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 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.
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