Cite this paper: Vietnam J Chem., 2021, 59(1), 57-68 Article DOI: 10.1002/vjch.202000100 Synthesis, characterization and biological activity of mixed ligand chelates of Ni(II) with pyridoxalthiosemicarbazone and dipeptides A Saritha1, Ch Venkata Ramana Reddy2*, B Sireesha3 Department of Chemistry, St Francis College for Women, Hyderabad, India 500016 Department of Chemistry, Jawaharlal Nehru Technological University Hyderabad, Hyderabad, India 500085 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., glycyl-glycine (gly-gly), glycyl-L-leucine (gly-leu), glycyl-L-tyrosine (gly-tyr) and glycylL-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 nonelectrolytic 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, antiinflammatory and antibacterial activities and the findings have been reported Keywords Mixed ligand chelates, DNA interaction, antibacterial, anti-inflammatory, antioxidant, molecular docking 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, 57 Wiley Online Library © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH Vietnam Journal of Chemistry 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 (gly-tyr) and glycyl-L-valine (glyval) 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 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-gly/0.3293 g (1.7 mmol) of glyleu/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 hours A red colored precipitate was obtained by adjusting the pH to 7-8 with a Ch Venkata Ramana Reddy et al 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)] C13H18N6O5SNi (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-1cm2M1 ,10-3, DMSO]: 07 [Ni(PLTSC-H)(gly-leu-H)]C17H26N6O5SNi (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.CO) 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)].H2O C20H26N6O7SNi (3): Anal Calcd.: C, 43.39; H, 4.70; N, 15.19 Found: C, 43.35; H, 4.66; N, 15.22 APCIMS (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 [Ω1 cm2M-1,10-3, DMSO]: 10 [Ni(PLTSC-H)(gly-val-H)] C16H24N6O5SNi (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.CO) 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 UVvisible 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 © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 58 Vietnam Journal of Chemistry by DNA After each addition of CT-DNA to the complex, the resulting solution was incubated for 10 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: Synthesis, characterization and biological… [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] 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 h Then, a loading buffer (1 µl) containing % 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 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] 2.3.2 Competitive DNA binding fluorescence studies 2.5 Antibacterial assay 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] = and incubated for 30 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] 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 for adsorption Using a sterile plastic borer of 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] 2.3.3 Viscosity studies The antioxidant activity of the mixed ligand Ni(II) complexes has been evaluated using 2,2-diphenyl-2picrylhydrazyl (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, mL) and the final volume was made to 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 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 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 mM Tris.HCl/50 mM NaCl buffer (pH 7.2), by 2.6 Antioxidant activity © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 59 Vietnam Journal of Chemistry 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 % BSA and the complexes in phosphate buffer at pH 7.4 were incubated at 37 ºC for 20 Then they were heated at 51 ºC for 30 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 = [(AcontrolAsample)/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 cocrystallized 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] RESULTS AND DISCUSSION 3.1 Characterization of complexes The mixed ligand complexes, [Ni(II)-PLTSCdipeptide] 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: 0515 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 Ch Venkata Ramana Reddy et al were recorded in APCI-positive mode The mass spectra provide information regarding the 1:1:1 (M:A:L) behavior of complexes The complexes to (Fig S1-S4) show peaks at m/z 428[M]+, 486[M+1]+, 553[M]+ and 472[M+1]+ respectively Complexes and 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 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 (16501600 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] © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 60 Synthesis, characterization and biological… Vietnam Journal of Chemistry Table 1: IR spectral data of the ligands and ternary complexes (cm-1) Ligand/ complex PLTSC gly-gly gly-leu ly-tyr gly-val ν(NH2) ν(NH) ν(C=N) 3296 3184 3286 3313 3257 3317 3361 3317 3251 3311 3184 1625 ν(COO-) asym sym - 3055 3178 3068 3188 3030 3188 3070 3184 1606 1612 1610 1610 1575 1616 1556 1616 1604 1616 1558 1591 1408 1419 1406 1388 1419 1386 1406 1388 ν(ArC-O) 1126 1184 1151 1147 1147 1145 ν(NH) bend ν(C=S) - 841 1533 1554 1514 1560 1517 1560 1519 1553 812 815 812 815 3.1.3 Thermal analysis 3.2 DNA binding studies The thermal stability of the mixed ligand complexes was studied by recording themogravimetric analysis The TG curves of the ternary complexes to (Fig S14-S17) showed a three step decomposition process An initial weight loss around 100 ºC is observed in the complexes 1, and 4, which may be due to the loss of moisture The weight loss at 115 ºC in the thermogram of complex 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 3.2.1 Electronic absorption spectroscopy 3.1.4 Electronic spectra The mixed ligand Ni(II) complexes, 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 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 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 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 which are presented in table The magnitude of the binding constant values suggest the decreasing order of binding as > > > 3.2.2 Competitive DNA binding studies Ethidium bomide (EB) is widely used as a sensitive fluorescence probe for DNA It intercalates strongly 61 Wiley Online Library © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH Vietnam Journal of Chemistry Ch Venkata Ramana Reddy et al 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 Complex Kb (M-1) Ni(II)-PLTSC-(gly-gly) 4.32105 Ni(II)-PLTSC-(gly-leu) 3.88105 Ni(II)-PLTSC-(gly-tyr) 6.20103 Ni(II)-PLTSC-(gly-val) 7.48106 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 The obtained data were then fit into the Stern-Volmer equation: I0/I = + 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 These values suggest strong interaction of the complexes with the DNA Table 3: Quenching constant (Kq) values of the complexes Complex Ni(II)-PLTSC-(gly-gly) Ni(II)-PLTSC-(gly-leu) Ni(II)-PLTSC-(gly-tyr) Ni(II)-PLTSC-(gly-val) Kb (M-1) 4.29104 4.11104 3.83104 4.09104 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 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, > > > 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 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 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 © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 62 Synthesis, characterization and biological… Vietnam Journal of Chemistry 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 Microorganism S aureus B subtilis 8 10 10 10 19 22 - P aeruginosa - - - - 20 - E coli - - - - 26 - 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 The inhibitory effect of the complexes on DPPH radical is also depicted in figure Table 5: The DPPH scavenging activity (IC50 in μM) of the metal complexes Compounds IC50 86.4 102.7 15.8 130.9 Vitamin C 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 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 represents the binding affinity of nickel complexes to All the complexes show good dock score ranging from -7.91 to -6.46 kcal/mol Complex showed the highest dock score of -7.91 kcal/mol Complex having highest binding constant values showed dock score of -7.08 kcal/mol Analysis of docking pose of complex was carried out to understand the binding mode Figure shows the dock pose of complex in the intercalation site of DNA, figure shows the interaction of metal complex with DNA bases Complex 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 3.7 Docking studies IC50 118.02 139.69 69.86 129.87 Diclofenac sodium 47.84 Molecular docking is a powerful technique in © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 63 Vietnam Journal of Chemistry Ch Venkata Ramana Reddy et al Table 7: Dock scores and calculated inhibition constants of the complexes Complexes Dock score (kcal/mol) Estimated Inhibition Constant, Ki (µM) -6.87 -7.91 -6.46 -7.08 9.23 1.6 18.3 6.46 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 antiinflammatory activity indicated that the complex [Ni-(PLTSC)(Gly-Val)] is more active than the other complexes studied 10 Acknowledgements The 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albumins, Polyhedron, 2017, 138, 258-269 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) © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 66 Vietnam Journal of Chemistry Synthesis, characterization and biological… Figure 2: Absorption spectra of mixed ligand Ni(II) complexes (20 µM) in phosphate buffer (pH 7.2) upon addition of CT-DNA (0-20 µM) Arrow indicates hypochromism with increase in concentration of DNA Inset: Plot of [DNA]/(εb–εf) versus [DNA] Figure 3: Quenched fluorescence spectra of EB bound CT DNA in the absence and presence of mixed ligand Ni(II) complexes to Arrow shows the decrease in emission intensity with the increase in complex concentration Inset: Plot of I0/I versus [Complex]/[DNA] © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 67 Vietnam Journal of Chemistry Synthesis, characterization and biological… Figure 4: Effect of increasing amounts of EB and mixed ligand complexes of Ni(II) on the relative viscosities of CT DNA at 25 °C Figure 5: Hydrolytic cleavage of supercoiled pBR 322 DNA (100 ng/μL) by the mixed ligand Ni(II) complexes Lane 1: DNA control, Lanes 2-4: DNA + (20, 40 and 60 µM resp.), Lanes 5-7: DNA + (20, 40 and 60 µM), Lanes 8-10: DNA + (20, 40 and 60 µM) and Lanes 11-13: DNA + (20, 40 and 60 µM) Figure 6: DPPH scavenging activity of mixed ligand Ni(II) complexes Figure 7: (a) Dockpose of complex into intercalation site of DNA (DNA in ribbon form) showing hydrogen bond interaction and π-π interaction with DNA bases (b) Dockpose of complex into intercalation site of DNA showing two hydrogen bond interaction with DNA base G13 and π-π interaction with DNA bases G13 and G5 © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 68 ... 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. .. Spectroscopic Characterization and biological activity of mixed ligand complexes of 34 S Sakat, A R Juvekar, M N Gambhire Invitro antioxidant and anti-inflammatory activity of Ni(II) with 1,10-phenanthroline... Vietnam Journal of Chemistry Synthesis, characterization and biological? ?? Figure 4: Effect of increasing amounts of EB and mixed ligand complexes of Ni(II) on the relative viscosities of CT DNA at