Transition-metal binding site of bleomycin A2. A carbon-13 nuclear magnetic resonance study of the zinc(II) and copper(II) derivatives.

The 13C NMR spectra at 25.2 MHz of the Zn(II) and Cu(II) complexes of the antitumor antibiotic bleomycin A2 are discussed. Complexation of the drug to Zn(II) causes 38 of the 52 resonance lines of bleomycin A2 to shift to new positions. All but ten of these shifted lines have been assigned in the Zn(II) bleomycin complex. Although the specific donor sites of the drug cannot be identified from the 13C NMR data, the analysis clearly shows that the pyrimidine-imidazole portion of the molecule is affected by chelation. This finding is in agreement with the previously reported metal-binding site of the antibiotic. The analysis also shows that carbon atoms which have large through-bond distances from the binding site can experience substantial chemical-shift changes upon metal binding. Complexation of the drug to Cu(II) eliminates 23 resonances from the spectrum of the molecule. All of these resonances emanate from carbon atoms which are located in the pyrimidine-imidazole portion of the drug.     

Copper(I) . bleomycin. A structurally unique oxidation-reduction active complex

Cu(I) and Cu(II) form stable 1:1 complexes with bleomycin (BLM). The affinity of both metals for the drug is greater than that of Fe(II). Cu(I) . BLM A2 binds to calf thymus DNA with about the same affinity as Fe(II) . BLM, as judged by DNA-induced fluorescence quenching of the bithiazole moiety of BLM. Based on 1H NMR and potentiometric titration data, the Cu(I) complexes of BLM are shown to have geometries very different than those of other BLM . metal(II) complexes studied thus far. As Cu(I) . BLM is oxidation-reduction active, its geometry is of importance in defining the structural requirements for BLM activity.     

A spectroscopic investigation of the metal binding site of bleomycin A2. The Cu(II) and Zn(II) derivatives.

Using a combination of ultraviolet-visible absorption, 1H NMR and ESR techniques we have established that N(1) of the imidazole and N(1) of the pyrimidine residues of bleomycin A2 bind to Cu(II) and Zn(II). The observations coupled with the earlier results that the alpha-amino group of the alpha-amino carboxamide function and the carbamoyl moiety are also Cu(II)-ligating groups makes it possible to reconstruct the detailed geometry and stereochemistry of the metal binding site of bleomycin A2.     

Deglyco-bleomycin. Degradation of DNA and formation of a structurally unique Fe(II) . CO complex

In analogy with bleomycin, deglyco-bleomycin B2 has been found to form a stable, diamagnetic complex with Fe(II) and CO. Although the stoichiometry of this complex appeared to be the same as that formed with bleomycin, the geometry of the deglyco-bleomycin complex was fundamentally different, especially as regards orientation of the beta-aminoalanine moiety. In the presence of Fe(II) and O2, deglyco-bleomycin A2 and deglyco-bleomycin B2 were found to release [3H]thymine from radiolabeled PM-2 DNA; when employed at limiting concentrations, deglyco-bleomycin A2 and B2 gave about half as much [3H]thymine release as the respective bleomycins. In view of the spectral evidence (Burger, R. M., Horwitz, S. B., Peisach, J., and Wittenberg, J. B. (1979) J. Biol. Chem. 254, 12299-12302) that Fe(II) . bleomycin . CO has the same geometry as the complex formed by initial association of bleomycin, Fe(II), and O2, the accumulated data suggest strongly that all metal complexes of bleomycin (derivatives) capable of DNA degradation need not have the same geometry.     

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.     

Studies on the chemical reactivity of copper bleomycin

The copper(II) complex of the clinically used antitumor agent bleomycin (Blm) has cytotoxic as well as antitumor properties. To understand the relationship of the bleomycin ligand, copper bleomycin, and other possible metal complexes of this agent, kinetic studies of the formation of Cu(II)Blm, ligand substitution reactions of CuBlm with ethylenediaminetetraaletic acid, and the redox reaction of CuBlm with thiols have been completed and interpreted along with previous studies of the thermodynamic stability of Cu2+ with bleomycin. Cu(II)Bm is found to be kinetically and thermodynamically stable in ligand substitution processes and is only slowly reduced and dissociated by sulfhydryl reagents. The rate constant of reduction of the complex by 2-mercaptoethanol (2-ME) at pH 7.4 and 25 degrees C is 9.5 X 10(-3) M-1 sec-1, explaining the inhibition of Fe2+-dependent strand scission of DNA by Cu2+ in the presence of 2-ME. CuBlm forms in preference to Fe(II)Blm and cannot be reduced and dissociated rapidly enough by thiols to liberate Blm and form the reactive iron complex. In agreement with the observed chemical stability of CuBlm, it is also shown that the complex is stable in human plasma and in the presence of Ehrlich cells suspended in ascites fluid. Interestingly, little CuBlm enters these cells to carry out cytotoxic reactions. Finally, it is shown that both Cu2+ and Zn2+, at equivalent concentrations to Fe2+, effectively inhibit the strand scission of DNA by Fe(II)Blm plus oxygen. However, at substoichiometric amounts of Cu2+, the ferroxidase activity of Blm enables the drug to remain effective in the strand-scission reaction, despite the lowered Cu-free Blm/Fe2+ ratio. These results are discussed in light of the proposed mechanism of action of 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.     

Antimicrobial and mutagenic activity of some carbono- and thiocarbonohydrazone ligands and their copper(II), iron(II) and zinc(II) complexes.

Several mono- and bis- carbono- and thiocarbonohydrazone ligands have been synthesised and characterised; the X-ray diffraction analysis of bis(phenyl 2-pyridyl ketone) thiocarbonohydrazone is reported. The coordinating properties of the ligands have been studied towards Cu(II), Fe(II), and Zn(II) salts. The ligands and the metal complexes were tested in vitro against Gram positive and Gram negative bacteria, yeasts and moulds. In general, the bisthiocarbonohydrazones possess the best antimicrobial properties and Gram positive bacteria are the most sensitive microorganisms. Bis(ethyl 2-pyridyl ketone) thiocarbonohydrazone, bis(butyl 2-pyridyl ketone)thiocarbonohydrazone and Cu(H2nft)Cl2 (H2nft, bis(5-nitrofuraldehyde)thiocarbonohydrazone) reveal a strong activity with minimum inhibitory concentrations of 0.7 microgram ml-1 against Bacillus subtilis and of 3 micrograms ml-1 against Staphylococcus aureus. Cu(II) complexes are more effective than Fe(II) and Zn(II) ones. All bisthiocarbono- and carbonohydrazones are devoid of mutagenic properties, with the exception of the compounds derived from 5-nitrofuraldehyde. On the contrary a weak mutagenicity, that disappears in the copper complexes, is exhibited by monosubstituted thiocarbonohydrazones.     

Bis-bidentate bridging ligands containing two N,O-chelating pyrazolyl-phenolate units; double helical complexes with Co(II), Cu(II) and Zn(II)

Two ligands have been prepared in which N,O-bidentate chelating pyrazolyl-phenolate units, based on 3-(2-hydroxyphenyl)pyrazole, are connected via methylene linkages to aromatic (1,4-phenylene or 3,3′-biphenylene) spacers. In each case the two N,O-donor units are too far apart to chelate to a single metal ion. Complexes of both ligands with Co(II), Cu(II) and Zn(II) were prepared and structurally characterised; in all cases the complexes are dinuclear double helicates M₂L₂, with each four-coordinate metal ion bound by a chelating unit from each of the two ligands in the complex. For Co(II) and Zn(II) the two M(NO) planes at each metal are close to perpendicular, indicative of a geometry which may be described as approximately distorted tetrahedral; for the Cu(II) complexes the angle between the two Cu(NO) planes is less, indicative of a distortion towards a more planar coordination geometry.     

Metal coordination core of bleomycin : comparison of metal complexes between bleomycin and its biosynthetic intermediate.

On the basis of electron spin resonance results, the 1:1 Cu(II), Co(II), Co(II)-O₂, and Ni(III) complexes of bleomycin(BLM) have been compared with the corresponding metal complexes of its biosynthetic intermediate(P-3A). The present study suggests that (1) P-3A is an useful ligand for the clarification of metal-binding sites of BLM; (2) the secondary amine, pyrimidine ring nitrogen, deprotonated peptide nitrogen of histidine residue, and histidine imidazole groups as planar ligand donors, and the α-amino group as axial donor, are substantially important for metal-coordination of BLM; and (3) the sugar and bithiazole portions of BLM probably contribute to stabilization of Co(II)-O₂ adduct complex and axial sixth coordination of Cu(II) and Ni(III) complexes.     

Structural study of copper(I)-bleomycin

Previous NMR studies on Cu(I)-bleomycin have suggested that this adduct has a geometry distinct from Fe(II)BLM. The coordination chemistry of this bleomycin derivative has been investigated through the extension of the NMR data reported previously, and the use of molecular dynamics calculations. The data collected from the NMR experiments support the coordination to the metal center of the primary and secondary amines in beta-aminoalanine and the pyrimidine ring. The detection in the NMR spectra of the signal derived from the amide hydrogen in beta-hydroxyhistidine indicates that this amide is protonated in Cu(I)-bleomycin, precluding participation of the pyrimidinyl carboxamide nitrogen in the coordination of Cu(I), as previously reported. Three-dimensional solution structures compatible with the NMR data have been assayed for Cu(I)-bleomycin for the first time by way of molecular dynamics calculations, and two models showing four and five coordination have been found to be those that better fit the experimental data. In both models the primary amine in beta-aminoalanine is coordinated such that it is located on the same side, with respect to the coordination cage, as the peptide linker fragment. This result seems important for the favored models to be compatible with either their possible oxidation to become one of the reported structures for Cu(II)BLM, or their transformation into Fe(II) adducts able to cause DNA damage.     

Some bivalent metal complexes of Schiff bases containing N and S donor atoms

Schiff bases have been synthesized by the reaction of p-nitrobenzaldehyde, o-nitrobenzaldehyde and p-toluyaldehyde with 4-amino-5-mercapto-1,2,4-triazole. The ligands react with Co(II), Ni(II) and Zn(II) metals to yield (1:1) and (1:2) [metal:ligand] complexes. Elemental analyses, IR, 1H NMR, electronic spectral data, magnetic susceptibility measurements, molar conductivity measurements and thermal studies have investigated the structure of the ligands and their metal complexes. The electronic spectral data suggests octahedral geometry for Co(II), Ni(II) and Zn(II). The antibacterial activities of the ligands and their metal complexes have been screened in vitro against three Gram-positive (Staphylococcus aureus, Staphylococcus epidermidis and Bacillus subtilis) and two Gram-negative (Salmonella typhi and Pseudomonas aeruginosa) organisms. The coordination of the metal ion had a pronounced effect on the microbial activities of the ligands and the metal complexes have higher antimicrobial effect than the free ligands.     

Comparison of the vibration mode of metals in HNO3 by a partial least-squares regression analysis of near-infrared spectra

The near-infrared (NIR) spectra of such metals as Cu(II), Mn(II), Zn(II) and Fe(III) in HNO(3) in the 700-1,860 nm region were subjected to a partial least-squares regression analysis and leave-out cross-validation to develop chemometric models. The models yielded a coefficient of determination in cross validation of 0.9744 [Cu(II)], 0.9631 [Mn(II)], 0.9154 [Zn(II)] and 0.741 [Fe(III)]. The regression coefficients for Cu(II), Mn(II) and Zn(II), but not for Fe(III), showed strong negative peaks at around 1,050-1,200 nm, a zone where spectral bands have been reported to decrease with increasing pH value. A positive peak at around 710-750 nm, which may have been due to water absorption, was observed in regression coefficients of Cu(II), Mn(II) and Zn(II) but not in Fe(III), while a negative peak was observed in that for Fe(III) at around 710-750 nm. These results indicate that the divalent cations [Cu(II), Mn(II) and Zn(II)] showed different absorption in the NIR region from the trivalent cation [Fe(III)], suggesting that the vibration mode of water, which mirrors the interaction between cations and water, may be influenced by valency.     

Comparison of the Vibration Mode of Metals in HNO3 by a Partial Least-Squares Regression Analysis of Near-Infrared Spectra

The near-infrared (NIR) spectra of such metals as Cu(II), Mn(II), Zn(II) and Fe(III) in HNO₃ in the 700–1860 nm region were subjected to a partial least-squares regression analysis and leave-out cross-validation to develop chemometric models. The models yielded a coefficient of determination in cross validation of 0.9744 [Cu(II)], 0.9631 [Mn(II)], 0.9154 [Zn(II)] and 0.741 [Fe(III)]. The regression coefficients for Cu(II), Mn(II) and Zn(II), but not for Fe(III), showed strong negative peaks at around 1050–1200 nm, a zone where spectral bands have been reported to decrease with increasing pH value. A positive peak at around 710–750 nm, which may have been due to water absorption, was observed in regression coefficients of Cu(II), Mn(II) and Zn(II) but not in Fe(III), while a negative peak was observed in that for Fe(III) at around 710–750 nm. These results indicate that the divalent cations [Cu(II), Mn(II) and Zn(II)] showed different absorption in the NIR region from the trivalent cation [Fe(III)], suggesting that the vibration mode of water, which mirrors the interaction between cations and water, may be influenced by valency.     

Complexes of salicylaldehyde acylhydrazones: cytotoxicity, QSAR and crystal structure of the sterically hindered t-butyl dimer

A series of acylhydrazones of salicylaldehyde and their transition metal complexes, predominantly copper(II), have been prepared and characterized. The crystal structure of the Cu(II) complex of the sterically hindered t-butyl derivative contains a phenolato bridged dimer with the ligand coordinated as a tridentate moiety. QSAR analyses of the cytotoxicity of the chelators and their Cu(II) complexes reveals that solubility is the dominant factor for activity. Compounds display a maximum with respect to lipophilicity, allowing optimization of the bioactivity for both the ligands and their complexes. Copper complexes are significantly more cytotoxic than the metal-free ligands and complexes of other metals: Cu > Ni > Zn = Mn > Fe = Cr > Cr > Co.     

Synthesis and characterization of a series of new mixed ligand complexes of manganese(III), iron(III), nickel(II), copper(II) and zinc(II) with schiff bases of N,N-diethylamino-dithiocarbamate as ligands

Twenty new bioactive complexes of Mn(III), Fe(III), Ni(II), Cu(II) and Zn(II) have been prepared containing Schiff bases of N,N-diethylaminodithio- carbamate as ligands. These complexes have been characterized by elemental analyses, IR and UV-Vis spectroscopy as well as by magnetic susceptibility measurements. The spectra of the complexes suggest that the ligands are coordinated to the metal ions via the sulfur atoms of the dithiocarbamato group.     

Demonstration of nitrogen coordination in metal--bleomycin complexes by electron spin--echo envelope spectroscopy

We have studied the Cu(II), Co(II), and Fe(III) complexes of the antineoplastic drug bleomycin by using electron spin--echo envelope spectroscopy. For all three complexes, nitrogen coordination of the metal ions is demonstrated. For the Cu(II)-- and Co(II)--drug complexes, we have been able to identify imidazole as a metal ligand.     

Binding of exogenously added carbon monoxide at the active site of the iron-only hydrogenase (CpI) from Clostridium pasteurianum.

A site for the binding of exogenously added carbon monoxide has been identified at the active site of the Fe-only hydrogenase (CpI) from Clostridium pasteurianum. The binding and inhibition of carbon monoxide have been exploited in biochemical and spectroscopic studies to gain mechanistic insights. In the present study, we have taken advantage of the ability to generate an irreversibly carbon monoxide bound state of CpI. The crystallization and structural characterization of CpI inhibited in the presence of carbon monoxide indicates the addition of a single molecule of carbon monoxide. The ability to generate crystals of the carbon monoxide bound state of the hydrogenase that are isomorphous to those of the native enzyme has allowed for a direct comparison of the crystallographic data and an unambiguous identification of the site of carbon monoxide binding at the active site of CpI. Carbon monoxide binds to an Fe atom of the 2Fe subcluster at the site of a terminally bound water molecule in the as crystallized native state of CpI that has been previously suggested to be a potential site of reversible hydrogen oxidation. Binding of carbon monoxide at this site results in an active site that is coordinately saturated with strong ligands (S, CO, and CN), providing a rational potential mechanism for inhibition of reversible hydrogen oxidation at the active site of CpI.     

Inactivation of glutamine synthetase by a purified rabbit liver microsomal cytochrome P-450 system

Several mixed-function oxidation systems catalyze inactivation of Escherichia coli glutamine synthetase and other key metabolic enzymes. In the presence of NADPH and molecular oxygen, highly purified preparations of cytochrome P-450 reductase and cytochrome P-450 (isozyme 2) from rabbit liver microsomes catalyze enzyme inactivation. The inactivation reaction is stimulated by Fe(III) or Cu(II) and is inhibited by catalase, Mn(II), Zn(II), histidine, and the metal chelators o-phenanthroline and EDTA. The inactivation of glutamine synthetase is highly specific and involves the oxidative modification of a histidine in each glutamine synthetase subunit and the generation of a carbonyl derivative of the protein which forms a stable hydrazone when treated with 2,4-dinitrophenylhydrazine. We have proposed that the mixed-function oxidation system (the cytochrome P-450 system) produces Fe(II) and H2O2 which react at the metal binding site on the glutamine synthetase to generate an activated oxygen species which oxidizes a nearby susceptible histidine. This thesis is supported by the fact that (a) Mn(II) and Zn(II) inhibit inactivation and also interfere with the reduction of Fe(III) to Fe(II) by the P-450 system; (b) Fe(II) and H2O2 (anaerobically), in the absence of a P-450 system, catalyze glutamine synthetase inactivation; (c) inactivation is inhibited by catalase; and (d) hexobarbital, which stimulates the rate of H2O2 production by the P-450 system, stimulates the rate of glutamine synthetase inactivation. Moreover, inactivation of glutamine synthetase by the P-450 system does not require complex formation because inactivation occurs when the P-450 components and the glutamine synthetase are separated by a semipermeable membrane. Also, if endogenous catalase is inhibited by azide, rabbit liver microsomes catalyze the inactivation of glutamine synthetase.     

Specificity of transport of bleomycin and cobalt-bleomycin in L5178Y cells

The mechanism of transport of [³H]peplomycin (PEP), a new member of bleomycin group antibiotics, was studied in cultured L5178Y mouse leukemic cells. Cobalt ions enhanced the uptake of PEP, but Cu, Zn, Fe(II) and Fe(III) had no effect. The initial rate of uptake of cobalt chelated PEP [PEP(Co)] was several times higher than that of free or Cu-chelated PEP and was temperature independent. A double reciprocal plot of the data demonstrated both saturable (K_m = 4.5 μM, V_max = 1.3 × 10^?18 mole/min/cell) and non-saturable components of the uptake of PEP(Co). The saturable component was inhibited specifically by cobalt chelated bleomycin analogs. PEP-chelates with metals other than cobalt, such as PEP(Cu) were metabolically unstable. These results suggest that bleomycin enters into cells as a metal chelate through a specific transport site.