Chemical reactivities of bleomycin.

Ferric bleomycin was tested for its ability to catalyze a set of six oxidative reactions characteristic of the heme-containing proteins, cytochrome P-450 and chloroperoxidase. These reactions included peroxyacid decarboxylation and aliphatic hydroxylation as typical cytochrome P-450 chemistries. Peroxyacid-supported oxygen evolution and hydrogen peroxide-mediated chlorination were utilized as characteristic chloroperoxidase reactivities. A typical peroxidative reaction and heteroatom dealkylation, common to both O2 activating enzymes, were also studied. Bleomycin was found to catalyze peroxidation of o-dianisidine. The ferric drug complex was found competent in carrying out N-demethylation of N,N-dimethylaniline when peroxides or peroxyacids or iodosobenzene were used as the oxidants. N-Demethylation was not achieved when N,N-dimethylaniline-N-oxide was substituted as the oxidant under similar conditions. Hydroxylation of cumene and decarboxylation of phenylperacetic acid were not found to be catalyzed by bleomycin. Oxygen evolution from m-chloroperbenzoic acid and chlorination of monochlorodimedone from chloride ion and hydrogen peroxide were found to be catalyzed by bleomycin. Cytochrome P-450cam was also evaluated for O2 evolution, and halogenation activity and was found not to demonstrate such reactivities. The results of this initial survey, along with those of previous studies, appear to indicate that the chemical reactivity of bleomycin can be more closely aligned with the reactivities demonstrated by chloroperoxidase than those of cytochrome P-450.

The bleomycins constitute a family of glycopeptide antibiotics which differ only in their terminal amide functional groups (1) and are employed clinically for the treatment of certain carcinomas and lymphomas (2). The therapeutic efficacy of the bleomycins is believed to be related to their ability to degrade DNA i n uiuo (3), a process that has been shown, i n uitro, to proceed in the presence of appropriate metal ions and a source of oxygen (4-8).
These antineoplastic agents are isolated as one-to-one cop-* This study was supported by National Institutes of Health Grant GM31756 to S. G. S. Some portions of this work have been presented at the 36th Southeastern Regional Meeting of the American Chemical Society, October 24-26, 1984, Raleigh, NC and the 69th Annual Federation of The American Societies for Experimental Biology Meeting, April 21-26, 1985, Anaheim, CA. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Recipient of Career Development Award AM 01160.
per complexes from cultures of Streptomyces uerticillus @,lo) but are found to readily form complexes with a variety of metals (11). Extensive investigations on these metallo-drug complexes, particularly the iron complexes, have led to the observation of several parallels between the bleomycins and the cytochromes P-450, a family of heme-containing monooxygenases (12). Although structurally dissimilar, with bleomycin lacking a conjugated polyaromatic structure like heme and thiolate ligation to the metal (11, 13), iron drug complexes, reminiscent of the cytochromes P-450, are readily formed which can bind dioxygen and carry out site-specific oxidations (5,14). Both systems activate dioxygen by parallel pathways (12,15) and combine with O2 to form an oxygenated complex (12,16). Similarities between the two systems in terms of the electronic parameters, crystal field analysis of certain intermediates of the activation cycle, and interactions with various oxygen analogues have been established using a variety of spectral methods including Mossbauer (171, 'H NMR (18), EPR (15, [19][20][21], and optical spectroscopies (20,22). Further interrelations have been observed relating to aerobic activation with NADPH cytochrome P-450 reductase (23)(24)(25), anaerobic activation by peroxides and other oxygen surrogates (5,15), the effects of certain inhibitors (20,22), and the reactivity towards certain olefinic substrates (5, 7). Bleomycin is not unique in its complementarity with cytochrome P-450. Chloroperoxidase, a heme protein isolated from the mold Caldariomyces fumago that catalyzes biological halogenation reactions, also demonstrates remarkable correlations with cytochrome P-450 in terms of its physiochemical properties as evaluated by optical absorption (26-29), ESR (29), Mossbauer (30, 31), resonance Raman (32, 33), and magnetic circular dichroism spectroscopies (34). Therefore, indirectly, bleomycin can also be likened to chloroperoxidase.
Based on the strong physical evidence which has indicated the similarities between bleomycin and the cytochromes P-450, and circuitously chloroperoxidase, we have sought to carry out a preliminary survey of the ability of ferric bleomycin to catalyze some of the reactions catalyzed by the two heme proteins. The results of this initial evaluation show that the chemical reactivities demonstrated by bleomycin appear to align more closely with those demonstrated by chloroperoxidase than those of cytochrome P-450.

Bleomycin sulfate (BlenoxanZ) was a gift of Bristol Laboratories
and was used without further purification. A molecular weight of 1550 was assumed for the drug and = 1.45 X lo' M" cm". Ferric bleomycin was prepared as described by Burger et al. (20).
Decarboxylation and Hydroxylation Assays-Phenylperacetic acid was synthesized as previously reported (37,38). All peracids and alkyl peroxides were quantitated by iodometric titration (39) or by the assay of Cotton and Dunford (40).
Assays for decarboxylation and hydroxylation were attempted in both phosphate buffer and methanol under a variety of conditions. Typical reactions involved 53.6 nmol of bleomycin in 300 pl of solvent with 100-to 1000-fold excess of phenylperacetic acid in the decarboxylation assays and 53.6 mmol of cumene supported by a 100-to 500fold excess of peracid or 500-to 1000-fold excess of H202 in the decarboxylation reactions incubated for up to 32 h. Reaction mixtures were diluted to 2 ml with 20 mM phosphate buffer at pH 7.0 and extracted with an equal volume of CHC13. Reaction extracts were evaluated by GC on 3% OV-17.
Oxygen Euolution-Formation of oxygen was monitored by a Clarktype oxygen electrode (Yellow Springs Instrument Co.) analogous to the method reported by Manthey and Hager (41). Assays were carried out in 20 mM phosphate buffer, pH 7.0. m-Chloroperbenzoic acid (Aldrich) was purified to >98% by recrystallization from ethyl ether/ petroleum ether and dissolved in methanol before use daily. The other peracids and alkylperoxides were scavenged for H202 with nanomolar amounts of catalase (Sigma) prior to assay. Electrode membranes were changed and the reaction chamber volume was calibrated weekly for the bleomycin assays and daily for the enzyme assays.
Halogenation Assay-The standard assay mixture for chloroperoxidase and cytochrome P-450, involves 100 mM potassium phosphate and 50 mM potassium chloride of pH 3.0 and 7.0, respectively, containing 0.16 pmol of monochlorodimedone and saturating amounts of hydrogen peroxide. The reaction was started by addition of a suitable amount of enzyme in a total volume of 1 ml. Reactions with bleomycin were carried out in 1 ml of chloride-saturated methanol containing 0.16 pmol of monochlorodimedone. The bleomycin reactions were started by addition of saturating amounts of hydrogen peroxide. Reactions were monitored as described by Hager et al. (42).
Demethylation Assay-Reactions were carried out in 300 p1 of methanol containing 53.6 nmol of bleomycin, 53.6 pmol of N,N-dimethylaniline and were started by addition of 53.6 *mol of the respective oxidant. The DMA was omitted in reactions involving DMANO, and reactions with iodosobenzene were carried out in 1 ml of methano1:water (1:l). After an incubation time of 10 min, 56.1 pmol of internal standard, 1,2,4-trichlorobenzene, was added and the reaction was effectively stopped by addition of 1.7 ml of 20 mM phosphate buffer, pH 7.0, followed by extraction with 2 ml of CHC13. The reaction extracts were quantitatively evaluated by GC on Carbowax 20 M against redistilled commercial standards. The iodosobenzene and DMANO were prepared as previously reported (43, 44). Ethylhydroperoxide was scavenged for H202 prior to assay with picomolar quantities of catalase.
Peroxidatiue Assay-Assays were carried out in 100 mM phosphate buffer of pH 3.0 for chloroperoxidase and pH 7.0 for cytochrome P-450,., and bleomycin. Each 1-ml assay contained 0.22 pmol of odiansidine, saturated with respect to H202 and containing suitable amounts of enzyme or bleomycin to give an adequate signal. Reactions were treated as previously described (45).

RESULTS AND DISCUSSION
In a recent study, McCarthy and White (46) demonstrated that cytochrome P-450 was unique, among several heme proteins, in its ability to mediate decarboxylation of peroxyacids and aliphatic hydroxylation. When bleomycin was incubated with phenylperacetic acid using a variety of incubation conditions, GC analysis of the reaction mixtures were unable to detect any benzyl alcohol, the decarboxylation product of the peroxyacid. In exDeriments incubating ferric bleomvcin with under various reaction conditions, the hydroxylated products, 2-phenyl-2-propanol or 2-phenyl-1-propanol, was not detected by GC evaluation.
Among the unique reactions catalyzed by chloroperoxidase is the evolution of oxygen from substituted peroxides and peroxyacid (47). Oxygen evolution from meta-chloroperbenzoic acid was found to be readily catalyzed by bleomycin. Results shown in Fig. 1 compare the moles of oxygen evolved as a function of the moles of bleomycin or chloroperoxidase present in the reaction mixture at a constant m-CPBA concentration. The leveling off of the oxygen evolution at the higher levels of bleomycin is attributed to insufficient excess of the peroxyacid to maintain the psuedo-first order reaction conditions. Chloroperoxidase is also found to show the same leveling effect at much higher enzyme concentrations (data not shown). Fig. 2 shows the oxygen evolved as a function of the m-chloroperbenzoic acid concentration at a fixed bleomycin level. This relationship was found to be linear over the concentrations of peroxyacid studied. The lower limit was determined by the sensitivity of the O2 electrode used in the assay, while the upper limit was a function of the solubility ofthe m-CPBA. Oxygen evolution could not be detected when the rn-CPBA was replaced by peracetic acid, phenylperacetic acid, ethyl hydroperoxide, or t-butyl hydroperoxide in the bleomycin system. Cytochrome P-450,,, was not found to catalyze O2 evolution from the various alkyl peroxides and peroxyacids at any detectable levels in our hands, which may be a result of this enzyme's rapid autooxidation rate.
Chloroperoxidase also has the capability of catalyzing the peroxidative formation of a carbon-halogen bond in the presence of hydrogen peroxide, chloride ion, and a suitable halogen acceptor. Bleomycin, in a methanol system, was also found to be able to utilize chloride ion and hydrogen peroxide to halogenate monochlorodimedone to dichlorodimedone ( Table  I). The reaction was found to be dependent on chloride ion, hydrogen peroxide, and bleomycin. The reaction product was Under the assay conditions used, no NMA could be detected for the iodosobenzene assays, -although NMA was detected a t longer incubation times or a t higher levels of oxidant.
confirmed as dichlorodimedone, with a trace amount of dimedone noted by GC analysis against synthesized standards. No reactivity was found when cytochrome P-450,,, was evaluated for its ability to catalyze the same reaction under conditions similar to those of the chloroperoxidase assay. The observed initial rates reported for the different systems in Table I cannot be compared quantitatively; however, empirically they appear to indicate that bleomycin is much less efficient than chloroperoxidase in catalyzing this reaction.
Bleomycin was found to catalyze N-demethylation of N,Ndimethylaniline, a reaction that has previously been shown to be efficiently mediated by cytochrome P-450 (48) and chloroperoxidase (49). Several oxidants were found to be able to support the N-demethylation reaction ( Table 11). The initial velocity of the reaction was found to be dependent on the identity of the oxidant under these conditions, which are not necessarily optimal for each oxidant. In a recent report, Murugesan and Hecht (50) have also reported the N-demethylation of N,N-dimethylaniline with iodosobenzene and with ascorbate and 02. N-Demethylation was not achieved in systems where N,N-dimethylaniline-N-oxide was substituted as the oxidant. This observation parallels the results of Hollenberg and co-workers (49) on chloroperoxidase. Cytochromes P-450,, and P-450LM, have been reported to efficiently utilize the exogenous oxidant DMANO in the N-demethylation of DMA (51). Both heme proteins and bleomycin were found to carry out the peroxidation of o-dianisidine. Based on observed initial rates, chloroperoxidase was found to catalyze this reaction most efficiently (1.83 X lo3 nmol of H202 consumed/min/ nmol of chloroperoxidase) followed by bleomycin (3.39 X 10" nmol of H202 consumed/min/nmol of bleomycin) and cytochrome P-450,., (1.57 x nmol of H202 consumed/min/ nmol of cytochrome P-450), respectively. In each of the above cases, the reaction was found to be dependent on H202 and the respective catalyst. Again, empirically, chloroperoxidase is much more effective in catalyzing this reaction than bleomycin. The low activity of the cytochrome P-45OEa,, can be attributed to the high substrate specificity and rapid autoxidation rate of the enzyme. Table I11 summarizes the results of this initial survey. The table shows that, for the cross-section of oxidative reactivities studied, bleomycin demonstrates the same reactivity as chloroperoxidase, including the two rather unique chloroperoxidase reactivities of halogenation and peroxyacid (m-CPBA)-mediated oxygen evolution.
The unique new reactivities of bleomycin shown in this initial evaluation definitely indicate that further and more extensive studies are merited. 14.