Fe III BLM

ACS Symposium Series American Chemical Society Washington, DC, 1980. 

In an effort to more clearly understand the mechanism by which bleomycin degrades DNA, three new bleomycin analogues were synthesized, 2-4. All three analogues were formed by the condensation of the primary amine function of BLM-A2 with either benzoyl chloride (2) or a sulfonyl chloride (3,4) using Schotten-Baumann conditions (l ). The compounds were purified via chromatographic means were characterized using H and nmr and po-tentiometric titration methods. Particularly noteworthy in the titration curves of the derivatives is the absence of inflections at pH values of 2.9 and 7.4. For BLM-Az, inflections at these pH values correspond to the loss of a proton from the protonated, but hydrogen bonded, secondary amine function (Eq. 1) and a loss of a proton from the protonated primary amine function respectively (Eq. 2). 

In contrast to BLM-A2, neither of the new derivatives facilitated the oxidation of Fe(II) to Fe(III) in the atmosphere (10). Esr studies of aqueous solutions containing equimolar amounts Fe(II)(C 0i,)2 and the analogues, 2-4 at pH, 7.0 showed the lack of formation of Fe(III). Similar observations have been reported for 

Using electrochemistry, uv-visible absorption, esr, and C nmr spectroscopy, and with the help of three new bleomycin analogues, the Fe(II) and Fe(III) binding sites of the antitumor antibiotic bleomycin have been located. The drug appears to utilize the amine-pyrimidine-imidazole region for iron binding. The physical properties of the metal complexes and how those properties relate to the proposed mechanism of action of the anticancer agent is also discussed. 

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The cytotoxicity of BLM is believed to result from its ability to bind iron, activate oxygen, and form an activated BLM (Fe-114) (556) which cleaves DNA and possibly RNA (557). The ability of the Fe(II)-BLM complex to bind to oxygen and produce oxygenated BLM species such as 02-Fe(III)-BLM or 02-Fe(II)-BLM may be due to the presence of delocalized 77-electrons around the iron and the strong iron-pyrimidine 77-back-bonding (558, 559). Oxygenated BLM accepts an additional electron to form activated low-spin ferric-peroxide-BLM (Oi -Fe(III)-BLM) (558, 559). The structural features of Fe-BLM responsible for DNA (or RNA) degradation remain unclear (560). Bleo-... 

However, spectroscopic studies of activated BLM indicate that it is not an Fev=0 species. It exhibits an S - 1/2 EPR spectrum with g values at 2.26, 2.17, and 1.94 [15], which is typical of a low-spin Fe111 center. This low-spin Fem designation is corroborated by Mossbauer and x-ray absorption spectroscopy [16,19], Furthermore, EXAFS studies on activated BLM show no evidence for a short Fe—0 distance, which would be expected for an iron-oxo moiety [19], These spectroscopic results suggest that activated BLM is a low-spin iron(III) peroxide complex, so the two oxidizing equivalents needed for the oxidation chemistry would be localized on the dioxygen moiety, instead of on the metal center. This Fe(III)BLM—OOH formulation has been recently confirmed by electrospray ionization mass spectrometry [20] and is supported by the characterization of related synthetic low-spin iron(III) peroxide species, e.g., [Fe(pma)02]+ [21] and [Fe(N4py)OOH]2+ [22], The question then arises whether the peroxide intermediate is itself the oxidant in these reactions or the precursor to a short-lived iron-oxo species that effects the cytochrome P-450-like transformations. This remains an open question and the subject of continuing interest. 

The natural product bleomycin mediates strand scission in the presence of iron and oxygen by a related mechanism (6, 12, 14) however, no diffusible intermediate is generated. Electrochemical and related studies showed that the mechanism of iron bleomycin (Fe-BLM) involves first reduction of Fe(III)-BLM, reaction of Fe(II)-BLM with 02, and reduction of the Fe(II)-BLM-02 adduct to activated Fe-BLM (15). 

Using Reaction 5 to generate H02 -Fe(iii)Blm, it has been shown that this species is competent to initiate strand scission or, at least, is the only observable precursor of a form that starts the process of DNA degradation. Indeed, if DNA is not present, this species begins to attack its own structure in a reaction that inactivates it for subsequent reactions with DNA. ° Considering the various outcomes of the reaction of H02"-Fe(iii)Blm with DNA as overall redox reactions (Figure 2), base release involves a simple stoichiometry of reaction ... 

As this C4 radical is converted into a hydroperoxide, an electron is required from some source in the reaction mixture. According to the other proposal, H02 -Fe(iii)Blm undergoes heterolytic cleavage to produce hydroxide and a perferryl species that does the hydrogen abstraction. The net reaction is the same as shown in Reaction 7 ... 

In this model, the perferryl species is one of the products. Thus, in neither hypothetical pathway of single strand scission is Fe(iii)Blm generated. Instead, a reactive species remains. How either ferryl or perferryl iron centers are reduced to Fe(ni) is unresolved. 

The conformational relationship between the metal domain of several metallob-leomycins and DNA has been probed by ESR studies of Fe(iii)Blm, NO-Fe(ii)Blm, and 02-Co(ii)Blm bound to oriented fibers of DNA. Remarkably, the dioxygen ligand of 02-Co(ii)Blm and the nitrosyl group of NO-Fe(ii)Blm, which largely bear the unpaired spins in these complexes, are rigorously constrained to a plane approximately perpendicular to the helix axis. These results provide clear evidence that the metal domain in these adducts, like the DNA domain, must be closely associated with DNA, and not loosely tethered to the polymer. They also indicate that Fe- and Co-Blm species bind to DNA with similar steric constraints. 

Fe(III)BLM exhibited narrow resonance lines for carbon atoms... 

C, 41-49 and C, 51-55 (Figure 2c). Broad unassignable resonances were observed at 64 and 71 ppm but the sugar region was simpler than was observed for Fe(II)BLM. In addition, broad and apparently shifted resonances were found at 14, 34, and 101 ppm. The nmr spectrum of Fe(III)BLM also contained a number of weak but narrow resonances at 14.2, 15.2, 33.9, 34.2 and 188.5 ppm. These resonances have not been previously observed in the spectrum of the drug or any of its metalloderivatives and they may be due to bleoinycin fragments which occur upon oxidation of Fe(lI)BLM (16). 

Since DNA degrading by BLM has been shown to be sensitive to the nature and concentration of the buffer ions which are present in solution ( ), we examined the effects of various buffers of the esr spectra and the electrochemical properties of the Fe(III) complexes. Electrochemistry allows a clear view of how one of the metal ligating sites of BLM, the 4-amino pyrimidine moiety, behaves in the presence of buffer ions. If Fe(III)BLM is synthesized from the free drug and Fe(III)(CJlO,j)3 in the absence of buffers, electrochemistry shows that the 4-amino pyrimidine remains bound to the metal ion within the pH range 4-9 where esr shows that low... 

Figure 6, The ESR spectrum of Fe(III)BLM taken immediately after oxidation of Fe(II)BLM with molecular oxygen...

Figure 7. The relative amounts o/ fAl high and (9) low spin Fe(III)BLM as a function of pH as determined by ESR...

One family of antitumorigenic compounds used in practice are the bleomycins, natural products that contain iron isolated from Streptomyces cultures. Bleomycin sulfate is used in combination chemotherapy for treatment of head and neck cancer. The mechanism of action of bleomycin is believed to include the binding of oxygen to form 02-Fe(II)-BLM. This complex can accept an electron to produce an active species, 02 -Fe(III)-BLM, which can cleave DNA and RNA, ultimately killing the cancer cell. 

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