Spectroscopic approaches to the study of the antineoplastic agent bleomycin

Abstract

Bleomycin (BLM) is a glycopeptide antibiotic used clinically in the treatment of various neoplasms. Although the drug's in vivo mechanism of action remains unproven, it has been shown that BLM is able to bind iron and activate oxygen, forming a species, activated BLM, which is kinetically competent to cleave DNA. Several spectroscopic approaches have been used to characterize the drug's mechanism of oxygen activation and interaction with DNA.;Resonance Raman spectroscopy was used to investigate the structure of iron bleomycin in the free and DNA-bound states. The observation of an Fe-OH stretching vibration provides the first experimental evidence for hydroxide ligation to iron in Fe(lll)BLM. The data also suggest the involvement of a delocalized {dollar}\pi{dollar}-electron system in FeBLM analogous to that in the porphyrin macrocycle and may partially explain the similar spectroscopic characteristics and reactivity of iron-bleomycin and heme systems. Furthermore, the spectrum of Fe(lll)BLM bound to the hexamer d(CGCGCG) indicates that a hydrogen bond is formed between the oligonucleotide and the hydroxide bound to iron. Interestingly, there is no evidence for a similar bond when Fe(lll)BLM is bound to d(ATATAT). These findings are of particular interest since BLM-mediated DNA degradation occurs preferentially at GC sequences.;Electrospray mass spectrometry has been used to characterize activated BLM, a short-lived species that is kinetically competent in the cleavage of DNA. It is formed from Fe(ll)BLM in an O{dollar}\sb2{dollar}-dependent reaction or from Fe(lll)BLM by reacting with H{dollar}{lcub}\rm\sb2O\sb2{rcub}.{dollar} The structure of activated BLM has eluded researchers for over a decade. By coupling a rapid mixing device to an electrospray ionization source, the mass spectrum of activated BLM was obtained and provides compelling evidence that activated BLM is a ferric peroxide complex.;The reaction of iodosylbenzene with Fe(lll)BLM has also been investigated by electrospray and tandem mass spectrometry. Contrary to suggestions from model studies, activated BLM was not produced in this reaction and, instead, several intermediates were identified which contain hypervalent iodine as the reactive center. These results indicate that, at least for the case of Fe(lll)BLM, iodosylbenzene does not act as a 'single oxygen atom donor' as is often believed, but instead forms hypervalent iodine-containing compounds.;Lastly, continuous wave and pulsed EPR spectroscopy have been used to elucidate the structures of metal-BLM complexes bound to DNA. EPR spectra have been obtained which confirm resonance Raman spectroscopic results that indicate Fe(lll)BLM assumes a more rigid structure when bound to DNA. Furthermore, ESEEM spectroscopy of Cu(ll)BLM bound to 4{dollar}\sp\prime{dollar}- ({dollar}\sp2{lcub}\rm H{rcub}\rbrack{dollar}-DNA reveals that the copper ion is approximately 4A away from the C4{dollar}\sp\prime{dollar} hydrogen atom and suggests that the proximity of the metal site to the C4{dollar}\sp\prime{dollar} position is responsible for the specific abstraction of a hydrogen atom from that position that is the initial step in BLM-mediated DNA degradation. ftn*All degree requirements completed in 1994, but degree will be granted in 1996.