Synthesis, molecular structure, biological properties and molecular docking studies on MnII, CoII...

Accepted Manuscript

Synthesis, molecular structure, biological properties and molecular docking studies onMnII, CoII and ZnII complexes containing bipyridine-azide ligands

Vijayan Thamilarasan, Arumugam Jayamani, Nallathambi Sengottuvelan

PII: S0223-5234(14)00892-7

DOI: 10.1016/j.ejmech.2014.09.073

Reference: EJMECH 7380

To appear in: European Journal of Medicinal Chemistry

Received Date: 18 February 2014

Revised Date: 21 September 2014

Accepted Date: 23 September 2014

Please cite this article as: V. Thamilarasan, A. Jayamani, N. Sengottuvelan, Synthesis, molecularstructure, biological properties and molecular docking studies on MnII, CoII and ZnII complexescontaining bipyridine-azide ligands, European Journal of Medicinal Chemistry (2014), doi: 10.1016/j.ejmech.2014.09.073.

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http://dx.doi.org/10.1016/j.ejmech.2014.09.073

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ACCEPTED MANUSCRIPT

Synthesis, molecular structure, biological properties and molecular docking studies on

MnII, CoII and ZnII complexes containing bipyridine-azide ligands

Vijayan Thamilarasana, Arumugam Jayamania, Nallathambi Sengottuvelanb*

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ACCEPTED MANUSCRIPT1

Synthesis, molecular structure, biological properties and molecular docking studies on

Mn II , CoII and ZnII complexes containing bipyridine-azide ligands

Vijayan Thamilarasana, Arumugam Jayamania, Nallathambi Sengottuvelanb*

aDepartment of Industrial Chemsitry, *bDDE, Alagappa University, Karaikudi -630003,

Tamilnadu, India

Abstract

Metal complexes of the type Mn(bpy)2(N3)2 (1), Co(bpy)2(N3)2.3H2O (2) and

Zn2(bpy)2(N3)4 (3) (Where bpy = 2,2-bipyridine) have been synthesized and characterized by

elemental analysis and spectral (FT-IR, UV-vis) studies. The structure of complexes (1-3) have

been determined by single crystal X-ray diffraction studies and the configuration of ligand-

coordinated metal(II) ion was well described as distorted octahedral coordination geometry for

Mn(II), Co(II) and distorted square pyramidal geometry for Zn(II) complexes. DNA binding

interaction of these complexes (1-3) were investigated by UV–vis absorption, fluorescence

circular dichroism spectral and molecular docking studies. The intrinsic binding constants Kb of

complexes 1, 2 and 3 with CT-DNA obtained from UV–vis absorption studies were 8.37x104,

2.23x105 and 5.52x104 M-1 respectively. The results indicated that the three complexes are able to

bind to DNA with different binding affinity, in the order 2 > 1 > 3. Complexes (1-3) exhibit a

good binding propensity to bovine serum albumin (BSA) proteins having relatively high binding

constant values. Gel electrophoresis assay demonstrated the ability of the complexes 1 - 3

promote the cleavage ability of the pBR322 plasmid DNA in the presence of the reducing agent

3-mercaptopropionic acid (MPA) but with different cleavage mechanisms: the complex 3 cleaves

DNA via hydrolytic pathway (T4 DNA ligase assay), while the DNA cleavage by complexes 1

and 2 follows oxidative pathway. The chemical nuclease activity follows the order: 2 > 1 > 3.

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The effects of various activators were also investigated and the nuclease activity efficacy

followed the order MPA > GSH > H2O2 > Asc. The cytotoxicity studies of complexes 1-3 were

tested in vitro on breast cancer cell line (MCF-7) and they found to be active.

Keywords: Manganese(II), Cobalt(II) and Zinc(II) complexes, X-ray crystal structure, Bovine

Serum Albumin, Molecular Docking, DNA binding and cleavage activity, cytotoxic activity.

* Corresponding authors. Tel./fax: +91 9488260744 (N. Sengottuvelan).

E-mail address: [email protected].

Present address: DDE, Chemistry, Alagappa University, Karaikudi -630003, Tamilnadu, India

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1. Introduction

The design and development of small- or medium-sized potential therapeutic agents to

target nucleic acid cleavage can lead synthesis of novel therapeutic agents for cancer, viral

diseases and can act as tool for molecular biology [1-3]. Small molecules interact with double-

stranded DNA in a number of ways [4-7] and in most cases it is main non-covalent binding

modes such as, intercalation, major/minor groove binding and electrostatic binding interaction.

The rationale of our strategy is to design polypyridyl-metal-azide complexes which prove to

possess pronounced biological and better pharmacological activity. Bipyridyl containing azide

complexes are well known, but there is no information on the solid state structure versus

biological activity. Amongst the metal ions chosen, we have opted for biocompatible endogenous

metal ions such as Mn, Co and Zn which play essential role in biological living system [8-10]. In

particular, the complexes based on essential metals are less toxic than those with non-essential

ones. The most important operation formed by manganese in nature is the photolytic oxidation of

water to dioxygen within the oxygen evolving complex (OEC) of photosystem (II) (PSII), found

in the photosynthetic apparatus of green plants and certain cyanobacteria [11,12]. It is also a

cofactor or required metal ion for many enzymes, such as superoxide dismutase (SOD),

glutamine synthetase and arginase [13]. It is known that cobalt(III) azide complexes undergo

photoreduction [14,15] and are used for reduction of nitric oxide and nitrous acid [16].

Structurally characterized cobalt complexes have been studied as hydrolytic agents for DNA

cleavage [17,18] and others showing antitumor, anti-proliferative [19], antimicrobial [20],

antifungal [21,22], antiviral [23] and antioxidant activity [24]. Zinc complexes with diverse

biological activity viz. antibacterial [25], anti-inflammatory [26], for the treatment of Alzheimer

disease [27] and anti-proliferative, antitumor activity [28] have been structurally characterized.

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The azide-pseudohalide ion has been shown to link with two or more metal ions in various

symmetric or asymmetric [29] modes: µ-1,1 (end-on), µ-1,3 (end-to-end), µ-1,1,3, and others.

The coordination mode of the azido ligand depends on the nature and oxidation state of the

central metal ion, as well as the nature of the other coordinated ligands.

In order to have better insight on the factors of DNA binding and cleavage mechanism,

Manganese(II), cobalt(II) and zinc(II) complexes of 2,2’-bipyridine and azide ligands were

synthesized and characterized. The single crystal structural analyses reveal that metal adopt

distorted octahedral MN6 coordination environments for complexes 1 and 2; distorted square

pyramidal MN5 coordination environment for complex 3. All the complexes (1-3) possess

extended aromatic π-π systems due to the coordination of azide ligands with metal atoms. In the

present study, the DNA-binding behaviors of the complexes (1-3) were explored by absorption,

emission and circular dichroisim spectroscopies. Computer-aided molecular docking studies

were performed to visualize the binding mode of the drug candidate at the molecular level. Their

abilities to induce cleavage of pBR322 DNA and in vitro cytotoxicity on breast cancer cell line

(MCF-7) were also investigated.

2. Experimental

2.1. Materials

2,2 -bipyridyl, manganese(II) acetate tetrahydrate, cobalt(II) acetate tetrahydrate,

zinc(II) acetate dihydrate (Sigma-Aldrich), sodium azide (Alfa Aesar) were used as received.

The solvents used were of reagent grade. Tris, agarose and ethidium bromide were purchased

from Sigma. Calf thymus DNA (CT DNA) and Supercoiled plasmid pBR322 DNA (Genei) were

utilized as received. Doubly distilled water was used to prepare buffers.

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2.2. Physical measurements

Elemental analysis was carried out using a Carlorerba-1106 microanalyzer. The infrared

spectra were recorded by Perkin–Elmer FT-IR spectrophotometer with a KBr disc. The

electronic spectra were recorded using Shimadzu UV-3101PC spectrophotometer. The

fluorescence study was carried out by Elico SL 174 spectrofluorometer. The CD spectra were

recorded on Jasco J-810 spectropolarimeter.

2.3.Synthesis of complexes

2.3.1 [Mn(bpy)2(N3)2] (1)

2,2 -bipyridyl (0.32 g, 2 mmol) in methanol (10 mL) was mixed with sodium hydroxide

(2 mmol) and the resulting solution was stirred for 15 min at room temperature. Further

(CH3.CO2)2Mn.4H2O (0.25 g, 1mmol) in methanol (10 mL) was added in to the solution. To the

reaction mixture sodium azide (0.13 g, 2 mmol) was added, followed by stirring for 3 h. Finally

resultant brown colored precipitate was recrystallized in DMF. Five days later round-shaped

brown crystals suitable for X-ray diffraction study were obtained. Yield: 70 %. Anal. Calc. for

C20H16N10Mn: C, 53.22; H, 3.57; N, 31.03. Found: C, 53.48; H, 3.40; N, 30.51%. FT-IR, (ν,

cm−1) (KBr Disc): 3370br, 2076s, 1316s (br, broad; s, sharp). UV-vis in DMF [λmax/nm (εmax/

mol-1 cm-1)]: 280 (3870), 390 (40) 539 (1). Conductivity (ΛM/Ω-1cm2 mol-1) in DMF: 11.

Caution! Azide complexes are potentially explosive. Only small amounts of material should be

prepared, and these should be handled with care.

2.3.2. [Co(bpy)2(N3)2].3H2O (2)

The complex 2 was obtained by the procedure described above for the complex 1 using

(CH3.CO2)2Mn.4H2O instead of (CH3.CO2)2Co.4H2O (0.25 g, 1mmol). The resulting dark green

colored precipitate formed was recrystallized in DMF. Nine days later needle-shaped green

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crystals were obtained. Yield: 62 %. Anal. Calc. (%) for C20H22N10O3Co: C, 47.16; H, 4.35; N,

27.50. Found (%): C, 46.79; H, 4.50; N, 27.27. FT-IR, (ν, cm−1) (KBr Disc): 3433 br, 2055s,

1315s. UV-vis in DMF [λmax/nm (εmax/ mol-1 cm-1)]: 268 (2260), 305 (4390), 347 (1420), 557

(20), 638(25). Conductivity (ΛM/Ω-1cm2 mol-1) in DMF: 16.

2.3.3. [Zn2(bpy)2(N3)4] (3)

The complex 3 was also obtained from the same procedure as described above by using

(CH3.CO2)2Zn.2H2O (0.22 g, 1mmol) instead of (CH3.CO2)2Mn.4H2O. The white colored

precipitate formed was recrystallized in DMF. Six days later, round-shaped colorless crystals

were obtained. Yield: 70 % Anal. Calc. (%) for C20H16N16Zn2: C, 39.30; H, 2.64; N, 36.67.

Found (%): C, 39.42; H, 2.78; N, 36.44. FT-IR, (ν, cm−1) (KBr Disc): 3417br, 2084s, 2050s,

1342s. UV-vis in DMF [λmax/nm (εmax/ mol-1 cm-1)]: 280 (3600), 394(20). Conductivity (ΛM/Ω-

1cm2 mol-1) in DMF: 14.

2.4. Crystal structure determination and refinement

The X-ray diffraction measurements were made on a Bruker APEX II CCD area detector

diffractometer (Mo-Kα radiation, graphite monochromator). Semi-empirical absorption

corrections were carried out using the program SADABS [30]. The structures were solved by

direct methods using the program SHELXS-97. The refinement and further calculations were

carried out using SHELXL-97. The C-bound H-atoms were included in calculated positions and

treated as riding atoms using SHELXL-97 default parameters. The non-H atoms were refined

anisotropically, using weighted fullmatrix least-squares on F2. Software packages APEX2 (data

collection), SAINT (cell refinement and data reduction) and SHELXTL (molecular graphics and

publication material) were also used [31]. A summary of the crystal data, experimental details

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and refinement results have been given in Table 1 and selected bond lengths and bond angles are

listed in Table 2.

2.5. DNA-binding studies

The DNA stock solution was prepared by dilution of CT DNA to buffer (containing 5

mM Tris–HCl/NaCl at pH 7.2) followed by exhaustive stirring at 4 °C for 4 days, and kept at

4 °C for no longer than a week. The stock solution of CT DNA gave a ratio of UV absorbance at

260 and 280 nm (A260/A280) of 1.89, indicating that the DNA was sufficiently free of protein

contamination [32]. The DNA concentration was determined by the UV absorbance at 260 nm

after 1:20 dilution using ε = 6600 M-1cm-1. Using the electronic absorption spectral method, the

relative binding of the complexes (1-3) to CT DNA was studied. Absorption titration

experiments were made using different concentration of DNA, while keeping the complex

concentration as constant. The intrinsic binding constants, Kb, of the compounds with CT DNA

have been determined using the absorption spectral technique for the complexes.

The competitive binding studies of each complexes (1-3) with EB (EB =3,8-diamino-5-

ethyl-6-phenyl-phenanthridinium bromide) have been investigated with fluorescence

spectroscopy in order to examine, whether the compound is able to displace EB from its CT

DNA–EB complex. The CT DNA–EB complex was prepared by adding 3.3 µM EB and 20 µM

CT DNA in buffer (5 mM NaCl and 5 mM Tris HCl at pH 7.4). The sample was excited at ~510

nm and its emission was observed at 596 nm. The intercalating effect of complexes 1–3 with the

DNA–EB complex was studied by adding a certain amount of a solution of the complexes step

by step into the solution of the DNA–EB complex. The influence of the addition of each

complex to the DNA–EB complex solution has been obtained by recording the variation of

fluorescence intensity in emission spectra.

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CD spectra of CT DNA in the absence and presence of complexes in the 1:1 molar ratio

of the CT DNA to the complexes were obtained in the wavelength range of 220–320 nm in 5

mM Tris–HCl buffer (pH 7.2). Each measurement was the average of three repeated scans and

the background was subtracted from all of the reagents by using a corresponding solution

without CT DNA as a reference solution. The concentration of CT-DNA and the complexes were

100 and 50 µM respectively.

2.6. Albumin binding studies

The protein binding study was performed by tryptophan fluorescence quenching

experiments using bovine serum albumin (BSA, 3 mM) in buffer containing 15 mM trisodium

citrate and 150 mM NaCl at pH 7.0. The quenching of the emission intensity of tryptophan

residues of BSA at 343 nm was monitored using bpy and their complexes 1-3 as quenchers with

increasing concentration. Fluorescence spectra were recorded in the wavelength range of 300 –

480 nm at an excitation wavelength of 295 nm. The fluorescence spectra of bpy and its

complexes were recorded under the same experimental conditions and a maximum emission

appeared at 328 nm. Therefore, the quantitative studies of the serum albumin fluorescence

spectra were performed after their correction by subtracting the spectra of the compounds.

2.7. Molecular docking studies

The rigid molecular docking studies were performed by using HEX 8.0 software [33].

The coordinates of complexes (1 – 3) were taken from their respective crystal structure as a CIF

file and was converted to the PDB format using Mercury software (http://www.ccdc.cam.ac.uk/).

The crystal structure of the B–DNA dodecamer d(CGCGAATTCGCG)2 (PDB ID: 1BNA) was

downloaded from the protein data bank (http://www.rcsb.org./pdb). All calculations were carried

out on an Intel pentium4, 2.4 GHz based machine running MS Windows XP SP2 as operating

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system. Visualization of the docked pose has been done by CHIMERA

(www.cgl.ucsf.edu/chimera).

2.8. DNA cleavage studies

Cleavage of supercoiled pBR322 DNA by the complexes was studied by agarose gel

electrophoresis. In a typical experiment, the reaction mixtures (25 µL total volume) containing

the supercoiled plasmid pBR 322 DNA (300 ng), dissolved in 50 mM Tris(hydroxymethyl)

aminomethane hydrochloride (Tris–HCl) buffer, 50 mM NaCl, were treated with the metal(II)

complexes (100 and 200 µM). The reaction mixtures were incubated at 37 °C for 4 h, and then

quenched by the addition of 5 µL loading buffer (0.25% bromophenol blue, 0.25% xylene

cyanol, 30% glycerol), samples were loaded on 0.8 % agarose gel containing ethidium bromide

(1 µg/mL) in TAE (Tris-acetate-EDTA) buffer. The gel was run at 50 V for 1.30 h and

photograph has been taken under UV light. The proportion of DNA in each fraction was

quantitatively estimated from the intensity of each band with the BioRad Gel Doc XR system

using the Labwork software. Anaerobic run was performed in a stopcock-equipped cuvette after

extensive purging of the reaction mixture with N2 prior to the addition of N2- purged complexes.

To enhance the DNA cleaving ability by the complexes, activators MPA, hydrogen peroxide,

glutathione (GSH) and ascorbate (Asc) were used. Moreover, cleavage mechanism was further

investigated by using scavengers for the hydroxyl radical species (1mM DMSO) and the singlet

oxygen species (1 mM NaN3) and (1 mM L-histidine), (1mM EDTA). All the experiments were

carried out in triplicate under the same conditions.

DNA ligation experiments were carried out follows: after the incubation of pBR322 DNA

with 50 µM of complex 3 in the presence of MPA, and incubating pBR322 DNA with EcoRI for

8 h, at 37 °C, the cleavage product, i.e. linear form, was purified by DNA Gel Extraction Kit.

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Then the mixture of 3 µL T4 ligase (4 units) and 1 µL 10× ligation buffer containing 1 mM ATP,

2 µL of the solution containing DNA cleavage fragment linearized by 3 and EcoRI and 6 µL

H2O, were incubated for 20 h at 16 °C. Afterwards, the ligation products were electrophoresed,

stained and imaged.

2.9. Cell culture

The MCF-7 human breast cancer cell line was obtained from National Center for Cell

Sciences (NCCS), Pune, India. The cell line was cultured in the Dulbecco’s Modified Eagles

medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 200 mM L-gulutamine,

100 U/mL penicillin, and 10 mg/mL streptomycin in a humidified atmosphere consisting of 5%

CO2 at 37º C.

2.10. Evaluation of cytotoxicity

The cytotoxic effect of complexes 1-3 against MCF-7 cells was evaluated by MTT [3-(4,

5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Briefly, MCF-7 cells were

seeded (5 × 104 cells/well) in a 96-well plate and kept in CO2 for attachment and growth for 24 h.

Then, the cells were treated with various concentrations of complex dissolved in DMSO (0.1-100

µM) and incubated for 24 h. After incubation, the culture medium was removed and 15 µL of the

MTT solution (5 mg/mL in PBS) was added to each well. Following 4 h incubation in dark,

MTT was discarded and DMSO (100 µL/well) was added to solubilize the purple formazan

product. The experiment was carried out in triplicates and the medium without complex served

as control. The absorbance was measured colorimetrically at 570 nm using an ELISA microplate

reader. The percentage of cell viability was calculated using the following formula and expressed

as IC50 = (OD value of treated cells)/ (OD value of untreated cells (control) ×100

The IC50 value is calculated using linear regression from excel.

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3. Results and discussion

3.1. Description of the crystal structures

3.1.1. Structure of [Mn(bpy)2(N3)2] (1)

The structural building block of 1 is mononuclear, neutral molecule [Mn(bpy)2(N3)2] (Fig.

1a). The metal ions are adopting distorted octahedral coordination geometry with the two azide

groups in cis positions. Although a large number of Mn(II)-azido complexes are known, only

limited number of mononuclear [MnL2(N3)2] compounds with L being a heterocyclic N,Nʹ-

bidentate ligand, have been structurally characterized to date. Like complex 1 they all have

distorted octahedral coordination geometries with azido groups in cis positions [34-36]. The Mn–

N(azido) bond lengths in these compounds range from 2.12 Ǻ to 2.21 Ǻ, with the present

complex in the lower end [2.147 (16) Ǻ]. A close to linear azido group [N–N–N angle of

178.03°] is also a consistent feature in these mononuclear compounds. The degrees of

asymmetry ∆d (∆d is the difference between the two N-N distances within an azido group) of

0.025 is found for N3–N4–N5 azido ligand. The most striking feature of 1 is that the single

molecule is linked to adjacent molecules through the C–H…N hydrogen bonds and extends to a

3D supramolecular structure (Fig. 2a). [C(2)–H(2)…N(5), 2.6 Ǻ, ∠C(2)–H(2)…N(5), 146.92˚;

C(9)–H(9)…N(5), 2.74 Ǻ, 117.90˚; C(10)–H(10)…N(5), 2.63 Ǻ, 124.29˚; C(4)–H(4)…N(3),

2.67 Ǻ, 178.02˚; C(7)–H(7)…N(3), 2.66 Ǻ, 166.71˚; C(7)–H(7)…N(4), 2.49 Ǻ, 142.30˚]. The

shortest distance between parallel bpy ligands from adjoining chains is about 3.323 Å,

suggesting the presence of strong interchain π-π stacking interactions, which extend the structure

into an interesting 3D supramolecular array.

3.1.2. Structure of [Co(bpy)2(N3)2].3H2O (2)

The structure of 2 (Fig.1b) shows that complex is neutral, with two positive charges at the

central Co ion neutralized by two negative charges of (N3)-. The cobalt atom is a six-coordinate

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and is surrounded by four N atoms of two bpy ligands and two N atoms of two azide ligands

show a distorted octahedral geometry and three uncoordinated lattice water molecules are at the

unit-cell corners. The Co–N distances are different from each other and lie in a wide range

[1.938(3) – 1.958(3) Ǻ]. The bpy ligands and the chelate rings are almost planar. The angle N1–

Co1–N3, N2–Co1–N8, N4–Co1–N5 is virtually linear with bond angles 177.85(13)˚,

173.76(14)˚, 174.88(14)˚, respectively. In the structure, the azides are coordinated in a quasi-

linear fashion as seen from its N5–N6–N7, N8–N9–N10; 176.72˚, 177.03˚. The degrees of

asymmetry ∆d of 0.071 and 0.066 found for N5–N6–N7, N8–N9–N10 azido ligands,

respectively. The crystal packing is mainly stabilized by inter-molecular hydrogen bonding. The

hydrogen bonding of C(17)–H(17)…N(10), 2.42Ǻ, ∠C(17)–H(17)…N(10), 139.06˚ with

terminal azido groups of adjacent complexes, giving rise to one-dimensional polymeric chain.

The 1D chains are self-assembled to form a molecular sheet via C–H–N(azido) and π–π

interactions between bipyridine rings, and are described in Fig. 2b. The robustness of the

supramolecular geometry has been enhanced through intermolecular hydrogen bonding

interactions between the N atoms of the azide groups and the amino H atoms of the pyridine ring

[C(10)–H(10…N(6), 2.5 Ǻ, ∠C(10)–H(10)…N(6), 143.80˚; C(10)–H(10…N(7), 2.66 Ǻ,

∠C(10)–H(10)…N(6), 130.30˚] which, lead to the construction of a three-dimensional network

structure (Fig. 2b). We would like to mention here that the similar complexes of

[Co(bpy)2(N3)2]+ with anions such as p-toluenesulfonate [37] nitrate [38], perchlorate [39] and

chloride [40] were isolated and molecular structures were determined by X-ray crystallography.

3.1.3. Structure of [Zn2(bipy)2(N3)4] (3)

Each zinc(II) atom is coordinated by two nitrogen atoms from a bipyridine ligand and

two nitrogen atoms from two (µ-1,1 EO) azido bridges and one terminal azide nitrogen, resulting

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distorted square pyramidal environment with ZnN5 chromophore.(Fig. 1c) There are two

different types of azido ligands, one as a terminal ligand and other in the single end-on (µ-1,1

EO) mode as a bridge between metal ions. The Zn–Zn separation through the EO bridge is Zn1-

Zn2 = 3.510 Ǻ and 3.516 Ǻ. In the structure, the azide are coordinated in a quasi-linear fashion

as seen from its N5–N6–N7, N8–N9–N10, N11–N12–N13, N14–N15–N16; 178.33˚, 176.05˚,

179.75˚, 177.56˚. The distortion parameters of the square pyramidal geometry are τ = 0.35 and

0.32 for the Zn1 and Zn2, respectively. The apical Zn–N(azido) distance (2.172 Ǻ) is

significantly longer than the basal Zn–N(azido) distance (av. 2.100 Ǻ), indicating a rather weak

axial coordinative interaction. The two binuclear units are bridged by single end-on azido (N14–

N15–N16) bridge, which is disposed in an asymmetric apical–basal fashion with Zn–Nav distance

is 2.127 Ǻ, giving rise to a one-dimensional polymeric chain with azide-bridged bonds (Fig. 2c).

The zig-zag chains are propagating along the c-axis. Similar type of end on azide bridged chiral

copper(II) complex, [Cu(N3)2(L)] (L = (1R)-6,6-dimethyl-5,7-methano-2-(2-pyridinyl)-4,5,6,7-

tetrahydro quinoline) has been reported earlier [41] and the Zn–N–Zn bond angle is 111.97˚. An

intramolecular interaction between the coordinated azide ligand having the non-bonded contacts

N1…N11 (3.091 Ǻ), N8…N11(2.88 Ǻ), N3…N12 (3.044 Ǻ). In the crystal lattice, the dinuclear

units are packed with weak C–H…N hydrogen bonding. The nitrogen atoms (N7, N10, and N13)

from terminal azide ligands are hydrogen bond acceptors and C–H (H7, H12, H14 and H17)

hydrogens of the organic ligand skeleton are donors [C(7)–H(7…N(7), 2.4 Ǻ, ∠C(7)–

H(7)…N(7), 166.95˚; ∠C(12)–H(12)…N(10), 2.59 Ǻ, 177.15˚; ∠ C(17)–H(17)…N(10), 2.48 Ǻ,

171.85 ˚; ∠C(14)–H(14)…N(13), 2.50 Ǻ, 154.27˚]. The 1D chains are self-assembled into

molecular sheets via strong π–π interactions between aromatic bipyridine rings and are described

in Figs. 2c. The bipyridine ring (Ar) of one molecule in one 1D chain interacts with bipyridine

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ring from the adjacent molecule of a neighboring chain. Thus, bipyridine (A) – bipyridine (B) (A

stands for one 1D chain and B stands for the adjacent 1D chain) are separated (C4…C13) by

3.271 Ǻ. The hydrogen bonding interaction and strong π–π interactions between aromatic

bipyridine rings lead to the construction of a three-dimensional network structure.

3.2. IR and UV/Visible Spectroscopy

The points of interest in the IR spectra of the 1-3 complexes lie mainly in the bands due

to the azido groups. The strongest intensity feature in the spectra of 1-3 appears in the region

2050-2090 cm−1. A strong peak at 2033 cm−1 is observed in the IR spectrum of sodium azide.

The shift towards higher wave numbers of the νas stretching of the azide in 1-3 supports the

coordination of this anion. In addition, the occurrence of medium and sharp intensity peaks in the

region 1315 to 1342 cm−1 [νs(N3)] suggest the presence of terminally bound azide [42, 43]. The

asymmetrical stretch, νasym(N3), appears split as a strong band at ca. 2084 and 2050 cm−1 which

are in good agreement with the simultaneous existence of terminal and EO bridging modes in the

complex 3 [44, 45]. A broad absorption band around 3433 cm-1 appears for 2 due to the νsym and

νasym vibration of the OH group of the water molecule.

The UV-vis spectra of the complexes (1-3) in DMF show two to four absorption bands.

The lower wavelength bands (260–305 nm) correspond to π - π* transition of the aromatic rings.

The bands between 340 and 400 nm are due to n - π* transitions. The spectrum of 1 also shows

low-intensity absorption band at 539 nm associated with d–d transitions [45, 46]. In the visible

region, weaker absorptions near 557 and 637 nm for the Co(II) complex are assigned as

4T1g(F)→4T2g (P) and 4T1g(F) →4A2g, for d–d transitions, consistent with Co in an octahedral

environment [47, 48]. The molar conductivity measurement of complexes 1-3 in DMF solution

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(ca. 10-3 M) is in the range of 11 –16 Ω-1 cm2 mol-1 at 25 °C, indicating neutral electrolytic

behavior.

3.3. DNA binding studies

3.3.1. Absorption spectral studies

Transition metal complexes can bind with DNA via both covalent (via replacement of a

labile ligand of the complex by a nitrogen base of DNA such as guanine N7) and/or noncovalent

(intercalation, electrostatic or groove binding) interactions [49, 50]. Absorption titration can be

used to observe the interaction of molecules with DNA. Fig. 3 shows the absorption spectra of

complexes (1-3) in the absence and presence of increasing amounts of CT-DNA. The absorption

bands of 1 at 236 and 278 nm exhibited hypochromism of 11 and 13 % and bathochromism shift

of about 0 and 8 nm respectively, indicating that the interaction with CT DNA results in the

direct formation of a new complex with double-helical CT DNA [51]. The observed

hypochromism could be attributed to stacking interaction between the aromatic chromophore of

the complexes and DNA base pairs, which is consistent with the intercalative binding mode,

while the red-shift is an evidence of the stabilization of the CT DNA duplex [52].

Bathochromism shift indicates the π* orbital of the intercalated ligand can couple with the π

orbital of the base pairs, thus decreasing the π-π* transition energy [53]. In the UV spectrum of

2, the respective bands at 247 and 302 nm exhibited hyperchromisms of 15 and 8 % and blue

shifts of 2 and 1 nm suggesting the tight binding to CT DNA. Additionally, the hyperchromism

of both bands are accompanied by a blue-shift of 2 nm which is an indicative of stabilization.

The behavior of complex 3 is quite similar (hyperchromism and blue-shift) to 2. Complex 3 at

243 and 295 nm exhibited the respective hyperchromism of 18 and 4 % with the blue shifts of

about 1 and 0 nm respectively. The hyper and hypsochromism of complexes 2 and 3 indicate the

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DNA-stabilization and the strong binding to CT DNA, possibly due to electrostatic interaction or

to the partial uncoiling of DNA helix structure, exposing more bases of DNA indicative of strong

binding of complexes to CT DNA [54]. Moreover, hyperchromic effect reflects the

corresponding changes in the secondary structure of DNA in its conformation after the complex-

DNA interaction. The results derived from the UV titration experiments suggest that all the

complexes can bind to CT DNA, although the exact mode of binding cannot be merely proposed

by UV spectroscopic titration studies. The existence of hypochromism could be considered as

evidence for the binding of the complexes involving intercalation between the base pairs of CT

DNA cannot be ruled out [55, 56]. It may be noticed from the spectra of 2 and 3 that the

complexes have more pronounced hyperchromism of 22% accompanied by a blue-shift has been

observed. Therefore, complexes 1–3 exhibit quite different behavior upon addition of CT DNA.

In order to compare the affinity of the three complexes toward DNA quantitatively, the

binding constant Kb of the three complexes to CT DNA was determined by using the following

equation [54],

DNA

DNA

1K

where [DNA] is the concentration of DNA in base pairs, the apparent absorption

coefficients εa, εf and εb correspond to Aobsd/[complex], the extinction coefficient for the free

complex and the extinction coefficient for the complex in the fully bound form, respectively. In

the plots of [DNA]/(εb–εf) versus [DNA], Kb is given by the ratio of the slope to the intercept.

The binding constants (Kb) of complexes (1-3) are calculated as 8.37x104, 2.23x105 and 5.52x104

M-1, respectively and the calculated binding constants are varied in the order: 2 > 1 > 3. The Kb

values for 1 and 3 are of lower magnitude than that of the classical intercalator EB-DNA (Kb =

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1.23 (±0.07) x 105 M-1) [57], but are comparable with some reported non-intercalators [58]. In

case of complex 2 the Kb value is higher than that of other classical intercalators.

3.3.2. Fluorescence spectroscopic studies

Fluorescence quenching measurements can be used to monitor metal binding [59]. EB

emits intense fluorescent light in the presence of CT DNA due to its strong intercalation between

the adjacent DNA base pairs. It was previously reported that the enhanced fluorescence could be

quenched by the addition of another molecule [55, 60]. The study involves the addition of the

complexes to DNA pretreated with EB ([DNA]/[EB] = 5.7) and the measurement of the intensity

of emission. The emission spectra of EB bound to DNA in the absence and presence of

complexes (1-3) are shown in Fig. 4(a-c). The intensity of the emission band at 596 nm of the

DNA–EB system decreased (up to 79 % of the initial EB–DNA fluorescence intensity for 1, up

to 46 % for 2 and up to 88 % for 3) upon the addition of each complex 1–3 at diverse r values

(Fig. 4d), showing that each Mn(II), Co(II) and Zn(II) complex competes with EB to bind with

DNA. According to linear Stern–Volmer equation: [55, 61]

1 KQ

where Io and I are the emission intensities in the absence and the presence of the

quencher, respectively, [Q] is the concentration of the quencher (complexes 1–3) and KSV is the

Stern–Volmer constant which can be obtained by the slope of the diagram Io/I vs [Q] (Fig. 4e)

and is often used to evaluate the quenching efficiency for each compound. The Stern–Volmer

plot illustrates the quenching of EB bound to DNA by complexes 1–3 is in good agreement (R ~

0.98) with Stern–Volmer equation, which proves that the partial replacement of EB bound to

DNA results in a decrease of fluorescence intensity [62]. The calculated KSV values of complexes

(1-3) are 5.41 x 103, 6.63 x 103 and 1.17 x 103 M-1, respectively and varies in the order: 2 > 1 > 3.

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The relatively high KSV values of the complexes show that they can displace EB and bind to the

DNA [54, 63]. From Figs. 4(a-c), we know that the three complexes bind to DNA in the series of

KSV order 2 > 1 > 3. This information also illustrates that the intercalation ability of cobalt

complex has better effect than manganese and zinc on the fluorescence intensity of EB-DNA

being quenched. Thus, it can be confirmed that the reactions of the four intercalary complexes

between the adjacent DNA base pairs have taken place [64].

3.3.3. Circular dichroic spectral studies

The conformational changes of CT-DNA induced by complexes (1- 3) were monitored by

CD spectroscopy in buffer at room temperature (Fig. 5). The observed positive band at 275 nm

and the negative band at 245 nm in the CD spectrum are due to the base stacking and the right-

handed helicity of B-DNA, respectively [65]. While groove binding and electrostatic interaction

of small molecules with DNA showed a little or no perturbations on the base stacking and

helicity bands, intercalation enhances the intensities of both the bands, stabilizing the right-

handed B conformation of CT DNA. With the addition of 1 to the solution of CT-DNA produced

shifts (3 nm) for the positive CD signal as well as an enhancement of CD ellipticity at 272 nm.

The increase in positive and a decrease in negative ellipticity indicate strong conformational

changes [65]. On the other hand in the presence of complexes 2 or 3, decrease in the intensity of

the positive band and an increase in the negative band of DNA have been observed. These

changes are revealed the non-intercalative mode of binding of these complexes and offer support

to their groove binding nature [66, 67]. Groove binding and electrostatic interaction of the

complexes with DNA have been shown to bring about only marginal changes in the intensity of

negative band as well as the positive band of DNA. Moreover in terms of DNA interaction, it has

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been found that complex 2 was more effective as compared to other complexes because of higher

Kb and Ksv values and circular dichroism spectra.

3.3.4. Interaction of the complexes with serum albumin proteins

Bovine serum albumin (BSA) (containing two tryptophans, Trp-134 and Trp-212)

solutions exhibit a strong fluorescence emission with a peak at 343 nm, due to the tryptophan

residues, when excited at 295 nm [68]. The interaction of bpy and complexes 1-3 with serum

albumin has been studied from tryptophan emission-quenching experiments. The changes in the

emission spectra of tryptophan in BSA are primarily due to the change in protein conformation,

subunit association, substrate binding or denaturation. Bpy and its complexes 1-3 exhibited a

maximum emission at 328 nm under the same experimental conditions and the SA fluorescence

spectra have been corrected before the experimental data processing. Addition of bipyridine or

its complexes 1-3 to BSA results in fluorescence quenching (up to 77% of the initial

fluorescence intensity of BSA for bpy, 66% for 1, 47% for 2 and 70% for 3, as calculated after

the correction of the initial fluorescence spectra, Fig. 6) due to possible changes in protein

secondary structure of BSA indicating the binding of the compounds to BSA [69]. The Stern-

Volmer and Scatchard graphs may be used in order to study the interaction of a quencher with

serum albumins. According to Stern-Volmer quenching equation [50,70]:

1 τ 1 KQ

where I0 = the initial tryptophan fluorescence intensity of SA, I = the tryptophan

fluorescence intensity of SA after the addition of the quencher, kq = the quenching rate constants

of SA, KSV = the dynamic quenching constant, τ0 = the average lifetime of SA without the

quencher, [Q] = the concentration of the quencher respectively,

K τ

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and, taking as fluorescence lifetime (τ0) of tryptophan in SA at around 10-8 s, the dynamic

quenching constant (KSV, M-1) can be obtained by the slope of the diagram I0 /I vs [Q] (Fig. S1),

and subsequently the approximate quenching constant (kq, M-1 s-1) may be calculated. The

calculated values of Ksv and kq for the interaction of the compounds with BSA are given in

Table 3 and indicate a good BSA binding propensity of the compounds with bpy and complex 2

exhibiting the highest BSA quenching ability. The kq values are higher than diverse kinds of

quenchers the existence of a static quenching mechanism [71, 72]. Using the Scatchard equation

∆ ⁄ ∆

where n is the number of binding sites per albumin and K is the association binding

constant, K (M-1), may be calculated from the slope in plots (∆I0/I)/[Q] versus (∆I/I0) (Fig. S2)

and n is given by the ratio of the y intercept to the slope. It is obvious (Table 3) that the

coordination of bipyridine to Co(II) results in an increased K value for BSA with complex 2

exhibiting the highest K value among the other complexes.

The Stern-Volmer equation applied for the interaction with BSA in Fig. S1 shows that the

curves have fine linear relationship (r = 0.97-0.99). The calculated values of KSV and kq are given

in Table 3 and indicate their good BSA binding propensity with complex 2 exhibiting the highest

BSA quenching ability. From the Scatchard graph (Fig. S1), the association binding constant to

BSA of each compound has been calculated (Table 3) with complexes exhibiting higher K values

than free bpy. The n value of bpy presents increases when it is coordinated to Mn(II) Co(II) and

Zn(II) in complexes 1-3 (Table 3). Comparing the affinity of complexes 1-3 for BSA (K values),

it is obvious that complexes 1-3 show higher affinity than bpy which presents lower affinity for

BSA. Additionally, Co (II) complex exhibit higher binding affinity for BSA than Mn(II) and

Zn(II) complexes which is in a similar way to that of DNA binding studies.

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3.3.5. Molecular docking with DNA

In order to rationalize the observed spectroscopic results and to get more insight into the

intercalation modality, the metal(II) complexes (1-3) were successively docked within the DNA

duplex of sequence d(CGCGAATTCGCG)2 dodecamer (PDB ID: 1BNA) in order to predict the

chosen binding site along with preferred orientation of the ligand inside the DNA minor groove.

It is well known that the interactions of chemical species with the minor groove of B–DNA differ

from those occurring in the major groove, both in terms of electrostatic potential and steric

hindrance, because of the narrow shape of the former. In contrast to major groove, small

molecules preferentially interact with the minor groove due to little steric interference.

Additionally, minor–groove binding molecules have aromatic rings connected by single bonds

that allow for torsional rotation in order to facilitate into the curvature of groove with

displacement of water molecules. The minimum energy docked pose (Fig. 7a to 7c) revealed that

complexes snuggly fitted into the curve contour of the targeted DNA in the minor groove and is

situated within G–C (~13.2Ǻ) region, and slightly bends the DNA in such a way that a part of the

planar heterocyclic rings makes favorable stacking interactions between DNA base pairs and

lead to van der Waals interaction and hydrophobic contacts with DNA functional groups that

define the groove [73, 74]. The resulting relative binding energy of docked bpy and metal

complexes 1–3 with DNA were found to be –161.86, –270.80, –281.23 and –296.86 eV,

respectively.

3.4. Nuclease activity

The ability of complexes to cleave supercoiled DNA was determined by agarose gel

electrophoresis. When circular plasmid DNA in the presence of an inorganic molecule is

subjected to electrophoresis, relatively fast migration will be observed for the intact supercoiled

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form (Form I). If scission occurs on one strand, the supercoil will relax to generate a slower

moving open circular form (Form II). If both the strands are cleaved, a linear form (Form III)

that migrates in between Form I and II will be generated. The DNA cleavage ability of

complexes (1- 3) were investigated using pBR322 DNA in aqueous buffer solution (50 mM Tris-

HCl/50 mM NaCl, pH 7.2) at 37 °C for 4h. No DNA cleavage was observed for the control in

which metal complex was absent, or incubation of the plasmid DNA with the metal complex (1

or 2 or 3) or with activators MPA or H2O2 (Fig.8(a-c), lane 1-4) . Complexes (1-3) can induce

the obvious cleavage of the plasmid DNA when 1 or 2 or 3 coexists with MPA. At the

concentration of 100 µM, complexes (1-3) can almost promote the complete conversion of DNA

from Form I to Form II. As depicted in figures 8a and 8b, lane 6 & 5 for complexes 1 & 2

respectively with the increase in concentration of complexes (1-2) from 100 µM to 200 µM,

supercoiled DNA decreases and at final, completely converted to nicked (Form II) and linear

form (Form III). The probability of double strand scission is enhanced when concentration of

complex (1 or 2) increases. Complex 2 exhibited higher cleavage than 1 and 3. However,

complex 3 exhibited much lower cleaving efficiency for pBR322 DNA, even at the concentration

of 200 µM and it cannot promote the conversion of DNA from form II to form III (Fig. 8c, lane

8). The chemical nuclease activity follows the order: 2 > 1 > 3. The different DNA-cleavage

efficiency of the complexes may be due to the different binding affinity of the complexes to

DNA [75]. This suggestion has been supported by their Kb values. This type of chemical

nuclease activity order for examined divalent metal ions can be explained on the basis that the

general series found by Irving and Williams [76] for the stability of complexes.

The nuclease efficiency of metal complexes is usually dependent on activators. Thus,

besides MPA, other activators such as H2O2 (Fig. 8(a-c)) glutathione (GSH) (Fig. 8d) ascorbate

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(Asc) (data not shown) were also used to investigate the DNA cleavage activity of complexes (1-

3). The nuclease activity efficacy followed the order MPA > GSH > H2O2 > Asc. The complexes

(1-3) exhibited a remarkable DNA cleavage activity with different activators [77].

The reactive oxygen species were also investigated, as shown in Fig. 9a and 9b. The

strong inhibitions of DNA cleavage to complex 2 was observed in the presence of the hydroxyl

radical scavengers KI and DMSO, indicating that hydroxyl radicals are responsible for the

cleavage(fig, 9a, lane 4 and 8). The singlet oxygen scavengers like L-hisidine and NaN3, failed to

inhibit cleavage, (Fig.9b, lane 3 and 7) suggesting that singlet oxygen and hydrogen peroxide are

not likely to be the reactive oxygen species in Co complex. In case of complexes 1 and 3 the

hydroxyl radical scavengers KI and DMSO do not inhibit the cleavage process (Fig. 9a) but

addition of singlet oxygen quenchers inhibits the cleavage process (Fig. 9b) and suggest that

singlet oxygen is responsible for the cleavage of Mn(II) and Zn(II) complexes.

Direct evidence of DNA hydrolysis was obtained further from ligation experiments. It is

well known that in DNA hydrolytic cleavage 3-OH and 5 -OPO3 (5ʹ-OH and 3 -OPO3)

fragments remain intact and that these fragments can be enzymatically ligated [78, 79]. The

linear DNA recovered was subjected to overnight ligation reaction with T4 DNA ligase. The

result after electrophoresis (Fig. 10) shows that the linear DNA fragments cleaved by 3 can be

religated by T4 ligase just like the linear DNA mediated by EcoRI. Hence, this result indicates

that the process of DNA cleavage by 3 is a hydrolysis that takes place by a reaction similar to

that of the natural enzyme EcoRI.

3.5. Cytotoxic activity

Cytotoxic potential of newly synthesized complexes (1-3) was investigated on human

breast cancer cell (MCF-7). The complexes (1-3) were applied in range of concentration

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0.1-100 µM for MCF-7 and left for 48 h. The activities of the complexes were determined by

MTT test in vitro and the results were expressed in terms of IC50 values. The positive results

obtained from DNA binding, protein binding and DNA cleavage studies of the new water soluble

complexes 1-3 encouraged us to test its cytotoxicity against a panel of human breast cancer cell

line by the MTT assay. The relations of inhibition rates and complexes concentrations against

human breast cancer cell (MCF-7) were shown in Fig.11. As shown in Fig.11, complex 2 has

stronger inhibition ratios than complexes 1 and b against tested human cancer cells at low

concentrations. The inhibition effects were further enhanced by increasing the concentration of

complexes. At the concentration of 100 µM, inhibition rates of the complexes 1-3 against human

breast cancer cells reached 70.9, 98.1 and 23.7 % respectively. The values of IC50 for the

complexes 1-3 were 56.1, 13.2 and100 µM. Also, the cytotoxicity of complexes (1-3) follows

order 2 > 1 > 3. It is commonly believed that the biological activities of anticancer metal

complexes are dependent on their ability to bind DNA and damage its structure resulting in the

impairment of its function, which is followed by inhibition of replication and transcription

processes and eventually cell death, if the DNA lesions are not properly repaired. The

cytotoxicity of complex 2 is higher than other complexes due to their ability to effect changes in

the secondary structure of DNA, and their stronger DNA binding affinity. The type of metal ion

may be another reason for their different anticancer activity [80]. This is due to the fact that

cobalt complexes have the capacity to reduce the energy status in tumors, as well as to enhance

tumor hypoxia, which also influences their antitumor activities.

4. Conclusions

Metal(II) complexes (1-3) consists of bipyridine and azide were synthesized and

characterized by spectroscopic studies. Molecular structure of complexes (1-3) were determined

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by X-ray crystallography afforded distorted octahedral coordination geometry for Mn(II), Co(II)

and distorted square pyramidal geometry for Zn(II) complexes. The DNA binding and cleaving

capabilities of these complexes (1-3) were investigated by absorption, fluorescence spectroscopy

and CD measurements. The corroborative results of these experiments validate that complex 1

bind to CT DNA via intercalation while complexes 2 and 3 preferentially via groove binding

propensity. Computer-aided molecular docking studies validated the interaction in the minor

groove of DNA helix. The gel electrophoresis assay demonstrated that the complexes 1-3

promote the cleavage ability of the pBR322 plasmid DNA but with different cleavage

mechanisms: the complex 3 cleaves DNA via hydrolytic pathway (further validated by T4 DNA

ligase assay), while the DNA cleavage by complexes 1 and 2 follows oxidative pathway. The

chemical nuclease activity follows the order: 2 > 1 > 3. This type of chemical nuclease activity

order explained on the basis that the general series found by Irving and Williams for the stability

of the complexes. The in vitro cytotoxicity of these complexes (1-3) on breast cancer cell line

(MCF-7) indicates that complexes have the potential to act as effective anticancer drugs in the

order: 2 > 1 > 3.

Acknowledgments: We are thankful to the University Grant Commission, New Delhi (project

No. 40-46/2011 (SR)) and DST (DST-SR/FT/CS-049/2009) for the financial support. The

authors thank the STIC Cochin University of Technology, Cochin and Center for Research

Facilities in IIT-Madras, Chennai-600036 for single crystal X-ray diffraction analysis of the

complexes. The help and support provided by Dr. P. G. Aravindan during the course of the

crystallographic work is gratefully acknowledged.

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Supplementary Material: Crystallographic data in CIF format for the manganese(II), cobalt(II)

and zinc(II) complexes have been deposited at the Cambridge Crystallographic Data Centre,

CCDC No. 952618, 945665 and 945930 respectively. Copies of CIFs are available free of

charge from The Director, CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK (fax: -/44-1223-

336-033; email:[email protected] or http://www.ccdc.cam. ac.uk).

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Figure Captions: Fig. 1. OPTEP views of the molecular structure and atom labeling scheme of complexes 1-3. Fig.2. Crystal packing diagrams of complexes 1-3 were showing the π-π stacking interaction. Fig. 3. Absorption spectra of complexes (1-3) (10–5 M) in 5 mM Tris–HCl /20mM NaCl buffer at pH 7.2 in the absence and presence of increasing amounts of CT-DNA. Fig. 4(a-c). Emission spectra of EB bound to DNA in the presence of complexes (1-3) ([EB] = 3.3 µM, [DNA] = 20 µM, [complex] = 0–25 µM, λex = 510 nm). (d). Plot of EB relative fluorescence intensity at λem = 596 nm (I/I0 (%)) vs r (r = [compound]/[DNA]) for bpy and its complexes 1-3 in buffer solution (5 mM NaCl and 5 mM Tris–HCl at pH 7.4). (e). The Stern–Volmer plot illustrating the quenching of EB bound to DNA by complexes 1–3. Fig. 5. CD spectra of CT-DNA (1.0 x 10-4 M), and the interaction with 1, 2 and 3 ([complex]/[DNA] = 0.5). All the spectra were recorded in 5 mM Tris–HCl/NaCl buffer, pH 7.2 and 25 °C. Fig. 6. Plot of % relative fluorescence intensity at λem = 351 nm (I/I0 (%)) vs r (r = [compound]/[BSA]) for bpy and its complexes 1-3 in buffer solution (150 mM NaCl and 15 mM trisodium citrate at pH 7.0). Fig. 7. Molecular docked model of complexes 1, 2 and 3 with DNA dodecamer duplex of sequence d(CGCGAATTCGCG)2(PDB ID: 1BNA).

Fig. 8. Agarose gel electrophoresis of SC pBR322 DNA (0.2µg, 33.3 µM) in presence complexes 1–3 in 50 mM Tris–HCl/50mM NaCl buffer (pH 7.2). (a) Lane 1, DNA control; lane 2, DNA + H2O2 (1 mM); lane 3, DNA + MPA (1 mM); lane 4, DNA + 1 (200 µM); lane 5, DNA + MPA (1 mM) + 1 (100 µM); lane 6, DNA + MPA (1 mM) + 1 (200 µM); lane 7, DNA + H2O2 (1 mM)+ 1 (100 µM); lane 8, DNA + H2O2 (1 mM) + 1 (200 µM). (b) Lane 1, DNA control; lane 2, DNA + MPA (1 mM); lane 3, DNA + 2 (200 µM); lane 4, DNA + MPA (1 mM) + 2 (100 µM); lane 5, DNA + MPA (1 mM) + 2 (200 µM); lane 6, DNA + H2O2 (1 mM); lane 7, DNA + H2O2 (1 mM) + 2 (100 µM); lane 8, DNA + H2O2 (1 mM) + 2 (200 µM). (c) Lane 1, DNA control; lane 2, DNA + H2O2 (1 mM); lane 3, DNA + MPA (1 mM); lane 4, DNA + 3 (200 µM);

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lane 5, DNA + H2O2 (1 mM) + 3 (100 µM); lane 6, DNA + H2O2 (1 mM) + 3 (200 µM); lane 7, DNA + MPA (1 mM) + 3 (100 µM); lane 8, DNA + MPA (1 mM) + 3 (200 µM). (d). Cleavage of SC pBR322 DNA (0.2µg, 33.3 µM) by complexes 1–3 with various activators; Lane 1, DNA control; lane 2, DNA + GSH (1 mM); lane 3, DNA + GSH (1 mM) + 1 (200 µM); lane 4, DNA + GSH (1 mM) + 2 (200 µM); lane 5, DNA + GSH (1 mM) + 3 (200 µM).

Fig. 9. Agarose gel electrophoresis of SC pBR322 DNA (0.2µg, 33.3 µM) in presence and complexes 1–3 with various inhibitors in 50 mM Tris–HCl/50mM NaCl buffer (pH 7.2). (a) Lane 1, DNA control; lane 2, DNA + mercaptopropionic acid + KI; lane 3-5, DNA + mercaptopropionic acid + KI + 1-3 (100µM) respectively; lane 6, DNA + mercaptopropionic acid + DMSO; lane 7-9, DNA + mercaptopropionic acid + DMSO + 1-3 (100µM) respectively. (b). Lane 1, DNA + mercaptopropionic acid + NaN3; lane 2-4, DNA + mercaptopropionic acid + NaN3 + 1-3 (100µM) respectively; lane 5, DNA + mercaptopropionic acid + L-histidine; lane 6-8, DNA + mercaptopropionic acid + L-histidine + 1-3 (100µM) respectively.

Fig. 10. Agarose gel electrophoresis for ligation of pBR322 DNA linearized by 3: lane 1: Lambda DNA / Hind III Marker; lane 2: DNA control; lanes 3 and 4: pBR322 DNA linearized by EcoRI without and with T4 DNA ligase; lanes 5 and 6: pBR322 DNA linearized by 3 without and with T4 DNA ligase.

Fig. 11. Cytotoxic effect of complexes 1-3 against MCF-7 at different concentration (0.1-100 µM). Cell viability decreased with increasing concentration of complexes 1-3.

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Table -1: Crystallographic data and structure refinement parameters for complexes 1 - 3.

1 2 3 Empirical formula C20H16MnN10 C20 H22 Co N10 O3 C20 H16 N16 Zn2 Formula weight 451.37 509.41 611.23 Temperature (K) 293(2) 296(2) 296(2) Wavelength (Å) 0.71073 0.71073 0.71073 Crystal system, space group Orthorhombic, Pbcn Triclinic, P-1 Triclinic, P-1 a (Å) 14.6090(8) 8.2713(12) 6.5538(12) b(Å) 8.1852(4) 11.7726(16) 10.8448(14) c (Å) 16.9512(7) 12.4019(17) 16.885(3) α (°) 90 91.358(6) 96.316(4) β (°) 90 92.213(5) 95.365(5) γ (°) 90 108.456(5) 90.497(5) Volume (Å3) 2026.98(17) 1143.8(3) 1187.4(3) Z, calculated density (mg m-3) 4, 1.479 2, 1.479 2, 1.710 Absorption coefficient (mm-1) 0.682 0.795 2.067 F(000) 924 526 616 Crystal size (mm) 0.30 x 0.30 x 0.25 0.35x0.30x0.20 0.35 x 0.30 x 0.20 θ range for data collection (°) 2.40 to 24.99 1.64 to 28.16 1.22 to 28.09 Limiting indices, h,k,l -17≤ h≤ 17, -9≤ k≤8,

-13≤l≤20 -10≤ h≤ 10, -15≤ k≤14, -14≤l≤16

-8≤ h≤ 6, -13≤ k≤ 11, -22≤ l≤ 22

Reflections collected / unique 9673 / 1779 8637 / 5376 8869 / 5428 Rint 0.0311 0.0204 0.0335 Data / restraints / parameters 1779 / 0 / 142 5376/9/323 5428 / 0 / 343 Goodness-of-fit on F2 1.045 1.032 1.115 Final R indices [I > 2σ (I)] R1 = 0.0283, wR2 =

0.0710 R1 = 0.0672,wR2 = 0.2093 R1 = 0.0779, wR2 = 0.2121

R indices (all data) R1 = 0.0376, wR2 = 0.0777

R1 = 0.0836, wR2 = 0.2308 R1 = 0.0908, wR2 = 0.2228

Largest difference peak and hole / e Å-3

Max. and min. transmission

0.176 and -0.171 0.8568 and 0.8136

2.625 and -0.508 0.8572 and 0.7683

3.413 and -1.358 0.6826 and 0.5315

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Table 2．Selected Bond Lengths (Å) and Bond Angles (°) for complexes

1 2 3

Bond distances (Ǻ) Bond angles (°) Bond distances (Ǻ) Bond angles (°) Bond distances (Ǻ) Bond angles (°)

Mn(1)-N(1) 2.3225(15) N(3)-Mn(1)-N(3)#1 92.46(10) Co(1)-N(1) 1.938(3) N(1)-Co(1)-N(3) 177.85(13) Zn(1)-N(5) 2.053(6) N(5)-Zn(1)-N(11) 97.7(2)

Mn(1)-N(2) 2.2730(16) N(3)-Mn(1)-N(2)#1 89.56(7) Co(1)-N(3) 1.941(3) N(1)-Co(1)-N(8) 91.35(14) Zn(1)-N(11) 2.081(5) N(5)-Zn(1)-N(2) 93.0(2)

Mn(1)-N(3) 2.1472(17) N(3)#1-Mn(1)-N(2)#1 162.35(6) Co(1)-N(8) 1.940(4) N(3)-Co(1)-N(8) 88.51(15) Zn(1)-N(2) 2.112(5) N(11)-Zn(1)-N(2) 147.0(2)

Mn(1)-N(3)#1 2.1472(17) N(3)-Mn(1)-N(2) 162.35(6) Co(1)-N(5) 1.946(4) N(1)-Co(1)-N(5) 89.48(14) Zn(1)-N(1) 2.154(5) N(5)-Zn(1)-N(1) 168.5(2)

Mn(1)-N(2)#1 2.2729(16) N(3)#1-Mn(1)-N(2) 89.56(7) Co(1)-N(4) 1.955(3) N(3)-Co(1)-N(5) 92.67(15) Zn(1)-N(14) 2.172(5) N(11)-Zn(1)-N(1) 93.7(2)

Mn(1)-N(1)#1 2.3225(15) N(2)#1-Mn(1)-N(2) 93.81(8) Co(1)-N(2) 1.958(3) N(8)-Co(1)-N(5) 92.55(16) Zn(2)-N(8) 2.062(6) N(2)-Zn(1)-N(1) 76.7(2)

N(3)-Mn(1)-N(1)#1 102.16(7) N(1)-Co(1)-N(4) 95.61(14) Zn(2)-N(14) 2.088(5) N(5)-Zn(1)-N(14) 87.0(3)

N(3)#1-Mn(1)-N(1)#1 91.49(6) N(3)-Co(1)-N(4) 82.24(14) Zn(2)-N(4) 2.112(5) N(11)-Zn(1)-N(14) 108.1(2)

N(2)#1-Mn(1)-N(1)#1 70.96(6) N(8)-Co(1)-N(4) 87.85(15) Zn(2)-N(3) 2.142(5) N(2)-Zn(1)-N(14) 103.5(2)

N(2)-Mn(1)-N(1)#1 95.31(5) N(5)-Co(1)-N(4) 174.88(14) Zn(2)-N(11)#1

2.160(5) N(1)-Zn(1)-N(14) 90.49(19)

N(3)-Mn(1)-N(1) 91.49(6) N(1)-Co(1)-N(2) 82.69(14) N(8)-Zn(2)-N(14) 98.9(2)

N(3)#1-Mn(1)-N(1) 102.16(7) N(3)-Co(1)-N(2) 97.38(14) N(8)-Zn(2)-N(4) 92.0(2)

N(2)#1-Mn(1)-N(1) 95.31(5) N(8)-Co(1)-N(2) 173.76(14) N(14)-Zn(2)-N(4) 147.91(19)

N(2)-Mn(1)-N(1) 70.96(6) N(5)-Co(1)-N(2) 89.25(15) N(8)-Zn(2)-N(3) 166.5(2)

N(1)#1-Mn(1)-N(1) 160.31(8) N(4)-Co(1)-N(2) 90.89(14) N(14)-Zn(2)-N(3) 94.6(2)

N(4)-Zn(2)-N(3) 75.81(19)

N(8)-Zn(2)-N(11)#1 86.0(3)

N(14)-Zn(2)-N(11)#1 106.7(2)

N(4)-Zn(2)-N(11)#1 104.06(19)

N(3)-Zn(2)-N(11)#1 91.32(19)

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Table 3. The BSA binding constants and parameters (Ksv, kq, K, n) derived for bpy and complexes 1-3

Compound KSV (M-1) kq (M-1s-1 K (M-1) n

bpy 5.7 x 103 5.7 x 1011 0.1470 0.0920

[Mn(bipy)2(N3)2] (1) 1.15 x 104 1.15 x 1012 0.0215 0.1359

[Co(bipy)2(N3)2].3H2O (2) 2.35 x 104 2.35 x 1012 0.0253 1.0024

[Zn2(bipy)2(N3)4] (3) 1.03 x 104 1.03 x 1012 0.0159 0.2553

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Highlights

Manganese(II) (1), Cobalt(II) (2) and Zinc(II) (3) complexes with the bipyridine and

azide ligands.

Metal adopt distorted octahedral for 1, 2 and distorted square pyramidal geometry for 3.

Complexes (1-3) were able to bind to DNA and protein in the order 2 > 1 > 3.

Chemical nuclease activity are in the order: 2 > 1 > 3.

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Synthesis, molecular structure, biological properties and molecular docking studies on MnII, CoII...

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Transcript of Synthesis, molecular structure, biological properties and molecular docking studies on MnII, CoII...