nickel(ii) complexes of 3,4-bis(2-pyridylmethylthio...

CHAPTER-VI

Nickel(II) complexes of

3,4-bis(2-pyridylmethylthio)-

toluene and halide/pseudohalides:

synthesis, crystal structure,

antibacterial activity, DNA and

BSA binding study

Nickel(II) complexes of 3,4-bis(2-pyridylmethylthio)toluene ........ DNA and BSA binding study Chapter -VI

151

CHAPTER-VI

Nickel(II) complexes

of 3,4-bis(2-pyridylmethylthio)toluene and

halide/pseudohalides: synthesis, crystal structure,

antibacterial activity, DNA and BSA binding study

ABSTRACT

A series of hexacoordinated nickel (II) complexes formulated as [Ni(L3)Cl2] (1) and

pseudohalides (X), formulated as [NiII(L3)X2] (where X = azide (2a), cyanate (2b) and

isothiocyanate (2c)) with a N2S2 donor ligand (L3 = 3,4-bis(2-

pyridylmethylthio)toluene) has been synthesized and isolated in pure form. The

complexes were characterized by physico-chemical and spectroscopic methods, 1, 2a

and 2b also by single crystal X-ray diffraction analyses. The structural study shows the

nickel ion in a distorted octahedral geometry that comprises the tetradentate N2S2 ligand

ring and pseudohalides in cis positions. Biological activity of complexes towards calf

thymus DNA and bovine serum albumin (BSA) has been examined with the help of

absorption and fluorescence spectroscopy tools. Antibacterial activity of complexes has

also been studied by agar disc diffusion method against some species of pathogenic

bacteria (Escherichia coli, Vibrio cholerae, Streptococcus pneumonia, Shigella specis

and Bacillus cereus).


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VI.1. Introduction The bioinorganic chemistry of nickel metalloenzymes continues to attract

considerable interest. Recent advances include the synthesis of geometrically distorted

nickel(II) complexes with ligands having mixed N and S donor set. The interest is due

to the fact that nickel is present in the active sites of several important classes of

metalloproteins, either as homo or hetero-binuclear species. It is still a challenge task

the prediction of the binding preference of polydentate ligands, especially when mixed

donor atom species are involved. In particular, complexes of nickel ion with nitrogen

based heterocyclic ligands have been widely investigated owing to their potential

applications as functional solid materials, photosensitization reactions [1], bioinorganic

chemistry [2], medicine [3], catalysis [4] and great variety of biological activities as

antimalarial, antibacterial, antitumoral, antiviral activities etc.[5, 6] which have often

been related to the chelating ability of ligands towards trace metal ions.

In this chapter, an account of coordination chemistry and the reactivity of nickel(II)

complexes of a ligand (L3 = 3,4-bis(2-pyridylmethylthio)toluene) having N2S2 donor

sites and chloride or different pseudohalide-anions will be illustrated. The detailed solid

state structures of three nickel(II) complexes, viz. [Ni(L3)Cl2] (1), [Ni(L3)(N3)2] (2a)

and [Ni(L3)(NCO)2] (2b), have been established by single crystal X-ray crystallography.

Binding study of 1 with calf thymus DNA and BSA is also described. Antibacterial

activity of complexes studied by agar disc diffusion method showed the comparable

inhibition activity of the nickel(II) complexes against some pathogenic bacteria namely

Escherichia coli, Vibrio cholerae, Streptococcus pneumonia, Shigella specis and

Bacillus cereus.

VI.2. Results and Discussion VI.2.1. Ligand and Complexes

The ligand, [3,4-bis(2-pyridylmethylthio)toluene] (L3) was synthesized by

refluxing the reaction mixture of toluene-3,4-dithiol with 2-picolyl chloride in ethanol

medium in presence of sodium ethoxide The organic product was extracted into

dichloromethane and the compound was obtained as yellow viscous liquid by

evaporating the solvent. The complex (1) was obtained from the reaction of the

nickel(II) chloride with equimolar amount of organic moiety in the methanol medium


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and other complexes (2a-2c) were obtained in good yield from the reaction of nickel(II)

acetate, organic ligand in the methanol medium followed by the addition of respective

aqueous solution of pseudohalides to the reaction mixture viz. Scheme VI.1. The

organic moiety (L3) acts as a tetradentate neutral ligand with four N2S2 donor centers in

the complexes, 1, (2a-2c).

Scheme VI.1. Ligand structure and synthetic route of the complexes

In these complexes (1, 2a-2c), the ligand (L3) acts as a tetradentate neutral ligand

with four N2S2 donor centers with central nickel(II) ion. Microanalytical data (Table VI.1)

and Nickel analyses have been used to establish the composition of these complexes.

All the complexes are soluble in common organic solvents and conductivity

measurement of the complexes in dimethyl sulphoxide showed conductance values in

range of 44-50 (Λo, ohm-1 cm2 mol-1) which suggests that all complexes are non-

electrolytes. The magnetic moments (µ) at room temperature of these complexes are

3.10, 3.06, 3.08 and 3.09 which indicate all complexes are high spin distorted octahedral

geometry.

SH

SH

N

Cl

NaOEt / EtOH

Reflux

S S

N N

L3

NiCl2

MeOH/ Reflux/ 3h[Ni(L3)Cl2]

Ni(OAC)2, 4H2O MeOH/Reflux/3h

MIxtureN3

-

NCO-

SCN -[Ni(L3)(N3)2]

Ni(L3)(NCO)2]

[Ni(L3)(SCN)2]

1

2a2c

2b

L3


154

Table VI.1. Microanalyticala and physicochemical data for the Ni(II) complexes

Compds.

Elemental analysesa % Magnetic momentb , B.M.

Conductance c

Λo, mho. mol-1cm-1 C H N Ni

1 48.75

(48.78)

3.84

(3.86)

5.98

(5.94)

12.53

(12.56)

3.06 46

2a 47.44

(47.42)

3.74

(3.72)

23.30

(23.28)

12.19

(12.22)

3.10 44

2b

52.43 (52.45)

3.74 (3.76)

11.65 (11.63)

12.19 (12.23)

3.08

48

2c

49.21

(49.19)

3.51

(3.54)

10.99

(10.96)

11.32

(11.34)

3.06 50

aCalculated values are in parenthesis; bat room temperature; cin DMSO

VI.2.2. Structural studies

The molecular structure of complexes 1, 2a and 2b with the atom numbering

scheme are shown in Figs.VI.1 respectively, Crystal data and details of refinement data

for complexes given in Table VI.2. While the selected bond lengths and bond angles

appear in Table VI.3. The X-ray crystal structure analyses of compounds 1, 2a and 2b

confirm the formation of neutral complexes where the nickel(II) ion is coordinated by

the tetradentate donor organic moiety L3 by means of the N2S2 donors. The ligand

embraces the metal in such a way that pyridine rings are in trans positions, while the

two thioether-S donors are located in the equatorial plane beside two chloride anions (in

complex 1), azide (in 2a) andcyanate( in 2b) which are at mutual cis positions, and

complete the distorted octahedral coordination sphere around metal ion. In these

complexes the Ni-N(py) bond distances (range 2.113(5)-2.144(3) Å) are slightly longer

than the Ni-N(pseudohalide) ones that vary from 2.097(5) to 2.109(5) Å, while the Ni-

S(1) bond lengths in complex 1 is higher than pseudohalides between 2.4174(19)-

2.3951(14)Å but Ni-S(2) bond length in NiCl2 complexe is lower than pseudohalides

complexes vary from 2.3887(18)- 2.415(2).Coordination bond angles slightly deviated

from ideal octahedral geometry, the more evident are the N(1)-Ni-N(2) angles of


155

164.18(19)° and 165.27(14)° in 1 and 2a, respectively that confirm the strain of the

ligand upon coordination.

1 2a

2b

Fig.VI.1. ORTEP diagram of complex 1, 2a and 2b with labeling scheme VI.2.3.Spectral studies

V.2.3.1. IR Spectra

The infrared spectral data of all the complexes represented in (Table VI.4) exhibit

characteristic strong to medium intensity band in the region of 1468-1472 cm-1 and 758-

761 cm-1, which are assigned to C=N stretching and C-S respectively. An intense band

centred at 2047 cm-1 assignable to N3 (2a), 2183 cm-1

assignable to NCO (2b),) and an

intense band centred at 2081 assignable to NCS (2c) [7-9]. This observation supports the

presence of the ligand frame containing the pyridine ring and the C-S bond, C-S is


156

generally observed in free ligand in the range of 780-790 cm-1. IR spectra of complexes

1 and 2 are given in Figs.VI.2 and 3 respectively.

Table VI.2. Crystal data and details of refinement data for complex 1, 2a and 2b

1 2a 2b

Empirical Formula C19 H18 Cl2 N2 Ni S2 C19 H18 N8 Ni S2 C21 H18 N4 Ni O2 S2

Formula Weight 468.08 481.24 481.22

Crystal System monoclinic monoclinic monoclinic'

Space group I 2/a P 21/c P 21/c

a (Å) 14.538(4) 14.640(3) 14.473(4)

b (Å) 19.804(4) 20.671(4) 20.889(5)

c (Å) 13.695(3) 14.070(3) 14.151(3)

(o) 90.00 90.00 90.00

(o) 97.69(3) 95.43(3) 95.18(3)

(o) 90.00 90.00 90.00

Volume (Å3) 3907.4(16) 4238.8(15) 4260.7(18)

Z 8 8 8

calc (g/cm3) 1.591 1.508 1.500

F(000) 1920 1984 1984

range(deg) 1.75- 24.71 1.40-24.40 1.41-24.10

(Mo K) (mm-1) 1.486 1.136 1.132

Collected reflns 5520 50823 51031

Independent reflns 1136 3199 1953

Observed data

[I > 2.0 σ(I)] 1667 1989 1953

R1 [I > 2.0 σ(I)] 0.0401 0.0399 0.0516

wR2 [I > 2.0 σ(I)] 0.0790 0.0877 0.1132

Goodness-of-fit 0.672 0.773 0.687


157

Table VI.3. Selected bond distances (Å) and bond angles (o) for 1, 2a and 2b

1 2a 2b

Bond lengths(Å)

Ni- N(1) 2.113(5) Ni- N(1) 2.144(3) Ni- N(1) 2.134(5)

Ni- N(2) 2.109(5) Ni- N(2) 2.114(3) Ni- N(2) 2.097(5)

Ni- Cl(1) 2.3850(17) Ni- N(3) 2.049(4) Ni- N(3) 2.034(7)

Ni- Cl(2) 2.3615(18) Ni- N(4) 2.042(4) Ni- N(4) 1.985(8)

Ni-S(1) 2.4174(19) Ni- S(1) 2.3951(14) Ni- S(1) 2.405(2)

Ni- S(2) 2.3887(18) Ni- S(2) 2.4068(13) Ni- S(2) 2.415(2)

Bond angles (°)

N(2) –Ni- N(1) 164.18(19) N(2)- Ni- N(1) 165.27(14) N(2)- Ni- N(1) 164.7(2)

N(2)- Ni- Cl(2) 97.61(15) N(4)- Ni- N(3) 95.96(17) N(4)- Ni- N(3) 97.3(3)

N(1)- Ni –l(2) 93.60(13) N(4)- Ni- N(2) 93.08(17) N(4)- Ni- N(2) 96.0(3)

N(2)- Ni – l(1) 91.78(13) N(3)- Ni- N(2) 92.21(14) N(3)- Ni- N(2) 93.5(3)

N(1)- Ni- Cl(1) 97.59(17) N(4)- Ni- N(1) 98.45(15) N(4)- Ni- N(1) 94.6(3)

Cl(2)- Ni-Cl(1) 98.47(6) N(4)- Ni- N(1) 98.45(15) N(3)- Ni- N(1) 96.1(3)

N(2)- Ni- S(2) 82.76(15) N(4)- Ni- S(1) 90.75(13) N(4)- Ni- S(1) 87.1(2)

N(1) –Ni- S(2) 84.87(14) N(3)- Ni- S(1) 173.30(12) N(3)- Ni- S(1) 175.5(2)

Cl(2)- Ni –S(2) 173.61(7) N(2)- Ni- S(1) 87.57(10) N(2)- Ni- S(1) 87.0(16)

Cl(1)- Ni –S(2) 87.89(6) N(1)- Ni- S(1) 83.17(11) N(1)- Ni- S(1) 82.4(17)

N(2) –Ni- S(1) 86.94(13) N(4)- Ni- S(2) 174.64(13) N(4)- Ni- S(2) 173.6(2)

N(1) -Ni –S(1) 82.46(17) N(3)- Ni- S(2) 86.40(12) N(3)- Ni- S(2) 88.8(2)

Cl(2) -Ni –S(1) 87.29(6) N(2)- Ni- S(2) 82.00(12) N(2)- Ni- S(2) 81.6(17)

Cl(1)- Ni –S(1) 174.22(6) N(1)- Ni- S(2) 86.08(9) N(1)- Ni- S(2) 86.6(16)

S(2)- Ni –S(1) 86.36(6) S(1)- Ni- S(2) 86.94(4) S(1)- Ni- S(2) 86.8(7)


158

4000 3500 3000 2500 2000 1500 1000 50030

40

50

60

70

80

90

%T

Wavenumber(cm-1)

Table VI.4. IR data of the nickel(II) complexes(1-4)

Fig.VI.2. IR spectrum of

complex 1

Fig.VI.3. IR spectrum of

complex 2b

VI.2.3.2. UV-Vis Spectra

The electronic absorption spectra of the complexes 1 and 2a- 2c were recorded at

room temperature using DMF as solvent (Fig.VI.4) and the data are tabulated in Table

VI.5. Each complex shows the absorptions ranging from 350 to 450 nm, which are not

Compds. IR data (cm-1)

(C=N) (C=S) (Ni-Cl) (N3-) (NCS-) (NCO-)

1 1472 760 287 - - -

2a 1468 761 - - - 2183

2b 1470 758 - 2047 - -

2c 1468 760 - - 2081 -


159

observed for the free ligand. It suggests that the absorption bands of all complexes are

assignable to the LMCT transition from the pyridinic-N of organic moiety to the nickel

centre. All the spectra of complexes bands lower than 400 nm are due to intramolecular

* and n * transitions for the aromatic ring. In octahedral nickel (II)

complexes, three spin allowed transitions are expected from the energy level diagram

for d8 ions due to 3A2g 3T1g (P), 3A2g 3T1g(F), 3A2g 3T2g transitions, which are

observed at low to high wavelengths respectively. Here the bands at 472 nm and 518

nm, which may be assigned to 3A2g3T1g(P) and 3A2g 3T1g(F) transition respectively.

Again the intensity of the peak at around 664 nm observed due to the 3A2g 3T2g

transition. These

observations suggest an

octahedral geometry of the

Ni (II) ion.

Fig.VI.4. A representative UV-Vis spectrum of Ni(II) complex 1 in DMF.

VI.2.4. Electrochemistry

The redox properties of the complexes were examined by cyclic voltammeter using

a Pt-disk working electrode and a Pt-wire auxiliary electrode in dry dimethylformamide

using [n-Bu4N]ClO4 (0.1 M) as the supporting electrolyte. Voltammetric data are given

in Table VI.5. The cyclic voltammogram of all [Ni(L)X2] exhibit quasi-reversible

where NiII/NiI [10] redox couples are centred at ca. -(1.01 - 1.03) V and -(0.40-0.43) V

versus Ag/AgCl. The voltammetric parameters were studied in the scan rate interval 50–

400 mV.s-1. The ratio between the cathodic peak current and the square root of the scan


160

rate is approximately constant. The peak potential shows a small dependence on the

scan rate. The ratio Ipc to Ipa is close to unity. From these data, it can be deduced that the

redox couple is related to a quasi-reversible one-electron transfer process controlled by

diffusion.

Table VI.5. UV-Vis spectral and electrochemical data

Compd. nm () (, dm3 mol-1 cm-1)a Electrochemical dataa,b

Epa V Epc V E1/2 V ipc/ipa

1 274(9412), 360(4976), 470(2103),

652(987)

-0.43 -1.02 -0.725 1.13

2a 272(9875), 358(4531), 482(2099),

654(874)

-0.40 -1.03 -0.715 1.11

2b 274(10,127), 354(5274), 474(2254), 652(168)

-0.42 -1.02 -0.72 1.13

2c 271(9678), 356(5128), 472(2314), 650(84)

-0.40 -1.01 -0.705 1.12

aIn DMF bscan rate of 100 mVs-1

VI.2.5. DNA-binding studies

The mode of interaction of all the complexes (1-2) with calf thymus DNA (CT-

DNA) is in the same fashion and the investigation by using absorption and emission

spectra indicates the groove binding interaction [11-15]. For this reason, here only

details study of the interactions of complex 1 with DNA has been discussed as follows.

VI.2.5.1. Spectrophotometric study

Electronic absorption spectroscopy is an effective method to examine the binding

modes of metal complexes with DNA. In general, binding of the nickel (II) complex to

the CT-DNA helix is examined by an increase of the absorption band (c.a. 264 nm) of

Nickel (II) complex. This increasing absorbance indicates that there is the involvement

of strong interactions between complex and the base pairs of DNA [16]. The absorption

spectra of the Nickel (II) complex 1 in the absence and presence of CT-DNA are given

in (Fig.VI.5). The extent of the hyper-chromism in the charge transfer band is generally

consistent with the strength of interaction [17-20].


161

In order to further illustrate the binding strength of the Nickel (II) complex with

CT-DNA, the intrinsic binding constant Kb was determined from the spectral titration

data using the following equation [21]:

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

where [DNA] is the concentration of DNA, f , a and b correspond to the

extinction coefficient, respectively, for the free Nickel (II) complex, for each addition of

DNA to the Nickel(II) complex and for the Nickel (II) complex in the fully bound form.

Fig.VI.5. Electronic spectral titration of complex 1 with CT-DNA at 266 nm in tris-HCl

buffer. Arrow indicates the direction of change upon the increase of DNA

concentration

From the [DNA]/(a-f) versus[DNA] plot (Fig.V.6), the binding constant Kb for

the Nickel (II) complex 1 was estimated to be 1.01 × 106 M-1 (R = 0.97281 for four

points) indicating in terms of groove binding.

V.2.5.2. Spectroflurimetric study

Fluorescence intensity of EB bound to CT-DNA at excitation wavelength of 522

nm shows a decreasing trend with the increasing concentration of the complex 1

(Fig.VI.7). The quenching of EB bound to DNA by the complex 1 is in agreement with

the linear Stern–Volmer equation [22]:

I0/I = 1 + Ksv[Q]

Where I0 and I represent the fluorescence intensities in the absence and

presence of quencher, respectively. Ksv is a linear Stern–Volmer quenching constant, Q

is the concentration of quencher. The Ksv value calculated from the plot (Fig.VI.8) of


162

I0/I versus [complex] for the complex 1 is 2.09× 104 (R = 0.95661 for four points),

suggesting a strong affinity of the complex 1 to CT-DNA.

Fig.VI.6. Plot of [DNA]/(a-f) vs .[DNA] for the absorption titration of CT-DNA with

the nickel (II) complex 1 in Tris-HCl buffer

Fig.VI.7. Emission spectra of the CT-DNA–EB system in tris–HCl buffer upon the

titration of the nickel(II) complex 1. ex = 522 nm. Arrow shows the intensity

change upon the increase of the complex concentration.

Number of binding sites can be calculated from fluorescence titration data using the

following equation [23]

log [(I0 - I)/I] = logK + nlog[Q]


163

here, K and n is the binding constant and binding site of complex 1 to CT-DNA

respectively.

Fig. VI.8. Plot of I0/I vs. [complex] for the titration of nickel (II) complex 1 with CT-

DNA-EB system in Tris-HCl buffer

The number of binding sites (n) determined from the intercept of log[(I0 – I)/I]

versus log[Q] is 0.91 which indicates less association of the complex 1 to the number of

DNA bases, also suggesting strong affinity of the complex 1 through surface or groove

binding.

V.2.5.3. Electrochemical study

Electrochemical investigations is useful technique to analyse metal-DNA

interactions over spectroscopic methods [24,25]. The binding nature of the Nickel(II)

complex 1 with DNA, has been shown in (Fig.VI.9) Cyclic voltammograms of the

nickel(II) complex 1 in the absence and presence of CT-DNA is exhibited significant

shifts in the anodic and cathodic peak potentials followed by decrease in both peak

currents, indicating the interaction existing between the nickel(II) complex and CT-

DNA. Equilibrium binding constants KR/K0 can be calculated by using the shift value of

the formal potential (ΔE0) of Ni(II)/Ni(I) according to the following equation [26].

ΔE0 = Eb0 – Ef

0 =0.0591 log(KR/K0)

where Eb0and Ef

0 are the formal potentials of the bound and free complex forms,

respectively, and KR and K0 are the corresponding binding constants for the binding of

reduction and oxidation species to DNA, respectively. Ratio of equilibrium binding

constants, KR/K0 is calculated to be 1.07 which indicate the strong binding of DNA with

reduced form over oxidised form of nickel complex.


164

Fig.VI.9. Cyclic voltammograms of complex 1 in Tris–HCl buffer in the absence

(a) and presence (b) of CT-DNA. v = 1 V s-1.

VI.2.6. Protien (Bovine serum albumin) binding experiments

VI.2.6.1. Absorption characteristics of BSA-complex 1

The absorption spectra of BSA in the absence and presence of Ni(II) complex

1(other complexes give same binding interaction) at different concentrations were

studied.(Fig.VI.10) From this study we observed that upon increasing the concentration

of the complex the absorption of BSA increases regularly. It is may be due to the

adsorption of BSA on the surface of the complex. From these data the apparent

association constant (Kapp) determined of the complexes with BSA has been determined

using the following equation, [21].

1/(Aobs – A0) = 1/(Ac – A0) + 1/ Kapp(Ac – A0)[comp]

Where, Aobs is the observed absorbance of the solution containing different

concentrations of the complex at 280 nm, A0 and Ac are the absorbances of BSA and

the complex at 280 nm, respectively, with a concentration of complex, and Kapp

represents the apparent association constant. The enhancement of absorbance at 280 nm

was due to absorption of the surface complex, based on the linear relationship between

1/(Aobs - A0) vs reciprocal concentration of the complex with a slope equal to 1/Kapp(Ac -

A0) and an intercept equal to 1/(Ac - A0) (Fig.VI.11). The value of the apparent

association constant (Kapp) determined from this plot is 2.73× 104 M-1(R = 0.98362 for

four points)


165

Fig.VI.10. Electronic spectral titration of complex 1 with BSA at 280 nm in tris-HCl

buffer. Arrow indicates the direction of change upon the increase of BSA

concentration.

Fig.VI.11. Plot of 1/(A - A0) vs. 1/[Complex 1] resulting from the electronic spectral

titration with BSA at 280 nm in tris-HCl buffer

VI.2.6.2. Fluorescence quenching of BSA by the complex 1

The effect of increasing the concentration of the complex, the fluorescence

emission spectrum of BSA were studied and represented in Fig.VI.12. With the addition

of complex, BSA fluorescence emission is quenched. The fluorescence quenching is

described by the Stern–Volmer relation [22] described above. A linear plot [Fig.VI.13]

between I0/I against [complex] was obtained and from the slope we calculated the KSV

as 1.15 × 103 (R = 0.9908 for four points).


166

Fig.VI.12. Fluorescence quenching of BSA in the presence of various concentrations of

the complex 1, [complex] = 0, 1, 2, 3 and 4 × 6.25 ×10-6 M.

Fig.VI.13. Plot of I0/I against [complex 1] in case of fluorescence quenching of BSA

VI.2.7. Antibacterial activity

Antibacterial activity of the lignd and corresponding complexes are recorded in

Table VI.6. From the antibacterial studies it is inferred that, all complexes have higher

activity than ligand. The increased activity of the metal chelates is due to lipophilic

character of the metal complexes. This increased lipophilicity enhances the penetration

of complexes into the lipid layer of the bacterial cell membranes and blocks the metal

binding sites in enzymes of microorganisms. These complexes also disturb the

respiration process of the cell and thus block the synthesis of proteins, which restricts

further growth of the microorganisms.


167

Table VI.6. Antibacterial data of the L and its Ni (II) complexes (100 µg/ ml)

VI.3. Experimental VI.3.1. Materials and Physical Measurements.

All chemicals and reagents were obtained from commercial sources and used as

received, unless otherwise stated. Solvents were distilled from an appropriate drying

agent. The elemental (C, H, N) analyses were performed on a Perkin Elmer model 2400

elemental analyzer. Nickel analysis was carried out by Varian atomic absorption

spectrophotometer (AAS) model-AA55B, GTA using graphite furnace. Electronic

absorption spectra were recorded on a JASCO UV-Vis/NIR spectrophotometer model

V-570. IR spectra (KBr discs, 4000–300 cm-1) were recorded using a Perkin-Elmer

FTIR model RX1 spectrometer. The room temperature magnetic susceptibility

measurements were performed by using a vibrating sample magnetometer PAR 155

model. Molar conductances (M) were measured in a systronics conductivity meter 304

model using ~10-3 mol.L-1 solutions in appropriate organic solvents. Electrochemical

measurements were performed using computer-controlled CH-Instruments (Model No-

CHI620D), All measurements were carried out under nitrogen environment at 298 K

with reference to SCE electrode in dimethyl sulphoxide using [n-Bu4N]ClO4 as

supporting electrolyte. The fluorescence spectra of EB bound to DNA were obtained in

the Fluorimeter (Hitachi-2000).

Treatment

Inhibition zone in mm

E.coli V.cholerae Shigella sp. S.pneumoniae Bacillus cereus

DMF (control) 0 0 0 0 0

L 16 12 14 14 13

1 22 18 20 18 21

2a 19 16 18 15 16

2b 20 17 16 16 17

2c 18 15 17 15 15

Bore diameter 06 06 06 06 06


168

VI.3.2. Preparation of the ligand (L3)

The synthesis of ligand L3 was prepared by the following procedure; An ethanolic

solution of 2-picolyl chloride, hydrochloride (5.0 mmol) was added to toluene-3,4-

dithiol (5.0 mmol) in dry ethanol containing sodium ethoxide (10.0 mmol) at low

temperature (0o-5oC). Then this mixture was allowed to stir at room temperature for 0.5

h and then it was refluxed for 3 h. The mixture was cooled to room temperature, water

was added and finally the ethanol was off by rotary evaporator. The product was

extracted into dichloromethane and dried by using NaHSO3. The product, [3,4-bis(2-

pyridylmethylthio)toluene] was obtained as a yellow viscous liquid by removing the

dichloromethane by rotary evaporator. Finally the products were verified by 1H NMR

spectroscopy.

VI.3.3. Preparation of the complexes

VI.3.3.1. Preparation of [Ni(L3)(Cl)2] (1)

To prepare this nickel (II) complex (1) a common procedure was followed as

described below, using nickel chloride and the organic ligand (L3) in equimolar

ratio(1:1). A methanolic solution of L3 (338 mg, 1.0 mmol) was mixed with 1.0 mmol

of nickel chloride (240.37 mg) in methanolic solution with stirring condition, and the

mixture was refluxed for 4 h. The product was collected by filtration and washing with

cold methanol and water, and dried. The pure crystallized product was obtained from

methanol.

VI.3.3.2. Preparation of [Ni(L3)(X)2] (2a-2c)

The complexes were synthesized following a common procedure, as described

below. To a methanolic solution of nickel acetate (248.0 mg, 1.0 mmol) was added to

the solution of the organic compound (L3) (338 mg, 1.0 mmol) in methanol (10 ml) and

the resulting solution was stirred for 2 h at ambient temperature. The reaction mixture

was refluxed for 3 h. To this solution aqueous solution of potassium azide (2.0 mmol)

(for 2a), sodium cyanate (2.0 mmol) (for 2b) and sodium thiocyanate (2.0 mmol) (for

2c) was added and stirring was continued for another 1h. The volume of the solution

was reduced at room temperature by slow evaporation. The product was collected by

washing with cold methanol and water; and dried. The pure crystallized product was

obtained from methanol.


169

VI.3.4. DNA binding experiments

Tris–HCl buffer (pH 7.0)solution prepared using deionized and sonicated HPLC

grade water (Merck) was used in all the experiments involving CT-DNA. The CT-DNA

used in the experiments was sufficiently free from protein as the ratio of UV absorbance

of the solutions of DNA in tris–HCl at 260 and 280 nm (A260/A280) was almost ≈1.9

[27]. The concentration of DNA was determined with the help of the extinction

coefficient of DNA solution at 260 nm of 6600 Lmol-1 cm-1 [28]. Absorption spectral

titration experiment was performed by keeping constant the concentration of the Ni (II)

complex and varying the CT-DNA concentration.

In the ethidium bromide (EB) fluorescence displacement experiment, 5 µL of the

EB tris–HCl solution (1 mmol L-1) was added to 1 mL of DNA solution (at saturated

binding levels) [29], stored in the dark for 2 h. Then the solution of the Ni (II) complex

was titrated into the DNA/EB mixture and diluted in tris–HCl buffer to 5 mL to get the

solution with the appropriate Ni (II) complex/CT-DNA mole ratio. Before

measurements, the mixture was shaken up and incubated at room temperature for 30

min. The fluorescence spectra of EB bound to DNA were obtained at an emission

wavelength of 522 nm in the Fluorimeter (Hitachi-2000).

VI.3.5. Protien (bovine serum albumin) binding experiments

The sample was placed in quartz cuvettes of 1 cm optical path. In these

experiments, 3mL of BSA solution were poured into the cell. The samples were

carefully degassed using pure nitrogen gas for 15 min. Emission spectra were recorded

after addition of the appropriate concentration of the Ni (II) complexes in the same

buffer. The samples were excited at 280 nm.

VI.3.6. In vitro antibacterial assay

The biological activities of synthesized ligand (L3) and its Ni (II) complexes have

been studied for their antibacterial activities by agar well diffusion method [30-33]. The

antibacterial activities were done at 100 µg/mL concentrations of different compounds

in DMF solvent by using four pathogenic gram negative bacteria (Escherichia coli,

Vibrio cholerae, Streptococcus pneumoniae, Shigella sp) and one gram positive

pathogenic bacteria (Bacillus cereus). DMF was used as a negative control. The Petri

dishes were incubated at 37 ◦C for 24 h. After incubation plates were observed for the


170

growth of inhibition zones. The diameter of the zone of inhibition was measured in

millimetres.

VI.3.7. X-ray crystal structure analyses

Crystal data and details of data collection and refinement for complex 1, 2a and 2b

were summarized in Table 1. Suitable single crystals for X-ray diffraction analysis of 1

were grown at ambient temperature by slow evaporation of a methanolic solution.

Diffraction data of 1 was collected at room temperature on a Nonius DIP-1030H

system, by using Mo-Kα radiation (λ = 0.71073 Å). Cell refinement, indexing and

scaling of the data set were performed using programs Denzo and Scalepack [34]. The

structure was solved by direct methods and subsequent Fourier analyses and refined by

the full-matrix least-squares method based on F2 with all observed reflections [35]. The

contribution of hydrogen atoms at calculated positions were included in final cycles of

refinement. All the calculations were performed using the WinGX System, Ver 1.80.05

[36].

VI.4. Epilogue The synthesis and characterization of four new mononuclear Ni (II) complexes with

a N2S2 donor set have been performed. The complexes1, 2a and 2b have been

characterized spectroscopically and by X-ray diffraction. These data indicate that the

nickel ion is in octahedral geometry with pyridine rings are in trans positions, while the

two thioether-S donors are located in the equatorial plane beside two chloride anions (in

complex 1), azide (in 2a) and cyanate( in 2b) which are at mutual cis positions, and

complete the distorted octahedral coordination sphere around metal ion. The interaction

of the complex 1 with calf thymus DNA and BSA has been investigated by using

absorption, emission spectra and the results indicate that the interaction of complex 1 to

calf thymus DNA is groove binding mode. From the antibacterial studies it could be

inferred that metal complexes have higher activity than ligand due chelation. All four

Ni(II) complexes have higher antibacterial activity than ligand L3 against five

pathogenic bacteria (Escherichia coli, Vibrio cholerae, Streptococcus pneumonia,

Shigella sp. and Bacillus cereus).


171

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