Comparative binding properties of metallobleomycins with DNA 10-mers.

Properties of the interaction of bleomycin (Blm) and metallobleomycins [M = Zn, Cu(II), Fe(III), and HO(2)-Co(III)] with site-specific and nonspecific DNA oligomers, d(GGAAGCTTCC)(2) (I) and d(GGAAATTTCC)(2) (II), respectively, were investigated. With both 10-mers association constants increased in the series Blm A(2), ZnBlm A(2), Cu(II)Blm A(2), Fe(III)Blm A(2), and HO(2)-Co(III)Blm A(2). Generally, the metallobleomycins were bound with a modestly higher affinity to I. One-dimensional (1)H NMR spectra of the imino proton region of I in the presence of this series of compounds revealed that Blm and Zn- and CuBlm bind in fast exchange on the NMR time scale, while the Fe and Co complexes bind in slow exchange. Blm, ZnBlm, and Cu(II)Blm caused little perturbation of the UV circular dichroism spectrum of I or II. In contrast, Fe(III)Blm and HO(2)-Co(III)Blm induced hypochromic effects in the CD spectrum of I and altered the spectrum of II to a smaller extent. On the basis of these results, the DNA binding structures and properties of Blm A(2), ZnBlm A(2), and CuBlm A(2) differ substantially from those of Fe(III)Blm A(2) and HO(2)-Co(III)Blm A(2).     

Properties of the binding of copper by bleomycin

The glycopeptide, bleomycin, binds metal ions including Cu²⁺. It is the copper complex of this material that is isolated from Streptomyces verticillus. Both free ligand and copper complex are excellent antitumor agents in animals. The biochemical and pharmacological relationship between these compounds has not been established. The present study begins an analysis of the chemistry and biochemistry of copper-bleomycin with structural and equilibrium properties of the complex. Potentiometric and fluorometric titrations of bleomycin confirm three acidic groups with pK_a values of 7.50, 4.93, and 2.72. The conjugate nitrogen bases of these groups, comprise three of the binding sites for Cu²⁺ according to similar titrations of copper-bleomycin. The fourth is a conjugate base of an acid with a very large pK_a that cannot be measured by these techniques. The participation of a fourth such group is inferred from both proton release studies of the binding of metal and ligand above pH 8 and from several studies of the thermodynamic stability of copper bleomycin. At low pH binding of copper to bleomycin occurs in two steps, as observed by several independent techniques which monitor either the metal or the ligand. Log stability constants for the reactions Cu²⁺ + H_kBlm ? CuH_k-nBlm + nH⁺ and CuH_k-nBlm ? CuH_k-n-rBlm + rH⁺ are 1.32 and ?4.31, respectively, with n of 2.21 in the first equation and r of 2.07 in the second equation. The derived logarithm of the pH independent stability constant for copper bleomycin multiplied by the protonation constant for the unknown fourth ligand in the binding site is 12.16. This agrees closely with values obtained from measurements of conditional formation constants. One of the groups which binds in the second reaction is the substituted pyrimidine.     

Bleomycin may be activated for DNA cleavage by NADPH-cytochrome P-450 reductase

In the presence of NADPH and O2, NADPH-cytochrome P-450 reductase was found to activate Fe(III)-bleomycin A2 for DNA strand scission. Consistent with observations made previously when cccDNA was incubated in the presence of bleomycin and Fe(II) + O2 or Fe(III) + C6H5IO, degradation of DNA by NADPH-cytochrome P-450 reductase activated Fe(III)-bleomycin A2 produced both single- and double-strand nicks with concomitant formation of malondialdehyde (precursors). Cu(II)-bleomycin A2 also produced nicks in SV40 DNA following activation with NADPH-cytochrome P-450 reductase, but these were not accompanied by the formation of malondialdehyde (precursors). These findings confirm the activity of copper bleomycin in DNA strand scission and indicate that it degrades DNA in a fashion that differs mechanistically from that of iron bleomycin. The present findings also-establish the most facile pathways for enzymatic activation of Fe(III)-bleomycin and Cu(II)-bleomycin, provide data concerning the nature of the activated metallobleomycins, and extend the analogy between the chemistry of cytochrome P-450 and bleomycin.     

Copper-dependent cleavage of DNA by bleomycin

DNA strand scission by bleomycin in the presence of Cu and Fe was further characterized. It was found that DNA degradation occurred readily upon admixture of Cu(I) or Cu(II) + dithiothreitol + bleomycin, but only where the order of addition precluded initial formation of Cu(II)--bleomycin or where sufficient time was permitted for reduction of the formed Cu(II)--bleomycin to Cu(I)--bleomycin. DNA strand scission mediated by Cu + dithiothreitol + bleomycin was inhibited by the copper-selective agent bathocuproine when the experiment was carried out under conditions consistent with Cu chelation by bathocuproine on the time scale of the experiment. Remarkably, it was found that the extent of DNA degradation obtained with bleomycin in the presence of Fe and Cu was greater than that obtained with either metal ion alone. A comparison of the sequence selectivity of bleomycin in the presence of Cu and Fe using 32P-end-labeled DNA duplexes as substrates revealed significant differences in sites of DNA cleavage and in the extent of cleavage at sites shared in common. For deglycoblemycin and decarbamoylbleomycin, whose metal ligation is believed to differ from that of bleomycin itself, it was found that the relative extents of DNA cleavage in the presence of Cu were not in the same order as those obtained in the presence of Fe. The bleomycin-mediated oxygenation products derived from cis-stilbene were found to differ in type and amount in the presence of added Cu vs. added Fe. Interestingly, while product formation from cis-stilbene was decreased when excess Fe was added to a reaction mixture containing 1:1 Fe(III) and bleomycin, the extent of product formation was enhanced almost 4-fold in reactions that contained 5:1, as compared to 1:1, Cu and bleomycin. The results of these experiments are entirely consistent with the work of Sugiura [Sugiura, Y. (1979) Biochem. Biophys. Res. Commun. 90, 375-383], who first demonstrated the generation of reactive oxygen species upon admixture of O2 and Cu(I)--bleomycin.     

Kinetics of reaction of DNA-bound Fe(III)bleomycin with ascorbate: interplay of specific and non-specific binding

The aerobic redox reaction of Fe(III)bleomycin (Blm) and ascorbate was examined in the absence of DNA and in the presence of 7.5 and 25 calf thymus DNA base pairs per-drug molecule, in order to investigate the effect of DNA binding on the properties of FeBlm activation and DNA strand cleavage. Under these successive conditions, the rate of initial reduction of Fe(III)Blm became progressively slower and biphasic. Using 7.5 base pairs per-molecule of FeBlm, 2-3 times as much drug reacted in the faster step as with the larger DNA to drug ratio. In each case, the more rapid process was identified with the reaction of high spin Fe(III)Blm-DNA. With the smaller ratio, dioxygen consumption, formation of HO(2)-Fe(III)Blm-DNA, and production of DNA strand breaks as measured by the formation of base propenal were largely rate limited by the initial reaction of ascorbate with Fe(III)Blm-DNA. After a burst of reaction with the larger ratio of base pairs to Fe(III)Blm, a small fraction of the total Fe(III)Blm, representing high spin Fe(III)Blm, entered a steady state as HO(2)-Fe(III)Blm-DNA. Thereafter, reaction of dioxygen and base propenal formation occurred slowly with similar first-order rate kinetics. In order to explain these results, it is hypothesized that the metal domain-linker of Fe(III)Blm adopts two conformations with respect to DNA. One, at specific binding sites, is relatively unreactive with ascorbate. The other, present at non-specific sites as HPO(4)-Fe(III)Blm, is readily reactive with ascorbate to generate HO(2)-Fe(III)Blm-DNA. At the larger base pair to drug ratio, movement of Fe(III)Blm between specific and non-specific sites to generate HO(2)-Fe(III)Blm is a necessary part of the mechanism of strand scission.     

Copper(I)-bleomycin: structurally unique complex that mediates oxidative DNA strand scission

Copper(I)-bleomycin [Cu(I) X BLM] was characterized in detail by 13C and 1H NMR. Unequivocal chemical shift assignments for Cu(I) X BLM and Cu(I) X BLM X CO were made by two-dimensional 1H-13C correlated spectroscopy and by utilizing the observation that Cu(I) X BLM was in rapid equilibrium with Cu(I) and metal-free bleomycin, such that individual resonances in the spectra of BLM and Cu(I) X BLM could be correlated. The binding of Cu(I) by bleomycin involves the beta-aminoalaninamide and pyrimidinyl moieties, and possibly the imidazole, but not N alpha of beta-hydroxyhistidine. Although no DNA strand scission by Cu(II) X BLM could be demonstrated in the absence of dithiothreitol, in the presence of this reducing agent substantial degradation of [3H]DNA was observed, as was strand scission of cccDNA. DNA degradation by Cu(I) X BLM was shown not to depend on contaminating Fe(II) and not to result in the formation of thymine propenal; the probable reason(s) for the lack of observed DNA degradation in earlier studies employing Cu(II) X BLM and dithiothreitol was (were) also identified. DNA strand scission was also noted under anaerobic conditions when Cu(II) X BLM and iodosobenzene were employed. If it is assumed that the mechanism of DNA degradation in this case is the same as that under aerobic conditions (i.e., with Cu(I) X BLM + O2 in the presence of dithiothreitol), then Cu X BLM must be capable of functioning as a monooxygenase in its degradation of DNA.     

Structures of HO(2)-Co(III)bleomycin A(2) bound to d(GAGCTC)(2) and d(GGAAGCTTCC)(2): structure-reactivity relationships of Co and Fe bleomycins

HO(2)-Co(III)bleomycin is a model for HO(2)-Fe(III)bleomycin, which initiates single and double strand cleavage of DNA. In order to enlarge the understanding of its structure and reactivity, three-dimensional structures of HO(2)-Co(III)bleomycin bound to two DNA oligomers, d(GAGCTC)(2) (I) and d(GGAAGCTTCC)(2) (II), that have 5'-GC-3' binding sites, have been determined by nuclear magnetic resonance (NMR) methods. Besides previously recognized determinants of binding selectivity, a probable hydrogen bond was detected between the pyrimidinyl acetamido NH(2) and the carbonyl of cytosine base paired to G at the recognition site. Another hydrogen bond between the NH of the dimethylsulfonium R group and N7 of guanine opposite cytosine at the GC site may contribute to specification of the pyrimidine. Substitution of G with inosine shifted HO(2)-Co(III)Blm A(2)[bond]I and Fe(III)Blm[bond]I into fast exchange on the NMR time scale, supporting the role of the 2-amino group in site specification for each molecule. The conformationally stable metal-domain linker established a close-packed adduct with the minor groove in which the hydroperoxide ligand occupies a sterically constrained pocket that is isolated from the solvent. The hydroperoxide group is directed toward one of the two cytosine H4' hydrogens but is sterically blocked from access to the other by the drug. These findings enlarge the structural understanding of selective binding of Co(III)/Fe(III)Blm species at G-pyrimidine sites. They also rationalize the instability of a number of ligands bound to Co(III)/Fe(III)Blm at specific binding sequences and the relative unreactivity of Fe(III)Blm[bond]I with ascorbate as well as its lack of interaction with spin labels.     

Orientation of non-blue cupric complexes on DNA fibers.

Three different orientations of non-blue, type 2 cupric complexes on DNA fibers are obtained from EPR data. The cupric complex of bleomycin, CuBlm, binds as described previously (Shields, H., McGlumphy,C., and Hamrick, P., J., Jr. (1982) Biochim. Biophys. Acta 697, 113-120), except possibly with more restricted motion. The square plane of CuBlm makes an angle of about 65 degrees with the fiber axis. The tridentate complex 2-formylpyridine monothiosemicarbazonato Cu2+ binds with its planar structure perpendicular to the fiber axis. In contrast, other tridentate cupric complexes of tripeptides, CuGHK and CuGHG, bind with the square plane parallel to the fiber axis. The bound forms of Cu(GHK) and Cu(GHG) are determined mostly by the GH moiety in the complex; the contribution of lysine in defining the orientation of the copper moiety is minimal. Thus, the structure of the ligand determines the orientation of these complexes on DNA.     

Regulation of peroxo-bridge formation between cobalt(II)-carnosine complexes by histidine: Possible involvement in polycythemia

The mechanisms by which histidine stabilizes the cobalt(II)-carnosine complex from oxidation to cobalt(III) in aqueous solution are investigated with ¹H-nmr, laser Raman, and Fourier transform-infrared spectroscopy. Histidine has at least three effects on the cobalt(II)-carnosine complex. First, over the concentration range of at least 5 to 250 mM, histidine stabilizes the cobalt(II)-carnosine complex from oxidation by excluding solvent molecules from the equatorial coordination positions. Second, at the upper end of this concentration range, histidine reduces the strained nonplanarity of the equatorial coordination positions around the cobalt(II) ion that results from tridentate chelation by carnosine. Bidentate ligation by histidine causes the carnosine to bind as a bidentate ligand also. Third, bidentate ligation of two carnosine molecules to the equatorial coordination positions of Co(II) ion places the β-alanyl residues inthe vicinity of the two axial coordination positions and thereby inhibits the binding of molecular oxygen. Substitution of a molecule of histidine for one of these two carnosine molecules makes an axial coordination position available for binding oxygen. The first two effects are expected to stabilize the cobalt(II) ion from rapid oxidation, whereas the third effect is expected to give long-term stability of the peroxo-bridged complex. Since bidentate ligation of histidine is favored over monodentate ligation only when the concentration of Co(II) ion is not limiting and is inhibited by high concentrations of carnosine in the same solution, the results presented provide a possible explanation for the observation that the stability of the Co(II) complexes toward oxidation and their ability to bind molecular oxygen depend on both the relative and absolute concentrations of Co(II) ion, carnosine, and histidine in solution. Furthermore, these results provide additional support to the suggestion that the high activity of carnosinase in kidney is involved in part in regulation of the oxygen sensor in this organ.     

DNA strand scission by iron complexes of meso-tetra(N-methylpyridyl)porphines

The DNA strand scission activities of three positional isomers of Fe(III) meso-tetra(N-methylpyridyl)porphine (Fe(III)TnMPyP, where n = 2, 3 or 4) have been investigated using PM2 DNA as a substrate. A significant degree of strand scission activity was noted in the presence of oxygen without the addition of a reducing agent. This activity was probably due to the presence of reducing agents in the agarose gels used to separate the DNA forms, as higher levels were recorded with reducing agents added to the strand scission mixture. The relative order of strand scission activity in the absence of added reducing agents was found to be Fe(III)T2MPyP greater than Fe(III)T4MPyP greater than Fe(III)T3MPyP. Comparative studies were also made with Fe(II)bleomycin. High concentrations of some reducing agents inhibited strand scission. Oxygen was required to produce optimal strand scission activity for all three porphyrins. It was also noted from spectroscopic measurements that the reduced porphyrins were degraded in the presence of oxygen. Studies with a series of potential strand scission inhibitors suggest that hydrogen peroxide and possibly peroxy radicals are intermediates in the reaction mechanism, while diffusible hydroxyl radicals appear to be excluded. However, superoxide radicals cannot be ruled out.     

The oxidation of cat,human, and the cat-human hybrid hemoglobins α2Humanβ2Cat and α2Catβ2Human by copper(II)

The heme iron of the β chains of mammalian hemoglobins are rapidly and selectively oxidized in the presence of excess Cu(II) ions in a reaction that requires the presence of a free -SH groups on the β globin chain. The presence of freely reactive -SH groups on the α chains of cat and sheep hemoglobins does not alter the course of this reaction: only the β hemes are oxidized rapidly by Cu(II) in these hemoglobins. Two equivalents of copper are required for the rapid oxidation of the two β chain hemes per mole of cat hemoglobin, in contrast with the four equivalents that are required for reaction with human hemoglobin. The human-cat hybrid hemoglobins, α₂^Humanβ₂^Cat and α₂^Catβ₂^Human, required two and four equivalents of copper/mol, respectively, for the reaction. Thus, the kinetics and stoichimetry of the reaction are determined by the nature of the β subunit. Analysis of the esr spectra of the products of the reaction of Cu(II) with these hemoglobins indicate that human hemoglobin and the hybrid α₂^Catβ₂^Human contain tight binding sites for two equivalents of Cu(II) that are not involved in the oxidation reaction and are not present in cat hemoglobin or α₂^Humanβ₂^Cat. Cat β globin like others (sheep, bovine) that lack the tight binding site, has no histidine residue at 2β. It has phenylalanine in this position. These results support the suggestion of Rifkind et al. (Biochemistry 15,5337[1976]) that the tight binding site is near the amino terminal region of the β chain and is associated with histidine 2β.     

Degradation of structurally modified DNAs by bleomycin group antibiotics

Bleomycin-mediated DNA strand scission has been shown to be diminished at certain sequences in proximity to 5-methylcytidines. We have investigated the molecular basis of this observed diminution using selective bleomycin (BLM) modifications at the C-terminus. Of the four different bleomycin congeners investigated, only bleomycin A2 and bleomycin BAPP were substantially affected by cytidine methylation. We have also examined the effect of other DNA modifications on bleomycin-mediated strand scission. Methylation at the N6 position of adenosine resulted in diminution of DNA cleavage by all four bleomycin congeners. The presence of bulky 5-(glucosyloxy)methyl groups in the major groove of T4 DNA had little effect on the efficiency of DNA strand scission mediated by bleomycin A2 or B2, suggesting the absence of important steric interactions between Fe(II).BLM and DNA in the major groove. In contrast, DNA cleavage mediated by bleomycin congeners was very sensitive to a major DNA conformational change, the B----Z transition. Salt and MgCl2 titrations of the DNA copolymers poly(dG-dC).poly(dG-dC) and poly(dG-MedC).poly(dG-MedC) demonstrated that bleomycin A2 and B2 did not cleave Z-DNA efficiently. In addition, circular dichroism titrations of these copolymers revealed that both bleomycin congeners increased the cation concentration necessary to induce the B----Z transition, implying that bleomycin preferentially binds to and stabilizes B-form DNA. These results are consistent with a model in which cytidine methylation at appropriate sequences of DNA is sufficient to induce subtle conformational changes that render the helix unreceptive to cleavage by some bleomycin congeners.     

Iron(II)-ethylenediaminetetraacetic acid catalyzed cleavage of RNA and DNA oligonucleotides: similar reactivity toward single- and double-stranded forms

Fe(II)-EDTA catalyzes the cleavage of nucleic acids with little or no base-sequence specificity. We have now studied the preference of this reagent in catalyzing the cleavage of single- versus double-stranded nucleic acid structures. Three RNA and two DNA molecules, each expected to contain both single- and double-stranded regions, were synthesized and their structures characterized by enzymatic digestion using secondary structure specific nucleases. Fe(II)-EDTA catalyzed nearly uniform strand scission along the entire length of each molecule; no correlation with secondary structure was observed. The homopolymer sequence dA30:dT30, embedded in a mixed-sequence context to promote exact register of the homopolymer tract, was cleaved to an extent similar to that of flanking sequences. The reactions were relatively insensitive to K+, Na+, and Mg2+ in the range 10-100 mM and were quenched by Tris-HCl buffer. We conclude that the Fe(II)-EDTA-catalyzed strand scission reaction does not discriminate between typical single- and double-stranded regions, which simplifies the interpretation of experiments in which the reaction is used to probe the tertiary structure of RNA molecules [Latham, J. A., & Cech, T. R. (1989) Science 245, 276-282].     

Asymmetric structure of a three-arm DNA junction

We present here experimental evidence that three-arm branched DNA molecules form an asymmetric structure in the presence of Mg2+. Electrophoretic mobility and chemical and enzymatic footprinting experiments on a three-arm branched DNA molecule formed from three 16-mer strands are described. The electrophoretic mobilities of three species of a three-arm junction in which pairs of arms are extended are found to differ in the presence of Mg2+: one combination of elongated arms migrates significantly faster than the other two. This effect is eliminated in the absence of Mg2+, leading us to suggest that the three-arm DNA junction forms an asymmetric structure due to preferential stacking of two of the arms at the junction in the presence of Mg2+. The pattern of self-protection of each 16-mer strand of the core complex exposed to Fe(II).EDTA and DNase I scission is unique, consistent with formation of an asymmetric structure in the presence of Mg2+. We conclude that three-arm junctions resemble four-arm junctions in showing preferential stacking effects at the branch site. Comparison of the scission patterns of linear duplexes and the branched trimer by the reactive probes methidiumpropyl-EDTA.Fe(II) [MPE.Fe(II)] and Cu(I)-[o-phenanthroline]2 [(OP)2CuI] further indicates that the branch point represents a site of enhanced binding for drugs, as it does in the four-arm case. Reaction with diethyl pyrocarbonate (DEPC), a purine-specific probe sensitive to conformation, is enhanced at the branch site, consistent with loosening of base pairing or unpairing at this point.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular interaction between bleomycin and amsacrine in the presence of cupric ions

The antineoplastic activity of m-AMSA [4'-(9-acridinylamino)-methanesulfon-m-anisidide] has been related to its ability to produce oxygenated free radical during its oxidation to a quinonimine form, in the presence of cupric ions. It has been demonstrated here that the rate of the oxidation is greatly increased by the addition of bleomycin (Blm), another antitumor agent, which is able to complex metallic ions. The catalytic role of Blm has been established on the basis of kinetics measurements and the occurrence of an intermediary ternary complex Blm-m-AMSA-Cu(II) has been demonstrated by circular dichroism and polarography experiments.     

Charge dependence of Fe(II)-catalyzed DNA cleavage.

The effect of charge of the Fe(II) reagent used to induce DNA strand cleavage reactions in the presence of a source of reducing equivalents is investigated using two oligonucleotide models. The first consists of the two strands dA20 and dT20, and an equimolar complex between them. The second is a short four-arm branched DNA complex composed of four 16-mer strands. In the former case, cleavage of the 1:1 complex by three reagents with different formal charge, Fe(II).EDTA2-, Fe(II).EDDA and Fe2+, is comparable in rate to that of the individual dT20 and the dA20 strands. While the three reagents show similar cleavage rates for the duplex and single stranded molecules, they give distinctive cutting patterns in the DNA tetramer, consistent with the presence of a site of excess negative charge at the branch point. Scission induced by Fe(II).EDTA2- shows lower reactivity at the branch site relative to duplex controls, whereas Fe(II)2+ shows enhanced reactivity. Formally neutral Fe(II).EDDA shows weak loss of cutting reactivity at the branch. The position of attack by Fe(II)2+ in the branched tetramer is shifted with respect to those of Fe(II).EDTA2- or Fe(II).EDDA; a slower migrating species is also detected in the scission of dA20.dT20 duplex by Fe(II) reaction. These results suggest that the Fe(II)2+ reaction proceeds by a different mechanism from the other agents. The difference in cutting profiles induced by the neutral and negatively charged chelated complexes is consistent with a local electrostatic repulsion of a negatively charged source of radicals, not a positively charged one.     

Raman spectroscopy of an O(2)-Co(II)bleomycin-calf thymus DNA adduct: alternate polymer conformations.

Bleomycin (Blm) is an antitumor agent which binds to specific sequences of DNA and as HO(2)-Fe(III)Blm causes single and double strand cleavage. In the present investigation, binding of O(2)-Co(II)Blm to a native DNA polymer, calf thymus DNA, was examined using conventional Raman spectroscopy. O(2)-Co(II)Blm is a model for O(2)-Fe(II)Blm, the direct precursor of HO(2)-Fe(III)Blm. Although the DNA polymer retained a predominant B-form structure, Raman spectral evidence was obtained for localized structural changes to A, C and Z-DNA forms. The presence of these alternate DNA forms within B-DNA implied the presence of B/A, B/C and B/Z junctions. The observed changes in DNA secondary structure were attributed to perturbation of structural water resulting from binding of O(2)-Co(II)Blm within the minor groove.     

Studies on the effects of the antitumor agent camptothecin and derivatives on DNA. Camptothecin potentiated cleavage of DNA by bleomycin in vitro

Addition of sodium camptothecin (2a, Fig. 1) in comparable low concentrations to the glycopeptide antitumor antibiotic bleomycin (BLM) leads to enhanced rates of single-strand scission of PM2-covalently closed circular DNA, whereas sodium camptothecin alone has no effect. A similar enhancement of DNA scission by sodium camptothecin is produced with the 1 : 1 bleomycin-iron complex alone or in conjunction with NADPH as an additional reductant. The interpretation that camptothecin may substitute for the reducing requirement of the antibiotic is supported by its oxidation at 37°C by the 1 : 1 bleomycin iron complex, by iron salts or more efficiently by hydrogen peroxide to the known hemiacetal (3, Fig. 1).Electrochemical studies of 2a, its analogues and selected model compounds established that the α-pyridone ring D is most susceptible to a one-electron reduction at a reversible potential of ?0.95 ± 0.01 V. The reduced camptothecin is a transient species readily capable of donating an electron. This process may by compatible with a coupled reduction of the sequestered Fe(III) in the glycopeptide antibiotic necessary for the expression of antibiotic and antitumor properties. The results may provide a mechanistic rationale for the observed potentiation of the antitumor activity of bleomycin by camptothecin in vivo.     

Synthetic analogues and biosynthetic intermediates of bleomycin. Metal-binding, dioxygen interaction, and implication for the role of functional groups in bleomycin action mechanism

In order to clarify the role of bleomycin functional groups in action mechanism, the metal-binding, dioxygen activation, and DNA cleavage of several synthetic analogues and biosynthetic intermediates of bleomycin have been investigated. The present results support that 1) the beta-aminoalaninepyrimidine-beta-hydroxyhistidine portion of the bleomycin molecule substantially participates in the Fe(II) and dioxygen interactions, 2) the transposition of the pyrimidine (or pyridine) and imidazole groups in the Fe(II)-coordination is essential for the effective binding and activation of molecular oxygen by the bleomycin ligands, and 3) the gulose-mannose moiety plays an important role as an environmental factor for the efficient dioxygen reduction and DNA cleavage, although the sugar portion does not contribute significantly to the nucleotide specificity in the DNA strand scission. Certain oligopeptides are able to mimic the metal-binding and dioxygen activation by bleomycin, but not induce the effective DNA cleavage. Probably, the bithiazole DNA interaction site of bleomycin delivers the iron/dioxygen chemistry to particularly the DNA (formula, see text) nucleotide sequences.     

Ligational behavior of a novel biologically active donor toward Cu(II) and Ni(II) ions and potentiation of its antibacterial activity by chelation with metal ions

The chelating behavior of a new multidentate ligand with tuberculostatic activity toward Cu(II) and Ni(II) ions has been studied. This ligand 3-(2-carboxyhydrazine)phenylimino-2-oximobutane(H2C POB) is found to chelate the above metal ions in both its keto and enol forms. The probable structures of all the complexes and the location of the bonding sites have been established through magnetic and spectroscopic (infrared, electronic) studies. The Cu(II) complex of the enol form exhibits subnormal magnetic moment at room temperature, indicating the probable existence of some sort of super exchange phenomenon in the system. The ligand itself and a few of its Cu(II) complexes have been found to exert powerful in vitro antibacterial activity toward some tuberculosis mycobacteria, such as Mycobacterium flae, Mycobacterium smegmatis, and Mycobacterium H37Rv.