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The formal C1'-oxidation product, 2-deoxyribonolactone, is formed as a result of DNA damage induced via a variety of agents, including gamma-radiolysis and the enediyne antitumor antibiotics. This alkaline labile lesion may also be an intermediate during DNA damage induced by copper-phenanthroline. Oligo-nucleotides containing this lesion at a defined site were formed via aerobic photolysis of oligonucleotides containing a photolabile ketone, and were characterized by gel electrophoresis and electrospray mass spectrometry (ESI-MS). Treatment of oligo-nucleotides containing the lesion with secondary amines produces strand breaks consisting of 3'-phosphate termini, and products which migrate more slowly in polyacrylamide gels. MALDI-TOF mass spectrometry analysis indicates that the slower moving products are formal adducts of the beta-elimination product resulting from 2-deoxyribonolactone and one molecule of amine. The addition of beta-mercapto-ethanol to the reaction mixture produces thiol adducts as well. The stability of these adducts suggests that they cannot be the labile species characterized by gel electrophoresis in copper-phenanthroline-mediated strand scission. The characterization of these adducts by mass spectrometry also provides, by analogy, affirmation of proposals regarding the reactivity of nucleophiles with the beta-elimination product of abasic sites. Finally, the effects of this lesion and the various adducts on DNA repair enzymes are unknown, but their facile generation from oligonucleotides containing a photolabile ketone suggests that such issues could be addressed.

The compound 3-amino-1,2,4-benzotriazine 1,4-dioxide (1, tirapazamine; also known as SR4233, WIN 59075, and tirazone) is a clinically promising anticancer agent that selectively kills the oxygen-poor (hypoxic) cells found in tumors. When activated by one-electron enzymatic reduction, tirapazamine induces radical-mediated oxidative DNA strand cleavage. Using the ability to generate a single deoxyribose radical at a defined site in an oligonucleotide, we recently provided direct evidence that, in addition to initiating the formation of DNA radicals, tirapazamine can react with these radicals and convert them into base-labile lesions [Daniels et al. (1998) Chem. Res. Toxicol. 11, 1254-1257]. The rate constant for trapping of a C1'-radical in single-stranded DNA by tirapazamine was shown to be approximately 2 x 10(8) M(-1) s(-1), demonstrating that tirapazamine can substitute for molecular oxygen in radical-mediated DNA strand damage reactions. Because reactions of tirapazamine with DNA radicals may play an important role in its ability to damage DNA, we have further characterized the ability of the drug and its metabolites to convert a C1'-DNA radical into a base-labile lesion. We find that tirapazamine reacts with a C1'-radical in double-stranded DNA with a rate constant of 4.6 x 10(6) M(-1) s(-1). The mono-N-oxide (3) stemming from bioreductive metabolism of tirapazamine converts the C1'-radical to an alkaline-labile lesion more effectively than the parent drug. Compound 3 traps a C1'-radical in single-stranded DNA with a rate constant of 4.6 x 10(8) M(-1) s(-1) and in double-stranded DNA with a rate constant of 1.4 x 10(7) M(-)(1) s(-)(1). We have also examined the rate and mechanism of reactions between the C1'-radical and representatives from two known classes of "oxygen mimetic" agents: the nitroxyl radical 2,2,6, 6-tetramethylpiperidin-N-oxyl (4, TEMPO) and the nitroimidazole misonidazole (5). TEMPO traps the C1'-radical in single-stranded DNA (7.2 x 10(7) M(-1) s(-1)) approximately 3 times less effectively than tirapazamine, but 2 times as fast in double-stranded DNA (9.1 x 10(6) M(-1) s(-1)). Misonidazole traps the radical in single- (6. 9 x 10(8) M(-1) s(-1)) and double-stranded DNA (2.9 x 10(7) M(-1) s(-1)) with rate constants that are roughly comparable to those measured for the mono-N-oxide metabolite of tirapazamine. Finally, information regarding the chemical mechanism by which these compounds oxidize a monomeric C1'-nucleoside radical has been provided by product analysis and isotopic labeling studies.

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R-Deoxynucleotides are potentially deleterious lesions when produced in DNA. They are presumably formed in part via misrepair of the respective C1′-nucleotide radicals by thiols. However, the selectivity and extent to which these lesions are formed via this pathway has not been ascertained. Using the ability to independently generate deoxyuridin-1′-yl (4) at a defined site in a biopolymer, we have determined that thiol trapping in duplex DNA occurs with high stereoselectivity from the R-face, resulting in restoration of the naturally occurring -deoxynucleotide. The observed stereoselectivity of thiol trapping in duplex DNA suggests that 4 is intrahelical. The rate constant for hydrogen atom donation to 4 is reduced 2-3-fold in doublestranded DNA compared to single-stranded DNA. This decrease is attributed to the relative inaccessibility of the C1′-position in duplex DNA. The combination of these two properties of 4 indicates that, at O 2 concentrations present in aerated water, R-deoxynucleotide formation should constitute a minor component of the reactivity of C1′-radicals. Accordingly, the chemical biology of other lesions derived from formal damage at C1′-position could be significant.R-Deoxynucleotides, such as R-deoxyadenosine (1), are produced during γ-radiolysis of DNA under anaerobic conditions. 1 R-Deoxyadenosine (1) has been shown to be premutagenic in vitro, inducing DNA polymerase to incorporate deoxyadenosine and deoxycytidine opposite it. 2 A variety of pathways have been proposed to account for R-deoxynucleotide formation, some of which require high fluxes of reactive intermediates. 3 One pathway leading to anomerization that does not require reaction of two reactive intermediates with a single nucleotide involves reversible ring opening of an initially formed C4′-radical, which is subsequently reduced. Chemical evidence for such a ring opening in a model system has been reported recently. 4 R-Deoxynucleotides may also be formed under oxidative stress conditions from hydrogen atom donation by thiol to the radical resulting from formal C1′-hydrogen atom abstraction. 5-7 The C1′-radical which undergoes misrepair may be produced either by hydrogen atom abstraction from the native nucleotide by species such as hydroxyl radical, via internucleotidyl hydrogen atom abstraction, or via deprotonation from C1′ of the nucleobase cation radical. 7-9 Unlike other lesions, such as thymidine C5-hydrate (2) and the thymidine glycols (3),(1) Lesiak, K.; Wheeler, K. T.

The effects of bulky ortho,ortho' groups on the reactions of aryl bromides and areneselenols with tributylstannane have been studied. Bulky ortho,ortho' groups accelerate the reaction of the bromides with the stannane but retard the reactions of the selenols. On the other hand, ab initio and force field calculations show that introducing bulky ortho substituents into selenols causes a greater increase in strain than in the corresponding bromides. Two possible explanations for the divergent reactivity patterns are advanced. On one hand, it is possible that bromine abstraction by stannyl radicals from aryl bromides proceeds in a single step through a linear transition state whereas the abstraction of SeH from the selenols involves a T-shaped, hypervalent intermediate. Alternatively, it may be that both reactions are concerted with the bromine abstraction having a late transition state and the SeH abstraction an early one. Approximate second-order rate constants for the reaction of tributylstannane with a range of hindered aryl bromides are derived from competition reactions. 2,4,6-Tri-tert-butylbenzeneselenol is able to function moderately well as a catalyst for the stannane-mediated reactions of vinyl bromides. The X-ray crystal structure of bis(2,4,6-triisopropylphenyl) diselenide is presented.

9-[1-(Phosphonomethoxycyclopropyl)methyl]guanine (PMCG, 1), representative of a novel class of phosphonate nucleosides, blocks HBV replication with excellent potency (EC(50) = 0.5 microM) in a primary culture of HepG2 2.2.15 cells. It exhibits no significant cytotoxicity in several human cell lines up to 1.0 mM. It does not inhibit replication of human immunodeficiency virus (HIV-1) or herpes simplex virus (HSV-1) at 30 microM. Many purine base analogues of 1 also exhibit inhibitory activity against HBV, but at 30 microM, pyrimidine analogues do not. 1 is 4 times more potent than 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA), which was used as a positive control (EC(50) = 2.0 microM). The characteristic cyclopropyl moiety at the 2'-position of 1 was prepared by titanium-mediated Kulinkovich cyclopropanation. 1 was modified to give the orally available drug candidate, PMCDG Dipivoxil (2). Compound 2 exhibited excellent efficacy when administered at 5 mg per kg per day in a study with woodchucks infected with woodchuck hepatitis B virus (WHBV). Drug candidate 2 has successfully completed phase I clinical trials and is currently undergoing phase II clinical studies for evaluation of efficacy.