Ruthenium complex intercalation in DNA

   Ruthenium(II) polypyridyl complexes have attracted comprehensive attention due to their significance in the development of new therapeutic agents and novel nucleic acid structural probes. Ru(II) complexes could potentially be modified to exhibit pH-dependent DNA damage, showing increased selectivity towards cancer cells. A comparative study of the DNA binding properties of a carboxyl group- and a salicylic group-containing Ru(II) complexes is reported. 2,2'-bipyridine (bpy) is chosen as the ancillary ligand of the two complexes since the ‘‘parent’’ complex [Ru(bpy)3]2+ binds extremely to doublestrand DNA so that we can reasonably examine the effects of CAIP and HCIP structures on the DNA binding properties of these two complexes. The salicylic group-containing Ru(II) complex can form an intramolecular hydrogen bond between a deprotonated carboxyl group and an adjacent hydroxyl group to extend the planarity and the p system, and thus bringing about clearly enhanced affinity to the DNA, as revealed by spectrophotometric methods and viscosity measurements [11].

Fig.1 Ruthenium(II) complexes of [Ru(bpy)2(CAIP)]Cl2 and [Ru(bpy)2(HCIP)]Cl2 (where bpy = 2,2'-bipyridine, CAIP = 4-carboxyl-imidado[4,5-f][1,10]-phenanthroline, HCIP = 3-hydroxyl-4-carboxyl-imidado[4,5-f][1,10]- phenanthroline).

  UV–visible absorption spectroscopy
   With increasing of DNA concentration, the MLCT transition bands of the two complexes at 458 and 456 nm exhibit hypochromism of 20.8% and 21.4%, as well as bathochromism ~2 and ~5 nm, respectively. These spectral characteristics suggest that the complexes might bind to DNA by an intercalative mode. After intercalating the base pairs of DNA, the π* orbital of the intercalated ligand could couple with π orbit of base pairs, thus decreasing the π–π* transition energy, and further resulting in the bathochromism. On the other hand, the coupling π* orbital was partially filled by electrons, thus decreasing the transition probabilities, and concomitantly, resulting in the hypochromism. To compare quantitatively the binding strengths of the complexes, the intrinsic binding constants K with DNA are determined from the decay of the absorbance at 458 nm for complex [Ru(bpy)2(CAIP)]2+ and 456 nm for [Ru(bpy)2(HCIP)]2+ [11].

Fig.2 Absorption spectra of the complexes upon addition of DNA (a) for [Ru(bpy)2(CAIP)]2+; (b) for [Ru(bpy)2(HCIP)] 2+.

   The extent of the hypochromism in MLCT bands and intrinsic binding constants commonly parallel the intercalative binding strength so we can deduce that [Ru(bpy)2(HCIP)]2+ intercalate into DNA more strongly than [Ru(bpy)2(CAIP)]2+ does [11].

  Luminescence studies
   The magnitudes of emission enhancement for [Ru(bpy)2(HCIP)]2+ are much larger than that for [Ru(bpy)2(CAIP)]2+, therefore we can also infer that [Ru(bpy)2(HCIP)]2+ inserts more deeply into DNA than [Ru(bpy)2(CAIP)]2+ does, which can be further supported by steady–state emission quenching experiment. [Fe(CN)6]4- is used as the quencher in emission quenching experiment. As illustrated in Fig. 3, in the absence of DNA, the emission of the two complexes was efficiently quenched by the quencher (curves a and b). In the presence of DNA, the slopes of the plots are remarkably decreased and the slope for [Ru(bpy)2(HCIP)]2+ (curve d) is smaller than that for [Ru(bpy)2(CAIP)]2+ (curve c). The anion [Fe(CN)6]4- has been shown to be able to distinguish differently bound ruthenium(II) species [11].

Fig.3 Emission quenching of the complexes with increasing concentrationsof [Fe(CN)6]4-at [Ru] = 3.47 lM, [DNA]/[Ru] = 40:1.   Curve a for free [Ru(bpy)2(CAIP)]2+, curve b for free [Ru(bpy)2(HCIP)]2+,curve c for [Ru(bpy)2(CAIP)]2+ + DNA, and curve d for [Ru(bpy)2(HCIP)]2+ + DNA.

   When bound to DNA the complex can be protected from the quencher because highly negatively charged [Fe(CN)6]4-would be repelled by the negative DNA phosphate backbone, hindering quenching of the emission of the bound complex. The slope can be taken as a measure of binding affinity. So we can conclude that [Ru(bpy)2(HCIP)]2+ intercalates into DNA more strongly than [Ru(bpy)2(CAIP)]2+ does [11].

   Viscosity measurements
   To clarify the nature of the interaction between the complexes and DNA, we used viscosity measurements. The viscosity of a DNA solution is sensitive to the addition of organic drugs and metal complexes bound by intercalation. Intercalation is expected to lengthen the DNA helix as the base pairs are pushed apart to accommodate the bound ligand, leading to an increase in the DNA viscosity. In contrast, a partial, non-classical intercalation of ligand could bend (or kink) the DNA helix, reduce its effective length, and concomitantly, its viscosity. We examined the effect on the specific relative viscosity of DNA upon addition of the complexes. The viscosities of DNA bound to the complexes increase with the increment of the complexes. The experimental results suggest that the complexes intercalate the base pairs of DNA. From the larger changes in viscosity caused by the addition of [Ru(bpy)2(HCIP)]2+, we can also see that [Ru(bpy)2(HCIP)]2+ intercalates into DNA more effectively than [Ru(bpy)2(CAIP)]2+ does [11].

   In conclusion, the distinct changes in specific viscosity of DNA upon addition of the Ru(II) complexes, and hypochromicity at different UV–visible absorption bands, and the enhancements in integrated emission intensities of the Ru(II) complexes and the inefficient emission quenching of the Ru(II) complexes by [Fe(CN) 6]4- upon addition of DNA all indicate the effective intercalation of [Ru(bpy)2(CAIP)]2+ and [Ru(bpy)2(HCIP)]2+into DNA, and the stronger binding affinity to DNA of [Ru(bpy)2(HCIP)]2+ than that of [Ru(bpy)2(CAIP)]2+. The enhanced DNA affinity observed for Ru(bpy)2HCIP)]2+ over [Ru(bpy)2(CAIP)]2+ is ascribed to the formation of an intramolecular hydrogen bond between a deprotonated carboxyl group and an adjacent hydroxyl group on [Ru(bpy) 2(HCIP)]2+ which extending the planarity and π system of HCIP [11].