IS DNA TOPOISOMERASE INVOLVED IN THE UV EXCISION REPAIR PROCESS? NEW EVIDENCE FROM STUDIES WITH DNA INTERCALATING AND NON-INTERCALATING ANTITUMOR AGENTS

Abstract— The effects of selected DNA intercalating and non-intercalating drugs on the UV excision repair process in human fibroblasts have been examined. 9-Amino acridine, acridine orange, quinacrine, doxorubicin (adriamycin), ethidium bromide and actinomycin-D all inhibited the removal of pyrimidine dimers from cellular DNA by inhibiting the incision process as monitored by the nick translation assay and by an endonuclease-sensitive site assay. These agents also partially inhibited incision by the M. luteus endonuclease in an in vitro system. This is the only class of compounds tested to date that appears to block this early step of repair in mammalian cells. The DNA topoisomerase inhibitors, m -amsacrine and VP-16 (etoposide) and the bacterial gyrase inhibitors nalidixic acid and oxolinic acid were shown not to inhibit UV repair. As shown previously, however, novobiocin does block dimer removal and we show here that it is a potent inhibitor of the M. luteus UV endonuclease. While it has recently been demonstrated that many DNA intercalating agents block the strand-passing activity of DNA topoisomerase II giving rise to protein associated DNA strand breaks, the finding that the specific inhibitors of topoisomerase, m -AMSA and VP-16, do not inhibit repair, even though they block this strand passing activity, strongly suggests that inhibition of DNA topoisomerase is not associated with inhibition of DNA repair.     

Solid-phase synthesis for the identification of high-affinity bivalent lectin ligands

The development of carbohydrate-based therapeutics has been frustrated by the low affinities that characterize protein-carbohydrate complexation. Because of the oligomeric nature of most lectins, the use of multivalency may offer a successful strategy for the creation of high-affinity ligands. The solid-phase evaluation of libraries of peptide-linked multivalent ligands facilitates rapid examination of a large fraction of linker structure space. If such solid-phase assays are to replicate solution binding behavior, the potential for intermolecular bivalent binding on bead surfaces must be eliminated. Here we report the solid-phase synthesis and analysis of peptide-linked, spatially segregated mono- and bivalent ligands for the legume lectin concanavalin A. Bead shaving protocols were used for the creation of beads displaying spatially segregated binding sequences on the surface of Tentagel resins. The same ligands were also synthesized on PEGA resin to determine the effect of ligand presentation on solid-phase binding. While we set out to determine the lower limit of assay sensitivity, the unexpected observation that intermolecular bivalent ligand binding is enhanced for bivalent ligands relative to monovalent ligands allowed direct observation of the level of surface blocking required to prevent intermolecular bivalent ligand binding. For a protein with binding sites separated by 65 A, approximately 99.9% of Tentagel(1) surface sites and 99.99% of the total sites on a PEGA bead must be blocked to prevent intermolecular bivalent binding. We also report agglutination and calorimetric solution-phase binding studies of mono- and bivalent peptide-linked ligands.     

Opening the [4 + 2 + 2] cycloadducts of bicyclo[2.2.1]hepta-2,5-dienes (norbornadienes) to cis-fused bicyclo[5.3.0]decanes

Tetracyclo[5.4.0.0.(2,4)0(3,7)]undec-9-enes, prepared by the transition-metal-catalyzed [4 + 2 + 2] homo-Diels-Alder reactions of norbornadiene and 1,3-butadienes, can be opened using either acid-promoted or Zeise's dimer-mediated cyclopropane ring cleavage, ultimately leading to cis-fused bicyclo[5.3.0]decanes (perhydroazulenes). Stereoselective functionalization of the olefin unit in the tetracycloundecenes to an alcohol or diol prior to ring opening is tolerated by the Zeise's dimer ([Pt(C(2)H(4))Cl(2)](2)) catalyst to yield highly functionalized bicyclo[5.3.0]decanes, which form the core structure of numerous sesquiterpenes.     

Quantitative chromatographic method for the determination of low levels of nitrilotriacetic and ethylenediaminetetraacetic acids in diethylenetriaminepentaacetic acid

A quantitative method for determination of low levels (0.05%, w/w) of nitrilotriacetic and ethylenediaminetetraacetic acids in diethylenetriaminepentaacetic acid is described. Palmitic acid is added to the chelator as an internal standard before esterification with methanol containing 2%(v/v)H2SO4. The methyl esters of palmitic, nitrilotriacetic, and ethylenediaminetetraacetic acids are first separated from diethylenetriaminepentaacetate by silicic acid column chromatography and are subsequently quantitated by gas-liquid chromatography. The method is both accurate and reproducible with less than 10% relative error. Thin-layer chromatographic separations of the methyl esters, and quantitation at the 1% level, are also described.     

Chemistry and biology of diazonamide A: first total synthesis and confirmation of the true structure

With the addition of a tenth ring, the exchange of an oxygen atom for a nitrogen in the heart of the molecule, and a different terminal residue, the revised architecture for diazonamide A (1) provided an even more challenging molecular puzzle for chemical synthesis than its predecessor. In this article, we detail the first successful total synthesis of diazonamide A, an endeavor which not only verified its proper connectivities and established the stereochemistry of its previously unassignable C-37 chiral center, but which also was attended by the development of several new synthetic strategies and tactics.     

Column selectivity in reversed-phase liquid chromatography. V. Higher metal content (type-A) alkyl-silica columns

Retention measurements involving 16 test solutes have been carried out for 38 type-A alkyl-silica columns and three bonded-zirconia columns. These measurements have been analyzed in terms of a model previously developed for type-B columns, so as to yield values of five column selectivity parameters (H, S*, A, B, C) for each type-A column. Overall differences in selectivity between type-A and -B columns can be related to the average values of H, S*, etc. for each column type. Compared to type-B columns, type-A columns provide generally stronger retention for carboxylic acids, while solutes that are more hydrophobic or less bulky are more retained on type-B columns. Hydrogen-bond acceptors (e.g. aliphatic amides) and cations (e.g. protonated bases) are strongly retained on type-A versus type-B columns. Compared to type-B columns, bonded-zirconia columns show much increased retention of cations and reduced retention of hydrogen-bond acceptors. Because of relatively large differences in the selectivity of bonded-zirconia, type-A, and type-B columns, it will prove difficult to find columns of different type (e.g. a type-A and a type-B column) which have equivalent selectivity. Type-A columns also tend to be more different from each other (in terms of selectivity) than is the case for type-B columns. As a result, the replacement of a given type-A column by an "equivalent" type-A column also appears unlikely, except for samples that do not contain ionized compounds.     

Carboxylate binding to be(2+) in proteins and influence of the dielectric environment

To gain insight into the interaction of Be2+ ions with negatively charged protein residues, the free energy changes associated with the replacement of water molecules in the first hydration shell of with one and two acetate anions were computed for the gas phase reactions using ab initio methods at the MP2 and DFT-B3LYP computational levels. Both unidentate and bidentate modes of coordination of the carboxylate group with the Be2+ ion are considered. Continuum dielectric calculations were then performed to estimate the corresponding free energy changes in several environments of varying dielectric strength. Environments with dielectric constants of 2 and 4, which represent a protein interior, and 78, which corresponds to water, were used. It is found that the free energy changes for the substitution reactions decrease in magnitude with increasing dielectric strength, in agreement with similar results reported for Mg2+, Ca2+, and Zn2+ (Dudev et al. J. Phys. Chem. B 2000, 104, 3692). However, unlike Mg2+, Ca2+, and Zn2+, the free energy change for single-anion or concerted two-anion substitution reactions with remains negative and indicates the reactions are still favorable in the high dielectric aqueous environment. It is also found that the unidentate mode of binding is favored over the bidentate mode, and this is attributed, in part, to the introduction of hydrogen bonds between one carboxylate oxygen and a water molecule within the cluster when unidentate binding with Be2+ is involved.     

Preparation of compounds in the new dipyrrolo[3,4-b:3′,4′-e]-pyridine series from 1-benzylidene-2,3-dioxopyrrolidines. A variation of the hantzsch synthesis

Treatment of easily prepared l-substituted-4-benzylidene-2,3-dioxopyrrolidines with ammonium formate produces 1,2,4,6,7,8-hexahydro-2,6-disubstituted-8-aryldipyrrolo [3,4-b:3′,4′-e]pyridine-3,5-diones (II), usually in yields of 50 to 60%. Aromatization of the dihydropyridine ring of the hexahydroderivatives II yields corresponding 1,2,6,7-tetrahydro-2,6-disubstituted-8-aryldipyrrolo[3,4-b:3′,4′-e]pyridine-3,5-diones (III). These compounds appear to be the first to incorporate the dipyrrolo[3,4-b:3′,4′-e]pyridine ring system.