Synthesis and DNA interactions of a bis-phenothiazinium photosensitizer

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 mu M of 3 in comparison to 1 mu M of the parent phenothiazine methylene blue (MB). At concentrations of phenothiazine ranging from 5 to 0.5 mM, 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 mu M of 3 and 10 mu M 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 Delta T-m 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.