Herein in continuation of our interest in
Herein, in continuation of our interest in designing new DNA photocleavage agents , we report the synthesis, characterization and DNA binding and cleaving properties of a novel compound: trans-N,N-dimethyl-10,11-di(pyridin-4-ium)-10,11-dihydro-9,12-dioxobenzo[e]phenanthrene tetrafluoroborate (1). As early as in 1947, it was shown by Schönberg that 9,10-phenanthrenequinone and other ortho-quinone derivatives form a dihydrodioxin photoproduct when mixed with substituted alkenes under visible light irradiation . Exposure of dihydrodioxin solutions to UV light can result in the reverse reaction of the photochemical release of the ortho-quinone and corresponding alkene . This reaction, therefore, was successfully implemented to photochemically mask highly reactive ortho-quinone species , . It is known that 9,10-phenanthrenequinone, a component of diesel exhaust particles–carcinogenic air pollutant, can induce the oxidative DNA damage by enhancing the production of reactive oxygen species . The mechanism of the ortho-quinone release was investigated in previous studies ,  in which the formation of an ortho-quinone radical anion, dihydrodioxin radical cation and Reactive Oxygen Species (ROS) was demonstrated. These species are well known to cause oxidative DNA damage, which may additionally contribute to DNA damaging properties of 1 , , , , [24a]. The interaction of 1 with DNA was described by a number of spectroscopic analytical tools, such as UV-Vis Aminoallyl-dUTP (DNA titration and DNA optical melting experiments), circular dichroism (CD) and fluorescence. Intercalative binding mode of 1 was unambiguously demonstrated by viscometric analysis. Photolysis of the compound in question with both visible and UV light was performed to better understand the photochemistry of 1 that is involved in DNA cleavage. ФX 174 photocleavage assay was used to evaluate DNA damage triggered by 1.
Main advantages of pyridinium containing dihydrodioxins are: a) ease of synthetic preparation from commercially available compounds; b) dihydrodioxins present a masked form of reactive ortho-quinones, release of which can be controlled by irradiation by different wavelengths of light; c) phenanthrene dihydrodioxin has a higher solubility in biological media comparing to the previously studied more hydrophobic pyrene dihydrodioxin derivative ; d) combination of flat aromatic moiety with positively charged pyridinium rings allows for both intercalative and electrostatic interactions of 1 with DNA.
Results and discussion
Conclusions A novel photoactivated DNA cleaving agent 1 was synthesized, characterized and its 9,10-phenanthrenequinone photochemical release was investigated. The interaction of 1 with DNA was studied by means of different analytical methods. Viscometric titration experiment demonstrated a gradual increase of the relative viscosity of the CT-DNA upon addition of 1. This result in combination with the UV-Vis absorption titration experiments which showed a significant bathochromic shift and hypochromic effect showed the intercalative fashion of DNA binding. The binding constant of 1 was derived from UV-Vis spectroscopic data and evaluated to be of 104M−1 order of magnitude that is comparable to the values of binding constants of typical intercalators. UV-Vis and CD Spectroscopy studies provided data to calculate the binding site size of 1. Both methods were in a good agreement with each other and indicated a 3.7 base pairs binding site. No significant difference of binding of 1 to either polyGC or polyAT double-stranded DNA sequences was observed. However, binding constant for polyAT appeared to be slightly higher than the one for polyGC. The conclusion on the intercalative nature of binding mode of 1 to DNA was supported by fluorescence titration of 1 by CT-DNA. The DNA addition resulted in quenching of the fluorescence of 1 due to the insertion of phenanthrene moiety of 1 between DNA base pairs. Changes in DNA structure and properties associated with interaction with 1 were studied by means of CD spectroscopy and DNA melting temperature analysis. CD spectroscopy experiments showed that addition of 1 to CT-DNA causes a partial conformational transition from B-form to Z-form of DNA molecule. DNA optical melting experiments demonstrated that 1 contributes greatly to the stabilization of the DNA duplex.