Molecular recognition of DNA by Hoechst benzimidazoles: Exploring beyond the pyrrole-imidazole-hydroxypyrrole polyamide-pairing code

A series of three-ring analogs of the minor-groove-binding molecule Hoechst 33258 (1), consisting of benzimidazole (B), imidazopyridine (P), and hydroxybenzimidazole (H) monomers, have been synthesized in order to investigate both their sequence specificity and binding modes. MPE⋅FeII Footprinting has revealed the preference of both PBB and BBB ligands for 5′-WGWWW-3′ and 5′-WCWWW-3′ tracts, as well as A⋅T-rich sequences. Affinity-cleavage titrations show no evidence for a 2 : 1 binding mode of these Hoechst analogs. Importantly, all derivatives are oriented in one direction at each of their binding sites. The implications of these results for the design of minor-groove-binding small molecules is discussed.