| The bleomycins are a group of structurally related glycopeptide antibiotics originally isolated from Streptomyces verticillus that mediate sequence selective oxidative damage of DNA and RNA. Deglycobleomycin, which lacks the carbohydrate moiety, cleaves DNA analogously to bleomycin itself, albeit to a lesser extent, and has been used as a successful model for understanding the functional domains of bleomycin. Although several structural modifications to bleomycin and deglycobleomycin have been performed in order to understand the roles of the functional domains of bleomycin, a bleomycin or deglycobleomycin analogue with enhanced biochemical activity has yet to be described. The successful synthesis of a deglycobleomycin on a solid support has permitted the rapid solid phase synthesis of 108 unique deglycobleomycin analogues through parallel solid phase synthesis. Each of the deglycobleomycin analogues was synthesized in high yield having purity greater than 60% as determined by HPLC integration. The solid phase synthesis of the deglycobleomycin library provided nearmilligram to milligram quantities of each deglycobleomycin, thereby permitting characterization by 1H NMR and high-resolution mass spectrometry. Each analogue demonstrated supercoiled plasmid DNA relaxation above background cleavage; the library contained two unique analogues that demonstrated plasmid relaxation to a greater extent than the parent molecule. The two improved deglycobleomycin analogues also demonstrated similar sequence selectivity compared to the parent molecule.; The ability to screen solid phase combinatorial libraries against a biological target while the ligand remains attached to the resin has become increasingly important with the advent of rapid parallel synthesis. While colorimetric enzyme binding assays exist for solid phase substrates, a DNA cleavage assay for resin-bound substrates has not been reported. A novel method for the assay of resin-bound deglycobleomycins was developed which utilizes agarose-suspended beads and molecular DNA beacons. By utilizing fluorescence resonance energy transfer of the beacons, the beads containing covalently attached active deglycobleomycin analogues can be seen visually as a result of the molecular DNA beacon cleavage, thereby permitting the identification of deglycobleomycin analogues having different DNA cleaving potencies. |
