Studies on 3,4,9,10-perylenetetracarboxylic acid diimide based ligands as G-quadruplex DNA interactive agents

G-quadruplex DNA is a diverse family of structures found at the ends of eukaryotic telomeres that have been implicated in several biological processes, including telomere maintenance. A number of molecular scaffolds have been shown to interact with G-quadruplex DNA, but there is little information concerning the requirements for selective G-quadruplex interaction. Here, we detail our investigations into one particular class of molecules, the 3,4,9,10- perylenetetracarboxylic acid diimides (PTCDIs), as G-quadruplex selective agents. We focus on the synthesis, self-association, G-quadruplex DNA binding and selectivity, and cellular effects in order to further our understanding of Gquadruplex DNA interaction. Three classes of 3,4,9,10-perylenetetracarboxylic acid diimides were prepared and examined: PTCDIs bearing basic sidearm substituents, PTCDIs bearing charged sidearm substituents, and PTCDI-based ligands with altered chromophores. The PTCDIs bearing basic sidearm substituents were found to aggregate in a pH-dependent manner. This aggregation mediates the observed Gquadruplex DNA selectivity of these ligands. The PTCDIs with cationic or anionic sidearms are water-soluble and do not extensively aggregate, but exist as dimers in high ionic strength buffers. The cationic PTCDI is found to have a relatively high affinity, but less selectivity for G-quadruplex DNA structures over double-stranded DNA when compared to the anionic PTCDI. The cationic PTCDI also demonstrates the strongest inhibition of telomerase of the ligands tested in a cell-free primer extension assay. The preparation and characterization of PTCDI-based ligands with altered chromophores is more challenging. Stericmediated twisting of the chromophore attenuates the self-association of the molecules, but the affinity for DNA and G-quadruplex DNA selectivity of these ligands is dominated by the charge of the PTCDI. Benzannulation of the PTCDI chromophore may be accomplished when the PTCDI sidearm substituents are highly lipophilic. This fact limits the charge of the ligands and results in low solubility in aqueous buffers that preclude detailed analysis of this class of compound. A novel flow-cytometric fluorophore displacement assay is also introduced. Fluorophore displacement in fixed and permeabilized A549 cells is demonstrated using the charged PTCDIs as well as the DNA-binding ligand mitoxantrone; the implications for whole cell analysis are discussed.