A Supramolecular Interaction of a Ruthenium Complex With Calf-Thymus DNA: A Ligand Binding Approach by NMR Spectroscopy

Abstract

Lawsone itself exhibits interesting biological activities, and its complexation with a metal center can improve the potency. In this context a cytotoxic Ru-complex, [Ru(law)(dppb)(bipy)] (law = lawsone, dppb = 1,4-bis(diphenylphosphino)butane and bipy = 2,2'-bipyridine), named as CBLAU, was prepared as reported. In this work, NMR binding-target studies were performed to bring to light the most accessible interaction sites of this Ru-complex toward Calf-Thymus DNA (CT-DNA, used as a model), in a similar approach used for other metallic complexes with anti-cancer activity, such as cisplatin and carboplatin. Advanced and robust NMR binding-target studies, among them Saturation Transfer Difference (STD)-NMR and longitudinal relaxometry (T₁), were explored. The ¹H and ³¹P -NMR data indicate that the structure of Ru-complex remains preserved in the presence of CT-DNA, and some linewidth broadening is also observed for all the signals, pointing out some interaction. Looking at the binding efficiency, the T₁ values are highly influenced by the formation of the CBLAU-DNA adduct, decreasing from 11.4 s (without DNA) to 1.4 s (with DNA), where the difference is bigger for the lawsone protons. Besides, the STD-NMR titration experiments revealed a stronger interaction (K_D = 5.9 mM) for CBLAU-DNA in comparison to non-complexed lawsone-DNA (K_D = 34.0 mM). The epitope map, obtained by STD-NMR, shows that aromatic protons from the complexed lawsone exhibits higher saturation transfer, in comparison to other Ru-ligands (DPPB and bipy), suggesting the supramolecular contact with CT-DNA takes place by the lawsone face of the Ru-complex, possibly by a spatial π-π stacking involving π-bonds on nucleic acids segments of the DNA chain and the naphthoquinone group.

Keywords: CT-DNA; NMR; anti-cancer; binding interactions; lawsone; ruthenium complex.

Figure 1

¹H-NMR spectra obtained at 600 MHz (for ¹H nucleus) and at 298 K for the characterization of the Ru-complex CBLAU (A) and the free ligand (lawsone) in (B).

Figure 2

Chemical structure for the Ru-complex (CBLAU) addressed in this work: the numbers 1 (black) correspond to the ligand lawsone; the number 2 (red) to aromatic protons of DPPB ligand; the number 3 (blue) to aliphatic hydrogens (butyl unit) between the two DPPB units and the number 4 (pink) to the hydrogens from the Bipy ligand.

Figure 3

¹H-NMR spectra obtained for CBLAU (5 mM): (A) with (red) and (B) without (black) CT-DNA (50 μM) at 600 MHz (for ¹H nucleus) and 298 K.

Figure 4

³¹P-NMR spectra obtained for CBLAU (5 mM): (A) with (red) and (B) without (black) CT-DNA (50 μM) at 161.96 MHz (for ³¹P nucleus) in DMSO:buffer (5:95 v/v) at 298 K.

Figure 5

Off-resonance STD spectrum (A) and STD diff-spectrum (B) obtained by applying 10.0 s of saturation time and used for the calculation of A_STD and consequent epitope map, highlighting the contribution (proximity) of each group present on CBLAU (5 mM), bipy (58.6%) (pink), butyl protons between the DPPB units (14.7%) (blue), lawsone (100.0%) (black), and DPPB (39.7%) (red) to CT-DNA (50 μM) surface.

Figure 6

Built-up curves of A_STD against saturation time at 298 K and 600 MHz (¹H nucleus) for CBLAU (5 mM) (A) and for the individual lawsone (5 mM) (B) in the presence of CT-DNA (50 μM).

Figure 7

Lineweaver-Burk plot for the experimental STD-NMR concentration experiments obtained for CBLAU and used to estimate the dissociation constant (K_D) for each group: lawsone (4.8 mM) (A), bipy (7.2 mM) (B), DPPB (7.4 mM) (C) and butyl group (4.3 mM) (D) and the average K_D value (5.9 mM) for this molecule (CBLAU) in interaction to CT-DNA.