NMR Evidence of Sequence Specific DNA Binding by a Cobalt(III)-Bleomycin Analog with Tethered Acridine

Attachment of acridine-9-carboxamido-N′-(3-propyl)imidazole (8, Int-A) to the Co(III) complex of a designed ligand PMAH that mimics the metal-binding domain of the antitumor drug bleomycin (BLM) has afforded the hybrid molecule [Co(PMA)(Int-A)]Cl₂ (7). This structurally characterized compound inflicts DNA strand scission under UV light much like the Co(III)-BLMs. It has been shown previously that UV irradiation of the [Co(PMA)]²⁺ unit gives rise to a ligand-based radical which, in aqueous medium, rapidly collapses into ^·OH radical, the species actually responsible for the DNA photocleavage observed with the [Co(PMA)X]ⁿ⁺ complexes. In this paper we report that attachment of the intercalator acridine to the DNA-cleaving [Co(PMA)]²⁺ unit not only allows the resulting hybrid molecule 7 to bind to DNA quite strongly but also results in preferential DNA photocleavage at 5′GG—N3′ sites. Specific interactions between 7 and the DNA substrate have been studied by high-field NOESY and COSY experiments using the self-complimentary oligonucleotide [d(GATCCGGATC)]₂ (9) which contains a GG—N site. Intercalative interaction between the acridine moiety of 7 and the duplex 9 is indicated by both broadening and upfield shifts of the acridine protons in the 7–9 (1:1) complex. Specific NOEs between the nonexchangeable base protons and the sugar protons of 9 also demonstrate that the acridine moiety of 7 intercalates into the G—G step of 9 and does so (a) with only the outer half (the N-containing side) of the acridine moiety within the base pairs and (b) from the major groove side of the duplex. Interestingly, no intermolecular NOE between the protons of the [Co(PMA)]²⁺ unit of 7 and those of 9 is observed in the NOESY spectra of the 7–9 (1:1) complex. Also, the resonances of the [Co(PMA)]²⁺ unit are strongly affected by the salt concentration. The latter two observations indicate that the [Co(PMA)]²⁺ unit of 7 binds to the phosphate backbone of 9 via electrostatic interaction. The products of photocleavage of radiolabeled 9 with 7 suggest that the acridine moiety of 7 binds to the G—G site of 9 and allows photodamage at the GG—N sites by the tethered [Co(PMA)]²⁺ unit.