Deglycobleomycin: solid-phase synthesis and DNA cleavage by the resin-bound ligand

[structure: see text] A greatly improved solid-phase synthesis of deglycobleomycin using a Dde-based linker is reported. The resin-bound deglycobleomycin could be completely deblocked and assayed for DNA plasmid relaxation, sequence-selective DNA cleavage, and light production from a molecular beacon.     

Solid-phase synthesis of bleomycin A(5) and three monosaccharide analogues: exploring the role of the carbohydrate moiety in RNA cleavage

The solid-phase synthesis of bleomycin A5 (BLM A5) and three monosaccharide analogues is presented. The monosaccharide analogues incorporated alpha-d-mannose, alpha-l-gulose, and alpha-l-rhamnose moieties in lieu of the disaccharide normally present in BLM A5. Also explored were the abilities of each of the monosaccharide congeners to cleave a 53-nt RNA. The elaboration of these carbohydrate-modified bleomycin analogues helps to define the role of the disaccharide moiety during the RNA cleavage event. The relatively facile solid-phase synthesis of bleomycin A5 and each of the carbohydrate analogues constitutes an important advance in the continuing mechanistic studies of bleomycin.     

Conformationally constrained analogues of bleomycin A5

The bleomycin (BLM) group antitumor antibiotics are glycopeptide-derived natural products shown to cause sequence selective lesions in DNA. Prior studies have indicated that the linker region, composed of the methylvalerate and threonine residues, may be responsible for a conformational bend in the agent required for efficient DNA cleavage. We have synthesized a number of conformationally constrained methylvalerate analogues and incorporated them into deglycobleomycin A(5) congeners using our recently reported procedure for the solid phase construction of (deglyco)bleomycin and its analogues. These analogues were designed to probe the effects of conformational constraint of the native valerate moiety. Initial experiments indicated that the constrained molecules, none of which mimic the conformation proposed for the natural valerate linker, possessed DNA cleavage activity, albeit with potencies less than that of (deglyco)BLM and lacking sequence selectivity. Further experiments demonstrated that these analogues failed to produce alkali-labile lesions in DNA or sequence selective oxidative damage in RNA. However, two of the conformationally constrained deglycoBLM analogues were shown to mediate RNA cleavage in the absence of added Fe(2+). The ability of the analogues to mediate the oxygenation of small molecules was also assayed, and it was shown that they were as competent in the transfer of oxygen to low molecular weight substrates as the parent compound.     

Solid-phase synthesis of bleomycin group antibiotics. Elaboration of deglycobleomycin A(5)

The solid-phase syntheses of two deglycobleomycin A(5) analogues were achieved using a commercially available polystyrene resin containing triphenylmethyl-linked spermidine. The final products were deblocked and released from the resin, analyzed, and purified by C(18) reversed phase HPLC and characterized by high-field (1)H NMR spectroscopy and mass spectrometry. The purified products relaxed supercoiled plasmid DNA in a concentration-dependent fashion and to the same extent as authentic material derived from natural BLM A(5).     

Total synthesis of deamido bleomycin a(2), the major catabolite of the antitumor agent bleomycin

Metabolic inactivation of the antitumor antibiotic bleomycin is believed to be mediated exclusively via the action of bleomycin hydrolase, a cysteine proteinase that is widely distributed in nature. While the spectrum of antitumor activity exhibited by the bleomycins is believed to reflect the anatomical distribution of bleomycin hydrolase within the host, little has been done to characterize the product of the putative inactivation at a chemical or biochemical level. The present report describes the synthesis of deamidobleomycin demethyl A(2) (3) and deamido bleomycin A(2) (4), as well as the respective aglycones. These compounds were all accessible via the key intermediate N(alpha)-Boc-N(beta)-[1-amino-3(S)-(4-amino-6-carboxy-5-methylpyrimidin-2-yl)propion-3-yl]-(S)-beta-aminoalanine tert-butyl ester (16). Synthetic deamido bleomycin A(2) was shown to be identical to the product formed by treatment of bleomycin A(2) with human bleomycin hydrolase, as judged by reversed-phase HPLC analysis and (1)H NMR spectroscopy. Deamido bleomycin A(2) was found to retain significant DNA cleavage activity in DNA plasmid relaxation assays and had the same sequence selectivity of DNA cleavage as bleomycin A(2). The most significant alteration of function noted in this study was a reduction in the ability of deamido bleomycin A(2) to mediate double-strand DNA cleavage, relative to that produced by BLM A(2).     

Analogues of bleomycin: synthesis of conformationally rigid methylvalerates

[structure: see text]. Several conformationally rigid analogues of the methylvalerate subunit contained within the linker domain of the antitumor antibiotic bleomycin have been prepared. These compounds have been protected in a fashion suitable for the solid-phase synthesis of bleomycin. Bleomycin congeners containing these analogues should facilitate a more detailed understanding of the nature of the conformational bend that the methylvalerate moiety is thought to impart to the natural product.     

TRAM linker: a safety-catch linker for the traceless release of acrylamides

[reaction: see text] A novel safety-catch linker for the solid-phase synthesis of small-molecule libraries containing electrophilic reactive groups has been developed. Upon cleavage from solid support, the linker generates a Michael acceptor (an acrylamide) on each library member. Utilization of a two-resin system in the final cleavage step provides crude products in high purity, allowing direct use in biological assays following filtration and evaporation.     

RNA cleavage and inhibition of protein synthesis by bleomycin

Bleomycin is a clinically used antitumor antibiotic long thought to function therapeutically at the level of DNA cleavage. Recently, it has become clear that bleomycin can also cleave selected members of all major classes of RNA. Using the computer program COMPARE to search the database established by the Anticancer Drug Screening Program of the National Cancer Institute, a possible mechanism-based correlation was found between onconase, an antitumor ribonuclease currently being evaluated in phase III clinical trials, and the chemotherapeutic agent bleomycin. Following these observations, experimentation revealed that bleomycin caused tRNA cleavage and DNA-independent protein synthesis inhibition in rabbit reticulocyte lysate and when microinjected into Xenopus oocytes. The correlation of protein synthesis inhibition to the previously reported site-specific RNA cleavage caused by bleomycin supports the thesis that RNA cleavage may constitute an important element of the mechanism of action of bleomycin.     

Resonance Raman studies of HOO-Co(III)bleomycin and Co(III)bleomycin: identification of two important vibrational modes, nu(Co-OOH) and nu(O-OH)

Bleomycin is an antitumor agent whose cytotoxicity is dependent on its ability to bind DNA in the nucleus and effect double-stranded DNA cleavage, which is difficult for the cell to repair. In order for this DNA cleavage to occur, bleomycin must, through a series of reactions, form a low-spin Fe(III) complex, the putative "activated" form of the drug, HOO-Fe(III)bleomycin. The relative strengths of the bonds in the Fe(III)-OOH linkage have not been determined due to the weakness of the hydroperoxo-to-iron(III) charge-transfer transition. The much more stable HOO-Co(III)bleomycin has often been studied as a structural analogue of HOO-Fe(III)bleomycin, and hence, an understanding of the relative bond strengths in the Co-OOH linkage may serve to enhance our understanding of the analogous Fe-OOH linkage. In this report, we present resonance Raman data that identify two important vibrational modes in the Co-OOH linkage, the stretching modes, nu(Co-OOH) and nu(O-OH). Both of these vibrational modes were found to be unperturbed by complexation of the drug with calf thymus DNA. Advantage was also taken of the isostructural realtionship between Fe-bleomycin and Co-bleomycin to analyze and assign the high-frequency modes for HOO-Co(III)bleomycin and Co(III)bleomycin (A(2) and B(2)). These data could be useful for future studies of photoactivated Co-bleomycin and Co-bleomycin analogues in an attempt to characterize oxygen-independent DNA damage pathways.     

Practical solid-phase parallel synthesis of Delta(5)-2-oxopiperazines via N-acyliminium ion cyclization

A practical solid-phase strategy for the synthesis of Delta(5)-2-oxopiperazines via N-acyliminium ion cyclization has been developed. A key step in the library synthesis is tandem acidolytic cleavage with subsequent in situ iminium formation, followed by stable enamide transformation. This approach is exemplified by the preparation of a 192-member pilot library using bromoacetal resins without further purification.     

Solid-phase synthesis of 3-aminopyrrole-2,5-dicarboxylate analogues

An efficient strategy has been developed for the solid-phase parallel synthesis of 3-aminopyrrole-2,5-dicarboxylate analogues. A library of twenty-nine 2,3,5-trisubstituted pyrroles has been synthesized on Wang resin by a 5-6 step process. The attachment of (2S,4R)-4-hydroxy-N-(PhF)proline cesium salt (PhF = 9-(9-phenylfluorenyl)) to Wang bromide resin, followed by alcohol oxidation, produced the resin-bound 4-oxo-N-(PhF)prolinate as the pyrrole precursor. Resin-bound 3-aminopyrroles were synthesized by treatment of the oxo-N-(PhF)prolinate resin with different secondary amines and diversified at the 2-position by acylation with trichloroacetyl chloride and haloform reactions with primary amines. 3-Aminopyrrole-2,5-dicarboxylates were isolated in 81-99% purity and 51-99% yields after cleavage from the resin using TFA or sodium methoxide.     

Directed library of anilinogeranyl analogues of farnesyl diphosphate via mixed solid- and solution-phase synthesis

[reaction: see text]. A directed library of anilinogeranyl diphosphate analogues of the isoprenoid farnesyl diphosphate has been prepared by solid-phase organic synthesis using a traceless linker strategy in moderate yield in three steps: reductive amination, bromination, and treatment with ((n-Bu)4N)3HP2O7.     

Tentoxin as a scaffold for drug discovery. Total solid-phase synthesis of tentoxin and a library of analogues

[reaction: see text] A solid-phase method for the synthesis of tentoxin has been developed. Two key steps-dehydration and N-alkylation-are carried out while the peptide is anchored to the resin. The method, which has been validated by the preparation of a library of tentoxin analogues, should be applicable to the generation of further libraries that have the tentoxin scaffold structure, as well as other structures containing N-alkylated didehydroamino acids.     

DNA-bleomycin interaction: Nucleotide sequence-specific binding and cleavage of DNA by bleomycin

Biosynthetic intermediates and synthetic analogues of bleomycin (BLM) have been investigated for their metal binding, dioxygen activation, and DNA cleavage. Molecular O₂ was activated by the Fe(II) complex of a synthetic model ligand. Nucleotide sequence specificities in DNA cleavage by the BLM-Fe(II) and deglyco-BLM-Fe(II) complexes were almost identical. It has been shown that (1) the β-aminoalanine-pyrimidine-β-hydroxyhistidine portion of BLM is essential for the metal binding and dioxygen activation and (2) the bithiazole moiety contributes to the specific binding to guanine base of DNA.     

Solid phase library synthesis of cyclic depsipeptides: aurilide and aurilide analogues

A solid-phase combinatorial synthesis approach toward the cyclic depsipeptide aurilide (1) and related analogues is described. The peptide moiety 2 was assembled on trityl linker-functionalized SynPhase Crowns using an Fmoc strategy. Optimization of the tetrapeptide assembly 5 was carried out using parallel multiple synthesis and LC/MS analysis. The aliphatic moiety 3a was coupled with the solid-supported 2 using DIC/HOBt. Following deprotection and cleavage of linear precursor 26, macrocyclization was achieved under high dilution conditions. Removal of the methylthiomethyl protecting group provided aurilide (1) in 11% overall yield. Synthesis of a combinatorial library of aurilide derivatives 4 was accomplished with a similar protocol using the TranSort technique.     

Tandem oligonucleotide synthesis on solid-phase supports for the production of multiple oligonucleotides

More than one oligonucleotide can be synthesized at a time by linking multiple oligonucleotides end-to-end in a tandem manner on the surface of a solid-phase support. The 5'-terminal hydroxyl position of one oligonucleotide serves as the starting point for the next oligonucleotide synthesis. The two oligonucleotides are linked via a cleavable 3'-O-hydroquinone-O,O'-diacetic acid linker arm (Q-linker). The Q-linker is rapidly and efficiently coupled to the 5'-OH position of immobilized oligonucleotides using HATU, HBTU, or HCTU in the presence of 1 equiv of DMAP. This protocol avoids introduction of phosphate linkages on either the 3'- or 5'-end of oligonucleotides. A single NH(4)OH cleavage step can simultaneously release the products from the surface of the support and each other to produce free 5'- and 3'-hydroxyl termini. Selective cleavage of one oligonucleotide out of two sequences has also been accomplished via a combination of succinyl and Q-linker linker arms. Tandem synthesis of multiple oligonucleotides is useful for producing sets of primers for PCR, DNA sequencing, and other diagnostic applications as well as double-stranded oligonucleotides. Tandem synthesis of the same sequence multiple times increases the yield of material from any single synthesis column for maximum economy in large-scale synthesis. This method can also be combined with reusable solid-phase supports to further reduce the cost of oligonucleotide production.     

Solid-phase synthesis of a thymidinyl dipeptide urea library

A thymidinyl dipeptide urea library with structural similarity to the nucleoside peptide class of antibiotics was designed and synthesized. To generate the library, a solid-phase synthesis was developed starting from 5'-azidothymidine attached to a polystyrene butyl diethylsilane (PS-DES) resin support. This study describes the prelibrary solid-phase synthesis development including maximum loading capacity optimization, selection of orthogonal functionalized side-chain protection strategies, synthesis of a 64-member test library, and optimization of the final cleavage step. Using the optimized procedures, we synthesized a 1000-member library in a 50 micromol quantity using IRORI-directed sorting technology in MiniKans, producing the target library in good yields and purity.     

Synthesis and DNA cleavage evaluation of epoxypiperidine derivatives bearing a dehydroamino acid unit

Epoxypiperidine derivatives bearing a dehydroamino acid unit were designed and synthesized as novel DNA alkylating agents based on the structure of azinomycins. A relaxation assay of plasmid DNA revealed that the epoxypiperidine derivative 3 has a DNA cleavage activity. Based on the studies, it would appear that the electron density of the amino group of epoxypiperidines plays a critically important role in the DNA cleavage.     

A new trick (hydroxyl radical generation) for an old vitamin (B12)

Photolysis of hydroxocobalamin in the presence of plasmid DNA (pBR322) results in DNA cleavage. Temporal control of hydroxyl radical production and DNA strand scission by hydroxocobalamin was demonstrated using a 2-deoxyribose assay and a plasmid relaxation assay, respectively. The light-driven hydroxocobalamin-mediated catalytic formation of hydroxyl radicals was demonstrated using radical scavenging studies of DNA cleavage and via recycling of a hydroxocobalamin-resin conjugate several times without loss of efficacy.