Synthesis and structure of a new ternary monocopper

Full paper Received: 6 January 2016

Revised: 1 March 2016

Accepted: 27 March 2016

Published online in Wiley Online Library: 23 May 2016

(wileyonlinelibrary.com) DOI 10.1002/aoc.3497

Synthesis and structure of a new ternary monocopper(II) complex containing mixed ligands of 2,2′-diamino-4,4′-bithiazole and picrate: in vitro anticancer activity, molecular docking and reactivity towards DNA Xi-Ling Wanga†, Kang Zhenga†, Ling-Yang Wanga, Yan-Tuan Lia*, Zhi-Yong Wua and Cui-Wei Yanb* A new ternary monocopper(II) complex with co-ligands of 2,2′-diamino-4,4′-bithiazole (dabt) and picrate (pic), namely [Cu(dabt)(pic)2], has been synthesized and characterized using elemental analyses, molar conductance measurements, infrared and electronic spectral studies and single-crystal X-ray diffraction. The crystal structure analyses revealed that the copper(II) ion has a {CuN2O4} distorted octahedral coordination environment. The hydrogen bonding interactions contribute to a three-dimensional supramolecular structure in the crystal. The reactivity towards herring sperm DNA showed that the copper(II) complex can interact with DNA in the mode of intercalation. The molecular docking of the complex with DNA sequence d(ACCGACGTCGGT)2 demonstrated that the copper(II) complex is stabilized by hydrogen bonding interaction. The in vitro anticancer activities suggested that the copper(II) complex is active against selected tumor cell lines. The effects of the two co-ligands in the copper(II) complex on DNA-binding events and in vitro anticancer activity are preliminarily discussed. Copyright © 2016 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web site. Keywords: ternary copper(II) complex; crystal structure; DNA interaction; anticancer activity

Introduction

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Since DNA has been identified as one of the primary targets for some metal-based antitumor drugs, the investigation of interactions of metal complexes with DNA is of intense current interest. Interest in this area not only stems from attempts to gain some insight into the mechanisms involved in the site-specific recognition of DNA, but also to understand the reactive models for protein–nucleic acid interactions and probes of DNA structure, and to get information about drug design and tools of molecular biology.[1–3] Cisplatin, as one of the most widely used metal-based antitumor drugs targeting DNA, is active in the treatment of several types of cancers.[4,5] However, the significant side effects originating from its binding mode to DNA due to the formation of covalent cross-links impede its clinical success.[6] Therefore, it is essential to develop more efficacious, target-specific, less toxic and non-covalently DNA-binding non-platinum-based drugs.[7] In the context, many investigations have focused on the selection of metal ions and the design of ligands. The medicinal properties of metal complexes depend on the nature of the metal ions and the ligands, and metal ions present in metal complexes not only accelerate the drug action but also increase the effectiveness of the organic ligands. Taking into account that copper is an essential element for human and most aerobic organisms, and copper-based complexes may be less toxic for normal cells with respect to cancer Appl. Organometal. Chem. 2016, 30, 730–739

cells, this makes copper complexes the most promising leads for next-generation metal-based anticancer agents as alternatives to cisplatin as anticancer drugs.[8–10] For this reason, many copper(II) complexes with 2,2′-bipyridine (bpy) or 1,10-phenanthroline as ligands have been synthesized and their mode of DNA binding as well as anticancer activity have been investigated.[11] Comparatively few studies of mixed-ligand complexes with 2,2′-diamino-4,4′-bithiazole (dabt) as ligand have been reported to date. However, those complexes with five-membered heterocycles such as thiazole and its derivatives (dabt) as ligands have attracted much attention due to the excellent biological activities that have been reported,[12–15] which also stimulated us to design and synthesize new ternary complexes with dabt as co-ligand to evaluate

* Correspondence to: Yan-Tuan Li, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, PR China. E-mail: [email protected] Cui-Wei Yan, College of Marine Life Science, Ocean University of China, Qingdao 266003, PR China. E-mail: [email protected] †

These authors contributed equally to this work

a School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, PR China b College of Marine Life Science, Ocean University of China, Qingdao 266003, PR China

Copyright © 2016 John Wiley & Sons, Ltd.

A new ternary monocopper(II) complex and understand the factors of the DNA-binding property and in vitro anticancer activity.[12] On the other hand, picrate (pic), due to its peculiar structure, involving phenolato and nitro groups that are non-coplanar with themselves and with the benzene ring, could be a good candidate to act as mono- and polydentate ligand to build metal complex systems. Hence, many metal complexes with pic as ligands have been reported.[16–18] However, to the best of our knowledge, so far, a ternary copper(II) complex containing mixed ligands of pic and dabt has not been reported. Thus, it is of considerable interest to synthesize and study the DNA-binding properties and anticancer activities of this kind of ternary copper (II) complex in order to gain insight into the structure–activity relationship. Our group has been engaged in the design, synthesis and activity evaluation of ternary copper(II) complexes with various mixed ligands.[19] To further study the effect of various co-ligands in ternary copper(II) complexes on DNA-binding properties and in vitro anticancer activities, as an extension of that investigation, in the work reported in this paper, we selected dabt and pic as co-ligands, and present here the synthesis and structure of a new ternary copper(II) complex with in vitro anticancer activity. The reactivity towards DNA of the complex has been studied both theoretically and experimentally. The main results suggest that different coligands in ternary monocopper(II) complexes may play an important role in the DNA-binding events and in vitro anticancer activities.

Experimental Materials and Physical Measurements All reagents were of AR grade and obtained commercially. Cu(pic)2
6H2O was prepared according to literature methods.[20] Doubly distilled water was used to prepare buffers. Ethidium bromide (EB) and herring sperm DNA (HS-DNA) were purchased from Sigma Corp. and used as received. Carbon, hydrogen and nitrogen elemental analyses were conducted with a PerkinElmer model 240 elemental analyzer. Infrared (IR) spectra were recorded using KBr pellets with a Nicolet Impact 470 FT-IR spectrophotometer in the range 400–4000 cm1. UV–visible spectra were recorded using a 1 cm path length quartz cell with a Cary 300 spectrophotometer. Fluorescence was measured with an Fp-750w fluorimeter. A CHI 832 electrochemical analyzer (Shanghai CHI Instrument, Shanghai, China) in connection with a glassy carbon working electrode, a saturated calomel reference electrode and a platinum wire counter electrode was used for electrochemical measurements. The glassy carbon electrode surface was freshly polished to a mirror prior to each experiment with 0.05 μm α-Al2O3 paste and then cleaned in water for 5 min. Viscosity measurements were carried out using an Ubbelohde viscometer immersed in a thermostatic water bath maintained at 289 (± 0.1) K.

Synthesis of [Cu(dabt)(pic)2]

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Crystal Structure Determination The X-ray diffraction experiment for the complex was carried out using a Bruker APEX area-detector diffractometer with graphite monochromatic Mo Kα radiation (λ = 0.71073 Å) at a temperature of 296 K. The crystal structure was solved by the directed method followed by Fourier syntheses. Structure refinement was performed by full-matrix least-squares procedures using SHELXL-97 on F2.[21] The hydrogen atoms on carbon atoms were placed in calculated positions with C–H = 0.93 (aromatic) then treated as riding, with Uiso(H) = 1.2 Ueq (C atoms). The hydrogen atoms on amino groups were found in a difference Fourier map and were refined freely. Crystal data and structural refinement parameters are summarized in Table 1, and selected bond distances and bond angels are listed in Table 2. DNA Interaction Studies of Copper(II) Complex Molecular docking calculations

Automated docking was used to preliminarily predict the DNA-binding affinity as well as the preferred orientation of the copper(II) complex binding to DNA. AutoGrid4 and AutoDock4 were used to set up and perform blind docking calculations.[22] It should be pointed out that HS-DNA used in the experimental work was too large for current computational resources to use; therefore, the structure of sequence d(ACCGACGTCGGT)2 (PDB id: 423D, a familiar sequence used in oligodeoxynucleotide studies) obtained from the Protein Data Bank (http://www.rcsb.org/pdb) at a resolution of 1.60 Å was constructed in the Autodock4 package to study the DNA-binding properties of the complex. Due to unavailability of standard parameters for copper(II) in the Autodock4 parameter file, the parameters of Cu(II) were set as vdW radii of 0.96 Å and vdW well depth of 0.01 kcal mol1.[23] The coordinates of the Table 1. Crystal data and details of structural determination for the complex Empirical formula

CuC18H10N10O14S2

Formula weight Crystal system Space group a (Å) b (Å) c (Å) β (°) 3 V (Å ) Z 3 D (calcd) (g cm ) 1 μ (Mo Kα) (mm ) F (000) Crystal size (mm) Temperature (K) Radiation (Å) θ range for data collection (°) Total, unique data, R(int) Observed data [I > 2σ(I)] R, ωR2, S Max., av. shift/error

718.02 Monoclinic P21/c 10.721(3) 15.165(4) 15.963(4) 96.964(5) 2576.2(11) 4 1.851 1.104 1444 0.09 × 0.14 × 0.32 296 Mo Kα, 0.71073 1.91–27.56 15 037, 5905, 0.0387 4113 0.0453, 0.1195, 1.025 0.001, 0.000

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A methanol aqueous solution (5 ml) of Cu(pic)2
6H2O (0.0627 g, 0.1 mmol) was added dropwise into a methanol solution (5 ml) containing dabt (0.0199 g, 0.1 mmol). After the reaction system was stirred at 333 K for 6 h, the resulting dark green solution was filtered and green cubic crystals of the complex suitable for X-ray analysis were obtained by slow evaporation at room temperature after

10 days. Yield: 0.0509 g (71%). Anal. Calcd for CuC18H10N10O14S2 (%): C, 30.08; H, 1.40; N, 19.51. Found (%): C, 30.05; H, 1.42; N, 19.53.

X.-L. Wang et al. Table 2. Selected bond distances (Å) and bond angles (°) for the complex Cu1–O1 Cu1–O8 Cu1–N1 O1–Cu1–O2 O1–Cu1–O9 O2–Cu1–O9 O1–Cu1–N1 O2–Cu1–N1 O8–Cu1–N1 O9–Cu1–N1 N1–Cu1–N2

1.941(2) 1.974(2) 1.985(3) 77.13(10) 98.17(11) 162.15(8) 94.11(10) 111.49(10) 163.38(9) 85.86(9) 82.65(11)

Cu1–O2 Cu1–O9 Cu1–N2 O1–Cu1–O8 O2–Cu1–O8 O8–Cu1–O9 O1–Cu1–N2 O2–Cu1–N2 O8–Cu1–N2 O9–Cu1–N2

2.425(3) 2.340(2) 1.966(3) 92.40(9) 84.87(9) 78.08(8) 163.31(11) 88.78(9) 95.23(10) 97.91(10)

complex were taken from the crystal structures as a CIF file and converted to the PDB format using Mercury software.[24] Receptor (DNA) and ‘the ligand’ (monocopper(II) complex) files were prepared using AutoDock Tools. The heteroatoms including water molecules were deleted. Polar hydrogen atoms and Kollman charges were added to the receptor molecule. All other bonds were allowed to be rotatable. In the docking analysis, the binding site was assigned across the minor and major grooves of the DNA molecule, which was enclosed in a box with number of grid points in x × y × z directions of 68 × 68 × 62 and a grid spacing of 0.375 Å. Initially, AutoGrid was run to generate the grid map of various atoms of ‘the ligand’ and the receptor. After the completion of the grid map, AutoDock was run using parameters as follows: GA population size, 150; maximum number of energy evaluations, 2 500 000; number of generations, 27 000. A total of 50 runs were carried out. A maximum of 50 conformers were considered for each molecule, and the root-mean-square cluster tolerance was set to 2.0 Å in each run. All calculations were performed with an Intel Core i7-based machine running GNU/Linux as operating system. For each of the docking cases, the lowest energy docked conformation, according to the AutoDock scoring function, was selected as the binding mode. Visualization of the docked pose was done using the PyMOL (PyMOL Molecular Graphics System, Version 1.3, Schrödinger LLC) molecular graphics program. DNA-binding experiments

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All experiments involving HS-DNA were performed in Tris–HCl buffer solution (pH = 7.16), which was prepared using deionized and sonicated triple-distilled water. Solution of DNA in Tris–HCl buffer gave a ratio of UV absorbance at 260 and 280 nm, A260/A280, of ca 1.9, indicating that the DNA was sufficiently free of protein.[25] The concentration of the prepared DNA stock solution was determined according to its absorbance at 260 nm. The molar absorption coefficient, ε260, was taken as 6600 l mol1 cm1.[26] Stock solution of DNA was stored at 277 K and used after no more than four days. Concentrated stock solution of the copper(II) complex was prepared by dissolving the complex in dimethylsulfoxide (DMSO) and diluted suitably with Tris–HCl buffer to required concentrations for all experiments. Absorption spectral titration experiment was performed by keeping the concentration of the copper(II) complex constant while varying the HS-DNA concentration. Equal solution of HS-DNA was added to the copper(II) complex solution and reference solution to eliminate the absorbance of HS-DNA itself. In the EB fluorescence displacement experiment, 5 μl of EB Tris–HCl solution

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(1 mmol l1) was added to 1 ml of HS-DNA solution (at saturated binding levels),[27] and stored in the dark for 2 h. Then the solution of the copper(II) complex was titrated into the DNA/EB mixture and then diluted in Tris–HCl buffer to 5 ml. Before measurements, the mixture was shaken up and incubated at room temperature for 30 min. The fluorescence spectra of EB bound to HS-DNA were obtained at an emission wavelength of 584 nm using a spectrofluorimeter, keeping the concentration of HS-DNA constant while varying the concentration of the copper(II) complex, and the inner-filter effect (IFE) was corrected according to a literature method.[28] The electrochemical titration experiment was also performed by keeping the concentration of the complex constant while varying the HS-DNA concentration, using a solvent of 50 mM NaCl/Tris–HCl buffer solution at pH = 7.16.[29] All voltammetric experiments were performed in a singlecompartment cell. The supporting electrolyte was 50 mM NaCl/Tris–HCl buffer solution at pH = 7.16.[29] Solutions were deoxygenated by purging with nitrogen gas for 15 min prior to measurements; during measurements a stream of nitrogen gas was passed over the solution. In viscosity measurements, HS-DNA sample approximately 200 base pairs in length was prepared by sonication to minimize complexities arising from DNA flexibility.[30] Flow times were measured with a digital stopwatch, and each sample was measured three times, and an average flow time was calculated. Relative viscosities for HS-DNA in the presence and absence of the complex were calculated from the relation η = (t  t0)/t0, where t is the observed flow time of DNA-containing solution and t0 is that of Tris–HCl buffer alone. Data were presented as (η/η0)1/3 versus binding ratio,[31] where η is the viscosity of DNA in the presence of the copper(II) complex and η0 is the viscosity of DNA alone. In Vitro Antitumor Activity Evaluation by SRB Assay In vitro anticancer activities of the tested copper(II) complex together with cisplatin were evaluated against selected cell lines by using the sulforhodamine B (SRB) assay. All cells were cultured in RPMI 1640 supplemented with 10% (v/v) fetal bovine serum, 1% (w/v) penicillin (104 U ml1) and 10 mg ml1 streptomycin. Cell lines were maintained at 310 K in a 5% (v/v) CO2 atmosphere with 95% (v/v) humidity. Cultures were passaged weekly using trypsin–EDTA to detach the cells from their culture flasks. The copper(II) complex was dissolved in DMSO and diluted to the required concentration with culture medium when used. The content of DMSO in the final concentrations did not exceed 0.1%. At this concentration, DMSO was found to be non-toxic to the cells tested. Rapidly growing cells were harvested, counted and incubated at the appropriate concentration in 96-well microplates for 24 h. The copper(II) complex dissolved in culture medium was then applied to the culture wells to achieve final concentrations ranging from 103 to 102 μg ml1. Control wells were prepared by addition of culture medium without cells. The plates were incubated at 310 K in a 5% CO2 atmosphere for 48 h. Upon completion of the incubation, the cells were fixed with ice-cold 10% trichloroacetic acid (100 ml) for 1 h at 277 K, washed five times in distilled water and allowed to dry in air and stained with 0.4% SRB in 1% acetic acid (100 ml) for 15 min. The cells were washed four times in 1% acetic acid and air-dried. The stain was solubilized in 10 mM unbuffered Tris base (100 ml) and the optical density of each well was measured at 540 nm with a microplate spectrophotometer. The IC50

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A new ternary monocopper(II) complex values were calculated from the curves constructed by plotting cell survival (%) versus copper(II) complex concentration.

Results and Discussion Synthesis and General Properties of Copper(II) Complex The goal of the present work was to ascertain whether different coligands in ternary monocopper(II) complexes can affect DNA binding, as well as in vitro anticancer activities. For that, dabt and pic were chosen as the co-ligands. Indeed, elemental analyses and single-crystal X-ray diffraction indicate that the reaction of Cu (pic)2
6H2O with dabt in 1:1 mole ratio yielded the ternary complex, namely [Cu(dabt)(pic)2], in which both the two pic groups, as bidentate chelate ligands, are coordinated with the copper(II) center to form a neutral ternary complex. The copper(II) complex is insoluble in non-polar solvents, moderately soluble in methanol and acetonitrile, and very soluble in dimethylformamide (DMF) and DMSO to give stable solutions at room temperature. In the solid state the copper(II) complex is fairly stable in air allowing physical measurements. The molar conductivity values of the complex in DMF (12 S cm mol1) and H2O (15 S cm mol1) fall in the expected range for non-electrolytes,[32] indicating that the picrate anions are situated inside the metal coordination sphere, and thus the ternary copper(II) complex may stay intact as a whole neutral [Cu(dabt)(pic)2] molecule in solutions. The structure of the complex was characterized using spectroscopic and single-crystal structure X-ray analyses, as described in the following.

Structural Description of [Cu(dabt)(pic)2] The structure of the complex is illustrated in Fig. 1, in which the two pic ligands are in cis arrangement, and the central copper(II) ion is in a distorted octahedral coordination geometry with a {N2O4} chromophore. The axial Cu1-O2 and Cu1-O9 bonds are longer than the equatorial Cu1-O1 and Cu1-O8 bonds due to the Jahn–Teller effect (Table 2). The three ligands are all bidentate with bite angles of 77.13(10)° (O1–Cu1–O2), 78.08(8)° (O8–Cu1–O9) and 82.65(11)° (N1–Cu1–N2). The five-membered chelate ring of dabt ligand is planar. While the two six-membered rings are folded with puckering parameters[37] of Q [0.628(2) Å], θ [72.2(3)°], ϕ [24.8(3)°] (Cu1–O1– C7–C8–N5–O2) and Q [0.492(2) Å], θ [65.3(3)°], ϕ [10.1(4)°] (Cu1– O8–C13–C14–N8–O9). As shown in Fig. 2, hydrogen bonding interactions, classical and non-classical, dominate the crystal. Through those between the dabt ligand and the pic ligands of O1 (Table 3), the complex is assembled into a one-dimensional chain parallel to the direction of [1 0 1]. In addition, via the non-classical C17-H17


O9 interactions between the other pic ligands (O8), the complex is joined to

IR Spectra The IR spectrum taken in the region 400–4000 cm1 provides some information regarding the mode of coordination in the complex. The band at 1308 cm1 can be assigned to the skeletal vibration of the bithiazole ring of dabt ligand. Notably, the band associated with ν(C=N) vibration from the aromatic ring of the dabt ligand is shifted to 1521 cm1 in the complex,[33] indicating that the nitrogen atoms of dabt are coordinated to copper(II) ion in the copper (II) complex. Furthermore, free Hpic has νas(NO2) at 1555 cm1, which splits into two bands at 1577 and 1538 cm1 in the complex. In addition, a sharp band observed at 1341 cm1 due to νs(NO2) in the copper(II) complex suggests that one of the nitro group oxygen atoms of pic ligands takes part in coordination.[34] Moreover, the O-H out-of-plane bending vibration of the free Hpic at 1151 cm1 disappears, and the ν(C-O) vibration of pic at 1265 cm1 is shifted to higher frequency, 1278 cm1, due to the oxygen atom of the phenolate in pic ligand coordinating with the copper(II) ion,[35] which coincides with the molar conductance data.

Figure 1. View of the complex with atom numbering scheme. Displacement ellipsoids are drawn at 30% probability and hydrogen atoms are shown as small spheres of arbitrary radii. Dashed lines show hydrogen bonds.

Electronic Spectra

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Figure 2. One-dimensional classical hydrogen bonding chain parallel to [1 0 1]. Symmetry codes: (i)  x, 1  y, 1 z; (ii) 1  x, 1  y, 2  z.

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The electronic spectrum of the copper(II) complex was recorded in the UV–visible region (200–800 nm) using methanol as solvent. Notably, spectra obtained for the copper(II) complex at different concentrations obeyed the Beer–Lambert law, indicating that copper (II) complex stays intact at these concentrations,[36] which is consistent with the molar conductance data. Moreover, for the complex, two main absorption bands with varied intensities can be observed. An intense band at 238 nm and a weak band at 356 nm are attributed to dabt (π–π*) transitions.

X.-L. Wang et al. another chain parallel to the c axis (Fig. 3). These two kinds of chains interweave with each other to complete a three-dimensional supramolecular structure. Compared with our previously reported analogous complex, [Cu(bpy)(pic)2],[19] the only difference between them is their co-ligands. In the present [Cu(dabt)(pic)2] complex, dabt is the co-ligand, while bpy is the co-ligand in the [Cu(bpy)(pic)2] complex. As a result, the present copper(II) complex is crystallized in monoclinic form with the P21/c space group. However, the previously reported [Cu(bpy)(pic)2] complex is crystallized in triclinic form with the P-1 space group. Furthermore, these differences of the structure also have an effect on the properties described in the following.

DNA Interaction Studies of the Complex As is well known, DNA is a particularly good target for some antitumor reagents in organisms. These reagents can react with DNA thereby changing the replication of DNA and inhibiting the growth of tumor cells.[38] Therefore, investigations of the interaction between DNA and metal complexes are significant for understanding the mechanism of binding and for designing new metal-based drugs. Therefore, the non-covalent interactions of the copper(II) complex with DNA are explored with the aid of various techniques and methods.

Table 3. Hydrogen bonding geometries for the complex D–H


A N3-H3A


O1 i N3-H3B


O7 ii N4-H4B


O4 N4-H4A


O8 N4-H4A


O14 iii C17-H17


O9

D-H (Å)

H


A (Å)

D


A (Å)

D-H


A (°)

0.81(4) 0.81(4) 0.80(4) 0.86(4) 0.86(4) 0.93

2.14(4) 2.26(4) 2.39(4) 2.17(4) 2.58(4) 2.51

2.871(4) 3.021(5) 2.944(5) 2.896(4) 3.327(5) 3.292(4)

150(4) 156(3) 128(3) 142(3) 146(3) 141.3

Symmetry codes: (i)  x, 1  y, 1  z; (ii) 1  x, 1  y, 2  z; (iii) x, ½  y, z + ½.

Molecular docking of copper(II) complex with DNA dodecamer d (ACCGACGTCGGT)2 The molecular docking technique plays an important role in understanding drug–DNA interactions for rational drug design and discovery, as well as in mechanistic studies by placing a small molecule into the binding site of the target-specific region of DNA mainly in a non-covalent fashion.[39] In our experiment, molecular docking studies of the copper(II) complex with DNA duplex of sequence d(ACCGACGTCGGT)2 dodecamer were performed in order to preliminarily predict the binding ability along with the preferred orientation of sterically acceptable complex inside DNA. According to the docking results, summarized in Table 4, the binding free energy value is negative (ΔGbinding = 7.62 kcal mol1), suggesting that the binding of the copper(II) complex to the DNA is spontaneous. Moreover, the vdW_hb_desolv energy is dominant in the final energy, which mainly relates to the formation of hydrogen bonds between the copper(II) complex and DNA. Comparing with our previously reported analogous complex [Cu(bpy) (pic)2],[19] it can be found that the present complex has a lower binding free energy than that of the previous one (ΔGbinding = 5.06 kcal mol1), which implies that the present complex may have stronger DNA-binding affinity than the previously reported complex. In order to understand comprehensively the docking results, and furthermore to obtain information about the hydrogen bonding interactions of the copper(II) complex with DNA, the energyminimized docked pose of the copper(II) complex is illustrated intuitively in Fig. 4, and the hydrogen bonds between the complex and the DNA are listed in Table 5. As seen from Fig. 4, the copper(II) complex can form four hydrogen bonds with the DNA chains A and B, which is in agreement with the fact mentioned above that the vdW_hb_desolv energy is dominant in energy terms. Among the established four hydrogen bonds, it is notable that the amino group (-NH2) of co-ligand dabt can link to the phosphate backbone by strong N–H


O hydrogen bonding with a distance of 1.955 Å ( Table 5), stabilizing the binding of the copper(II) complex to the DNA. Meanwhile, there are three hydrogen bonds between the pic ligands and the base pairs of the DNA. Interestingly, if co-ligand dabt in the present copper(II) complex [Cu(dabt)(pic)2] is replaced by bpy as in our previously reported analogous complex [Cu(bpy) (pic)2], no hydrogen bonds between the [Cu(bpy)(pic)2] complex and DNA are observed. This fact demonstrates that the strong hydrogen bonding of dabt with the DNA phosphate backbone for the present copper(II) complex plays an important role in the DNA-binding events. Thus, the molecular docking prediction provides a preliminary understanding of the binding nature of the synthesized complex, and the positive results promote us to further study the binding mode and binding ability of the present copper (II) complex to DNA by the experimental studies described in the following. Electronic absorption titration

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Figure 3. One-dimensional non-classical hydrogen bonding chain parallel to the c axis. Symmetry codes: (iii) x, ½  y, z + 1/2; (iv) x, ½  y, z  ½.

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Monitoring the changes in absorption spectra of metal complexes upon addition of increasing amounts of DNA is one of the most widely used methods for determining overall binding events. The absorption spectra of the copper(II) complex in the absence and presence of HS-DNA are shown in Fig. 5. Both hypochromism and bathochromism (3 nm) are observed at the characteristic absorption peak of 238 nm after interaction with different concentrations of HS-DNA. These spectral characteristics suggest that the complex can interact with HS-DNA through the intercalation mode, due to

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A new ternary monocopper(II) complex Table 4. DNA docking results of the complex Binding free energy (ΔGbinding)a

vdW_hb_desolv energy (ΔGvdW+hb+desolv)

Electrostatic energy (ΔGelec)

Torsional free energy (ΔGtor)

7.62

5.45

1.86

0.31

ΔGbinding = ΔGvdW+hb+desolv + ΔGelec + ΔGtor.

a

Figure 5. Absorption spectra of complex upon titration of HS-DNA. Arrows indicate the change upon increasing DNA concentration (the red line for the absence of HS-DNA). Inset: plot of [DNA]/(εa  εf) versus [DNA] for the absorption titration of HS-DNA with the complex. Figure 4. Molecular docking of the complex with DNA sequence d (ACCGACGTCGGT)2. Wireframe model of DNA with the complex (ball and stick) and the hydrogen bonds (yellow dashes). The complex is interacting with both chain A and chain B.

Table 5. Hydrogen bonding interactions involving energy-minimized docked poses of d(ACCGACGTCGGT)2 with the complex Donor group (Y–H) H4B(complex) H7(G10)(DNA-chain A) H62(A13)(DNA-chain B) H7(A13)(DNA-chain B)

Acceptor group Z

Distance (Å)

OP2(G10)(DNA-chain A) O13(complex) O12(complex) O11(complex)

1.955 2.050 2.105 2.216

the intercalative mode involving a strong stacking interaction between an aromatic chromophore and the base pairs of DNA.[36] To evaluate quantitatively the affinity of the copper(II) complex toward HS-DNA, the intrinsic binding constant Kb was determined by monitoring the changes in absorbance at 238 nm for the copper(II) complex using the following equation:[40] ½DNA ½DNA 1 ¼ þ εa  εf εb  εf K b ðεb  εf Þ

(1)

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EB fluorescence displacement assay A competitive binding experiment was employed to clarify the binding mode of the complex with HS-DNA. The fluorescence intensity of DNA or EB in Tris–HCl buffer is very low. However, EB, which is one of the most sensitive fluorescence probes, emits intense fluorescence in the presence of DNA due to its strong intercalation between the adjacent DNA base pairs[43] and this enhanced fluorescence could be quenched by the addition of another molecule[44] owing to the decreasing binding sites of DNA available for EB.[44,45] Thus, EB can be used to probe the interaction of complexes with DNA. If the complex can intercalate into DNA, it will lead to a decrease in the binding sites of DNA available for EB, and hence to a quenching of fluorescence intensity of the EB–DNA system. It should be pointed out that the IFE,[28] which refers to the absorption of light at the excitation or emission wavelength by other compounds present in the solution, may also lead to a decrease in fluorescence intensity. The decrease in the fluorescence intensity of a solution induced by the occurrence of the IFE could cause a nonlinear relationship between the observed fluorescence intensity and the concentration of the fluorophore, as well as result in the introduction of large errors in the data interpretation. Therefore, it is necessary to consider the IFE and eliminate its interference with the results. In our study, the IFE was corrected with the

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where [DNA] is the concentration of DNA, εa, εf and εb correspond to the extinction coefficients for each addition of DNA to the copper(II) complex, for the free copper(II) complex and for the copper (II) complex in the fully bound form, respectively. From a plot of [DNA]/(εa  εf) versus [DNA] (inset in Fig. 5), the binding constant Kb is given by the ratio of the slope to the intercept. The Kb value

for the complex is 3.33 × 105 M1 (R = 0.9998 for six points), which is lower than observed for a classical intercalator such as EB– DNA,[41] but higher than some mononuclear copper(II) complexes,[42] indicating that the present copper(II) complex can strongly bind to HS-DNA by intercalation, and this may be related to its structural characteristics.

X.-L. Wang et al. following equation:[28]

Electrochemical titration

F corr ¼ F obs 10ðAex dex =2ÞþðAem dem =2Þ

(2)

where Fobs and Fcorr are the measured fluorescence and the correct fluorescence intensity, dex and dem are the cuvette pathlength in the excitation and emission direction (in cm) and Aex and Aem are the measured change in absorbance value at the excitation and emission wavelength, respectively. After removal of the IFE, the fluorescence intensity of EB–DNA at 584 nm exhibits a marked decrease with increasing concentration of the copper(II) complex, as illustrated in Fig. 6, suggesting that some EB molecules are released after exchange with the copper(II) complex, resulting in fluorescence quenching of EB. The quenching of EB bound to the DNA by the complex is in agreement with the Stern–Volmer equation:[46] I0 ¼ 1 þ K sv ½Q I

(3)

where I0 and I represent fluorescence intensities in the absence and presence of quencher, respectively. Ksv is referred to as the Stern–Volmer constant and [Q] is the concentration of quencher. In the quenching plot of I0/I versus [complex] (inset in Fig. 6), Ksv is given by the ratio of the slope to intercept as 3.74 × 105 (R = 0.9991 for seven points). Comparing with our previously reported analogous complex [Cu (bpy)(pic)2],[19] we find the Kb and Ksv values for the present complex are both higher than those for [Cu(bpy)(pic)2] (Kb = 2.35 × 105 M1; Ksv = 2.55 × 105). This may be attributed to the fact that the substituents (–NH2) on the thiazole ring of coligand dabt in [Cu(dabt)(pic)2] can serve as hydrogen bond acceptors, leading to stronger binding affinity between [Cu(dabt)(pic)2] and the base pairs of DNA[47] than [Cu(bpy)(pic)2], which is in agreement with the molecular docking calculations. These results reveal that the binding affinity of these ternary monocopper(II) complexes toward DNA may be controlled and tuned by changing the coligands. Indeed, further studies using various co-ligands are still required in order to get deeper insight into the interactions of these complexes with DNA.

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Figure 6. Fluorescence intensity of EB–DNA as the system was titrated with the complex. Arrow shows the direction of change upon increasing the complex concentration. Inset: plot of I0/I versus [complex] for the absorption titration of the complex.

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The application of the cyclic voltammetric technique to the study of interaction between metal complexes and DNA provides a useful complement to the previously used spectral studies.[48] Before studying the interaction between the complex and DNA, it is helpful to study the electrochemical behavior of the copper(II) complex in Tris–HCl buffer solution. The cyclic voltammograms of the complex with and without HS-DNA at different scan rates (from 0.06 to 0.20 V s1) in the potential range between 0.3 and 0.3 V were investigated, and the results are shown in Fig. 7. It can be seen that the oxidation and reduction peak currents (ipa and ipc) both exhibit a linear relationship versus the square root of scan rate, indicating that the electrode processes in the buffer solution are diffusion controlled.[49] In order to get a deeper insight into the electrochemical behavior, the dependences of cathodic currents of the copper(II) complex on scan rate (ν), as an example, were further investigated and the results are illustrated in Fig. 8(a). It is observed that the cathodic currents of the copper(II) complex both with and without HS-DNA are linear with the square root of the scan rate (ν1/2), while the slop of the Ipc–ν1/2 plot decreases distinctly after mixing with DNA, indicating the reduction in the apparent diffusion coefficient of copper(II) complex in the presence of DNA.[50] Thus, we can interpret the change in current upon DNA addition

Figure 7. (a) Cyclic voltammograms of the complex at scan rates of 60, 80, 1 100, 120, 140, 160, 180 and 200 mV s from inner to outer. (b) Cyclic voltammograms of the complex with DNA at scan rates of 60, 80, 100, 120, 1 140, 160, 180 and 200 mV s from inner to outer.

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A new ternary monocopper(II) complex in terms of the diffusion of an equilibrium mixture of free and DNAbound complex to the electrode surface. The cyclic voltammetric technique was further employed to study the interaction of the copper(II) complex with HS-DNA. After addition of HS-DNA, positive shifts of the redox peak potentials with decreasing peak current are clearly observed as shown in Fig. 8(b). The decrease of the voltammetric current in the presence of HSDNA may be attributed to slow diffusion of the copper(II) complex bound to HS-DNA. It can be seen from Fig. 8(b) that, in the absence of DNA, the copper(II) complex shows a quasi-reversible oneelectron redox process (Ipc/Ipa ≈ 1) corresponding to Cu(II)/Cu(I) with the cathodic (Epc) and anodic peak potential (Epa) being 0.1462 and 0.0448 V (ΔEp = 0.1014 V), respectively. The free formal potential (Ef°’), taken as the average of Epc and Epa, was found to be 0.0955 V. While in the presence of HS-DNA with R = 10 (R = [DNA]/[complex]) the voltammetric peak currents decreased apparently, suggesting that there exists an interaction between the copper(II) complex and DNA.[51] The decrease of the voltammetric currents in the presence of HS-DNA may be attributed to slow diffusion of the copper(II) complex bound to DNA. The cathodic and anodic peak potentials of the complex are found to be 0.1460 and 0.0200 V. Clearly, the peak-to-peak separation becomes larger, as ΔEp = 0.1260 V, suggesting that in the presence of HS-DNA the electron transfer process becomes less reversible for the copper(II) complex. The formal potential of the Cu(I)/Cu(II) couple in binding form (Eb°’) is 0.0830 V. Obviously, the Eb°’ value of the copper(II) complex is shifted towards positive region by 0.0125 V, indicating that the copper(II) complex could bind intercalatively to HS-DNA.[51] The separation between Eb°’ and Ef°’ can be used to estimate the ratio of binding constants for the reduced and oxidized forms to DNA using the following equation:[52]   KR (4) ΔE° ′ ¼ E b ° ′  E f ° ′ ¼ 0:059 log KO where KR and KO are the binding constants of Cu(I) and Cu(II) forms to DNA, respectively. The ratio of constants for the binding of Cu(I) and Cu(II) ions to HS-DNA is estimated to be 1.63, indicating that the reduced form of the complex interacts strongly than the oxidized one. In order to quantitatively evaluate the affinity of the ternary copper(II) complex toward HS-DNA, the apparent binding constant (K) of the copper(II) complex with HS-DNA was obtained from the following equation:[52] 1 K ð1  AÞ ¼ K ½DNA 1  i=i 0

Figure 8. Cyclic voltammograms of complex [Cu(dabt)(pic)2] in the absence and presence of HS-DNA. (a) Plots of cathodic peak currents of [Cu(dabt)(pic)2] in the absence (1) and presence (2) of DNA versus square 1/2 root of the scan rate (ν ). (b) Cyclic voltammograms of the complex in the absence (red curve) and presence (blue curve) of HS-DNA. (c) Plot of 1/ [DNA] versus 1/(1  i/i0).

where [DNA] is the concentration of DNA, i0 and i are the peak current without and with DNA and A is a proportionality constant. Notably, the hypothesis condition of using equation (5) is that a 1:1 ‘association complex’ forms between the copper(II) complex and HS-DNA, and meanwhile [DNA] is much larger than the total concentration of the copper(II) complex in solution.[53] In other words, if the experimental data correspond well to the equation, this may suggest that the proportion of the ‘association complex’ was 1:1. According to the experimental data shown in Fig. 8(c), linear equations of [DNA] versus 1/(1  i/i0) were obtained with a linear correlation coefficient of 0.9961 for seven points, which showed that the binding number was 1, and the result is consistent with the hypothesis mentioned above. In addition, the K value of the copper(II) complex with HS-DNA obtained from the intercept was 1.79 × 105 M1. Thus, based on the above results of electrochemical

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(5)

X.-L. Wang et al. experiments, the binding mode of the complex to HS-DNA is in line with intercalation under our experimental conditions, which is consistent with the spectral results discussed above.

Table 6. In vitro anticancer activity of the complexes Complex

IC50 value (μM) SMMC-7721

A549

Hep G2

L02

6.2 ± 1.2 8.4 ± 0.5 3.6 ± 0.2

8.4 ± 1.5 10.2 ± 1.1 5.8 ± 0.5

3.4 ± 0.9 4.5 ± 0.3 10.8 ± 1.0

398 ± 15 388 ± 17 192 ± 11

Viscosity measurements To further confirm the interaction mode of the copper(II) complex with HS-DNA, viscosity measurements were also carried out. Generally, in classical intercalation the DNA helix lengthens as base pairs are separated to accommodate the bound ligand leading to increased DNA viscosity, whereas a partial, non-classical intercalation causes a bend in the DNA helix reducing its effective length and thereby its viscosity. Therefore, viscosity measurement is regarded as the least ambiguous and the most critical means investigating the binding mode of metal complexes with DNA in solution and provides stronger arguments for intercalative binding mode.[54] As shown in Fig. 9, it is obvious that the relative viscosity of HSDNA increases with increasing concentration of the complex. The result further indicates that the copper(II) complex can bind to DNA in the mode of intercalation, which is consistent with the foregoing conclusions. On the basis of the above discussion of the DNA-binding events for the copper(II) complex, both theoretically and experimentally, it has been shown that the resulting binding energy of the docked copper(II) complex correlates well with the experimental DNAbinding affinity. Thus, it is evident that there is a mutual complement between the experimental measurements and molecular docked calculations. The positive results of DNA-binding studies for the complex prompt us to investigate the in vitro anticancer activity of the copper(II) complex. In Vitro Antitumor Activities With the purpose of evaluating the potential antitumor activities of the ternary copper(II) complex, cytotoxicity assays of the copper(II) complex against three cancer cell lines, namely human hepatocellular carcinoma SMMC-7721, human lung adenocarcinoma A549 and human liver carcinoma Hep G2, along with the human normal cell line hepatocyte L02, were conducted using the SRB method, and cisplatin was used as a positive control to assess the cytotoxicity of the tested complex. The results are expressed as the half maximal inhibitory concentration (IC50) in Table 6. As evident form the table, the copper(II) complex possesses the most potent inhibitory effect against Hep G2 cell line, and its inhibitory activity is about three times that of the well-known anticancer drug (cisplatin).

[Cu(dabt)(pic)2] [Cu(bpy)(pic)2]a Cisplatina a

The IC50 values of [Cu(bpy)(pic)2] and cisplatin have been previously reported.[19] All values are mean ± SD of three independent experiments.

And the IC50 values of the complex for the other cancer cell lines are much higher than those of the clinically applied antitumor drug cisplatin, indicating that the in vitro anticancer activities of the present complex are less than that of cisplatin. However, the inhibition of cell proliferation produced by the copper(II) complex on the same batch of cell lines is still rather active. Particularly, the IC50 value of the copper(II) complex against the human normal cell line hepatocyte L02 is higher than that of cisplatin, suggesting that the complex acts very specifically on the selected tumor cell lines. These results imply that the copper(II) complex may have potential to be developed as an anticancer agent. Comparing the IC50 values of the present complex [Cu(dabt) (pic)2] with that of our previously reported analogous complex [Cu(bpy)(pic)2], we found that the values of the present complex against the three cancer cell lines are lower than those of [Cu (bpy)(pic)2],[19] indicating that the present complex possesses better antitumor activities than the previous one under identical experimental conditions. More interestingly, it is worth noting that the order of the antitumor activities of the two ternary monocopper (II) complexes against the selected cancer cell lines is in accordance with their DNA-binding abilities, implying that the anticancer activities of these copper(II) complexes may be related to, or originate from, their ability to intercalate the base pairs of DNA. In other words, the two monocopper(II) complexes might target DNA primarily, leading to cell death. This fact indicates that the DNAbinding abilities and the in vitro antitumor activities may possibly be tuned through changing the co-ligands in these copper(II) systems, which may open vast perspectives in understanding the structure–activity relationships of these kinds of metal complexes, and providing an important insight into the field of DNA interactions.

Conclusions

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Figure 9. Effect of increasing amount of complex on relative viscosity of HS-DNA at 289 (± 0.1) K.

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In order to investigate the influence of different co-ligands in ternary monocopper(II) complexes on DNA-binding properties as well as cytotoxic activities, a new ternary complex, [Cu(dabt)(pic)2], has been synthesized and structurally characterized using single-crystal X-ray diffraction. The reactivity towards DNA as well as cytotoxic activities of the copper(II) complex have also been investigated. Comparing the DNA-binding properties and in vitro anticancer activities with another ternary copper(II) complex with similar structure, it is obvious that the different co-ligands in these ternary copper(II) complexes may influence the DNA-binding affinities and in vitro anticancer activities. It is interesting that the DNA-binding abilities of these ternary copper(II) complexes are consistent with their in vitro cytotoxic activities. These results attest that varying the coligands in the ternary copper(II) complexes can create some

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A new ternary monocopper(II) complex interesting differences, which results in some differences in the DNA-binding behaviors and cytotoxic activities. This is a step towards enabling the rational design of novel metallodrugs.

Acknowledgments This project was supported by the National Natural Science Foundation of China (no. 51273184) and the NSFC-Shandong Joint Fund for Marine Science Research Centers (grant no. U1406402).

References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22]

M. Chauhan, K. Banerjee, F. Arjmand, Inorg. Chem. 2007, 46, 3072. F. Arjmand, M. Aziz, Eur. J. Med. Chem. 2009, 44, 834. M. J. Clarke, Coord. Chem. Rev. 2003, 236, 209. T. Boulikas, M. Vougiouka, Oncol. Rep. 2003, 10, 1663. E. Wong, C. M. Giandomenico, Chem. Rev. 1999, 99, 2451. B. Neto, A. Lapis, Molecules 2009, 14, 1725. M. Frezza, S. Hindo, D. Chen, A. Davenport, S. Schmitt, D. Tomco, Q. P. Dou, Curr. Pharm. Design 2010, 16, 1813. Y. Ma, L. Cao, T. Kavabata, T. Yoshino, B. B. Yang, S. Okada, Free Radic. Biol. Med. 1998, 25, 568. F. Liang, C. Wu, H. Lin, T. Li, D. Gao, Z. Li, J. Wei, C. Zheng, M. Sun, Bioorg. Med. Chem. Lett. 2003, 13, 2469. J. Easmon, G. Purstinger, G. Heinisch, T. Roth, H. H. Fiebig, W. Holzer, W. Jager, M. Jenny, J. Hofmann, J. Med. Chem. 2001, 44, 2164. G. Psomas, C. P. Raptopoulou, L. Iordanidis, C. Dendrinou-Samara, V. Tangoulis, D. P. Kessissoglou, Inorg. Chem. 2000, 39, 3042. L. D. Wang, K. Zheng, Y. T. Li, Z. Y. Wu, C. W. Yan, J. Mol. Struct. 2013, 1037, 15. M. Mourer, N. Psychogios, G. Laumond, A.-M. Aubertin, J.-B. Regnouf-de-Vains, Bioorg. Med. Chem. 2010, 18, 36. Y. N. Tian, P. Yang, Chinese J. Chem. 1996, 14, 428. H. Sasaki, Tetrahedron Lett. 1994, 35, 4401. F. Arnaud-Neu, J. M. Harrowfield, S. Michel, B. W. Skelton, A. H. White, Supramol. Chem. 2005, 17, 609. A. P. Marchand, A. Hazlewood, Z. Huang, S. K. Vadlakonda, J. D. R. Rocha, T. D. Power, K. M. Majerski, L. Klaic, G. Kragol, J. C. Bryan, Struct. Chem. 2003, 14, 279. J. M. Harrowfield, B. W. Skelton, A. H. White, Aust. J. Chem. 1995, 48, 1311. K. Zheng, M. Jiang, Y. T. Li, Z. Y. Wu, C. W. Yan, J. Mol. Struct. 2014, 1058, 97. G. Brauer (Ed), Handbook of Preparative Inorganic Chemistry, Academic Press, New York, 1965. G. M. Sheldrick, Acta Crystallogr. A 2008, A64, 112. G. M. Morris, R. Huey, W. Lindstrom, M. F. Sanner, R. K. Belew, D. S. Goodsell, A. J. Olson, J. Comput. Chem. 2009, 30, 2785.

[23] A. Robertazzi, A. V. Vargiu, A. Magistrato, P. Ruggerone, P. Carloni, P. de Hoog, J. Reedijk, J. Phys. Chem. B 2009, 113, 10881. [24] C. F. Macrae, P. R. Edgington, P. McCabe, E. Pidcock, G. P. Shields, R. Taylor, M. Towler, J. Appl. Crystallogr. 2006, 39, 453. [25] J. Marmur, J. Mol. Biol. 1961, 3, 208. [26] M. E. Reichmann, S. A. Rice, C. A. Thomas, P. J. Doty, J. Am. Chem. Soc. 1954, 76, 3047. [27] J. B. Chaires, N. Dattagupta, D. M. Crothers, Biochemistry 1982, 21, 3933. [28] M. E. Pacheco, L. Bruzzone, J. Lumin. 2012, 132, 2730. [29] P. T. Selvi, H. Stoeckli-Evans, M. Palaniandavar, J. Inorg. Biochem. 2005, 99, 2110. [30] G. Cohen, H. Eisenberg, Biopolymers 1969, 8, 45. [31] J. K. Barton, J. M. Goldberg, C. V. Kumar, N. J. Turro, J. Am. Chem. Soc. 1986, 108, 2081. [32] W. J. Geary, Coord. Chem. Rev. 1971, 7, 81. [33] K. Nakamoto, Infrared and Raman Spectra of Inorganic and Coordination Compounds, 5th ed, Wiley, New York, 1997. [34] S. X. Liu, W. S. Liu, M. Y. Tan, K. B. Yu. J. Coord. Chem. 1996, 39, 105. [35] Y. L. Song, Y. T. Li, Z. Y. Wu, J. Inorg. Biochem. 2008, 102, 1691. [36] P. Siega, V. Vrdoljak, C. Tavagnacco, R. DreosInorg, Inorg. Chim. Acta 2012, 387, 93. [37] D. Cremer, J. A. Pople, J. Am. Chem. Soc. 1975, 97, 1354. [38] Y. B. Zeng, N. Yang, W. S. Liu, J. Inorg. Biochem. 2003, 97, 258. [39] R. Rohs, I. Bloch, H. Sklenar, Z. Shakked, Nucleic Acids Res. 2005, 33, 7048. [40] A. Wolf, G. H. Shimer, Jr., T. Meehan, Biochemistry 1987, 26, 6392. [41] A. Dimitrakopoulou, C. Dendrinou-Samara, A. A. Pantazaki, M. Alexiou, E. Nordlander, D. P. Kessissoglou, J. Inorg. Biochem. 2008, 102, 618. [42] R. Rao, A. K. Patra, P. R. Chetana, Polyhedron 2008, 27, 1343. [43] F. J. Meyer-Almes, D. Porschke, Biochemistry 1993, 32, 4246. [44] B. C. Baguley, M. L. Bret, Biochemistry 1984, 23, 937. [45] R. F. Pasternack, M. Cacca, B. Keogh, T. A. Stephenson, A. P. Williams, E. J. Gibbs, J. Am. Chem. Soc. 1991, 113, 6835. [46] O. Stern, M. Volmer, Z. Phys. Chem. 1919, 20, 183. [47] U. Bierbach, Y. Qu, T. W. Hambley, J. Peroutka, H. L. Nguyen, M. Doedee, N. Farrell, Inorg. Chem. 1999, 38, 3535. [48] S. Mahadevan, M. Palaniandavar, Inorg. Chem. 1998, 37, 693. [49] A. J. Bard, L. R. Faulkner, Electrochemical Methods, Wiley, New York, 1980. [50] M. T. Carter, M. Rodriguez, A. J. Bard, J. Am. Chem. Soc. 1989, 111, 8901. [51] Y. M. Song, P. J. Yang, M. L. Yang, J. W. Kang, S. Q. Qin, B. Q. Lv, L. F. Wang, Transit. Metal Chem. 2003, 28, 712. [52] G. C. Zhao, J. J. Zhu, J. J. Zhang, H. Y. Chen, Anal. Chim. Acta 1999, 394, 337. [53] Q. Feng, N. Li, Y. Jiang, Anal. Chim. Acta 1997, 344, 97. [54] L. Jin, P. Yang, J. Inorg. Biochem. 1997, 68, 79.

Supporting Information Additional supporting information may be found in the online version of this article at the publisher’s web site.

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