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The antitumor antibiotic bleomycin has long been believed to exert its therapeutic effects at the level of DNA cleavage. Recently, evidence has been presented to suggest that RNA cleavage may also be important and that one or more transfer RNAs may be involved. To define those tRNAs that may represent important loci for the action of bleomycin, we have fractionated chicken liver tRNAs and identified those isoacceptors most susceptible to oxidative cleavage by Fe(II).BLM. Two chicken liver tRNAs, tRNA3Lys and tRNAPhe, were found to be cleaved with exceptional facility by Fe(II).BLM, and both were cleaved predominantly at U66. The cleavage of tRNA3Lys was shown to be minimally affected by physiological concentrations of Mg2+. Chicken liver tRNA3Lys is identical in sequence with human tRNA3Lys. These findings support a possible role for a critical tRNA such as tRNA3Lys in the mechanism by which bleomycin mediates its antitumor activity.

The bleomycin (BLM) group antitumor antibiotics are glycopeptide-derived natural products shown to cause sequence selective lesions in DNA. Prior studies have indicated that the linker region, composed of the methylvalerate and threonine residues, may be responsible for a conformational bend in the agent required for efficient DNA cleavage. We have synthesized a number of conformationally constrained methylvalerate analogues and incorporated them into deglycobleomycin A(5) congeners using our recently reported procedure for the solid phase construction of (deglyco)bleomycin and its analogues. These analogues were designed to probe the effects of conformational constraint of the native valerate moiety. Initial experiments indicated that the constrained molecules, none of which mimic the conformation proposed for the natural valerate linker, possessed DNA cleavage activity, albeit with potencies less than that of (deglyco)BLM and lacking sequence selectivity. Further experiments demonstrated that these analogues failed to produce alkali-labile lesions in DNA or sequence selective oxidative damage in RNA. However, two of the conformationally constrained deglycoBLM analogues were shown to mediate RNA cleavage in the absence of added Fe(2+). The ability of the analogues to mediate the oxygenation of small molecules was also assayed, and it was shown that they were as competent in the transfer of oxygen to low molecular weight substrates as the parent compound.

The solid-phase synthesis of bleomycin A5 (BLM A5) and three monosaccharide analogues is presented. The monosaccharide analogues incorporated alpha-d-mannose, alpha-l-gulose, and alpha-l-rhamnose moieties in lieu of the disaccharide normally present in BLM A5. Also explored were the abilities of each of the monosaccharide congeners to cleave a 53-nt RNA. The elaboration of these carbohydrate-modified bleomycin analogues helps to define the role of the disaccharide moiety during the RNA cleavage event. The relatively facile solid-phase synthesis of bleomycin A5 and each of the carbohydrate analogues constitutes an important advance in the continuing mechanistic studies of bleomycin.

The bleomycin (BLM) group of antitumor antibiotics effects DNA cleavage in a sequence-selective manner. Previous studies have indicated that the metal-binding and bithiazole moieties of BLM are both involved in the binding of BLM to DNA. The metal-binding domain is normally the predominant structural element in determining the sequence selectivity of DNA binding, but it has been shown that replacement of the bithiazole moiety with a strong DNA binder can alter the sequence selectivity of DNA binding and cleavage. To further explore the mechanism by which BLM and DNA interact, a trithiazole-containing deglycoBLM analogue was synthesized and tested for its ability to relax supercoiled DNA and cleave linear duplex DNA in a sequence-selective fashion. Also studied was cleavage of a novel RNA substrate. Solid-phase synthesis of the trithiazole deglycoBLM A(5) analogue was achieved using a TentaGel resin containing a Dde linker and elaborated from five key intermediates. The ability of the resulting BLM analogue to relax supercoiled DNA was largely unaffected by introduction of the additional thiazole moiety. Remarkably, while no new sites of DNA cleavage were observed for this analogue, there was a strong preference for cleavage at two 5'-GT-3' sites when a 5'-(32)P end-labeled DNA duplex was used as a substrate. The alteration of sequence selectivity of cleavage was accompanied by some decrease in the potency of DNA cleavage, albeit without a dramatic diminution. In common with BLM, the trithiazole analogue of deglycoBLM A(5) effected both hydrolytic cleavage of RNA in the absence of added metal ion and oxidative cleavage in the presence of Fe(2+) and O(2). In comparison with BLM A(5), the relative efficiencies of hydrolytic cleavage at individual sites were altered.