Synthesis and DNA interactions of a bis-phenothiazinium photosensitizer

Abstract

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 °C and pH 7.0) was markedly enhanced by 1 μM of 3 in comparison to 1 μM of the parent phenothiazine methylene blue (MB). At concentrations of phenothiazine ranging from 5 to 0.5 μM, 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 μM of 3 and 10 μM 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 ΔT_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.