DESIGN AND BINDING OF A DISTAMYCIN-A ANALOG TO D(CGCAAGTTGGC)BULLET-D(GCCAACTTGCG) - SYNTHESIS, NMR-STUDIES, AND IMPLICATIONS FOR THE DESIGN OF SEQUENCE-SPECIFIC MINOR GROOVE BINDING OLIGOPEPTIDES

An oligopeptide has been synthesized in which an imidazole ring is substituted for the central pyrrole ring of distamycin A. Two-dimensional NMR spectroscopy was used to characterize the complex formed between 3-[1-methyl-4-[1-methyl-4-[1-methyl-4-(formylamino)pyrrole-2-carboxamido]imidazole-2-carboxamido]pyrrole-2-carboxamido]propionamidine hydrochloride (2-ImD) and d(CGCAAGTTGGC).d(GCCAACTTGCG). Titration of the AAGTT duplex with 2-ImD yielded a single complex with a ligand:DNA stoichiometry of 2:1. The nuclear Overhauser effect (NOESY) experiment in D2O was used to assign the aromatic and C1'H DNA protons and to identify intermolecular ligand-DNA contacts between nonlabile protons. The NOESY experiment in H2O was used to assign the imino and amino DNA protons and the amide protons of the ligands and to identify ligand-DNA contacts involving these labile protons. These data indicate that two ligand molecules bind simultaneously to the minor groove of the central 5'-AAGTT-3' sequence in a head-to-tail orientation. Molecular modeling, using 35 ligand-DNA distance constraints derived from a semiquantitative analysis of the NOESY data, shows that the imidazole N3 of one of the ligands forms a hydrogen bond with the C2 amino group of the guanine in the binding site. Additionally, the titration of d(CGCAAATTGGC).d(GCCAATTTGCG) with 2-ImD was performed. No specific complex was detected by NMR spectroscopy between 2-ImD and the AAATT duplex. This result emphasizes the importance of the imidazole N3 atom of 2-ImD in the recognition of the AAGTT binding site.