DNA Binding and Cleavage, Stopped-Flow Kinetic, Mechanistic, and Molecular Docking Studies of Cationic Ruthenium(II) Nitrosyl Complexes Containing "NS 4 " Core.

This work aimed to evaluate in vitro DNA binding mechanistically of cationic nitrosyl ruthenium complex [RuNOTSP] + and its ligand (TSPH 2 ) in detail, correlate the findings with cleavage activity, and draw conclusions about the impact of the metal center. Theoretical studies were performed for [RuNOTSP] + , TSPH 2 , and its anion TSP -2 using DFT/B3LYP theory to calculate optimized energy, binding energy, and chemical reactivity. Since nearly all medications function by attaching to a particular protein or DNA, the in vitro calf thymus DNA (ctDNA) binding studies of [RuNOTSP] + and TSPH 2 with ctDNA were examined mechanistically using a variety of biophysical techniques. Fluorescence experiments showed that both compounds effectively bind to ctDNA through intercalative/electrostatic interactions via the DNA helix's phosphate backbone. The intrinsic binding constants (K b ), (2.4 ± 0.2) × 10 5 M -1 ([RuNOTSP] + ) and (1.9 ± 0.3) × 10 5 M -1 (TSPH 2 ), as well as the enhancement dynamic constants (K D ), (3.3 ± 0.3) × 10 4 M -1 ([RuNOTSP] + ) and (2.6 ± 0.2) × 10 4 M -1 (TSPH 2 ), reveal that [RuNOTSP] + has a greater binding propensity for DNA compared to TSPH 2 . Stopped-flow investigations showed that both [RuNOTSP] + and TSPH 2 bind through two reversible steps: a fast second-order binding, followed by a slow first-order isomerization reaction via a static quenching mechanism. For the first and second steps of [RuNOTSP] + and TSPH 2 , the detailed binding parameters were established. The total binding constants for [RuNOTSP] + (K a = 43.7 M -1 , K d = 2.3 × 10 -2 M -1 , ΔG 0 = -36.6 kJ mol -1 ) and TSPH 2 (K a = 15.1 M -1 , K d = 66 × 10 -2 M, ΔG 0 = -19 kJ mol -1 ) revealed that the relative reactivity is approximately ([RuNOTSP] + )/(TSPH 2 ) = 3/1. The significantly negative ΔG 0 values are consistent with a spontaneous binding reaction to both [RuNOTSP] + and TSPH 2 , with the former being very favorable. The findings showed that the Ru(II) center had an effect on the reaction rate but not on the mechanism and that the cationic [RuNOTSP] + was a more highly effective DNA binder than the ligand TSPH 2 via strong electrostatic interaction with the phosphate end of DNA. Because of its higher DNA binding affinity, cationic [RuNOTSP] + demonstrated higher cleavage efficiency towards the minor groove of pBR322 DNA via the hydrolytic pathway than TSPH 2 , revealing the synergy effect of TSPH 2 in the form of the complex. Furthermore, the mode of interaction of both compounds with ctDNA has also been supported by molecular docking.