This paper describes the synthesis of a new 9-aminomethylanthracene dye N-substituted with a pyridinylpolyamine side chain (4). The effects of NaCl and KCl on anthracene/DNA interactions were then studied, with the goal of simulating the conditions of high ionic strength that a DNA photosensitizer might encounter in the cell nucleus (~150 mM of NaCl and 260 mM of KCl). As exemplified by methylene blue (5), the expected effect of increasing ionic strength is to decrease DNA binding and photocleavage yields. In contrast, the addition of 150 mM of NaCl in combination with 260 mM of KCl to photocleavage reactions containing micromolar concentrations of 4 triggers the conversion of supercoiled, nicked, and linear forms of pUC19 plasmid into a highly degraded band of DNA fragments (350 nm hν, pH 7.0). Circular dichroism spectra point to a correlation between salt-induced unwinding of the DNA helix and the increase in DNA photocleavage yields. The results of circular dichroism, UV-vis absorption, fluorescence emission, thermal denaturation, and photocleavage inhibition experiments suggest that the combination of salts causes a change in the DNA binding mode of 4 from intercalation to an external interaction. This in turn leads to an increase in the anthracene-sensitized production of DNA-damaging reactive oxygen species.

We report the synthesis of new photonuclease 4 consisting of two acridine rings joined by a pyridine-based copper binding linker. We have shown that photocleavage of plasmid DNA is markedly enhanced when this ligand is irradiated in the presence of copper(II) (419 nm, 22 degrees C, pH 7.0). Viscometric data indicate that 4 binds to DNA by monofunctional intercalation, and equilibrium dialysis provides an estimated binding constant of 1.13 x 105 M-1 for its association with calf thymus DNA. In competition dialysis experiments, 4 exhibits preferential binding to GC-rich DNA sequences. When Cu(II) is added at a ligand to metal ratio of 1:1, electrospray ionization mass spectrometry demonstrates that compound 4 undergoes complex formation, while thermal melting studies show a 10 degrees C increase in the Tm of calf thymus DNA. Groove binding and intercalation are suggested by viscometric data. Finally, colorimetric and scavenger experiments indicate that the generation of Cu(I), H2O2, and superoxide contributes to the production of DNA frank strand breaks by the Cu(II) complex of 4. Whereas the strand breaks are distributed in a relatively uniform fashion over the four DNA bases, subsequent piperidine treatment of the photolysis reactions shows that alkaline labile lesions occur predominantly at guanine.

Substituted 1-aminocyclohexene-2,4-dicarbonitriles were obtained by reaction of a,P-unsaturated nitriles (two equivalents) with benzyl cyanide. By recrystallization from ethanol one diastereomeric racemate

We report the synthesis and characterization of N,N-bis[(7-dimethylamino)phenothiazin-5-ium-3-yl]-4,4-ethylenedipiperidine diiodide (3), consisting of two photosensitizing phenothiazinium rings attached to a central ethylenedipiperidine linker. At all time points (10, 30, 60 min) and all wavelengths (676, 700, 710 nm) tested, photocleavage of pUC19 plasmid DNA (22 degrees C and pH 7.0) was markedly enhanced by 1 microM of 3 in comparison to 1 microM of the parent phenothiazine methylene blue (MB). At concentrations of phenothiazine ranging from 5 to 0.5 microM, the photocleavage levels produced by compound 3 were consistently higher than the cleavage produced using approximately twice the amount of MB (e.g., 710 nm irradiation of 5 microM of 3 and 10 microM of MB cleaved the plasmid DNA in 93% and 71% yields, respectively). Scavenger assays provided evidence for the involvement of singlet oxygen and, to a lesser extent, hydroxyl radicals in DNA damage. Analysis of photocleavage products at nucleotide resolution revealed that direct strand breaks and alkaline-labile lesions occurred predominantly at guanine bases. While compound 3 and MB were both shown to stabilize duplex DNA, the DeltaTm values of calf thymus (CT) and C. perfringens DNAs were approximately three fold higher in the presence of compound 3. Finally, viscometric data indicated that CT DNA interacts with compound 3 and MB by a combination of groove binding and monofunctional intercalation, and with compound 3 by a third, bisintercalative binding mode.