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The enediynes exemplify nature's ingenuity. We have cloned and characterized the biosynthetic locus coding for perhaps the most notorious member of the nonchromoprotein enediyne family, calicheamicin. This gene cluster contains an unusual polyketide synthase (PKS) that is demonstrated to be essential for enediyne biosynthesis. Comparison of the calicheamicin locus with the locus encoding the chromoprotein enediyne C-1027 reveals that the enediyne PKS is highly conserved among these distinct enediyne families. Contrary to previous hypotheses, this suggests that the chromoprotein and nonchromoprotein enediynes are generated by similar biosynthetic pathways., Calicheamicin [γ.sub.1.sup.I] (Fig. 1) from Micromonospora echinospora ssp. calichensis is the most prominent member of the nonchromoprotein enediyne family and, as an antitumor agent, is > 5000 times as potent [...]

Synthetic calicheamicin oligosaccharide derivatives were designed fro pyrimidine recognition.

Research is presented demonstrating the development of a synthetic unified route to 3-hex-en-1,5-diynes. The method is used to synthesize cyclic and linear enediynes.

Two related methods to synthesize (plus or minus)-calicheamicinone, the aglycon of the calicheamicin antitumor antibiotic, are reported. Both compounds represent difficult synthetic targets and are of interest with regard to the mode of action of enediyne antitumor agents. The syntheses showed high levels of steroselectivity and that a calicheamicinone of unnatural stereochemistry may be more efficient at producing double strand cuts in DNA, than natural stereochemical material.

Previous research has shown that the calicheamicin oligosaccharide domain exhibits impressive binding properties. To determine the structural basis for this ability, nuclear magnetic resonance spectroscopy and molecular dynamics calculations are used to solve the solution structure of the 1:1 complex between the head-to-head dimer and the oligonucleotide d2. Results indicate that carbohydrates can be useful in generating DNA target specificity and may control the behavior of nucleic acids.

The synthetic oligosaccharide moiety of the antibiotic calicheamicin and the head-to-head dimer of this oligosaccharide, were examined if they can prevent transcription factor function. These carbohydrates were discovered to prevent the binding of transcription factors to DNA depending on the sequence. The can also inhibit transcription by polymerase II in vitro. The dimer is a stronger inhibitor than the monomer.

The total syntheses of two anticancer drugs, calicheamin gamma1(sup I) and dynemicin A, has been achieved through two separate pathways. Both procedures revealed new insights into the chemistry of cyclic enediyne synthesis such as the feasibility of using enediyne anions for cyclization reactions and the glycal formation of rare carbohydrates. The natural products were first isolated from Micromonospora echinospora spp. calichensis and M. chersina, respectively.

The solution structures of calicheamicin [[gamma]1.sup.1], its cycloaromatized analog (calicbeamicin [epsilon]), and its aryl tetrasaccharide complexed to a common DNA hairpin duplex have been determined by NMR and distance-refined molecular dynamics computations. Sequence specificity is associated with carbohydrate-DNA recognition that places the aryl tetrasaccharide component of all three ligands in similar orientations in the minor groove at the d(T-C-C-T).d(A-G-G-A) segment. The complementary fit of the ligands and the DNA minor groove binding site creates numerous van der Waals contacts as well as hydrogen bonding interactions. Notable are the iodine and sulfur atoms of calicheamicin that hydrogen bond with the exposed amino proton of the 5'- and 3'-guanines, respectively, of the d(A-G-G-A) segment. The sequence-specific carbohydrate binding orients the enediyne aglycone of calicheamicin [[gamma]1.sup.1] such that its C3 and C6 proradical centers are adjacent to the cleavage sites. While the enediyne aglycone of calicheamicin [gamma]1.sup.1] is tilted relative to the helix axis and spans the minor groove, the cycloaromatized aglycone is aligned approximately parallel to the helix axis in the respective complexes. Specific localized conformational perturbations in the DNA have been identified from imino proton complexation shifts and changes in specific sugar pucker patterns on complex formation. The helical parameters for the carbohydrate binding site are comparable with corresponding values in B-DNA fibers while a widening of the groove is observed at the adjacent aglycone binding site.

Asymmetric allylboration of isopropylidene-N-Boc-L-serinal with two different allylboronates modified by tartrate esters results in the formation of an amino sugar of Calicheamicin gamma(sub 1)(super I) and its erythro diasteromer, respectively. A high degree of purity of the starting material ensures good diastereoselectivity of this reaction. The nature of the solvent used determines the conformations of the intermediate methyl pyranosides.

The binding of calicheamicin to three tetranucleotide sequences in the AT-region of a 142 base pair DNA restriction fragment and their subsequent cleavage is dependent on the salt between 150 and 125 mM concentrations of NaCl. This salt sensitivity is confirmed by variations in values of deltaG from -7.6 to -8.9 Kcal mol-1. Heterostatic forces add to the energetics of the calicheamicin-DNA sequences association.

The binding affinity between calicheamicin and a single TCCT.AGGA site within a synthetic duplex DNA is measured by kinetic analysis. DNA binding affinity of the carbohydrate domain is analyzed by competitive binding analysis involving calicheamicin and the calicheamicin oligosaccharide. Iodine substituent on the aromatic ring of the carbohydrate domain interacts with the exocyclic C2-NH2 group or both guanine residues at the target site.

Duplex d(GCATCCTAGC).d(GCTAGGATGC) reacts with calicheamicin and calicheamicin oligosaccharide domain to produce two complexes. NMR spectroscopic analyses show that the oligosaccharide domain of calicheamicin influences binding and sequence recognition in the DNA microgroove. Two species identified in the NMR spectra imply two different binding modes which permit each radical to reach and abstract its target. A structural model for the calicheamicin complex is derived by molecular dynamics calculation.

The calicheamicin gamma1 and glutathione reaction in the presence of double-stranded DNA yields four products generated by S-S bond exchange. The calicheamicin gamma1 bisulfide group and glutathione thiol function react to give the S-S bond product. There is no evidence of DNA catalysis of thiol function in water.

The enantioselective total synthesis of the naturally occurring antipode of the calicheamicin aglycon, (-)-calicheamicinone, was accomplished. An intramolecular 1,3-dipolar cycloaddition reaction of an alkynyl nitrite oxide was used to incorporate the nitrogen atom of the urethane. Asymmetry was introduced into the molecule through an asymmetric allylboration reaction. The intramolecular alkylation of an aldehyde was used to bridge the enediyne residue.

Schmidt's trichloroacetimidate procedure was used to achieve the stereoselective glycosidation, which combined the suitably functionalized aglycon 3 with the oligosaccharide fragment 2, in the first total synthesis of calicheamicin gamma (sub 1)(super I). The required alcoxylamine 32 and its A-4 isomer 32-epi were produced by the stereoselective reduction of oxime 31, obtained from the coupling product, with NACNBH3 in the presence of BF3.OEt2, during the synthetic process.

The calicheamicin gamma (sub 1)(super I) oligosaccharide portion was totally synthesized for the first time in the form of its methyl glycoside. The stereochemical difficulties associated with a (3,3)-sigmatropic realignment of an allylic thionoimidazolide, were overcome by the easy identification and examination of the synthetic challenge of the B-ring.

The bicyclo(7.3. 1)tridecenediyne core structure of the antitumor antibiotics esperamicin and calicheamicin can be constructed via the complexation of the 10,11-acetylenic bond as its derived eta2 Co(CO)6 adduct. This complexation results in the cation being aligned axially to the enol pi-system in the cyclohexanone ring, with the products unable to undergo cycloaromatization. Cyclohexane-1,2-dione yields 13-ketobicyclo(7.3.1)tridecenediyne in ten steps. The Co2(CO)6-eta2 propargylic cation methodology may also be used in the preparation of the core structures of dynemicin and neocarzinostatin.

The 13-ketobicylco(7.3.1)tridecenediyne core structure of calicheamicin and esperamicin is reported. The core structure is functionalized in a sequential, oxidative manner, which allows the insertion of the 1,2 double bond and 3-oxygen substitution. The 4beta-hydroxyl substituent allows the intramolecular introduction of the nitrogen functionality at C2. However, the cyclic carbamate formed is too resistant to hydrolysis to be of any synthetic use. Allylic trisulfide may be attached to the 13-keto moiety by Wittig chemistry.

Degradation and spectroscopic studies are performed to elucidate the chemical structure of the antitumor antibiotic calicheamicin gamma1. The structural assignment of the glycosidic chain results from the methanolysis studies on calicheamicin gamma1 and N-acetylcalicheamicin gamma1. Chemical and spectroscopic evidence support the enediyne structure of calicheamicinone the existence of a 1,4-dehydrobenzene diradical as a reaction intermediate. The enediyne system is revealed to be a major determinant of the DNA-damaging capabilities of the calicheamicins.

A conceptual different approach to the synthesis of calicheamicinone is discussed. The approach is based on the intramolecular alkenyl nitrile oxide dipolar cycloaddition reaction which leads to the incorporation of the full functionality of the agyclon and which agrees with enantioselective synthesis. The methodology offers an easy access to the study of the calicheamicin- and esperamicin-type aglycon skeletons in their optically active forms.

Although extensive effort has been applied toward understanding the mechanism by which enediynes cleave DNA, a continuous assay for this phenomenon is still lacking. In fact, with the exception of assays for DNase, continuous assays for most DNA cleavage events are unavailable. This article describes the application of 'molecular break lights' (a single-stranded oligonucleotide that adopts a stem-and-loop structure and carries a 5'-fluorescent moiety, a 3'-nonfluorescent quenching moiety, and an appropriate cleavage site within the stem) to develop the first continuous assay for cleavage of DNA by enediynes. Furthermore, the generality of this approach is demonstrated by using the described assay to directly compare the DNA cleavage by naturally occurring enediynes [calicheamicin and esperamicin), non-enediyne small molecule agents (bleomycin, methidiumpropyl-EDTA-Fe(II), and EDTA-Fe(II]), as well as the restriction endonuclease BamHI. Given the simplicity, speed, and sensitivity of this approach, the described methodology could easily be extended to a high throughput format and become a new method of choice in modern drug discovery to screen for novel protein-based or small molecule-derived DNA cleavage agents. calicheamicin | esperamicin | assay | molecular beacon | bleomycin

Calicheamicin-homeodomain conjugate is found to be an effective sequence-specific DNA cleavage and mapping tool.

A gene (calC) from Micromonospora echinospora has been discovered and characterized.

Calicheamicin γ.sup.I/.sub.1: An Antitumor Antibiotic That Cleaves Double-Stranded DNA Site Specifically Calicheamicin γ.sup.I/.sub.1 (Structure 1) (1, 2) has been recently isolated from fermentations of Micromonospora echinospora ssp. calichensis and is [...]

Experiments were performed to synthesize a 10-membered enediyne ring based on an eta2Co2(CO)6-propargylic aldol cyclization. Commercially available 5-methoxysalicylic acid was used to prepare the phenol in four standard steps. The preparation of aglycon calicheamicinone from 5-methoxysalicylic acid obtained 1.4% overall yield at an average of 86% per step. Although the route described was the most efficient synthesis performed, modification of the method to provide an enantioselective synthesis can be done by the application of asymmetric induction methodology.

Calicheamicin oligosaccharide dimeric forms are synthesized for a molecular recognition program in biological systems. Specific, high-affinity binding of the two variables to specific duplex DNA sequences are established through competitive bidding experiments. Oligosaccharide-DNA interactions displayed characteristic site selectivities that indicated need for additional research on DNA attachments and related issues.

Calicheamicin gamma1 I was complexed to different recognition sites and studied.

Calicheamicin γ.sub.1/.sup.I and DNA: Molecular Recognition Process Responsible for Site-Specificity AN IMPORTANT AND INTRIGUING ASPECT of the phenomenally potent antitumor agent calicheamicin γ.sub.1/.sup.I (structure 1a) is its interaction in vitro [...]