HIGH-FIELD NMR AND RESTRAINED MOLECULAR MODELING STUDIES ON A DNA HETERODUPLEX CONTAINING A MODIFIED APURINIC ABASIC SITE IN THE FORM OF COVALENTLY-LINKED 9-AMINOELLIPTICINE

Two-dimensional NMR methods were used to model the possible solution s tructure of an intercalative complex of 9-aminoellipticine (Aell), a p olycyclic pyridocarbazolamine, covalently bound to an apurinic ring-op ened deoxyribose site of a duplex DNA fragment in the reduced Schiff b ase form. The required oligonucleotide single strand containing covale ntly attached aminoellipticine was obtained by reductive amination in the presence of sodium cyanoborohydride. The combined NMR-energy minim ization methods were employed to refine the model structures of two di stinct forms, intrahelical and extrahelical, of a control 9-mer duplex DNA, d(CGTG.dr.GTGC).d(GCACTCACG), which contains an apurinic site po sitioned opposite a dT residue on the complementary strand. The model structure of an aminoellipticine conjugate with the same DNA sequence, derivatized via the aforementioned covalent attachment, was also obta ined by incorporating intermolecular drug-DNA and intra- and internucl eotide NOE-derived proton-proton distance estimates as restraints in e nergy minimization routines. The indole ring system of aminoellipticin e, which is inserted at the apurinic site, intercalates between and is parallel to flanking GC base pairs. The pyridinic ring of aminoellipt icine, in protonated form, also stacks between cytidine and thymidine bases on the complementary strand, which is consistent with the observ ation that the normal sequential NOE connectivity at the 5'-C-13-T-14 step is broken and indeed diverted through the ellipticine moiety, e.g ., C-13-Aell-T-14 connectivities through the Aell-H4/CSMe protons. Int erestingly, the partial stacking of the pyridinic ring is observed onl y between the 5'-CT step vs an adjacent 5'-TC step, owing to inherentl y weak stacking interactions associated with the former. In the absenc e of any potential groups that can participate in electrostatic or hyd rogen-bonding interactions with the nucleic acid, pi-pi stacking and h ydrophobic contacts at the intercalation site appear to be the importa nt factors in determining stability and conformation of the aminoellip ticine-DNA conjugate. Stacking interactions in such a bistranded inter calative complexation of aminoellipticine apparently govern the format ion of a single intrahelical form of a right-handed B-type DNA duplex. The overall structural features lead us to propose working models for an enzyme-like DNA cleavage activity of 9-aminoellipticine and the ob served inhibition of the AP endonuclease-dependent DNA excision-repair pathway.