A Synthetic Nucleoside Probe that Discerns a DNA Adduct from Unmodified DNA

Selective pairing of engineering nucleosides in DNA duplexes provides a potential means to probe structurally modified DNA bases (i.e., DNA adducts) and address challenges associated with correlating adduct chemical structure with biological impact. The current study provides the first example of a thermodynamically stable DNA base pair that is comprised of a biologically relevant carcinogen−DNA adduct and a synthetic nucleoside probe. O6-Benzylguanine is a mutagenic DNA adduct; molecular modeling indicates that a novel diaminonaphthyl-derived nucleoside (dNap):O6-benzylguanine base pair may be stabilized by a combination of hydrogen-bonding and hydrophobic interactions. The nucleoside dNap was synthetically incorporated into oligonucleotides, and a series of duplexes were evaluated by thermal denaturation studies. The bulky DNA adduct O6-benzylguanine forms a highly stable and orthogonal base pair with dNap. Data indicate π-stacking potential, self-pairing capacity, isomeric selectivity, 1:1 duplex stoichiometry, and a B-form DNA structure. Further studies are required to understand the physical determinants of adduct:probe pair stability for the design of probes for diverse forms of DNA damage and for the development of adduct−probe-based molecular techniques

Catalytic Asymmetric Cyclocarbonylation of Nitrogen-Containing Enynes

The asymmetric Pauson−Khand type cyclization of nitrogen-containing enynes using carbon monoxide and a catalytic amount of (EBTHI)TiMe2 was examined. The influence of the nitrogen substituent and the concentration of the catalyst on the enantioselectivity of this cyclization was explored, and it has been found that enynes with an octyl-, benzyl-, or allylamino group, positioned β to the alkyne and the olefin, are cyclized with a high degree of enantioselectivity. Substrates with either bulky and/or electron-withdrawing nitrogen substituents are converted to products in low to moderate enantioselectivity

Monocyclopentadienyltitanium Aryloxide Complexes: Preparation, Characterization, and Application in Cyclization Reactions

A variety of monocyclopentadienyltitanium aryloxide complexes were prepared, characterized by X-ray crystallography, and used to catalyze or mediate cyclization reactions. Structural characterization allowed for the comparison of steric parameters of various 2,6-disubstituted aryloxide ligands. Transformations of dienes and enynesincluding the catalysis of a 1,6-diene cycloisomerization and the intramolecular Pauson−Khand reactionwere investigated. A titanium metallacycle was prepared from a sterically hindered enyne containing a trisubstituted olefin. The Pauson−Khand reaction of trimethylsilyl-substituted enynes was promoted to generate α-silylcyclopentenones

A Synthetic Nucleoside Probe that Discerns a DNA Adduct from Unmodified DNA

Selective pairing of engineering nucleosides in DNA duplexes provides a potential means to probe structurally modified DNA bases (i.e., DNA adducts) and address challenges associated with correlating adduct chemical structure with biological impact. The current study provides the first example of a thermodynamically stable DNA base pair that is comprised of a biologically relevant carcinogen−DNA adduct and a synthetic nucleoside probe. O6-Benzylguanine is a mutagenic DNA adduct; molecular modeling indicates that a novel diaminonaphthyl-derived nucleoside (dNap):O6-benzylguanine base pair may be stabilized by a combination of hydrogen-bonding and hydrophobic interactions. The nucleoside dNap was synthetically incorporated into oligonucleotides, and a series of duplexes were evaluated by thermal denaturation studies. The bulky DNA adduct O6-benzylguanine forms a highly stable and orthogonal base pair with dNap. Data indicate π-stacking potential, self-pairing capacity, isomeric selectivity, 1:1 duplex stoichiometry, and a B-form DNA structure. Further studies are required to understand the physical determinants of adduct:probe pair stability for the design of probes for diverse forms of DNA damage and for the development of adduct−probe-based molecular techniques

Monocyclopentadienyltitanium Aryloxide Complexes: Preparation, Characterization, and Application in Cyclization Reactions

A variety of monocyclopentadienyltitanium aryloxide complexes were prepared, characterized by X-ray crystallography, and used to catalyze or mediate cyclization reactions. Structural characterization allowed for the comparison of steric parameters of various 2,6-disubstituted aryloxide ligands. Transformations of dienes and enynesincluding the catalysis of a 1,6-diene cycloisomerization and the intramolecular Pauson−Khand reactionwere investigated. A titanium metallacycle was prepared from a sterically hindered enyne containing a trisubstituted olefin. The Pauson−Khand reaction of trimethylsilyl-substituted enynes was promoted to generate α-silylcyclopentenones

Synthesis of Deoxytetrahydrouridine

The α-hydroxyamido functionality of 2′-deoxytetrahydrouridine (dTHU) makes this seemingly simple and generally useful compound difficult to obtain. Reported synthetic strategies produce extremely poor yields and multiple products, and full characterization data is not available. Described herein is a two-step approach for synthesizing dTHU in increased yields and purity; stability concerns are also addressed. Catalytic reduction (5% Rh/alumina) of 2′-deoxyuridine, followed by reduction with sodium borohydride as a limiting reagent, produces dTHU and limits formation of side products. Evidence was obtained for formation of a methoxy-substituted analogue during purification. By this strategy, dTHU of >95% purity can be obtained in 40% yield on a 150 mg scale

Susceptibility of the Antioxidant Selenoenyzmes Thioredoxin Reductase and Glutathione Peroxidase to Alkylation-Mediated Inhibition by Anticancer Acylfulvenes

Selenium, in the form of selenocysteine, is a critical component of some major redox-regulating enzymes, including thioredoxin reductase (TrxR) and glutathione peroxidase (Gpx). TrxR has emerged as an anticancer target for drug development due to its elevated expression level in many aggressive human tumors. Acylfulvenes (AFs) are semisynthetic derivatives of the natural product illudin S and display improved cytotoxic selectivity profiles. AF and illudin S alkylate cellular macromolecules. Compared to AFs, illudin S more readily reacts with thiol-containing small molecules such as cysteine, glutathione, and cysteine-containing peptides. However, a previous study indicates that the reactivity of AFs and illudin S with glutathione reductase, a thiol-containing enzyme, is inversely correlated with the reactivity toward small molecule thiols. In this study, we investigate mechanistic aspects underlying the enzymatic and cellular effects of the AFs and illudin S on thioredoxin reductase. Both AF and HMAF were found to inhibit mammalian TrxR in the low- to submicromolar range, but illudin S was significantly less potent. TrxR inhibition by AFs was shown to be irreversible, concentration- and time-dependent, and mediated by alkylation of C-terminus active site Sec/Cys residues. In contrast, neither AFs nor illudin S inhibits Gpx, demonstrating that enzyme structure-specific small molecule interactions have a significant influence over the inherent reactivity of the Sec residue. In human cancer cells, TrxR activity can be inhibited by low micromolar concentrations of all three drugs. Finally, it was demonstrated that preconditioning cells by the addition of selenite to the cell culture media results in an enhancement in cell sensitivity toward AFs. These data suggest potential strategies for increasing drug activity by combination treatments that promote selenium enzyme activity

Hydrogen-Bonding Interactions at the DNA Terminus Promote Extension from Methylguanine Lesions by Human Extender DNA Polymerase ζ

Chemically induced DNA lesions can become DNA replication substrates that are bypassed by low-fidelity DNA polymerases. Following nucleotide misinsertion opposite a DNA lesion, the extension step can contribute to preserving such errors and lead to genomic instability and cancer. DNA polymerase ζ, a B-family polymerase, is proficient as an extender polymerase that catalyzes elongation; however, the chemical factors that impact its DNA replication are not understood. This study addresses the question of how DNA polymerase ζ achieves extension by examining the ability of recombinant human DNA polymerase ζ to extend from a series of methylated guanine lesions. The influence of H-bonding was examined by placing structurally altered nucleoside analogues and canonical bases opposite G, O6-MeG, N1-MeG, and N2-MeG. We determined that terminal base pairs with the highest proclivity for H-bonding were most efficiently extended in both primer extension assays and steady-state kinetic analysis. In contrast, when no H-bonding was possible at the DNA terminus, the least efficient steady-state kinetics were observed. To evaluate H-bonding protein minor groove interactions that may underlie this phenomenon, we performed computational modeling with Escherichia coli DNA polymerase II, a homologue for DNA polymerase ζ. The modeling data together with the primer extension assays demonstrate the importance of having a carbonyl group on the primer strand that can interact with a lysine residue found to be conserved in many B-family polymerases, including human Pol ζ. These data provide a model whereby interbase H-bonding interactions at the DNA terminus promote lesion bypass and extension by human DNA polymerase ζ

Nucleobase-Dependent Reactivity of a Quinone Metabolite of Pentachlorophenol

Pentachlorophenol (PCP) is a possible human carcinogen detected widely in the environment. A quinone metabolite of PCP, tetrachloro-1,4-benzoquinone (Cl4BQ), is a reactive electrophile with the capacity to damage DNA by forming bulky covalent DNA adducts. These quinone adducts may contribute to chlorophenol carcinogenesis, but their structures, occurrence, and biological consequences are not known. Previous studies have indicated that several DNA adducts are formed in vivo in rats exposed to Cl4BQ, but these adducts were not identified structurally. In the present study, we have elucidated the structure of new agent-specific DNA adducts resulting from the reaction of dGuo, dCyd, and Thd with Cl4BQ. These have been characterized chemically by liquid chromatography−electrospray ionization mass spectrometry, HPLC, UV, and NMR analysis. Two dGuo adducts and one dCyd adduct resulting from the reaction of double-stranded DNA with Cl4BQ have been identified. The results indicate that, in the structural context of DNA, Cl4BQ reacts most readily with dGuo compared to the other DNA bases and that the mode of Cl4BQ reactivity is dependent on the base structure; i.e., multiple types of adducts are formed. Finally, DNA adducts consistent with Cl4BQ reactions are observed when DNA or dGuo is treated with PCP and a peroxidase-based bioactivating system

Tolerance of Base Pair Size and Shape in Postlesion DNA Synthesis

The influence of base pair size and shape on the fidelity of DNA polymerase-mediated extension past lesion-containing mispairs was examined. Primer extension analysis was performed with synthetic nucleosides paired opposite the pro-mutagenic DNA lesion <i>O</i>⁶-benzylguanine (<i>O</i>⁶-BnG). These data indicate that the error-prone DNA polymerase IV (Dpo4) inefficiently extended past the larger Peri:<i>O</i>⁶-BnG base pair, and in contrast, error-free extension was observed for the smaller BIM:<i>O</i>⁶<i>-</i>BnG base pair. Steady-state kinetic analysis revealed that Dpo4 catalytic efficiency was strongly influenced by the primer:template base pair. Compared to the C:G pair, a 1.9- and 79 000-fold reduction in Dpo4 efficiency was observed for terminal C:<i>O</i>⁶-BnG and BIM:G base pairs respectively. These results demonstrate the impact of geometrical size and shape on polymerase-mediated mispair extension