Highly Potent Fluorogenic Ligands for Triplex DNA: 5‐Substituted 2‐(Naphthalen‐2‐yl)‐4 <i>H</i> ‐Chromen‐4‐Ones

We previously reported two series of 5-substituted flavonoids that selectively bind to triplex DNA while interacting minimally with duplex DNA. Our studies suggest that flavonoids are a promising scaffold for designing triplex ligands, owing to their mild synthesis conditions and ease of structural modification. In the present work, we introduce a novel class of highly potent triplex-specific ligands [5-substituted 2-(naphthalen-2-yl)-4H-chromen-4-ones], developed by expanding the aromatic surface of the flavone moiety. UV thermal denaturation studies indicate these compounds strongly stabilize triplex DNAs, enabling their direct dissociation into random coils without transitioning through a duplex intermediate. Circular dichroism (CD) analysis reveals that these ligands induce much greater conformational changes in triplex DNA compared to duplex DNA. Fluorescent intercalator (FID) displacement assays cross-verify the importance of pendant chains for triplex binding. Viscosity measurements suggest that these compounds bind to triplex DNA via intercalation. Notably, a representative compound (3b) emits strong blue fluorescence upon binding to triplex DNA, allowing direct visualization under UV illumination and providing a convenient method for probing triplex DNA. Electrophoretic mobility shift assay confirms that 3b promotes triplex formation at submicromolar concentrations. Additionally, a plasmid-based assay developed in our lab demonstrates that 3b inhibits DraI endonuclease activity at low micromolar concentrations.