The preparation and examination of 3 and 4 and their unnatural epimers 6 and 7 by following a new and alternative synthesis of 2 was conducted to assess the role of the pyrimidine C4 amine of bleomycin A(2) (1) and deglycobleomycin A(2) (2). The agent 3 bearing a pyrimidine C4 dimethylamino substituent exhibited a substantially diminished DNA cleavage efficiency (10-15x) relative to 2 and the loss of the characteristic 5'-GC/5'-GT cleavage selectivity. The agent 4 in which the pyrimidine C4 amino group was removed exhibited an even greater diminished DNA cleavage efficiency (30x) relative to that for 2. For this agent, the characteristic cleavage selectivity is either slightly or significantly reduced depending on the assay conditions. Even in the instances where it was not substantially altered, the ability to detect it required a temperature of 4 versus 25-37 degrees C. This information and temperature dependence suggest a reduced binding interaction and are consistent with the participation of the pyrimidine C4 amine in one of two critical hydrogen bonds of a minor groove tripler-like recognition between the metal binding domain of 1 and 2 and guanine at the 5'-GC/5'-GT cleavage sites responsible for the characteristic cleavage selectivity. These observations have further implications on the potential origin of inherent 5'-GPy > 5'-APy cleavage selectivity of bleomycin A(2) itself. This cleavage preference of 5'-GPy > 5'-APy may be analogously attributed to a reduced binding affinity at the 5'-APy sites resulting from one versus two tripler-like hydrogen bonds to adenine.