Synthesis of key analogs of bleomycin A2 that permit a systematic evaluation of the linker region: identification of an exceptionally prominent role for the L-threonine substituent.

The synthesis of a full series of analogs 2b-k of deglycobleomycin A2 (2a) containing systematic variations in the linker domain of bleomycin A2 (1) is described. The agents 2b-k, which are not accessible through structural modification of 1 or 2a, constitute key substructure analogs incorporating deep-seated structural modifications in the linker domain capable of delineating the contribution of the individual backbone substituents to the DNA cleavage efficiency, characteristic DNA cleavage selectivity, and double strand to single strand DNA cleavage ratio. The comparative examination of the DNA cleavage properties of the Fe(II) and Fe(III) complexes of 2a-k upon activation by O2-thiol or H2O2, respectively, revealed several characteristic features and trends. First, none of the substituents affect the characteristic 5'-GC, 5'-GT > 5'-GA DNA cleavage selectivity of bleomycin A2. In contrast, an exceptionally prominent role for the L-threonine substituent and an important role for the C4-methyl substituent of the (2S,3S,4R)-4-amino-3-hydroxy-2-methylpentanoic acid subunit were observed on the DNA cleavage efficiency of the agents. Similarly, the L-threonine substituent was found to substantially increase the ratio of double strand to single strand DNA cleavage events (2-3 times). In a w794 DNA cleavage assay, shortening the linker region by two carbons resulted in an exceptionally large reduction in DNA cleavage efficiency (125 times) and provided an agent that was only 1.3 times more effective than Fe(III) indicating that this deep-seated modification essentially destroys the DNA cleavage capabilities of the agent. The L-threonine substituent contributes in an exceptional manner, and its removal resulted in a 25 times reduction in DNA cleavage efficiency. A substantial contribution was observed for the C4-methyl group on the 4-aminobutanoic acid subunit and its removal resulted in a 7 times reduction in DNA cleavage efficiency. Little effect for the C3-hydroxyl and C2-methyl substituents on the 4- aminobutanoic acid subunit was observed (0-2.5 times) and even their inversion of stereochemistry had little impact on DNA cleavage efficiency or selectivity. Notably, the magnitude of the previously unappreciated L-threonine substituent contribution to the DNA cleavage efficiency and on the ratio of double to single strand DNA cleavage events is the largest effect observed to date including the well recognized disaccharide potentiation (6 times) of the DNA cleavage properties. Consequently, the past role and relative importance of the L-threonine subunit and substituent has been underestimated. Moreover, the cumulative effect of the two important linker chain substituents clearly illustrate that the functional role of this domain is much more important than its simply serving as a linker.