A novel octahedral complex CoII(HAPP)(TFA)2 [hexaazaphenantholine‐cyclophane (HAPP), trifluoroacetate (TFA)] is a DNA bulge‐specific probe with single‐strand DNA cleavage activity in the presence of H2O2. This complex exhibits low affinity towards double‐stranded DNA and low reactivity toward single‐stranded DNA. Metal–HAPP complexes with different coordination number and ring size were synthesized and their selectivity and reactivity for DNA bulges were compared. The DNA sequence at the bulge site influences the intensity of cleavage at the bulge and the flanking sites after piperidine treatment. Cleavage specificity of CoII(HAPP)(TFA)2 was characterized extensively using scavenger reagents to quench the cleavage reaction and high‐resolution polyacrylamide gel electrophoresis. In addition, 3′‐phosphoglycolate cleavage products were trapped and analyzed by matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry. These data were used to deduce that the DNA cleavage pathway for CoIIHAPP2+ in the presence of H2O2 involves 4′‐H abstraction of the deoxyribose moiety.
