Phenanthridine–pyrene conjugates as fluorescent probes for DNA/RNA and an inactive mutant of dipeptidyl peptidase enzyme

Abstract

Two novel conjugate molecules were designed: pyrene and phenanthridine-amino acid units with a different linker length between the aromatic fragments. Molecular modelling combined with spectrophotometric experiments revealed that in neutral and acidic buffered water solutions conjugates predominantly exist in intramolecularly stacked conformations because of the π–π stacking interaction between pyrene and phenanthridine moieties. The investigated systems exhibited a pH-dependent excimer formation that is significantly red-shifted relative to the pyrene and phenanthridine fluorescence. While the conjugate with a short linker showed negligible spectrophotometric changes due to the polynucleotide addition, the conjugate with a longer and more flexible linker exhibited a micromolar and submicromolar binding affinity for ds-polynucleotides and inactivated a mutant of dipeptidyl peptidase enzyme E451A. Confocal microscopy revealed that the conjugate with the longer linker entered the HeLa cell membranes and blue fluorescence was visualized as the dye accumulated in the cell membrane.

Keywords

• dipeptidyl peptidase enzyme
• excimer
• molecular dynamics simulations
• phenanthridine
• polynucleotide
• pyrene