In Vitro Cytotoxic Activities, DNA-, and BSA-Binding Studies of a New Dinuclear Copper(II) Complex...

J BIOCHEM MOLECULAR TOXICOLOGYVolume 28, Number 2, 2014

In Vitro Cytotoxic Activities, DNA-, and BSA-BindingStudies of a New Dinuclear Copper(II) Complex withN-[3-(Dimethylamino)propyl]-N′-(2-carboxylatophenyl)-Oxamide as LigandJing Jiao,1 Man Jiang,2 Yan-Tuan Li,1 Zhi-Yong Wu,1 and Cui-Wei Yan3

1Marine Drug & Food Institute Ocean University of China, Qingdao, Shandong 266003, People’s Republic of China;E-mail: [email protected] Municipal Medical Group, Qingdao, Shandong 266011, People’s Republic of China3College of Marine Life Science, Ocean University of China, Qingdao 266003, People’s Republic of China; E-mail: [email protected]

Received 24 July 2013; revised 24 September 2013; accepted 12 October 2013

ABSTRACT: A new dinuclear copper(II) complexbridged by N-[3-(dimethylamino)propyl]-N′- (2-carbo-xylatophenyl)oxamide (H3dmapob), and endcappedwith 2,2′-diamino-4,4′-bithiazole (dabt), namely [Cu2

(dmapob)(dabt)(CH3OH)(pic)]·(DMF)0.75·(CH3OH)0.25

has been synthesized and characterized by elementalanalysis, molar conductivity measurement, infraredand electronic spectra studies, and single-crystal X-raydiffraction. In the crystal structure, both copper(II)ions have square–pyramidal coordination geometries.The Cu···Cu separation through the oxamido bridgeis 5.176(9) A. A two-dimensional supramolecularframework is formed through hydrogen bonds andπ–π stacking interactions. The reactivities towardherring sperm DNA and bovine serum albumin (BSA)show that the complex can interact with the DNA viaintercalation mode and bind to the BSA responsiblefor quenching of tryptophan fluorescence by thestatic quenching mechanism. The in vitro anticanceractivities suggest that the copper(II) complex is activeagainst the selected tumor cell lines. The influenceof different bridging ligands in dinuclear complexes

Correspondence to: Yan-Tuan Li and Cui-Wei Yan.Contract Grant Sponsor: National Natural Science Foundation

of China.Contract Grant Number: 21071133, 51273184, and 81202399.Contract Grant Sponsor: The Program for Science and Technol-

ogy of Shandong Province.Contract Grant Number: 2011GHY11521.Contract Grant Sponsor: Natural Science Foundation of

Qingdao City.Contract Grant Number: 11-2-4-1-(9)gch, 12-1-3-52-(1)-nsh, and

12-1-4-16-(7)-jch.Jing Jiao and Man Jiang contributed equally to this study.

C© 2013 Wiley Periodicals, Inc.

on the DNA- and BSA-binding properties as well asanticancer activities is preliminarily discussed. C© 2013Wiley Periodicals, Inc. J. Biochem. Mol. Toxicol. 28:47–59,2014; View this article online at wileyonlinelibrary.com.DOI 10.1002/jbt.21535

KEYWORDS: Crystal Structure; μ-Oxamido-Bridge; Di-copper(II) Complex; DNA and BSA Binding; In VitroCytotoxic Activities

INTRODUCTION

The design, synthesis, and reactivities towardDNA and protein of metal complexes are of current in-terest in connection with gaining some insight into themechanism involving in the site-specific recognition ofDNA and the reactive models for protein–nucleic acidinteractions, and obtaining information about the ra-tional design and synthesis of new types of anticancermedicine [1–3].

Cisplatin is one of the most successful metal-basedantitumor drugs targeting DNA [4, 5], but the side ef-fects limit its clinical application [4, 6]. The detailedmolecular mechanism of its side effects involves cova-lent binding to DNA. To find highly effective, target-specific, and less toxic drugs, much effort has been de-voted to the development of metal-based anticanceragents binding to DNA through a noncovalent in-teraction. Three binding modes of metal complexeswith DNA exist in a noncovalent way: electrostatic,groove, and intercalation. Many important applications

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48 JIAO ET AL. Volume 28, Number 2, 2014

of these complexes require that they can bind to DNAvia an intercalative mode that could induce cellulardegradation [7]. The planarity of the ligand and thepresence of aromatic systems are factors that favor theintercalation [8, 9]. In addition, proteins are importantchemical substances and major targets for many typesof medicines. Studies on binding of metal complexeswith protein also interpret the metabolism and trans-porting process [10, 11]. Bovine serum albumin (BSA)has been frequently selected as the protein model pri-marily due to its water-soluble nature, unusual bind-ing properties, and similarity to human serum albumin[12]. Thus, the reactivities of metal complexes towardDNA and protein BSA are important in understandingthe mechanism of binding and in the rational designof metallodrugs. So far, in the context of reactivitiesof transition metal complexes with DNA and protein,many investigations have focused on the selection ofmetal ions and the design of ligands [13]. Copper be-ing a bioessential transition metal ion [14], its com-plexes have been synthesized and extensively exploredin virtue of their strong interactions with DNA and cy-totoxic activities [15,16]. Comparing with mononuclearcopper(II) complexes [17–19], relatively few studies ondicopper(II) complexes have been reported [20]. How-ever, enhancement of DNA-binding activity for dicop-per(II) complexes together with the fact that dicoppercenters exist in a large amount of proteins that playparamount roles in biology [21] stimulates us to de-sign and synthesize new dicopper(II) complexes. Indesigning and synthesizing dinuclear systems, it isgenerally known that N,N′-bis(substituted)oxamidescould be good bridging ligand candidates [20, 22].Many oxamido-bridged polynuclear complexes withsymmetric N,N′-bis(substituted)oxamide ligands havebeen prepared, while only a few asymmetric N,N′-bis(substituted)oxamide polynuclear complexes havebeen reported because of the difficulties in their syn-thesis [23]. Exploration of the interactions of thesecomplexes with DNA and protein BSA encouragesus to design and synthesize new dicopper(II) com-plex with asymmetric N,N′-bis- (substituted)oxamideas a bridge to get an insight into the reactiv-ity of such complexes toward DNA and proteinBSA.

In the present study, a new μ-oxamido-bridgeddinuclear copper(II) complex of the formula [Cu2(dmapob)(dabt)(CH3OH)(pic)]·(DMF)0.75·(CH3OH)0.25,where H3dmapob denotes N-[3-(dimethylamino)propyl]-N′- (2-carboxylatophenyl)oxamide, and dabtrepresents 2,2′-diamino-4,4′-bithiazole, has beensynthesized and structurally characterized by single-crystal X-ray diffraction. In vitro anticancer activities,and the reactivity toward protein BSA and herringsperm (HS)-DNA were also studied.

MATERIALS AND METHODS

Materials and Reagents

The ligand H3dmapob was prepared according tothe literature [24]. All chemicals used in the synthe-sis were of reagent grade. Doubly distilled water wasused to prepare buffers. Ethidium bromide (EB), BSA,and HS-DNA were purchased from Sigma (St. Louis,Missouri) and used as received.

Apparatus and Measuring Techniques

The carbon, hydrogen, and nitrogen elementalanalyses were measured with a Perkin–Elmer ele-mental analyzer Model 240. Molar conductance wasmeasured with a Shanghai DDS-11A conductometer(ShangHai ShengKe Instrument Equipment Co., Ltd,Shanghai, People’s Republic of China). Infrared (IR)spectra were recorded on KBr pellets with a Nicoletmodel impact 470 FTIR spectrophotometer (AmericaNieolet Instrument, Beijing, People’s Republic ofChina) in the range 4,000∼400 cm−1. Electronic spec-tra were measured at room temperature on a Cary300 spectrophotometer (Ameirca Varian Instrument,California) equipped with quartz cuvettes of 1 cm pathlength. Fluorescence was measured on a FP-6200 fluo-rometer equipped with quartz cuvettes at ambient tem-perature. A CHI 832 electrochemical analyzer (Shang-hai CHI Instrument, Shanghai, People’s Republic ofChina) in connection with a glassy carbon working elec-trode (GCE), a saturated calomel reference electrode,and a platinum wire counter electrode was used for theelectrochemical measurement. The GCE surface wasfreshly polished to a mirror prior to each experimentwith 0.05-μm α-Al2O3 paste and then cleaned in wa-ter for 5 min. Viscosity measurements were carried outusing an Ubbelohde viscometer immersed in a thermo-static water bath maintained at 289 K.

Synthesis of the Dicopper(II) Complex

A methanol solution (5 mL) containingCu(pic)2·6H2O (0.0627 g, 0.1 mmol) was dropwiseadded to a methanol solution (5 mL) of H3dmapob(0.0146 g, 0.05 mmol) and piperidine (0.75 ml,0.15 mmol) at room temperature. The mixture wasstirred quickly for 30 min, and a methanol solution(5 mL) of dabt (0.01 g, 0.05 mmol) was then dropwiseadded to the mixture. After stirring for 8 h at 328 K,the reaction mixture was filtered, and the precipitatewas dissolved in component solvent (10 mL) ofdimethylformamide (DMF) and MeOH (1:5). Browncrystals suitable for X-ray analysis were obtained by

J Biochem Molecular Toxicology DOI 10.1002/jbt

Volume 28, Number 2, 2014 OXAMIDO-BRIDGED DICOPPER(II) COMPLEX 49

TABLE 1. Crystal Data and Details of the Structure Determi-nation for the Complex

Empirical formula[Cu2C27H28N10O12S2]·(CH4O)1/4·(C3H7NO)3/4

Formula weight 938.63Crystal system TriclinicSpace group P-1Unit cell dimensions (A, ◦) a = 12.165(14), b = 12.231(15),

c = 15.14(3)α = 98.76(2), β = 107.70(2),

γ = 112.252(17)Volume (A3) 1892(5)Z 2Calculated density (g·cm−3) 1.647μ(Mo-Kα) (mm−1) 1.312F(000) 961Crystal size (mm) 0.10 × 0.14 × 0.26Temperature (K) 296Radiation (Mo-Kα) (A) 0.71073Limiting indices −15 ≤ h ≤ 13, −14 ≤ k ≤ 15, −12 ≤

l ≤ 18Total unique data, R(int) 9258, 7271, 0.0254θ range for data collection 2.02◦ to 26.15◦Observed data [I > 2σ (I)] 4457R, ωR2, S 0.0836, 0.2878, 1.124Maximum average shift/error 0.000, 0.000

slow evaporation of the solution at room temperaturefor 2 weeks. Yield: 0.0384 g (82%). Anal. Calcd forCu2C29.5H34.25N10.75O13S2: C, 37.75; H, 3.68; N, 16.04.Found: C, 37.92; H, 3.65; N, 16.21.

Determination of Crystal Structure

The single crystal used for data collection ofthe dicopper(II) complex was selected and mountedon a Bruker APEX area-detector diffractometerwith graphite monochromatic Mo Kα radiation (λ =0.71073 A) at the temperature of 296 K. The crystalstructures were solved by the directed method followedby Fourier syntheses. Structure refinements were per-formed by full matrix least squares procedures usingSHELXL-97 on F2 [25]. The solvent methanol and DMFmolecules are disordered for occupying too-near posi-tions. Their occupancies were refined at the beginningand then fixed to 0.25 and 0.75, respectively. Seven re-strains to the two solvent molecules were employed toget reasonable geometries. The hydroxyl H atoms werefound in difference Fourier maps and then refined asriding. Other H atoms were placed in calculated posi-tions, with C–H = 0.96 (methyl), 0.97 (methylene), 0.93´A (sp2) and N–H = 0.86 A, then refined in riding mode,with Uiso(H) = 1.5 Ueq (methyl) and 1.2 Ueq (carrieratoms). Crystal data and structural refinement param-eters for the dicopper(II) complex are summarized inTable 1, and selected bond distances and angles arelisted in Tables 2 and 3.

TABLE 2. Selected Bond Distances (A) and Angles (◦) for theComplex

Cu1–O1 1.903(6) Cu1–O6 2.446(7)Cu1–N1 1.967(6) Cu1–N2 1.951(7)Cu1–N3 2.043(7) Cu2–O3 1.957(6)Cu2–O4 1.928(6) Cu2–O5 2.195(10)Cu2–N4 1.953(7) Cu2–N5 1.964(7)

O1–Cu1–O6 94.9(3) O1–Cu1–N1 90.2(3)O1–Cu1–N2 165.0(3) O1–Cu1–N3 88.0(3)O6–Cu1–N1 97.7(3) O6–Cu1–N2 99.5(3)O6–Cu1–N3 94.2(3) N1–Cu1–N2 84.0(3)N1–Cu1–N3 168.1(3) N2–Cu1–N3 94.9(4)O3–Cu2–O4 84.0(2) O3–Cu2–O5 92.6(3)O4–Cu2–O5 96.5(3) O3–Cu2–N4 95.7(3)O3–Cu2–N5 171.5(3) O4–Cu2–N4 171.1(3)O4–Cu2–N5 96.7(3) O5–Cu2–N4 92.4(3)O5–Cu2–N5 95.7(3) N4–Cu2–N5 82.3(3)

TABLE 3. Hydrogen-Bonding Geometries (A, ◦) for theComplex

D–H···A D–H H···A D···A D–H···A

O5–H5A···O2a 0.93 1.72 2.600(12) 158.0O14–H14···O7 0.82 2.08 2.84(4) 153.1N6–H6A···O3 0.86 2.08 2.822(10) 143.5N6–H6B···O13 0.86 2.02 2.68(3) 132.8N6–H6B···O14b 0.86 1.86 2.70(4) 166.5N7–H7A···O4 0.86 2.08 2.831(9) 146.2N7–H7B···O6c 0.86 2.07 2.787(9) 140.2

a Symmetry codes 1–x, 1–y, 1–z.b Symmetry codes –x, –y, 1–z.c Symmetry codes 1–x, –y, 1–z.

In Vitro Cytotoxic Activity Evaluation bySulforhodamine B Assays

In vitro cytotoxic activity of the dicopper(II) com-plex was evaluated against two cancer cell lines in-cluding SMMC-7721 and A549 by using the Sulforho-damine B (SRB) assay. All cells were cultured inRoswell Park Memorial Institute 1640 (RPMI 1640)supplemented with 10% (v/v) fetal bovine serum, 1%(w/v) penicillin (104 U/mL), and 10 mg/mL strep-tomycin. Cell lines are maintained at 310 K in a5% (v/v) CO2 atmosphere with 95% (v/v) humid-ity. Cultures were passaged weekly using trypsin-ethylenediaminetetraacetic acid to detach the cells fromtheir culture flasks. The dicopper(II) complex and cis-platin were dissolved in dimethyl sulfoxide (DMSO)and diluted to the required concentration with culturemedium. The content of DMSO in the final concen-trations did not exceed 0.1%. At this concentration,DMSO was found to be nontoxic to the cells tested.Rapidly growing cells were harvested, counted, andincubated at the appropriate concentration in 96-wellmicro-plates for 24 h. The tested complex dissolved inculture medium was then applied to the culture wellsto achieve final concentrations ranging from 10−3 to



102 μg/mL. Control wells were prepared by the addi-tion of a culture medium without cells. The plates wereincubated at 310 K in a 5% CO2 atmosphere for 48 h.Upon completion of the incubation, the cells were fixedwith ice-cold 10% trichloroacetic acid (100 mL) for 1 hat 277 K, washed five times in distilled water, allowedto dry in the air, and stained with 0.4% SRB in 1% aceticacid (100 mL) for 15 min. The cells were washed fourtimes in 1% acetic acid and air dried. The stain was solu-bilized in 10-mM unbuffered tris base (100 mL) and theoptical density of each well was measured at 540 nm ona microplate spectrophotometer. The IC50 values werecalculated from the curves constructed by plotting cellsurvival (%) versus the dicopper(II) complex concen-tration (μg/mL).

DNA-Binding Studies

All experiments involving HS-DNA were per-formed in tris(hydroxymethyl)aminomethane-HCl(Tris-HCl) buffer solution (pH 7.13). Solution of HS-DNA in Tris-HCl buffer gave the ratio of ultraviolet(UV) absorbance at 260 and 280 nm, A260/A280, of ca.1.9, indicating that HS-DNA was sufficiently free ofprotein [26]. The concentration of the prepared HS-DNA stock solution was determined according to itsabsorbance at 260 nm using ε260 = 6600 L·mol−1·cm−1.Stock solution of HS-DNA was stored at 277 K and usedafter no more than 4 days. Concentrated stock solutionof the dicopper(II) complex was prepared by dissolvingthe dicopper(II) complex in DMSO and diluted suit-ably with Tris-HCl buffer to required concentrationsfor all the experiments. Absorption spectral titrationexperiment was performed by keeping the concentra-tion of the dicopper(II) complex constant while varyingthe HS-DNA concentration. Equal solution of HS-DNAwas added to the dicopper(II) complex solution andreference solution to eliminate the absorbance of HS-DNA itself. In EB, fluorescence displacement experi-ment, 5 μL of the EB-Tris-HCl solution (1 mmol·L−1)was added to 1 mL of HS-DNA solution (at saturatedbinding levels) [27], stored in the dark for 2 h. Thenthe solution of the dicopper(II) complex was titratedinto the DNA–EB mixture and then diluted in Tris-HClbuffer to 5 mL, producing the solutions with the var-ied mole ratio of the dicopper(II) complex to HS-DNA.Before measurements, the mixture was shaken up andincubated at room temperature for 30 min. The fluores-cence spectra bound to HS-DNA were obtained at anemission wavelength of 584 nm in the fluorometer. Theelectrochemical titration experiments were performedby keeping the concentration of the dicopper(II) com-plex constant while varying the HS-DNA concentrationusing the solvent of Tris-HCl buffer. In viscosity mea-

surement, HS-DNA samples approximately 200 basepairs in length were prepared by sonication to mini-mize complexities arising from HS-DNA flexibility [28].Flow times were measured with a digital stopwatch,and each sample was measured three times, and anaverage flow time was calculated. Relative viscositiesfor HS-DNA in the presence and absence of the di-copper(II) complex were calculated from the relationN = (t – t0)/t0, where t is the observed flow time ofHS-DNA–containing solution and t0 is that of Tris-HClbuffer alone. Data were presented as (N/N0)1/3 versusbinding ratio [29], where N is the viscosity of HS-DNAin the presence of the dicopper(II) complex and N0 isthe viscosity of HS-DNA alone.

BSA-Binding Studies

All experiments involving BSA were performedin 50-mM NaCl/Tris-HCl buffer solution (pH 7.13);and solutions of BSA and the dicopper(II) complexwere prepared by dissolving them in the NaCl/Tris-HCl buffer solution to required concentrations, respec-tively. For UV-absorption experiment, a 5-mL solutionof BSA (10 μM) was titrated and BSA concentrationis kept while varying the concentration of the dicop-per(II) complex. Equal solution of the dicopper(II) com-plex was added to the reference solutions to eliminatethe absorbance of the complex itself. In the trypto-phan fluorescence quenching experiment, quenchingof the tryptophan residues of BSA [30] was done bykeeping the concentration of BSA constant while vary-ing the dicopper(II) complex (quencher) concentration,producing the solutions with the varied mole ratio ofthe quencher to BSA. The fluorescence spectra wererecorded at an excitation wavelength of 295 nm and anemission wavelength of 347 nm in the fluorometer aftereach addition of the quencher.

RESULTS

Synthesis and General Properties of theDicopper(II) Complex

In this study, our purpose was to obtain a newdicopper(II) complex with asymmetric N,N′-bis-(substituted)oxamide as a bridging ligand. For that,H3dmapob was chosen as bridging ligand becauseit can coordinate with metal ions through oxygensand nitrogens of oxamido, and oxygens of carboxyl.Meanwhile, dabt and copper picrate were used as theterminal ligand and metal salt, respectively. Inthe course of preparing the dicopper(II) complex,the use of piperidine as base makes the bridgingligand (H3dmapob) coordinate with copper(II) ionthrough the deprotonated oxamido nitrogen atoms.



Indeed, elemental analyses indicate that the reac-tion of H3dmapob with Cu(pic)2·6H2O and dabt in1:2:1 mole ratio yielded the dicopper(II) complexidentified as [Cu2(dmapob)(dabt)(CH3OH)(pic)]·-DMF0.75·(CH3OH)0.25.

The dicopper(II) complex is very soluble in DMFand DMSO to given stable solutions at room tempera-ture, moderately soluble in methanol and acetonitrile,and practically insoluble in carbon tetrachloride, chlo-roform, water, and benzene. In the solid state, the di-copper(II) complex is fairly stable in air so as to allowphysical measurements.

General Characterization of theDicopper(II) Complex

Molar Conductivity

The molar conductance value of the dicopper(II)complex (50 −1·cm2·mol−1 in DMF solution) fallsin the expected range for nonelectrolytic nature [31],suggesting that the dicopper(II) complex in solu-tions consists of a netural species [Cu2(dmapob)(dabt)(CH3OH)(pic)], in which the picrate group is involvedwith the coordination, and is consistent with the fol-lowing IR and electronic spectra analyses as well as thedetermination of the crystal structure.

IR Spectra

The IR spectrum taken in the region of4000∼400 cm−1 provides some information regardingthe mode of coordination in the dicopper(II) complexand is analyzed in a careful comparison with that ofthe bridging ligand (H3dmapob). In the IR spectrum,the carbonyl (C=O) stretching vibration at 1671 cm−1

for the free ligand is redshifted to 1642 cm−1 in the di-copper(II) complex, which indicates that the oxygen ofthe carbonyl takes part in coordinating with the cop-per(II) ions. This shift has often been used as definiteproof of an oxamido bridge [32]. On the other hand, theband at 1548 cm−1 associates with ν(C=N) vibration ofthe terminal ligand (dabt), suggesting that the nitro-gen atoms from dabt coordinate with copper(II) ions inthe dicopper(II) complex. Furthermore, the O–H out-of-plane bending vibration of the free Hpic at 1151 cm−1

disappears, indicating that the hydrogen atom of thehydroxyl is replaced. In addition, the inplane deforma-tion vibration of the phenoxy ν(C–O) of the picrateat 1265 cm−1 is shifted toward higher frequency at1309 cm−1 in the dicopper(II) complex, which indicatesthe coordination of the picrate via the phenolic oxygen[33], in accord with the molar conductance value of thedicopper(II) complex.

Electronic Spectra

To obtain further structural information, the elec-tronic spectrum of the dicopper(II) complex was mea-sured in the UV–visible region (200–800 nm) in DMSO.Spectrum obtained for the dicopper(II) complex at dif-ferent concentrations (1.0 × 10−4 to 1.0 × 10−6 M)obeyed the Beer–Lambert law, indicating that the di-copper(II) complex stays intact at these concentrations[34], which is consistent with the molar conductancemeasurement and IR data. For the dicopper(II) com-plex, two absorption bands were observed. The intenseband at 230 nm may be attributed to π–π* transitionsof dabt ligand in the dicopper(II) complex. Besides, theless intense band at 355 nm in the spectrum is typicalof charge transfer transition between picrate and metal[35].

The structure of the dicopper(II) complex is furtherconfirmed through the following single-crystal X-raydiffraction.

Structure Description of the Dicopper(II)Complex

As illustrated in Figure 1A, the dicopper(II) com-plex takes a cis-oxamide configuration, dmapob3− asbridging ligand, and dabt as terminal ligand. It alsocontains a coordinated picrate group and a coordinatedmethanol molecule. Besides, one dicopper(II) complexhas equivalently one-fourth methanol and three-fourthDMF as solvent molecules of crystallization. Both cop-per(II) ions separated by the oxamide group with adistance of 5.176(9) A adopt square–pyramidal coordi-nation geometries with the τ values [36] of 0.05 (Cu1)and 0.01 (Cu2). Atom Cu1 at the endo and atom Cu2at the exo site of dmapob3− ligand deviates 0.224(4)and 0.147(4) A off their basal plane, respectively. Theirapical positions are occupied by a pic− ion and amethanol molecule, respectively, with correspondingaxial Cu1–O6 and Cu2–O5 bond lengths of 2.446(7)and 2.195(10) A. The bonds Cu1–N1 [1.967(6) A] andCu1–N2 [1.951(7) A] are shorter than Cu1–N3 [2.043(7)A], which is consistent with the stronger donor abilitiesof the sp2 hybrid N atoms than the sp3 one.

The ligand dmapob3− bridges the two copper(II)ions in an usual chelating mode by the oxamide groupwith the bite angles of 84.0(3)◦ for atom Cu1 and 84.0(2)◦

for atom Cu2. In the five chelating rings around atomsCu1 and Cu2, the three five-membered ones are almostplanar and the two six- membered ones are folded. Theone involving propylene diamine fragment is close tohalf-chair conformation with the puckering parametersof Q = 0.496(12) A, θ = 35.5(12)◦ and ϕ = 205(2)◦. Whilethe one involving carboxylate group is boat, with Q =0.249(9) A, θ = 89(2)◦ and ϕ = 295.4(19)◦.



FIGURE 1. (A) An ORTEP view of [Cu2(dmapob)(dabt)(CH3OH)(pic)]·(DMF)0.75·(CH3OH)0.25 with the thermal ellipsoids at 20%probability level. H atoms are shown as small spheres of arbi-trary radii. Hydrogen bonds are shown as dotted lines, (B) a one-dimensional chain parallel to the b axis, formed through hydrogenbonds. Hydrogen bonds are shown as dotted lines. [Symmetry codes:(i) 1–x, 1–y, 1–z; (ii) 1–x, –y, 1–z], (C) the π–π stacking interactionsalong the a axis in the crystal. [Symmetry code: (iii) –x, –y, 1–z].

In the crystal, these complexes are connected byhydrogen bonds to form a one-dimensional strip par-allel to the b axis (Table 3, Figure 1B). Both sides ofthe strip are inlaid with dabt ligands and pic− ions.Moreover, along with the piling of the strips along the a

axis, there is an offset π–π stacking interaction betweenthe thiazole ring containing atom S1 and the benzenering of pic− ion at –x, –y, 1–z (iii), and vice versa (Fig-ure 1C). The angle of the thiazole ring and the ben-zene ring is 13.3(5)◦. The separations of the overlappedatoms to the opposite aromatic ring are 3.563(8) (S1iii),3.292(13) (C24iii), and 3.364(12) A (C25iii). The solventmethanol molecules also take part in the connectionof the strips using hydrogen bonds. Through the twokinds of interactions, these complexes finally are as-sembled to a two-dimensional layer extending alongthe a0b plane.

In Vitro Anticancer Activities of theDicopper(II) Complex

To explore the potential antitumor activities of thedicopper(II) complex, the cytotoxicity assays of thedicopper(II) complex and cisplatin against two can-cer cell lines human hepatocellular carcinoma SMMC-7721 and human lung adenocarcinoma A549 were con-ducted in our study. The IC50 values for SMMC-7721and A549 are 12.5 ± 0.3 and 17.9 ± 0.8 μg/mL for the di-copper(II) complex, and 5.4 ± 0.2 and 7.6 ± 0.4 μg/mLfor cisplatin.

DNA Interaction Studies

Electronic Absorption Titration

The application of electronic absorption spec-troscopy is one of the most useful techniques forDNA-binding studies of metal complexes. In general,the hypochromism and redshift are associated withthe binding of metal complexes to DNA helix dueto the intercalative mode involving a strong stackinginteraction between the aromatic chromophore of thecomplexes and the base pairs of the DNA [37]. Theabsorption spectra of the dicopper(II) complex in theabsence and presence of HS-DNA at different concen-trations are given in Figure 2A. As shown in this fig-ure, with increasing concentration of HS-DNA, the ab-sorption band at observed 230 nm presented significanthypochromism of 16.5% at a ratio of [DNA]/[complex]of 6, and accompanied a slight redshift of 3 nm. Theseresults suggest that the dicopper(II) complex is bindto HS-DNA via an intercalation mode that involvesπ–π stacking interactions between the aromatic chro-mophores and the DNA base pairs, which can berationalized by the following reasons. When the di-copper(II) complex intercalates to the base pairs of HS-DNA, the π* orbital of the intercalated ligand (dabt)in the dicopper(II) complex can couple with π orbitalof the base pairs, thus decreasing the π–π* transition



FIGURE 2. (A) Absorption spectra of the dicopper(II) complex upon the titration of HS-DNA. Arrow indicates the change upon increasing theHS-DNA concentration (the red line for the absence of HS-DNA), (B) plot of [DNA]/(εa–εf) versus [DNA] for the absorption titration of HS-DNAwith the dicopper(II) complex, (C) emission spectra of the HS-DNA-EB system upon titration of the dicopper(II) complex. Arrow shows thechange upon increasing complex concentration, (D) plot of I0/I versus [complex] for the titration of the complex to HS-DNA-EB system.

energy and leading to bathochromism. On the otherhand, the coupling π* orbital is partially occupied, thusdecreasing the transition probabilities and resulting inhypochromism.

To enable quantitative evaluation, the affinity ofthe dicopper(II) complex toward HS-DNA, the intrinsicbinding constant Kb of the dicopper(II) complex withHS-DNA is obtained by analyzing the absorption spec-tral data using the following equation [37]:

[DNA]/(εa − εf) = [DNA]/(εb − εf) + 1/Kb(εa − εf)(1)

where [DNA] is the concentration of HS-DNA and εa,εf, and εb correspond to the extinction coefficient, re-spectively, for addition of DNA to the dicopper(II) com-plex, for the free complex, and for the complex in thefully bound form. From the plot of [DNA]/(εa–εf) ver-sus [DNA], shown in Figure 2B, the binding constant

Kb for the dicopper(II) complex was estimated to be8.63 × 104 M−1 (R = 0.9992, for seven points), which islower than those of typical intercalators (e.g., EB-DNA,∼106 M−1) [38], and has the same level as those of someDNA intercalative dicopper(II) complexes (∼104 M−1)[20], but higher than some mononuclear copper(II)complexes [18].

Fluorescence Titration

The EB fluorescence displacement experimentswere used to further investigate the interaction modeof the dicopper(II) complex with HS-DNA. The intrin-sic fluorescence intensity of DNA is very low, and thatof EB in the Tris buffer is also not high due to quench-ing by the solvent molecules. However, with the addi-tion of another molecule, which binds to DNA morestrongly than EB, the fluorescence intensity of EB can



be quenched due to the decrease of the binding sites ofDNA available for EB [39]. Thus, EB can be used to aprobe the interaction of complexes with DNA. In ourexperiment, as illustrated in Figure 2C, the fluorescenceintensity of EB at 584 nm shows a remarkable decreas-ing trend with the increasing concentration of the di-copper(II) complex, suggesting that the EB moleculesare displaced from their HS-DNA binding sites and arereplaced by the dicopper(II) complex. This observationis often the characteristic of intercalation [40].

To quantify the binding strength of the dicopper(II)complex with HS-DNA, the linear Stern–Volmer equa-tion was employed:

I0/I = 1 + Ksv[Q] (2)

where I0 and I represent the fluorescence intensi-ties in the absence and presence of quencher, respec-tively. Ksv is a linear Stern–Volmer quenching con-stant and Q is the concentration of quencher. Fromthe quenching plot of I0/I versus [Q], illustrated inFigure 2D, the Ksv value for the dicopper(II) complexis obtained by the ratio of the slope to intercept as7.68 × 104 (R = 0.9948, for eight points).

Cyclic Voltammetric Studies

The application of cyclic voltammetric techniqueto the study of interaction between metal complexesand DNA provides a useful complement to the pre-viously used spectral studies [41]. To further clarifythe interaction mode between the dicopper(II) com-plex and HS-DNA, the cyclic voltammetric techniqueis employed, and the results of cyclic voltammogramof the dicopper(II) complex in the absence and pres-ence of HS-DNA are shown in Figure 3. It can be seenfrom this figure that in the absence of HS-DNA (redline), the dicopper(II) complex has been found to showa couple of waves corresponding to Cu(II)/Cu(I) withthe cathodic (Epc) and anodic peak potential (Epa) be-ing –0.346 and –0.095 V, respectively. The separation ofanodic and cathodic peaks (�Ep) is found to be 0.251V, indicating a quasireversible one-electron redox pro-cess (Ipc/Ipa ≈ 1) in the dicopper(II) complex. The for-mal potential of the Cu(I)/Cu(II) couple in free form(Ef

o′), taken as the average of Epc and Epa is –0.221 V.While in the presence of HS-DNA (blue line) with R = 5(R = [DNA]/[complex]), the cyclic voltammogramsof the dicopper(II) complex exhibited certain shifts inthe anodic and cathodic peak potentials followed bydecrease in both peak currents, indicating the inter-action existing between the dicopper(II) complex andHS-DNA [42]. The drop of the voltammetric current inthe presence of HS-DNA may be attributed to the in-creased difficulty in diffusion of the dicopper(II) com-plex bound to HS-DNA. The cathodic and anodic peak

FIGURE 3. Cyclic voltammograms of the dicopper(II) complex inthe absence (the red line) and presence (the blue line) of HS-DNA.

potentials are found to be at –0.383 and –0.009 V, re-spectively. The peak-to-peak separation becomes largerwith �Ep = 0.374 V, suggesting that in the presenceof HS-DNA the electron-transfer process seems to be-come less reversible for the dicopper(II) complex. Theformal potential of the Cu(I)/Cu(II) couple in bindingform (Eb

o′) is –0.196 V. It is noteworthy that the Ebo′

value of the complex is shifted toward positive regionby 0.025 V, indicating that the dicopper(II) complexcould bind intercalatively to HS-DNA. The separationbetween Eb

o′ and Efo′ can be used to estimate the ra-

tio of binding constants for the reduced and oxidizedforms to DNA using the equation as follows [43]:

Eo ′b − Eo ′

f = 0.059log[KCu(I)/KCu(II)] (3)

where KCu(I) and KCu(II) are the binding constants ofCu(I) and Cu(II) forms to DNA, respectively. The ra-tio of constants for the binding of the Cu(I) and Cu(II)ions to HS-DNA was estimated to be 2.65 for the di-copper(II) complex, suggesting that the reduced formof the dicopper(II) complex interacts more stronglythan the oxidized one. These results are in agreementwith the above spectral observations.

Viscosity Measurements

Viscosity measurement, which is sensitive to thechanges in the length of DNA molecule, is regardedas the least ambiguous and the most critical test ofevaluating the binding mode of metal complexes withDNA in solution, and provides stronger arguments forintercalative binding mode [44]. A classical intercala-tion model results in lengthening the DNA helix, asbase pairs are separated to accommodate the bound



FIGURE 4. Effect of the increasing amount of the dicopper(II) com-plex on the relative viscosity of HS-DNA at 289 (±0.1) K, [DNA] =0.1 mM.

ligand leading to the increase of HS-DNA viscosity. Incontrast, a semi-intercalation of ligand could bend (orkink) DNA helix, reduce its effective length, and con-comitantly its viscosity [45]. To further clarify the inter-action mode of the dicopper(II) complex with HS-DNA,viscosity measurements were carried out on the DNAby varying the concentration of the added dicopper(II)complex. The results of the effect of the dicopper(II)complex on the viscosities of HS-DNA are shown inFigure 4. As illustrated in the figure, on increasing theamount of the dicopper(II) complex, the relative vis-cosity of HS-DNA increased steadily, showing that thedicopper(II) complex binds to HS-DNA in the mode ofintercalation. Thus, the results obtained from viscositystudies are consistent with our foregoing conclusions.

BSA-Binding Properties

Fluorescence Quenching Studies

Generally, the fluorescence of protein is causedby three intrinsic characteristics of the protein, namelytryptophan, tyrosine, and phenyl alanine residues. Ac-tually, the intrinsic fluorescence of many proteins ismainly contributed by tryptophan alone. The emissionintensity depends on the degree of exposure of the twotryptophan side chains [46], Trp-134 and Trp-212, to thepolar solvent. The effect of the dicopper(II) complex onBSA fluorescence intensity is shown in Figure 5A. Asillustrated in this figure, it is obvious that BSA has astrong fluorescence emission peaked at 347 nm. WhenBSA was titrated with different concentrations of the di-copper(II) complex, a remarkable intrinsic fluorescence

decrease of BSA was observed, indicating that the in-teraction of the dicopper(II) complex with BSA couldcause changes in the protein’s secondary structure lead-ing to changes in tryptophan environment of BSA [47].Quenching of fluorescence of BSA by the complexesis either by a static or a dynamic mechanism. Staticquenching refers to quenching through fluorophore–quencher complex formation, and dynamic quenchingrefers to a process that fluorophore and quencher comeinto contact during the transient existence of the excitedstate.

The quenching mechanism of the complexes withBSA was probed using the Stern–Volmer equation:

I0/I = 1 + Ksv[Q] = 1 + Kq τ0[Q] (4)

I0 and I represent the fluorescence intensities in the ab-sence and presence of quencher, respectively. Q is theconcentration of quencher. Ksv is a linear Stern–Volmerquenching constant. Kq is the biomolecular quenchingrate constant, τ 0 is the average lifetime of the fluo-rophore in absence of the quencher as 10−8 s. From thequenching plot of I0/I versus [complex] (Figure 5c), theKsv and Kq values for the complex are 3.27 × 104 M−1

and 3.27 × 1012 M−1·s−1 (R = 0.9915 for nine points),respectively.

UV Absorption Spectra Studies

Ultraviolet absorption measurement is also a sim-ple method to explore the structural changes in proteinsand the type of quenching of BSA by the complexes [48].Dynamic quenching has no function on the absorptionspectrum because it only affects the excited states of thefluorophore. However, static quenching will frequentlyresult in perturbation of the absorption spectrum of thefluorophore with fluorophore–quencher complex for-mation in the ground state [49]. As can be seen fromFigure 5B, BSA has two absorption peaks, one strongabsorption located at 227 nm, which reflects the frame-work conformation of protein, and another weak ab-sorption at 278 nm due to the aromatic amino acid. Thetwo absorption peaks of BSA at 227 and 278 nm pre-sented hypochromism and accompanied the obviousredshifts at 227 nm with the addition of the dicopper(II)complex.

Binding Constant and Number of Binding Sites

For the static quenching interaction, the bindingconstant (K) and the number of binding sites (n) can bedetermined according to the following equation [50]:

log[(F0 − F )/F ] = log K+nlog[Q] (5)

where F0 and F represent the fluorescence intensitiesin the absence and presence of quencher, respectively.



FIGURE 5. (A) Emission spectra of BSA upon the titration of the dicopper(II) complex. Arrow shows the change upon the increasing complexconcentration, (B) absorption spectra of BSA upon the titration of the dicopper(II) complex. Arrow indicates the change upon increasing thecomplex concentration, (C) plot of I0/I versus [complex] for the titration of the complex to BSA, (D) plot of log[(F0–F)/F] versus log[Q] for thetitration of the complex to BSA.

K is the binding constant of the dicopper(II)complex with BSA, n is the number of binding sites.As can be seen in Figure 5D, K and n of the dicopper(II)complex can be obtained. The binding constant K is9.6 × 107 M−1 (R = 0.9985 for eight points), and thebinding sites n is 1.50. The results show that there is astrong binding force with one binding site between thedicopper(II) complex and BSA.

DISCUSSION

If we compare Kb and Ksv values of the presentdicopper(II) complex with those of our previously re-ported analogous dicopper(II) complex [Cu2(apopoxd)(dabt)](ClO4)·2H2O [20], where H3apopoxd standsfor N-(2-aminopropyl)-N′-(2-oxidophenyl)oxamide,we find that the Kb and Ksv values of the presentdicopper(II) complex are both higher than those ofthe previously reported ones, indicating that thepresent dicopper(II) complex can bind to HS-DNAmore strongly by intercalation. From the structure ofthe two dicopper(II) complexes’ point of view, it is

obvious that the two dicopper(II) complexes have thesame metal ion and teminal ligand (dabt), and themain differences between them are the structures ofthe bridging ligands, which resulted in an increase inthe DNA-binding affinity of the present dicopper(II)complex. On the other hand, the in vitro cytotoxicactivities of the present dicopper(II) complex areless than those of cisplatin, but the inhibition of cellproliferation produced by the present dicopper(II)complex on the same batch of cell lines and underidentical experimental conditions is still rather active.Furthermore, on comparison of the IC50 values of thetwo dicopper(II) complexes, we found that the IC50values of the present copper(II) complex are lower thanthose of our previously reported analogous complex[20], indicating that the former demonstrated betteractivities than the latter under identical experimentalconditions. Interestingly, the order of the in vitro anti-cancer activities of the two dicopper(II) complexes tothe selected cancer cell lines is in accordance with theirDNA-binding abilities, implying that the anticanceractivities of the two dicopper(II) complexes may beclosely related to or originated from their ability tointercalate the base pairs of the DNA. In other words,



the two dicopper(II) complexes might target DNAprimarily, leading to cell death. And this fact indicatesthat the DNA binding and the in vitro anticanceractivities could possibly be tuned through varying thestructure of bridging ligands in dicopper(II) systems.This strategy opens vast perspectives in understandingthe anticancer activities and DNA-binding behaviorsof this kind of metal complexes.

In the BSA-binding studies, the rate constant (Kq)of protein quenching initiated by the dicopper(II) com-plex is greater than maximum collision quenching con-stant (2.0 × 1010 M−1·s−1) [51] of various kinds ofquenchers to biomacromolecule, which suggests thatthe possible quenching mechanism is a static quench-ing process. In addition, the change of the absorptionspectra of BSA with increasing concentration of thecomplex implies that the fluorescence quenching be-tween the complex and BSA is mainly the static quench-ing by forming a complex-BSA ground state system[49, 52], which reconfirms our observation madethrough fluorescence studies.

CONCLUSIONS

To examine the effect of structural variationof the bridging ligand in dicopper(II) systems onDNA-binding properties and in vitro anticancer ac-tivities, and furthermore, to gain an insight intothe structure–activity relationship and the reactivityof this kind of complexes toward protein BSA, anew dicopper(II) complex identified as [Cu2(dmapob)(dabt)(CH3OH)(pic)]·(DMF)0.75·(CH3OH)0.25 has beensynthesized and structurally characterized by usingH3dmapob as bridging ligand and terminating dabt.The in vitro cytotoxicity of the dicopper(II) complexsuggests that it is active against the selected tumor celllines. The reactivity toward HS-DNA and protein BSArevealed that the dicopper(II) complex can interact withHS-DNA in the mode of intercalation, and the dicop-per(II) complex binds to protein BSA responsible forquenching of tryptophan fluorescence by static quench-ing mechanism. Comparing the DNA-binding prop-erties and cytotoxicities with other dicopper(II) com-plexes [20] containing different bridging ligands revealthat the in vitro anticancer activities of these dicop-per(II) complexes are in accordance with their DNA-binding abilities. The results imply that the in vitro an-ticancer activities of these dicopper(II) complexes maybe associated with or originate from their ability to in-tercalate the base pairs of the DNA. In other words, theDNA-binding abilities and in vitro anticancer activitiescould possibly be tuned through varying the structuresof bridging ligands in these dicopper(II) systems. Suchstrategy should be valuable in understanding the re-

lationship of DNA-binding behaviors and anticanceractivities of this class of complexes as well as laying afoundation for the rational design of novel, powerfulagents for probing and targeting nucleic acids, which isexpected to provide an important insight into the fieldof DNA and protein interactions.

SUPPLEMENTARY MATERIAL

Crystallographic data (excluding structure factors)for the structure reported in this work have been de-posited with the Cambridge Crystallographic DataCenter and allocated the deposition number CCDC910451.

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