Alkyl chain length effect on construction of copper(II) complexes with tridentate Schiff base ligand and DNA interaction

Abstract Two tridentate Schiff base ligands were synthesized by condensation of equimolar amounts of benzoylacetone and 2-amino-1-ethanol or 3-amino-1-propanol, H2L1 and H2L2, respectively. The reaction of the Schiff base ligands with Cu(CH3COO)2 in methanol leads to (CuL1)4, 1 and (CuL2)2, 2. In the tetranuclear cubane species, the tridentate H2L has both a chelating and a bridging mode, after double deprotonation of the enolic OH groups. The copper(II) centers are five-coordinate with a NO4 donor set from the ligands. The coordination geometry around each copper ion is essentially square pyramidal with one nitrogen and two oxygens from one ligand and two oxygens of adjacent ligands from the next unit of the cubane. In dinuclear 2, H2L2 has chelating and bridging modes after double deprotonation of the enolic OH groups. The dianionic form of the Schiff base coordinates forming a six-membered chelate ring with Cu(II). Two such monomeric CuL2 entities are eventually linked through the alkoxo bridges to produce dinuclear 2. The absorption spectra strongly suggest that 2 interacts with CT-DNA. Both 1 and 2 appear to be more efficient than the parent compound in DNA cleavage.


ABSTRACT
Two tridentate Schiff base ligands were synthesized by condensation of equimolar amounts of benzoylacetone and 2-amino-1-ethanol or 3-amino-1-propanol, H 2 L 1 and H 2 L 2 , respectively. The reaction of the Schiff base ligands with Cu(CH 3 COO) 2 in methanol leads to (CuL 1 ) 4 , 1 and (CuL 2 ) 2 , 2. In the tetranuclear cubane species, the tridentate H 2 L has both a chelating and a bridging mode, after double deprotonation of the enolic OH groups. The copper(II) centers are five-coordinate with a NO 4 donor set from the ligands. The coordination geometry around each copper ion is essentially square pyramidal with one nitrogen and two oxygens from one ligand and two oxygens of adjacent ligands from the next unit of the cubane. In dinuclear 2, H 2 L 2 has chelating and bridging modes after double deprotonation of the enolic OH groups. The dianionic form of the Schiff base coordinates forming a six-membered chelate ring with Cu(II). Two such monomeric CuL 2 entities are eventually linked through the alkoxo bridges to produce dinuclear 2. The absorption spectra strongly suggest that 2 interacts with CT-DNA. Both 1 and 2 appear to be more efficient than the parent compound in DNA cleavage.
Herein, we report the synthesis, spectroscopic characterization, structural aspects, and DNA interaction of one tetranuclear cubane (CuL 1 ) 4 and one dinuclear (CuL 2 ) 2 Schiff base complex. Two Schiff base ligands, H 2 L 1 and H 2 L 2 (with ONO donors), were obtained from 1 : 1 condensation of benzoylacetone and 2-amino-1-ethanol or 3-amino-1-propanol, respectively (scheme 1). The coordination sphere around copper(II) can be modified easily and leads to a number of Cu(II) complexes with different nuclearity.
The syntheses of 1 and 2 were previously reported by the template method and their magnetic properties were studied. Molecular weight measurements in chloroform on two different structures for these complexes have been suggested [30]. However, the synthesis of the free Schiff base ligands and crystallographic data of the complexes have not been reported.

Reagents
All chemicals were used as supplied by Merck and Fluka without purification. Supercoiled pBR322 DNA was obtained from Sigma-Aldrich and stored at 4 °C. The tris(hydroxymethyl)aminomethane-HCl (Tris-HCl) buffer was prepared in doubly distilled water.

Physical measurements
Infrared spectra were taken with an equinox 55 Bruker FT-IR spectrometer using KBr pellets from 400-4000 cm −1 . Absorption spectra of the ligands were determined in methanol and for 1 and 2 in DMF using a GBC UV-visible Cintra 101 spectrophotometer with 1 cm quartz cells, from 200-800 nm at 25 °C. elemental analyses (C, H and N) were performed using a CHNS-O 2400II PeRKIN-eLMeR elemental analyzer.

DNA binding
Interaction of the complexes with calf thymus CT-DNA was studied in Tris-HCl buffer (5.5 mM Tris-HCl, pH 7.2) containing 50 mM NaCl at room temperature. The solution was kept for over 24 h at 4 °C. The resulting, somewhat viscous, solution was clear and particle-free. CT-DNA in the buffer medium gave a ratio of UV absorbance at 260 and 280 nm of 1.8 : 1, indicating that the DNA was sufficiently free of protein [31]. The DNA concentration was measured from its absorption intensity at 260 nm using the molar absorption coefficient (ε) value of 6600 M −1 cm −1 as reported [32]. Stock solutions of complexes were freshly prepared by dissolving the complexes in DMF and diluting them with the buffer. The amount of DMF was kept at 10% (by volume) for each set of experiments and has no effect on the experimental Scheme 1. synthesis of the copper(ii) complexes.
results. Absorption spectral titrations were performed while maintaining a constant complex concentration and varying the nucleic acid concentration. This was achieved by dissolving an appropriate amount of the complexes (40 μM) and DNA stock solutions (0-54 μM) while maintaining the total volume constant (1 mL). Spectral bands were recorded after successive addition of CT-DNA. The Tris-HCl buffer was used as a blank to make preliminary adjustments. The intrinsic binding constant (K b ) of the complex to CT-DNA was determined from the spectral titration data using the following equation [33]: where [DNA] is the concentration of CT-DNA in base pairs and the apparent absorption coefficients ε a , ε f and ε b correspond to A obs /[DNA], the absorbance for the free-Cu(II) complex (unbound), and the absorbance for the fully bound complex, respectively. A plot of [DNA]/(ε a −ε f ) versus [DNA] gave a slope 1/(ε b −ε f ) and a intercept 1/K b (ε b −ε f ), so the value of K b can be determined from the ratio of the slope to the intercept.

DNA cleavage
Cleavage of plasmid DNA was monitored using agarose gel electrophoresis following the literature method [34][35][36]. Supercoiled pBR322 DNA (0.1 mg mL −1 , 1.5 μL) in Tris buffer (pH 7.2, 5.0 mM Tris-HCl, 20 mM NaCl) with 50 mM NaCl was treated with varying concentrations of the copper(II) complexes (40-600 μM). The concentration of the complexes in the buffer corresponded to the quantity after the dilution of the complex stock to the 20 μL final volume using Tris buffer. The samples were incubated for 2 h at 37 °C, followed by addition of the loading buffer containing 12.5% bromophenol blue, 25% xylene cyanol, 1% tris, and the solution was finally loaded on agarose (1%) containing 10 mg mL −1 ethidium bromide (eB). The electrophoresis was carried out at 60 V for 3 h in Tris-acetate-eDTA. Afterward, electrophoresis bands were visualized by UV light and photographed. The cleavage mechanism of pBR322 DNA was measured in the presence of H 2 O 2 (oxidizing agent), DMSO (hydroxyl scavenger), and NaN 3 (singlet oxygen scavenger).

X-ray crystallography
Diffraction images were measured at 200 K on a Nonius Kappa CCD diffractometer using Mo Kα, graphite monochromator (λ = 0.71073 Å), and data extracted using the DENZO/SCALEPACK package [37]. The structures were solved by direct methods with SIR92 [38]. The structures were refined on F 2 by full matrix least-squares using the CRySTALS program package [39]. Hydrogens were added at calculated positions and then refined with soft restraints on the bond lengths and angles to regularize their geometry. In 1 the hydrogens were then refined with riding constraints, whereas in 2 they were allowed to refine freely. Atomic coordinates, bond lengths and angles, and displacement parameters have been deposited at the Cambridge Crystallographic Data Center. Crystallographic details for 1 and 2 are summarized in table 1.

Syntheses of (CuL 1 ) 4 , 1 and (CuL 2 ) 2 , 2
Complexes 1 and 2 were synthesized by a general method. Copper(II) acetate monohydrate (2 mmol 0.396 g) was slowly added to a methanol solution (30 mL) of the appropriate Schiff base (2 mmol, 0.41 g for 1 and 0.438 g for 2). The resulting solution was stirred for 2 h at room temperature. The resulting dark green precipitates were collected by filtration and dried in air.

Synthesis and characterization of the complexes
Syntheses of 1 and 2 are schematically represented in scheme 1. The tridentate Schiff base ligands were obtained by condensation of benzoylacetone and amino alcohol under reflux. The reaction of Table 1. Crystallographic data of 1 and 2.
18.1, 9.39, 2.80 and x = F/F max . copper(II) acetate with an equimolar amount of H 2 L 1 in methanol leads to tetranuclear 1. When H 2 L 2 was used for the synthesis, dinuclear 2 was formed. The most significant IR bands for the ligands and complexes are given in the experimental section. The IR spectra of the free Schiff bases show a band at 1602 cm −1 , assigned as νC=N. IR spectra of 1 and 2 show a decrease in νC=N in comparison with the free ligand, which indicates coordination of the imine nitrogen to copper [12,41,42]. The fairly broad band of medium intensity at 3357 and 3379 cm −1 corresponds to intramolecular hydrogen bonding in the free ligands, and this band disappears for the copper complexes. The lack of OH groups in the copper complexes indicates that the OH groups have been deprotonated and are coordinated to copper.
The electronic spectra of the copper complexes in DMF show a broad band at 638 (for 1, log ε = 1.40) and 584 nm (for 2, log ε = 1.26) and a sharper signal at 346 (for 1, log ε = 3.64) and 345 nm (for 2, log ε = 3.66), which arise from a spin-allowed d-d transition of the copper(II) ion (d 9 electronic configuration) and a charge transfer transition, respectively [43,44].

Crystal structure of 1
The molecular structure of 1 is shown in Figure 1. Selected bond lengths and angles, as well as interatomic distances are summarized in table 2. Complex 1 crystallizes in trigonal space group R3. In the copper complex, H 2 L 1 has both chelating and bridging modes after double deprotonation of the enolic OH groups. The dianionic form of the Schiff base ligand coordinates to the metal center through the imine nitrogen and deprotonated alkoxo oxygens. By a self-assembly process, four monomeric CuL 1 entities are linked through alkoxo bridges to produce the tetranuclear cubane, 1.
The Cu-O and Cu-N bond lengths in the equatorial plane are 1.908-1.984 Å and 1.920-1.940 Å, respectively (table 2), which agree with similar compounds [12,14,41]. The apical oxygens show longer Cu-O(alkoxo) bond lengths than the equatorial oxygens [43,48]. The elongation of the Cu-O axial bonds is due to a pseudo-Jahn-Teller distortion of the d 9 copper center. The lengths of the bonds between copper and the donor atoms are within the range of values normally found for such systems [14,19,49,50]. The bridging bond angles of Cu-O(alkoxo)-Cu are 89.26-112.25° (table 2). It is evident from the different Cu-O body diagonal distances as well as from the unequal metal-metal distances (table 2) that the cubane core is not a regular one, but distorted. In tetranuclear 1, the dihedral angles of adjacent phenyl rings are between 51 and 55°.
In the absence of suitable polar hydrogens there is no hydrogen bonding in the intra-and intermolecular interaction of the complex. However, the 3-D lattice structure of 1 shows the presence of intermolecular non-covalent C-H⋯N=C and C-H(Ph)⋯C-H(Ph) interactions ( Figure 2).

Crystal structure of 2
The molecular structure of 2 is shown in Figure 3. Selected bond lengths and angles, as well as interatomic distances are summarized in table 2; 2 crystallizes in the monoclinic space group P2 1 /n. In 2, similar to 1, H 2 L 2 is both chelating and bridging after double deprotonation of the enolic OH groups. The dianionic form of the ligand coordinates, forming six-membered chelate rings with Cu(II). Two such monomeric CuL 2 entities are eventually linked through the alkoxo bridges to produce the dinuclear 2.
The dinuclear unit is formed by two Cu(II) ions labeled Cu1 and Cu1* (symmetry code: −x + 1, −y + 1, −z + 1), bridged by the two μ 2 -alkoxo oxygens of the Schiff base ligands. Coordination about each copper ion is square planar with one nitrogen and two oxygens from the Schiff base and one oxygen from the next unit of the dimer. The Cu-O, Cu-N, and two Cu-O bridge bond distances are 1.9071(12), 1.9455(13), 1.9313 (11), and 1.9197(11) Å, respectively, in agreement with those observed for similar compounds [12,14,49,51]. As usually found in oxo-bridged Cu(II) complexes [12,14,52,53], the Cu-O bridge bond distances (1.9313 (11)  , indicating that there is distortion from ideal square planar geometry. The geometry of copper is best described as distorted square planar with τ 4 index 0.141. The τ parameter is τ 4 = [360°−(α + β)]/141°, where a and b are the largest angles around the central metal in the complex, τ 4 = 1 for a regular tetrahedron and τ 4 = 0 for a regular square planar [54]. The copper ions lie 0.043 Å above the NO 3 donor planes.
In 2 there are no intramolecular interactions. The crystal packing of the complex shows the presence of intermolecular non-covalent interactions between CH 2 units of the propylene to form a 2-D planar network parallel to the ab plane ( Figure 4).

DNA binding studies
UV-visible spectroscopy was performed to study the interaction of the complexes with DNA keeping the complex concentration constant (40 μM) and varying the concentration of CT-DNA (0-54 μM). The change in absorbance values at 260 nm was used to evaluate the intrinsic binding constant K b . The absorption spectra of 2 in the absence and presence of CT-DNA (at constant concentration of complex) are shown in Figure 5. Hypochromicity of the DNA at 265 nm without a shift in band position is observed with the addition of CT-DNA.
The hypochromism (decrease of absorbance) indicated a strong interaction between the complexes and CT-DNA mainly through intercalative binding [55,56]. The non-covalent intercalative binding of the compound to the DNA helix can lead to large hypochromism due to the strong stacking interaction between the aromatic moiety of the complex and the base pairs of DNA [57,58]. Complex 2 binds to DNA through intercalation and resulted in hypochromism due to the stacking effect of π electrons which leads to a decrease in the peak intensities in the UV spectra of complex [59,60].
From the binding constant value, it was clear that 2 had good interaction with CT-DNA. However, 1 and free ligands H 2 L 1 and H 2 L 2 exhibited no significant DNA bonding.

DNA cleavage with 1
DNA cleavage was analyzed by monitoring the conversion of supercoiled DNA (form I) to nicked circular DNA (form II) and linear DNA (form III). 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 [43,44]. At micro-molar concentrations, for 3 h incubation periods, in the absence of H 2 O 2 (an oxidizing agent) 1 exhibited no significant nuclease activity ( Figure 6, lanes 2-5). However, 1 displays efficient cleavage of pBR322 circular plasmid DNA converting form I to form II and form III in the presence of hydrogen peroxide (Figure 6, lane 6), indicating an oxidative cleavage process [36].  (Figure 7, lane 7). The control results suggest the complex to be more efficient than copper salt and ligand for the chemical nuclease activity.
To elucidate the cleavage mechanism of DNA, the cleavage was investigated in the presence of hydroxyl radical (OH • ) scavenger (DMSO, 1.4 mM) and a singlet oxygen ( 1 O 2 ) quencher (NaN 3 , 1 mM). The experiments reveal that NaN 3 and DMSO significantly inhibited the cleavage activity of Cu(II) complex ( Figure 6, lanes 7 and 8), indicating that singlet oxygen and hydroxyl radical play significant roles in the DNA cleavage reaction [68,69]. The result suggests that 1 cleaves DNA via oxidative cleavage [35,36]. From the observed results, it was concluded that 2 effectively cleaved the DNA as compared to control DNA and H 2 L 2 alone. Upon increasing the concentration of 2 more cleavage activity was observed ( Figure 8, lanes 2-5). This shows that the concentration of optimal value led to extensive degradations, resulting in conversion of the supercoiled form (Form I) into a nicked circular form (Form II). The activity for DNA cleavage without an oxidant is often a hydrolytic rather than an oxidative mechanism [43].

DNA cleavage with 2
Also, the nuclease activity of the complex was investigated in the presence of H 2 O 2 as an oxidant and a hydroxyl radical scavenger, DMSO, and a singlet oxygen quencher, NaN 3 . In the presence of H 2 O 2 , 2 shows higher activity and supercoiled DNA (Form I) is cleaved to form a mixture of Form II and Form III (Figure 8, lane 6). The presence of DMSO did not significantly reduce the efficiency of DNA cleavage ( Figure 8, lane 7), ruling out the possibility of the involvement of diffusible hydroxyl radicals in the cleavage in the presence of H 2 O 2 [39,40]. However, the addition of the singlet quencher, NaN 3 significantly inhibited the cleavage activity of 2 ( Figure 8, lane 9). The results suggest that singlet oxygen is responsible for the DNA cleavage in the presence of H 2 O 2 [43].

Conclusion
We have synthesized a tetranuclear copper(II), 1, and a dinuclear, 2, complex with two tridentate Schiff base ligands. The complexes were structurally characterized by single crystal X-ray diffraction analysis. Complex 1 has a cubane Cu 4 O 4 core in which copper and oxygen are present at alternative vertices. The coordination geometry about each copper ion is square pyramidal. Complex 2 contains a coplanar Cu 2 O 2 core and the coordination geometry around the copper(II) is four-coordinate square planar. Complex 1 exhibits significant DNA cleavage activity in the presence of H 2 O 2 as an oxidant. The binding of the complexes with CT-DNA was studied using UV-vis spectroscopy. The absorption spectra indicate that 2 interacts with CT-DNA. Both complexes are more efficient than their parent compound in DNA cleavage.

Supplementary material
The deposition numbers of 1 and 2 are CCDC 1045677 and 1045678, respectively. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, by emailing data-request@ccdc.cam.ac.uk, or by contacting the Cambridge Crystallographic Data Center, 12 Union Road, Cambridge CB2 1ez, UK; Fax +44 1223 336033.