Chemical synthesis of radiolabeled bleomycin a₂ and Its Binding to DNA

A method for the preparation of biologically active [³H]- and [¹³C]bleomycin A₂ is described. Demethyl Cu(ll):bleomycin A₂, isolated after pyrolysis of Cu(ll):bleomycin A₂₍ was methylated with either [³H]- or [¹³C]methyl iodide, which resulted in Cu(ll): bleomycin A₂ labeled in the dimethylsulfonium moiety. Copper was removed by treatment with dithizone in chloroform, and structures were verified by thin-layer chromatography and ¹H and ¹³C nuclear magnetic resonance spectroscopy. Copper-free [³H] and [¹³C]bleomycin A₂ are active in the degradation of DNA in vitro. Gel exclusion chromatography and equilibrium dialysis were used to determine the apparent equilibrium constants for binding of [³H]bleomycin A₂ and Cu(ll):[³H]bleomycin A₂ to calf thymus DNA, noncovalently associated polydeoxyguanylate: polydeoxycytidylate, and noncovalently associated polydeoxyadenylate:polydeoxythymidylate. In 2.5 mM sodium phosphate buffer, pH 7.0, binding data obtained by gel filtration with calf thymus DNA reveal an apparent equilibrium constant for [³H]bleomycin A₂ of 5.7*10⁵/mol and for Cu(ll):[³H]bleomycin A₂ of 3.9*10⁵/mol. One molecule of [³H]bleomycin A₂ binds for every 3.7 base pairs in DNA, and one molecule of Cu(ll):[³H]bleomycin A₂ binds for every 2.8 base pairs in DNA. Analysis of binding data with calf thymus DNA, noncovalently associated polydeoxyguanylate:polydeoxycytidylate, and non-covalently associated polydeoxyadenylate:polydeoxythymidy-late obtained by equilibrium dialysis reveals, in each instance, 2 types of binding sites for both the copper and metal-free form of the antibiotic. For those sites in calf thymus DNA with tighter binding affinity, the apparent equilibrium constant for [³H]bleomycin A₂ was 6.8 * 10⁶/mol and for the Cu(ll):[³H]bleomycin A₂ complex, 4.4 * 10⁵/mol. As seen with calf thymus DNA, the affinity of [³H]bleomycin A₂ is slightly greater than that of Cu(ll):[³H]bleomycin A₂ for the synthetic DNAs, although more of the copper form of the drug binds to these polymers.