Journal of Photochemistry and Photobiology B: Biology 140 (2014) 321–331

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Synthesis and structure elucidation of a copper(II) Schiff-base complex: In vitro DNA binding, pBR322 plasmid cleavage and HSA binding studies Sartaj Tabassum a,⇑, Musheer Ahmad b, Mohd Afzal a, Mehvash Zaki a, Parimal K. Bharadwaj b,⇑ a b

Department of Chemistry, Aligarh Muslim University, Aligarh, UP 202002, India Department of Chemistry, Indian Institute of Technology, Kanpur, UP 208016, India

a r t i c l e

i n f o

Article history: Received 10 May 2014 Received in revised form 31 July 2014 Accepted 24 August 2014 Available online 1 September 2014 Keywords: In vitro DNA binding HSA binding pBR322 DNA cleavage Molecular docking

a b s t r a c t New copper(II) complex with Schiff base ligand 4-[(2-Hydroxy-3-methoxy-benzylidene)-amino]-benzoic acid (H2L) was synthesized and characterized by spectroscopic and analytical and single crystal X-ray diffraction studies which revealed that the complex 1 exist in a distorted octahedral environment. In vitro CT-DNA binding studies were performed by employing different biophysical technique which indicated that the 1 strongly binds to DNA in comparison to ligand via electrostatic binding mode. Complex 1 cleaves pBR322 DNA via hydrolytic pathway and recognizes minor groove of DNA double helix. The HSA binding results showed that ligand and complex 1 has ability to quench the fluorescence emission intensity of Trp 214 residue available in the subdomain IIA of HSA. Ó 2014 Elsevier B.V. All rights reserved.

1. Introduction DNA is a primary intracellular target of anti-cancer drugs because it regulates many biochemical processes that occur in the cellular system, many small molecules exert their anticancer activities by binding with DNA, thereby altering DNA replication, blocking the division of cancer cells and resulting in the cell death [1–3]. The different loci present in the DNA are involved in various regulatory processes such as gene expression, gene transcription, mutagenesis and carcinogenesis [4]. Similarly, DNA cleavage reaction is also considered of prime importance as it proceeds by targeting various constituents of DNA viz., the nucleic bases, deoxyribose sugar moiety and phosphodiester linkage involving oxidative and hydrolytic mechanisms. The hydrolytic cleavage of phosphodiester bond in DNA offers important advantages as compared to the oxidative DNA cleavage because nucleic bases and deoxyribose sugar moiety are not modified and no additional reagents is necessary when they are hydrolytically cleaved which allows the cleaved fragments to be religated enzymatically [5]. Therefore, the interaction of small metal complexes targeting nucleic acids is of immense interest for the development of new therapeutic modulaties for cancer chemotherapy owing to the fact that many present treatment regimes (platinum-based drugs) in chemotherapy have failed or fall short either in terms of efficiency

⇑ Corresponding authors. Tel.: +91 9358255791. E-mail address: [email protected] (S. Tabassum). http://dx.doi.org/10.1016/j.jphotobiol.2014.08.015 1011-1344/Ó 2014 Elsevier B.V. All rights reserved.

or toxicity problems associated with them [6–8]. Among the transition metals, copper plays a peculiar role in chemotherapy and provided encouraging perspectives [9–11], that interacts directly with DNA, causing site-specific damage which leads to generation of reactive oxygen species (ROS) under physiological conditions. Copper being redox active exists in biologically accessible +2/+1 oxidation states and is an important co-factor of several enzymes involved in cellular respiration, antioxidant defence, neurotransmission, connective tissue biosynthesis and cellular iron metabolism [12]. Furthermore, copper also plays a peculiar role in chemotherapy since it accumulates in tumors due to selective permeability through cancer cell membranes to copper compounds. Because of this, a number of copper complexes were screened for anti-cancer activity and some of them were found active both in vivo and in vitro [13]. Schiff-bases, in particular hydroxyl-substituted Schiff-bases, have drawn considerable attention, owing to their free radical scavenging [14] and anticancer activities [15]. The interaction of the Schiff-base transition metal complexes with DNA is of particular interest due their use as new structural probes in nucleic acid chemistry as well as development of therapeutic agents [16]. Reedjik and co-workers reported the DNA binding and cleaving ability of a novel Cu(II) Schiff base complex which showed promising cytotoxic effects on HL 60 (leukemia) cancer cells [17]. Recently, Lou et al. have prepared a novel cytotoxic copper(II) complex with Schiff base ligand which exhibited potential antitumor activities on human breast cancer cell (MCF-7 cells) [18]. However, the studies on the DNA-binding mechanism of Schiff-base copper(II) complexes derivated from Schiff

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base ligand 4-[(2 Hydroxy-3-methoxy-benzylidene)-amino]-benzoic acid have not been explored. Herein, we describe the synthesis, spectroscopic characterization and single crystal X-ray studies of copper(II) complex containing Schiff base ligand H2L (Scheme 1). In vitro DNA binding profile of ligand (H2L) and complex 1 with CT-DNA was carried out by employing UV–vis and fluorescence spectroscopic techniques. Complex 1 exhibits high chemical nuclease activity cleaving supercoiled pBR322 DNA via hydrolytic pathway. The affinity of 1 towards human serum albumin (HSA) was also investigated to understand the carrier role of plasma protein for such compound in blood under physiological conditions. 2. Experimental section 2.1. Reagents and materials Reagent grade o-vanillin, 4-aminobenzoic acid, Cu(NO3)23H2O were acquired from Aldrich and used as received. All other chemicals and solvents were obtained from S.D. Fine Chemicals, India. Solvents were purified following standard procedures prior to use. Supercoiled plasmid DNA pBR322 (Genei) were utilized as received.

X-ray diffractometer. Electronic spectrum was recorded on UV1700 PharmaSpec UV–vis spectrophotometer (Shimadzu) in DMSO cuvettes of 1 cm path length. Data were reported in kmax/nm. Fluorescence measurements were determined on a RF-5301 PC spectrofluorophotometer (Schimadzu). 2.3. Synthesis 2.3.1. Synthesis of the ligand 4-[(2-Hydroxy-3-methoxy-benzylidene)amino]-benzoic acid (H2L) The ligand H2L was synthesized by a mixture of o-vanillin (2.0 g, 0.013 mol) and 4-aminobenzoic acid (1.80 g, 0.013 mol) in 40 mL absolute ethanol was stirred in a 100 mL round-bottomed flask for 12 h whereby orange colored precipitate was obtained. This precipitate was filtered, washed with ethanol and then airdried. Yield: 75% (2.64 g and 0.0097 mol); m.p: 254–255 °C). 1H NMR (CDCl3, 500 MHz): 10.21(s, 1H; ACOOH), 8.92(s, 1H; HC@N); 7.98(d, 2H; ArH); 7.56(d, 2H; ArH); 6.88(d, 2H; ArH); 6.48(s, 1H; ArH); 5.74(s, 1H; OH); 3.81(s, 3H; Me); 13C NMR (CDCl3, DMSOd6, 125 MHz): 165.4, 153.5, 152.3, 151.1, 131.6, 131.2, 124.4, 121.8, 120.8, 119.6, 119.6, 113.0, 56.3; ESI-MS: m/z [M  1] 270.08; calculated 271.08; IR (KBr, cm1): 2942(m), 2548(m), 1687(s), 1594(m), 1575(s), 1471(s), 1424(s), 1313(s), 1287(s), 1255(s), 1200(s), 1170(m), 970(s), 861(s), 781(s), 733(s). (See Supporting Information Figs. S6–S9)

2.2. Methods and instrumentation Microanalyses for the compounds were performed using a CE440 elemental analyzer (Exeter Analytical Inc.). Infrared spectra were obtained (KBr disk, 400–4000 cm1) on a Perkin-Elmer Model 1320 spectrometer. 1H NMR and 13C NMR spectra were recorded on a JEOL-ECX 500 FT (500 and 125 MHz, respectively) instrument in CDCl3 and DMSO-d6 respectively. ESI mass spectra were recorded on a WATERS Q-TOF Premier mass spectrometer. Thermogravimetric analyses (TGA) were obtained on a Mettler Toledo Star System (heating rate of 5 °C/min). Powder X-ray diffraction patterns for the compounds (Cu Ka radiation, scan rate 3°/min, 293 K) were collected on a Bruker D8 Advance Series 2 powder COO H O

COO H

H

2.4. Single-crystal X-ray Studies

H O

EtOH + H 2O , RT

N

O

H O

CH 3

NH 2

O CH 3

COOH COO H

EtOH + H 2O , RT N

H O O CH 3

Cu (NO 3)2

2.3.2. Synthesis of Complex {[Cu(H2L)2(H2O)2]2NO32H2O} (1) Hot solution of H2L (50 mg, 0.18 mmol) in 6 mL EtOH and Cu(NO3)23H2O (90 mg, 0.37 mmol) in 5 mL distilled water were mixed. The resultant solution was reduced in volume by heating on hot plate and filtered under hot condition which on slow evaporation yielded blue colored block shaped crystals of 1 in 56% (83 mg, 0.10 mmol) yield. The crystals obtained were washed with acetone and air-dried. Anal. Calcd. for C30H34N4O18Cu: C, 44.92; H, 4.27; N, 6.98%. Found: C, 44.84; H, 4.19; N, 6.89%. ESI-MS: m/z 803.11[M + 1]; IR (KBr, cm1): 3465(s), 2930(m), 1592(s), 1547(s), 1467(m), 1450(s), 1386(s), 1313(m), 1237(s), 1191(s), 1104(m), 1081(m), 977(m), 791(s), 746(s), 706(m). (See Supporting Information Figs. S10–S11).

N

H

CH 3

O

O Cu

O CH 3 H

O

N

COO H Scheme 1. Synthetic scheme of H2L and complex 1. Counter anion/cation and solvent molecules have been omitted for clarity.

Single crystal X-ray data of complex 1 were collected at 100 K on a Bruker SMART APEX CCD diffractometer using graphite monochromated Mo Ka radiation (k = 0.71073 Å). The linear absorption coefficients, scattering factors for the atoms and the anomalous dispersion corrections were referred from the International Tables for X-ray Crystallography [19]. The data integration and reduction were worked out with SAINT software [20]. Empirical absorption correction was applied to the collected reflections with SADABS [21], and the space group was determined using XPREP [22]. The structure was solved by the direct methods using SHELXTL-97 [23] and refined on F2 by full-matrix least-squares using the SHELXTL-97 programme package [24]. Only a few H atoms could be located in the difference Fourier maps in the structure. The rest were placed at calculated positions using idealized geometries (riding model) and assigned fixed isotropic displacement parameters. All non-H atoms were refined anisotropically. Several DFIX and DANG commands were used for fixing some bond distances in complex 1. The crystal and refinement data are collected in Table 1. Selective bond distances and angles are given in Table S1. 2.5. DNA binding and cleavage experiments DNA binding experiments include absorption spectral traces, emission spectroscopy and viscosity conformed to the standard

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3.1. Electronic spectra

Table 1 Crystal and structure refinement data for complex 1.

a

Parameters

1

Formula Fw (g mol1) Crystal system Space group a (Å) b (Å) c (Å) a (deg) b (deg) c (deg) U (Å3) Z qcalc (g/cm3) l (mm1) F (0 0 0) Crystal size (mm) Temp (K) Measured reflns Unique reflns h Range (deg)/completeness (%) GOFa Final Rb indices [I > 2r(I)] Rb indices (all data) Largest diff. peak/hole (e Å3)

C30H34N4O18Cu 802.15 Triclinic P1 6.918(5) 11.068(4) 11.529(5) 104.271(5) 102.278(5) 91.544(5) 833.0(8) 1 1.599 0.745 415 0.18  0.13  0.10 100(2) 4383 1993 1.87 to 25.49/0.966 0.990 R1 = 0.0691 wR2 = 0.1299 R1 = 0.1095 wR2 = 0.1505 0.711/0.442

GOF is defined as

323

nP h  i o1=2 w F 20  F 2c =ðn  pÞ where n is the number of data

and p is the number of parameters. n o P P P 2 P 2 1=2 b R ¼ f kF 0 j  jF c k= jF 0 jg; wR2 ¼ ½wðF 20  F 2c Þ = wðF 20 Þ .

The UV–vis spectrum of complex 1 was recorded in DMSO at room temperature. The electronic spectra of complex 1 exhibited high energy band at 291 nm attributed to the intraligand n–p* transition of azomethine group of coordinated Schiff base ligand while a shoulder at 344 nm was assigned to LMCT transition. However, low energy broad asymmetrical d–d band in the visible region was observed at 586 nm consistent with the square planar geometry around Cu(II) ion. The complex exhibited similar spectral features as expected on the basis of X-crystallographic data discussed below. 3.2. TGA and PXRD studies In order to examine the thermal stabilities of complex 1, thermal analysis was carried out in a N2 atmosphere at the rate of 5 ° C per minute. Complex 1 shows a weight loss of 9.0% (expected = 8.98%) within the temperature range 80–240 °C that corresponds to the release of two coordinated and two lattice water molecules (Fig. S1). The complex was found to be stable up to 250 °C, where after the framework starts to decompose. Powder X-ray diffraction (PXRD) pattern of complex 1 matches well with the simulated patterns obtained from the single crystal X-ray data (Fig. S2). This confirms that the crystals are truly representative of the bulk phase. The differences in intensity could be due to the different orientation of the powder samples. 3.3. Crystal structure description

methods and practices previously adopted by our laboratory [25– 28]. While measuring the absorption spectra an equal amount of DNA was added to both the compound solution and the reference solution to eliminate the absorbance of the CT DNA itself, and Tris buffer was subtracted through base line correction. 2.6. Molecular docking studies The rigid molecular docking studies were performed using HEX 6.1 software [29], an interactive molecular graphics program for calculating and displaying feasible docking modes of pairs of protein, enzymes and DNA molecule. Structures of the complex were sketched by CHEMSKETCH (http://www.acdlabs.com) and convert it into pdb format from mol format by OPENBABEL (http:// www.vcclab.org/lab/babel/). The crystal structure of the B-DNA dodecamer d(CGCGAATTCGCG)2 (PDB ID: 1BNA) and humanserum-albumin (PDB ID: 1h9z) were downloaded from the protein data bank. All calculations were carried out on an Intel pentium4, 2.4 GHz based machine running MS Windows XP SP2 as the operating system. Visualization of the docked pose has been done using CHIMERA and PyMol molecular graphics programs.

Single crystal X-ray structural study shows that the complex 1 crystallizes in the triclinic crystal system. The structure was successfully solved and converged in the space group P  1. The asymmetric unit consists of half metal ion (having half occupancy and sit on the two-fold axis of symmetry), one H2L ligand, one metal coordinated water and one nitrate anion and one aqua molecule in the lattice (Fig. 1a). The metal shows slight distorted octahedral CuO6 coordination environment with ligation from two ethereal oxygen (CuAO = 2.313(3) Å) and two hydroxyl O atoms (CuAO = 1.935(3) Å) and two water O atoms (CuAO1w = 1.994(4) Å) from two different H2L ligand moieties. Both coordinated methoxy and hydroxyl O sit on the equatorial plane while coordinated water molecules were occupying axial position. A 3D supramolecular structure (Fig. 1b) is generated which was stabilized by multipoint H-bonding (NO 3   OwH2 = 1.824–2.269 Å, COOH  OwH2 = 1.750–1.908  Å and AOCH3  COOH = 2.716 Å) and strong p–p (3.380–3.400 Å) aromatic stacking arrangement between two neighboring phenyl moieties (Fig 2a and b). Selected Hydrogen bond distances and bond angles for complex 1 were provided in supporting information (Table S2). 4. DNA binding studies

3. Results and discussion 4.1. Absorption spectral studies New copper(II) complex of Schiff base ligand 4-[(2 Hydroxy-3methoxy-benzylidene)-amino]-benzoic acid (H2L) was synthesized and characterized by analytical and spectral studies. The formulation of the complex was further confirmed by determination of the X-ray crystal structure which revealed that complex 1 exist in a slightly distorted octahedral coordination environment and soluble in common organic solvents like ethanol, methanol, DMF and DMSO. The complex 1 was stable towards air and moisture and soluble in DMSO and DMF. Molar conductance value of complex 1 in DMSO (1  103 M) at 25 °C suggest its 1:1 electrolyte nature (63 X1 cm2 mol1).

DNA is the essential carrier of genetic information which was concerned with most cancers resulting from DNA damage; therefore, DNA binding is one of the most critical steps for the action of a large number of metal based anticancer drugs. A variety of small molecules interact reversibly with DNA, primarily through three modes: (i) electrostatic interactions (ii) binding interactions with grooves of DNA double helix; and (iii) intercalation between the stacked base pairs of native DNA. The electronic absorption spectrum of ligand H2L and complex 1 in DMSO-buffer mixture exhibit an intense transition at 265 nm, attributed to the intraligand

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Fig. 1. Schematic view of (a) asymmetric unit (wire-frame model) and (b) 3D supramolecular framework in complex 1.

Fig. 2. Representation of supramolecular interactions (a) H-bonding and (b) p–p in complex 1.

transition. On incremental addition of DNA of varying concentration (0–4.66)  105 M to ligand H2L and metal based chemotherapeutic candidates complex 1, there was an enhancement of intraligand absorption band ‘hyperchromism’ of 84.3% and 89.7% with blue shift of 7 nm consistent with non-covalent interaction (Fig. 3). The observed hyperchromic effect with blue shift suggests that complex bind to DNA by external contact possibly via electrostatic binding [30,31]. These spectral features suggest that hyperchromism results from slight change in conformation of DNA due to the cleavage of its secondary structure.

Copper complex probably bind to the double-helical DNA in different binding fashions depending on the structure, charge and type of ligands. Since, DNA possesses several hydrogen bonding sites which are accessible both in major and minor grooves [32]. So it is likely that AOH and AC@N groups of H2L ligand form hydrogen bonding with the base pairs of DNA helix. To compare quantitatively the binding affinity of ligand H2L and complex 1, the intrinsic binding constant Kb was calculated by monitoring the changes in absorbance in the intraligand band at corresponding kmax with increasing concentration of DNA. The intrinsic binding

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Fig. 3. Absorption spectra of (a) Ligand H2L and (b) complex 1 in 5 mM Tris HCl/50 mM NaCl buffer upon the addition of calf thymus DNA; Inset: Plots of [DNA]/ (ea–ef)  1010 M2 cm vs. [DNA]  105 M for the titration of CT DNA with ligand H2L ( ) and complex 1 ( ), experimental data points; full lines, linear fitting of the data. [Ligand/Complex] = 6.67  106 M, [DNA] = (0–4.66)  105 M. Arrow shows change in intensity with increasing concentration of DNA.

constant Kb values of H2L and complex 1 were found to be 2.07  104 and 4.25  104 M1. The results suggest that complex 1 has better prospect to act as a good chemotherapeutic agent as compared to ligand H2L. Interestingly, the intrinsic binding Kb value is much higher in magnitude in comparison to our previous copper complex (Kb value 3.55  104 M1) [33] which demonstrate the remarkably higher binding strength of complex 1 to DNA due to the external contact (electrostatic binding). 4.2. Fluorescence spectral studies In the absence of CT DNA, ligand H2L and complex 1 emit intense luminescence when excited at 265 nm in 0.01 Tris-HCl/ 50 mM NaCl buffer at room temperature with an emission maximum appearing at 340 nm. On addition of increasing amount of CT DNA emission intensities of H2L and complex 1 decreases by 2.34 and 4.61 times than those in the absence of DNA and saturates at a ratio of [DNA]/[H2L/1] is 1.5, respectively (Fig. 4). The quenching of the luminescence excited state of 1 was attributed to energy or electron transfer from the guanine base of DNA to the MLCT of complex as reported in the case of [Co(bzimpy)2], [Ru(bzimpy)2]2+, [Ru(TAP)3]2+ and [Ru(bpz)3]2+ [34–37]. The binding constant (K) values for H2L and complex 1 determined from Scatchard were calculated to be 1.73  104 and 2.87  104 M1, respectively, consistent with the results obtained from absorption titration.

sensitive fluorescence probes that show no apparent emission intensity in buffer solution because of solvent quenching. In fact the EB fluorescence intensity will be enhanced in the presence of DNA because of its intercalation into the helix, and it was quenched by the addition of another molecule either by replacing the EB and/or by accepting the excited-state electron of the EB through a photoelectron transfer mechanism [38]. The extent of reduction of the emission intensity gives a measure of the binding propensity of the complex to CT-DNA. The fluorescence quenching of EB bound to CT-DNA by ligand H2L and complex 1 was shown in Fig. 5. The fluorescence quenching spectra illustrated that upon increasing the concentration of the compounds the emission band at 591 nm exhibited hypochromism up to 44.71%, and 45.89% of the initial fluorescence intensity accompanied by red shift of 22 and 18 nm for H2L and complex 1, respectively. The observed decrease in the fluorescence intensity with a strong red shift clearly indicates that the EB molecules are displaced from their DNA binding sites and are replaced by the compounds under investigation [39]. Since EB was not completely displaced, partial intercalation through the planar portion of the complex (benzene ring) in addition to the electrostatic mode of binding cannot be ruled out. Furthermore, the quenching extents were evaluated quantitatively by employing Stern–Volmer equation. The Ksv value for ligand H2L and complex 1 was found to be 1.61 and 2.46, respectively revealing that part of the EB was displaced from their DNA binding sites, which is consistent with the above electronic absorption result.

4.3. EB displacement assay Ethidium bromide (EB) fluorescence displacement experiments were also performed in order to investigate the interaction mode of ligand H2L and complex 1 with CT-DNA. EB is one of the most

4.3.1. Viscosity titration measurements To further confirm the binding mode of ligand H2L and complex 1 with calf thymus DNA (CT DNA), viscosity titration

Fig. 4. Emission spectra of (a) Ligand H2L and (b) complex 1 in Tris-HCl buffer (pH 7.2) in the presence and absence of CT DNA at room temperature. Arrow shows change in intensity with increasing concentration of DNA.

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Fig. 5. Emission quenching spectra of CT DNA bound ethidium bromide in the presence of (a) Ligand H2L and (b) complex 1, in buffer 5 mM Tris-HCl/50 mM NaCl, pH = 7.2 at 25 °C. Arrow shows change in intensity with increasing concentration of ethidium bromide.

measurements was carried out to examine the effect of ligand H2L and complex 1 on the relative viscosity of DNA by keeping [DNA] = 7.6  106 constant and increasing [complex]/[DNA] ratios from 0.7 to 4. When the DNA helix was intercalated by planar molecules such as by classical intercalator EB, base pairs are separated to accommodate the binding molecule, resulting in the lengthening of the DNA helix and subsequently increased DNA viscosity. On contrary complex bound to DNA through groove binding do not alter the relative viscosity of DNA whereas complex that bound electrostatically will produce bend or kink the DNA helix, reducing its effective length and its viscosity, concomitantly [40]. On addition of increasing amount of ligand H2L and complex 1, the relative viscosity of CT DNA decreases as depicted in Fig. 6. These results suggested that ligand H2L and complex 1 was bound to CT DNA through electrostatic interaction which leads to decreases in the relative viscosity.

4.4. Fluorescence quenching studies with HSA Human serum albumin (HSA), the most abundant protein in plasma, is a monomeric multidomain macromolecule, representing the main determinant of plasma oncotic pressure and the main modulator of fluid distribution between body compartments. HSA displays an extraordinary ligand and metal-drug complex binding capacity, providing a depot and carrier for many endogenous and exogenous compounds. Therefore, emission quenching experiment was carried out to understand the interaction of ligand H2L and complex 1 with HSA (Fig. 7). The intrinsic fluorescence of HSA was due to the presence of mainly tryptophan residues.

Molecules when excited at 295 nm specifically bind to albumin in the region containing this Trp 214 residue causing fluorescence quenching [41]. The fluorescence spectra of HSA in the absence and presence of H2L and complex 1 as a quencher in Tris-HCl buffer (pH 7.4) were monitored with an excitation wavelength of 293 nm. On addition of increasing concentration of H2L and complex 1 (3.33  106 to 26.6  105 M) to fixed amount of HSA, intrinsic fluorescence intensity of HSA decreased gradually at 362 nm up to 67% and 70% accompanied by a marked red shift of 8–9 nm in the tryptophan emission maxima of HSA. The observed red shift was mainly due to the increase in the polarity of the microenvironment around the tryptophan residue [42]. These results indicated strong protein-binding ability of ligand H2L and complex 1 inducing conformational changes in HSA, because the intramolecular forces involved to maintain the secondary structure could be altered (affecting the tryptophan residues of HSA) together with decreased hydrophobicity indicating that Trp residues were more exposed to solvent [43]. In order to speculate the possible fluorescence quenching mechanism of HSA in presence of H2L and complex 1, Stern–Volmer equation was applied:

Fo ¼ 1 þ K q so ½Q  ¼ 1 þ K sv ½Q  F

where Fo and F are the fluorescence intensities in the absence and presence of quencher, while Kq, Ksv, and [Q] are the quenching rate constant of the biomolecules, the Stern–Volmer quenching constant, the average life time of the molecule without quencher (so = 108 s) and the concentration of the quencher, respectively. The Stern–Volmer plots of Fo/F vs [Q] for the quenching of HSA fluorescence by H2L and complex 1 was depicted in Fig. S3 and the calculated KSV and Kq values were found to be 2.58  103, 7.63  103 M1 and 2.58  1012, 7.63  1012 M1 s1 respectively. The observed Kq value was larger than the limiting diffusion constant Kdif of the biomolecules (Kdif = 2.0  1010 M1 s1) [44], indicating that the fluorescence quenching was caused due to the specific interaction of H2L and complex 1 with HSA, consistent with the static quenching mechanism [45]. For static quenching, the Scatchard equation was employed to calculate the binding constant and number of binding sites [46]:

log

Fig. 6. Effect of increasing amount of ligand H2L ( ) and complex 1 ( ) on the relative viscosities (g/g0) of CT DNA in Tris-HCl buffer (pH 7.2).

ð7Þ

  Fo  F ¼ log k þ n log½Q  F

ð8Þ

where Fo and F are the fluorescence intensities of HSA in the absence and presence of quencher, k and n are the binding constant and the number of binding sites, respectively. Thus, a plot of log[(Fo  F)/F] versus log[Q] was used to determine K (binding constant) from the intercept on Y-axis and n (binding sites) from the slope (Fig. S4). From the corresponding Scatchard plot the k values

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327

Fig. 7. The fluorescence quenching spectra of HSA at different concentrations of (a) Ligand H2L and (b) complex 1 with the excitation wavelength at 293 nm in 5 mM Tris-HCl/ 50 mM NaCl buffer, pH 7.3, at room temperature: [HSA], 6.67  106 M; the concentration of ligand H2L and complex 1 was 3.33  106 to 26.6  105 M. Arrow shows the intensity changes upon increasing concentration of the quencher.

were found to be 0.0346 and 0.416 for H2L and complex 1 while the n was calculated to be and 0.40 and 1.46, respectively. The results revealed that the k value of complex 1 are within an optimum range; they are high enough to allow the binding of a compound to HSA and quite below the association constant of one of the strongest known non-covalent bonds of avidin-ligands interaction (K  1015 M1), suggesting a possible release from the serum albumin to the target cells. 4.5. DNA cleavage activity 4.5.1. DNA cleavage without added reductant Transition metal complexes have been extensively studied for their nuclease-like activity using the redox properties of the metal and dioxygen to produce reactive oxygen species to promote DNA cleavage, yielding direct strand scission or base modification. The ability of complex to cleave supercoiled (SC) pBR322 DNA was assayed with the aid of gel electrophoresis in the absence and/or presence of external agents. If one strand is cleaved, the SC DNA produces a slower-moving nicked circular (NC) form. If both strands are cleaved, the linear (L) form is generated which migrates in between the SC and NC forms [47]. Upon increasing concentration

of complex 1, the amount of SC form decreases gradually and there was partial conversion to NC form with simultaneous increase in the intensity of the latter form. It was clearly seen that even at 20 lM concentration complex 1 was found to promote the cleavage of DNA from supercoiled form (SC) to nicked circular form (NC) (Fig. 8a) revealing the single strand DNA cleavage. As the concentration of complex 1 was increased to 30 lM, there was significant conversion of SC form to NC form which clearly implicates the role of metal ion in the process of DNA cleavage. Quantification of the both the forms originating from SC and NC plasmids by using Vilber-INFINITY gel documentation system were depicted in Fig. 8b–d. It was remarkable to note that the amount of NC form in lane 6 was highest showing the maximum band area clearly indicating the concentration dependent cleavage of DNA by complex 1. 4.5.2. DNA cleavage in presence of activators The cleavage efficiency of complex 1 was assessed in the presence of H2O2, ascorbate (Asc), 3-mercaptopropionic acid (MPA) and glutathione (GSH). The cleavage activity was significantly enhanced (Fig. 9) in the presence of these activators and follows the order H2O2  MPA > GSH > Asc. Thus, complex 1 exhibited

Fig. 8. Agarose gel electrophoresis pattern for the cleavage of pBR322 plasmid DNA (300 ng) by complex 1 at 37 °C after incubation for 45 min at different concentration (a); Lane 1: DNA control; Lane 2: 10 lM complex 1 + DNA; Lane 3: 15 lM complex 1 + DNA; Lane 4: 20 lM complex 1 + DNA; Lane 5: 25 lM complex 1 + DNA; Lane 6: 30 lM complex 1 + DNA; (b) Quantification of band area in gel electrophoresis originating from Form I and Form II pBR322 plasmid DNA by complex 1 at different concentration. 2D projection of gel images for the cleavage of pBR322 plasmid DNA at different concentration of complex 1 for (c) Form I bands and (d) Form II bands. Arrow indicating decrease in relative intensity of Form I band.

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Fig. 9. Agarose gel electrophoresis pattern for the cleavage pattern of pBR 322 DNA (300 ng) by complex 1 at 37 °C after incubation for 45 min in presence of (a) different activating agents; Lane 1, DNA control; Lane 2, DNA + complex 1 + Asc (0.4 mM); Lane 3, DNA + complex 1 + H2O2 (0.4 mM); Lane 4, DNA + complex 1 + GSH (0.4 mM); Lane 5, DNA + complex 1 + MPA (0.4 mm); (b) Quantification of band area in gel electrophoresis originating from Form I and Form II pBR322 plasmid DNA by complex 1 in the presence of various activating agents. 2D projection of gel images for the cleavage of pBR322 plasmid DNA by complex 1 in presence of different activating agents (c) Form I bands and (d) Form II bands.

significant DNA cleavage activity in the presence of H2O2 and MPA, followed by complete degradation of DNA. 4.5.3. DNA cleavage in the presence of reactive oxygen species Metallonucleases could cleave DNA by oxidative and hydrolytic processes, and therefore provide advantages over conventional enzymatic nucleases in that they are smaller in size and can reach

more sterically hindered regions of macromolecules. Therefore, cleavage mechanism of pBR322 DNA induced by complex 1 was investigated (Fig. 10) and clarified in the presence of hydroxyl radical scavenger 0.4 M DMSO and EtOH (lanes 2 and 3), singlet oxygen quencher NaN3 (0.4 M) and superoxide quencher SOD (4 units) (lanes 4 and 5) under our experimental conditions. On addition of DMSO and EtOH (lane 2 and 3), significant inhibition in the DNA

Fig. 10. Agarose gel electrophoresis pattern for the cleavage pattern of pBR322 DNA (300 ng) by complex 1 at 37 °C after incubation for 45 min in presence of (a) different radical scavengers; Lane 1, DNA control; Lane 2, DNA + complex 1 + DMSO (0.4 mm); Lane 3, DNA + complex 1 + EtOH (0.4 mM); Lane 4, DNA + complex 1 + NaN3 (0.4 mM); Lane 5, DNA + complex 1 + SOD (0.25 mM); (b) Quantification of band area in gel electrophoresis originating from Form I and Form II pBR322 plasmid DNA by complex 1 in the presence of various radical scavengers. 2D projection of gel images for the cleavage of pBR322 plasmid DNA by complex 1 in presence of different radical scavengers; (c) Form I bands and (d) Form II bands.

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cleavage activity was observed, suggesting that hydroxyl radical as the reactive species involved in the DNA strand scission. Thus, hydrolytic cleavage of the sugar phosphate backbone mediated by hydroxyl radicals could be rationalized. However, addition of NaN3 did not show significant quenching of the DNA cleavage and even in presence of SOD the cleavage reaction was quite enhanced (L form appears). These results revealed that singlet oxygen and superoxide anion was not involved in the cleavage process (Lane 4 and 5). Since, the complex 1 was able to cleave DNA in the absence of any reducing agent, which implies that DNA might be cleaved by a discernible hydrolytic pathway. Hydrolytic pathways usually depend on the Lewis acidity of the central metal ion, which serves to activate the phosphodiester bonds towards nucleophilic attack via charge neutralization. Also the presence of aqua ligands in the complex provide an inbuilt nucleophile in the complex, which attack the phosphorus atom thereby leading to direct hydrolysis of the diester bonds. The complex contain coordinated water molecule, which facilitates the nucleophilic attack of water oxygen to phosphorus, followed by a five-coordinate phosphate intermediate and subsequent rearrangement of the phosphate allows the DNA to be cleaved readily.

4.5.4. Molecular docking with DNA Molecular docking studies of ligand H2L and complex 1 with DNA duplex of sequence d(CGCGAATTCGCG)2 dodecamer (PDB ID: 1BNA) was performed in order to predict the binding site along with preferred orientation of the ligand (Fig. 11). The docked model reveals that ligand H2L and complex 1 was fitted into the curved contour of the targeted DNA minor groove within GAC rich region, and slightly bends the DNA in such a way that planar part of the aromatic rings makes favorable stacking interactions between DNA base pairs and leads to van der Waals interaction and hydrophobic contacts with the DNA functional groups which define the stability of groove [48]. Moreover, the o-vanillin ring of the complex 1 arranged in a parallel fashion with respect to the deoxyribose groove walls of the DNA and was stabilized by hydrogen bonding

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(2.8–3.0 Å) between OH group of carboxylic acid with N3 and anomeric oxygen of deoxyribose of G16B and water mediated hydrogen bond with O2 of C10A. The resulting relative binding energy of docked H2L and complex 1 with DNA were found to be 218.9 and 258.6 eV respectively, which was complement for the experimental results obtained from spectroscopic investigations. 4.5.5. Molecular docking with HSA Descriptions of the crystalline structure of albumin have revealed that human serum albumin (HSA) consists of a single polypeptide chain of 585 amino acid residues and comprises three structurally homologous domains (I–III): I (residues 1–195), II (196–383), and III (384–585) that assemble to form a heart-shaped molecule (Fig. 12). The principal regions of ligand binding sites of

Fig. 12. X-ray crystallographic structure of HSA (PDB ID: 1h9z). The domains and subdomains were displayed with different color, the every subdomain and classical binding site were marked in the corresponding location.

Fig. 11. Molecular docked model of ligand H2L and complex 1 with DNA [dodecamer duplex of sequence d(CGCGAATTCGCG)2 (PDB ID: 1BNA)].

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Fig. 13. Molecular docked model of (a) H2L ligand and (b) complex 1 (sphere representation) located within the hydrophobic pocket in subdomain IIA of HSA.

HSA are located in hydrophobic cavities in subdomains IIA and IIIA, corresponding to site I and site II, respectively and sole tryptophan residue (Trp-214) of HSA was in subdomain IIA. Despite very high stability, HSA is a flexible protein with the 3D structure susceptible to environmental factors such as pH and ionic strength, [49]. There was a large hydrophobic cavity in subdomain IIA to accommodate the drug molecule, which play an important role in metabolism and transportation of biomolecule. Herein, molecular docking studies of H2L (Fig. S5) and complex 1 with molecular target HSA was performed to understand their binding affinity and binding location (Fig. 13). The docked conformation showed that the ligand was located within the binding pocket of subdomain IIA of the HSA, and adjacent to hydrophobic residues Phe223, Arg222, Arg218, Ala215, Trp214, Leu238, Arg257, Leu260, and Ala291. However, complex 1 was half-surrounded within subdomain IIA hydrophobic cavity, and it was in close proximity to hydrophobic residues, such as Lys195, Trp214, Ala215, Arg218, Leu219, Val216, Val216, Ala291 and Glu292, of subdomain IIA of HSA, suggesting the existence of hydrophobic interaction between them. Furthermore, there are also a number of specific electrostatic interactions and hydrogen bonds in the proximity of the ligand play an important role in stabilizing the molecule. 5. Conclusion In this work, we have designed and synthesized new copper complex as a discrete with Schiff-base ligand H2L. The in vitro DNA binding studies of H2L ligand and complex 1 revealed an electrostatic mode of binding as well as strongly bind to minor groove of DNA while moderate affinity towards the subdomain IIA of HSA, consistent with molecular docking investigations. The gel electrophoresis results showed that complex 1 cleaves supercoiled plasmid pBR322 DNA in a concentration dependent manner and mechanistic investigations proves the hydrolytic cleavage mechanism. These studies suggested that a synergistic combination of ligand and metal ion was important in the design of a potential chemotherapeutic drug targeting DNA, in addition to their interaction with HSA which was helpful to understand the distribution and transportation of drugs at molecular level. 6. Abbreviations

UV–vis CT DNA Tris EB H2L

UV–visible Calf thymus DNA Tris(hydroxymethyl)aminomethane Ethidium bromide 4-[(2-Hydroxy-3-methoxy-benzylidene)amino]-benzoic acid

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