| Summary: | This work aimed to evaluate in vitro DNA binding mechanistically of cationic nitrosyl ruthenium complex [RuNOTSP]<sup>+</sup> and its ligand (TSPH<sub>2</sub>) in detail, correlate the findings with cleavage activity, and draw conclusions about the impact of the metal center. Theoretical studies were performed for [RuNOTSP]<sup>+</sup>, TSPH<sub>2</sub>, and its anion TSP<sup>−2</sup> 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]<sup>+</sup> and TSPH<sub>2</sub> 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<sub>b</sub>), (2.4 ± 0.2) × 10<sup>5</sup> M<sup>−1</sup> ([RuNOTSP]<sup>+</sup>) and (1.9 ± 0.3) × 10<sup>5</sup> M<sup>−1</sup> (TSPH<sub>2</sub>), as well as the enhancement dynamic constants (K<sub>D</sub>), (3.3 ± 0.3) × 10<sup>4</sup> M<sup>−1</sup> ([RuNOTSP]<sup>+</sup>) and (2.6 ± 0.2) × 10<sup>4</sup> M<sup>−1</sup> (TSPH<sub>2</sub>), reveal that [RuNOTSP]<sup>+</sup> has a greater binding propensity for DNA compared to TSPH<sub>2</sub>. Stopped-flow investigations showed that both [RuNOTSP]<sup>+</sup> and TSPH<sub>2</sub> 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]<sup>+</sup> and TSPH<sub>2</sub>, the detailed binding parameters were established. The total binding constants for [RuNOTSP]<sup>+</sup> (K<sub>a</sub> = 43.7 M<sup>−1</sup>, K<sub>d</sub> = 2.3 × 10<sup>−2</sup> M<sup>−1</sup>, ΔG<sup>0</sup> = −36.6 kJ mol<sup>−1</sup>) and TSPH<sub>2</sub> (K<sub>a</sub> = 15.1 M<sup>−1</sup>, K<sub>d</sub> = 66 × 10<sup>−2</sup> M, ΔG<sup>0</sup> = −19 kJ mol<sup>−1</sup>) revealed that the relative reactivity is approximately ([RuNOTSP]<sup>+</sup>)/(TSPH<sub>2</sub>) = 3/1. The significantly negative ΔG<sup>0</sup> values are consistent with a spontaneous binding reaction to both [RuNOTSP]<sup>+</sup> and TSPH<sub>2</sub>, 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]<sup>+</sup> was a more highly effective DNA binder than the ligand TSPH<sub>2</sub> via strong electrostatic interaction with the phosphate end of DNA. Because of its higher DNA binding affinity, cationic [RuNOTSP]<sup>+</sup> demonstrated higher cleavage efficiency towards the minor groove of pBR322 DNA via the hydrolytic pathway than TSPH<sub>2</sub>, revealing the synergy effect of TSPH<sub>2</sub> in the form of the complex. Furthermore, the mode of interaction of both compounds with ctDNA has also been supported by molecular docking.
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