Furthermore, catalase-like activity and the interaction with plasmid DNA (pBR322 DNA) employing gel electrophoresis of the compounds are also investigated. Biological and catalytic activities may be related to the redox properties of complexes. Moreover, some metals are toxic in elevated concentration so many investigations have attempted to develop sensors to determine their presence with high selectivity. The possible biomimetic activity and the potential application as sensors for metal complexes can be evaluated from the electrochemical behavior. We have also studied the electrochemical behavior of complexes by cyclic voltammetry, because their applications as biomimetic complexes and as copper and ruthenium sensors are related to the redox properties. EXPERIMENTAL Materials The oxime ligands 1-p-diphenylmethane-2-hydroxyimino-2(4-chloroanilino)-1-ethanone (1) and 1-p-diphenylmethane-2hydroxyimino-2-(4-toluidino)-1-ethanone (2) were synthesized in our laboratory according to the procedure described previously.[23] All solvents, 4-chloroaniline, 4-toluidine, and metal salts [RuCl3 ·H2 O, Cu(CH3 COO)2 ·H2 O] and other chemicals used for the synthesis and physical measurements were purchased from Aldrich (Germany), J. T. Baker (Holland), and Merck (Germany) and used as received. Physical Measurements Microanalyses were carried out using a LECO 932 CHNS analyzer (USA). Copper and ruthenium were analyzed on a Perkin-Elmer Optima 5300 DV ICP-OES spectrometer (USA). IR spectra (KBr pellets, 4000–400 cm–1) were measured on a Shimadzu IRPrestige-21 FT-IR spectrophotometer (Japan). Magnetic susceptibility measurements were carried out at room temperature on a Sherwood Scientific Magnetic Susceptibility Balance (Model MX1, England). Conductivity measurements were carried out using 10–3 M freshly prepared dimethylformamide (DMF) solutions on Optic Ivymen System conductivity meter (Spain). Melting point determinations were performed with a digital melting point instrument (Electrothermal model IA 9100, UK). The thermogravimetric analyses (TG and DTG) of the complexes were measured on a Perkin-Elmer Diamond TGA thermal analyzer (USA). The experiments were carried out in dynamic nitrogen atmosphere (20 mL min–1) with a heating rate of 10◦ C min–1 in the temperature range 20–1000◦ C. Preparation of Complexes Synthesis of Mononuclear Cu(II) Complexes For [Cu(Ln)2 (H2 O)2 ] (3 and 4), the mononuclear Cu(II) complexes were prepared as reported in similar literature.[22,24] A stoichiometric amount of Cu(CH3 COO)2· H2 O (0.20 g, 1 mmol) in EtOH was added to a hot solution of the bidentate ligand
945
(2 mmol, 0.72 g HL1, and 0.68 g HL2) in absolute EtOH, and the reaction mixture was boiled under reflux with stirring for 2 h. On slow evaporation of EtOH the desired complex was obtained as powder. The precipitate was separated by filtration, washed with cold EtOH followed by Et2 O, and finally dried over P2 O5 . For [Cu(L1)2 (H2 O)2 ] (3), brown complex; yield: 42%; m.p.: 190◦ C. Anal. Calcd. for [C42 H36 N4 O6 Cl2 Cu]: C, 60.98; H, 4.38; N, 6.77; Cu, 7.68. Found: C, 60.64; H, 3.98; N, 6.54; Cu, 7.40%. M (DMF solution, ohm–1 cm2 mol–1): 16.0; µeff = 1.56 B.M.; FTIR (KBr, cm–1): 3369 b (NH), 3448 b (OH), 1670 w (C O), 1600 m (C N), 1415 m (C-N), 1219 m (NO), 503 w (Cu-O). For [Cu(L2)2 (H2 O)2 ] (4), light brown complex; yield: 61%; m.p.: 202◦ C. Anal. Calcd. for [C44 H42 N4 O6 Cu]: C, 67.21; H, 5.38; N, 7.13; Cu, 8.08. Found: C, 67.09; H, 5.02; N, 6.83; Cu, 7.86%. M (DMF solution, ohm–1 cm2 mol–1): 28.0; µeff = 1.32 B.M.; FTIR (KBr, cm–1): 3336 b (NH), 3448 b (OH), 1655 w (C O), 1601 m (C N), 1419 m (C-N), 1266 w (NO), 513 w (Cu-O). Synthesis of Mononuclear Ru(III) Complexes For [Ru(Ln)2 Cl(H2 O)] (5 and 6), the Ru(III) complexes were prepared as reported in similar literature.[16,18] In all cases, analytical (C, H, N) and thermogravimetric data (H2 O) for the isolated compounds fit well with the reported formulas. A stoichiometric amount of RuCl3· H2 O (1 mmol, 0.22 g) in EtOH was added to a hot solution of the desired ligand (2 mmol, 0.72 g HL1, and 0.68 g HL2) in absolute EtOH, and the reaction mixture was boiled under reflux with stirring for 2 h. On slow evaporation of EtOH the desired complex was obtained as powder. The deeply colored precipitates were separated by filtration, washed with cold EtOH followed by Et2 O, and dried over P2 O5 . For [Ru(L1)2 Cl(H2 O)] (5), black complex; yield: 31%; m.p.: 210◦ C. Anal. Calcd. for [C42 H34 N4 O5 Cl3 Ru]: C, 57.18; H, 3.88; N, 6.35; Ru, 11.46. Found: C, 56.98; H, 3.87; N, 5.93; Ru, 10.99%; M (DMF solution, ohm–1 cm2 mol–1): 33.0; µeff = 1.38 B.M.; FTIR (KBr, cm–1): 3320 b (NH), 3441 b (OH), 1601 s (C N), 1413 w (C-N), 1295 w (NO), 581 w (Ru-O). For [Ru(L2)2 Cl(H2 O)] (6), black complex; yield: 38%; m.p.: 215◦ C. Anal. Calcd. for [C44 H40 N4 O5 ClRu]: C, 62.82; H, 4.79; N, 6.66; Ru, 12.01. Found: C, 62.38; H, 4.51; N, 6.43; Ru, 11.59%. M (DMF solution, ohm–1 cm2 mol–1): 26.0; µeff = 1.63 B.M.; FTIR (KBr, cm–1): 3320 b (NH), 3438 b (OH), 1601 s (C N), 1419 m (C-N), 1276 w (NO), 581 w (Ru-O). Cyclic Voltammetry Measurements Cyclic voltammograms were run on Autolab PGSTAT 302N Potentiostat/Galvanostat controlled by GPES 4.9 version software (Ecochemic, the Netherlands). The three-electrode measurements were carried out with a Pt wire as an auxiliary electrode, a platinum (Pt) electrode of 2 mm diameters as a working electrode, and an Ag/AgCl reference electrode. Catalase Activity The catalytic activity of the ligands (1 and 2) and their complexes (3 and 4) with hemolysate toward the disproportionation
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F. KARIPCIN ET AL.
of hydrogen peroxide was investigated by measuring the pressure of evolved oxygen during the course of the reaction. Blood collected from bovine with EDTA was centrifuged (15 min, 2500 g). Preparation of the hemolysate was done as described by Ninfali.[25] In a typical experiment, the hemolysate (20 µL) was added to 2900 µL of sodium phosphate buffer saline (PBS, 0.01 M, pH 7.4) and 100 µL for per complex and 100 µL of an aqueous solution of H2 O2 (9.7 M H2 O2 ) was added to start the reaction. The pressure of the evolved dioxygen following the reduction of hydrogen peroxide was monitored manometrically at 1 min time intervals. In cases where imidazole (100 µL) was added this was introduced into the reaction vessel before the addition of H2 O2 (in the absence of the imidazole the complexes were either inactive or very weak catalysts for this reaction). Cleavage of pBR322 DNA For the agarose gel electrophoresis experiments, 0.5 µg/µL supercoiled pBR322 DNA (0.5 µl) was treated with 1 µL of 1 mM the tested ligands and their complexes in DMF and 2 µL of 0.1M Tris–HCl (pH 8.0) buffer in the absence and presence of 2 µL of 5.0 mM hydrogen peroxide as a co-oxidant reagent. After incubation at 37◦ C for 2 h, 1 µL of loading buffer (0.25% bromophenol blue, 0.25% xylene cyanol, 30% glycerol in H2 O) 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 L–1 EDTA). Gels were stained with ethidium bromide (1 mg mL–1) 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 DNR Minibis Pro 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. RESULTS AND DISCUSSION The condensation of 1-p-diphenylmethane-2-hydroxyimino2-chloro-1-ethanon with 4-toluidine or 4-chloroaniline gave the ligands.[23] Characterization of Metal Complexes The complexes are insoluble in water and common organic solvents but show maximum solubility in DMF and DMSO at room temperature. The complexes are thermally stable at least up to about 200◦ C. Attempts to isolate crystals suitable for X-ray diffraction were unsuccessful. Therefore, elemental analysis, spectroscopic techniques, conductivity, and thermal and magnetic susceptibility techniques were employed in order to determine the structural characteristics of the complexes. The analytical data of the complexes indicate 1:2 metal-ligand stoichiometry (Figure 1). The values of the molar conductance
R1
C
X2
O
N C
M
C R
O
N
O
O
C
X1
R:
R1 :
R1
-NH
Cl
for 1
-NH
CH3
for 2
CH2
R
for 1 and 2
FIG. 1. The mononuclear Cu(II) (3 and 4) and Ru(III) (5 and 6) complexes of the ligands (1 and 2). M = Cu(II), Ru(III); X1 = X2 = H2 O in Cu(II) complexes. X1 = H2 O, X2 = Cl in Ru(III) complexes.
in DMF in 10–3 M solutions are in the range 16–33 ohm–1 cm2 mol–1, suggesting a nonelectrolytic nature for these complexes. IR Spectra The IR spectra of the free ligands and their complexes exhibited various bands in the 400–4000 cm–1 region. The IR spectra of the ligands showed medium-to-strong bands in the 3241–3242 cm–1 range, assignable to ν(OH) of oxime groups; the broad nature of these bands and their low wavenumbers suggested the presence of hydrogen bonding.[27] The spectrum of the ligands exhibited fairly strong bands at 3370 (1) and 3389 (2) cm–1 attributable to the ν(NH) vibration of the aromatic amine groups. The medium sharp band at 1242–1246 cm–1 was due to the N–O stretching vibrations.[1,2] In the IR spectra of the ligands, the stretching vibrations of C N were observed at 1601 cm−1. The C O vibrations for the ligands were observed as medium bands at 1675–1680 cm–1.[1,2,24] The characteristic absorption bands of the ligands were shifted on complex formation, and new vibrational bands characteristic of the complex appeared. The broad stretching bands due to the -OH groups of the free ligands did not appear in the IR spectra of the complexes indicating the deprotonation of the -OH groups and the formation of M–O bonds. This was supported by the appearance of a new band in the region 503–581 cm−1 assigned to ν(M–O).[1,2,24] All of the complexes showed strong and broad bands in the 3438–3448 cm–1 region. These bands are attributed as asymmetric and symmetric stretching modes of water and their center of gravity near 3400 cm–1 implies coordination of water molecules to the metal ions.[27] In the complexes, C O group bands became extinct, indicating coordination through the C O group. Vibrational evidence for the coordination of the oximato group in the complexes is provided by the higher frequency band of N–O at ca. 1219–1295 cm–1.[3] Thus, the IR spectra of the ligands and their metal complexes give strong evidence for the complexation of the ligands.
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CU(II), RU(III) OF A NEW SCHIFF BASE
Magnetic Studies The room temperature magnetic moments of the complexes showed that all of the complexes are paramagnetic. The measured magnetic moments of the mononuclear ruthenium(III) complexes are 1.38 and 1.63 µB for (5) and (6), respectively. Magnetic susceptibility measurements showed that these complexes are one-electron paramagnetics, which corresponds to the +3 oxidation state of ruthenium (low spin d5, S = 1/2) in these complexes.[1,2] The magnetic moments of the mononuclear copper(II) complexes (3) and (4) are 1.32–1.56 µB. This values are in agreement with a spin value of 1.73 µB to Cu(II) complexes.[28] Electrochemistry Cyclic voltammetric studies were performed for all the complexes at a Pt working electrode. The supporting electrode used was 0.1 M tetrabutylammonium perchlorate (TBAP) in DMF solution. The concentration of the complexes was 1.10–3 M. The solutions were degassed with a continuous flow of N2 gas before scanning. As the ligands used in this work are not reversibly reduced in the applied potential range (Figure 2), we believe that reduction processes observed for these complexes are metal centered only. The cyclic voltammograms of four complexes exhibit a reversible reduction peak and a reversible oxidation peak at a scan rate of 50 mVs–1. All the complexes showed only a reversible reduction wave in the –0.5 to –1.5 V range. The reduction of each complexes are characterized by well-defined waves with Ef values in the range –0.36 to –0.96 V. Complex 3 (Epc = –0.45 V) reduces at less negative potential compared with complex 4 (Epc = –0.63 V). Similarly, complex 5 (Epc = –0.96 V) reduces at less negative potential compared with complex 6 (Epc = -1.07 V). This may be due to distortion in geometry that arises due to axial coordination of the anions to the copper and ruthenium ion.[29] Ef values for complexes 3 and 4 are –0.36 and –0.55, respectively. For ruthenium complexes also the change in redox potential is observed with change in geometry. Ef values for complexes 5 and 6 are –0.87 and –0.96, respectively. 0.03x10-4 0
i/A
-0.03x10
(a)
-4
-0.05x10-4 -0.08x10-4
TABLE 1 Cyclic voltammetric dataa of copper(II) and ruthenium(III) complexes Complex 3 4 5 6
Epc (V)
Epa (V)
Ef (V)
Ep (mV)
–0.45 –0.63 –0.96 –1.07
–0.28 –0.47 –0.79 –0.85
–0.36 –0.55 –0.87 –0.96
170 160 170 220
a Working electrode, platin electrode; reference electrode, silver/silver chloride electrode; Supporting electrolyte, TBAP (0.10 M); Scan rate, 50 mVs–1; Ef = 0.5 (Epa + Epc ); Ep = (Epa – Epc ), where Epa and Epc are anodic and cathodic potentials, respectively.
The peak-to peak separation (Ep value) ranging from 160 to 220 mV reveals that is process is quasireversible.[30] The voltammetric data are given in Table 1. Thermal Studies In thermogravimetry (TGA) the change in weight of a complexTable 2 was recorded as a function of temperature during heating. The TGA curve was also supported by the derivative thermogravimetry (DTG) curves. All the complexes show a gradual mass loss indicating decomposition by fragmentation with an increase in temperature. For the Ru(III) and Co(II) complexes the mass loss starts at 160–180◦ C temperature range and there is a sharp inflexion between 160 and 230◦ C indicating the presence of water molecules that are coordinated to the metal ion. Above 230◦ C the complexes decompose in a gradual manner rather than with the observed sharp decomposition, which may be due to fragmentation and thermal degradation of the organic moiety. In the Ru(III) complexes, second decomposition step may be attributed to the liberation of Cl atom and amine and diphenylmethane groups of the ligand molecules.[22,23] Ruthenium complexes show residues not conforming to the corresponding a metallic residue such as metal oxides or metallic ruthenium, even at 1000◦ C, indicating that the decomposition of the organic moiety remains incomplete even at this temperature. In the case of the Cu(II) complexes, above 230◦ C the complexes show loss of mass in a gradual manner. The continuous loss of weight is observed up to 910–935◦ C; after that the weight of the product of the complexes remains constant. The final weight losses in these cases agree with the formation of the respective metallic copper. The presence of coordinated water molecules in the metal complexes further corroborates the assumption made on the basis of the infrared spectral studies.
(b)
-0.10x10-4 -0.13x10-4 -1.10
-0.85
-0.60
-0.35
-0.10
E/ V
FIG. 2. Cyclic voltammograms of the ligand 1 (a) and ligand 2 (b) (color figure available online).
Catalase-Like Activity The effects of the ligands (1, 2) and their complexes (3–6) to catalase activity of hemolysate to disproportionate H2 O2 into H2 O and O2 was examined in N,N-dimethylformamide at ambient temperature. The catalytic activity of hemolysate was investigated in the presence and absence of the base imidazole and the compounds by measuring the pressure of evolved
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F. KARIPCIN ET AL.
TABLE 2 Time course of oxygen evolution in molecules of H2 O2 disproportionated by the compounds 1–6 and the hemolysate with added imidazole (100 µL) at ambient temperature Time (min)
1
2
3
4
5
6
1 Last (max)
423 2041
423 2831
1098 3987
1002 4102
693 3139
578 3293
sition of H2 O2 is enhanced in the presence of a heterocyclic base such as imidazole because of its strong π -donating ability.[31] On the other hand heterocyclic bases themselves cause only a very slight disproportionation of the peroxide. As a result of catalase-like activity studies, the present copper(II) complexes 3 and 4 have found that a high disproportionation efficiency and show very similar catalase activity, possibly indicating that a similar active species is formed.
oxygen during the course of the reaction. Because none of the compounds exhibited catalytic activity on its own. All the complexes display catalytic ability for the disproportionation of H2 O2 in the presence of imidazole but the activity of copper(II) complexes (3, 4) are relatively higher than the other complexes. Table 2 shows the rate of evolution of oxygen from the respective reactions for the compounds with the hemolysate first minute and last minute. All the compounds showed activity for the catalytic decomposition of H2 O2 in the presence of imidazole. Examination of Table 2 shows that over the first minute of complex 3 appears to be the most efficient catalyst, with 1,098 molecules of peroxide disproportionate by one molecule of the complex. The ligands appear to be the least efficient catalyst over the first minute with one molecule of the compound with the hemolysate the knocking down just 423 molecules of the peroxide. But, comparison of the total number of molecules of H2 O2 disproportionate by one molecule of compounds with the hemolysate shows that complex 4 is the most effective catalyst with 4102 molecules. The time course of the O2 evolution is shown in Figure 3. The H2 O2 disproportionate efficiency of the complexes in the presence imidazole according to the total number of molecules of H2 O2 disproportionate by one molecule follows the order 4 > 3 > 6 > 5 > 2 > 1. The ligands (1, 2) and the ruthenium(III) complexes (5, 6) are less effective for the catalytic decomposition of when compared with the copper(II) complexes (3, 4). In the absence of heterocyclic base, the complexes decompose hydrogen peroxide slowly but the decompo-
DNA Cleavage Activity The cleavage of supercoiled form of pBR322 DNA with the ligands (1 and 2), their copper(II) (3 and 4) and ruthenium(III) (5 and 6) complexes was studied in the absence or presence of H2 O2 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) in aerobic condition. When circular plasmid DNA is subjected to electrophoresis, 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.[32,33] The results of the gel electrophoresis separations of plasmid pBR322 DNA by the ligands (1 and 2) and their complexes (3–6) in the absence or presence of H2 O2 are depicted in the Figure 4. Control experiments were applied using only DNA and DNA+H2 O2 . As shown in Figure 4, incubation of the pBR322 DNA at 37◦ C for 2 h with 1 µg of the compounds cause the conversion of Form I to Form II and Form III. The supercoiled (Form I) DNA was cleaved to Form II and Form III in the absence of H2 O2 of the ligands (Lanes 3–4) and all the complexes (Lanes 5–8). The cleavage efficiency after incubation for 2 h in the absence of H2 O2 , follows the order 4 > 3 > 6> 5 > 1> 2. The cleavage percentages are listed in Table 3. These results indicate that the examined complexes induces very similar conformational changes on supercoiled DNA as conversion of supercoiled form to nicked form than linear form
250
p(bar)
200
H+P H+P+ m
150
1 2
100
3 4
50
5 6
0 0
5
10
15
20
25
30
Time (min)
FIG. 3. Time courses of dioxygen evolution in the disproportionation of H2 O2 by hemolysate compounds (1–6) mixture in DMF. [hemolysate] = 40 µL, [H2 O2 ] = 9.7 M, [compound] = 1 mM, [PBS] = 0.01 M, pH = 7.4, ambient temperature, H = hemolysate; P = H2 O2 ; im = imidazole (color figure available online).
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CU(II), RU(III) OF A NEW SCHIFF BASE
TABLE 3 DNA cleavage data of pBR322 plasmid DNA (0.1 µg) by 1–6 Lane no 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Reaction conditions
Ink. Time (hour)
Form I %SC
Form II %NC
Form III %LC
Form III %SF
DNA DNA + H2 O2 DNA + 1 DNA + 2 DNA + 3 DNA + 4 DNA + 5 DNA + 6 DNA + 1+ H2 O2 DNA + 2+ H2 O2 DNA + 3+ H2 O2 DNA + 4+ H2 O2 DNA + 5+ H2 O2 DNA + 6+ H2 O2
2 2 2 2 2 2 2 2 2 2 2 2 2 2
91.9 86.9 75.9 77.4 64.7 65.5 65.4 68.4 67.8 65.2 ND ND 61.9 59.0
9.1 13.1 9.6 12.1 13.9 11.3 16.1 11.2 10.1 12.6 7.8 10.3 11.3 9.8
ND ND 14.5 10.5 21.4 23.2 18.5 20.4 22.1 22.2 74.3 73.5 26.9 31.2
ND ND ND ND ND ND ND ND ND ND 17.9 16.2 ND ND
SC = supercoiled; NC = nicked circular; LC = linked circular; SF = smaller fragment; ND = not detected.
in a sequential manner. But (6) and (5) are less effective than complexes (4) and (3). On the other hand, the pBR322 DNA treated with the ligands (1 and 2) showed less changes in the form levels compared with the complexes. Namely, the ligands alone are less effective. The different DNA cleavage efficiency of the ligands and the complexes may be due to the different binding affinity of the complexes to DNA.[34,35] The degradation of pBR322 DNA is also dependent on cooxidant used. The cleavage mechanism of pBR322 DNA induced by the compounds (1–6) was investigated (Figure 3) and clarified in the presence of H2 O2 as a cooxidant (Lanes 9–14). In the ligands, the intensity of the circular supercoiled DNA (Form I) band was found decrease, while that of nicked (Form II) and linear DNA (Form III) bands increase apparently (Lane 9 and 10) in the presence of H2 O2 . The copper(II) complexes (3 and 4), the cleavage is found to be much more efficient, they cleaved the supercoiled pBR322 DNA into Form II, Form III, and much smaller fragments and the circular supercoiled DNA (Form I) bands were disappeared completely (Lanes 11 and 12). But the activities of the ruthenium(III) complexes (5 and 6) were weaker than the copper(II) complexes. It could not cleave
FIG. 4. Gel electrophoresis diagram showing the cleavage data of pBR322 plasmid DNA (0.1 µg) by the ligands and their 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 + H2 O2 ; lanes 3–8, pBR322 plasmid DNA + the compounds; lanes 9–14, pBR322 plasmid DNA + the compounds + H2 O2 (the compounds = 1, 2, 3, 4, 5, and 6, respectively).
all linear DNA into smaller fragments. These observations suggest that the complexes mediated cleavage reaction proceed via oxidative pathway mechanism and imply that the singlet oxygen playing a role in the cleavage chemistry. In the presence of H2 O2 all the complexes (3–6) are remarkably degrading the pBR322 DNA by oxidative (O2 -dependent pathway) cleavage mechanism using the singlet oxygen as the reactive species.[33,35] These results are similar to that observed for some Cu(II) and Ru(III) complexes as chemical nuclease.[33,36–39] CONCLUSION The oxime ligands 1-p-diphenylmethane-2-hydroxyimino-2(4-chloroanilino)-1-ethanone (1) and 1-p-diphenylmethane-2hydroxyimino-2-(4-toluidino)-1-ethanone (2) and their Cu(II) and Ru(III) complexes were synthesized and characterized by elemental, thermal analyses, ICP-OES, magnetic susceptibility, conductivity measurements, FT-IR, and cyclic voltammetry. The magnetic measurements, infrared and thermal data provided evidence for the structures of the isolated complexes. Cyclic voltammetry studies evidenced one quasireversible reduction wave for all the complexes in the cathodic region. In addition we have tested the DNA cleavage activity of the ligand and the complexes. The DNA cleavage results showed that the copper and ruthenium complexes can effectively cleave supercoiled DNA to form nicked or linear DNA by performing single strand and double strand scissions under aerobic conditions. The copper complexes cleaved the supercoiled pBR322 DNA into much smaller fragments in the presence of hydrogen peroxide as co-oxidant. In addition we have tested the catalytic activity of the complexes toward the disproportionation of hydrogen peroxide. The catalytic results indicated that the activity of copper(II) complexes (3 and 4) is relatively higher than the other complexes.
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