Synthesis, structure information, DNA/BSA binding ...

Journal of Photochemistry & Photobiology, B: Biology 160 (2016) 278–291

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Synthesis, structure information, DNA/BSA binding affinity and in vitro cytotoxic studies of mixed ligand copper(II) complexes containing a phenylalanine derivative and diimine co-ligands B. Annaraj, C. Balakrishnan, M.A. Neelakantan ⁎ Chemistry Research Centre, National Engineering College, K.R.Nagar, Kovilpatti 628 503, Tamil Nadu, INDIA

a r t i c l e

i n f o

Article history: Received 25 December 2015 Received in revised form 28 March 2016 Accepted 18 April 2016 Available online 21 April 2016 Keywords: Copper(II) complex Phenylalanine derivative Diimine coligands DNA binding and cleavage Cytotoxicity Molecular docking

a b s t r a c t Binary [Cu(PAIC)(H2O)2]·H2O (1) and mixed ligand [Cu(PAIC)(L)]·2H2O complexes, where PAIC = phenylalanine imidazole carboxylic acid, L = diimine coligands [2,2′-bipyridine (bpy) (2) and 1,10-phenanthroline (phen) (3)] have been synthesized and fully characterized by analytical and spectral techniques. The X-ray structure of [Cu(PAIC)(phen)]·2H2O (3) shows a N4O coordination with square pyramidal geometry around the copper (II) atom. The spin Hamiltonian parameters calculated for the complexes account for the distorted square planar structure and rules out the possibility of a trigonal bipyramidal structure. Interaction of the complexes (1–3) with calf thymus DNA (CT DNA) was studied by using different techniques (absorption titration, fluorescence quenching and thermal melting) and the studies suggest that these complexes bind to CT DNA through intercalation. The DNA-binding affinity of the complexes has further been explained by DFT computational results. Binding activity of Bovine serum albumin (BSA) reveals that the complexes can strongly quench the intrinsic fluorescence of BSA through a static quenching mechanism. DNA cleavage experiments using plasmid DNA pUC 19 show that the complexes exhibit efficient chemical nuclease activity even in the absence of any external additives. The cytotoxicity of the complexes against human normal cell line (HBL 100) and human breast cancer cell line (MCF-7) shows that metal complexation of the ligands results in a significant enhancement in the cell death of MCF-7. Finally, docking studies on DNA and protein binding interactions were performed. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Metal complexes that can bind and react with specific DNA sites under physiological conditions are of substantial interest in the development of anticancer agents [1]. Metal complexes exert antitumor effects by binding and cleaving the DNA [2]. When compared to natural DNA binding enzymes, artificial nucleases are found to have low molecular weight and less reactive conditions [3,4]. Moreover, some of the transition metal complexes are under the process of clinical trials [5]. Hence, developing and studying new transition metal complexes for their use as potential anticancer agents are the interest of present investigation. Due to the biocompatibility and redox nature, copper complexes play a major role in the field of metal based chemotherapeutics [6]. Copper is important for the functioning of different enzymes and proteins and engaged in respiration, energy metabolism and DNA synthesis [7]. It has the ability to produce reactive oxygen species that may affect the oxidative

⁎ Corresponding author. E-mail addresses: [email protected], [email protected] (M.A. Neelakantan).

http://dx.doi.org/10.1016/j.jphotobiol.2016.04.021 1011-1344/© 2016 Elsevier B.V. All rights reserved.

damage in the DNA and interfere with the redox-related cellular signaling pathways [8]. Many copper complexes have been prepared as artificial nucleases possessing higher reactivity in the cleavage of DNA. The imidazole functionality plays a significant role in many structures within the human body. The uses of imidazole ring as a bioagent revolve around its ability to bond to metals as a ligand and its ability to hydrogen bond with drugs and proteins. L Phenylalanine is an important aromatic amino acid, changes into tyrosine in vivo and makes essential hormones, such as norepinephrine and epinephrine. The derivatives of phenylalanine are important in pharmaceutical field, e.g. in the case of Parkinson's drug L-3,4dihydroxyphenylalanine (L-DOPA).The major structural components of proteins are amino acid and peptide units which are mainly used to recognize particular sequence of DNA and stabilize [9,10]. Under biological conditions, amino acids bind to transition metal ions via the carboxylate-O and the amino-N donor atoms, thereby forming a thermodynamically stable five-membered chelate ring [11]. For these reasons, we are interested in the development of synthetic amino acid derivatives. Amide bond is present in a number of modern pharmaceuticals and biologically active compounds. Furthermore, the favorable properties of amides, such as high polarity,

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λmax, nm (ε, dm3 mol−1 cm− 1) 242 (9450), 288 (8800), 641 (200). FT-IR (KBr, cm−1), 1594 (–C = N), 1632 (O–CO), 1669 (C = Oamide), 2931 (C–H). ESI mass: m/z 527.51 (M + H)+.

stability and conformational diversity make –CONH as one of the most popular and reliable functional groups [12]. We previously reported a few Cu(II) complexes with strong affinity towards DNA and high nuclease activity [13–16]. The present investigation discusses the synthesis and characterization of copper(II) mixed ligand complexes containing a phenylalanine derivative and diimine coligands. Diimine heterocyclic bases are important class of coligand which can be used to tune electronic property, chemical reactivity and rigid structure of the metal complexes [17]. The binding constant (Kb) and quenching constant (Kq) for the interaction of copper complexes with CT DNA and BSA under physiological conditions have been determined. Molecular docking studies have also been carried out to validate the binding nature. The in vitro cytotoxicity of the complexes against human normal cell line (HBL 100) and human breast cancer cell line (MCF-7) has been evaluated.

2.1.3. Synthesis of [Cu(PAIC)(Phen)]·2H2O (3) Complex 3 was prepared by adopting the procedure used for the synthesis of 2. 1,10-phenanthroline (Phen) (0.18 g, 1 mmol) was used instead of bipyridine. The blue colored solution obtained was kept aside for slow evaporation. Needle shaped single crystal formed was filtered with cold methanol and diethyl ether. Chemical Formula: C26H25CuN5O5 calculated (%): C, 56.67; H, 4.57; N, 12.71. Found (%): C, 56.12; H, 4.55; N, 12.63. UV–vis (in methanol): λmax, nm (ε, dm3 mol−1 cm−1) 234 (9310), 268 (10,450), 640 (150). FT-IR (KBr, cm−1), 1599 (–C = N), 1638 (O–CO), 1663 (C = Oamide), 2930 (C–H). ESI mass: m/z 551.48 (M + H)+.

2. Experimental Section

2.2. Single Crystal X-ray Crystallography

2.1. Materials and Methods

The single crystal X-ray diffraction data were recorded using the ω scan method on a Bruker SMART APEX II area detector using graphite monochromated Mo Kα radiation (λ = 0.71073 Å). The unit cell parameters were determined from 36 frames (0.5° phi-scan), which were measured from three different crystallographic zones using the method of difference vectors. The intensity data were collected with an average fourfold redundancy per reflection and optimum resolution (0.75 Å). The intensity data collection, frame integration, LP correction and decay correction were done using SAINT-NT (version 6.0) software. Empirical absorption correction (multi-scan) was performed using SADABS [19]. The structure was solved by direct methods using the SHELXS-97 program and refined by fullmatrix least-squares of SHELXL-97 [20]. The non-hydrogen atoms were refined with anisotropic displacement parameters. All hydrogens were placed at the calculated positions and refined as riding, using isotropic displacement parameters. The geometric calculations were performed by using PARST program [21]. The linear absorption coefficient for Mo Kα radiation is 0.946 mm− 1.

Chemicals used in this study were purchased from Sigma Aldrich, USA and used as received. Solvents were procured from the commercial suppliers and purified before being used according to standard methods. Calf thymus (CT) DNA and supercoiled pUC19 DNA (CsCl purified) were purchased from Genei, Bangalore and stored at 4 °C. Tris(hydroxymethyl)-aminomethane–HCl (Tris–HCl) and methanol were supplied by Merck, India. Tris–HCl buffer solution was prepared using triple distilled water. The C, H and N microanalyses were performed on an Elemental Vario EL III CHNOS elemental analyzer. Molar conductance was measured using Systronics 304 conductometer. IR spectra on KBr pellets were recorded on a Shimadzu FT IR-8400 spectrometer in the range of 4000–400 cm−1. The absorption spectra were recorded at room temperature using Shimadzu-2450 UV–visible spectrophotometer. The emission spectra were recorded using Jasco-8300 spectrophotometer. ESI-MS were obtained from Thermo Finnigan LCQ 6000 advantage max ion trap mass spectrometer. The X-band EPR spectra of 1–3 in DMSO at room temperature and liquid nitrogen temperature were obtained on a Varian spectrometer using tetracyanoethylene (TCNE) as the “g” (2.0027) marker. Single crystal data were collected from Bruker Kappa Apex II single crystal diffractometer and SHELXS-97 program was used for solving the structure. 2.1.1. Synthesis of [Cu(PAIC)(H2O)2]·H2O (1) Phenylalanine imidazole carboxylic acid (PAIC/L) was synthesized as reported in our earlier work [18]. PAIC (0.27 g; 1 mmol) was dissolved in methanol (10 mL) and added dropwise to a methanolic solution (10 mL) of (CH3COO)2 Cu·H2O (0.198 g; 1 mmol). The solution was stirred for 2 h at room temperature and blue colored precipitate formed was filtered to obtain complex 1. The product was washed with cold methanol and diethyl ether. Chemical Formula: C14H19CuN3O6, calculated (%): C, 43.24; H, 4.92; N, 10.81. Found (%): 43.12; H, 4.89; N, 10.73. UV–vis (in methanol): λmax, nm ( , dm3 mol−1 cm− 1) 242 (8960), 261 (9568), 659 (275). FT-IR (KBr, cm−1), 1590 (–C = N), 1635 (O–CO), 1664 (C = Oamide), 2934 (C–H). ESI mass: m/z 389.29 (M + H)+. 2.1.2. Synthesis of [Cu(PAIC)(Bipy)]·2H2O (2) Complex 2 was prepared by stirring a methanolic solution of Cu(CH3COO)2·H2O (0.198 g, 1 mmol) with PAIC (0.27 g, 1 mmol) for half an hour at room temperature. Then, bipyridine (Bipy) (0.16 g, 1 mmol) in methanol was slowly added to the reaction mixture and stirred well for 2 h. Blue colored precipitate formed was filtered and washed with ice cold methanol and diethyl ether and dried in vacuum. Chemical Formula: C24H25CuN5O5 calculated (%): C, 54.69; H, 4.78; N, 13.29. Found (%): C, 53.98; H, 4.72; N, 13.24; UV–vis (in methanol):

2.3. DNA Binding Experiments DNA binding of copper complexes (1–3) was studied using absorption spectroscopy, fluorescence spectroscopy and thermal denaturation techniques. 2.3.1. Absorption Spectral Studies The interaction of complexes (1–3) with calf thymus (CT DNA) was studied by absorption spectroscopic method. The absorption titrations were carried out in Tris–HCl buffer (5 mM Tris–HCl, pH 7.2) containing 5% methanol solution. The solution of CT-DNA gave the ratio of UV absorbance at 260 nm and 280 nm (260/280 = 1.86) representing that the DNA was sufficiently free of protein [22]. The concentration of CT DNA was calculated from the absorption intensity at 260 nm with a molar extinction coefficient value of 6600 M−1 cm−1 [23]. Absorption titrations were carried out by measuring the absorption of metal complexes (40 μM, methanol: water) in the range of 250–320 nm with varying concentration of CT DNA (0–120 μM). While measuring the absorption, equal amount of DNA was added to both the complex solution and the reference solution to eliminate the absorbance of DNA itself. Before recording the spectrum, each sample was equilibrated. The intrinsic binding constant, Kb was determined by the following equation. ½DNA ½DNA 1 ¼ þ ðεa −ε f Þ ðεb −ε f Þ K b ðεb −ε f Þ where [DNA] is the concentration of DNA in base pairs, εa, εf, and εb are the extinction coefficients of the apparent, free and bound metal

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complexes, respectively. The plot of [DNA]/(εa − εf) versus [DNA] gave a slope and the intercept which are equal to 1/(εb – εf) and 1/Kb(εb – εf), respectively; Kb is the ratio of the slope to the intercept.

The output of the AutoDock was further analyzed by PyMOL software package [26]. 2.6. DNA Cleaving Studies

2.3.2. Fluorescence Studies (Competitive Binding Studies) The competitive ethidium bromide (EB) binding studies have been undertaken to determine the relative binding abilities of the metal complexes to CT DNA by fluorescence spectral technique. Fluorescence intensities of DNA bound EB was measured at 595 nm with an excitation wavelength of 520 nm in the absence and presence of copper complexes (0–100 μM) in Tris–HCl buffer (5 mM Tris–HCl, pH 7.2). The effect of copper complexes on the emission intensity of DNA bound EB was used to determine the binding properties of metal complexes. The binding constant was calculated from the slopes of the lines in the plot of the fluorescence intensity versus copper complex concentration. According to the classical Stern–Volmer equation,

The cleavage and condensation of plasmid DNA, pUC 19 were monitored using agarose gel electrophoresis. 1% agarose gels containing 0.5 μg/mL of ethidium bromide as staining agent were prepared in Tris-boric acid-ethylenediamine tetraacetic acid buffer (TBE). The reaction mixtures were prepared (20 μM of DNA, 40 μM of complex) and suitably incubated and finally quenched by the addition of loading buffer containing 40% sucrose, 0.5 M EDTA and 0.2% bromophenol blue. Aliquots of the reaction mixture were then loaded on to the agarose gel. The gels run at 100 V for approximately 2 h in TBE buffer. The DNA bands were photographed under UV by LARK gel documentation system.

F 0 = F ¼ K q ½Q  þ 1:

2.7. Evaluation of Cytotoxicity and IC50

where F0 is the fluorescence intensity in the absence of the quencher, F is the fluorescence intensity in the presence of the quencher, Kq is the quenching constant and [Q] is the quencher concentration. The Kq value is obtained as a slope from the plot of F0/F versus [Q]. The binding constant (Kb) and the number (n) of binding sites for ligand and complexes with DNA can be determined by the following equation [24],

The in vitro cytotoxicity assay of copper(II) complexes (1–3) was assessed against human normal cell line (HBL 100) and human breast cancer cell line (MCF-7) using the micro-titration colorimetric method [27]. The tetrazolium salt 3-[4,5-dimethylthiazol-2-yl]2,5diphenyl tetrazolium bromide (MTT) was used to determine cell viability in assays of cell proliferation and cytotoxicity. To attain a final concentration of 0.5 mg/mL, MTT powder was dissolved in Dulbecco's PBS (stock solution of MTT (5 mg/mL)). The stock solution was filtered and sterilized through a 0.25 μM filter and stored at − 20 °C. MTT was reduced in metabolically active cells to yield an insoluble purple formazan product. Cells were harvested from maintenance cultures in the exponential phase and counted by a hemocytometer using trypan blue solution. The cell suspensions were dispensed (100 μL) in triplicate into 96-well culture plates at optimized concentrations of 1 × 104 cells/mL after 48 h recovery period to form formazan crystal reaction. For the calculation of IC50 value the dose-dependent curves were conducted with a series of different concentrations (0–150 μM). The cells were incubated at 37 °C for 4 h and then the medium is aspirated and replaced with 100 μL DMSO to dissolve the control wells. The culture plates were shaken for 5 min and the absorbance of each well was read at 570 nm using ELISA multi-well plate reader (Thermo Multiskan EX, USA). The relative viability of the treated cells compared to the control cells is expressed as % of cytoviability, using the following equation,

log½ð F 0 –F Þ=F  ¼ logK b þ n log½Q : where Kb and n are the binding constant and the number of binding sites in base pairs. The plots of log[(F0 − F)/F] versus log[Q]. 2.3.3. Thermal Denaturation Experiments Thermal denaturation experiments were carried out with the Shimadzu-2450 spectrophotometer equipped with digital temperaturecontroller (CYBERLAB). The temperature is slowly increased from 30 °C to 90 °C at a rate of 3 °C min−1. The absorbance at 260 nm is recorded for CT DNA (200 μM) in the absence and presence of copper(II) complexes (20 μM) at an interval of 5 °C. The Tm values were determined from the plot of relative absorbance (A/A30°) vs. temperature, where A is the observed absorbance and A30 is the absorbance at 30 °C. 2.4. Protein Binding Studies Protein binding studies were performed with excitation wavelength of 280 nm and the corresponding emission at 341 nm. The excitation and emission slit widths and scan rates were maintained constant for all of the experiments. A stock solution of BSA was prepared in 50 mM phosphate buffer (pH = 7.2) and stored in the dark at 4 °C for further use. A concentrated stock solution of the compounds was prepared as mentioned for the DNA binding experiments, except that the phosphate buffer was used instead of a Tris–HCl buffer for all of the experiments. 2.5. Molecular Docking Studies Molecular docking was carried out by using AutoDock 4.2 program [25]. The PDB format of metal complexes was obtained from their optimized geometry using Gaussian G09 program. The crystal data of BDNA and BSA protein were obtained from Protein Data Bank (PDB) identifier 3V9D (A-DNA) and 1BNA (BDNA). The water molecules and other unsupported elements (e.g. Na, K, Hg, etc.,) were removed from the DNA and BSA protein. Gasteiger charges were added to the metal complexes by Autodock Tools (ADT) before subjecting to docking analysis. The docking calculations were carried out using Lamarckian genetic algorithm (LGA) [23]. The binding area was focused on the macromolecules (DNA/BSA) and a grid box size of 126 × 126 × 126 was created along the x, y and z axes, i.e., blind docking was performed.

%of viability ¼

OD value of sample treated cells  100 OD value of untreated cells

2.8. AO/EB Staining Assay Ethidium bromide/acridine orange staining was carried out by the method of Gohel et al. [28]. MCF-7 cells were plated at a density of 5 × 104 in 6-well plates. They were allowed to grow at 37 °C in a humidified CO2 incubator until it reaches 70–80% confluent. Then the cells were treated with IC50 concentrations of complexes 1–3 for 24 h. The culture medium was aspirated from each well and the cells were gently rinsed twice with PBS at room temperature. Then equal volumes of cells from control and metal complexes treated were mixed with 100 μL of dye mixture (1:1) of ethidium bromide and acridine orange and viewed immediately by fluorescence microscopy. 3. Results and Discussion The present investigation aims to ascertain the effect of planar diimine coligands in the mixed ligand complexes on DNA and BSA binding events and in vitro anticancer activities. The elemental

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Scheme 1. Synthetic scheme of copper (II) complexes (1–3).

analyses, IR and electronic spectral studies, and single-crystal X-ray diffraction demonstrate that the reaction of PAIC with copper salt and diimine yielded 1:1:1 complex. The general synthetic pathway for the complex formation is given in Scheme 1. The complexes are soluble in methanol, acetonitrile, DMSO and DMF. The observed molar conductance values in methanol (15–22 Ω mol− 1 cm2) indicate non-electrolytic nature of the complexes. The stability of the metal complexes in solution was studied by UV– vis measurements. The UV–vis spectra of the metal complexes (1–3) in methanol-buffer were monitored for 5 days at room temperature. Only a slight decrease in the absorption was observed in the region of 200– 500 nm. The d–d transition observed around 650 nm experienced 3% decrease in intensity without any change in the wavelength. This clearly demonstrates the stability of the metal complexes in methanol-buffer [29].

3.1. Description of the Structure The crystal structure of complex 3 is shown in Fig. 1 and the parameters are given in Table 1. The complex is crystallized in monoclinic system with P2(1)/c space group. The ORTEP diagram shows N4O coordination with approximate square pyramidal geometry around copper center. Some selected bond distances and bond angles are given in Tables 2 and S1. The unit cell packing diagram of 3 is shown in Fig. 2. In the complex PAIC acts as a tri-dentate ligand occupying three equatorial positions of basal plane of the square with bond lengths Cu(1)–N(3), Cu(1)–N(5) and Cu(1)–O(1) as 1.996(2)Å, 1.926(2) Å and 1.980(2)Å, respectively. The secondary ligand 1,10-phenanthroline occupies one equatorial as well as one axial position with Cu(1)– N(1) and Cu(1)–N(2) bond distances of 2.035(2)Å and 2.265(2) Å, respectively. Such shortening of equatorial Cu–N1 bond compared to the

Fig. 1. Crystal structure of complex, 3 (CCDC No. 1,400,382).

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Table 1 Crystallographic data of complex, 3. Parameters

Complex 3

Identification code Empirical formula Formula weight Temperature Wavelength Crystal system, space group Unit cell dimensions

shelxl C26 H21 Cu N5 O6 563.02 296(2) K 0.71073 Å Triclinic, P-1 a = 9.5067(5) A α = 76.954(3)° b = 11.1824(8) A β = 79.419(3)° c = 12.7868(10) A γ = 79.667(3)° 1288.16(15) A3 2, 1.452 Mg/m3 0.898 mm−1 578 0.25 × 0.20 × 0.20 mm 1.65 to 28.08 °. −12 b= h b= 12, −14 b= k b= 14, −16 b=l b= 16 11,071/6047 [R(int) = 0.0250] 96.60% Semi-empirical from equivalents 0.8408 and 0.8066 Full-matrix least-squares on F^2 6047/0/344 1.126 R1 = 0.0444, wR2 = 0.1157 R1 = 0.0650, wR2 = 0.1380 0.548 and −0.382 e·A−3

Volume Z, calculated density Absorption coefficient F(000) Crystal size Theta range for data collection Limiting indices Reflections collected/unique Completeness to theta = 28.08 Absorption correction Max. and min. transmission Refinement method Data/restraints/parameters Goodness-of-fit on F^2 Final R indices [I N 2sigma(I)] R indices (all data) Largest diff. peak and hole

apical Cu–N2 bond is a common phenomenon and reported in the literature [30,31]. The Cu–N axial distance is elongated with respect to Cu–N basal distance by 0.23 Å is typical for d9 Cu(II) centers with square pyramidal geometry [32]. The bond angles between two coordinates in square are 93.22(9), 99.34(13), 82.00(9) and 82.45(9) for each O(1)– Cu(1)–N(1), N(1)–Cu(1)–N(3), N(5)–Cu(1)–N(3) and O(1)–Cu(1)– N(5), respectively. The sum of the square angles is 357.01°, which is slightly deviated from the ideal square (360°). The axial nitrogen (N5) shows small angle towards N(1)–Cu(1)–N(3) plane compared to other planes. This is due to the rigidity of phenanthroline ring and accounts for the distortion in the geometry. The trigonality index (τ) provides a measure of the degree of distortion in square-based pyramidal versus trigonal bipyramidal geometry. The formula for five coordinate complex is τ = β − α/60, where β and α, are the largest coordination angles, and its value varies from 0 for square pyramidal to 1 for trigonal bipyramidal [33]. The τ value of complex 3 is 0.061, which shows the existence of square pyramidal geometry around the Cu(II) ion. Every complex molecule is connected to alternate molecule through hydrogen bonding with O1, O2 and O3 atoms of ligand. All the bond lengths agree well with similar CuII complexes with square pyramidal geometry reported in the literature [34,35]. The 1D coordinating polymer chains in 3 are involved in extensive intermolecular hydrogen bonding with the lattice oxygen atoms giving

Table 2 Selected bond lengths (Å) and bond angles (°) of complex, 3. Parameters

Bond length & bond angles

N(1)–Cu(1) N(2)–Cu(1) N(3)–Cu(1) N(5)–Cu(1) O(1)–Cu(1) N(5)–Cu(1)–N(3) N(5)–Cu(1)–O(1) O(1)–Cu(1)–N(3) N(1)–Cu(1)–N(2) N(1)–Cu(1)–N(3)

2.035(2) 2.265(2) 1.999(2) 1.926(2) 1.9798(19) 82.15 82.45 99.05 77.26 99.34

Fig. 2. Unit cell packing diagram of complex, 3.

rise to a 2D supramolecular arrangement. The three oxygen atoms of 3 (imidazole carbonyl group, phenylalanine carbonyl group and metal bonded oxygen atom) are involved in connecting the 1D chain to generate the 2D network. In this hydrogen bonding network, the bond length between the metal coordinated oxygen atom (O1) and the lattice oxygen atom (O1S) and (O3S) is 2.948 Å and 2.875 Å, respectively. Also, hydrogen bonding exists between uncoordinated oxygen atom of phenylalanine (O2) and the lattice oxygen atom (O3S) (2.775 Å). Through hydrogen bonding O3S acts as a bridge between O2 and O3. Thus the oxygen atoms O3S and O1S act as donor as well as acceptor. The oxygen atom (O2S) plays a significant role by bridging the lattice oxygen atom O3S and uncoordinated phenylalanine oxygen atom (O2) (O2S—O3S distance: 2.888 Å) (O2S—O3S distance: 2.846 Å) from different 1D chains. This forms the inter-chain hydrogen bonding and extends the dimensionality (Fig. S1). 3.2. IR Spectra IR spectrum of PAIC shows characteristic amide N–H bending frequency at 1555 cm−1, which is disappeared in the spectra of metal complexes, demonstrates that amide group coordinates with the metal ion after deprotonation (Fig. S2). It is further evidenced by the lower shifting of amide I band (C=O) of PAIC (1673 cm−1) in the spectra of the complexes. The asymmetric and symmetric carboxylate stretching frequencies of PAIC are shifted to lower values and the magnitude of separation between the two vibrations (N 200 cm−1) suggesting the coordination of carboxylate group with metal ion in unidentate fashion [36]. The imidazole ring C=N stretching of PAIC at 1602 cm−1 is shifted to lower value (1595 cm−1) in the complex confirms the involvement of imidazole nitrogen into the coordination of metal ions. The coordination of PAIC to the Cu(II) ion through the oxygen and nitrogen atoms is substantiated by the observation of new weak bands around 422 cm−1

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Fig. 3. UV–visible spectra of (a) PAIC (L) and its copper(II) complexes (200–500 nm); (b) d–d transition of copper(II) complexes (1–3) in methanol (400–900 nm).

and 578 cm − 1 , [37]. Medium intensity band is observed around 1093 cm− 1 due to the C–N stretching vibration of diimine coligands. The coordination of the biby/phen nitrogen atoms incomplexes 2 & 3 is indicated by the heterocyclic ring breathing frequencies in the fingerprint region of 600–1400 cm − 1 [38]. The experimental values were compared with the computed vibrational frequencies of the optimized structure at DFT level (Table S2 and Fig. S3). The calculated frequencies are higher than the experimental values. The reason for the discrepancy between the experimental and computed spectra

is that the experimental value is an anharmonic frequency, while the calculated value is a harmonic frequency. 3.3. Electronic Absorption Spectral Studies The electronic absorption spectra of the complexes were recorded in methanol at room temperature. The ligand PAIC shows two absorption bands at 215 nm and 258 nm corresponding to π–π* transition of aromatic chromophores (Fig. 3). These bands were

Fig. 4. Absorption spectra of L and 1–3, upon addition of CT DNA. Inset: plots of [DNA]/(εa − εb) vs. [DNA].

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shifted to higher wavelength (red shift) in the spectra of metal complexes (Table S3) indicating that PAIC forms complexes with metal ions. An intense band observed in the range 298–305 nm has been assigned to the transfer of electrons from nitrogen to Cu(II) (LMCT transition) demonstrating the involvement of diimine nitrogen atom in coordination with the metal ion. In the visible region, complexes 1–3 (20 × 10− 4 M) show a less intense band at 659, 641 and 640 nm respectively. This d–d band is attributed to 2B1 → 2E transition characteristic of Cu(II) ions in a square pyramidal environment [39]. The result implies that the structure of the complex is stable even in the solution state. 3.4. ESI Mass Spectral Studies Formation of the ligand (PAIC) and its copper complexes was confirmed by ESI-MS studies. Mass spectrum of PAIC which shows a peak at m/z 274, is assigned to its [M + H]+ molecular ion. Copper complexes 1–3 show peak at m/z 389.29, 527.51 and 551.48, respectively corresponding to the molecular mass [M + H]+, [M + H]+ and [M + H] + (Figs. S4–S6). The molecular ion peaks for copper complexes have half intensity peaks due to isotopic distribution of copper ( 63 Cu and 65Cu) [40]. The m/z values indicate that all the complexes are stable in solution state. The mass spectra of the ligand and metal complexes are in good agreement with the computed mass value of proposed structure. 3.5. EPR Spectral Studies The X-band EPR spectra of 1–3 were recorded in DMSO at liquid nitrogen temperature and room temperature (Figs. S7 and S8). The spectra at room temperature show isotropic spectrum in the high field region due to the tumbling motion of the molecules. However in the frozen state, complexes exhibit well resolved three out of four peaks corresponding to the hyperfine splitting of copper nucleus (I = 3/2). The fourth component is masked by the broad perpendicular component. The hyperfine lines are not resolved in the perpendicular region. Spin Hamiltonian parameters of 1–3 are given in the Table S4. The magnetic moment value (1.88 B.M) calculated from the equation μeff = g[s(s + 1)]1/2 using experimental giso value agrees well with the measured value of 1.79–1.88 B.M indicating that the solid structure of the complexes are retained in the solution. The magnetic moment value obtained corresponding to the one unpaired electron indicates that the complexes are mononuclear [41]. All the complexes show typical axial spectrum at 77 K with g || N g⊥. The g || N g accounts for the distorted square planar structure and rules out the possibility of a trigonal bipyramidal structure, which would be expected to have g N g ||. The trend of g || N g N ge observed in the complexes shows that the unpaired electron lies predominantly in the d 2x 2– y orbital with Cu(II) having 2B 1 as the ground state. The A || values of the complexes are in the range of 165–171 cm− 1 indicating tetragonally distorted geometry for the complexes. In the present investigation, g || values are in the range of 2.21–2.28 revealing covalent nature of the metal ligand bond. Thus, the coordination polyhedron consists of one phenanthroline/ bipyridine nitrogen, amide nitrogen, imidazole nitrogen, and carboxylato oxygen of PAIC which form the base of the pyramid, and the remaining nitrogen of the heterocyclic base occupies the apical position of the square pyramid. The covalent bonding parameters α 2 (in-plane σ bonding), β 2 (in-plane π bonding) and γ2 (out of plane π bonding) were also determined. The α2 value of 0.5 indicates that the complete covalent bonding nature, while the value of α 2 equal to 1.0 suggests that ionic bonding character [42]. The α2 values determined indicate that the complexes have covalent character in the ligand environment (Table S4). The β 2 and γ2 values indicate that there is an interaction in the out of plane π bonding between

Table 3 Binding constant values of copper(II) complexes with DNA. Complex

Kb (M−1) × 104

Kq (M−1) × 104

Kapp (M−1) × 104

n

L 1 2 3

1.01 ± 1.0 1.15 ± 0.8 2.20 ± 0.1 2.60 ± 0.3

1.4 ± 0.5 2.1 ± 0.2 2.5 ± 0.6 2.9 ± 0.9

1.9 ± 0.8 2.6 ± 1.1 3.1 ± 0.7 3.5 ± 0.2

0.98 1.02 1.11 1.16

the metal ion and ligand. This is also confirmed by orbital reduction factors, KII and K⊥. The trend that KII b K⊥ implies a considerable inplane π bonding and KII N K⊥ shows out of plane π bonding between metal ion and ligand [43]. The resulted KII and K⊥ values suggest that the out of plane π bonding in metal ligand interaction (Table S4).

4. Biological Studies 4.1. DNA Binding Studies UV–visible spectral measurements were performed to study the electronic perturbation of metal complexes during their interaction with DNA. The absorption spectra of the complexes were recorded in the absence and presence of incremental concentration of CT DNA. The absorption spectra of complexes (1–3) in the presence of DNA are shown in Fig. 4. Upon addition of DNA with complex, hypochromism is observed at 263 nm along with red shift (2–5 nm). It is observed because of the intercalation of metal complexes into the double helix structure of DNA [44]. The calculated binding constant values are shown in Table 3. The binding constant values are in the order 1 b 2 b 3. The lower binding constant values of binary complex (1) compared to the mixed ligand complexes (2 and 3) are due to the absence of planar heterocyclic bases in the structure. The higher DNA binding ability of the phen complex (3) compared to its bpy analogue (2) is due to the presence of an extended aromatic phenyl ring (in phen), which might facilitate partial intercalation of the base moiety through non-covalent interaction with the DNA bases. Moreover, the binding constant values fall in the same range as that for mixed ligand Cu(II) complexes containing bipyridine and phenanthroline ligands such as ([Cu(dipica)(phen)]2+, Kb = 2.7 × 103) [45], (Cu(pmdt)(phen)]2 + Kb = 3.1 × 104, [Cu(pmdt)(bpy)]2+ Kb = 2.2 × 104) [46], ([Cu(II)(DPPA)(bpy)].5H2O, Kb = 1.2 × 105, [Cu(II)(DPPA)(phen)]·5H2O, Kb = 1.3 × 105) [47],

Fig. 5. Melting curves of CT DNA upon addition of complexes 1–3.

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Fig. 6. Fluorescence titration of L and its copper complexes (0–100 μM) with EB bounded CT DNA.

(Cu(mef)2(bipy)], Kb = 9.03 × 104, [Cu(mef)2(phen)], Kb = 6.95 × 105) [48].

4.2. Thermal Denaturation Studies Thermal denaturation studies can conveniently be used in predicting the nature of binding of the complexes to DNA and their relative binding strength [49]. A high ΔTm value is suggestive of an intercalative mode of binding of the metal complex to DNA, while a low value (1–3 °C) indicates a non-intercalative binding mode [50]. The melting curves of CT DNA (200 mM) in the absence and presence of metal complexes (1– 3) (20 μM) are presented in Fig. 5. Tm corresponds to the breaking of hydrogen bonds between the base pairs present in the double stranded DNA to form the single stranded DNA. In the present experimental conditions, Tm value of DNA is 66 ± 0.5 °C. The ΔTm values of metal complexes 1 (4 °C), 2 (6 °C), and 3 (7 °C) suggest, that all the complexes bind to DNA through intercalative mode.

4.3. EB Displacement Assay The quenching experiments based on the displacement of ethidium bromide (EB) from CT DNA will provide information about the relative binding affinity of the metal complexes with DNA. The conjugate planar EB has weak fluorescence intensity in Tris–HCl buffer. But, the fluorescence intensity of EB is greatly increased when intercalated into the base pairs of double-stranded DNA. The additions of ligand (L) and metal complexes 1–3 to EB–DNA adduct causes appreciable reduction in the emission intensity, indicating that these compounds competitively bind to CT DNA. The emission spectra of EB–DNA adduct in the absence and presence of ligand and complexes 1–3 are given in Fig. 6. The Stern–Volmer plots and Scatchard plots of the fluorescence titrations of L and complexes with DNA are given in Figs. S9 and S10. Intercalators and groove binders can quench the emission of DNA bound EB by replacing EB and/or by accepting the excited state electron of EB through a photo-electron transfer mechanism [51]. Increasing concentrations of L and 1–3 to EB–DNA causes a reduction in emission

Table 4 Frontier molecular orbital energies (εi/a.u.) of L and 1–3.

L 1 2 3

HOMO + 2

HOMO + 1

HOMO

LUMO

LUMO + 1

LUMO + 2

ΔεL–H

ΔεL–NH

Δε

−0.2462 −0.2306 −0.2095 −0.2090

−0.2409 −0.2224 −0.1987 −0.1981

−0.2373 −0.2122 −0.1825 −0.1820

−0.0327 −0.0460 −0.0869 −0.0874

−0.02054 0.0084 −0.0586 −0.0820

0.00458 0.0113 −0.0439 −0.0363

0.2045 0.1661 0.0956 0.0946

0.2082 0.1763 0.1118 0.1106

0.2063 0.1712 0.1037 0.1026

ΔεL–H = energy difference between LUMO and HOMO. ΔεL–NH = energy difference between LUMO and HOMO + 1. Δε = (ΔεL–H + ΔεL–NH)/2.

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Table 5 Selected calculated bond lengths (Ǻ) and bond angles and dihedral angles (°) of 1–3.

m

Complex

Cu–Nm

N–Cu–Nm

(C–N)m

Cu–Nco

N–Cu–Nco

(C–N)co

Dihedral angle

1 2 3

1.946 1.987 1.882

84.313 109.948 118.075

1.344 1.314 1.300

– 2.150 1.943

– 79.508 82.080

– 1.358 1.363

−0.429(m) −0.350(m) 0.330(m)

– −0.150(co) 0.183(co)

= main ligand; co = co-ligand.

intensity of ca. 23%, 35%, 40%, and 46%, respectively. The quenching constants (Kq) calculated from the Stern–Volmer plots (Table 3) indicate that the quenching efficiency of complex 3 is higher than those of other compounds, which is in accordance with the absorption titrations data. Further, the binding constant (Kapp) value obtained for the compounds using the following equation, KEB [EB] =Kapp [compound] where the compound concentration has the value at a 50% reduction of the fluorescence intensity of [EB], KEB = 1.0 × 107 M−1 and [EB] = 2.5 μM. The quenching constants and binding constants of the ligand and Cu(II) complexes suggest that the interaction of all the compounds with DNA should be of intercalation [52]. On the basis of absorption spectroscopic studies, we concluded that the free ligand and copper(II) complexes can bind to CT DNA via an intercalative mode and also the Cu(II) complexes bind to CT DNA strongly than the free ligand. 4.4. Molecular Geometry Optimization and Theoretical Explanation of DNA Binding The ground-state geometries of complexes 1–3 were optimized using density functional theory (DFT) with a Becke 3-parameter LeeYang-Parr (B3LYP) [53] hybrid functional and a 6-31G (d) basis set.

Optimized structures of Cu(II) complexes are depicted in Fig. S11. The experimental and calculated geometrical parameters of complex 3 are presented in Table S1. It clearly shows that the calculated geometrical parameters are in good agreement with the experimental values. Based on the structure of complex 3, we have optimized the structure of complexes 1 and 2 with the same level of theory. All binary and ternary complexes are having slight distortion in geometry from perfect square pyramidal. The experimental studies suggest that the metal complexes bind to DNA through intercalative mode. In the intercalation mode, the π–π interaction between the base pairs of DNA and the metal complex is important. The metal complex–DNA system is too large in size to compute by DFT. Hence, the interaction between the metal complexes and DNA was analyzed by applying the frontier-molecular orbital theory (FMO). According to the FMO, in a reaction an electron easily moves from the HOMO of a reactant to the LUMO of another reactant. In DNA the electron cloud is predominantly populated on HOMO and HOMO + 1, whereas the electronic cloud of the LUMO and LUMO + 1 is mainly distributed on the intercalative ligands, bpy and phen of the metal complexes. This type of electron cloud distribution helps the orbital overlap between the HOMO of DNA and the LUMO of the complexes in the intercalative mode. The binding interaction is of the order εL (L) N εL (1) N εL (2) N εL (3) (Table 4). The HOMO–LUMO diagram of the compounds is given in Fig. S12. The planarity area of an

Fig. 7. Fluorescence spectra of BSA (1 μM) in the presence of increasing amounts of L and 1–3 (0–20 μM).

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Fig. 8. Molecular docking image of, (a) 1; (b) 2; (c) 3 with DNA.

intercalative ligand of the metal complex is also a significant aspect [54]. The planarity area of the intercalative ligand is larger, the π–π stacking interaction between the intercalative ligand and base pairs of DNA is stronger. This gives the better binding affinity between the complexes with DNA. The LUMO energy and planarity area of complex 3 are higher than those of complex 1 which clearly explain the higher binding affinity of complex 3 when compared with the complex 1 (Table 5). 4.5. Protein Binding Studies The electronic absorption titration of BSA with ligand and its metal complexes was done to predict the type of quenching process. The additions of the compounds to BSA lead to an increase in absorption

intensity without affecting the position of absorption wavelength (Fig. S13). These results show that the type of interaction between compounds and BSA was a static quenching process [55]. Fluorescence quenching measurements have been widely used to study the interaction of metal complexes or small molecules with proteins [56]. Changes in the emission spectra of BSA are common in response to its conformational transitions/substrate binding. Interaction of synthesized metal complexes (1–3) with BSA was studied at room temperature using solution of BSA (1 μM) and titrated with various concentrations of metal complexes (0–20 μM). BSA showed a strong fluorescence emission peak at 341 nm upon excitation (λex 280 nm) and the fluorescence intensity was gradually decreased with the addition of metal complexes due to their interaction with BSA (Figs. 7 and S14, S15). Moreover,

Table 6 Docking results of copper(II) complexes with BSA protein (kcal/mol). Complex

Binding energy (ΔGbinding)

Vdw_hb_desolv energy (ΔGvdW+hb+desolv)

Electrostatic energy (ΔGelec)

Total internal energy (ΔGtotal)

Torsional free energy (ΔGtor)

Unbound system's energy (ΔGunb)

1 2 3

−7.3 −8.3 −8.5

−7.2 −8.1 −8.2

−0.52 −0.61 −0.64

−1.22 −1.08 −0.89

0.41 0.39 0.28

−1.23 −1.10 −0.95

ΔGbinding = ΔGvdW+hb+desolv + ΔGelec + ΔGtotal + ΔGtor − ΔGunb.

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Fig. 9. Molecular docking image of, (a) 1; (b) 2; (c) 3 with BSA.

they showed a slight red shift in the emission spectra, implying that the microenvironment around the chromophore of BSA was altered after the addition of each complex that bound to BSA [55]. Hence, dynamic quenching only influenced the excited state of fluorophore but did not change the absorption spectrum. On the other hand, the formation of a non-fluorescence ground state complex provokes a change in the absorption spectrum of fluorophore, suggesting that the probable quenching mechanism of BSA by the complex or ligand is a static quenching process [57]. The fluorescence quenching data have been analyzed with Stern–Volmer equation and the quenching constant (Kq) was calculated using the plot of F0/F versus [Q]. The calculated values of quenching constant related to the interaction of complexes indicating the potential BSA binding propensity of the complexes (Table S5).

binding energies of the docked metal complexes are − 8.1, − 8.2 and −8.4 kcal mol−1. These are consistent with the results obtained from spectral studies and showed greater binding affinity of metal complexes with DNA. 4.7. Molecular Modeling Studies with BSA Protein The molecular docking method was used to get the preferred binding location and help the understanding of the complexes–protein interaction. The two principal binding sites in the BSA molecule are in the nearness of Trp134 and Trp213 [58], where Trp213 is placed within

4.6. Molecular Docking Studies with DNA The mode of interaction of metal complexes with DNA was theoretically calculated by molecular docking studies using duplex DNA d(CGCGAATTCGCG)2 dodecamer (PDBID: 355D). Minimum energy conformation was obtained from docking of the complexes (1–3) with d(CGCGAATTCGCG)2 dodecamer. Fig. 8 shows the docked structure of the metal complexes with DNA. Further, resulting structures are stabilized by van der Waals and hydrogen bond interaction with DNA. Important hydrogen bond donor atoms and acceptor atoms are given in the Table S6. It reveals that the amide and carboxylic oxygen atoms are playing as important donor atoms for hydrogen bonding. Relative

Fig. 10. Cleavage of super coiled pUC 19 DNA (20 μM) by complexes 1–3 in a buffer containing 5 mM Tris HCl/50 mM NaCl at pH = 7.2 and 37 °C with an incubation time of 2 h. Lane 1, DNA; lane 2, DNA + 1 (40 μM); lane 3, DNA + 2 (40 μM ); lane 4, DNA + 3 (40 μM).

B. Annaraj et al. / Journal of Photochemistry & Photobiology, B: Biology 160 (2016) 278–291 Table 7 Cleavage data of SC pUC19 DNA (20 μM) by complexes, 1–3 (40 μM) for an incubation time of 2 h. Form (%) Lane number

Reaction conditions

1 2 3 4

DNA DNA + 1 DNA + 2 DNA + 3

SC

NC

98.1 43 12 82

1.9 57 88 8

289

induced by the complex bound to SC DNA provides evidence for the intercalation mode of interaction between the complexes and DNA. The percentage of SC and NC forms cleaved by the complexes are presented in the Table 7. Interestingly, binary complex (1) is found to cleave the DNA like mixed ligand complexes (with diimine backbone). This trend may be justified by the following reasons. Lewis acidity of the central metal ion and aqua ligand of the complex are activating the phosphodiester bond towards nucleophilic attack followed by hydrolysis of ester [60]. 4.9. Cytotoxicity Studies

Table 8 In vitro cytotoxicity assay for complexes 1–3 against human breast cancer lines (MCF-7). IC50 values are in μM. (Data are mean ± SD of three replicates each). Complex

IC50 (μM)

L 1 2 3

142 ± 0.23 42.8 ± 1.4 39.5 ± 1.0 40.1 ± 0.5

a hydrophobic binding pocket and Trp134 is located on the surface in the hydrophilic region of the molecule. According to the above quenching experiment, all complexes have only one binding site in BSA. In order to find out which one of the two binding sites is favored by the complexes to bind to BSA, all complexes were docked into the 3D structure (PDB ID: 4F5S) of BSA using AutoDock. The docking results are listed in Table 6. From the results, the more preferential binding of the complexes is Trp-134 than Trp-213, which is located on the surface in the hydrophilic region of the BSA protein molecule. Besides, the binding free energies of complexes 1, 2 and 3, it can be concluded that the binding capacity of complex 3 with BSA protein is better than that of complexes 1 and 2, which agrees with the results get from the experimental studies. The docked structures of the complexes are shown in Fig. 9 and hydrogen bonding residues and nearby amino acids are given in Table S7. 4.8. DNA Cleavage Studies The DNA cleaving ability of the metal complexes (1–3) was tested against supercoiled plasmid pUC19 DNA by electrophoresis in Tris– HCl buffer medium. The plasmid DNA was incubated with 1–3 for an hour at 37 °C leading to cleavage of supercoiled (SC) form to nicked (NC) form (Fig. 10). In the absence of metal complexes, no distinct cleavage pattern was observed in the DNA (lane 1). When the metal complexes (1–3) were added to the DNA, nicked form is observed (lanes 2–4). So, all the complexes are having the tendency to cleave the DNA from SC form to NC form even in the absence of activating agents. Since the complex brings about DNA cleavage in the absence of any external agents, the mechanism could only be through a hydrolytic pathway [59]. In addition, the amount of helical unwinding

The results of DNA interaction studies encouraged us to study the ability of the metal complexes to control the growth of cancer cells. In order to test the cytotoxicity of PAIC and copper(II) complexes, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed in human breast (MCF7) and normal cell (HBL100) cell lines. The concentrations of the metal complexes used in this study were 15–150 μM. The IC50 values of the complexes (1–3) are given in the Table 8. It is clear that all complexes are exhibiting moderate in vitro cytotoxicity against the selected tumor cell line. Moreover, the cytotoxicity of metal complexes is higher than that of free ligand. It may be due to the process of complexation which results in higher cytotoxicity. The IC50 values are somewhat lesser than famous anticancer drug (cis-platin IC50 = 26.7 μM). A similar observation has been reported for the mixed ligand copper(II) complexes [61]. All the complexes are exhibiting better cytotoxic effect towards MCF-7 cancer cells without affecting the growth of normal cells. Further biochemical study is needed to understand the complete mechanism of action. 4.10. AO/EB Staining Assay Type of cell death caused by our compounds was accessed by AO/EB dual staining assay. In this assay, the cells were stained with acridine orange (AO) and ethidium bromide (EB). AO can enter into the living cell and emit green fluorescence at the time of binding with DNA. On the other hand, EB enters only through modified cell membrane of dead cells and emit red fluorescence. It is very difficult to differentiate the alive and dead cells by staining with individual stain. But mixture of stains could be used to discriminate the cells. The IC50 concentration of compounds has been used for this study. The untreated normal cells clearly showed its well organized nuclei by emitting green fluorescence. On other hand, the cells treated with complexes 1–3 showed red fluorescence along with green fluorescence (Fig. 11). It reveals that all the complexes cause cell damage by affecting the cell membrane integrity. 5. Conclusions To develop metal complexes as potential anticancer agents, binary and mixed ligand phenylalanine derivative Cu(II) complexes containing diimine co-ligands have been synthesized and characterized.

Fig. 11. Fluorescence images of MCF-7 cells treated with complexes 1–3 (IC50 concentration) for 24 h. Cells were co-stained with AO/EtBr (green & red). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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The Cu(II) complex containing 1,10-phenanthroline as coligand has been structurally characterized by X-ray crystallography which shows monoclinic system with N4O coordination of approximate square pyramidal geometry. This work provides insight into the binding and cleaving nature of the complexes with DNA. The studies reveal that complexes interact with DNA through intercalation mode and cleave the DNA hydrolytic pathway. Experimental and docking studies reveal that all the complexes interact effectively with BSA protein. All the complexes are exhibiting better cytotoxic effect towards MCF-7 cancer cells without affecting the growth of normal cells. Acknowledgment Financial supports received from the Department of Science and Technology (DST), New Delhi, India (EMR-II/2014/000081) and BRNS, BARC-DAE, Mumbai, India (35/14/03/2014-BRNS) is gratefully acknowledged. SAIF-STIC, Cochin is acknowledged for CHNS and SCXRD analysis. Appendix A. Supplementary data Spectral data relevant to this article are given in the supplementary data. Crystallographic data are deposited in the CCDC (1400382) and can be downloaded from the website www.ccdc.cam.ac.uk/. Supplementary data associated with this article can be found in the online version, at http://dx.doi.org/10.1016/j.jphotobiol.2016.04.021. References [1] R. Kramer, Bioinorganic models for the catalytic cooperation of metal ions and functional groups in nuclease and peptidase enzymes, Coord. Chem. Rev. 182 (1999) 243–261. [2] F. Mancin, P. Scrimin, P. Tecillab, U. Tonellato, Artificial metallonucleases, Chem. Commun. (2005) 2540–2548. [3] J.A. Cowan, Metal activation of enzymes in nucleic acid biochemistry, Chem. Rev. 98 (1998) 1067–1088. [4] A. Sreedhara, J.A. Cowan, Catalytic hydrolysis of DNA by metal ions and complexes, J. Biol. Inorg. Chem. 6 (2001) 337–347. [5] B.C. Bales, T. Kodama, Y.N. Weledji, M. Pitie, B. Meunier, M.M. Greenberg, Mechanistic studies on DNA damage by minor groove binding copper–phenanthroline conjugates, Nucleic Acids Res. 33 (2005) 5371–5379; G.L. Fernandes, J.A. Parrilha, L.J.M. Lessa, M.M. Santiago, F. Kanashiro, F.S. Boniolo, A.J. Bortoluzzi, N.V. Vugman, M.H. Herbst, A. Horn Jr., Synthesis,crystal structure, nuclease and in vitro antitumor activities of a new mononuclearcopper(II) complex containing a tripodal N3O ligand, Inorg. Chim. Acta 359 (2006) 3167–3176. [6] M. Marzano, F. Pellei, F. Tisato, C. Santini, Copper complexes as anticancer agents, Anti Cancer Agents Med. Chem. 9 (2009) 185–211. [7] C. Marzano, M. Pellei, F. Tisato, C. Santini, Copper complexes as anticancer agents, Anti Cancer Agents Med. Chem. 9 (2009) 185–211. [8] N. Thanki, J.M. Thornton, J.M. Goodfellow, Distributions of water around amino acid residues in proteins, J. Mol. Biol. 202 (1988) 637–657. [9] R.S. Kumar, S. Arunachalam, DNA binding and antimicrobial studies of polymer– copper(II) complexes containing 1,10-phenanthroline and L-phenylalanine ligands, Eur. J. Med. Chem. 44 (2009) 1878–1883. [10] P. Manikandan, B. Epel, D. Goldfarb, Structure of copper(II)–histidine based complexes in frozen aqueous solutions as determined from high-field pulsed electron nuclear double resonance, Inorg. Chem. 40 (2001) 781–787. [11] (a) J.E. Letter Jr., John E. Baumann Jr., Thermodynamic study of the complexation reactions for a series of amino acids related to serine with copper(II) and nickel(II), J. Am. Chem. Soc. 92 (1970) 437–442; (b) T.B. Freedmann, D.A. Young, M.R. Oboodi, L.A. Nafie, Vibrational circular dichroism in transition-metal complexes. 3. Ring currents and ring conformations of amino acid ligands, J. Am. Chem. Soc. 109 (1987) 1551–1559. [12] V.R. Pattabiraman, J.W. Bode, Rethinking amide bond synthesis, Nature 480 (2011) 471–479. [13] V. Selvarani, B. Annaraj, M.A. Neelakantan, S. Sundaramoorthy, D. Velmurugan, Synthesis, characterization and crystal structures of copper(II) and nickel(II) complexes of propargyl arm containing N2O2 ligands: antimicrobial activity and DNA binding, Polyhedron 54 (2013) 74–83. [14] S. Thalamuthu, B. Annaraj, S. Vasudevan, S. Sengupta, M.A. Neelakantan, DNA binding, nuclease, and colon cancer cell inhibitory activity of a Cu(II) complex of a thiazolidine-4-carboxylic acid derivative, J. Coord. Chem. 66 (2014) 1805–1820. [15] L. Subha, C. Balakrishnan, S. Thalamuthu, M.A. Neelakantan, Mixed ligand Cu(II) complexes containing o-vanillin-L-tryptophan Schiff base and heterocyclic nitrogen bases: synthesis, structural characterization, and biological properties, J. Coord. Chem. 68 (2015) 1021–1039.

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