Accepted Manuscript Synthesis, Spectral Characterization and DNA bindings of Tridentate N2O donor Schiff base Metal(II) Complexes Sellamuthu Kathiresan, Thangavel Anand, Subramanian Mugesh, Jamespandi Annaraj PII: DOI: Reference:

S1011-1344(15)00139-6 http://dx.doi.org/10.1016/j.jphotobiol.2015.04.016 JPB 10013

To appear in:

Journal of Photochemistry and Photobiology B: Biology

Received Date: Revised Date: Accepted Date:

20 February 2015 8 April 2015 16 April 2015

Please cite this article as: S. Kathiresan, T. Anand, S. Mugesh, J. Annaraj, Synthesis, Spectral Characterization and DNA bindings of Tridentate N2O donor Schiff base Metal(II) Complexes, Journal of Photochemistry and Photobiology B: Biology (2015), doi: http://dx.doi.org/10.1016/j.jphotobiol.2015.04.016

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Synthesis, Spectral Characterization and DNA bindings of Tridentate N2O donor Schiff base Metal(II) Complexes

1 2 3

4 Sellamuthu Kathiresana, Thangavel Anandb, Subramanian Mugeshb and Jamespandi Annaraja* a

5 Department of Materials Science, School of Chemistry, Madurai Kamaraj University, Madurai6 625021, Tamil Nadu, India b

7 Department of Microbial Technology, School of Biological Sciences, Madurai Kamaraj 8 University, Madurai-625 021, Tamil Nadu, India 9 *Corresponding author: Tel: +91- 8098478272 10 E-mail address: [email protected] 11 12 Abstract: 13 To evaluate the biological preference of synthetic small drugs towards DNA target, new metal 14 based chemotherapeutic agents of Cu(II), Co(II), Ni(II) and Zn(II), 2,4-diiodo-6-((pyridin-215 ylmethylimino)methyl)phenol (L) Schiff base complexes (1, 2, 3 & 4) having N,N,O donor 16 system respectively were synthesized and thoroughly characterized. The IR results confirmed the 17 tridentate binding of the ligand with metal centre during complexation and reflects the proposed 18 structure. The density function theory calculations were also used to further investigate the 19 electronic structure and properties of ligand and complexes. The preliminary investigation of 20 herring Sperm (HS-DNA) interaction propensity of complexes 1-4 were carried out in Tris-HCl 21 buffer at pH 7.1 to demonstrate their mode of interactions. The obtained results reveal that these 22 complexes significantly interact with DNA on the grooves, further, this observed mode of 23 interactions was also confirmed by molecular docking evaluations. The complexes 1-4 were also 24 screened for antimicrobial evaluations which demonstrated that their significant activity against 25 various human pathogens. The cleavage studies with pBR322 plasmid DNA revealed higher 26 nuclease activity of 1 as compared to other complexes.

27 Keywords: Schiff base, DFT, DNA interaction, DNA cleavage, Molecular docking, Antibacterial 28 investigation. 29 Abbreviations 30 HS DNA

Herring Sperm DNA

31 EtBr

Ethidium bromide

32 DFT

Density functional theory

33 AFM

Atomic force microscopy

34 1. Introduction 35 Tridentate Schiff bases with NNO donor atoms are well recognized to coordinate with various 36 metal ions and have concerned a great deal of interest in recent years due to their rich 37 coordination chemistry [1-3]. They are also used as catalysts in polymer and dyes industry, 38 besides some uses as antifertility and enzymatic agents as well as designing metal complexes 39 related to synthetic and natural oxygen carriers [4]. Though, various Schiff base metal complexes 40 have been widely studied and displayed a wide range of biological applications such as 41 anticancer, antibacterial, antiviral and antifungal agents [5,6] chelating ligands containing N, N, 42 and O donor atoms show broad biological activities and are of special interest because of the 43 ways in which they are bonded to the metal ions [7]. It is known that existence of metal ions 44 bonded to biologically active compounds may enhance their activities [8,9]. 45 Salicylaldehyde derivatives, with one or more halogens in the aromatic ring type Schiff base 46 metal complexes have a wide range of antibacterial and antifungal activities [10,11]. Metal 47 complex oriented research is an emerging field of research due to the postulate of new metal 48 based antimicrobial compounds [12,13]. Important features that can be correlative with adept 49 antimicrobial activities are the lipophilicity and incursion of the complexes via the lipid 50 membrane. In this work, we have chosen a Schiff base derivative of iodo-substituted

51 salicyladehyde component in synergy with Cu(II), Co(II), Ni(II) and Zn(II) metal ions with 52 picolylamine appendage to yield 1-4 (Scheme 1) as potential antibacterial and DNA cleaving 53 agents. The interaction studies of synthesized complexes with HS-DNA, employing biophysical 54 experiments were evaluated to demonstrate their possible mode of interaction towards the 55 molecular drug target DNA. The observed results denoted that all these complexes interact on the 56 groove of DNA sugar phosphate back bone. The cleavage activity by these complexes using gel 57 electrophoresis studies also exhibited their impressive chemical nuclease activity towards 58 supercoiled pBR322. These results would be helpful to understand the binding mode of this kind 59 of complexes to DNA. The ligand and complexes 1-4 were evaluated for their antibacterial 60 activity against E. coli, MRSA, Klebsilla pneumonia, Salmonella typhii and Proteus mirabilis 61 which revealed that the varying inhibitory effects on the growth of bacterial strains. Antibacterial 62 activity of complex 2 against E. Coli was found to be most effective using AFM analysis. Finally, 63 computer-aided molecular docking studies were carried out to validate the spectroscopic studies 64 which also revealed that the complexes interact on the DNA grooves. 65 2. Experimental section 66 2.1 Materials 67 Caution! Perchlorate salts of metal complexes are potentially explosive and therefore should be 68 prepared in a small quantity. 69 All the metal salts were purchased from Merck except cobalt perchlorate hexahydrate which was 70 purchased

from Sigma Aldrich, India. The starting materials for the ligand 3,5-

71 diiodosalicyaldehyde and 2-picolylamine (Sigma Aldrich), Herring sperm DNA (SRL), Tris 72 (hydroxymethyl)aminomethane-HCl (Tris-HCl) (SRL) and sodium chloride (SRL), Ethidium 73 bromide (Sigma Aldrich) were used as received. Double distilled water was used to prepare 74 buffer solution.

75 2.2. Methods and instrumentation 76 Open capillary method was used to determine the melting points of all the synthesized ligand and 77 its metal complexes. Carbon, hydrogen and nitrogen contents were determined using Vario III 78 CHNS analyser. Infrared spectra of the ligand and complexes were recorded using KBr pellets on -1

79 a JASCO FT-IR 410 double beam infrared spectrophotometer in the range of 4000 to 400 cm . 1

80 The H-NMR spectra were recorded on a Bruker 300 MHz spectrometer in CDCl3 solution using 81 TMS as the internal standard. Electrospray Ionization Mass Spectrometry (ESI-MS) analysis was 82 performed in the positive ion mode on a liquid chromatography-ion trap mass spectrometer (LCQ 83 Fleet, Thermo Fisher Instruments Limited, USA). UV-vis spectral measurements were recorded 84 using UV-8453 Agilent Spectrophotometer in the range 190-1100 nm. The fluorescence spectra 85 were made on Agilent Cary Eclipse fluorescence spectrophotometer. The X-band electron 86 paramagnetic resonance (EPR) spectra were recorded on a Jeol-300MHz EPR spectrometer in the 87 solid state at room temperature (RT). Circular dichroism spectroscopy was performed on a 88 JASCO J-810 Spectropolarimeter at room temperature using a 1 mm quartz cuvette. Cyclic 89 voltammetric measurements were carried out on a CH electrochemical analyser. A three electrode 2

90 cell comprising Ag/AgCl, platinum wire and glassy carbon (GC with surface area of 0.07 cm ) 91 were used as reference, counter and working electrode respectively. A cyclic voltagram has been 92 recorded for a blank solution to check the purity of the supporting electrolyte and the solvent. 93 AFM analysis was carried out using AGS100, ATE RESEARCH. 94 2.3. Synthesis 95 2.3.1. Synthesis of 2,4-diiodo-6-((pyridine-2-ylmethylimino)methyl)phenol (L) 96 The ligand (L) was prepared by a slow addition of 2-picolyamine (0.1446g, 1 mmol) in methanol 97 to 3,5-diiodosalicyaldehyde (0.5 g, 1 mmol). The mixture was stirred for 30 min at 30°C and the 98 progress of reaction was monitored by TLC. The obtained yellow precipitate was filtered off,

99 washed thoroughly with methanol and dried in vacuo: Yield: 84%; M.P: 1100C; Color: yellow. 100 IR (KBr, cm-1): 3448 (-OH), 1633(CH=N), 1573(m), 1462(w) (C=C), 1437 (C=N), 1429, 101 1356(m), 1276(m) (C-O). 102

1

H NMR (300 MHz, CDCl3) δ:14.70 (s, 1H, OH), 8.56 (d, J = 4.8 Hz, 1H, ArH), 8.32 (s, 1H,-

103 C=N), 8.04 (d, J = 2.1 Hz, 1H, ArH), 7.69 (td, J = 7.7, 1.8 Hz, 1H, ArH), 7.55 (d, J = 2.1 Hz, 1H, 104 ArH), 7.33 (d, J = 7.8 Hz, 1H, ArH), 7.24 – 7.16 (m, 1H, ArH), 4.93 (s, 2H, -CH2). UV-vis 105 (MeOH, nm) 235, 344, 406; ESI-MS (MeOH) Found m/z= 464.75 [M+H] (calculated m/z = +

106 463.89 for M ). 107 2.4. Synthesis of metal complexes: 108 2.4.1. Synthesis of [C13H9ClCuI2N2O]Cl.2H2O (1) 109 The complex was prepared by a general synthetic method as follows, a hot methanolic solution of 110 Schiff base ligand (0.100 g, 1 mmol) was slowly added to methanolic solution of CuCl2.2H2O 111 (0.037 g, 1 mmol). The reaction mixture was refluxed on a water bath for 2-3 h. The obtained 112 product was filtered off, washed several times with cold methanol, and dried in vacuo. Color: 0

113 dark green, Yield: 76%. M.P: 208-210 C. Anal. Calc. for C13H9ClN2OI2Cu (%): C, 27.78; H, -1

114 1.61; N, 4.98, Found: C, 27.74; H, 1.60; N, 5.02 IR: (KBr, cm ):1620 (CH=N), 1570(m), 115 1487(m) (C=C), 1435 (C=N), 582(M-O), 464 (M-N). UV-vis (MeOH, nm) 244, 396, 638; ESI+

+

116 MS (MeOH): Found m/z = 560 [M] , 526 [M-Cl] (calculated m/z = 560.78). 117 2.4.2. Synthesis of [C26H18CoI4N4O2]2(ClO4).6H2O (2) 118 The complex was synthesized by a similar procedure as described for complex 1 with 119 Co(ClO4).6H2O (0.040 g, 0.5 mmol). Color: green, Yield: 68%. M.P: 214-2160C. Anal. Calc. for 120 C26H18I4N4O2Co (%): C, 31.70; H, 1.84; N, 5.69, Found: C, 31.64; H, 1.80; N, 5.73 IR: (KBr, cm121

1

): 3446(OH), 1626(CH=N), 1562(m), 1489(w) (C=C), 1423 (C=N), 1406(s), 1307(w), 1284(m),

122 557(M-O), 491(M-N). UV-vis (MeOH, nm) 256, 410, 618; ESI-MS (MeOH) Found m/z = 123 984.54 [M]+ (calculated m/z = 984.69). 124

2.4.3. Synthesis of [C13H9ClI2N2ONi]Cl.6H2O (3)

125 The complex was synthesized by a similar procedure as described for complex 1 with 0

126 NiCl2.6H2O (0.051 g, 1 mmol). Color: brown, Yield: 64%. M.P: 164-166 C. Anal. Calc. for 127 C13H9ClI2N2ONi (%): C, 28.02; H, 1.63; N, 5.03, Found: C, 28.02; H, 1.63; N, 5.03. IR: (KBr, -1

128 cm ): 1626 (CH=N), 1577 (C=C), 1425 (C=N), 543 (M-O), 493 (M-N). UV-vis (MeOH, nm) +

129 237, 289, 403; ESI-MS (MeOH) Found m/z = 552.78 [M-Cl+CH3OH] (calculated m/z = 552.84). 130 2.4.4. Synthesis of [C13H9ClI2N2OZn] (4) 131 The complex was synthesized by a similar procedure as described for complex 1 with ZnCl2 132 (0.030 g, 1 mmol). Color: dark yellow, Yield: 72%. M.P:176-1780C. Anal. Calc. for. 133 C13H9ClI2N2OZn (%): C, 27.69; H, 1.61; N, 4.97, Found: C, 27.73; H, 1.57; N, 5.04. IR: (KBr, 134 cm-1): 1622(CH=N), 1573 (C=C), 1437 (C=N), 538 (M-O), 488 (M-N). UV-vis (MeOH, nm) +

135 245, 396; ESI-MS (MeOH) Found m/z = 526.84 [M-Cl+H] (calculated m/z = 526.81). 136 2.5. Chemistry 137 The synthesized metal complexes are solid materials with distinguished colour, stable at room 138 temperature and possess high melting point (>160° C). The metal complexes are insoluble in 139 water and poorly soluble in common organic solvents but completely soluble in DMF and 140 DMSO. The evidence for the formation of tridentates NNO donor nature of the Schiff base ligand 141 (L) was confirmed by using various spectral techniques. Elemental analysis and analytical data 142 were well agreed with the proposed composition of Schiff base ligand (L) and its metal 143 complexes. These data of metal complexes suggest that the metal to ligand ratio of the complexes 144 is 1:1 stoichiometry of the type [M(L)(Cl)] for Cu(II), Ni(II) and Zn(II) complexes respectively

145 and 1:2 stoichiometry of the type [(M)(L)2] for Co(II) complex, where L stands for deprotonated 146 ligand. 147 2.6. DNA binding and cleavage experiments 148 2.6.1. Electronic absorption spectra 149 The concentration of HS-DNA per nucleotide was measured by using its known extinction -1

-1

150 coefficient at 260 nm (~6600 M cm ). Tris-HCl buffer was used for the absorption experiment. 151 The electronic absorption spectra of metal complexes were recorded in the absence and presence 152 of HS-DNA in MeOH:buffer solution. Absorption titrations were done with fixed complex -4

153 concentration (in 10 M) and by varying the DNA concentration (0-300 µM) [14,17]. 154 2.6.2. Fluorescence spectra 155 The fluorescence spectra was recorded at room temperature using 10-4 M DNA and 10 -5 M EtBr. 156 The [DNA]/[EtBr] ratio of 10:1 was prepared by addition of 250 µL EtBr (10-3 M) and 1250 µL 157 DNA (2 X 10 -3 M) into 25 mL Tris-HCl buffer solution at (pH=7.1) [15]. The decrease in the 158 fluorescence intensity at ~600 nm with an excitation wavelength of 525 nm was recorded with 159 increasing amounts (0-30 µM) of different metal complexes. 160 2.6.3. CD spectra 161 Circular dichroic spectrum of DNA in the presence and absence of metal complexes was recorded 162 on a JASCO J-810 (163-900 nm) spectropolarimeter using a quartz cuvette with 1 mm optical 163 path length at increasing concentration of complexes to DNA solution (r = 0.3-0.9). Each sample 164 solution was scanned in the range of 220-300 nm. Every CD spectrum was collected after 165 averaging over at least four accumulations using a scan speed of 100 nm min-1 and a one second 166 response time from which the buffer background had been subtracted. [DNA] =100 µM [16]. 167 2.6.4. Cyclic voltammetry

168 The cyclic voltammogram were carried out with a three electrode apparatus using a CH 169 electrochemical analyser in MeOH:buffer mixture as solvent system with the fixed concentrations 170 of the complexes in the absence and presence of increasing concentration of HS-DNA. The shifts 171 in the values of ∆Ep (separation of the anodic and cathodic peak potentials), changes observed in 172 the ipc/ipa (the ratio of the cathodic to anodic peak currents) values, and the formal potential (E1/2) 173 changes explain the binding ability of the complexes with HS-DNA. 174 2.7. DNA cleavage study 175 The DNA cleavage experiments were performed using supercoiled pBR322 plasmid DNA (2µL, 176 10µM) in Tris-acetate-EDTA (TAE) buffer which was treated with metal complexes (30µM) and 177 the extent of cleavage to its nicked circular (NC) form was determined by agarose gel 178 electrophoresis. The samples were incubated for 3 h at 37°C in a dark chamber and were added to 179 loading buffer containing 25% bromophenol blue, 0.25% xylene cyanol, 30% glycerol (3µL) and 180 the solution was finally loaded on 1.0 % agarose gel containing 0.5l g mL-1 ethidium bromide 181 (EtBr). The electrophoresis experiments were carried out in a dark chamber for 2 h at 110V in 182 Tris–acetic acid–ethylenediaminetetraacetic acid (TAE) buffer and the bands were photographed 183 under UV light. 184 2.8. Antibacterial activity 185 All the synthesized ligand and complexes 1-4 were screened for their in vitro antibacterial 186 activity against human bacterial pathogens (E. coli, MRSA, Klebsilla pneumonia, Salmonella 187 typhii and Proteus mirabilis) procured from Apollo Hospital, Madurai. The agar well diffusion 188 method [18] was adopted for measuring the antibacterial assays. Briefly, all cultures were 189 routinely maintained on NA (nutrient agar) and incubated at 37°C for overnight. The culture was 190 centrifuged at 1000 rpm and pellets were re-suspended and diluted in sterile Normal Saline 5

191 Solution to obtain viable count of 10 cfu/ml. Volume of 0.1 ml diluted bacterial culture 192 suspension was spread uniformly with the help of spreader on NA plates. Early log phase (8 hrs)

193 bacterial cultures were made using sterile cotton swab and labelled. Wells were made in the agar 194 media with the help of a metallic cork borer. Desired concentration of (100µl) ligand and 195 metal(II) complexes from the stock solution, 1 mg/ml was dissolved in DMSO and added in the 196 respective wells. Other wells were treated with 100µl of Streptomycin and DMSO of 1 mg/ml 197 was served as a positive and negative control. The plates were then incubated for 24 h at 37°C 198 and the resulting zones of inhibition (mm) were measured. Bacterial culture growth inhibition 199 was compared with the positive control Streptomycin drug. 200 2.8.1 AFM 201 The maximum inhibitory activity shown by the complex was taken for the further cell wall 202 rupture analysis under AFM technique. 5 ml of E. coli culture treated with 100 µg of cobalt(II) 203 complex and E. coli with positive control culture in the absence of cobalt(II) metal complex were 204 kept separately under incubation at 37°C for 6 hours. After incubation, a loop full of culture was 205 placed in glass cover slip and allowed to air dry at room temperature for overnight and then the 206 changes occurred in the E. coli cell wall analysis was carried out. 207 2.9 Computational details 208 The geometries of the ligand and complexes were optimized using density functional theory 209 (DFT) by B3LYP [19] combined with 6-31G and LANL2DZ [20] basis sets. Molecular frontier 210 orbital HOMO (highest occupied molecular orbital), LUMO (lowest unoccupied molecular 211 orbital) and the optimized structures were visualized with Gaussian 03 package. 212 2.9.1. Molecular docking 213 The rigid molecular docking studies were performed by using HEX 8.0 software [21] which is an 214 interactive molecular graphics program to calculate and display feasible docking modes of a pairs 215 of protein, enzymes and DNA. Structures of the complexes were sketched and converted into pdb

216 format from mol format by ChemDraw (http://www.cambridgesoft.com) software. The structure 217 of HS-DNA (PDB id: 423D, sequence d[CCGTCGACGG]2) was retrieved from Protein Data 218 Bank (http://www.rcsb.org./pdb) and DNA-metal complexes were analysed to study nature of 219 interactions [22]. All calculations were carried out on an Intel Core 2 Duo, 1.86 GHz based 220 machine running MS Windows 7 as operating system. 221 3. Result and discussions 1

222 3.1. H NMR spectra 1

223 The H NMR spectra of the Schiff base ligand was recorded in CDCl3 solution using TMS as an 224 internal standard. The observed spectrum showed signals in the δ 7.2-8.6 (m) region for the 225 aromatic protons. Schiff base has been evidenced by the presence of C=N azomethine proton 226 signal appeared at δ 8.3 ppm (Supplementary files Fig: S1). Peaks viewed at δ 4.9 and 14.7 ppm 227 were attributed to the methylene and phenolic –OH protons respectively. 228 3.2 IR Spectra 229 The IR spectra provide valuable information regarding the coordinating sites of ligand. The IR 230 spectra of the complexes were compared with that of the free ligand to determine the changes that 231 might have taken place during the complexation. The ligand exhibited a band at 1633 cm

-1

-1

232 assigned to the azomethine nitrogen. A stretching vibration band occurs at 1437 cm due to the -1

233 picolylamine nitrogen and a broad band was observed for the ligand at 3448 cm due to phenolic 234 –OH stretching group. In the IR spectra of complexes, the stretching vibration band appeared at 235 1633 cm-1 for the azomethine group of free ligand which underwent a negative shift to the lower 236 frequency ~1618-1626 cm-1 indicating that the coordination of azomethine nitrogen and 237 picolylamine nitrogen to the central metal ion in all the complexes. The band at 3448 cm-1 238 corresponding to the free –OH group was disappeared in the complexes due to the deprotonation 239 on complexation. These observed results clearly indicated that the coordination of ligand to the

240 metal centre has occurred as proposed in the present structure. Moreover, the non-ligand bands in 241 the region 540-590 cm-1 and 460-500 cm-1 are newly observed due to the formation of M-O and 242 M-N bonds respectively [23], the M-Cl stretching bands appeared below 400 cm-1 region [24] 243 further confirmed the complex formation. In conclusion that these data suggest an NNO tridentate 244 behaviour of the ligand. 245 3.3 Electronic spectra 246 Electronic spectra of the ligand and its complexes were measured in methanol. The electronic 247 absorption spectra of ligand displayed high-energy bands at 344 and 406 nm, corresponds to 248 π→π* transition of the aromatic ring and n–π* transitions of the C=N groups, respectively. 249 However, these bands were shifted to 344–244 and 406–396 nm in the complexes attributing to 250 intraligand and LMCT transitions of coordinated ligand. Additionally, the electronic spectra of 251 Cu(II) complex displayed low intensity broad band at ca. 638 nm, in the visible region 252 attributable to d–d (2B1g→2Eg) transition and suggesting square planar environment [25]. In the 253 case of Co(II) complex a lowest energy band appeared at 618 nm due to d-d (4T1g(F)→4A2g) 254 transition responsible for its octahedral environment [26]. The electronic spectrum of Ni(II) 255 complex displayed d-d transitions at 403 nm assigned to a square planar geometry [27]. In 256 contrast Zn(II) complex does not exhibit any d-d band because of its completely filled d10 257 transition (Supplementary file Fig: S2). 258 3.4 Mass and EPR spectral studies 259 The ESI-mass spectra of synthesized ligand and their complexes were recorded and the obtained 260 molecular ion peaks confirm the proposed formulae. The mass spectrum of the ligand shows a 261 molecular ion peak at m/z = 464 [M+H] which coincides with the formula weight of the ligand. 262 The ESI mass spectrum of the [Cu(L)Cl], [Co(L)2], [Ni(L)Cl] and [Zn(L)Cl] complexes showed +

+

+

263 molecular ion peaks at m/z = 526 [M-Cl] ; 984 [M] ; 553 [M-Cl+CH3OH] and 527 [M-Cl+H]

+

264 respectively. Whereas in [Co(L)2] complex have shown at 984 m/z which evidenced that the 265 biligated structure of the complex and spectra are shown in (Supplementary files Fig: S3-S7). 266 The ESR spectrum of Cu(II) complex provides information on the metal ion environment within 267 the complex, i.e., the geometry and nature of the ligating sites of the Schiff base and metal. The 268 solid state X–band EPR spectrum of copper complex was recorded at room temperature under the 269 frequency of 9.1 GHz with magnetic field strength of 3000 ± 1000 G using tetracyanoethylene as 270 a field marker (Supplementary file Fig: S8). The spin Hamiltonian parameters for the Cu(II) 271 complex is used to derive the ground state and the observed spectrum of copper ion in the 9

272 complex was determined to be d system [28]. 273 3.5 Cyclic voltammetry studies 274 The cyclic voltammogram (CV) experiments were performed at room temperature in DMSO 275 under nitrogen atmosphere in the potential range -1.4 to +1.4 V. The cyclic voltammogram of 276 [Cu(L)Cl] showed a well-defined redox process corresponding to the CuII/I couple. It exhibits a 277 cathodic peak at Epc = 0.299 V and the respective anodic peak appeared at Epa = 0.468 V. The 278 peak-to-peak separation (∆Ep) was 0.169 V, is more than 59 mV, indicating the quasi-reversible 279 redox character. The formal potential E1/2 taking average of Epa and Epc, is 0.384 V. The obtained 280 ipa/ipc value, 1.316 its simple one electron transfer [29]. Similarly the cyclic voltammogram of 281 redox process in Co(II), Ni(II) and Zn(II) complexes exhibits cathodic peak at Epc= 0.196, 0.606. 282 0.066 volts and respective anodic peak at Epa = 0.288, 0.774, 0.269 volts (Supplementary file Fig. 283 S9). The observed ∆Ep values for the cobalt, nickel and zinc complexes were 0.092, 0.168, 0.203 284 V respectively, indicating their quasi-reversible redox character (Table. 1). 285 4. Biochemistry 286 4.1 DNA binding studies

287 DNA binding is the important step for chemical nuclease activity of the metal complexes. Thus, 288 the binding propensity of the title metal(II) complexes 1-4 towards HS-DNA was studied using 289 various spectroscopic methods. 290 4.1.1. Absorption spectral studies 291 Electronic absorption spectroscopy is one of the most useful techniques to understand the drug– 292 DNA binding studies. Upon addition of increasing concentrations of HS-DNA to the complexes 293 [Cu(II), Co(II), Ni(II) and Zn(II)] with fixed concentration (300 µM), an increase in the 294 absorbance (hyperchromic effect) of the intraligand absorption band was observed with a minor 295 red shift in the absorption maxima (Fig. 1). The observed spectral behavior obviously rule out 296 intercalative binding of the complexes to DNA, since intercalation leads to hypochromism in the 297 spectral bands. This ‘hyperchromic effect’ was attributed to contraction and overall damage 298 caused to the secondary structure of DNA double helix [30]. Hyperchromism with less or no shift 299 in absorbance is consistent with groove binding, therefore in these complexes it can be attributed 300 to external contact (surface binding) with the DNA duplex. Also the extent of hyperchromism is 301 commonly consistent with the groove or electrostatic binding indicating the unwinding of the 302 DNA double helix [31]. These observations may indicate that the interactions between DNA and 303 the complexes may occur via a non-classical intercalation, probably through electrostatic and/or 304 groove binding modes [32]. The intrinsic binding constant (Kb) was calculated according to the 305 following equation. 306

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

307 Where εa the extinction coefficient observed for the charge transfer absorption at a given DNA 308 concentration, εf, the extinction coefficient of the complex free in solution, εb, the extinction 309 coefficient of the complex when fully bound to DNA. A plot of [DNA]/ (εa-εf) versus [DNA], 310 gives Kb as the ratio of the slop to the intercept. The intrinsic binding constant values of all the

311 complexes with HS-DNA have been summarized in the Table: 2 and are observed in the 312 following order: 313

[Cu(L)]Cl > [Ni(L)]Cl >[Zn(L)]Cl > [Co(L)2].

314 This observed order also stated that the copper(II) complex was more intensively bound with 315 DNA and also illustrated that its biological importance compared to other metals. 316 4.1.2. Circular dichroism studies 317 The structural changes of HS-DNA due to the interactions with complexes were measured using 318 circular dichroism (CD) spectroscopy. It is a powerful, sensitive and sophisticated tool to identify 319 the conformational changes occurred in DNA during the interaction of small molecules. The so320 called right handed B form of free helical DNA exhibits a positive band at 275 nm due to base 321 stacking and a negative band at 245 nm due to helicity. The CD spectra of HS-DNA (100 µM) in 322 the presence of the title complexes (30 µM) showed a remarkable decrease in positive and a 323 slight changes in the negative bands which clearly indicates that the significant interactions have 324 occurred between them without causing any conformational changes to it even after interaction 325 with a higher concentration of the complex [33,43]. The decreased intensity in the negative band 326 (Fig. 2) suggests that the complexes can unwind the DNA helix and reduce its stability to a 327 certain extent. These changes are indicative of a non-intercalative DNA-binding mode of the 328 complexes, probably through groove and/or on the surface [34]. The decreasing intensity of both 329 positive and negative bands were more compare to other complexes, may be due to the more 330 binding surface area on the sugar phosphate backbone. The bi-ligated structure of the cobalt 331 complex may stick on the phosphate backbone like a butterfly sit on the twisted ladder (it is 332 obvious that the DNA structure is look alike a twisted ladder), therefore, due to this strong 333 binding and increased surface area may causes for the more decreasing intensity of the DNA

334 band observed at 277 nm. This bi-ligated structure of the cobalt(II) complex also may be one of 335 the possible reason for its major groove mode of interaction. 336 4.1.3. Fluorescence spectra 337 The ethidium bromide (EtBr) displacement evaluation was used to measure the relative binding 338 of complexes to HS-DNA. The competitive DNA binding constants (Kapp) of complexes were 339 measured by monitoring changes in emission intensity at 603 nm of ethidium bromide (EtBr) 340 bound to HS-DNA as a function of added complex concentration. EtBr is a conjugate planar 341 molecule with very weak fluorescence intensity due to fluorescence quenching of the free EtBr 342 by solvent molecules but it is greatly enhanced when EtBr is specifically intercalated into the 343 adjacent base pairs of double stranded DNA [35]. The enhanced fluorescence can be quenched 344 upon the addition of the second molecule which could replace the bound EtBr or break the 345 secondary structure of the DNA (due to stronger binding affinity to DNA than EtBr). On addition 346 of increasing concentration of complexes to HS-DNA pretreated with EtBr ([DNA]/[EtBr] = 1) a 347 significant reduction in the emission intensity (Fig. 3) was observed, indicating that the 348 replacement of the EtBr by these complexes, which results in a decrease of the binding constant 349 of ethidium bromide to DNA. As there was incomplete quenching of the EtBr–induced emission 350 intensity, the intercalative binding mode was ruled out. The extent of quenching of the emission 351 intensity gives a measure of the binding propensity of the interacting molecule to HS-DNA [36]. 352 The quenching of EtBr bound to DNA by the complexes in agreement with the linear Stern353 Volmer equation [37]. 354

I0 / I = Ksq [Q] + 1

355 Where, Io is the emission intensity in the absence of a quencher, I is the emission intensity in the 356 presence of a quencher, Ksq is the quenching constant, and [Q] is the quencher concentration. The 357 Ksq value is obtained as a slope from the plot of I0/I versus [Q]. The quenching plots in Fig. 3

358 illustrate that the quenching of EtBr bound to HS-DNA by the complexes are in good agreement 359 with the linear Stern-Volmer equation. The Ksq values were found to be 0.281, 0.129, 0.260 and 360 0.240 for 1, 2, 3 and 4 respectively, following the order 1 > 3 > 4 > 2. 361 The Kapp values (apparent binding constant) were also calculated for the complexes 1, 2, 3 and 4 362 using the literature method [38].

KEtBr [EtBr] = Kapp [Complex]

363

364 Where [complex] corresponds to 50% reduction of emission intensity of EtBr-bound DNA, and 7

-1

365 KEtBr = 1.0 X 10 M , [EtBr] = 10 µM. The emission intensity of EtBr-bound DNA system was 366 plotted against different concentration of metal complexes. The complex concentrations 367 corresponding to 50% reduction of emission intensity were found and further used to calculate 368 the Kapp for each complex. The apparent DNA binding constants for complexes 1, 2, 3 and 4 were 369 found as 2.0 ×106 M-1, 0.48 ×10 6 M-1, 0.67 ×106 M-1 and 0.50 × 106 M-1 respectively. Thus, both 370 intrinsic and apparent binding parameters clearly showed that all these complexes have a good 371 binding propensity for the HS-DNA under the discussed experimental conditions. These observed 372 values are also in well agreement with the Kb values observed from the absorption spectral 373 techniques. 374 4.1.4. Cyclic voltammetry studies 375 Amongst the various electroanalytical techniques in general, cyclic voltammetry (CV) is by far 376 the most versatile electrochemical method as many redox active metal complexes which are not 377 amenable to various spectroscopic methods, either because of weak absorption bands (forbidden 378 d-d transitions) or because of the overlap of electronic transitions with those of

the DNA

379 molecule. The cyclic voltammogram of [Cu(L)Cl] in the absence of HS-DNA showed a Cu II/I 380 redox couple with cathodic peak potential at Epc of -0.068 V and anodic peak potential Epa at 381 0.058 V (Supplementary file: Table. 1). The separation of the anodic cathodic peak potentials,

382 ∆Ep is 0.126 V, and the ipa/ipc, is 1.43, indicates that the reaction of the complex on the glassy 383 carbon electrode surface was quasi-reversible redox process. The formal potential E1/2, is -0.005 384 V in the absence of DNA. The addition of increasing concentrations of DNA to [Cu(L)Cl] causes 385 a considerable decrease in the voltammetric current coupled with a slight shift in the E1/2 (-0.009 386 V) to a less negative potential as illustrated in Fig. 4. These changes can be attributed to diffusion 387 of the metal complex bound to the large, slowly diffusing DNA [39,40]. From this data it is 388 understood that the synthesized complex interact with DNA through a non-intercalative manner 389 may be on the grooves. 390 Differential pulse voltammogram (DPV) of [Cu(L)]Cl complex has also shown a decreasing 391 current along with a slight potential shift were observed due to the addition of increasing 392 concentrations of DNA (Fig. 5). 393 The ratio of equilibrium constants, k2+/k+ for the binding of Cu(I) and Cu(II) forms of complexes 394 to DNA can be estimated from the net shift in E1/2, assuming reversible electron transfer. For a 395 Nernstian electron transfer in system in which both the oxidized and reduced forms associated 396 with a third species such as DNA in solution, Scheme 3 can be applied for a more detailed n+

n+

397 account. Here, CuL -DNA represents the CuL

complex bound to DNA having n+ oxidation

398 state of the copper. Thus for one electron transfer process,

Ebo’ – Efo’ = 0.059log (K+/K2+)

399 o’

400 where, Ef

and Ebo’ are the formal potentials of the CuL+/CuL2+ couple in the free and bound

401 forms, respectively. Thus from the shift in E1/2 values, K2+/K+ ratios have been calculated. 402 Scheme 3

2+

CuL

+ e-

CuL

K2+

CuL

+

o' Ef

K+ 2+

- DNA

+ e-

CuL

+

o' - DNA E b

403 404 For all these complexes, K+ is higher than K2+ suggest that the DNA preferentially stabilize the 405 Cu(I) form over the Cu(II), as expected. The possible specificity of these complexes towards the 406 pyrimidine or purine bases of the DNA is still undergoing with the help of biologists. 407 4.2. DNA cleavage studies 408 To scrutinize whether the DNA interactions and conformational changing properties of the title 409 complexes were associated with additional pharmacological performance, chemical nuclease 410 activity assay was also performed by incubating them with pBR322 plasmid DNA as a substrate 411 in a medium of 5 mM Tris–HCl/50 mM NaCl buffer at pH 7.2 for 2 h at 37 °C. The ability of 412 complexes to cleave supercoiled (SC) pBR322 DNA was assayed with the aid of gel 413 electrophoresis in the absence and/or presence of external agents. It is known that plasmid DNA 414 cleavage produces relaxation of the supercoiled circular conformation to the nicked circular 415 and/or linear conformations. When circular pBR322 DNA is subjected to gel electrophoreses, 416 relatively fast migration is observed for the intact supercoiled form (Form I). If scission occurs on 417 one strand (nicking), the supercoiled form will relax to generate a slower-moving open circular 418 form (Form II). If both strands are cleaved, a linear form (Form III) is generated that migrates in 419 between Forms I and II. 420 Transition metal complexes have been reported as the inhibitors of DNA repairing enzymes. The 421 DNA cleavage ability of complexes 1-4 was studied by agarose gel electrophoresis using 422 supercoiled pBR322 plasmid DNA as a substrate due to their high binding ability (Fig. 6). A 423 significant conversion of supercoiled form (Form I) of pBR322 DNA to nicked form (Form II)

424 was observed for all the complexes (Lanes 6–10). However, all these complexes were able to 425 convert supercoiled form (Form I) to nicked open circular form (Form II) with the fixed 426 concentration (Fig. 6, Lanes 6–10) without the appearance of linear form (Form III) of pBR322 427 plasmid DNA which indicated that no double strand DNA cleavage was observed. The delivery 428 of metal ion to the helix, in locally generating oxygen or hydroxide radicals, yields an efficient 429 cleavage reaction [41]. 430 4.2.1. Effects of additives on plasmid DNA cleavage 431 The cleavage tests were performed under aerobic conditions with H2O2 as a co-oxidant at a fixed 432 complex concentration. Neither H2O2 nor the metal(II) salts yielded substantial strand scission 433 (lans 1-5) when applied separately, also the present complexes individually did not produced any 434 significant cleavage (data not shown). These experimental facts demonstrated that a combination 435 of both the metal complexes and H2O2 are required to display effective cleavage of plasmid 436 DNA. Fig. 6 reveals that cleavage of pUC19 DNA induced by complexes 3–6 in the presence of 437 H2O2 results in the conversion of Form I (lane 1) into Form II (lane 2). The intensity of the 438 supercoiled SC (Form I) band diminished gradually and converted to nicked form (NC) (Form II) 439 of DNA by the metal complexes in the presence of H2O2 [42]. This results in oxidative attack on 440 the deoxyribose moiety at C-1 hydrogen, leading to a series of elimination reactions that ruptures 441 the phosphodiester backbone. This observation illustrated that these metal complexes behave as 442 efficient chemical nuclease for the cleavage of double strand DNA may be due to the formation 443 of hydroxyl radicals. It is obvious that Cu(II) (Fig. 6; lane 6) has more ability to cleave the 444 supercoiled plasmid DNA when compared to that of other complexes. According to the previous 445 report [43], it is believed that the DNA cleavage ability of complexes is due to the reaction of 446 metal ions in the presence of H2O2 which produces diffusible hydroxyl radicals or molecular 447 oxygen at ease, which damage DNA through Fenton-type chemistry. 448 4.3. Antibacterial activity

449 The development of new bacterial resistant strains to current antibiotics has become serious 450 problem in public health. Therefore, there is a strong motivation to develop new bactericides. So, 451 nowadays pharmaceutical industries are looking for synthesizing the alternative compounds 452 which act as drug. Currently much attention has been focus on the synthesis of new metal 453 complexes and evaluating their antibacterial activity. In the present work, we desire to explore the 454 activity of the Schiff base ligands and its metal(II) complexes against five different human 455 pathogens. The diameter of the zone of inhibition (mm) was used to compare the antimicrobial 456 activity of the all complexes with the commercial drug (streptomycin). The results revealed that 457 complexes exhibited varying degree of inhibitory effects on the growth of bacterial strains may 458 be due to the effect of the metal ion on the cell metabolism. A large number of reports were 459 illustrated that the significance of Co(II) complexes as antibacterial agents, presumably due to 460 their aqueous stability, availability, and ease of synthesis [44]. Even though DMSO is an 461 antimicrobial solvent, here we performed our antibacterial tests by using DMSO as a negative 462 control. Further, control experiments using DMSO alone do not show any antibacterial effect. 463 The observed remarkable activity against E. coli with zone inhibitory diameter of 21 mm 464 (Supplementary file Fig. S10), by cobalt(II) complex and showed maximum zone of inhibition 465 than the positive control streptomycin. [45,46] Copper, nickel and zinc complexes also shown 466 significant activity towards MRSA, Klebsilla pneumonia, Salmonella typhii and Proteus mirabilis 467 compared to the positive control streptomycin. Whereas the ligand has shown very negligible 468 activity compared to all complexes and the results have been summarized in (supplementary file 469 Table. S2). Hence, it is noticed that the complexes are more potent bactericides than the free 470 Schiff bases. This higher antimicrobial activity of the metal complexes compared to Schiff bases 471 may be due to the change in structure due to coordination and chelating tends to make metal 472 complexes act as more powerful and potent bacteriostatic agents, thus inhibiting the growth of the 473 microorganisms. Moreover, coordination reduces the polarity of the metal ion mainly because of

474 the partial sharing of its positive charge with the donor groups within the chelate ring system 475 formed during the coordination. This process, in turn, increases the lipophilic nature of the central 476 metal atom, which favours its permeation more efficiently through the lipid layer of the 477 microorganism, thus destroying them more aggressively. The results show that cobalt(II) 478 complexes show higher antibacterial activity than other complexes. The variation in the activity 479 of complexes against different microorganisms depends either on the impermeability of the cells 480 of the microbes or differences in ribosomes in microbial cells [47]. 481 4.3.1. AFM 482 The observed antibacterial activity suggests that the cobalt(II) complex showing high inhibitory 483 activity through agar diffusion technique in E. coli culture plates. To confirm this higher activity, 484 100 µg was inoculated for 24 hours culture broth. After incubation for 6 hours at 37°C, the 485 culture was analysed under Scanning Probe Microscope. The appeared morphology changes of E. 486 coli cell was not altered in the absence of cobalt(II) complex, whereas the culture which treated 487 with complex showed significantly ruptured cells as illustrated in the Fig. 7, indicates that 488 complex has complete inhibitory activity on E. coli cells. This depicts that the complex binds 489 with the cell wall and ruptured the cell membrane due to its oxidative stress on the E. Coli. 490 Thereby the complex could enter into the nucleus, consequently degraded the chromosomal DNA 491 by preventing protein synthesis which is essential for all cellular metabolism [48]. 492 4.4. DFT calculation 493 The frontier molecular orbitals, called HOMO and LUMO. The HOMO represents the ability to 494 donate an electron, LUMO as an electron acceptor represents the ability to obtain an electron. 495 These orbitals play an important role in the electric properties and determine the way of 496 molecules interacts with other species. Both HOMO and LUMO are the main orbital taking part 497 in chemical reactions. While the energy of the HOMO is directly related to the ionization 498 potential, LUMO energy is directly related to the electron affinity. Also, the frontier orbital gap

499 between HOMO and LUMO, represents stability of structure [49,50]. The HOMO and LUMO 500 energy gap of ligand -0.3183, complexes 1, 2, 3 and 4 are -0.3457, -0.4953, -0.3002 and -0.3028 501 respectively (Fig 8,9). 502 4.5. Molecular docking 503 The binding efficacy of metal complexes and HS-DNA were determined using E-total value 504 generated by the HEX 8.0. The complexes showed relatively low binding values to the HS-DNA. 505 Energetically favourable docked possess were obtained from the rigid molecular docking 506 experiments carried out between the optimized energy-minimized structures (Fig. 10) for the 507 ligand and complexes 1, 2, 3 and 4 with DNA duplex of sequence d(CGCGAATTCGCG)2 508 dodecamer (PDB ID: 1BNA). All the metal complexes found to be binds on the grooves, among 509 the metal complexes, complex 2 shown potent effect on DNA (-339.66 kjmol-1) and bind on the 510 major groove [51], whereas, complex 1 has the highest binding affinity on the minor groove with 511 -269.99 k jmol-1. Each of the metal complexes have variable binding site on HS-DNA. The 512 geometry of metal complexes bound DNA and their sequences were shown in (Supplementary 513 file: Table. 3). 514 5. Conclusion 515 In the present work Cu(II), Co(II), Ni(II) and Zn(II) complexes have been synthesized using a 516 Schiff base as a ligand. They were thoroughly characterized by various spectral and 517 electrochemical methods. Preliminary DNA binding studies of metal complexes 1-4 were carried 518 out by spectral and electrochemical techniques. The obtained results suggested that the present 519 metal complexes bind with HS-DNA on the grooves. The binding affinity of these complexes 520 with HS-DNA followed the order 1>3>4>2, complex 1 displaying a higher binding as compared 521 to other complexes. The DNA cleavage activity is performed using supercoiled pBR322 DNA 522 and the observed results indicate that the complexes have significant potential to cleave pBR322

523 DNA. Antibacterial activity reveals that the cobalt(II) complex has shown remarkable inhibitory 524 activity among the complexes compared to the standard drug streptomycin against E. coli. 525 Further its (E.Coli) cell morphology changes due to the inhibition of cell growth via cell wall 526 rupture by the Co(II) complexes was analysed with AFM technique. 527 Acknowledgements 528 Mr. SK and Dr. JA gratefully acknowledge the Department of Science and Technology, New 529 Delhi [Grant No: SB/FT/CS-175/2011] for financial support. 530 References: 531 [1] C. Rajarajeswari, M. Ganeshpandian, M. Palaniandavar, A. Riyasdeen, M.A. Akbarsha, Mixed 532

ligand copper(II) complexes of 1,10-phenanthroline with tridentate

533

phenolate/pyridyl/(benz)imidazolyl Schiff base ligands: covalent vs non-covalent DNA

534

binding, DNA cleavage and cytotoxicity, J. Inorg. Biochem. 140 (2014) 255-268.

535 [2] H. Khanmohammadi, M. Pass, K. Rezaeian, G. Talei, Solvatochromism, spectral properties 536

and antimicrobial activities of new azo–azomethine dyes with N2S2O2 donor set of

537

atoms, Molecular. Structure. 1072 (2014) 232-237.

538 [3] J.R. Anacona, N. Noriega, J. Camus, Synthesis, characterization and antibacterial activity of a 539

tridentate Schiff base derived from cephalothin and sulfadiazine, and its transition metal

540

complexes, Spectrochim. Acta A. 137 (2015) 16-22.

541 [4] A. Nasser, M.A. Alaghaz, M.E. Zayed, S.A. Alharbi, Synthesis, spectral characterization, 542

molecular modeling and antimicrobial studies of tridentate azo-dye Schiff base metal

543

complexes, J. Mol. Struct. 1084 (2015) 36-45.

544 [5] J. Charo, J.A. Lindencrona, L.M. Carlson, J. Hinkula, R. Kiessling, Protective efficacy of a 545

DNA influenza virus vaccine is markedly increased by the coadministration of a Schiff base-

546

forming drug, J. Virol. 78 (2004) 11321-11326.

547 [6] H.L. Singh, S. Varshney, A.K. Varshney, Synthesis and spectroscopic studies of organotin(IV) 548

complexes of biologically active Schiff bases derived from sulpha drugs, Appl. Organometal.

549

Chem. 14 (2000) 212-217.

550 [7] M. Thankamony, K. Mohanan, Synthesis, spectral studies, thermal decomposition kinetics, 551

reactivity and antibacterial activity of some lanthanide(III) nitrate complexes of 2-(N-indole-2-

552

one)amino-3-carboxyethyl-4,5,6,7-tetrahydrobenzo[b]thiophene, Indian Journal of Chemistry

553

A. 46 (2007) 247-251.

554 [8] B. Halli, V. B. Patil, Synthesis, spectral characterization and DNA cleavage studies of Co(II), 555

Ni(II), Cu(II), Zn(II), Cd(II) and Hg(II) complexes with benzofuran-2-carbohydrazide Schiff

556

bases, Indian Journal of Chemistry A. 50 (2011) 664-669.

557 [9] K. Shivakumar, M. B. Halli, Synthesis, characterization and antibacterial studies on metal 558

complexes with a naphthofuran thiosemicarbazide derivatives. J. Coord. Chem. 59 (2006)

559

1847-1856.

560 [10] L.C. Felton, J.H. Brewer, Action of Substituted Salicylaldehyde on Bacteria and Fungi, 561

Science. 105 (1947) 409-410.

562 [11] A. Roth, E.T. Spielberg, W. Plass, Kit for Unsymmetric Dinucleating Double-Schiff-Base 563

Ligands: Facile Access to a Versatile New Ligand System and Its First Heterobimetallic

564

Copper- Zinc complex, Inorg. Chem. 46 (2007) 4362-4364.

565 [12] A. Scozzafava, C.T. Supuran, Carbonic Abhydrase and Matrix Metalloproteinase Inhibitors: 566

Sulfonylated Amino Acid Hydroxamates with MMP Inhibitory Properties Act as Efficient

567

Inhibitors of CA Isoenzymes I, II and IV and N-Hydroxysulfonamides Inhibit Both These

568

Zinc Enzymes, J. Med. Chem. 43 (2000) 3677-3687.

569 [13] S.A. Rice, M. Givskov, P. Steinberg, S. Kjelleberg, Bacterial Signals and Antagonists: The 570

Interaction between Bacteria and Higher Organisms, J. Mol. Microbiol. Biotechnol. 1 (1999)

571

23-31.

572 [14] P.P. Netalkar, S.P. Netalkar, S. Budagumpi, V.K. Revankar, Synthesis, crystal structures and 573

characterization of late first row transition metal complexes derived from benzothiazole core:

574

Anti-tuberculosis activity and special emphasis on DNA binding and cleavage property,

575

European Journal of Medicinal Chemistry. 79 (2014) 47-56.

576 [15] Y.C. Liu, Z.F. Chen, L.M. Liu, Y. Peng, X. Hong, B. Yang, H.G. Liu, H. L.C. Orvig, 577

Divalent later transition metal complexes of the traditional chinese medicine (TCM)

578

liriodenine: coordination chemistry, cytotoxicity and DNA binding studies, Dalton Trans.

579

(2009) 10813-10823.

580 [16] X. Bing Fu, D. Dan Liu, Y. Lin, W. Hu, Z.W. Maoand Xue-Yi L, Water-soluble DNA minor 581

groove binders as potential chemotherapeutic agents: synthesis, characterization, DNA

582

binding and cleavage, antioxidation, cytotoxicity and HSA interactions, Dalton Trans. 43

583

(2014) 8721-8737.

584 [17] J. Annaraj, S. Srinivasan, K.M. Ponvel, PR. Athappan, Mixed ligand copper(II) complexes of 585

phenanthroline/bipyridyl and curcumin diketimines as DNA intercalators and their

586

electrochemical behavior under Nafion® and clay modified electrodes, Journal of Inorganic

587

Biochemistry. 99 (2005) 669–676.

588 [18] A.K. Sadana, Y. Mirza, K.R. Aneja, O. Prakash, Hypervalent iodine mediated synthesis of 1589

aryl/hetryl-1,2,4-triazolo[4,3-a] pyridines and 1-aryl/hetryl 5-methyl-1,2,4-triazolo[4,3-

590

a]quinolines as antibacterial agents, Eur. J. Med. Chem. 38 (2003) 533–536.

591 [19] C. Lee, W. Yang, R.G Parr, Development of the Colle-Salvetti correlation-energy formula 592

into a functional of the electron density, Phys. Rev. B 37 (1988) 785–789.

593 [20] P.J. Hay, W.R. Wadt, Ab initio effective core potentials for molecular calculations. Potentials 594

for the transition metal atoms Sc to Hg, J. Chem. Phys. 82 (1985) 270-283.

595 [21] F. Arjmand, I. Yousuf, M. Afzal, L. Toupet, Design and synthesis of new Zn(II) nalidixic 596

acid–DACH based Topo–II inhibiting molecular entity: Chemotherapeutic potential validated

597

by its in vitro binding profile, pBR322 cleavage activity and molecular docking studies with

598

DNA and RNA molecular targets, Inorg. Chim. Acta. 421 (2014) 26-37.

599 [22] K. Zheng, F. Liu, X. Mei Xu, Y. Tuan Li, Z. Y. Wuand Cui-Wei Yan, Synthesis, structure 600

and molecular docking studies of dicopper(II) complexes bridged by N-phenolato-

601

N′-[2-(dimethylamino)ethyl]oxamide: the influence of terminal ligands on cytotoxicity and

602

reactivity towards DNA and protein BSA, New J. Chem. 38 (2014) 2964-2978.

603 [23] K. Nakamato, Infrared and Raman Spectra of Inorganic and Coordination Compounds, Wiley 604

Interscience, New York, 1971.

605 [24] P.F. Rapheal, E. Manoj, M.R.P. Kurup, Copper(II) complexes of N(4)-substituted 606

thiosemicarbazones derived from pyridine-2-carbaldehyde: Crystal structure of a binuclear

607

complex, Polyhedron. 26 (2007) 818-828.

608 [25] R. Kannappan, S. Tanase, I. Mutikainen, U. Turpeinen, J. Reedijk, Square-planar copper(II) 609

halide complexes of tridentate ligands with π–π stacking interactions and alternating short and

610

long Cu ⋯ Cu distances, Inorganica Chimica Acta. 358 (2005) 383-388.

611 [26] M. Kalita, P. Gogoi, P. Barman & B. Sarma, Nickel(II), copper(II), and cobalt(II) complexes 612

derived from a new unsymmetrical ONS donor Schiff base ligand: synthesis, characterization,

613

crystal structure, and catalytic activities, Journal of Coordination Chemistry. 67 (2014) 2445-

614

2454.

615 [27] D.H. Shi, Z. L. Cao, W.W. Liu , R.B. Xu , N. Zhang & Q. Zhang , L. L. Gao, Synthesis and 616

Crystal Structures of Schiff Base Nickel(II) Complexes With Biological Activity, Synthesis

617

and Reactivity in Inorganic, Metal-Organic, and Nano-Metal Chemistry, 42 (2012) 1128-

618

1131.

619 [28] A.M. Herrera, R.J. Staples, S.V. Kryatov, A.Y. Nazarenko, E.V. Akimova, Nickel(II) and 620

copper(II) complexes with pyridine-containing macrocycles bearing an aminopropyl pendant

621

arm: synthesis, characterization, and modifications of the pendant amino group, Dalton Trans.

622

(2003) 846-856.

623 [29] H. Okawa, M. Tadokoro, Y. Aratake, M. Ohba, K. Shindo, M. Mitsumi, M. Tomono, D.E. 624

Funton, Dicopper(II,II) and dicopper(I,II) complexes of a series of dinucleating macrocycles,

625

Journal of the Chemical Society, Dalton Transactions. (1993) 253-258.

626 [30] Y. An, M.–L. Tong, L.–N. Ji, Z.–W. Mao, Double–strand DNA cleavage by copper 627

complexes of 2,2′–dipyridyl with electropositive pendants. Dalton Trans. (2006) 2066–2071.

628 [31] X.B. Fu, D.D. Liu, Y. Lin, W. Hu, Z.W. Maoand Xue-Yi L, Water-soluble DNA minor 629

groove binders as potential chemotherapeutic agents: synthesis, characterization, DNA

630

binding and cleavage, antioxidation, cytotoxicity and HSA interactions, Dalton Trans. 43

631

(2014) 8721-8737.

632 [32] S. Tabassum, M. Ahmad, M. Afzal, M. Zaki, P.K. Bharadwaj, Synthesis and structure 633

elucidation of a copper(II) Schiff-base complex:In vitro DNA binding, pBR322

634

plasmid cleavage and HSA binding studies, Journal of Photochemistry and Photobiology

635

B: Biology 140 (2014) 321-331.

636 [33] A. Meenongwa, R.F. Brissos, C. Soikum, P. Chaveerach, P. Gamez, Y. Trongpaniche, U. 637

Chaveerach, DNA-interacting and biological properties of copper(II) complexes from

638

amidino- O-methylurea, New J. C hem., 39 (2015) 664-675.

639 [34] L. Tjioe, A. Meininger, T. Joshi, L. Spiccia, and B. Graham, Efficient Plasmid DNA 640

Cleavage by Copper(II) Complexes of 1,4,7-Triazacyclononane Ligand Featuring Xylyl-

641

Linked Guanidinium Groups, Inorg.Chem. 50 (2011) 4327-4339.

642 [35] C. Metcalfe and J. A. Thomas, Kinetically inert transition metal complexes that reversibly 643

bind to DNA, Chem. Soc. Rev. 32 (2003) 215-224.

644 [36] Y.B. Zeng, N. Yang, W.S. Liu, N. Tang, Synthesis, characterization and DNA-binding 645

properties of La(III) complex of chrysin, J. Inorg. Biochem. 97 (2003) 258-264.

646 [37] P. li, M. Niu, M. Hong, S. Cheng, J.M. Dou, Effect of structure and composition of nickel(II)

647

complexes with salicylidene Schiff base ligands on their DNA/protein interaction and

648

cytotoxicity, Journal of Inorganic Biochemistry. 137 (2014) 101-108.

649 [38] S.S. Bhat, A.A. Kumbhar, H. Heptullah, A.A. Khan, V.V. Gobre, S.P. Gejji and V.G. 650

Puranik, Synthesis, Electronic Structure, DNA and Protein Binding, DNA Cleavage, and

651

Anticancer Activity of Fluorophore-Labeled Copper(II) Complexes, Inorganic Chemistry. 50

652

(2011) 545-558.

653 [39] Q.X. Wang, F. Gao, K. Jiao, Voltammetric Studies on the Recognition of a Copper Complex 654

to Single and Double-Stranded DNA and Its Application in Gene Biosensor, Electroanal. 19

655

(2008) 2096-2101.

656 [40] N. Raman, A. Selvan, P. Manisankar, Spectral, magnetic, biocidal screening, DNA binding 657

and photocleavage studies of mononuclear Cu(II) and Zn(II) metal complexes of tricoordinate

658

heterocyclic Schiff base ligands of pyrazolone and semicarbazide/thiosemicarbazide based

659

derivatives, Spectrochimica Acta, Part A. 76 (2010) 161-173.

660 [41] G. Prativel, M. Pitie, J. Benadou, B. Meunier, Furfural as a Marker of DNA Cleavage by 661

Hydroxylation at the 5′ Carbon of Deoxyribose, Angew. Chem. Int. Ed. Eng. 30 (1991) 702-

662

704.

663 [42] E. Gao, Y. Sun, Q. Liu, L. Duan, An anticancer metallobenzylmalonate: crystal structure and 664

anticancer activity of a palladium complex of 2,2′-bipyridine and benzylmalonate, J. Coord.

665

Chem. 59 (2006) 1295-1300.

666 [43] P. Krishnamoorthy, P. Sathyadevi, R.R. Butorac, A.H. Cowley, N.S.P. Bhuvanesh, N. 667

Dharmaraj, Copper(I) and nickel(II) complexes with 1 : 1 vs. 1 : 2 coordination of ferrocenyl

668

hydrazone ligands: Do the geometry and composition of complexes affect DNA

669

binding/cleavage, protein binding, antioxidant and cytotoxic activitie, Dalton Trans. 41

670

(2012) 4423-4436.

671 [44] E.L. Chang, C. Simmers, D.A. Knight, Cobalt Complexes as Antiviral and Antibacterial

672

Agents, Pharmaceuticals. 3 (2010) 1711-1728.

673 [45] V.P. Singh, P. Gupta, Synthesis, characterization and biocidal activities of some metal(II) 674

complexes with diacetyl salicylaldehyde acyldihydrazones, J. Coord. Chem. 59 (2006) 1483-

675

1494.

676 [46] K. Singh, Y. Kumar, P. Puri, M. Kumar, C. Sharma, Cobalt, nickel, copper and zinc 677

complexes with 1,3-diphenyl-1H-pyrazole-4-carboxaldehyde Schiff bases: Antimicrobial,

678

spectroscopic, thermal and fluorescence studies, European Journal of Medicinal Chemistry.

679

52 (2012) 313-321.

680 [47] R. Nagar, Syntheses, characterization and microbial activity of some transition metal 681

complexes involving potentially active O and N donor heterocyclic ligands, J. Inorg.

682

Biochem. 40 (1990) 349-356.

683 [48] Nazzaro. F, Fratianni. F, De Martino. L, Coppola. R, & De Feo. Effect of essential oils on 684

pathogenic bacteria. Pharmaceuticals. 6 (2013) 1451-1474.

685 [49] M. Kurtomglu, M.M. Dagdelen, S. Toroglu, Synthesis and Biological Activity of Novel 686

(E,E)- vic-dioximes, Transit. Met. Chem. 31 (2006) 382-388.

687 [50] K. Fukui. Role of frontier orbitals in chemical reactions, Science. 218 (1982) 747-754. 688 [51] M. Shamsi, S. Yadav, F. Arjmand, Synthesis and characterization of new transition metal 689

{Cu(II), Ni(II) and Co(II)} L-phenylalanine–DACH conjugate complexes: In vitro DNA

690

binding, cleavage and molecular docking studies, Journal of Photochemistry and

691

Photobiology B: Biology. 136 (2014) 1-11.

692 693 694 695 696

Scheme 1. Synthetic route for the preparation of ligand L.

Scheme 2. Synthetic route for the preparation of complexes.

Tables Table 1. Voltammertric behaviour of metal(II) complexes 1-4 (in V). Complexes

[Cu(L)]Cl [Co(L)2] [Ni(L)]Cl [Zn(L)]Cl

Epc

Epa

0.468 0.288 0.774 0.269

0.299 0.196 0.606 0.066

∆Ep

E1/2

ipa/ipc

00.169 00.092 00.168 00.203

0.384 0.242 0.690 0.168

1.316 0.649 1.206 1.299

Table 2. The electronic absorption spectral properties of Cu(II), Co(II), Ni(II) and Zn(II) complexes.

N No

λmax

Compound

∆λ (nm)

Free

Bound

H%a

Kb( M-1) b

1

1

[Cu(L)]Cl

389.0

393.0

4.0

18.8

1.34 X 105

2

2

[Co(L)2]

413.0

416.0

3.0

17.7

1.02 X 10 4

3

[Ni(L)]Cl

392.6

393.4

0.8

15.6

5.04 X 104

4

[Zn(L)]Cl

405.2

406.2

1.0

24.7

1.27 X 10 4

4 a

H% = [Afree – Abound)/] X 100%.

b

Kb = DNA binding constants were determined from the UV-Vis absorption spectral titration.

697

Figure captions

698 Fig. 1. Electronic absorption spectra of (a) Cu (b) Co (c) Ni (d) Zn complexes in the absence 699

and presence of increasing amount of HS-DNA. Arrows show the changes in absorbance

700

with respect to increase in the DNA concentration. Plot between [DNA] and [DNA] / (εa-εf).

701 Fig. 2. Circular dichroism spectra of HS-DNA in the absence and presence of complexes in Tris702

HCl buffer (pH 7.1) at room temperature. [Complex]= 30 µM, [DNA] = 100 µM.

703 Fig. 3. Emission spectrum of EtBr bound to DNA in the presence of complexes (a) 1 (b) 2 (c) 3 704

(d) 4 in Tris-HCl buffer (pH=7.1). Arrows indicate the intensity changes upon increasing

705

concentration of the complexes. -4

706 Fig. 4. Cyclic voltammogram of 10 M solution of [Cu(L)]Cl with increasing amount of DNA 707

in MeOH:buffer mixture at a scan rate of 100 mV s-1. The arrow indicates upon

708

increasing the amount of DNA.

709 Fig. 5. Difference pulse voltammogram of 10-4 M of [Cu(L)]Cl with increasing amount of DNA 710

at scan rate of 100 mV s-1. The arrow indicates upon increasing the amount of DNA.

711 Fig. 6. Gel electrophoresis pattern showing cleavage of pBR322 supercoiled DNA (10 µM) by 712

the Cu(II), Co(II), Ni(II) and Zn(II) complexes (60 µM) in the presence of H2O2 (100

713

µM). Lane 1: DNA alone; lane 2: DNA+H2O2; Lane 3: CuCl2+ DNA+H2O2; Lane 4:

714

NiCl2+DNA+H2O2; Lane 5: ZnCl2+DNA+ H2O2; Lane 6: [Cu(L)Cl]+DNA+H2O2; Lane

715

7: [Co(L)2]+DNA+H2O2; Lane 8: [Ni(L)Cl]+DNA+H2O2; Lane 9

716

[Zn(L)Cl]+DNA+H2O2; Lane 10: L+DNA+H2O2.

717 Fig. 7. AFM image of (a) control: Rod shape of E. coli and (b) Treated sample: Cobalt(II) 718

complex induces cell disruption of E. coli.

719 Fig. 8. The optimized molecular structures of the investigated ligand and its metal complexes. 720 Fig. 9. Frontier molecular orbitals optimized at the B3LYP/LANL2DZ level of theory. 721 Fig. 10. Molecular docked model of L and complexes (a) 1 (b) 2 (c) 3 and (d) 4 with DNA 722

dodecamer duplex of sequence d(CGCGAATTCGCG)2 (PDB ID: 1BNA).

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Fig. 5

Fig. 6

Fig. 7

Fig. 8

Fig. 9

Fig. 10

723

CuL

2+

+ e-

CuL

K2+

CuL

+

o' Ef

K+ 2+

- DNA

+ e-

CuL

+

o' - DNA E b

724 725 726

Scheme 3. Nernstian electron transfer of both the oxidized and reduced forms of complexes associated with the third species (DNA).

Research Highlights 

Synthesis of metal(II) complexes with N, N and O donor Schiff base ligand.



Ligand and its metal(II) complexes have been completely characterized.



These metal(II) complexes may bound on the grooves of HS-DNA.



All the complexes shows significant cleavage of pBR322 DNA.



AFM study displayed a ruptured cell wall morphology of E. Coli by Co(II) complex.