OPTIMIZING THE TARGETED CHEMICAL NUCLEASE ACTIVITY OF 1,10-PHENANTHROLINE-COPPER BY LIGAND MODIFICATION

Our interest in improving the efficiency of targeted scission reagents has prompted us to study the influence of ring substituents on the nu clease activity of 1,10-phenanthroline -copper conjugated to oligonucl eotides and DNA-binding proteins. Since methyl substitution at all but the 2 and 9 positions enhances the copper-dependent chemical nuclease activity of 1, 10-phenanthroline, we have compared the reactivity of conjugates prepared from 5-(aminomethyl)-1,10-phenanthroline (MOP) to those of conjugates prepared from 5-amino-1,10-phenanthroline (amino-O P). Tethering MOP derivatives to the Escherichia coli Fis protein enha nces DNA scission several-fold at the weaker cleavage sites initially observed with conjugates prepared from amino-OP. However, scission eff iciency is not increased at the stronger cleavage sites, or when sciss ion is targeted to single-stranded DNA by a complementary oligonucleot ide. These results are consistent with a change in the rate-determinin g step for cleavage associated with the differential accessibility of the DNA-bound coordination complex to solvent and reductant. Although the free bis cuprous complex of 2,9-dimethyl-1,10-phenanthroline (neoc uproine) is redox-inactive, an oligonucleotide tethered to neocuproine through C5 of the phenanthroline ring efficiently cleaves a complemen tary DNA sequence. These results establish that the nucleolytic specie s in targeted scission is the 1:1 cuprous complex and suggest that the oxidative reaction proceeds through a copper-ore intermediate rather than a metal-coordinated peroxy species. However, substituents at the 2 and 9 positions of the ligand will often hinder close approach of th e phenanthroline -copper moiety to the oxidatively sensitive ribose as shown by the preference of the oligonucleotide-targeted chimera for c leavage of single-stranded regions and the failure of neocuproine-DNA- binding protein chimeras and a C2-tethered chimera to cleave DNA.