Syntheses: Synthesis of [C H ON Cl CuSn]Cl - INFLIBNETshodhganga.inflibnet.ac.in/bitstream/10603/12890/9/09_chapter 3.pdf · Syntheses: Synthesis of [C 16H 22ON 5Cl ... heterobimetallic

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Syntheses:

Synthesis of [C16H22ON5Cl4CuSn]Cl

To a methanolic solution of Phenylglycine chloride hydrochloride (10 mM, 2.06 g) 10

mM (1.70 g) of CuCl2.2H2O in MeOH was added dropwise. The fluorescent green

solution obtained was left to stir for 1h. An equimolar amount of dichlorodimethyl-

bis(4-pyrazole N2) tin (IV), dissolved in 5 mL MeOH was added to the stirring

solution and refluxed for 3h. The completion of the reaction was monitored by thin

layer chromatography. The solution was cooled to room temperature and left for two

days, a green colored crystalline product obtained was filtered, washed with CHCl3

and dried in vacuo.

Yield: 68.0 %. mp; 242 ±3 ºC Anal. Calcd. for [C16H22ON5Cl4CuSn]Cl: C, 29.12; H,

3.36; N, 10.61, Found: C, 29.15; H, 3.35; N, 10.52; Selected IR data (KBr, ν cm-1)

3317 (NH2); 1747 (C=O); 1388 (C-H); 1236 (C-N); 1043 (C-C); 765 (Ar); 496 (Cu-

N). Molar Conductance: M (1·10-3 M, H2O): 140.00 -1cm2 mol-1 (1:1 electrolyte).

UV-vis (1·10-3 M, H2O max / nm) 258 nm, 340 nm, 590 nm. ESI-MS (m/z+) 624

[C16H22ON5Cl4CuSn]+.

Synthesis of [C16H22ON5Cl4NiSn]Cl

The complex [C16H22ON5Cl4NiSn]Cl was synthesized using NiCl2.6H2O (2.37 g, 10

mM) by a similar method as described for [C16H22ON5Cl4CuSn]Cl. The yellow

colored product obtained was filtered, washed with CHCl3 and dried in vacuo.

Yield: 58%; mp; 242 ±5 ºC. Anal. Calcd. for [C16H22ON5Cl4NiSn]Cl: C, 29.41; H,

3.40; N, 10.72. Found: C, 29.36; H, 3.41; N, 10.63. Selected IR data (KBr, ν cm-1)

3342 (NH2); 1720 (C=O); 1388 (C-H); 1296 (C-N); 1043 (C-C);778 (Ar); 502 (Ni-

N); 1H NMR (400 MHz, DMSO-d6, 25 ºC, δ) 2.68 (-CH3); 3.83 (-CH); 6.35, 6.43 ( H

phenyl, pyrazole ring); 7.80, 8.05 (pyrazole NH, NH2); 13C NMR (100 MHz DMSO-

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d6, 25 ºC, δ) 50.82-77.11 (CH3, CH2); 125.72-129.63(Ar); 165.96 (C=O); 119Sn NMR

(400 MHz DMSO- d6, 25 ºC, δ) -218.83; Molar Conductance, M (1·10-3 M, H2O):

145.00 -1cm2 mol-1 (1:1 electrolyte) UV-vis (1·10-3 M, H2O, max /nm) 260 nm, 344

nm 578 nm. ESI-MS (m/z+) 619 [C16H22ON5Cl4NiSn]+.

Results and discussion

The complexes [C16H22ON5Cl5CuSn] and [C16H22ON5Cl5NiSn] were synthesized by

reacting dichlorodimethyl-bis(4-pyrazole N2) tin(IV) [188], with the corresponding

1:1 metal complex of phenylglycinechloride hydrochloride as shown in scheme 3.

Scheme 3. Proposed structure of complexes [C16H22ON5Cl4CuSn]Cl and [C16H22ON5Cl4NiSn]Cl.

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Figure 35. Three dimensional ball and Stick model of complex [C16H22ON5Cl4CuSn]Cl; Colour scheme: C grey, N blue, Cl green, O red, Cu(II), Dark green purple Sn (IV). For clarity H atoms are omitted. Complex [C16H22ON5Cl4NiSn]Cl was synthesized only for the NMR structure

elucidation. The mass spectrometric and elemental analyses were consistent with

proposed molecular formulae of the complexes. Molar conductance measurements

suggest that complexes [C16H22ON5Cl4CuSn]Cl and [C16H22ON5Cl4NiSn]Cl behave

as 1:1 electrolytes. The complexes are soluble in polar organic solvents, DMSO,

MeOH and H2O. AFM, TEM and XRD studies proved that the nano-sized

heterobimetallic Cu-Sn complex has the ability to form DNA condensates. To validate

specific DNA binding of the designed molecule various spectroscopic studies were

done and binding parameters were calculated.

Infrared Spectroscopy

The IR spectra of the complexes [C16H22ON5Cl4CuSn]Cl and [C16H22ON5Cl4NiSn]Cl

revealed a characteristic broad band at 3317-3342 cm-1 due to the stretching vibrations

of the -NH2 and NH groups of the phenylglycine chloride and pyrazole ligands. The

frequency shift of the (-NH2) towards lower region in comparison to that observed

for free phenylglycine chloride ligand (3400 cm-1) confirms its coordination to the

metal ion [189]. Similarly, the participation of C=O group in complex formation was

ascertained by the shift of the band at 1720 and 1747 cm-1 which appears at 1765 cm-1

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in the ligand. Other ligand skeletal bands observed in the range 765-778 cm-1, 1155-

1162 cm-1, 1043 cm-1, were ascribed to the out of plane –CH bending of aromatic

rings , -CH3, and –CH2 groups, respectively. Bands appearing near 2900 cm-1 were

attributed to stretching vibrations of the -CH3 group [190a,b].

Nuclear magnetic Resonance spectroscopy

To further elucidate the structure of the heterobimetallic complexes, the diamagnetic

complex [C16H22ON5Cl5NiSn] was characterized by NMR spectroscopy. The 1H

NMR and 13C NMR spectra show aliphatic and aromatic signals with chemical shifts

in accordance with the proposed structure as shown in figure 36 and 37 respectively.

The 1H NMR spectrum of complex [C16H22ON5Cl4NiSn]Cl contained multiplets at

6.35 ppm and 7.80- 8.05 ppm consistent with the presence of the pyrazole and the

aromatic ring of amino acid derivative, respectively [191]. A sharp doublet at 3.87

ppm indicated the proton attached to the chiral carbon next to the benzene ring and

the singlet at 2.68 ppm was associated with the methyl protons [192].

The 13C NMR spectrum reveals distinct signals in the region 50.82, 54.02, 77.11 ppm

representing the -CH2 and -CH3 carbons [193]. The aromatic ring carbon signals were

observed at 125.72 ppm and 129.63 ppm [194]. The peak at 165.96 ppm corresponded

to the carbonyl carbon.

Figure 36. 1H NMR of complex [C16H22ON5Cl4NiSn]Cl in D2O at 25°C.

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Figure 37. 13C NMR of complex [C16H22ON5Cl4NiSn]Cl in D2O at 25°C.

119Sn NMR spectrum recorded a sharp peak at -218.83 ppm in agreement with the

hexa-coordinated environment of tin atom as shown in figure 38[195].

Figure 38. 119Sn NMR of complex [ C16H22ON5Cl4NiSn]Cl in D2O at 25°C

Electron paramagnetic resonance spectroscopy

The X-band electron paramagnetic resonance spectrum of complex

[C16H22ON5Cl4CuSn]Cl was recorded at a frequency of 9.1 GHz under the magnetic

field strength 3000±1000 gauss with tetracyanoethylene (TCNE) as field marker (g =

2.0027) at LNT. The spectrum of complex [C16H22ON5Cl5CuSn] as shown in figure

39 depicted a very broad axial symmetrical line shape with g|| = 2.401, g = 2.33 and

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gav = 2.35 computed from the formula gav2 = (g||

2+2g2) ∕3. These parameters are in

accordance with axially symmetrical square pyramidal Cu (II) systems [196]. The

trend g|| > g > 2 revealed that the unpaired electron is present in the dx2

-y2

orbital

[197]. For a covalent complex, g|| < 2.3 and for an ionic environment, g|| = 2.3 or more.

In the present complex g|| > 2.3 indicates an appreciable metal-ligand ionic character

[198].

Figure 39. X-band polycrystalline powder EPR spectrum of complex [C16H22ON5Cl4CuSn]Cl at room temperature. Electronic absorption spectra

The electronic spectra of the complexes [C16H22ON5Cl4CuSn]Cl and

[C16H22ON5Cl4NiSn]Cl were recorded in MeOH at room temperature. In the UV

region, both complexes [C16H22ON5Cl4CuSn]Cl and [C16H22ON5Cl4NiSn]Cl exhibit

intense absorption band at 258-260 nm and a shoulder at 340-344 nm attributed to π–

π* [199] or charge transfer transitions of the aromatic chromophore. In the visible

region, complex [C16H22ON5Cl4CuSn]Cl display a broad band at 590 nm assigned to

2B1g→2A1g ligand field transition [200]. Similarly, complex [C16H22ON5Cl4NiSn]Cl

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also displays a low energy metal centered d-d absorption band at 578 nm. These

values are consistent with square pyramidal geometry around Cu (II)/Ni (II) metal

ions [201].

XRD Measurements

Although single crystal of the complex was not obtained, however, the crystalline

nature of the complex was authenticated by XRD measurements. Figure 40 shows the

XRD pattern of the complex [C16H22ON5Cl4CuSn]Cl with peaks at 2θ scattering

angles of 27.22, 33.32, 36.33, 38.42, 44.11, 62.54 assigned to (001) (111), (201),

(200), (212) and (220) crystal planes respectively, characteristic of well ordered

tetragonal arrangement of tin atoms [202]. The lattice parameters are a = 5.79 and c =

0.5322 which are in good agreement with known lattice parameters a = 5.8316 and c

= 0.5455 indicating that the nano particles have the same crystal structure as that of

the bulk [203].

Figure 40. The XRD pattern nanoparticles of complex [C16H22ON5Cl4CuSn]Cl showing peaks at different scattered angles.

Intense reflections derived from (200), (212) planes reveal the existence of copper

atoms in a cubic geometry in good agreement with the known lattice parameters

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[204]. The average grain size of the powder sample was calculated to be 20-30 nm

according to half width of (200) diffraction peaks and the size of the DNA condensed

particles was found in the range of 60-80 nm according to half width of (200) using

Debye Sherrer formula [205].

DNA binding studies

DNA is the primary intracellular target of antitumor drugs. Interaction of the

complexes with DNA can induce DNA damage, which leads to blockage of cell

division and eventually cell death.

Absorption spectral studies

The interaction of the metal complex to DNA is often characterized through

absorption spectral titration followed by the changes in the absorbance and shift in the

wavelength. The absorption spectral traces of the Cu-Sn heterobimetallic complex

[C16H22ON5Cl4CuSn]Cl in the absence and presence of CT DNA are shown in figure

41. Upon the addition of CT DNA to complex [C16H22ON5Cl5CuSn] significant

hyperchromism with a red shift of 3 nm was observed at the intraligand bands.

Hyperchromism and hypochromism are the spectral changes typical of a metal

complex association with the DNA helix [206]. Hypochromism results from

contraction of DNA helix as well as change in its conformation while hyperchromism

results from the damage of DNA double helix structure [207]. The observed

hyperchromism results due to the larger positive charge of cations binding to DNA

presumably by a strong electrostatic attraction to the phosphate group of the DNA

backbone and thereby causing a contraction and overall damage to the secondary

structure of DNA [208]. Complex [C16H22ON5Cl4CuSn]Cl shows better prospects as

an antitumor agent due the presence of two different metal centers having different

specificity at the molecular level.

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The hard Lewis acid Sn (IV) atom binds electrostatically to the phosphate backbone

of DNA helix, while the Cu (II) centre may preferentially coordinate with the N7

position of

Figure 41. Absorption spectral traces of complex [C16H22ON5Cl4CuSn]Cl in Tris-HCl buffer upon addition of CT-DNA. Inset: plot of [DNA]/εa-εf vs [DNA] for the titration of CT-DNA with complex [C16H22ON5Cl4CuSn]Cl:complex [C16H22ON5Cl4CuSn]Cl= 0.40 x 10-4 M, [DNA]= 0.14 x 10-4 M - 0.84 x10-4 M; (▲) experimental data points; full lines, linear fitting of the data. guanine either due to the replacement of the labile chloride ligand or by direct

coordination. Base binding of the complex can perturb the hydrogen bonding between

the base pairs causing destabilization of DNA. To assess the binding ability of the

complex [C16H22ON5Cl4CuSn]Cl with CT DNA, the intrinsic binding constant was

determined using equation I [172,173]. The binding constant Kb value was found to

be 8.42 x104 M-1. The significant binding constant Kb value reflects the average of the

dual binding mode of complex [C16H22ON5Cl5CuSn] to DNA.

[DNA] / | a-f | = [DNA] / | b-f | + 1 / Kb | b-f | (I)

Fluorescence studies

The interaction of complex [C16H22ON5Cl4CuSn]Cl with CT DNA was also

investigated by using emission spectroscopy. Complex [C16H22ON5Cl4CuSn]Cl emits

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strong luminescence with maximum at 530 nm when excited at 260 nm in 0.01 mM

Tris–HCl/50 mM NaCl buffer at ambient temperature. Upon the addition of CT DNA

the intensity of the emission band at 530 nm enhances gradually, which indicates that

the complex could interact with DNA (figure 42). The increase in the emission

intensity usually depends on the degree of exposure of the complex molecule into

DNA double helix. The enhancement of the emission intensity is mainly due to the

change in the environment of the metal complex and is related to the extent to which

the complex is inserted into the hydrophobic environment inside the DNA helix

[209,210]. DNA being hydrophobic molecule restricts the mobility of the solvent

water molecules, at the binding site thereby preventing the quenching effect

particularly in an intercalative binding mode [211]. It is quite obvious that the

Figure 42. Emission spectra of the complex [C16H22ON5Cl5CuSn] in Tris-HCl buffer upon addition of CT DNA. Arrow shows intensity change upon increasing concentration of DNA. Inset: plot of r/Cf vs r: [complex (C16H22ON5Cl5CuSn)] = 0.06 x10-4 M, [DNA] = 0-0.33x10-4M.; (▲) experimental data points; full lines, linear fitting of the data.

complex [C16H22ON5Cl4CuSn]Cl is shielded by the DNA duplex efficiently from the

bulk solution and because of the reduced collision between the solvent and the

complex molecule, a decrease in the vibrational modes of relaxation results and hence

quenching does not occur [212]. The observed increasing emission intensity implies

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binding of the complex to the hydrophobic pocket of the DNA along the major or

minor grooves by electrostatic interactions with the phosphate groups [213].

From a plot of r/cf versus r the binding constant for complex [C16H22ON5Cl4CuSn]Cl

was calculated to be 3.42 x 104 M-1 which is in well agreement with the binding

constant values as determined by UV-vis titrations.

Cyclic Voltammetry

Cyclic voltammetry is a useful technique for studying the interaction of metal

complexes with CT DNA and understanding the nature of DNA binding. The cyclic

voltammogram of complex [C16H22ON5Cl4CuSn]Cl was recorded in MeOH at the

scan rate of 0.2 Vs-1over the potential range of 2.0-1.2V. The cyclic voltammetric

response of complex [C16H22ON5Cl4CuSn]Cl in the absence and in presence of CT

DNA is depicted in figure 43. The CV of complex [C16H22ON5Cl4CuSn]Cl exhibit a

quasireversible redox wave corresponding to CuII/CuI with Epc= -0.514 V and Epa= -

0.287 V.

Figure 43. Cyclic voltammogram (scan rate 0.2 Vs-1, MeOH, 25 °C) of (a) complex [C16H22ON5Cl4CuSn]Cl (b) complex [C16H22ON5Cl4CuSn]Cl in presence of CT DNA.

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For this couple, the difference between cathodic and anodic peak potential ΔEp and

the ratio of anodic and cathodic peak current are –0.227V and 0.98, respectively. The

formal electrode potential E1/2 taken as the average of Epa and Epc was -0.401 V in the

absence of DNA. Addition of CT DNA results in the significant shift in E1/2 and

reduction in peak currents (E1/2=-0.413 and ΔEp=-0.164 V respectively). The ratio of

Ipa/Ipc decreases to 0.84. The shift in formal potential and decreases in the current ratio

suggest strong binding of complex [C16H22ON5Cl4CuSn]Cl to CT DNA [214].

DNA Cleavage activity

The ability of the heterobimetallic complex [C16H22ON5Cl4CuSn]Cl to induce DNA

cleavage was assessed on pBR322 supercoiled plasmid DNA. The cleavage activity

was determined with 0.05-0.25 mM of the complex in 0.01mM Tris HCl/50 mM NaCl

buffer at pH 7.2 in a total volume of 30 µL containing pBR322 plasmid DNA, after an

incubation of 1h at 37 ºC. During electrophoresis when scission occurs on one strand

(nicking), the supercoil relaxes to generate Form II. When both strands are cleaved a

linear Form III is generated which migrates between Form I and Form II. In absence

of any exogenous reductant, the pBR322 plasmid DNA remains primarily supercoiled

(form I); however upon increasing the concentration of the metal complex the DNA

was converted to nicked DNA (Form II) although less efficiently without any

concurrent formation of linearized DNA (Form III), suggesting a single strand

breakage [215]. Complex [C16H22ON5Cl4CuSn]Cl unwinds the DNA duplex by

interacting with supercoiled form of pBR322DNA to form a DNA-complex adduct

and hence reduces the number of supercoils. The decrease in supercoils upon binding

of unwinding agents causes a decrease in the rate of migration through agarose gel

revealing the interaction and cutting effect of complex with DNA at a particular

concentration (figure 44a). The nuclease efficiency of the complex was also

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investigated in presence of ascorbate as an activator. The complex was found to

mediate the rapid degradation of the supercoiled (Form I) plasmid DNA to produce

nicked form (form II) in a concentration dependent manner. With the increase in the

concentration of the complex, the band intensity of form II increases while the band

intensity of Form I decreases indicating the DNA cutting efficiency of the complex

increases in the presence of reducing agent. No cleavage was observed beyond

background for ascorbate alone as shown in figure 44b. To better evaluate the

cleavage mechanism, experiment with the scavenging agent 5% DMSO was carried to

identify the intermediate hydroxyl radical (ROS species) that might form during

cleavage reaction.

Figure 44a. Gel Electrophoresis diagram showing cleavage of pBR322 supercoiled DNA(300 ng) by complex [C16H22ON5Cl4CuSn]Cl; Lane 1: DNA; Lane 2: 0.05 mM [C16H22ON5Cl4CuSn]Cl + DNA; Lane 3: 0.1 mM [C16H22ON5Cl4CuSn]Cl + DNA; Lane 4: 0.15 mM [C16H22ON5Cl4CuSn]Cl + DNA; Lane 5: 0.2 mM [C16H22ON5Cl4CuSn]Cl + DNA; Lane 6: 0.25 mM [C16H22ON5Cl4CuSn]Cl + DNA.

Figure 44b. Gel Electrophoresis diagram showing cleavage of pBR322 supercoiled DNA (300ng) by [C16H22ON5Cl4CuSn]Cl in presence of radical scavenger (5%DMSO) and ascorbic acid H2A (0.1-0.25 mmol) Lane 1: DNA; Lane 2: 0.05mM [C16H22ON5Cl4CuSn]Cl + DNA + DMSO; Lane 3: 0.1mM [C16H22ON5Cl4CuSn]Cl + DNA + DMSO + 0.1 mM H2A; Lane 4: 0.15mM [C16H22ON5Cl4CuSn]Cl + DNA + DMSO +0.15 mM H2A; Lane 5: 0.2 mM + DMSO + 0.2 mM H2A [C16H22ON5Cl4CuSn]Cl + DNA; Lane 6: 0.25mM [C16H22ON5Cl4CuSn]Cl + DNA + DMSO + 0.25 mM H2A.

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The result showed a little effect on the DNA cleavage ruling out the possibility of

DNA cleavage by hydroxyl radicals [216]. Based on these observation, it is proposed

that the Cu (II) complex is initially reduced to Cu (I) species by ascorbic acid, which,

then binds to DNA to form the Cu (I) complex-DNA adduct.

DNA Condensation

DNA condensation is an integral step involved in many DNA transactions such as

recombination, replication, transcription and repair [217]. To define the

pharmacological action of the drug candidate controllable drug-DNA condensation is

imperative to allow protected packaging from enzymatic degradation and

transportation across the biological barriers to a specific target cell. Complex

[C16H22ON5Cl4CuSn]Cl has the potential to induce DNA condensation due to strong

charge compensation of the anionic phosphate segments by the cationic

heterobimetallic core. In this study, DNA condensation to the nano particulate

structure by complex [C16H22ON5Cl5CuSn] under neutral conditions in aqueous

medium (Tris-HCl buffer/50mM NaCl pH 7.2) was achieved by evaporating an

equimolar mixture of complex [C16H22ON5Cl4CuSn]Cl and CT DNA. The increase in

the size of the complex nano particles after CT DNA condensation was determined by

XRD measurements. To analyze the formation of the drug-DNA condensates,

visualization techniques - transmission electron microscopy (TEM) and atomic force

microscopy (AFM) were employed.

TEM and AFM imaging of Complex-DNA condensates

To recognize the complex-DNA condensate morphology, TEM image of uncondensed

complex nanoparticles is given for comparison in figure 45a. The shape of the

particles is irregular but close to spherical. The particles have wide size distribution

and the average

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Figure 45a: TEM image of the nano particles of complex [C16H22ON5Cl4CuSn]Cl.

Figure 45 (b) TEM image indicating the condensation of CT DNA on nano particles of complex [C16H22ON5Cl4CuSn]Cl (c) TEM image indicating the coagulation of nano particles around CT DNA for 12h. particle diameter is in the range of 20-30 nm. The particle size increases almost thrice

when CT DNA was condensed on it (figure 45b and 45c). This clearly indicates that

the complex nanoparticles have a good ability to condense the free CT DNA (existing

as loose strands) to compact solid particles. To obtain the morphological and

structural information about the free complex [C16H22ON5Cl4CuSn]Cl and the

condensates, tapping mode AFM (atomic force microscopy) experiments were

performed using commercially etched silicon tips as AFM probes with typical

resonance frequency of 300 Hz (RTESP Veeco Innova II nanoscope). Two and three

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dimensional AFM images of the complex [C16H22ON5Cl5CuSn] and the condensate

materials are shown in figure 46 (a and b) and 47 (a and b). Figure 48a shows the

morphology of complex [C16H22ON5Cl4CuSn]Cl condensed with supercoiled pBR322

DNA at a particular time (6h) and figure 48b shows the three dimensional

morphology of the condensate material at the same time. Figure 49a shows the change

in the morphology of complex [C16H22ON5Cl4CuSn]Cl condensed with supercoiled

pBR322 DNA at a particular time (after 12h) and figure 49b shows three dimensional

morphology of the condensate material at the same time. The width of the condensate

material increases with respect to time however there is no change in the vertical

height. The condensation of CT DNA and supercoiled pBR322 DNA on the surface of

these nano particles was studied. The surfaces of these nanoparticles were uneven

structures, and after the DNA condensation the grooves were observed on the plane

surface with little pyramidal shapes of the condensate materials. It is clear from the

figure that the width of the structure in figure 49 (a and b) is about three times the

width of the structures in figure 48 (a and b). It clearly shows that with the

advancement of time, the complex [C16H22ON5Cl4CuSn]Cl has the tendency to

condense on the supercoiled pBR322DNA. Thus, the results are indicative of the good

DNA condensation ability of the heterobimetallic Cu-Sn complex.

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Figure 46. (a) Two dimensional AFM image of the nano particles of complex [C16H22ON5Cl4CuSn]Cl (b) Three dimensional AFM image of the nano particles of complex [C16H22ON5Cl4CuSn]Cl

Figure 47. (a)Two dimensional AFM image of the CT DNA condensed nano particles;50 µM nano complex [C16H22ON5Cl4CuSn]Cl + CT DNA in (1:1) ratio. (b) Three dimensional AFM image of the CT DNA condensed nano particles ; 50 µM nano complex [C16H22ON5Cl4CuSn]Cl + CT DNA in (1:1) ratio.

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Figure 48. (a) Two dimensional AFM image of the CT DNA condensed nano particles of complex [C16H22ON5Cl5CuSn]+ 30 µM nano complex [C16H22ON5Cl4CuSn]Cl pBR322 DNA in (1:1) ratio. (b) Three dimensional AFM image of the CT DNA condensed nano particles of complex [C16H22ON5Cl4CuSn]Cl 30 µM nano complex [C16H22ON5Cl4CuSn]Cl + pBR322 DNA + 30 µM Plasmid DNA for 6h. Figure 49. (a) Two dimensional AFM image of the CT DNA condensed nano particles of complex [C16H22ON5Cl4CuSn]Cl 30 µM nano complex [C16H22ON5Cl4CuSn]Cl pBR322 DNA in (1:1) ratio after 12h. (b) Three dimensional AFM image of the CT DNA condensed nano particles of complex [C16H22ON5Cl4CuSn]Cl 30 µM nano complex [C16H22ON5Cl4CuSn]Cl + pBR322 DNA + 30 µM Plasmid DNA for 12h.

(a) (b)

(a) (b)

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Conclusion

The nanoparticulate heterobimetallic complex has been designed and synthesized with

an aim to develop new efficacious chemotherapeutic agent. By using a combination of

spectroscopic techniques, AFM and TEM imaging techniques, we have validated the

DNA binding and condensation properties of nano complex. DNA binding

experiments reveal that complex [C16H22ON5Cl4CuSn]Cl is an avid DNA binding

agent due to the presence of two metal Cu (II) and Sn (IV) ions which provide a dual

mode of binding at the molecular target site and exhibit novelty due to preferential

selectivity towards DNA. The relatively small size, subtle lipophilicity of the ligand

framework, water solubility and balanced electrostatic interactions play an important

role to induce DNA condensation. The nanoparticulate size of the complex

[C16H22ON5Cl4CuSn]Cl makes it most desirable target –specific chemotherapeutic

drug for cancer inhibition and it warrants evaluation in cell based protocols or in gene

therapy.

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Syntheses:

Synthesis of [C8H14N2Cl2Sn]

The dichlorodimethylbis (imidazole) tin (IV) [C8H14N2Cl2Sn] was synthesized

according to the method reported in the literature [218].

Synthesis of [C16H22ON5Cl4CoSn]Cl

To a methanolic solution of Phenylglycine chloride hydrochloride (5 mM 1.03 g), (5

mM 1.19 g) of CoCl2.6H2O in MeOH was added drop wise. The violet blue colour

solution obtained was left to stir for 3h. An equimolar amount of [C8H14N2Cl2Sn]

dissolved in 5 mL MeOH was added to the stirring solution and refluxed for 8h. The

completion of the reaction was monitored by thin layer chromatography (TLC). The

solution was cooled to room temperature and left for two days, when a violet blue

coloured crystalline product was isolated, washed with CHCl3 and dried in vacuo.

Yield: 62%. mp; 254±3ºC, Anal. Calcd. for [C16H22ON5Cl4CoSn]Cl: C, 29.33; H,

3.38; N, 10.69, Found: C, 29.36; H, 3.41; N, 10.64; Selected IR data (KBr, ν cm-1)

3357 (NH2); 1713 (C=O); 1358 (C-H); 1225 (C-N); 1023 (C-C); 763 (Ar); 463 (Co-

N). Molar Conductance: M (1·10-3 M, DMSO): 89.00 -1cm2 mol-1 (1:1 electrolyte).

UV-vis (1X10-3 M, DMSO max, nm) 257 nm, 296 nm , 325 nm, 656 nm. ESI-MS

(m/z+) 655 [C16H22ON5Cl4CoSn]Cl

Synthesis of [C16H22ON5Cl4ZnSn]Cl The complex [C16H22ON5Cl4ZnSn]Cl was synthesized using anhydrous ZnCl2 (1.36g,

10 mM) by a similar method as described for [C16H22ON5Cl4CoSn]Cl. The white

coloured product obtained was filtered, washed with CHCl3 and dried in vacuo. Yield:

72%; mp; 259±2 ºC. Anal. Calcd. for [C16H22ON5Cl4ZnSn]Cl C, 29.04; H, 3.35; N,

10.58. Found: C, 29.11; H, 3.39; N, 10.61. Selected IR data (KBr, ν cm-1) 3392

(NH2); 1707 (C=O); 1381 (C-H); 1256 (C-N); 1013 (C-C);761 (Ar); 496 (Zn-N); 1H

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NMR (400 MHz, DMSO-d6, 25 ºC, δ) 2.58 (-CH3); 3.63 (-CH); 6.45, 7.93 (phenyl,

imidazole ring); 8.05, 8.21 (imidazole NH, NH2); 13C NMR (100 MHz DMSO- d6, 25

ºC, δ) 45.82-71.13 (CH3, CH2); 122.12-139.33(Ar); 159.23 (C=O); 119Sn NMR (400

MHz DMSO- d6, 25 ºC, δ) -560.23; Molar Conductance, M (1X10-3 M, DMSO):

114.00 -1cm2 mol-1 (1:1 electrolyte) UV-vis (1·10-3 M, DMSO, max /nm) 258 nm,

325 nm. ESI-MS (m/z+) 661 [C16H22ON5Cl4ZnSn]Cl

Results and discussion

The complexes [C16H22ON5Cl4CoSn]Cl and [C16H22ON5Cl4ZnSn]Cl were synthesized

according to the procedure described in the literature [219], by the reaction of

dichlorodimethyl bis(imidazole ) tin (IV) with the corresponding 1:1 metal complex

of phenylglycine chloride hydrochloride as shown in scheme 4. Scheme 4:

Step 1

Step 2

Scheme 4. Schematic representation of the complexes [C16H22ON5Cl4CoSn]Cl and [C16H22ON5Cl4ZnSn]Cl

N

NH

N

NH

SnH3C

ClClCH3

N

NH

CH2Cl2+Stirring

SnClCl

H3C CH3

(1)

2

N

HN

Co

H2NC C Cl

O

H

N

NH

SnH3CClCl

CH3H2NC C Cl

O

H CoCl2.6H2O+MeOH

+ 1 Reflux for 5h

Cl .Cl

77

Complex [C16H22ON5Cl4ZnSn]Cl was synthesized only for the NMR structure

elucidation, and the structure was determined by employing various spectroscopic

techniques such as elemental analysis, molar conductivity, UV-vis., IR, ESI-MS and

multinuclear NMR spectroscopy. The mass spectrometric and elemental analyses

were consistent with proposed molecular formulae of the complexes. Molar

conductance measurements suggest that complexes [C16H22ON5Cl4CoSn]Cl and

[C16H22ON5Cl4ZnSn]Cl behave as 1:1 electrolytes. The complexes are soluble in

MeOH, DMSO and DMF. The nano-sized dimension of the heterobimetallic complex

[C16H22ON5Cl4CoSn]Cl is supported by AFM, TEM and XRD measurements. To

validate specific DNA binding of the designed molecule various spectroscopic studies

were employed and binding parameters were calculated.

Infrared spectroscopy

The IR spectrum of the free phenylglycine chloride hydrochloride exhibits

characteristic bands of the amine (-NH2) at 3400 cm-1. However, on complexation

υ(N-H) shifts towards lower wave number, Complexes [C16H22ON5Cl4CoSn]Cl and

[C16H22ON5Cl4ZnSn]Cl exhibit the broad band at 3341 cm-1 –3342 cm-1 due to the

stretching vibrations of the -NH2 and NH groups of the phenylglycine chloride and

imidazole ligands. The frequency shift of the (-NH2) towards lower region in

comparison to that observed for free phenylglycine chloride ligand (3400 cm-1)

confirms its coordination to the metal ion [189]. The involvement of C=O in metal

complexes is evident from the shift in (C=O) stretch which appears at 1713 and

1707 cm-1 in comparison to free uncomplexed ligand band at 1765 cm-1. Other ligand

skeletal bands observed in the range 689–848cm-1, 1129-1179 cm-1, 1074 cm-1, were

ascribed to the out of plane –CH bending of aromatic rings, -CH3, and –CH2 groups,

respectively. Bands appearing near 2900 cm-1 were attributed to stretching vibrations

78

of the -CH3 group [190b,220]. The IR spectra of [C16H22ON5Cl4CoSn]Cl and

[C16H22ON5Cl4ZnSn]Cl reveal (M-N) and (M-O) stretching vibrations in the range

430-450 and 535-580 cm-1, respectively.

Nuclear magnetic Resonance spectroscopy

1H, 13C, 119Sn NMR was used for the characterization of the diamagnetic complexes

[C16H22ON5Cl4ZnSn]Cl. The 1H NMR and 13C NMR, and 119Sn NMR spectra show

aliphatic and aromatic signals with chemical shifts in accordance with the proposed

structure. The 1H NMR spectrum of complex [C16H22ON5Cl4ZnSn]Cl exhibits

multiplets at 7.36–7.62 ppm and 7.40- 8.75 ppm consistent with the presence of the

imidazole and the aromatic ring of amino acid, respectively [212]. A sharp doublet at

3.12 ppm indicated the proton attached to the chiral carbon next to the benzene ring

and the proton attached to the methyl group of tin atom gives a singlet at 1.01–1.23

ppm as shown in figure 50[221, 222].

Figure 50. 1H NMR of the complex [C16H22ON5Cl4ZnSn]Cl.

The 13C NMR spectrum reveals distinct signals in the region 50.82, 54.02, 77.11 ppm

representing the -CH and -CH3 carbons [223]. The aromatic ring carbon signals were

79

observed at 129.59-137.23 ppm [224]. The peak at 163.34 ppm corresponded to the

carbonyl carbon. 119Sn NMR spectrum recorded a sharp peak at -560.23 ppm in

agreement with the hexa-coordinated environment of tin atom as shown in figure

51[225].

Figure 51. 119Sn NMR of the complex [C16H22ON5Cl4ZnSn]Cl.

Mass spectral analysis

The complexes [C16H22ON5Cl4CoSn]Cl and [C16H22ON5Cl4ZnSn]Cl have been

unambiguously characterized through mass spectral analysis. The ESI mass spectrum

of complex [C16H22ON5Cl5CoSn], exhibits the molecular ion peak m/z at 655 which

was assigned to [C16H22ON5Cl4CoSn]Cl. The complex [C16H22ON5Cl4CoSn]Cl,

showed the prominent peaks m/z at 403 with a relative abundance of 60% which was

assigned to [C14H16ON5Cl2Co-3H+]. The fragmentation peaks obtained at m/z 266,

205 which was obtained by the successive expulsion of imidazole and cobalt metal

ion, respectively. The relatively 60% abundant peak m/z at 161 corresponding to

isotopic peak of free phenylglycine chloride was observed. Similar pattern was

obtained for the complex [C16H22ON5Cl4ZnSn]Cl which exhibits the molecular ion

peak m/z at 661.

80

XRD Measurements

XRD measurement of the powered sample of complex [C16H22ON5Cl4CoSn]Cl was

employed to determine the crystalline nature of the complex [C16H22ON5Cl4CoSn]Cl.

The XRD pattern obtained for the metal complex [C16H22ON5Cl4CoSn]Cl shows well

defined crystalline peaks which evidenced for the inherent crystalline nature of the

metal complex. Figure 52 shows the XRD pattern of the complex

[C16H22ON5Cl4CoSn]Cl with peaks at 2θ scattering angles of 23.23, 24.84, 34.23,

42.86, 48.29, 63.86 and 78.46 assigned to (100) (100), (002), (102), (111) and (202)

and (203) crystal planes respectively, characteristic of well ordered tetragonal

arrangement of tin atoms [203]. The

lattice parameters are a = 4.230 and c = 5.187, which are in good agreement with

known lattice parameters a = 4.109 and c = 5.180 indicating that the nano particles

have the same crystal structure as that of the bulk [204]. The average size of the

Figure 52. The XRD pattern nanoparticles of complex [C16H22ON5Cl4CoSn]Cl showing peaks at different scattered angles. powder sample of complex [C16H22ON5Cl4CoSn]Cl was calculated to be 15-20 nm

according to half width of (002) diffraction peaks and the size of the DNA condensed

particles was found in the range of 60-80 nm according to half width of (002) using

81

Debye Sherrer formula [203].A summary of the refined XRD consisting of unit cell

parameters are given in table 2 .

Table 2. Summary of the XRD data and the refinement parameters for compound [C16H22ON5Cl4CoSn]Cl.

Electronic absorption spectra

The electronic spectra of the complexes [C16H22ON5Cl4CoSn]Cl and

[C16H22ON5Cl4ZnSn]Cl were recorded in DMSO at room temperature. In the UV

region both complexes [C16H22ON5Cl4CoSn]Cl and [C16H22ON5Cl4ZnSn]Cl exhibit

intense absorption band at 256-258 nm and a shoulder at 325-329 nm attributed to

S.No

Compound

2

1. Formula

C16H22ON5Cl5CoSn

2. Molecular weight(gmol-1/)

655

3. Temperature(K)

527

4. Crystal System

Primitive

5. Space Group

P-6

6. Method Micro crystalline

7. Cell Parameters

a = 4.230 b = 4.230 c = 5.187 α = 90 β = 90 γ = 120

8. Z 1

9. Radiation

Cu-Kα

10. 2θ min-max 23.23-78.46

82

π–π* [199, 226] or charge transfer transitions of the aromatic chromophore. In the

visible region complex [C16H22ON5Cl4CoSn]Cl display a broad band at 656 nm,

which is consistent with square pyramidal geometry around Co (II) metal ions [227].

DNA binding studies

Absorption titration studies

The interaction of the metal complex to DNA is often characterized through

absorption spectral titration followed by the changes in the absorbance and shift in the

wavelength. The absorption spectral traces of the Co-Sn heterobimetallic complex

[C16H22ON5Cl4CoSn]Cl in the absence and presence of CT DNA are shown in figure

53. With increasing concentration of CT DNA (0-0.4 X 10-4 M), the absorption bands

of [C16H22ON5Cl4CoSn]Cl were affected, exhibiting hyperchromism (24%) and red

shift, respectively in absorption intensities. “Hyperchromic effect” arises mainly due

to the presence of positively charged transition metal ion and Sn (IV) which bind to

DNA via non-covalent interaction [228]. A strong hyperchromic effect with a

significant red shift in -* transition was observed for [C16H22ON5Cl4CoSn]Cl

suggesting that, this complex possess a higher propensity for DNA binding These

changes are typical of complexes bound to DNA through noncovalent interaction [208].

Since numerous naturally and man- made compounds contained Cobalt at two common

oxidation states Co(II) and Co(III). There is growing interest in investigating cobalt and

other transition metal complexes for their interaction with DNA [229]. This may be partly

influenced by the results of extensive investigation into two areas of research, viz. (i) the

binding specificity of small organic molecules for their possible modulation and

inhibition of DNA replication, transcription and recombination, and (ii) anticancer,

antiviral and antibacterial drugs [230]. The DNA specific interactions of complex

[C16H22ON5Cl4CoSn]Cl shows better prospects as an antitumor agent due the

presence of two different metal centers having different specificity at the molecular

83

level. The hard Lewis acid Sn (IV) atom binds electrostatically to the phosphate

backbone of DNA helix, while the Co+2 ion may preferentially coordinate with the N3

position of guanine either due to the replacement of the labile chloride ligand or by

direct coordination. Base binding of the complex can perturb the hydrogen bonding

between the base pairs causing destabilization of DNA.

Figure 53. Absorption spectral traces of complex [C16H22ON5Cl4CoSn]Cl in Tris-HCl buffer upon addition of CT-DNA. Inset: plot of [DNA]/εa-εf vs [DNA] for the titration of CT-DNA with complex [C16H22ON5Cl4CoSn]Cl. [Complex] = 0.067 X 10-4 M, [DNA]= 0.067 X10-4- 0.466 x10-4 M; (▲) experimental data points; full lines, linear fitting of the data.

To assess the binding ability of the complex [C16H22ON5Cl4CoSn]Cl with CT DNA,

the intrinsic binding constant was determined using equation I [170,171]. The binding

constant Kb value was found to be 5.14 x104 M-1. The significant binding constant Kb

value reflects the average of the dual binding mode of complex

[C16H22ON5Cl4CoSn]Cl to DNA.

[DNA] / |a-f| = [DNA] / |b-f| + 1/ Kb |b-f | (1)

To investigate the specific recognition of complex [C16H22ON5Cl4CoSn]Cl with the

nucleobases/ phosphate sugar backbone of DNA helix, we have carried out binding

studies of [C16H22ON5Cl4CoSn]Cl with 5'-GMP by UV-vis absorption titrations. On

84

addition of increasing amount of 5'-GMP, [C16H22ON5Cl4CoSn]Cl there was a sharp

increase in absorption intensity ‘hyperchromism’ and small blue shift of 2 nm (Figure

54).

Hyperchromic effect is the result of damage caused to the secondary structure of

DNA

Figure 54. Variation of UV-vis absorption for complex [C16H22ON5Cl4CoSn]Cl with increase in the concentration of 5′ GMP (0.067 X10-4 -0.466 X10-4 M) in buffer (5mM Tris–HCl/50 mM NaCl, pH= 7.2) at room temperature. Inset: plot of [5′ GMP]/(εa- εf) vs [DNA] for the titration of 5′ GMP.(■), experimental data points; full linear, linear fit of the data.[complex]= 0.33X10-6. through phosphate backbone interaction while small blue shift is attributed to the

coordinative interaction to N7 of 5'-GMP. This is an inherent characteristic feature of

transition metals.

Fluorescence spectral studies

The interaction of complex [C16H22ON5Cl4CoSn]Cl with CT DNA was also

investigated by using emission spectroscopy. Complex [C16H22ON5Cl4CoSn]Cl emits

luminescence with maximum at 344 nm when excited at 263 nm in 0.01 mM Tris–

HCl/50 mM NaCl buffer at ambient temperature. Upon the addition of CT DNA the

intensity of the emission band at 344 nm enhances gradually, which indicates that the

complex could interact with DNA (Figure 55). As DNA is a highly organized

macromolecular complex and its double helix provides a unique coat: core

85

environment comprising hydrophilic backbone of ribose phosphate around the

hydrophobic core of stacked bases [210, 211].

Figure 55. Emission spectra of the complex[C16H22ON5Cl4CoSn]Cl in Tris-HCl buffer upon addition of CT DNA. Arrow shows intensity change upon increasing concentration of DNA. [Complex] = 0.06 x10-4 M, [DNA] = 0―0.466 x 10-4 M. The enhancement of the emission intensity is mainly due to the change in the

environment of the metal complex and is related to the extent to which the complex is

inserted into the hydrophobic environment inside the DNA helix [231]. Since it is

found that the complexes with increased hydrophobicities exhibit greater increase in

emission intensities upon binding to polyectrolytes like DNA, It is therefore

concluded that the emission intensity increases due to the interaction between the

complex [C16H22ON5Cl4CoSn]Cl and DNA which is due the hydrophobicity of both

the complex [C16H22ON5Cl4CoSn]Cl and the DNA. An additional ionic strength

experiment showed the electrostatic mode of binding.

From a plot of r/cf versus r the binding constant for complex [C16H22ON5Cl4CoSn]Cl

was calculated to be 2.62 x 104 M-1 which is in well agreement with the binding

constant as determined by UV-vis titrations.

86

To evaluate the interacting strength of complex [C16H22ON5Cl4CoSn]Cl emission

quenching experiments using [Fe(CN)6]4- as quencher were also performed. In the

absence of DNA, emission intensity of the complex [C16H22ON5Cl4CoSn]Cl were

Figure 56 . Emission quenching curve of complex [C16H22ON5Cl4CoSn]Cl in absence and presence of DNA

efficiently quenched by [Fe(CN)6]4-. The plots of the complexes

[C16H22ON5Cl4CoSn]Cl gave the value of Ksv = 4.23 X 104 M-1 .In presence of DNA,

the slope was remarkably decreased to 1.92 X 104 for the complex

[C16H22ON5Cl4CoSn]Cl as shown in figure 56 . The greater decrease of the Ksv value

for the complex [C16H22ON5Cl4CoSn]Cl indicates the higher DNA binding propensity

of [C16H22ON5Cl4CoSn]Cl. These results are consistent with the electronic absorption

titration.

Effect of ionic strength

To determine the efficient and distinguishable binding modes between DNA and

small molecules, ionic strength is an important parameter. The addition of cation

weakens the surface binding interactions due to a competition for phosphate anion,

viz electrostatic binding and hydrogen bonding between the CT DNA and the

interacting molecules [232, 233]. The effect of ionic strength on complex –DNA

binding of [C16H22ON5Cl4CoSn]Cl was studied and the results so obtained revealed

87

the strong dependence of fluorescence intensity on ionic strength. Moderate

fluorescence quenching was observed in complex-DNA system for both the

complexes [C16H22ON5Cl4CoSn]Cl. The results indicate that the interaction between

complexes and DNA is predominantly electrostatic viz. DNA phosphate backbone.

Cyclic Voltammetry

The application of cyclic voltammetry to the study of metal complex-DNA interaction

provides a useful complement to the previously used methods of investigations, such

as UV-visible spectroscopy. Equilibrium constant (Kb) for the interaction of the metal

complexes with DNA can be obtained from the shifts in peak potentials, the number

of base pair sites involved in binding via intercalative, electrostatic or hydrophobic

interactions and from the dependence of the currents passed during oxidation or

reduction of the bound species on the amount of the added DNA [234]. The cyclic

voltammogram of complex [C16H22ON5Cl4CoSn]Cl was recorded in DMSO at the

scan rate of 0.3 Vs-1over the potential range of 2.0 — -0.8V. The cyclic voltammetric

response of complex [C16H22ON5Cl5CoSn] in absence and in presence of CT DNA is

depicted in figure 57.

Figure 57. Cyclic voltammogram (scan rate 0.3 Vs-1, DMSO, 25°C) of (a) complex [C16H22ON5Cl4CoSn]Cl (b) complex [C16H22ON5Cl4CoSn]Cl in presence of CT DNA.

a b

88

The cyclic voltammogram of [C16H22ON5Cl4CoSn]Cl in the absence of DNA reveal a

non- Nerstian but fairly reversible/quasireversible one electron redox process

involving Co3+/Co2+ couple as observed from peak potential separation of 0.149V

(0.59V for a one electron transfer process). On addition of CT DNA to the complex

[C16H22ON5Cl4CoSn]Cl, there was a significant shift in formal electrode potential E1/2

0.119mV for the complex. In addition to changes in formal potential, voltammetric

current Ipa / Ipc decreases from 0.89 to 0.71 and separation of peak potential ΔEp also

decreases from -0.313 to -0.334.

Furthermore, the significant shift in the electrode potentials and peak current ratios on

addition of CT DNA can be explained in terms of the diffusion of an equilibrium

mixture of free and DNA bound metal complexes to the electrode surface [208] thus

implying a strong binding of [C16H22ON5Cl4CoSn]Cl with CT DNA

DNA Cleavage activity

Upon gel electrophoresis of the reaction mixture, a concentration dependent DNA

cleavage was observed. A significant conversion of supercoiled pBR322 DNA to

Form II and Form III, was observed with increase in concentration of complex

[C16H22ON5Cl4CoSn]Cl (Figure 58). The most impressive cleavage feature observed

for complex [C16H22ON5Cl4CoSn]Cl is that, Form III DNA appears before the

disappearance of Form I DNA (lanes, 3, 4, 5 and 6). This phenomenon indicates that

the complex [C16H22ON5Cl4CoSn]Cl is capable of performing direct double-strand

scission [235], while many cobalt complexes are only able to cleave single strand

successively. The complex [C16H22ON5Cl4CoSn]Cl shows discernible DNA cleavage

with the increase in concentration, more intensified nicked (Form II) while linear

(Form III) was observed in traces. Minor groove binding agent (DAPI) [236] and

major groove binding agent (Methyl Green) [237] were used to probe the potential

89

interacting sites of complex [C16H22ON5Cl5CoSn] with pBR322 DNA. Figure 59

demonstrates that Co (II) complex [C16H22ON5Cl5CoSn] inhibits the methyl green

(Lane 9) as well as DAPI (Lane 10) of the DNA digestion, In these circumstances,

complex [C16H22ON5Cl5CoSn] may bind to both major as well as minor groove .

Figure 58 . Gel Electrophoresis diagram showing cleavage of pBR322 supercoiled DNA (300 ng) by complex [C16H22ON5Cl4CoSn]Cl ; Lane 1: DNA; Lane 2: [C16H22ON5Cl4CoSn]Cl 5 µM +DNA; Lane 3: [C16H22ON5Cl4CoSn]Cl 10µM + DNA; Lane 4: [C16H22ON5Cl4CoSn]Cl 15µM + DNA; Lane 5: [C16H22ON5Cl4CoSn]Cl 20µM + DNA; Lane 6: [C16H22ON5Cl4CoSn]Cl 25 µM + DNA; Lane 7: [C16H22ON5Cl4CoSn]Cl 30 µM + DNA; Lane 8: [C16H22ON5Cl4CoSn]Cl 35 µM + DNA

DNA cleavage in presence of activators

The nuclease efficiency of metal complexes is mainly dependent on activators [238].

Thus, further activity of metal complex [C16H22ON5Cl4CoSn]Cl has been done with

different activators viz; H2O2, ascorbate (Asc), 3-mercaptopropionic acid (MPA) and

glutathione (GSH). As shown in figure 59, the cleavage activity of Co (II) complexes

[C16H22ON5Cl4CoSn]Cl was significantly enhanced in presence of these activators.

[239]. However, in the presence of H2O2, the DNA bands of [C16H22ON5Cl4CoSn]Cl

completely diminished. The activating efficacy of [C16H22ON5Cl5CoSn] follows the

order H2O2>MPA>Asc≈GSH.

Form II Form III Form I

1 2 3 4 5 6 7 8 9

90

Figure 59. Gel Electrophoresis diagram showing cleavage of pBR322 supercoiled DNA (300 ng) by complex [C16H22ON5Cl4CoSn]Cl in presence of different activators and groove binders: Lane1: DNA; Lane 2: [C16H22ON5Cl4CoSn]Cl 35µM + DNA + EtOH; Lane 3: [C16H22ON5Cl4CoSn]Cl 35µM + DNA+ 5% DMSO; Lane 4: [C16H22ON5Cl4CoSn]Cl35 µM + DNA+(0.4mM) GSH; Lane 5: [C16H22ON5Cl4CoSn]Cl 35µM + DNA+(0.4mM) MPA; Lane 6: [C16H22ON5Cl4CoSn]Cl 35µM + DNA+(0.4mM) H2O2; Lane 7: [C16H22ON5Cl4CoSn]Cl 35µM + DNA +(0.4mM) NaN3; Lane 8: [C16H22ON5Cl4CoSn]Cl 35µM + DNA +(15unit) SOD; Lane 9: [C16H22ON5Cl4CoSn]Cl 35µM + DNA +(4 µM) DAPI ; Lane 10: [C16H22ON5Cl4CoSn]Cl 35µM + DNA+(1 µl of 0.01mg/ml) methyl green; Lane 11: [C16H22ON5Cl5CoSn] 35µM + DNA +(0.4mM) Ascorbate

DNA cleavage in presence of reactive oxygen species

Reactive oxygen species generated during the interaction between metal complexes

and dioxygen or redox reagents are believed to be a major cause of DNA damage

To probe the potential mechanism of DNA cleavage mediated by complex, some

standard radical scavengers were used prior to the addition of metal complexes to

DNA solution. On adding tert-butyl alcohol (hydroxyl radical scavenger) [240], to

[C16H22ON5Cl4CoSn]Cl, DNA cleavage is inhibited suggesting the possibility of

hydroxyl radical as one of the reactive species (figure 59, lane 2). Thus, free radicals

participate in the oxidation of the deoxyribose moiety, followed by hydrolytic

cleavage of the sugar phosphate back bone in the absence of the scavengers. On the

other hand, addition of NaN3 as 1O2 scavenger (figure 59, lane 7) [96], also reduced

the same extent of cleavage as in case of tert-butyl alcohol; reveal that 1O2 may also

be activated oxygen intermediate responsible for the cleavage. Addition of SOD, as a

superoxide anion radical (O2•‾) scavenger [104], to the reaction mixture shows no

significant quenching of the cleavage revealing that superoxide anion is not the active

Form II Form III Form I

1 2 3 4 5 6 7 8 9 10 11

91

species. These results suggest that in the DNA strand scission reaction caused by the

complex various diffusible oxygen intermediate species (hydroxyl radical or singlet

oxygen) are probably involved. Since the complex is able to cleave DNA in the

absence of any reducing agent, it may be assume that DNA might be cleaved by a

discernible hydrolytic path.

DNA Condensation

DNA condensation is an essential process to transport a therapeutic gene to its target

[241]. It includes the collapse of extended DNA chain into compact, orderly particles

containing one or more molecule. Many biologically relevant divalent metal ions are

capable of DNA condensation and play a critical role in controlling the compact state

of genomic nucleic acid. Polyamines are also required in DNA catenation

(interlocking) by topoisomerases, presumably for local condensing neighbouring

DNA segments [242]. Many small molecules (such as drugs, ligand etc) are thus able

to recognize and bind to single or double stranded DNA with high affinity and

selectivity which could induce DNA structural alterations. Several metal based drugs

affect a number of physiological and biochemical regulatory functions in humans and

have been reported to be able to bind with DNA and other nucleotides.

Complex [C16H22ON5Cl4CoSn]Cl has facilitates the DNA condensation due to

neutralization of negative charge of the DNA base pairs by the positive charge of the

cationic complex. In this study, DNA condensation to the nano particulate structure

by complex [C16H22ON5Cl4CoSn]Cl under neutral condition in aqueous medium

(Tris-HCl buffer/50mM NaCl pH 7.2) was achieved by evaporating an equimolar

mixture of complex [C16H22ON5Cl4CoSn]Cl and CT DNA which have the tendency to

bind and compact DNA more efficiently at a very low concentration. The increase in

the size of the complex nano particles after CT DNA condensation was determined by

92

employing various visualization techniques viz. Transmission electron microscopy

(TEM) and atomic force microscopy (AFM).

TEM and AFM imaging of Complex-DNA condensates

Transmission electron microscopy (TEM) and atomic force microscopy (AFM) are

useful technique for biological and chemical researches. These techniques have been

used here to analyze the morphology of the complex and complex-DNA condensates.

The TEM images of the complex nanoparticles are given in figure 60.

Figure 60. (a-b) TEM image of the nano particles of complex [C16H22ON5Cl4CoSn]Cl. The shape of the particles is irregular and the particles are of variable diameter

ranging from 15-22 nm. The particles size increases nearly 3-4 times when CT DNA

condensed to it indicating that the complex nanopatrticles can condense free CT DNA

(existing as loose strands) to compact solid DNA drug condensate as shown in figure

61 (a-d). Several numbers of drugs are unable to be effective due to the lack of the

information of their structural and morphological details.

b a

93

Figure 61(a-d) TEM image indicating the condensation of CT DNA on nano particles of complex [C16H22ON5Cl5CoSn] at different time intervals To obtain the morphological and structural information about the free

[C16H22ON5Cl4CoSn]Cl and the condensates, tapping mode AFM (atomic force

microscopy) experiments were performed using commercially etched silicon tips as

AFM probes with typical resonance frequency of 300 Hz (RTESP Veeco). Two and

three dimensional AFM images of the complex [C16H22ON5Cl4CoSn]Cl and the

condensate materials are shown in figure 62. The change in the morphological

structure in complex [C16H22ON5Cl4CoSn]Cl and its condensates with CT DNA

clearly validating that the complex is facilitating the DNA condensation, as the width

and the vertical height of the peaks increases. As the complex nanoparticles are of

different diameters and irregular in size and after condensation they appear to be

regular, very close to spherical as shown in figure 63, these results are indicative of

good DNA condensing ability of the complex.

94

Figure 62 (a-d). Two and Three dimensional AFM image of the nano particles of complex [C16H22ON5Cl4CoSn]Cl at different time interval with variable scales

a b

c d

95

Figure 63(a-d). Two and Three dimensional AFM image of the nano particles of complex –DNA condensates at different time interval with variable scales.

a b

c d

96

Molecular Docking

In order to understand further the binding modes obtained from spectroscopic studies,

rigid molecular docking experiment were performed by placing a small molecule into

the binding site of the target macromolecule mainly through non–covalent

interactions. Literature reveals that the forces maintaining the stability of DNA–

intercalator complex include van der Waals, hydrogen bonding, hydrophobic charge

transfer and electrostatic interactions [243]. A critical issue in docking operations

includes the prediction of correct binding pose and the accurate estimation of the

corresponding binding affinity. Despite the enormous size of the conformational

space for a given moiety, current docking methodologies have been successfully

employed by our group in reproducing putative binding modes. The metal complex

was restrained, by applying five dihedral and twenty-one distance restraints, to

disallow distortion. The initial docked structures were subjected to energy

minimization of -229.6 KJ / mol and examined for hydrogen bond interactions

between the metal complexes and the DNA.

The molecular modeling results so obtained indicate the change of total potential

energies of the process of complex [C16H22ON5Cl4CoSn]Cl docking between DNA

base pairs from major and minor groove as shown in figure 64. The total potential

energies of complex–DNA-binding system after intercalation are much less than those

of the system before intercalation, which may be associate with electrostatic

interaction and space matching. Furthermore, total potential energy of complex–

DNA-binding system formed by complex intercalating into C5G6 region from minor

groove is less than those of other systems, illustrating that the minor groove binding

of the complex in C5G6 region is the most preferential binding modeling of the

interactions between the complex and DNA base pairs [244].

97

Figure 64. The binding mode between the complex [C16H22ON5Cl4CoSn]Cl and B-DNA dodecamer d(CGCGAATTCGCG)2 (PDB ID: 1BNA) in minor groove.

Minor Groove

Complex 2

Major Groove

98

Conclusion

The in vitro DNA binding studies of novel heterobimetallic complex

[C16H22ON5Cl4CoSn]Cl reveal an electrostatic mode of binding as well as major /

minor groove binding. The complex [C16H22ON5Cl4CoSn]Cl exhibited an outstanding

ability to affect DNA double strand scission in oxidative as well as free hydroxyl

radical manner. Our spectroscopic results demonstrate that complex

[C16H22ON5Cl4CoSn]Cl is an avid DNA binder which is well corroborated with the

literature reports. The validation of DNA binding studies and DNA condensation was

carried out by using visualization techniques. Results from these visualization

techniques validate the size of the complex which leads it to be most specific target

drug for cancer inhibition.