Azinomycin B (also known as carzinophilin A) contains two electrophilic functional groups-an epoxide and an aziridine residue-that react with nucleophilic sites in duplex DNA to form cross-links at 5'-dGNT and 5'-dGNC sequences. Although the aziridine residue of azinomycin is undoubtedly required for cross-link formation, analogues containing an intact epoxide group but no aziridine residue retain significant biological activity. Azinomycin epoxide analogues (e.g., 5 and 6) are of interest due to their potent biological activity and because there is evidence that azinomycin may decompose in vivo to yield such compounds. To investigate the chemical events underlying the toxicity of azinomycin epoxides, DNA binding and alkylation by synthetic analogues of azinomycin B (6, 8, and 9) that comprise the naphthalene-containing "left half" of the antibiotic have been investigated. The epoxide-containing analogue of azinomycin (6) efficiently alkylates guanosine residues in duplex DNA. DNA alkylation by 6 is facilitated by noncovalent binding of the compound to the double helix. The results of UV-vis absorbance, fluorescence spectroscopy, DNA winding, viscometry, and equilibrium dialysis experiments indicate that the naphthalene group of azinomycin binds to DNA via intercalation. Equilibrium dialysis experiments provide an estimated binding constant of (1.3 +/- 0.3) x 10(3) M(-)(1) for the association of a nonalkylating azinomycin analogue (9) with duplex DNA. The DNA-binding and alkylating properties of the azinomycin epoxide 6 provide a basis for understanding the cytotoxicity of azinomycin analogues which contain an epoxide residue but no aziridine group and may provide insight into the mechanisms by which azinomycin forms interstrand DNA cross-links.