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The bleomycins (BLMs) are structurally related glycopeptide antibiotics isolated from streptomyces vetricillus that mediate the sequence-selective oxidative damage of DNA and RNA. Deglycoblemycin, which lacks the carbohydrate moiety cleaves DNA analogously to bleomycin itself and is used for analyzing the functional domains of bleomycin.

Research has been conducted on the bleomycin and metallobleomycins. The interaction properties of these agents with site-specific and nonspecific DNA oligomers have been investigated.

Research has been conducted on the Co-bleomycin coordination chemistry. Nuclear magnetic resonance spectroscopy has been used in investigating Co(II) in Co(II)-bleomycin coordination sphere.

Research on the metallobleomycins may lead to analogues of the drug that are more effective in treating cancer. Bleomycin is a natural product that breaks DNA but it requires metal ions and oxygen in order to do this. The structure of several metallobleomycins is disussed.

Research was conducted to examine two new bleomycin analogues whose synthesis, spectroscopy and crystal structures were analyzed. Their Cu(super II) complexes were isolated and characterized by X-ray crystallography. X-ray crystallography showed that the successive replacement of hydrogen atoms by methyl groups on the carbon atoms changed the values of tau from 0.087 to 0.37.

A study on the 455-amino-acid cysteine proteinase called bleomycin hydrolase (BH) demonstrated two-hybrid and in vitro binding of human BH (hBH) with ribosomal proteins L11 and L29. The experiment also illustrated hBH association with the ribosomal subcellular fraction from Chinese hamster ovary cells and binding of recombinant His-tagged hBH to isolated CHO ribosomes. The study strongly suggests that hBH exists in both free cytosolic and ribosome-bound forms.

Experiments were performed to illustrate the L-threonine side chain in bleomycin A2, a major component of the clinical antitumor drug Blenoxane. The studies provided evidences of a stable input of the L-threonine subunit on the 5'-GC,5'-GT cleavage selectivity in deglycobleomycin. The findings suggest the active role of the L-threonine side chain in promoting the preorganization of bleomycin A2 that facilitates successful DNA cleavage.

Experiments were performed to characterize the deglycobleomycin A2 analogs and to understand the mechanics of the valerate linker region. The findings suggest the significant contribution of the valerate subunit to DNA cleavage efficiency but has a minimal effect on the main 5'-GPy cleavage selectivity. However, the presence of a C3-hydroxy group proved the participation of the valerate subunit in an intermolecular H bond with nonbridging oxygen of a phosphate that enhances the binding affinity of a DNA-bound agent.

Research was conducted to prove the hypothesis that deglycobleomycin (dgBLM) binds to and degrades the self-complementary oligonucleotide d(CGCTAGCG)2 in a sequence-selective manner. A 1:1 complex of Zn(II)*dgBLM A2 with the DNA octanucleotide was investigated by NMR experiments and restrained molecular dynamics calculations to characterize the binding interaction. A binding model for the interaction with the octanucleotide was obtained.

A study was conducted to examine the cleavage of nucleic acids by bleomycin. The reactions allowing iron-bleomycin to attack DNA were considered in the study. The stopped-flow spectroscopy method was utilized to monitor bleomycin activity in vitro, wherein two kinetics events were observed optically after the addition of O2 to anaerobic Fe(II)-bleomycin. The cleavage of the activated bleomycin peroxide was also anticipated to produce the proximate DNA-attacking intermediate.

A study was conducted on the hydroperoxide form of cobalt deglycoBleomycin A2 (HOO-CodGBLM) and its complex with oligonucleotide d(CCAGGCCTGG). The similarities in model structures between HOO-CoBLM and its corresponding aglycon, and the lack of a carbamoyl amino group as a potential axial ligand in the latter, suggest that the primary amine of Beta-aminoalanine is a ligand in the parent compound. The results also indicate that deglycoBLM analogues can serve as a good model to analyze the ds cleavage process.

The intrinsic endopeptidase activity related to human bleomycin hydrolase (hBH) was described by using artificial substrates and by implementing intracellular dimerization of hBH. Two independent locations of homomeric interactions were identified via deductive domain analysis. The results revealed that 8 C-terminal amino acids, called bleomycin hydrolase-like domain, were not critical for aminopeptidase activity and but were important for bleomycin deamidation.

Research was conducted to examine the geometric and electronic structures of high-spin ferrous complexes of bleomycin (Fe11BLM) and a series of systematically perturbed BLM derivatives. Methods used in the investigation were low-temperature magnetic circular dichroism spectroscopy, optical absorption and circular dichroism. Results indicate that perturbing the axial primary amine significantly affects the degree of Fe-> pyrimidine band-bonding. This electronic stabilization is necessary for the proper functioning of the drug-dioxygen complex in forming activated BLM.

The anisotropic, distance-dependent dipolar hyperfine couplings from iron in ferric bleomycin (Fe(III)-BLM) and in activated bleomycin (Act-BLM) to 31P of substrate DNA were measured using Q-band electron nuclear double resonance (ENDOR). Results demonstrate the proximity of the iron to the (31P)phosphate of the DNA substrate and perturbation to the BLM structure, particularly as evidenced by proton ENDOR.

A novel and alternative synthetic approach was used to prepare and analyze derivatives of bleomycin A2 and deglycobleomycin A2 and their unnatural epimers for an evaluation of the role of their pyrimidine C4 amine group. A significantly reduced DNA cleavage efficiency was observed in the agent bearing a pyrimidine C4 dimethylamino substituent, while the agent where the pyrimidine C4 amino group was removed showed an even greater dimunition in DNA cleavage efficiency.

Analysis of bleomycin (BLM) activity in the beta-globin gene cluster indicates that intensity of BLM damage sites differs in purified DNA and intact cells. The chromatin structure probably affects BLM activity at the beta-globin promoter sequence. BLM activity is low at the site of protein-DNA interactions in intact cells. It is affected at nuclear factor binding sites in the beta-globin locus control region hypersensitive site 2 of intact cells. The activity of BLM is lowest in K562 cells with a ten-fold reduction in damage at the tandem nuclear factor-erythroid 2/AP1 DNA sequence element.

The covalent attachment of bithiazole to the Co(III) complex of a designed ligand PMAH is studied. The deprotonated ligand, which mimics the metal-binding locus of the antitumor drug bleomycin, uses five nitrogens located in various sites to bind Co(III). Two hybrid analogues are also identified. It is shown that sequence specificity is determined to a large extent by the metalated core of the hybrid analogues.

The solution structure of Co.Bleomycin (CoBLM) A2 green complexed with the self-complementary oligonucleotide d(CCAGGCCTGG) with a cleavage site at C6 was determined by 2D nuclear magnetic resonance spectroscopic processes and molecular dynamics computations. Molecular modeling indicates that the distal oxygen of the hydroperoxide is 2.5 angstroms from the 4'-hydrogen of C6. There may be several additional intramolecular hydrogen bonds between the hydroperoxide ligand and the peptide linker region.

The structure of homogeneous Co.Bleomycin (CoBLM) A2 green was studied by 2D nuclear magnetic resonance and molecular dynamics computations. The two possible axial ligands that may comprise the structure of CoBLM are the primary amine of the beta-aminoalanine and the carbamoyl nitrogen of the mannose. Coupling constants, NOE arguments, potential energy considerations, model analyses and evaluations of BLM analogs indicate that the primary amine of beta-aminoalanine is the more likely axial ligand.

13C spectra indicating 113Cd-13C spin-spin couplings show that nitrogen atoms from the primary amine, pyrimidine and imidazole are bound to 113Cd in 113Cd-bleomycin. Bound and free forms of the secondary amine nitrogen may be in equilibrium. There are at least two differently coordinated forms of cadmium in the molecule and there are at most four nitrogen atoms bound to Cd at low temperature. These anions can bind to major and minor forms of the structure and there is a minor species which does not associate with the chloride.

The cleavage of a DNA triple helix by Fe(II).bleomycin (BLM) occurs on both strands of the 32-base pair duplex at the duplex-triplex junction. Cleavage is highest at the 5'-duplex-triplex junction which is the favored binding site of intercalating agents. Molecular-modeling analysis of the triplex region reveals significant alterations in the minor groove width. The preference of BLM for the duplex-triplex junctions appears to be due to the shape of the minor groove.

Nuclear magnetic resonance experiments and retrained molecular dynamics calculations revealed that the antitumor antibiotic bleomycin (BLM) binds to and devalues the self-complementary octanucleotide d(CGCTAGCG)2 differently based on the DNA sequence. Sites that are rich in GC optimize partial intercalative binding while T4A5 sites optimize minor groove binding through providing favorable electrostatics.

The high-spin ferrous complexes of bleomycin (Fe(II)BLM) and the structural analog PMAH, Fe(II)PMA +, support a dissociative mechanism for oxygen reactivity. PMAH is a macrocyclic ligand with deprotonated amide, pyrimidine, imidazole and secondary and primary amine functionalities. An effective iron (II)-to-pyrimidine backbonding occurs in Fe(II)BLM and Fe(II)PMA + due to the presence of low energy charge transfer transition states. A low spin state of both ferrous complexes is obtained when they coordinate to pi-acceptor exogenous ligands, yielding long-lived oxygen intermediates.

X-ray absorption spectroscopy (XAS) enables the study of the oxidation and spin state of iron in activated Bleomycin (BLM). Activated BLM possesses a six-coordinate low-spin ferric complex and has no short Fe-O bond. Its oxygen activated intermediate has a structure similar to peroxy-ferric complexes, which are expected to play an important role in the oxygen activation of heme enzymes.

Mechanistic study of the reaction of ferric bleomycin (Fe(III)BLM) with iodosylbenzene (PhIO) by EPR spectroscopy and electrospray ionization mass spectrometry indicates hypervalent iodine I(III) as the oxidant. Product analysis reveals the identical nature of the products with those derived from the reaction between Fe(III)BLM and H2O2. Formation of the same products in the reaction between Zn(II)BLM and PhIO indicates that oxidation of BLM by PhIO does not require a metal ion.

DNA cleavage experiments using radio labeled DNA fragments show that a hybrid molecule (Co(PMA)(Int-A))Cl2 cleaves the DNA strand preferentially at 5'GG-N3' sites. High field 2-D NMR experiments help investigate the specific interactions of the hybrid molecule with a self-complimentary oligonucleotide having a GG-N site. Photoproduct analysis by high-resolution polyacrylamide gel electrophoresis shows that the acridine group of the hybrid-molecule attaches to G-G site of the oligomer to induce photodamage to the GG-N sites.

An extension of the total synthesis scheme of deglycobleomycin A(sub 2) and deglycobleomycin A(sub 1) yields various derivatives of glycobleomycin A(sub 2). The C2 acetamido sidechain influences the synthesis. DNA cleavage properties of Fe(II) or Fe(III) complexes of deglycobleomycin derivatives are analyzed and compared. Desimidazolyldeglycobleomycin A(sub 2) exhibits an altered and nonselective cleavage pattern.

The total synthesis of bleomycin A (sub 2) involves the incorporation of the disaccharide subunit 2-O-(3-O-carbamoyl-alpha-D-mannopyranosyl)- L-gulopyranose into the procedure. A diasteroselective alpha-O-glycosidation is the vital step for the synthesis of the disaccharide. Order of assemblage of subunits is critical for the total synthesis of bleomycin A(sub 2).

Inverse electron demand Diels-Alder reaction of 2,4, 6-tris(ethoxycarbonyl)- 1,3,5-triazine is the key step in the synthesis of pyrimidoblamic acid subunits. Optically-active N-acyloxazolidinones undergo an imine addition reaction to introduce the C2 acetamido sidechain to complete the synthesis of (-)pyrimidoblamic acid. The absolute configuration of the C2 acetamido chain is described and is extended for the diastereoselective synthesis of (-)descarboxamidopyrimidoblamic acid.

A concise, diastereocontrolled process yields bleomycin A(sub 2) subunits and optically-active tripeptide S, tetrapeptide S, and pentapeptide S. Absolute DNA binding constants reveal the influence of linking chain substituents on DNA binding strength and the binding site size of the agent. Calculated binding site sizes reveal that the tripeptide S subunit of bleomycin A(sub 2) is fully-bound to duplex DNA.

Electrospray mass spectrometric technique is employed to analyze the reaction between Fe(III) bleomycin (BLM) and H2O2. The reaction produces an activated BLM and has a mass similar to HOO-Fe(III) BLM. The increase in mass is due to two hydrogen peroxide-derived oxygen atoms and reveals the activated BLM as a ferric peroxide complex.

HO2-Co(III)bleomcyin A2 and Co(III)bleomycin A2 are synthesized during the oxidation of Co(III) bleomycin A2 which is performed by dioxygen compounds. Two dimensional nuclear magnetic resonance (NMR) spectral methods determine the H1 NMR chemical shift assignments for the protons of the two products. The proton chemical shift is different for both the products, indicating the presence of numerous metal-binding domains and peptide linkers in the products. The variation in connecting the products to DNA by the domains and linkers produces the difference in chemical shift assignments.

Chromatin reconstituted from a supercoiled plasmid and core histone was studied with respect to the stability of the abasic sites induced by bleomycin and neocarzinostatin. Spontaneous cleavage in chromatin was observed in all the drug-induced abasic sites. The mutagenic potential of bleomycin-induced abasic sites was determined by transfecting bleomycin-treated shuttle vectors into mammalian cells. The results suggest that the abasic sites have only a slight potential to produce base substitutions in mammalian cells and that a substantial fraction of the double-stranded breaks induced by bleomycin and neocarzinostatin result from spontaneous cleavage of abasic sites.

The sequence specific isotope effects of activated iron bleomycin (Fe BLM) on DNA cleavage at thymidine residues was studied after (4'-2H)thymidine was incorporated into DNA. A phosphorus-32 end label was used as a remote label for quantifying these effects. Results showing no isotope effect for DNA deuteriated at other positions demonstrates the high regiospecificity of the BLM-mediated carbon-hydrogen bond cleavage. For Fe BLM, the 4'-isotope effects were found to have a magnitude of close to 2-7 and were shown to be sensitive to changes in BLM structure and in the cleavage mechanism.

The lability of oxygen in activated bleomycin (BLM) was investigated with the use of electron paramagnetic resonance spectroscopy to measure the exchange of 17O between activated BLM and solvent. It was demonstrated that exchange does not occur between oxygen bound to activated BLM and that of solvent. It was postulated that the radical rebound mechanism of BLM-mediated DNA degradation may not be operative.

The bleomycin mimic ligands L1 and L2 are synthesized and their pH-dependent complexations with Cu(II), Fe(II) and Zn(II) are investigated via pH-metric titrations. The protonation constants for both ligands show the considerable influence exerted by the carbamide group. The complexation of L1 with Cu(II) forms (Cu(II)(H(sub -1)L1H(super 1+)))2+ at pH < 5 and (Cu(II)(H(sub -1)L1))1+ at pH > 6. Meanwhile, L1 and Zn(II) give (Zn(II)L1)2+ at pH < 6 and (Zn(II)(H(sub 1-)L1))1+ at pH > 8. Finally, the complexation of L2 with Cu(II) yields (Cu(II)(H(sub -1)L2))1+.

The reaction of ferric bleomycin, Fe(super III)BLM, with DNA in the presence of ascorbate was studied to resolve the BLM paradox. BLM is a chemotherapeutic agent causing DNA-breakage when activated by reducing agents. However, these same agents protect cellular DNA from BLM damage. Results suggest that activation of BLM and generation of redox-inactive BLM is facilitated by a single reaction. Moreover, BLM-induced DNA damage results only when BLM-DNA reaction occurs ahead of BLM reduction.

Desacetamidopyrimidoblamic acid was synthesized through the inverse-electron demand Diels-Alder reaction of 2,4,6-tris(ethoxycarbonyl)-1,3,5-triazine with 1-bis(benzylamino)-1-propyne. The Fe(II) complex of deglyco desacetamidobleomycin A2 was found to generate a single- and double-strand cleavage of phiX174 RFI DNA to generate relaxed and linear DNA, respectively. Furthermore, the Fe(II) complex was found to be 1/3 to 1/5 less effective than Fe(II)-deglycobleomycin A2 in the cleavage process.

Two bleomycin (BLM) monothiazoles have been tested for their effectivity in promoting the oxygenation of low molecular weight substrates and the oxidative degradation of DNA molecules. The results have shown that both BLM monothiazoles function well in activating oxygen in substrates such as naphthalene, N,N-dimethylaniline and styrene. However, they have been found ineffective in inducing the selective scission of DNA. These results indicate that the bithiazole structure is not needed in oxygen activation which is in contrast with DNA cleavage wherin both rings must be present to mediate the process.

Thereaction of the antitumor bleomycin with DNA in the formation of a base propenal and the 3'-phosphoglycolate and 5'-phosphate moieties was investigated, resulting in a postulate incorporating five elements. First is the stereospecific displacement of the 2'-pro-R proton. Second is the inclusionof one atom of 18O in the carbonyl group of glycolic acid from the second O2 equivalent. Third is the inclusion of 18O from solvent into the aldehyde group of base propenal. The fourth involves 3'-C-O bond cleavage and the fifth involves the more speedier liberation of the 2'-pro-R proton and DNA strand scission.

(Co PMA H2O)(NO3)2, (Co PMA N-Melm)(NO3)2 and (Co PMA Cl)Cl.EtOH cobalt complexes were successfully prepared from the aerobic oxidation of Co(II) salts and PMAH ligands. Structural characterization indicated the similarity of the three complexes with the metal-chelating moiety of the antibiotic bleomycin (BLM). Also, the reactivities and spectral properties of the Co(III)-BLM complexes resembled those of the Co(III)-PMA complexes. Further studies involving spin-trapping and DNA scission reactions were conducted to determine the behavior of the Co(III) complexes.

The copper(II) complex Cu(Py3P) was synthesized from the reaction of N,N'-bis(2-(2-pyridyl)ethyl)pyridine-2,6-dicarboxamide (Py3PH2) with copper(II) acetate hydrate in methanol. The crystal structure of the complex was made up of Cu(Py3P) units and described to be a distorted square-pyramidal with the Cu(II) ion joined by five nitrogen atoms. This distortion and the weak in-plane crystal field strength resulted to the small 154 G A value of the electron paramagnetic resonance spectrum of the Cu(II) complex.

Iron complexes incorporated with two ligands PMAH and PMCH which mimic the metal-binding domain of bleomycin activate dioxygen to produce a species which shows EPR spectra similar to that of the 'activated bleomycin.' Intramolecular hydrogen bonding associated with the secondary amine group of bleomycin has no influence on the activation of dioxygen.

Hairpin DNAs selected for their ability to bind to BLM [A.sub.5] are used for determining the relationship between high-affinity DNA binding sites and the cleavage efficiency and selectivity of BLM [A.sub.5] and deglycoBLM [A.sub.5] on these DNAs. The chemistry of DNA degradation is examined by treating selected hairpin DNAs with n-butylamine following cleavage with Fe(II).BLM [A.sub.5] or Fe(II).deglycoBLA [A.sub.5] to analyze the alkali labile pathway of DNA degradation by BLM.

The synthesis of the tri- and tetrapeptide S subunits of bleomycin A2 was undertaken. Tripeptide S was generated from the precursor (2S,3S, 4R)-N-(tert-butoxycarbonyl)-4-amino-3-hydroxy-2-methylpentanoic acid via a syn aldol addition reaction followed by methyl ester formation, N-BOC deprotection, S-methylation and acid-catalyzed deprotection. Tetrapeptide S was also derived in an analogous manner from the same precursor.

Inverse electron demand Diels-Alder cycloaddition of 1,1-diaminoethane with 2,4,6-tris(ethoxycarbonyl)-1,3, 5-triazine provides the pyrimidine nucleus for the synthesis of desacetamido P-3A, the simplest member of the bleomycin antitumor drugs. The synthetic study of P-3A seeks to prove that it is not the C2-acetamido chain but the pyrimidine nucleus itself that is involved in the chelation of copper(II). The chelation site indicates the point of O2 activation and DNA cleavage thought to be responsible for bleomycin's antitumor activity.

The MM2 calculations of five- and six-coordinate bleomycin-metal complexes have provided structural geometries that resemble those that are obtained from X-ray analysis. The Fe-bleomycin model features an axial NH2 coordination in the absence of hydrogen-bonding. However, the occurrence of O2 or O2H- ligand activation, which requires an axial imidazole group, leads to intramolecular hydrogen-bonding. The examination of the observed hydrogen-bonding in peroxide-containing Fe-bleomycin and Fe-PYML models can be used to determine the epoxidation properties of cis- and trans-stilbenes.

The biochemical evaluation of a 108-member deglycobleomycin library is presented. The results prove that the process can also be used for identifying bleomycin (BLM) analogues with altered properties.

The structure of 4'-oxidized abasic site (X) is determined by two-dimensional nuclear magnetic resonance (NMR) methods, molecular modeling and molecular dynamics simulations. Molecular dynamics simulations have shown that the normal abasic site is more flexible than the 4'-oxidized abasic site.

The possible reaction pathways for decay of the active bleomycin-Fe(III)-OOH complex, so-called bleomycin suicide are investigated using first-principles molecular dynamics simulations (Car-Parrinello method). Simulations performed both in a vacuum and in water show that a facile decaying process involves a homolytic O-O bond cleavage with an almost simultaneous hydrogen atom abstraction.