The Relationship between Ligand Aggregation and G-Quadruplex DNA Selectivity in a Series of 3,4,9,10-Perylenetetracarboxylic Acid Diimides

Abstract

Human telomeres are comprised of d(TTAGGG) repeats that are capable of forming G-quadruplex DNA structures. Ligands that bind to and stabilize these G-quadruplex DNA structures are potential inhibitors of the cancer cell-associated enzyme telomerase. Other potential biological uses of G-quadruplex targeting ligands have been proposed. One particularly challenging aspect of the contemplated uses of G-quadruplex targeting ligands is their selectivity for G-quadruplex DNA versus double-stranded DNA structures. We have previously reported the observation that two structurally related 3,4,9,10-perylenetetracarboxylic acid diimide-based G-quadruplex DNA ligands, PIPER [N,N'-bis(2-(1-piperidino)ethyl)-3,4,9,10-perylenetetracarboxylic acid diimide] and Tel01 [N,N'-bis(3-(4-morpholino)propyl)-3,4,9,10-perylenetetracarboxylic acid diimide], have different levels of G-quadruplex DNA binding selectivity at pH 7 as determined by absorbance changes in the presence of different DNA structures [Kerwin, S. M., Chen, G., Kern, J. T., and Thomas, P. W. (2002) Bioorg. Med. Chem. Lett. 12, 447-450]. Here we report that the less G-quadruplex DNA selective ligand PIPER can unwind double-stranded, closed circular plasmid DNA, as determined by a topoisomerase I assay. A model for the interaction of Tel01 with the G-quadruplex DNA structure formed by d(TAGGGTTA) was determined from NMR experiments. This model is similar to the previously published model for PIPER bound to the same G-quadruplex DNA and failed to provide a structural basis for the observed increased selectivity of Tel01 interaction with G-quadruplex DNA. In contrast, investigation into the aggregation state of Tel01 and PIPER as well as other 3,4,9,10-perylenetetracarboxylic acid diimide analogues bearing basic side chains demonstrates that ligand aggregation is correlated with G-quadruplex DNA binding selectivity. For all six analogues examined, those ligands that were aggregated at pH 7 in 70 mM potassium phosphate, 100 mM KCl, 1 mM EDTA buffer also demonstrated G-quadruplex DNA binding selectivity under these buffer conditions. Ligands that were not aggregated under these conditions display much lower levels of G-quadruplex DNA selectivity. The aggregation state of these ligands is extremely sensitive to the buffer pH. Tel01, which is aggregated at pH 7, is not aggregated at pH 6.4, where it demonstrates only modest G-quadruplex DNA binding selectivity, and PIPER in pH 8.5 buffer is both aggregated and highly G-quadruplex DNA-selective. To our knowledge, these studies demonstrate the first DNA structure selectivity as achieved through pH-mediated ligand aggregation. The potential impact of these findings on the selectivity of other classes of G-quadruplex DNA ligands is discussed.

Evaluation of binding of perylene diimide and benzannulated perylene diimide ligands to dna by electrospray ionization mass spectrometry

“…The PDI Tel12 displays only weak fluorescence and no RLS spectra. The weak fluorescence of Tel12 may be due to ligand dimerization, as has been noted for this PDI in 150 mM KCl phosphate buffer [37]; however, the fluorescence intensity of Tel12 in methanolic buffer is higher than that in the high-salt buffer (data not shown), indicating that dimerization is less extensive in the methanolic buffer. Because Tel12 has been found to dimerize, but does not produce a RLS spectrum, this ligand self-associates.…”

“…Chemicals PIPER, Tel01, Tel11, Tel12 were prepared as described previously [35,37,38]. The manuscript detailing the synthesis of Tel18, Tel32, and Tel34 is in preparation.…”

“…PDIs like Tel01 and PIPER are known to undergo pH-dependent self-association to form aggregates in solution [37][38][39]. The selectivity of PDIs towards Gquadruplex DNA depends on the aggregation state of these ligands, which, in addition to pH, may also be affected by ligand concentration, buffer ionic strength, and the presence of nonaqueous cosolvents [36,38].…”

“…The selectivity of PDIs towards Gquadruplex DNA depends on the aggregation state of these ligands, which, in addition to pH, may also be affected by ligand concentration, buffer ionic strength, and the presence of nonaqueous cosolvents [36,38]. Previous solution studies of the aggregation state and G-quadruplex DNA binding selectivity of PIPER [36,38], Tel01 [36,38], Tel11 [37], and Tel12 [37] were carried out under relatively high-salt (170 mM KCl) phosphate buffer. To determine the aggregation state of these and the other PDI ligands under the conditions of the ESI-MS analysis discussed later, the fluorescence and resonance light scattering spectra were determined in 3:1 25 mM ammonium acetate/methanol.…”

“…One promising class of quadruplex selective ligands are perylene diimides (PDIs) [35][36][37][38][39][40][41][42][43]. Recent solution studies of N,N=-bis [2-(1-piperidino)-ethyl]-3,4,9,10-perylenetetracarboxylic acid diimide (PIPER) [35] and N, N=-bis(4-morpholinylpropyl)-3,4,9,10-perylenetetracarboxylic acid diimide (Tel01) [38] indicate that these molecules exhibit G-quadruplex affinity by stacking on the faces of the terminal G-tetrads, thereby stabilizing the G-quadruplex structure.…”