Light-induced cleavage of DNA by a variety of metal complexes, predominantly ruthenium complexes have been extensively studied due to their potential application as therapeutic agents. The redox-active metal complexes were found to mediate nucleobase damage typically in the presence of oxidizing agents, such as hydrogen peroxide or per acids. Ru(II) polypyridyl complexes were found to photocleave DNA by sensitization of singlet oxygen. In others, when powerful oxidizing groups were attached to the Ru(II) polypyridyl complexes, the reaction with DNA involved primarily electron transfer. We describe a case of efficient light-induced direct strand scission of DNA by the modified tris(2,2'-bipyridyl)Ru(II) complex, Ru(II)-2, in which two hydroxamic acid groups are attached to one of three bipyridyl ligands (Fig 1). The photo-chemical DNA cleavage event results solely from the absorption of visible light and molecular oxygen is proposed as the quencher molecule in our system .
Fig.1 Molecular structures of the ruthenium complexes studied.
A number of derivatives of ruthenium tris(bipyridine) complexes were studied in relation to their photo-chemical behavior in the presence of DNA (Fig 1). Irradiation (λexc > 380 nm) of Ru(II)-2 in aqueous solution in the presence of super-coiled plasmid DNA (pLitmus28) results in DNA cleavage to fragments of markedly reduced size (as was demonstrated by gel electrophoresis and by imaging of plasmid DNA using atomic force microscopy (AFM)) .
When the solution of the DNA was either kept in the dark with or without Ru(II)-2 or irradiated in the absence of the metal complex, no smearing was observed. Some DNA nicking is observed only in the presence of light. This may have some contribution to the observed photo-chemistry. As some of the supercoiled plasmid is nicked, the relaxed form of the plasmid may be more readily accessible to the additional DNA damage exerted by the metal complex Ru(II)-2 .
In order to accentuate the unique photo-chemical behavior of Ru(II)-2, we have repeated the photo-chemical reaction with DNA using other Ru(II) complexes, namely, Ru(bipy)3 2+ and Ru(phen)2dppz2+ where dppz = dipyridophenazine and phen = phenanthroline (Fig 1). The latter is a well-established metallointercalator capable of preferentially oxidizing guanines via sensitization of singlet oxygen .
The photo-cleavage reaction was found to be responsive to the ionic strength of the solution; no effect was observed when Ru(II)-2 was dissolved in phosphate buffer (PBS) or in 10 mM solution MgCl2. We believe that this behavior stems from the electrostatic competition between Ru(II) and the other cations in solution for the polyanionic DNA backbone. Some DNA photo-cleavage was observed in a 10 mM NaCl solution that may be correlated to a less effective competition for the DNA polyanoin backbone in comparison to divalent Mg2+, which is expected to be more effective in inhibiting the electrostatic interaction between the DNA polyanion backbone and Ru(II)-2 .
In conclusion, efficient DNA photo-cleavage of plasmid DNA was obtained by irradiating the ruthenium complex Ru(II)-2 with visible light. A suggested mechanism for the reaction is presented in Scheme 1:
Schema 1. Proposed mechanism for the photo-physical behavior of Ru(II)-2 and the different species involved in the photo-degradation of he DNA.
Upon irradiation of Ru(II)-2 in the presence of oxygen, an electron transfer from Ru(II) to O2 takes place, resulting in the formation of a genuine cage complex consisting of Ru(III) and superoxide radical anion (Scheme1(a)). The DNA molecule disrupts the cage complex, liberating both Ru(III), which oxidizes the DNA molecule mainly at guanine bases and the superoxide radical. Since the observed nonspecific DNA cleavage cannot be attributed solely to Ru(III) oxidation, the involvement of the reactive OH radicals, which are very potent and non-specific DNA cleaving agents, is suggested. The superoxide radical, which is liberated from the cage complex, is not considered to be a very reactive species towards DNA. This species abstracts a proton from the adjacent hydroxamic acid, reacts with another hydroperoxy radical to yield hydrogen peroxide, initiating a cycle that leads to the production of OH radicals (Scheme 1(b)) .
We are currently synthesizing new molecules where one of the bipyridyl ligands will be modified in order to achieve intercalation into the DNA backbone. These molecules are planned to be examined on 32P labeled oligonucleotides, either free in solution or covalently linked to pre-designed duplexes. Since these systems are both water soluble and are activated by visible light, optimization of the molecular structure in this family of molecules is promising for developing novel agents in the field of photo-dynamic therapy .