Bleomycin B2 Research Articles

An effective validation of analytical method for determination of a polar complexing agent: the illustrative case of cytotoxic bleomycin

The effectiveness of highly polar agents in cancer treatment is well recognized, but their physicochemical properties make their analytical determination a demanding task. Their analysis requires peculiar sample preparation and chromatographic separation, which heavily impacts the precision of such an analytical method. As a case study, we chose a polar cytotoxic bleomycin, which is a mixture of complexing congeners with relatively high molecular mass, a fact that creates an added challenge in regard to its detection via electrospray mass spectrometry. These issues combined lead to a deprived method performance, so the aim of this study is manifold, i.e., to optimize, validate, and establish quality performance measures for determination of bleomycin in pharmaceutical and biological specimens. Quantification of bleomycin is done at diametrically different concentration levels: at the concentrations relevant for analysis of pharmaceutical dosage forms it is based on a direct reversed-phase HPLC-UV detection, involving minimum sample pretreatment. On the contrary, analysis of bleomycin in biological specimens requires phospholipid removal and protein precipitation followed by HILIC chromatography with MS/MS detection of bleomycin A2 and B2 copper complexes being the predominant species. This study further attempts to solve the traceability issue in the absence of certified reference standards, determines measurement uncertainty, investigates BLM stability and method performance characteristics, and, last but not least, provides an explanatory example of how a method quality assurance procedure should be established in case of an exceedingly complex analytical method.Graphical abstract

Two distinct rotations of bithiazole DNA intercalation revealed by direct comparison of crystal structures of Co(III)•bleomycin A2 and B2 bound to duplex 5′-TAGTT sites

Bleomycins constitute a family of anticancer natural products that bind DNA through intercalation of a C-terminal tail/bithiazole moiety and hydrogen-bonding interactions between the remainder of the drug and the minor groove. The clinical utility of the bleomycins is believed to result from single- and double-strand DNA cleavage mediated by the HOO-Fe(III) form of the drug. The bleomycins also serve as a model system to understand the nature of complex drug-DNA interactions that may guide future DNA-targeted drug discovery. In this study, the impact of the C-terminal tail on bleomycin-DNA interactions was investigated. Toward this goal, we determined two crystal structures of HOO-Co(III)•BLMA2 “green” (a stable structural analogue of the active HOO-Fe(III) drug) bound to duplex DNA containing 5′-TAGTT, one in which the entire drug is bound (fully bound) and a second with only the C-terminal tail/bithiazole bound (partially bound). The structures reported here were captured by soaking HOO-Co(III)•BLMA2 into preformed host–guest crystals including a preferred DNA-binding site. While the overall structure of DNA-bound BLMA2 was found to be similar to those reported earlier at the same DNA site for BLMB2, the intercalated bithiazole of BLMB2 is “flipped” 180˚ relative to DNA-bound BLMA2. This finding highlights an unidentified role for the C-terminal tail in directing the intercalation of the bithiazole. In addition, these analyses identified specific bond rotations within the C-terminal domain of the drug that may be relevant for its reorganization and ability to carry out a double-strand DNA cleavage event.

Two Distinct Rotations of Bithiazole DNA Intercalation Revealed by Direct Comparison of Crystal Structures of Co(III)•Bleomycin A2 and B2 Bound to Duplex 5´-TAGTT Sites

Two Distinct Rotations of Bithiazole DNA Intercalation Revealed by Direct Comparison of Crystal Structures of Co(III)•Bleomycin A2 and B2 Bound to Duplex 5´-TAGTT Sites

Enhancement of bleomycin production in Streptomyces verticillus through global metabolic regulation of N-acetylglucosamine and assisted metabolic profiling analysis

BackgroundBleomycin is a broad-spectrum glycopeptide antitumor antibiotic produced by Streptomyces verticillus. Clinically, the mixture of bleomycin A2 and bleomycin B2 is widely used in combination with other drugs for the treatment of various cancers. As a secondary metabolite, the biosynthesis of bleomycin is precisely controlled by the complex extra-/intracellular regulation mechanisms, it is imperative to investigate the global metabolic and regulatory system involved in bleomycin biosynthesis for increasing bleomycin production.ResultsN-acetylglucosamine (GlcNAc), the vital signaling molecule controlling the onset of development and antibiotic synthesis in Streptomyces, was found to increase the yields of bleomycins significantly in chemically defined medium. To mine the gene information relevant to GlcNAc metabolism, the DNA sequences of dasR-dasA-dasBCD-nagB and nagKA in S. verticillus were determined by chromosome walking. From the results of Real time fluorescence quantitative PCR (RT-qPCR) and electrophoretic mobility shift assays (EMSAs), the repression of the expression of nagB and nagKA by the global regulator DasR was released under induction with GlcNAc. The relief of blmT expression repression by BlmR was the main reason for increased bleomycin production. DasR, however, could not directly affect the expression of the pathway-specific repressor BlmR in the bleomycins gene cluster. With at the beginning of bleomycin synthesis, the supply of the specific precursor GDP-mannose played the key role in bleomycin production. Genetic engineering of the GDP-mannose synthesis pathway indicated that phosphomannose isomerase (ManA) and phosphomannomutase (ManB) were key enzymes for bleomycins synthesis. Here, the blmT, manA and manB co-expression strain OBlmT/ManAB was constructed. Based on GlcNAc regulation and assisted metabolic profiling analysis, the yields of bleomycin A2 and B2 were ultimately increased to 61.79 and 36.9 mg/L, respectively.ConclusionsUnder GlcNAc induction, the elevated production of bleomycins was mainly associated with the alleviation of the inhibition of BlmT, so blmT and specific precursor synthesis pathways were genetically engineered for bleomycins production improvement. Combination with subsequent metabolomics analysis not only effectively increased the bleomycin yield, but also extended the utilization of chitin-derived substrates in microbial-based antibiotic production.

Negative regulation of bleomycins biosynthesis by ArsR/SmtB family repressor BlmR in Streptomyces verticillus.

Bleomycin, a broad-spectrum antibiotic, has been widely used for various tumor treatments. However, its poor fermentation yield is not satisfactory for industrial production. Here, the ArsR/SmtB family regulator BlmR was characterized as a repressor of bleomycin production. As an autoregulator, BlmR was found to bind to a 12-2-12 imperfect palindrome sequence in its own promoter, and deletion of blmR led to a 34% increase of bleomycin B2 production compared with the wild-type strain. Using reverse transcription and quantitative PCR (RT-qPCR), blmT, which encoded a putative transporter, was identified as the target gene regulated by BlmR. Therefore, high-production strain was constructed by blmT overexpression in a blmR deletion strain, and the bleomycin B2 titer reached to 80 mg/L, which was 1.9-fold higher than the wild-type strain. Moreover, electrophoretic mobility shift assay (EMSA) showed neither metal-binding motifs nor redox switches in BlmR. In order to elucidate the regulatory mechanism, a model of BlmR was constructed by homology modeling and protein-protein docking. The BlmR-DNA complex was generated by protein-DNA docking with the assistance of site-directed mutagenesis and molecular dynamic (MD) simulation, which directly revealed several key amino acid residues needed for the maintenance and stabilization of the interface between BlmR and target DNA. The interface information could provide the configuration reference and seek the potential effectors that could interact with BlmR, thereby extending the regulation role of ArsR/SmtB family members on the improvement of antibiotic production.

Zorbamycin has a different DNA sequence selectivity compared with bleomycin and analogues

Bleomycin (BLM) is used clinically in combination with a number of other agents for the treatment of several types of tumours. Members of the BLM family of drugs include zorbamycin (ZBM), phleomycin D1, BLM A2 and BLM B2. By manipulating the BLM biosynthetic machinery, we have produced two new BLM analogues, BLM Z and 6′-deoxy-BLM Z, with the latter exhibiting significantly improved DNA cleavage activity. Here we determined the DNA sequence specificity of BLM Z, 6′-deoxy-BLM Z and ZBM, in comparison with BLM, with high precision using purified plasmid DNA and our recently developed technique. It was found that ZBM had a different DNA sequence specificity compared with BLM and the BLM analogues. While BLM and the BLM analogues showed a similar DNA sequence specificity, with TGTA sequences as the main site of cleavage, ZBM exhibited a distinct DNA sequence specificity, with both TGTA and TGTG as the predominant cleavage sites. These differences in DNA sequence specificity are discussed in relation to the structures of ZBM, BLM and the BLM analogues. Our findings support the strategy of manipulating the BLM biosynthetic machinery for the production of novel BLM analogues, difficult to prepare by total synthesis; some of which could have beneficial cancer chemotherapeutic properties.

Identification and quantification of bleomycin in serum and tumor tissue by liquid chromatography coupled to high resolution mass spectrometry

Bleomycin is a cytotoxic antibiotic available as a compost of structurally strongly related glycopeptides, which is in vivo found chelated with several metals. Its pharmacotherapy has merely been based on experimental dose – response data, whereas its biodistribution and pharmacokinetics remain fundamentally unknown. This is reasoned by an absence of a specific and sensitive mass spectrometry-based analytical method for its determination in biological tissues. We herein reveal the results of our study on the mass spectrometric behavior of two main bleomycin fractions A2 and B2, including their metal complexes, particularly the predominant copper chelates. In the electrospray ion source bleomycin forms double charged species, where for the metal-free fraction A2 and its copper complex m/z 707.76 and m/z 707.21 are seen, respectively. Hence, the second isotopic ion of the chelate (m/z 707.71) nearly coincides with the first isotopic ion of the metal-free fraction. This phenomenon can only be followed by high-resolution mass spectrometry, and is considered the plausible reason, why the attempts to determine bleomycin with mass spectrometry have been so scarce. The presented paper further describes a sensitive and selective liquid chromatography – mass spectrometry analytical method for determination of bleomycin in serum and tumor tissues. This newly developed method was employed for bleomycin pharmacokinetic studies in serum and tumors of laboratory animals. Additionally, the method was employed for determination of bleomycin pharmacokinetic parameters in elderly patients in order to determine the effective therapeutic window of electrochemotherapy with bleomycin.

HPLC-QTOF-MS Method for Identification and Determination of Bleomycin A2 and B2 Fractions

Bleomycin is a very important drug in oncology used as first-line treatment for many cancers. The development of high-performance liquid chromatography—tandem mass spectrometry method for the separation, identification, and determination of two major structurally related analogs of this antibiotic drug, namely bleomycin A2 and B2, is presented in this work. The proposed method is based on a hydrophilic interaction chromatography (HILIC) stationary phase that enables one to avoid an ion-pairing reagent in the separation system (formerly used in mobile phase for bleomycin) in order to properly separate these highly polar bleomycin analogs. The ion-pairing reagent-free HILIC separation system is suitable for coupling the chromatographic and mass spectrometry stages (for the first time for bleomycin). Some performance parameters, namely, limit of detection (ng/mL-pg/mL), limit of quantification, linearity, precision, and recovery/accuracy, were evaluated showing high reliability, selectivity, and sensitivity of the method. It was successfully applied for the quality drug control determining bleomycin A2 and B2 fractions in commercial pharmaceuticals (Bleomedac infusions). In addition, the high-performance liquid chromatography–quadrupole-time of flight mass spectrometry (HPLC-QTOF-MS) method was able to determine an accurate molecular weight of bleomycin A2 and B2 fractions, confirming an identity/quality of the (commercial) drug. The possibilities of the HPLC-QTOF-MS method to identify bleomycin A2 and B2 in model plasma samples were also demonstrated.

Stability of an extemporaneously prepared bleomycin–lidocaine–epinephrine intradermal admixture used in dermatology

Bleomycin (0.75 g/L)-lidocaine (3.5 g/L)-epinephrine (3.5 mg/L) admixture (BLE) is indicated for keloids treatment. Effect of short-term storage, influence of BLE components and temperature on efficacy and chemical compatibility of bleomycin were studied. The stability study of BLE was conducted in polypropylene infusion bags at 4℃ and 22℃ over a period of 7 days versus bleomycin diluted alone in normal saline and stored in the same conditions. Active Bleomycin fractions, bleomycin A2 (BA2) and bleomycin B2 (BB2) and inactive demethylbleomycin A2 (DBA2) were analyzed by a validated stability-indicating HPLC method. BA2 and BB2 remained stable for up to 7 days, in both types of bleomycin preparations, whatever the temperature (<10% loss of the initial peak response). In BLE, DBA2 fraction (2.2% at T0) decreased with time. Less degradation was noted with refrigeration (remained % at day 7: 0.86 ± 0.06% at 4℃ versus 0.43 ± 0.02% at 22℃; p = 0.0004). A degradation product (DP) was observed. More significant percentage (of the total of bleomycin fractions) was reached at room temperature (at day 7, 0.97 ± 0.03% at 4℃ versus 1.41 ± 0.06% at 22℃, p = 0.0004). DP attained an identification threshold of 0.5% between the third and fourth days at 4℃ and between the first and the second days at 22℃. A maximum of three days storage at 4℃ and only one day at 22℃ was approved for BLE.

Crystal structure of DNA-bound Co(III)·bleomycin B 2 : Insights on intercalation and minor groove binding

Bleomycins constitute a widely studied class of complex DNA cleaving natural products that are used to treat various cancers. Since their first isolation, the bleomycins have provided a paradigm for the development and discovery of additional DNA-cleaving chemotherapeutic agents. The bleomycins consist of a disaccharide-modified metal-binding domain connected to a bithiazole/C-terminal tail via a methylvalerate-Thr linker and induce DNA damage after oxygen activation through site-selective cleavage of duplex DNA at 5'-GT/C sites. Here, we present crystal structures of two different 5'-GT containing oligonucleotides in both the presence and absence of bound Co(III).bleomycin B(2). Several findings from our studies impact the current view of bleomycin binding to DNA. First, we report that the bithiazole intercalates in two distinct modes and can do so independently of well ordered minor groove binding of the metal binding/disaccharide domains. Second, the Co(III)-coordinating equatorial ligands in our structure include the imidazole, histidine amide, pyrimidine N1, and the secondary amine of the beta aminoalanine, whereas the primary amine acts as an axial ligand. Third, minor groove binding of Co(III).bleomycin involves direct hydrogen bonding interactions of the metal binding domain and disaccharide with the DNA. Finally, modeling of a hydroperoxide ligand coordinated to Co(III) suggests that it is ideally positioned for initiation of C4'-H abstraction.

Analytical and biological inequivalence of two commercial formulations of the antitumor agent bleomycin.

Bleomycin is an antitumor agent which is a mixture of glycopeptides containing at least 55-75% bleomycin A2 and 25-32% bleomycin B2 fractional composition. Two bleomycin formulations, bleomycin sulfate, USP (Blenoxane, Bristol-Myers Squibb Oncology, Princeton, N.J.) and bleomycin HCI (Tianjin Hebei Pharmaceutical, Tianjin, China) were compared analytically and biologically. Reverse-phase high-performance liquid chromatography (HPLC) analyses using the USP methodology showed that Blenoxane contained primarily (69%) bleomycin A2 and 29.3% bleomycin B2. In contrast, Tianjin-supplied bleomycin HCI contained 97% bleomycin A5 fraction. In vitro tumor cell growth inhibition assays showed equivalent activity in human OVCAR-3 ovarian cancer cells and slightly greater potency in murine L-1210 leukemia cells for the Tianjin formulation. In C57/B1 mice bearing B-16 melanoma tumors, Tianjin-supplied bleomycin produced slightly greater tumor growth inhibition at the expense of greater drug-induced lethality at higher dose levels. These studies show there are significant differences in two international bleomycin formulations. These compositional differences lead to altered biologic effects.

Degradation of DNA and structure-activity relationship between bleomycins A2 and B2 in the absence of DNA repair

The contribution of DNA repair to the net number of DNA breaks produced during chemical degradation of DNA was determined by using temperature-sensitive mutant cells deficient in ATP-dependent DNA ligase [poly(deoxyribonucleotide):poly(deoxyribonucleotide) ligase, EC 6.5.1.1]. In a very sensitive assay for determining lesions introduced into Saccharomyces cerevisiae DNAs, 2-14C- and 6-3H-prelabeled DNAs from ligase-proficient and ligase-deficient cells were sedimented together through precalibrated, isokinetic alkaline sucrose gradients. DNA ligation was slower after chemical degradation of DNA by bleomycin than after gamma irradiation. DNA breaks increased approximately linearly with drug concentrations, and were approximately equivalent for ligase-proficient and ligase-deficient cells. These results were unexpected because ligase-deficient, but not ligase-proficient, cells lacked the capacity to eliminate DNA breaks produced by bleomycin. The results indicated that DNA repair did not occur during the chemical degradation of DNA under the experimental conditions. Bleomycin B2 produced considerably more DNA breaks than bleomycin A2 over a range of concentrations in ligase-proficient cells, which tolerated higher numbers of DNA breaks in general than ligase-deficient cells. The chemical analogues are structurally identical except for their cationic C-terminal amine. The actual number of DNA breaks produced by bleomycin A2 or bleomycin B2, and not the concentration of bleomycin A2 or bleomycin B2 per se, determined the amount of cell killing. DNA repair is critical in quantitating DNA breaks produced by chemicals, but was ruled out as a factor in the higher DNA breakage by bleomycin B2 than bleomycin A2.

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.

Purification of bleomycin hydrolase with a monoclonal antibody and its characterization

We established a hybridoma clone that produced anti-bleomycin hydrolase antibody. The subclass of the monoclonal antibody was immunoglobulin M. The antibody significantly reacted with bleomycin hydrolase from rabbit tissues, mouse livers, sarcoma 180, and adenocarcinoma 755 but not significantly with that from MH 134 and Ehrlich carcinoma. The enzyme from L5178Y cells showed an intermediate reactivity. Bleomycin hydrolase was purified from rabbit liver by immunoaffinity with the monoclonal antibody and DEAE gel chromatography. Approximately 1300-fold-purified bleomycin hydrolase was obtained. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing on a polyacrylamide slab gel of purified bleomycin hydrolase showed a single band with an apparent Mr of 48K and an isoelectric pH of 5.2. The molecular weight of bleomycin hydrolase determined on gel filtration high-performance liquid chromatography was ca. 300K, suggesting a hexameric enzyme. The enzyme showed an optimum pH of 6.8-7.8 and gave a Vmax value of 6.72 mg min-1 mg-1 for peplomycin and 9.24 mg min-1 mg-1 for bleomycin B2 and a Km value of 0.79 mM for both substrates. The enzyme was inhibited by E-64, leupeptin, p-tosyl-L-lysine chloromethyl ketone, N-ethylmaleimide, Fe2+, Cu2+, and Zn2+ but was enhanced by dithiothreitol. The results suggest that bleomycin hydrolase is a thiol enzyme.

DNA damage and growth inhibition in cultured human cells by bleomycin congeners

Bleomycin is hypothesized to cause cell growth inhibition and cell death via DNA cleavage. We have attempted to determine if net DNA cleavage is directly related to growth inhibition by measuring whether both parameters vary in parallel. Of primary importance to these studies was use of several bleomycin congeners. We have shown that these congeners vary in their abilities both to inhibit cell growth and to cause DNA damage. Bleomycin B2, tallysomycin, and phleomycin were the most potent growth inhibitors, and bleomycin B2 caused the most DNA damage. N-Acetylbleomycin A2 was inactive in both assays. The net amount of DNA damage measured at two levels of growth inhibition was compared for each congener and was found to vary widely among the congeners. Similarly, the degree of growth inhibition at a given level of submaximal DNA damage was found to vary widely when individual congeners were compared to each other. Hence, growth inhibition and net DNA damage due to bleomycin are not directly correlated with each other when individual congeners are compared to each other.

DNA damage induced by bleomycin in the presence of dibucaine is not predictive of cell growth inhibition

Growth inhibition and cell killing by bleomycin are believed to be related to the ability of this antibiotic to cleave chromosomal DNA. Because bleomycin has an intracellular site of action, its ability to cross biological membranes must be critical to its overall effectiveness as an antitumor agent. The local anesthetic dibucaine acts to enhance membrane fluidity; therefore, the reported ability of this local anesthetic to modulate bleomycin effects on KB cells was investigated. Cells were treated with various bleomycin congeners in the presence or absence of dibucaine for 24 h. Dibucaine enhanced the inhibition of cell growth mediated by bleomycin A2, demethylbleomycin A2, bleomycin B2, and isobleomycin A2. N-Acetylbleomycin A2 did not inhibit cell growth in the absence of dibucaine, but it was inhibitory in the presence of dibucaine. Cells treated simultaneously for analysis of DNA breakage on alkaline sucrose gradients revealed that breakage was also enhanced in the presence of dibucaine. The degree of enhancement varied with dose and bleomycin congener. N-Acetylbleomycin A2 did not induce DNA breakage in either the absence or the presence of dibucaine. While growth inhibition and net DNA breakage correlated reasonably well in the absence of dibucaine for each bleomycin analogue tested, proportionality was lost in the presence of dibucaine, and very little DNA breakage was present when growth inhibition was complete. These observations imply that, at least in the presence of dibucaine, bleomycin may mediate growth inhibition at some locus in addition to chromosomal DNA and, also, that a given net amount of bleomycin analogue induced DNA damage per se does not produce a specific degree of growth inhibition.

Radioimmunoassay of bleomycins.

Rabbit antisera highly specific to the bleomycinic acid moiety of bleomycins were obtained by immunizing with a conjugate of copper-complex of bleomycin A5 and bovine serum albumin. These antisera not only reacted with bleomycin A5 but also with other bleomycins such as bleomycin A2, bleomycin B2 and peplomycin. The antisera showed little cross-reactivity with deamido-, depyruvamido- and decarbamoyl-bleomycins. Thus, these antisera were found to be highly specific for the intact bleomycinic acid moiety. One of the antisera was successfully applied to radioimmunoassay of bleomycin and peplomycin in mouse and human sera. The detection limit was 1 ng/ml. This radioimmunoassay is expected to be widely used for the determination of active bleomycins in biological and clinical samples.

Specificity of deoxyribonucleic acid cleavage by bleomycin, phleomycin, and tallysomycin.

The sites of cleavage of DNA by bleomycin A2, bleomycin B2, phleomycin, tallysomycin A, and Blenoxane (Bristol-Meyers) in reactions containing equimolar Fe2+ and atmospheric oxygen were analyzed by gel electrophoresis of 32P end labeled DNA fragments. Bleomycin A2 and bleomycin B2 reactions cleaved DNA at all sites with a frequency equal to that of Blenoxane. At high concentrations of bleomycin the site specificity of cleavage was unchanged. Bleomycin cleavage sites and phleomycin cleavage sites are a subset of sites cleaved in reactions containing tallysomycin A. The nature of 5' and 3' termini induced by bleomycin cleavage was investigated. Electrophoresis of bleomycin-induced fragments after alkaline phosphatase or polynucleotide kinase treatment indicated that 5' termini are phosphoryl groups but 3' termini are not simple phosphoryl groups. Analysis of bleomycin cleavage of single-stranded DNA substrate showed that cleavage occurs only in regions of potentially double-stranded looped-back sequences. Possible mechanisms for determination of bleomycin cleavage sequence specificity are discussed.

Structure-activity relationships involved in the site-specific fragmentation of linear duplex DNAs by talisomycin and bleomycin analogs.

The fragmentation of Hind III- and Pst I-digested PM2 DNA and of Hind III-digested pBR322 DNA by bleomycin A2 and B2 and talisomycins A, B, S2b, and S10b was investigated. As observed by electrophoresis on agarose gels, the ethidium bromide staining band patterns produced following incubation of the various restriction endonuclease-digested DNAs with the compounds were different for the bleomycin analogs and for the talisomycin analogs. Quantitation of the degree of fragmentation of various segments of linear PM2 DNA by either bleomycin A2 or talisomycin A indicated that certain segments of the PM2 genome serve as better substrates than other segments for double-strand fragmentation by either of the two antitumor antibiotics. These results show that in this system bleomycin and talisomycin analog treatment of linear PM2 or pBR322 DNA resulted in breakage of DNA, producing different-length DNA fragments, and demonstrate the importance of the two amino acids and the 4-amino-4,6-dideoxy-L-talose sugar, which are located near the bithiazole in talisomycin but absent in the bleomycin structure in conferring a different site-specificity for DNA fragmentation to talisomycin than to bleomycin.

Field desorption and fast atom bombardment mass spectrometry of bleomycins and their derivatives

Summary Earlier work using field desorption mass spectrometry for the characterisation of guanidino containing bleomycins is extended to include bleomycin A2 and related substances. The new technique of fast atom bombardment mass spectrometry is assessed and compared with field desorption for the analysis of these complex antibiotics. Bleomycin A2 is shown to be more reactive to acetic anhydride than bleomycin B2. A procedure for in situ derivatisation for fast atom bombardment studies is described and unexpected catalysis in the glycerol matrix is reported.