Synthesis, characterization and biological activity of mixed ligand chelates of Ni(II) with pyridoxalthiosemicarbazone and dipeptides

Trang 1

DOI: 10.1002/vjch.202000100

Synthesis, characterization and biological activity of mixed ligand chelates of Ni(II) with pyridoxalthiosemicarbazone and dipeptides

A Saritha 1 , Ch Venkata Ramana Reddy 2* , B Sireesha 3

1

Department of Chemistry, St Francis College for Women, Hyderabad, India 500016

2

Department of Chemistry, Jawaharlal Nehru Technological University Hyderabad, Hyderabad, India

500085

3

Department of Chemistry, Osmania University, Hyderabad 500001 Submitted April 30, 2020; Accepted August 18, 2020

Abstract

The synthesis, characterization of mixed ligand chelates of Ni(II), [NiAL] involving Pyridoxalthiosemicarbazone (A) and dipeptides (L) viz., glycine (gly-gly), L-leucine (gly-leu), L-tyrosine (gly-tyr) and glycyl-L-valine (gly-val) and their biological activities have been studied The complexes were characterized based on their elemental analysis, LC-MS, IR, UV-Vis spectral studies, magnetic moment, molar conductance and thermal analysis The mixed ligand complexes were formed with 1:1:1 (Ni:A:L) ratio The molar conductance data reveal the non-electrolytic nature of the metal chelates IR spectra show that the ligands are coordinated to the metal ion in a tridentate manner, involving O,N,S and O,N,N donor sites of ligands, A and L respectively Based on the analytical data, octahedral geometry has been proposed for the metal complexes DNA binding properties of the complexes have been investigated by UV-Vis and fluorescence spectroscopy and also by viscosity measurements The obtained results indicate that the complexes bind to DNA through intercalation mode, which is further validated by molecular docking studies The hydrolytic cleavage of the pBR322 DNA from supercoiled to nicked form, by the metal complexes was investigated by gel electrophoresis technique The metal complexes were also screened for their antioxidant, anti-inflammatory and antibacterial activities and the findings have been reported

Keywords Mixed ligand chelates, DNA interaction, antibacterial, anti-inflammatory, antioxidant, molecular

docking

1 INTRODUCTION

Polydentate Schiff base ligands and their transition

metal complexes are of great interest in coordination

chemistry,[1,2] due to their structural features and

biological activities Pyridoxalthiosemicarbazone

(PLTSC), a tridentate Schiff base ligand with O, N,

S donor atoms, forms stable metal chelates.[3] Binary

complexes of PLTSC with the transition metal ions

have been reported by various researchers.[4-7] A

major interest in the metal complexes of PLTSC

derives from their biological and chemotherapeutic

activities, such as suppressive effect on Friend

erythroleukemia cells (FLC), inhibition of reverse

transcriptase and cytotoxicity.[8-10]

Similarly, dipeptides are also versatile ligands for

complexation with many metal ions The solution

chemistry and synthesis of binary complexes of

dipeptides are reported, where in the common

binding sites of dipeptides include amino nitrogen,

peptide oxygen or peptide nitrogen and carboxylate oxygen.[11,12] The metal chelates of dipeptides were found to show various biological activities as antibacterial, anti-inflammatory and antitumor activities.[13-16]

The role of mixed ligand chelates in biological processes such as activation of enzymes, storage and transport of substances across the membranes is well established The potential ligands compete for the

metal ions in vivo which results in mixed ligand

chelation in biological fluids They act as antimicrobial, antioxidant, cytotoxic, anticancerous agents[17-20] and also as catalysts in various organic reactions.[21,22]

Though there are some reports on binary complexes of PLTSC and dipeptides, there are no significant studies on the mixed ligand complexes involving these ligands Accordingly, we report herewith the synthesis, characterization and biological activity studies as antibacterial,

Trang 2

antioxidant, anti inflammatory, DNA binding and

cleavage of the mixed ligand chelates of NiAL,

involving PLTSC (A) and dipeptides (L) viz.,

glycyl-glycine (gly-gly), glycyl-L-leucine (gly-leu),

glycyl-L-tyrosine tyr) and glycyl-L-valine

(gly-val)

2 MATERIALS AND METHODS

2.1 Materials and physical measurements

Pyridoxal hydrochloride, thiosemicarbazide,

dipeptides, CT DNA and pBR 322 DNA, DPPH,

Diclofenac Sodium were purchased from Sigma

Aldrich KH2PO4, K2HPO4 and nickel(II) chloride

hexahydrate were obtained from Merck, India

Muller Hinton Agar medium was purchased from

Himedia All other chemicals and solvents were of

analytical grade and were used without further

purification PLTSC was prepared by a known

procedure.[23]

Elemental (C,H,N) analysis was carried out on a

Thermo Finnigan 1112 elemental analyzer Mass

spectra of the complexes were recorded on a LCMS

2010A, Shimadzu spectrometer The other

spectroscopic measurements were made using the

instruments, IR: Shimadzu IR Prestige-21

Spectrometer (KBr, 4000-250 cm-1); UV-Vis:

Systronics UV-Vis Double beam spectrophotometer

2201; Fluorescence: Shimadzu Spectrofluorometer,

RF-5301 The molar conductivity of the freshly

prepared (10-3 M) solutions of complexes in DMSO

was measured using a Digisun digital conductivity

bridge Thermo gravimetric analysis (TGA) was

performed using Shimadzu TGA-50H in nitrogen

atmosphere in the temperature range from room

temperature to 1000 ºC with a heating rate of 20 ºC

per min Magnetic susceptibilities were measured at

room temperature on Faraday balance, model 7550

using Hg[Co(NCS)4] as an internal standard

Diamagnetic corrections were made using Pascal’s

constants.[24] Molecular docking study was carried on

Autodock 4.2 programme

2.2 Synthesis of mixed ligand Ni(II) complexes

0.4838 g (1.7 mmol) of PLTSC and 0.3293 g (1.7

mmol) of gly/0.3293 g (1.7 mmol) of

gly-leu/0.4168 g (1.7 mmol) of gly-tyr/0.3048 g (1.7

mmol) of gly-val was added simultaneously to an

aqueous solution containing 0.404 g (1.7 mmol) of

NiCl2.6H2O An immediate color change was

observed The mixture was refluxed over the steam

bath for 3 hours A red colored precipitate was

obtained by adjusting the pH to 7-8 with a

methanolic solution of ammonium hydroxide The solid obtained was filtered, washed several times with hot distilled water, followed by petroleum ether and finally air dried

[Ni(PLTSC-H)(gly-gly-H)] C 13 H 18 N 6 O 5 SNi (1): Anal Calcd (%): C, 36.36; H, 4.19; N, 19.58

Found: C, 36.32; H, 4.23; N, 19.55 APCI-MS (m/z):

428 [M+], (Fig S1) IR (KBr) cm-1: ν(C=N) 1606,

ν(Ar.C-O) 1151, ν(C=S) 812, ν(peptide –NH) 1554, ν(-COO- asym)

1618, ν(-COO- sym) 1388 UV-Vis (DMSO) λmax/nm:

259, 336, 400, 486 μeff (BM): 2.99 Λm [Ω-1cm2M

-1,10-3, DMSO]: 07

[Ni(PLTSC-H)(gly-leu-H)]C 17 H 26 N 6 O 5 SNi (2):

Anal Calcd (%): C, 42.06; H, 5.36; N, 17.31 Found: C, 42.01; H, 5.40; N, 17.28 APCI-MS (m/z):

486 [M+] (Fig S2) IR (KBr) cm-1: ν(C=N) 1612, ν (Ar.C-O) 1147, ν(C=S) 815, ν(peptide –NH) 1560, ν(-COO- asym) 1616,

ν(-COO- sym) 1388 UV-Vis (DMSO) λmax/nm: 262, 400,

489 μeff (BM): 2.96 Λm [Ω-1cm2M-1,10-3, DMSO]:

06

[Ni(PLTSC-H)(gly-tyr-H)].H 2 O

C 20 H 26 N 6 O 7 SNi (3): Anal Calcd.: C, 43.39; H, 4.70;

N, 15.19 Found: C, 43.35; H, 4.66; N, 15.22

APCI-MS (m/z): 553 [M+] (Fig S3) IR (KBr) cm-1: ν(C=N)

1610, ν(Ar.C-O) 1147, ν(C=S) 815, ν(peptide –NH) 1560, ν (-COO- asym) 1616, ν(-COO- sym) 1386 UV-Vis (DMSO)

λmax/nm: 259, 400, 489 μeff (BM): 3.01 Λm [Ω

-1cm2M-1,10-3, DMSO]: 10

[Ni(PLTSC-H)(gly-val-H)] C 16 H 24 N 6 O 5 SNi (4): Anal Calcd.: C, 40.76; H, 5.09; N, 17.83

Found: C, 40.72; H, 5.13; N, 17.80 APCI-MS (m/z):

472 [M+] (Fig S4) IR (KBr) cm-1: ν(C=N) 1610, ν (Ar.C-O) 1145, ν(C=S) 815, ν(peptide –NH) 1553, ν(-COO- asym) 1591,

ν(-COO- sym) 1388 UV-Vis (DMSO) λmax/nm: 271, 399,

482 μeff (BM): 2.89 Λm [Ω-1cm2M-1,10-3, DMSO]:

07

2.3 DNA binding studies

2.3.1 UV-Visible absorption titration

The DNA binding interaction of the metal

complexes 1-4 was measured in potassium

phosphate buffer solution (pH 7.2) The absorption ratio at 260 and 280 nm of Calf Thymus DNA (CT DNA) solutions was found as 1.9:1, which shows that the DNA is sufficiently free from protein The concentration of DNA was determined by UV-visible absorbance at 260 nm, using ε value of 6600

M-1cm-1 The titration experiments were performed

by maintaining the concentration of metal complexes constant at 20 µM, while the concentration of CT DNA was varied within 0-20

µM An equal quantity of CT DNA was also added

to the reference solution to eliminate the absorption

Trang 3

by DNA After each addition of CT-DNA to the

complex, the resulting solution was incubated for 10

min and the absorption spectra were recorded in the

wavelength range of 200-500 nm The binding

constants (Kb) were calculated from the

spectroscopic titration data using the equation:

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

where, [DNA] is the concentration of CT-DNA, εa,

εb,εf are the extinction coefficients of apparent,

bound and free complex respectively Kb of the

complex is calculated from the ratio of slope to

intercept in the plot of [DNA] vs [DNA]/(εa-εf).[25]

2.3.2 Competitive DNA binding fluorescence studies

Further support for the intercalative mode of binding

to DNA was obtained using fluorescence spectral

experiments, wherein the ability of a metal complex

to displace ethidium bromide (EB) from a DNA-EB

adduct was studied EB displacement experiments

were carried out by the addition of metal complex

solutions to a DNA and EB mixture in potassium

phosphate buffer solution (pH 7.2) The DNA was

pretreated with EB at a concentration ratio of

[DNA]/[EB] = 1 and incubated for 30 min at room

temperature Then the changes in fluorescence

intensities of EB bound to DNA at 605 nm were

recorded with an increasing amount of the complex

concentration from its 50 µM stock solution The

observed changes of fluorescence intensity with

increasing concentration of the quencher (complex)

were used to calculate the binding constant or Stern -

Volmer quenching constant Kq.[26,27]

2.3.3 Viscosity studies

The viscosity measurements were carried out on an

Ostwald viscometer, immersed in a thermostatic

water bath maintained at 25±1 ºC Concentration of

ternary metal complexes was varied by adding

increasing amounts from their 50 µM stock solution

to CT-DNA solution (300 µM) in phosphate buffer

(pH 7.2) Flow time was recorded using a digital

stopwatch in triplicate and an average flow time was

calculated Data are presented as plot of (η/η0)1/3

versus [complex]/[CT-DNA], where, η is the

viscosity of CT-DNA in the presence of complex

and η0 is the viscosity of CT-DNA alone.[28]

2.4 DNA cleavage studies

Interaction between pBR 322 plasmid DNA and the

mixed ligand Ni(II) complexes was examined in 5

mM Tris.HCl/50 mM NaCl buffer (pH 7.2), by

agarose gel electrophoresis experiments Freshly prepared complex solutions in DMSO (20, 40 and 60 µM) were incubated with plasmid DNA (300 ng/3 µl) at 37 ºC for 1 h Then, a loading buffer (1 µl) containing 1 % bromophenol blue and 40 % Sucrose was added and loaded onto a 0.8 % agarose gel containing EB (1 µg/ml) The samples along with the control DNA were subjected to electrophoresis

in TAE buffer (Tris-acetic acid-EDTA) at 60 V for 2

h The bands of DNA have moved on the agarose gel under the influence of electric field These bands were visualized by viewing the gel on a transilluminator and photographed.[29]

2.5 Antibacterial assay

The complexes were screened against Staphylococcu saureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa for their antibacterial

activity The tests were performed using well diffusion method The stock solutions of the complexes (1 mg/mL) were prepared in DMSO Petri plates containing 20 ml Mueller Hinton Agar medium were inoculated with 100 µL of 24 hour culture of the test bacterial strain and kept for 15 min for adsorption Using a sterile plastic borer of 8

mm diameter, wells were bored into the seeded agar plates and were loaded with a 100 µL solution of each metal complex The diameter of inhibition zone around each well was measured in mm after 24 h DMSO was used as negative control and amikacin (30 µg) was used as the standard.[30]

2.6 Antioxidant activity

The antioxidant activity of the mixed ligand Ni(II) complexes has been evaluated using 2,2-diphenyl-2-picrylhydrazyl (DPPH) radical assay The DPPH is a stable free radical with a λmax at 517 nm Stock solutions of the metal complexes were prepared in DMSO, from which different concentrations containing 50, 100, 150, 200 and 250 μg/mL were prepared by diluting with methanol 1ml of each solution was added to a solution of DPPH in methanol (0.4 mM, 1 mL) and the final volume was made to 3 mL with methanol.[31] DPPH solution in methanol was used as a positive control and methanol alone as a blank The solutions were thoroughly shaken and incubated at room temperature for 30 min in dark The decrease in absorbance of DPPH was measured at 517 nm Ascorbic acid was used as a standard All the measurements were made in triplicates The percentage of inhibition (I%) of free radical production by DPPH was calculated using the

Trang 4

formula, (I%) = [(A0 - Ac)/A0]  100 where A0 and

Ac are the absorbance in the absence and presence of

the metal complexes respectively.[32]

2.7 In vitro Anti-inflammatory activity

The in vitro anti-inflammatory activity of the mixed

ligand complexes was studied by using inhibition of

Bovine Serum Albumin (BSA) denaturation

technique as reported by Mizushima et al.[33] and

Sakat et al.[34] with minor modifications Reaction

mixtures consisting of 1 % BSA and the complexes

in phosphate buffer at pH 7.4 were incubated at 37

ºC for 20 min Then they were heated at 51 ºC for 30

min After cooling, the turbidity of the samples was

measured at 660 nm The percentage inhibition of

protein denaturation was calculated by using the

formula, Percentage inhibition = [(Acontrol-

Asample)/Acontrol]  100.[35-37]

2.8 Molecular Docking studies

Docking studies of the interaction of metal

complexes with DNA was carried out in Autodock

4.2 as reported.[38,39] Crystal structure of DNA was

downloaded from protein data bank

(www.rcsb.org)pdb id: 1N37,[40] which was prepared

by protein preparation wizard applying OPLS 2005

force field in Schrodinger suite A grid was prepared

around the intercalation site by selecting the

co-crystallized ligand Metal complexes were

constructed and optimized in ChemDraw These

were docked into DNA intercalation site using

Autodock 4.2 Molecular interaction diagrams are

obtained from PMV.[41]

3 RESULTS AND DISCUSSION

3.1 Characterization of complexes

The mixed ligand complexes,

[Ni(II)-PLTSC-dipeptide] obtained were amorphous, colored solids

and stable at room temperature They are soluble in

DMSO, DMF and insoluble in common organic

solvents The complexes gave satisfactory C, H, and

N analysis The molar conductivity values (Λm:

05-15 ohm-1cm2mol-1) of 10-3 M solutions of the

complexes in DMSO indicate the complexes to be

non-electrolytes The magnetic moment values (µeff:

2.89-3.01BM) of the complexes suggest the

presence of two unpaired electrons in Ni(II) ion

3.1.1 APCI-MS

The mass spectra of the mixed ligand complexes

were recorded in APCI-positive mode The mass spectra provide information regarding the 1:1:1

(M:A:L) behavior of complexes The complexes 1 to

4 (Fig S1-S4) show peaks at m/z 428[M]+, 486[M+1]+, 553[M]+ and 472[M+1]+ respectively

Complexes 1 and 2 also display peaks at m/z 450

and 508 respectively assigned to [M+23]+ The data are in good agreement with the stoichiometry of the ternary complexes in 1:1:1 (M:A:L) ratio and the proposed molecular formulae

3.1.2 IR spectra

All the complexes exhibited similar IR spectroscopic properties The comparison of IR spectra of ligands and the complexes (Fig S5-S13) support the coordination of PLTSC (A) and dipeptides (B) to Ni(II) ion Assignments of the characteristic IR bands of the ligands and the ternary complexes are presented in table 1 The PLTSC is coordinated in tridentate mode, through deprotonated phenolic oxygen, nitrogen of azomethine and sulphur of thioamide group The band attributed Py-NH+ due to the migration of Py-OH proton to Py-N in PLTSC[42]

at 2823 cm-1 has disappeared The shifts in the stretching vibrations arising from the C=N

(1650-1600 cm-1) agree well with the involvement of the azomethine nitrogen in the coordination The participation of the phenolic oxygen in coordination

is clear from a shift in the stretching frequency of ArC-O from 1126 to 1147 cm-1 A shift in the C=S band in the metal complexes from 841 cm-1 of PLTSC indicate the coordination of sulphur to the metal ion.[43]

Dipeptides are coordinated in tridentate mode via nitrogen of free amino group, nitrogen of peptide nitrogen and oxygen of deprotonated carboxyl group Existence of the free dipeptides as zwitter ions in the solid state is confirmed by a medium intensity band near 2100 cm-1 region, due to δ(NH3+) and ρ(NH3+) This band disappears upon coordination to the metal ion and the N-H stretching frequency of the amino group is shifted to higher frequencies compared to the free dipeptides.[44]

These spectroscopic observations indicate the binding of NH2 group to the Ni(II) The peptide -NH coordination is suggested by the shift in the stretching vibration (100-120 cm-1) around 3180

cm-1 and bending frequency by 20-40 cm-1 around

1550 cm-1 The difference in stretching frequencies

of carboxylate group [Δ = νas(COO–)-νs(COO–)], for all the complexes (above 200 cm-1) were larger than the Δ values of the free dipeptides (150-185 cm-1) indicating the coordination of carboxylate oxygen.[45]

Trang 5

Table 1: IR spectral data of the ligands and ternary complexes (cm-1) Ligand/

bend ν(C=S) asym sym

3184

1184

3.1.3 Thermal analysis

The thermal stability of the mixed ligand complexes

was studied by recording themogravimetric analysis

The TG curves of the ternary complexes 1 to 4 (Fig

S14-S17) showed a three step decomposition

process An initial weight loss around 100 ºC is

observed in the complexes 1, 2 and 4, which may be

due to the loss of moisture The weight loss at 115

ºC in the thermogram of complex 3 corresponds to

one mole of lattice water The first step of weight

loss was also accompanied by an endothermic peak

in DTA curve The second step of decomposition

occurred between 225-440 ºC in all the complexes,

suggesting the loss of ligand moieties From 440 ºC

onwards the complexes exhibited a similar

decomposition process to give metal oxide as the

final product

3.1.4 Electronic spectra

The mixed ligand Ni(II) complexes, 1 to4 are

characterized by three main bands in their electronic

spectra (figures S18-S21) at 258, 400 and 486 nm

The band at 258 nm comprises π-π* and n- π*

transitions of imine and pyridine ring of PLTSC

The band at 400 nm may be due to LMCT

transitions arising from N → Ni and SCN → Ni

charge transfer The low intense band at 486 nm

corresponds to 3A2g → 3T1g(P) transition In addition,

complex 4 also displays a band of low intensity at

750 nm due to 3A2g → 3T1g(F) transition The

electronic spectroscopic data coupled with the high

magnetic moment values (2.86 to 3.01 BM)

implying the octahedral geometry of the

complexes.[46] Based on the spectroscopic and

analytical data, the tentative structure of the mixed

ligand complexes is proposed as shown in figure 1

3.2 DNA binding studies

3.2.1 Electronic absorption spectroscopy

The uncontrolled proliferation of tumor cells can be blocked by targeting DNA by its interaction with small molecules Therefore, it is important to study the DNA binding properties of metal complexes An effective method to obtain the preliminary information about the binding mode of DNA and metal complexes is by UV-Visible spectroscopy In the present study, the concentration of the metal complex was kept constant to which DNA was added in increasing amounts Complexes bound to DNA through intercalation, often result in hypochromism with red-shift in their absorption spectra This happens due to the strong stacking interaction between the DNA base pairs and the aromatic chromophore of the complex The extent of hypo chromism suggests the strength of intercalation.[47] The absorption spectra of the ternary complexes in the absence and presence of DNA are presented in figure 2 The incremental addition of DNA to the metal complexes resulted in hypo chromism along with slight bathochromic shift This shows that the metal complexes are bound to CT DNA through intercalative mode The quantitative binding strengths of the metal complexes to DNA were determined by the calculation of binding constants (Kb) from equation 1 which are presented

in table 2 The magnitude of the binding constant

values suggest the decreasing order of binding as 4 >

1 > 2 > 3

3.2.2 Competitive DNA binding studies

Ethidium bomide (EB) is widely used as a sensitive fluorescence probe for DNA It intercalates strongly

Trang 6

between the adjacent DNA base pairs, which results

in intense fluorescence In the EB displacement

technique, a decrease in the fluorescence intensity

Table 2: Intrinsic binding constant (Kb) values of the

complexes

Ni(II)-PLTSC-(gly-gly) 4.32105

Ni(II)-PLTSC-(gly-leu) 3.88105

Ni(II)-PLTSC-(gly-tyr) 6.20103

Ni(II)-PLTSC-(gly-val) 7.48106

occurs from the displacement of bound EB from

DNA-EB adduct, by the addition of a quencher, due

to the reduction in the number available binding

sites for EB on the DNA Hence, this method

provides an indirect evidence for the intercalative

binding mode.[48] In the present study, a quenching in

the fluorescence emission intensity of EB bound to

DNA was observed with the addition of metal

complex solution, indicating that the EB molecules

are displaced by the added complexes from the DNA

binding sites The fluorescence spectra of EB bound

to DNA quenched by the ternary metal complexes

are shown in figure 3 The obtained data were then

fit into the Stern-Volmer equation:

I0/I = 1 + Kq.[Q] (2)

where, I0 and I are the fluorescence emission

intensities in the absence and presence of the

quencher respectively, Kq is the quenching constant

and [Q] is the concentration of the quencher The

value of Kq is given by the ratio of slope to intercept

in a plot of I0/I Vs [Q] The quenching constants for

the complexes are presented in table 3 These values

suggest strong interaction of the complexes with the

DNA

Table 3: Quenching constant (Kq) values of the

complexes

Ni(II)-PLTSC-(gly-gly) 4.29104

Ni(II)-PLTSC-(gly-leu) 4.11104

Ni(II)-PLTSC-(gly-tyr) 3.83104

Ni(II)-PLTSC-(gly-val) 4.09104

3.2.3 Viscosity studies

Though optical photophysical methods provide the

information about the binding modes of the metal

complexes to the DNA, hydrodynamic

measurements that are sensitive to the change in DNA length are considered to be the most effective means of a binding model in solution, in the absence

of crystallographic structural data According to the classical intercalation model, lengthening of DNA double helix takes place due to the separation of base pairs to accommodate the binding complexes, resulting in the increase of DNA viscosity In contrast, partial or non-classical intercalation of the complex leads to a bend or kink in the DNA helix, decreasing its viscosity.[49] The changes in the viscosity of DNA upon addition of mixed ligand

Ni(II) complexes, 1-4 are shown in figure 4 The

viscosity of DNA increased steadily with the increase in the concentration of the complexes, supporting the intercalation of the complexes between the DNA base pairs The binding ability of the complexes to increase the DNA viscosity follows

the order, 4 > 1 > 2 > 3 The observed results for

viscosity study are in accordance with the UV-Vis absorption titration results

3.3 Hydrolytic cleavage of DNA

The nuclease activity of the mixed ligand complexes

1-4 has been studied by the agarose gel

electrophoresis, using supercoiled pBR 322 DNA (100 ng/μL) in 5 mM Tris–HCl/50 mM NaCl buffer solution (pH 7.2), without any added reagents When plasmid DNA is subjected to electrophoresis, the fastest migration will be observed for the supercoiled (SC) form (Form I) If one strand is cleaved, the supercoiled form relaxes to give a slow moving open circular or nicked circular (NC) form (Form II) If both the strands are cleaved, a linear form (Form III) is generated which migrates at the rate between the forms I and II.[50] In the present investigation, pBR 322 plasmid DNA was incubated with three different concentrations (20, 40 and 60 µM) of the mixed ligand complexes, in TrisHCl buffer for 1hour at 37 °C All the complexes have cleaved the plasmid DNA effectively into nicked form It was also observed that the extent of DNA cleavage increased with the complex concentration The nuclease activity of the metal complexes on pBR 322 DNA is presented in figure 5

3.4 Antibacterial activity

Antibacterial activity of the metal complexes against some gram positive and gram negative bacteria such

as Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa was

evaluated by agar well diffusion method The zone

of inhibition values (mm) are presented in table 4

Trang 7

The complexes could not inhibit the growth of gram

negative bacteria and are moderately active against

gram positive bacteria The resistance of gram

negative bacteria is attributed to their outer membrane, which is an important barrier and provides protection against many antibiotics.[51]

Table 4: The zone of inhibition (mm) values for the antibacterial activity of the complexes

Inhibition zone (mm) Metal complexes (100 μg/well) Positive control

Amikacin (30 μg/well)

Negative control DMSO

3.5 Antioxidant activity

Many Ni(II) complexes bearing biologically

important ligands were reported to possess free

radical scavenging properties.[52] The DPPH assay is

extensively used in radical scavenging or hydrogen

donor ability of the tested samples.[53] The free

radical scavenging activity of the metal complexes

has been investigated using DPPH assay and the

corresponding IC50 values have been tabulated in

Table 5 The inhibitory effect of the complexes on

DPPH radical is also depicted in figure 6

Table 5: The DPPH scavenging activity (IC50 in μM)

of the metal complexes

Compounds

IC50 86.4 102.7 15.8 130.9 1.94

3.6 Anti-inflammatory activity

The anti-inflammatory activity of the

ternarycomplexes indicated mild inhibition of

bovine serum albumin denaturation The results are

compared with the standard drug Diclofenac sodium

The complexes exhibited less activity compared to

Diclofenac sodium Among the complexes

[Ni(PLTSC)(gly-tyr)] showed the highest inhibitory

efficiency The IC50 values (concentration of the

inhibitor required to reduce the denaturation of BSA

by half) of the complexes and the standard drug are

presented in table 6

3.7 Docking studies

Molecular docking is a powerful technique in

predicting the binding affinity and orientation of various molecules to their biological targets like DNA and proteins In the present study, molecular docking is carried out to investigate the extent and mode of binding affinity between DNA and mixed ligand Ni(II) complexes The dock score provided in table 7 represents the binding affinity of nickel

complexes 1 to 4 All the complexes show good

dock score ranging from -7.91 to -6.46 kcal/mol

Complex 2 showed the highest dock score of -7.91 kcal/mol Complex 4 having highest binding

constant values showed dock score of -7.08

kcal/mol Analysis of docking pose of complex 4

was carried out to understand the binding mode

Figure 7 shows the dock pose of complex 4 in the

intercalation site of DNA, figure 7 shows the interaction of metal complex with DNA bases

Complex 4 forms two hydrogen bonds with G13

base The pyridine moiety of the complex intercalates between the bases pairs G13:C4 and C12:G5, forming strong π-π interaction with DNA bases G13 and G5 All the complexes have the pyridine moiety hence show the similar kind of π-π interaction resulting in similar binding affinity values The results obtained in docking studies also support the DNA binding interaction observed in experimental techniques

Table 6: The anti-inflammatory activity (IC50 in μM)

of the metal complexes Compounds

sodium

IC50 118.02 139.69 69.86 129.87 47.84

Trang 8

Table 7: Dock scores and calculated inhibition

constants of the complexes

Complexes

Dock score (kcal/mol) -6.87 -7.91 -6.46 -7.08

Estimated Inhibition

Constant, Ki (µM)

9.23 1.6 18.3 6.46

4 CONCLUSION

The mixed ligand chelates of Ni(II) complexes with

PLTSC (A) and dipeptides (B) have been

synthesized and characterized by various

spectroscopic and analytical techniques The

interactions between the metal complexes and CT

DNA have been studied by UV-Visible absorbance

and fluorescence emission spectroscopic techniques,

and also by viscosity measurements The results

obtained indicated the intercalation of metal

complexes between the base pairs of DNA

Molecular docking studies on the interaction of the

metal complexes with DNA have also confirmed the

experimental results The complexes also exhibited

excellent hydrolytic cleavage of pBR 322

supercoiled DNA, which was studied by gel

electrophoresis The complexes displayed moderate

antibacterial activity against gram positive bacteria

The IC50 values of the metal complexes for anti

oxidant activity by DPPH method and

anti-inflammatory activity indicated that the complex

[Ni-(PLTSC)(Gly-Val)] is more active than the other

complexes studied

Acknowledgements The authors are thankful to

Osmania University, JNT University Hyderabad and

St Francis College for Women for providing the

research facilities to carry out this work

REFERENCES

1 M Tümer Polydentate Schiff-base ligands and their

Cd(II) and Cu(II) metal complexes: synthesis,

characterization, biological activity and

electrochemical properties, J Coord Chem., 2007,

60, 2051-2065

2 A Golcu, M Tümer, H Demirelli, A R Wheatley

Cd(II) and Cu(II) complexes of polydentate Schiff

base ligands: synthesis, characterization, properties

and biological activity, Inorg Chim Acta, 2005, 358,

1785-1797

3 M F Belicchi, G F Gasparri, E Leporati, C

Pelizzi, P Tarasconi, G Tosi Thiosemicarbazones as

co-ordinating agents Solution chemistry and X-ray

structure of pyridoxal thiosemicarbazonetrihydrate

and spectroscopic properties of its metal complexes,

J Chem Soc Dalton Trans., 1986, 2455-2461

4 M A S Chagas., C C Gatto Structural investigation of a novel Copper(II) complex with

pyridoxalthiosemicarbazone, ActaCryst., 2018, A74,

a12

5 C C Gatto, M A S Chagas, I J Lima, F M Andrade, H D Silva, G R Abrantes, E.P.S Lacerda Copper(II) complexes with pyridoxaldithiocarbazate and thiosemicarbazone ligands: crystal structure, spectroscopic analysis and

cytotoxic activity, Trans Met Chem., 2019, 44, 1-12

6 T Jakusch, K Kozma, E A Enyedy, N V May, A Roller, R Kowol, B K Keppler, T Kiss Complexes

of pyridoxalthiosemicarbazones formed with vanadium(IV/V) and copper(II): Solution equilibrium

and structure, Inorg Chim Acta, 2018, 472, 243-253

7 V M Leovac, L S Jovanovic, V S Jevtovic, G Pelosi, F Bisceglie Transition metal complexes with thiosemicarbazide-based ligand- Part LV: Synthesis and X-ray structural study of novel Ni(II) complexes with pyridoxalsemicarbazone and

pyridoxalthiosemicarbazone, Polyhedron, 2007, 26,

2971-2978

8 M F Belicchi, G F Gasparri, P Tarasconi, R Albertini, S Pinelli, R Starcich Synthesis, spectroscopic and structural characterization, and biological activity of aquachloro(pyridoxalthiosemicarbazone) Copper(II)

chloride, J Inorg Biochem., 1994, 53, 13-25

9 J S Casas, M C M C Rodrı ́guez-Argüelles, U Russo, A Sánchez, J Sordo, A Vázquez-López, S Pinelli, P Lunghi, A Bonati, R Albertini Diorganotin (IV) complexes of pyridoxalthiosemicarbazone: Synthesis,

spectroscopic properties and biological activity, J

Inorg Biochem., 1998, 69, 283-292

10 M F Belicchi, F Bisceglie, G Pelosi, P Tarasconi, R Albertini, P P Dall'Aglio, S Pinelli, Bergamo, G Sava Synthesis, characterization and biological activity of Copper complexes with pyridoxalthiosemicarbazone derivatives X-ray

crystal structure of three dimeric complexes, J Inorg

Biochem., 2004, 98, 301-312

11 C M Murdoch, M K Cooper, T W Hambley, W

N Hunter, H C Freeman Three Chromium(III)

complexes with dipeptide ligands, J Chem Soc

Chem Commun., 1986, 17, 1329-1331

12 S H Kim, R B Martin Noncovalent ligand-metal and ligand-ligand interactions in tridentate

(dipeptide)palladium(II) complexes, J Am Chem

Soc., 1984, 106, 1707-1712

13 M Nath, S Pokharia, G Eng, X Song, A Kumar New triorganotin(IV) derivatives of dipeptides as

anti-inflammatory-antimicrobial agents,Eur J Med

Chem., 2005, 40, 289-298

14 M Nath, S Pokharia, G Eng, X Song, M Gielen,

M Kemmer, M Biesemans, R Willem, D de Vos New organotin(IV) derivatives of dipeptides as

models for metal-protein interactions: in vitro anti‐

Trang 9

tumor activity, Appl Orgmet Chem., 2003, 17,

305-314

15 I Neundorf Metal complex-peptide conjugates: How

to modulate bioactivity of metal-containing

compounds by the attachment to peptides, Curr Med

Chem., 2017, 24, 1853-1861

16 S M S A Abdel-Mottaleb, E H Ismail Transition

metal complexes of mixed bioligands: Synthesis,

characterization, DFT modeling, and application, J

Chem., 2019, 2019, 3241061, 18 pages

17 V S Shivankar, R B Vaidya, S R Dhrwadkar, N

V Thakkar Synthesis, characterization, and

biological activity of mixed ligand Co(II) complexes

of 8‐hydroxyquinoline and some amino acids, Synth

React Inorg Met Org Chem., 2003, 33, 1597-1622

18 S H Sakr, H S Elshafie, I Camele, S A Sadeek

Synthesis, spectroscopic, and biological studies of

mixed ligand complexes of gemifloxacin and glycine

with Zn(II), Sn(II), and Ce(III), Molecules, 2018, 23,

1182

19 T K Pal, Md A Alam, S Paul, Md C Sheikh

Spectral, magnetic, thermal, antioxidant and

biological studies on new mixed ligand complexes,

J King Saud Univ Sci., 2019, 31, 445-451

20 A Temitayo, A Ezekiel, O Efere, O Idowu,

I David, J Johan Synthesis characterization and

biological activities of coordination compounds of

4-Hydroxy-3-nitro-2H-chromen-2-one and its

aminoethanoicacid and Pyrrolidine-2-carboxylic acid

mixed ligand complexes, Bioinorg Chem Appl.,

2017, 2017, 6426747

21 K Prashanthi, I Kiranmai, K K Sathish, K C

Vijay, Shivaraj Spectroscopic Characterization and

biological activity of mixed ligand complexes of

Ni(II) with 1,10-phenanthroline and heterocyclic

Schiff bases, Bioinorg Chem Appl., 2012, 2012,

948534, 8 pages

22 S S Chavan, S K Sawanth, V A Sawanth, G K

Lahiri Mixed-ligand complexes of copper(I) with

Schiff base and triphenylphosphine: Effective

catalysts for the amination of aryl halide, Inorg

Chem Commun., 2011, 14, 1373-1376

23 E W Y Tido, C Faulman, R Roswanda, A

Meetsma, P J V Koningsbruggen Tuning of the

charge in octahedral ferric complexes based on

pyridoxal-N substituted thiosemicarbazone ligands,

Dalton Trans., 2001, 39, 1643-1651

24 B N Figgis, J Lewis The Magnetochemistry of

complex,Mod Coord Chem., 1960, Inter Science,

New York

25 M N Patel, S H Patel, P B Pansuriya DNA

binding and cleavage by dinuclear Nickel(II)

complexes with neutral bidentate ligands and

ciprofloxacin, Med Chem Res., 2011, 20,

1371-1384

26 S S Bhat, A A Kumbhar, H Heptullah, A Khan,

V V Gobre, S P Gejji, V G Puranik Synthesis,

electronic structure, DNA and protein binding, DNA

cleavage, and anticancer activity of

fluorophore-labeled Copper(II) complexes, Inorg Chem., 2011,

50, 545-558

27 A K Srivastava, N Kumari, R A Khan, R Rai, G Rai, S Tabassum, L Mishra DNA cleavage activity and cytotoxicity of mononuclear and trinuclear Cu(II) complexes containing 1H-pyrazole-3,5-dicarboxylic

acid as ligand, Ind J Chem., 2013, 52A, 835-844

28 H Wu, T Sun, K Li, B Liu, F Rou, F Jia, J Yuan,

Y Bai Synthesis, crystal structure, and DNA-binding studies of a Nickel(II) complex with the

bis(2-benzimidazolymethyl)amine ligand, Bioinorg

Chem Appl., 2012, 2012, 609796

29 P R Reddy, A Shilpa Interaction of DNA with small molecules: Role of copper histidyl peptide complexes in DNA binding and hydrolytic cleavage,

Ind J Chem., 2010, 49A, 1003-1015

30 M Balouiri, M Sadiki, S K Ibnsouda Methods

for in vitro evaluating antimicrobial activity: A

review, J Pharm Anal., 2016, 6, 71-79

31 I P Ejidike, P A Ajibade Synthesis, characterization, antioxidant, and antibacterial studies

of some metal(II) complexes of tetradentate Schiff base ligand:(4E)-4-[(2-(E)-[1-(2,4-dihydroxyphenyl)ethylidene]aminoethyl)imino]penta

n-2-one, Bioinorg Chem Appl., 2015, 2015, 890734

32 C Sulpizio, S T R Müller, Q Zhang, L Brecker, A Rompel Synthesis, characterization, and antioxidant activity of Zn 2+ and Cu 2+ coordinated

polyhydroxychalcone complexes, Monatsh Chem.,

2016, 147, 1871-1881

33 Y Mizushima, M Kobayashi Interaction of anti-inflammatory drugs with serum proteins, especially

with some biologically active proteins, J Pharm

Pharmacol., 1968, 20, 169-173

34 S Sakat, A R Juvekar, M N Gambhire Invitro

antioxidant and anti-inflammatory activity of

methanol extract of Oxalis corniculata Linn, Int J

Pharm Pharm Sci., 2010, 2, 146-155

35 D Ashok, E V L Madhuri, M Sarasija, S Sreekanth, M Vijjulatha, D A Malini, S R Sagurthi Synthesis, biological evaluation and molecular docking of spirofurochromanone derivatives as anti-inflammatory and antioxidant

agents, RSC Adv., 2017, 7, 25710-25724

36 L A D Williams, A O'Connar, L Latore, O Dennis, S Ringer, J A Whittaker, J Conrad, B

Vogler, H Rosner, W Kraus The in vitro

anti-denaturation effects induced by natural products and non-steroidal compounds in heat treated (immunogenic) bovine serum albumin is proposed as

a screening assay for the detection of anti-inflammatory compounds, without the use of animals, in the early stages of the drug discovery

process, West Indian Med J., 2008, 57, 327-331

37 N H Grant, H E Alburn, C Keyzanauskas Stabilization of serum albumin by anti-inflammatory

drugs, Biochem Pharmacol., 1970, 19, 715-722

38 N Nagula, S Kunche, M Jaheer, R Mudavath, S Sivan Spectroanalytical, computational and in vitro biological studies of novel substituted quinolone

hydrazone and its metal complexes, J Fluoresc.,

Trang 10

2018, 28, 225-241

39 B Anupama, A Aruna, V Manga, S Sivan, M V

Sagar, R Chandrashekar Synthesis, spectral

characterization, DNA/protein binding, DNA

cleavage, cytotoxicity, antioxidative and molecular

docking studies of Cu(II)complexes containing Schiff

base-bpy/Phenligands, J Fluoresc., 2017, 27,

953-965

40 M S Searle, A J Maynard, H E L Williams DNA

recognition by the anthracycline antibiotic

respinomycin D: NMR structure of the intercalation

complex with d(AGACGTCT)(2), Org Biomol

Chem., 2003, 1, 60-66

41 M S Sanner Python: A programming language for

software integration and development, J Mol

Graphics Mod., 1999, 17, 57-61

42 V M Leovac, V S Jevtovic, L S Jovanovic, G A

Bogdanovic Metal complexes with Schiff-base

ligands pyridoxal and semicarbazide-based

derivatives, J Serb Chem Soc., 2005, 70, 393-422

43 L S Vojinović-Ješić, M V Rodić, B B Holló, S A

Ivković, V M Leovac, K M Szécsényi Synthesis,

characterization and thermal behavior of Copper(II)

complexes with pyridoxalthiosemi (PLTSC)- and

S-methylthiosemicarbazone (PLITSC), J Therm Anal

Calorim., 2016, 123, 2069-2079

44 G Facchin, E Kremer,E J Baran, E E Castellano,

O E.Piro, J Ellena, A J Costa-Filho, M H Torre

Structural characterization of a series of new

Cu-dipeptide complexes in solid state and in solution,

Polyhedron, 2006, 25, 2597-2604

45 E Katsoulakou, M Tiliakos, G Papaefstathiou,

A.Terzis, C Raptopoulou, G Geromichalos, K

Papazisis, R Papi, A Pantazaki, D Kyriakidis, P

Cordopatis, E Manessi-Zoupa Diorganotin(IV)

complexes of dipeptides containing the

α-aminoisobutyryl residue (Aib): Preparation, structural

characterization, antibacterial and antiproliferative

activities of [(n-Bu)2 Sn(H −1 L)] (LH =

H-Aib-L-Leu-OH, H-Aib-L-Ala-OH), J Inorg Biochem., 2008,

102, 1397-1405

46 A B P Lever Inorganic Electronic Spectroscopy,

2 nd ed., Elsevier Publishing Co., Amsterdam, 1984

47 R Natarajan, J Ramaraj, S Mariyyappan, B Raja,

R Y Chithu Synthesis, DNA binding and antimicrobial studies of novel metal complexes containing a pyrazolone derivative Schiff base,

Chem Papers, 2010, 64, 318-328

48 R Singh, R Jadeja, M Thounaajam, R Devkar, D Chakraborthy Synthesis, characterization, DNA binding and cytotoxicity studies of moxifloxacinato

complexes, Transi Metal Chem., 2012, 37, 541-551

49 T Deepthi, P Venugolan Synthesis, DNA-binding, and cytotoxic studies on three Copper(II) complexes

of unsymmetrical synthetic analogues of curcumin, J

Coord Chem., 2016, 69, 3403-3416

50 V Narendrula, K Marri, R Gali, N Ganji, S Daravath, Shivraj DNA interactions and biocidal activity of metal complexes of benzothiazole Schiff

bases: synthesis, characterization and validation J

Chem Sci., 2017, 129, 609-622

51 A H Delcour Outer membrane permeability and

antibiotic resistance, Biochim Biophys Acta, 2009,

1794, 808-816

52 S Perontsis, A Tialiou, A G Hatzidimitriou, A N Papadopoulos, G Psomas Nickel(II)-indomethacin mixed-ligand complexes: Synthesis, characterization, antioxidant activity and interaction with DNA and

albumins, Polyhedron, 2017, 138, 258-269

53 H Yapati, S R Devineni, S Chirumamilla, S Kalluru Synthesis, characterization and studies on antioxidant and molecular docking of metal

complexes of 1-(benzo[d]thiazol-2-yl)thiourea, J

Chem Sci., 2016, 128, 43-51

Corresponding author: Ch Venkata Ramana Reddy

Jawaharlal Nehru Technological University Hyderabad Hyderabad, Telangana State, India

E-mail: vrr9@jntuh.ac.in

Figures

Figure 1: Proposed structure of the mixed ligand Ni(II) complexes Where, R = H (Gly-Gly),

-CH2-CH(CH3)2 (Gly-Leu), -CH2-C6H4-OH (Gly-Tyr) and -CH(CH3)2 (Gly-Val)

Định dạng
Số trang	12
Dung lượng	0,94 MB