Alteration of the selectivity of DNA cleavage by a deglycobleomycin analogue containing a trithiazole moiety.

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.

On the chemistry of RNA degradation by Fe.bleomycin.

The chemistry of RNA degradation by Fe.bleomycin was studied using two RNA substrates that are modified efficiently at a small number of sites by the antitumor antibiotic. Cleavage of tRNAHis precursor transcript by Fe(II).BLM A2 was shown to require O2; cleavage was also observed when the same substrate was treated with Fe(III).BLM A2 + H2O2. Consistent with earlier observations made for DNA, the extent of tRNAHis precursor cleavage was greater for Fe(II).BLM A5 than for Fe(II).BLM A2; the least cleavage was obtained using Fe(II).BLM demethyl A2. By the use of 32P end labeled tRNAHis precursor transcript that was also 3H labeled within the uracil moieties, it was shown that release of uracil was nearly stoichiometric with tRNA strand scission by Fe(II).BLM A2. Nonetheless, treatment of the tRNAHis with hydrazine following BLM-mediated cleavage indicated formation of a new product that must have derived from a BLM-induced lesion. Also employed for characterization of BLM cleavage of RNA were the octanucleotides CGCTAGCG, C3-ribo-CGCTAGCG and C3-ara-CGCTAGCG. Analysis of the products of cleavage indicates that Fe.BLM is capable of mediating cleavage by abstraction of a H atom either from C-4' H or c-1' H of the chimeric oligonucleotides.