Investigating Nucleic Acid Damage Processes via Independent

NOVEMBER 1998 VOLUME 11, NUMBER 11 © Copyright 1998 by the American Chemical Society

Invited Review Investigating Nucleic Acid Damage Processes via Independent Generation of Reactive Intermediates† Marc M. Greenberg Department of Chemistry, Colorado State University, Fort Collins, Colorado 80523 Received July 24, 1998

Contents 1. Introduction 1.1. Why Focus Directly on the Reactive Intermediates Involved in Nucleic Acid Damage? 2. Nucleobase Reactive Intermediates 2.1. Pyrimidin-6-yl Radicals 2.2. Pyrimidine Olefin Cation Radicals 2.3. Pyrimidin-5-yl Radicals 3. DNA Damage Derived from C1′-Hydrogen Atom Abstraction 3.1. Reactivity of C1′-Radicals 3.2. Formation of 2′-Deoxyribonolactone Lesions 3.3. Formation of Direct Strand Breaks 4. DNA Damage Derived from C4′-Hydrogen Atom Abstraction 4.1. Formation of Direct Strand Breaks 4.1.1. Criegeé Rearrangement 4.1.2. Schulte-Frohlinde Cleavage and Grob Fragmentation 4.1.2.1. Monomer and Model Studies 4.1.2.2. Oligonucleotide Studies 4.2. Formation of Alkaline Labile Lesions 4.3. Using Independently Generated Reactive Intermediates To Probe Electron Transfer in DNA 5. Conclusions

1. Introduction 1235 1236

1236 1236 1237 1238 1239 1239 1240 1241 1242 1242 1242 1243 1243 1244 1245 1245

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It is likely that this review article is not the only contribution to this issue of Chemical Research in Toxicology in which some aspect of DNA damage is addressed. The magnitude of the importance of DNA damage in the etiology and treatment of a range of diseases has provided a tremendous impetus for scientific investigations into this subject. Chemistry plays a vital role in understanding DNA damage. The elements of theory, mechanism, and synthesis have been utilized in studies on nucleic acid damage, as well as in determining how to selectively damage this biopolymer. Many elegant mechanistic studies have been carried out on individual DNA-damaging agents, including the most general nucleic acid damaging process, γ-radiolysis (1-3). During this decade, a number of groups have employed an approach in studies on nucleic acid damage in which organic chemistry was used to independently generate reactive intermediates involved in these processes. This challenging physical organic chemistry strategy has simplified studies of DNA damage by enabling chemists to directly study what are sometimes common intermediates produced by different damaging agents, without the possible complications introduced by the actual damaging agent. The ability to focus on the reactivity of discrete reactive intermediates

† For simplicity, chemical structures which appear as monomers and in biopolymers are assigned the same identification number.

10.1021/tx980174i CCC: $15.00 © 1998 American Chemical Society Published on Web 10/22/1998

1236 Chem. Res. Toxicol., Vol. 11, No. 11, 1998

has enabled chemists to resolve mechanistic disparities, and has given rise to surprising observations that reveal that DNA damage processes are more complex than previously thought. 1.1. Why Focus Directly on the Reactive Intermediates Involved in Nucleic Acid Damage? γ-Radiolysis has been used to treat tumors for more than a century, and DNA damage is a principle source of its cytotoxic effects. A great deal has been learned about the chemistry of DNA damage using pulse radiolysis and various forms of ionizing radiation (1, 4, 5). However, mechanistic studies of biopolymers can be hampered using these methods due to the simultaneous generation of multiple reactive intermediates and the lack of control over where these intermediates are produced in the biopolymer. For instance, the possibility that an initially formed reactive intermediate can result in multiple lesions (DNA damage amplification), including doublestrand breaks, is an important contentious issue, whose resolution would benefit from the ability to generate reactive intermediates in a controlled manner at a defined site in a biopolymer (6-9). Independent generation of reactive intermediates involved in nucleic acid damage also facilitates separation of the chemical processes that generate the species from subsequent effects of a damaging agent on the reactivity of the intermediate. This is particularly important for determining why different damaging agents give rise to distinct lesions via common reactive intermediates. Unambiguous generation of reactive intermediates at a defined site in a biopolymer can also reveal complexities in DNA damage that would otherwise be masked due to the generation of multiple lesions in a small region of the nucleic acid. Finally, in the course of any mechanistic investigation, particularly ones as complicated as those involved in nucleic acid damage, there is always the possibility of discovering new reaction processes. In the realm of nucleic acid damage, these processes could have significant relevance to human health.

Greenberg

repaired, or give rise to alkaline labile lesions and direct strand breaks. Other antitumor compounds exert their effects on DNA by alkylating the nucleobases (e.g., 5-7) (16-18). While some antitumor compounds generate

nucleobase radicals and radical ions, the formation of these reactive intermediates represents minor pathways (19, 20). In contrast, nucleobase-centered reactive intermediates constitute a significant component of DNA damage resulting from the direct and indirect (solvated electrons, hydrogen atoms, and hydroxyl radicals) effects of ionizing radiation (1). Trapping of a nucleobase radical (or its respective peroxyl radical) by an exogenous hydrogen atom donor produces a damaged nucleoside which could be mutagenic and/or cytotoxic (21-24). It is also possible to form such lesions via electron transfer processes, followed by reaction with nucleophiles or electrophiles. However, to produce a direct strand break (as opposed to an alkaline labile lesion) in DNA, the radical center must be transferred to the sugar backbone. In principle, this may occur in an intra- or internucleotidyl fashion. Internucleotidyl hydrogen atom abstraction is an example of DNA damage amplification, and is not known to occur during DNA damage induced by antitumor compounds. Consequently, examination of the reactivity of nucleobase radicals presents a platform from which the organic chemist may discover new DNA damage pathways. 2.1. Pyrimidin-6-yl Radicals. Ionizing radiation has yielded indirect evidence in support of DNA damage amplification involving spin transfer from nucleobases to the carbohydrate moieties (25-30). These experiments have led to the proposal that internucleotidyl hydrogen atom abstraction is the rate-limiting step in strand scission at neutral pH and under anoxic conditions (Scheme 1). Intranucleotidyl hydrogen atom abstraction is disfavored due to poor stereoelectronic overlap. Steady state ESR studies on the hydroxyl radical adducts of pyrimidine nucleosides indicate that these species are Scheme 1

2. Nucleobase Reactive Intermediates A variety of antitumor antibiotics and radiomimetic agents damage nucleic acids by direct (or formal) hydrogen atom abstraction from the carbohydrate backbone (e.g., 1-4) (10-15). The radical species formed can be

Invited Review

Chem. Res. Toxicol., Vol. 11, No. 11, 1998 1237 Scheme 2

Scheme 4

Scheme 3

stable on the millisecond time scale, consistent with this prediction (31). However, these experiments do not preclude their participation in intranucleotidyl hydrogen atom abstraction, because kinetic studies of nucleic acid cleavage (under anaerobic conditions) indicate that the half-life for such a process is greater than 0.1 s (29, 30). More quantitative estimation of the rate constant for intranucleotidyl hydrogen atom abstraction within the C5-hydroxyl radical adduct of thymidine (8) was obtained by independently generating 8 photolytically from 10 via photoinduced single-electron transfer from N-methylcarbazole (eq 1) (32, 33). The rate constant for intramolecu-

lar hydrogen atom abstraction was estimated by constructing a competition between an intermolecular deuterium atom donor, whose bimolecular rate constants could be estimated on the basis of literature values for reaction with other alkyl radicals, and the intramolecular process of interest (Scheme 2). 2H NMR analysis of intact nucleosides (e.g., 12), and mass spectral determination of the isotopic content of free nucleobases (13, Scheme 3), led to the conclusion that intramolecular hydrogen atom abstraction within 8 was not kinetically competent to be involved in nucleic acid strand scission (34). The relevance of the results obtained using this nucleoside to the issue of intranucleotidyl hydrogen atom abstraction in biopolymers, where the 3′- and 5′-hydroxyl groups are replaced by phosphato groups, is supported by calculations on 1-amino-2-deoxy-3,5-diphosphateribofuranose. These calculations indicate that intramolecular hydrogen atom abstraction should be less favorable thermodynamically in biopolymers than in the free nucleoside, and therefore presumably even slower (35, 36). 2.2. Pyrimidine Olefin Cation Radicals. The question of intranucleotidyl versus internucleotidyl hydrogen atom abstraction by olefin cation radicals, as well as the interconversion of nucleobase hydroxyl radical adducts

and olefin cation radicals, has also been addressed (31, 37, 38). There are a number of observations that suggest that olefin cation radicals and nucleobase radical adducts react in a similar fashion, and in some instances, one may pass through the other during a multistep DNA damage process. The olefin cation radicals of the pyrimidines and the nucleobase radicals, resulting from the direct and indirect effects of ionizing radiation, respectively, produce a similar profile of degradation products. The connection between the direct and indirect effects of ionizing radiation is further strengthened by determination that the efficiency and rate of strand scission from the two pathways are affected similarly by pH and an exogenous hydrogen atom donor (dithiothreitol) (37). These observations were rationalized by proposing that as the pH is lowered, the increased rate constant for strand scission in poly(U) is due to an increase in the rate of formation of 15 from the C5-hydroxyl radical adduct (14) (29). Steady state ESR analysis of the reaction products of HO• with poly(U) was consistent with this proposal (Scheme 4). At pH 7, the C5-hydroxyl radical adduct (14) was observed, but at pH e4, 16 was observed. Although these observations support this proposal, one should not lose sight of the fact that steady state ESR experiments may not reflect reaction dynamics. The results of ESR studies on monomeric olefin cation radicals are somewhat variable, but there is evidence to support radical transfer from the nucleobase to the sugar in some systems (31, 39). Clearly, the equilibrium between the hydroxyl radical adduct and the cation radical lies on the side of the former, and the extent of participation of the cation radical in strand scission is determined by the competition between hydration and hydrogen atom abstraction (intra- or internucleotidyl). Results from experiments on monomeric pyrimidine olefin cation radicals of deoxyribonucleosides indicate that hydration is much faster than intramolecular hydrogen atom abstraction. However, as mentioned above, results from pH studies on strand scission suggest that dehydration of hydroxyl radical adducts may be the ratelimiting step in strand scission, implying that the olefin cation radical rapidly abstracts hydrogen atom(s) from

1238 Chem. Res. Toxicol., Vol. 11, No. 11, 1998 Scheme 5

an adjacent nucleotide (37). The viability of olefin cation radical formation at neutral pH was investigated using independently generated 8 (eq 1) (33). Since the initial hydroxyl group in 8 was not derived from the solvent, as is the case when it is produced during γ-radiolysis, it was possible to use H218O as a label for detecting the intermediacy of 17 (Scheme 5). No [18O]12 was detected following sequential glycolysis, persilylation, and mass spectral analysis of 13 (Scheme 3). Furthermore, elevated levels of thymine were not observed upon formic acid treatment, indicating that thymidine C6-hydrate (18) was not formed via hydration of the olefin cation radical. Finally, using trapping of 8 by 2,2,6,6-tetradeuteriocyclohexa-1,4-diene (11) as a kinetic clock, it was shown that dehydration of 8 at neutral pH is not kinetically competent to be involved in strand scission (Scheme 5). 2.3. Pyrimidin-5-yl Radicals. While intranucleotidyl hydrogen atom abstraction by C6-radical adducts of pyrimidines is not a consideration, internucleotidyl hydrogen atom abstraction is a possibility. Computational studies on 5,6-dihydrothymidin-5-yl (19), which is produced via the direct and indirect effects of ionizing radiation (eq 2), concluded that this radical should be more likely to abstract a hydrogen atom from an adjacent nucleotide than the regioisomeric C6-radical (40). This

prediction was based upon the proximity of atoms in a calculated ground state geometry and not upon energetics. Nonetheless, these calculations were supported by ESR experiments from which it was concluded that 19 abstracts the C1′-, C2′-, and C4′-hydrogen atoms of adjacent nucleotides (41, 42). This proposal was recently tested in studies in which 5,6-dihydrothymidin-5-yl (19) was independently generated via Norrish type I photocleavage of 20 (eq 2) (43). Chemical trapping with nonexchangeable hydrogen atom donors and O2 in aque-

Greenberg

ous solvents confirmed that the radical is produced from 20 in high yield. The feasibility for internucleotidyl hydrogen atom transfer from the carbohydrate components of nucleic acids to 19 was initially investigated using 2-propanol (21) and the dimethylacetal of glycoaldehyde (22) as exogenous hydrogen atom donors (eq 3).

The acetal and alcohol were chosen as models of the C1′and C4′-positions of the deoxyribose ring, respectively. The reactivity of 19 was examined in the presence of high concentrations (5 M) of each hydrogen atom donor so the potential effective molarity of an adjacent sugar ring in a biopolymer could be mimicked. However, no evidence for hydrogen atom abstraction from these traps was obtained. The ability of 19 to effect strand damage in DNA via hydrogen atom abstraction was investigated further by independently generating the radical from 20 within biopolymers (44, 45). Since 20 is a dihydropyrimidine and unstable to concentrated ammonium hydroxide, it was site specifically incorporated into chemically synthesized DNA using a general deprotection method that eliminates the use of strong base (46, 47). Gel electrophoretic analysis of 32P-labeled oligonucleotides containing 20 that were irradiated under anaerobic conditions known to form 19 did not indicate formation of any direct strand breaks, or alkaline labile lesions (44). These observations confirmed the results of experiments on monomeric 19, in which it was concluded that the radical is incapable of amplifying DNA damage via internucleotidyl hydrogen atom abstraction. Although tumor cells are often hypoxic, the formation of peroxyl radicals can potentially compete with hydrogen atom abstraction by alkyl radicals. Oxygen trapping occurs at close to diffusion-controlled rates (∼2 × 109 M-1 s-1), whereas rate constants for hydrogen atom abstraction are typically several orders of magnitude lower. Hence, peroxyl radical formation can compete with hydrogen atom abstraction, even at low O2 concentrations. The general formation of peroxyl radicals within γ-irradiated biopolymers in vitro was confirmed using ESR. Time-resolved ESR and conductivity measurements corroborate their involvement in hydrogen atom abstraction, the putative rate-limiting step of strand scission. Although the specific identity of the peroxyl radical(s) involved in the hydrogen atom abstraction step(s) has (have) not been determined, time-resolved light scattering, UV transient experiments, and product studies are consistent with strand scission resulting from this pathway (1, 30, 48-53). More direct examination of one particular peroxyl radical was carried out in experiments in which 5,6dihydrothymidin-5-yl (19) was independently generated from 20 under aerobic conditions (44, 45). In contrast to experiments carried out under anaerobic conditions, independent generation of 19 in the presence of O2 produced direct strand breaks and alkaline labile lesions at the 5′-adjacent nucleotide in single-stranded oligo-

Invited Review


Scheme 8

reminiscent of the reactivity of peroxyl radicals derived from aromatic substrates, and was confirmed using two spectrophotometric assays for superoxide. Epinephrine oxidation (eq 4) and cytochrome c reduction (eq 5) were inhibited by the addition of superoxide dismutase, indicating that the observed phenomena could be attributed to O2-•. Semiquantitative analysis of superoxide forma-

Scheme 7

nucleotides. Alkaline labile lesions were produced approximately 10 times more frequently than direct strand breaks. Kinetic isotope effect experiments suggested that 23 selectively abstracts the C1′-hydrogen atom of the 5′adjacent nucleotide (Scheme 6). The kinetic isotope effect experiments were corroborated by product studies (ratio of direct vs alkaline labile lesions, enzymatic end group analysis) of an independently generated C1′-nucleotide radical (see below) at a defined site in an oligonucleotide (54). Preferential cleavage of the C1′ hydrogen bond in favor of the slightly stronger C4′ hydrogen bond was also evident from product studies (35, 36). For instance, 3′phosphoglycolate termini, normally indicative of DNA cleavage initiated by C4′-hydrogen atom abstraction, were not observed. Instead, 3′- and 5′-phosphates were the exclusive end groups produced in all biopolymers containing 20. The rate constant or efficiency of DNA damage amplification by 23 in the biopolymer has not been determined. However, competitive kinetics experiments on monomer 19 revealed one pathway which may compete with internucleotidyl hydrogen atom abstraction by the peroxyl radical (23) (55). Thymidine is observed when 19 is generated in the presence of O2 under radical chain conditions from 24, but not under degassed conditions (Scheme 7). Thymidine is believed to result from elimination of hydroperoxyl radical from 23. This behavior is

tion using the cyctochrome c assay accounted for >50% of the expected O2-• based upon the amount of thymidine formed. The ratio of rate constants (1.3 × 10-2 M) for elimination of O2-• versus intermolecular hydrogen atom abstraction from Bu3SnH was estimated by measuring the ratio of 5,6-dihydro-5-hydroxythymidine (12) versus thymidine as a function of tributyltin hydride concentration (Scheme 7). Thymidine was the major product despite the presence of Bu3SnH at a concentration as high as 10 mM. Assuming that the bimolecular rate constant for trapping of 23 by Bu3SnH was 5 × 103 M-1 s-1, the rate constant for superoxide elimination was estimated to be ≈65 s-1. The correlation between the bimolecular trapping of 23 by Bu3SnH and internucleotidyl hydrogen atom abstraction from a neighboring deoxyribose ring in a polynucleotide remains to be determined.

3. DNA Damage Derived from C1′-Hydrogen Atom Abstraction In duplex DNA, the C1′-hydrogen atom is deeply embedded in the minor groove, and is relatively inaccessible to a freely diffusing reactive species compared to the slightly more strongly bonded C4′-hydrogen atom (36). Despite its relatively poor accessibility, the C1′position of a nucleotide is believed to be directly attacked by a variety of DNA-damaging agents, and is even believed to be abstracted by a vinyl radical of an adjacent nucleotide that lies in the major groove of duplex DNA (2, 10, 12, 56). 3.1. Reactivity of C1′-Radicals. The C1′-radical follows two general reaction pathways that involve the formation of either direct strand breaks or alkaline labile lesions (Scheme 8) (2). Quenching of the radical by thiol

1240 Chem. Res. Toxicol., Vol. 11, No. 11, 1998 Scheme 9

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Photolysis of 31 under aerobic conditions in the presence of varying concentrations of β-mercaptoethanol enabled estimation of the rate of hydrogen atom abstraction by 27 from the thiol which was ∼4 × 106 M-1 s-1 (Scheme 9) (59). Similarly, generation of 27 at a defined site in an oligonucleotide (33) enabled the estimation of a rate constant for trapping of the radical by β-mercaptoethanol by measuring the dependence of total strand damage (alkaline labile lesions and direct strand breaks) on thiol concentration (54). While the absolute rate

can result in the formation of premutagenic R-deoxynucleotides (R-28) (57, 58). Alkaline labile lesions are typically characterized by formation of the 2′-deoxyribonolactone (29) (2, 12). Direct strand breaks most often give rise to the formation of 3′- and 5′-phosphate termini, and can be accompanied by the formation of the 5-methylene furanone (30) (10). In fact, formation of 29 and/ or formation of the furanone 30, the elimination product that is presumably derived from it, are often used as flags for hydrogen atom abstraction from the C1′-position of nucleotides. The mechanisms by which these products are formed, as well as the factors that control which reaction channel the common radical is funneled through, have until recently been less well understood. The design of molecules that enable one to independently generate the C1′-nucleoside radicals (and nucleotide radical within the biopolymer) from chemically synthesized nonnative nucleosides has proven useful for studying these processes, as have the study of model compounds of metastable products (59, 60). Independent generation of 27 at a defined site in an oligonucleotide from 31 demonstrated that in the absence of additional factors, alkaline labile lesions are formed 8-10 times more often than direct strand breaks (54). In addition to being of importance in its own right, this observation supports the recently proposed mechanism for DNA damage amplification via the initial formation of 5,6-dihydrothymidin5-yl (19, Scheme 6) (45). Kinetic measurements of radical reactivity provide valuable mechanistic insight. The first reported laser flash photolysis study of a C1′-nucleoside radical (27) indicates that O2 traps this species at a rate (2 × 109 M-1 s-1) comparable to those of typical alkyl radicals (eq 6) (61). Thus far, information on rate

constant measured for thiol trapping of 27 in the polymer was slightly higher (∼1 × 107 M-1 s-1), the conclusion drawn regarding the formation of R-deoxyuridine from 27 remains constant. Both sets of experiments indicate that R-deoxynucleotide (R-28) formation via hydrogen atom abstraction by the anomeric nucleotide radical will account for only a minor amount of the reaction products from this radical under aerobic conditions in the presence of physiological levels (∼7 mM) of thiol. In fact, the 4-5% of R-deoxynucleotides that can be attributed to the anomeric nucleotide radical may be an overestimate, because the thiol most likely present in vivo, glutathione, may react with DNA radicals even more slowly than β-mercaptoethanol due to its negative charge (51, 64). Furthermore, the stereoselectivity of thiol trapping of anomeric radicals (e.g., 27) in duplex DNA (which are unknown) may also affect the efficiency of R-deoxynucleotide formation. 3.2. Formation of 2′-Deoxyribonolactone Lesions. The results of experiments involving thiol trapping of 27 suggest that, barring other trapping processes, 2′-deoxyribonolactone (29) should be a major product arising from C1′-radical formation during γ-radiolysis. Lesion 29 may also arise via a variety of other DNA damage processes, some of which do not proceed through the C1′radical (62, 63). For instance, a family of oxoruthenium complexes (e.g., 34) induce DNA damage via direct twoelectron oxidation to yield the C1′-carbocation, which ultimately yields the lactone upon trapping by H2O (65, 66).

constants for other processes involving 27 has been obtained indirectly via competitive kinetics (Scheme 9) (59). This approach was employed for estimating the extent of R-deoxynucleotide (R-28) formation under aerobic conditions. These premutagenic lesions have been detected during γ-radiolysis of DNA under anoxic conditions. Determination of the relative rates for these two trapping reactions by measuring the amounts of the respective products produced under aerobic γ-radiolysis conditions in the presence of thiols is complicated by the possibility that the products can be formed via pathways that do not involve the C1′-radical, and that a multitude of products are produced by other active pathways during γ-radiolysis (62, 63). Independent generation of 27 eliminates these complications.

Direct oxidation of the C1′-radical to the carbocation (35) is one of three general pathways by which this reactive intermediate can produce the deoxyribonolactone alkaline labile lesion (Scheme 10). Although the oxidation potentials (Eox) of these radicals are unknown, estimates based upon similarly substituted systems suggest that the values for C1′-radicals of nucleotides could be as low as 0.3 V (Figure 1) (67). Potential oxidants include DNAdamaging agents such as Cu(OP)2 (1). H218O labeling studies of strand damage mediated by 1 are consistent with this pathway. Some metal-mediated DNA damage processes proceeding through the C1′-radical offer an alternative pathway to 2′-deoxyribonolactone lesions (29) (68). Metalloporphyrins (e.g., 36) are believed to oxidize organic substrates via metal-oxo species which hydroxylate the organic substrates via an oxygen rebound mechanism (69). In this mechanism, the oxygen bound to the

Invited Review


metal abstracts the hydrogen atom from the substrate. The resulting metal-hydroxyl group traps the initially formed carbon radical, whereby the oxygen of the metalhydroxyl group becomes associated with the oxygenated substrate. Investigation of how 36 produces 2′-deoxyribonolactone lesions (29) in DNA was carried out using 18O labeling (69). Analysis of the furanone (30) released

upon heating enabled Meunier to distinguish between the three possible reaction pathways available to the anomeric radical (Scheme 10). The incorporation of 50% 18O in the furanone (30) was fully consistent with the oxygen rebound mechanism. The most general pathway from a C1′-radical to the alkaline labile deoxyribonolactone lesion involves trapping this radical with O2. Until recently, the subsequent transformation of the peroxyl radical into the lactone lesion (29) was unclear. One viable mechanism involves reduction by thiols. In some instances, reaction of two peroxyl radicals was suggested as a pathway for lactone formation (70). However, DNA peroxyl radicals are neither freely diffusible nor likely to be formed in sufficiently high concentration to be within proximity of one another. Unimolecular decomposition of an intermediate peroxyl radical (32) via heterolytic cleavage of the C1′-oxygen bond could account for the formation of 2′deoxyribonolactone (29) lesions. In related molecules, laser flash photolysis studies utilizing conductivity detection estimated the loss of superoxide (O2-•) from 37 to be >7 × 104 s-1 (eq 7) (70). More recent experiments

Figure 1. Oxidation potentials of radicals analogous to the C1′radical of nucleosides (67).

utilizing the respective peroxyl radical derived from the dimethyl acetal of acetaldehyde 38 as a model for the C1′peroxyl radical of a nucleoside indicated that superoxide is cleaved with a rate constant of ∼6.5 × 104 s-1 at pH 5.0 (eq 8) (71). Considering the biological importance of O2-•, which in the presence of NO or redox active metal ions is transformed into more reactive agents (peroxy nitrite and hydroxyl radical, respectively) which can further damage DNA, such a process could be biologically significant (6, 72). Since the lactone (29) is still produced at the original site of the radical (27), this process represents an example of DNA damage amplification. Indeed, qualitative evidence for superoxide production from 32 was gleaned from the epinephrine spectrophotometric assay (eq 4) which results in the formation of adrenochrome (26) (55). Quenching of the formation of 26 in the presence of superoxide dismutase supported the proposal that superoxide is responsible for the oxidation of epinephrine (25). A more quantitative indirect analysis of superoxide release from 32 was provided by isotopic labeling. When 31 was irradiated in H218O under aerobic (16O2) conditions, the ratio of [16O]29:[18O]29 varied linearly with the concentration of β-mercaptoethanol (55). If it is assumed that the bimolecular trapping rate constant by thiol is between 5 × 103 and 5 × 104 M-1 s-1, the rate of loss of superoxide is estimated to be >1.4 s-1. These relative rates result in only 4% of 32 eliminating O2-• in the presence of 5 mM β-mercaptoethanol. However, in light of recent estimates for GSH trapping of DNA peroxyl radicals (108 s-1. These experiments were corroborated by studies on a series of bis-phosphates (e.g., 55) in which the ratio of kelim/kT was determined by measuring the ratio of 58:57 (Scheme 15) (91). The increase in kelim/kT with increasing solvent polarity signifies an even greater increase in kelim than originally assumed, because hydrogen atom transfer from thiols to alkyl radicals is accelerated in polar protic solvents (92). In addition, experiments involving 55 demonstrate that

the 3′-phosphate cleaves faster than the 5′-phosphate (88). Expansion of the scope of these model studies revealed previously unrecognized structural effects on the rate constant of the fragmentation process. The formal phosphate elimination is correlated with the basicity of the nucleobase (93). Also, the retarding effect on the rate constant for phosphate elimination by an inductively electron-withdrawing alkoxy group at the C2′-position of a nucleotide implies that C4′-hydrogen atom abstraction in RNA may result in quite different levels and types of damage (94). The above experiments are consistent with, but do not necessitate, the formation and trapping of an olefin cation radical. The stereochemistry of the substitution products observed by Giese can be rationalized by either an SRN2 reaction (involving attack by the pendant nucleobase, 59, eq 12) or the steric influence of the nucleobase on the reactivity of the cation radical formed via an SRN1 pathway. Credibility was provided to the former mech-

anism by computational studies (95). However, product studies using diastereomeric C-nucleosides that are incapable of providing anchimeric assistance (SRN2) support the SRN1 pathway (eq 13) (96). Diastereomeric

radical precursors (60 and 61) yield identical product mixtures upon photolysis, indicating that they proceed through a common intermediate (62). In contrast, laser flash photolysis experiments on systems that lack heteroatom substitution of the radical cation show no evidence of discrete olefin cation radical formation (97). Instead, the product radicals resulting from formal


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Scheme 16

nucleophilic trapping of the unobserved transient species were found to increase in a manner consistent with bimolecular attack on the initially formed radical. These results were in contrast to a related study in which olefin cation radicals were detected during laser flash photolysis studies (98). The relevance of these recent model studies to DNA is uncertain, because the substrates did not contain polarizable substituents similar to those present in nucleic acids. In addition, one should note that bimolecular substitution of the phosphate diester by water via an SRN2 mechanism is inconsistent with the stereochemical results obtained by Giese in nucleotide model systems, as well as CIDNP experiments on other model compounds (96, 99). Clarification and adjudication of the disparate observations obtained by these groups await further experimentation. 4.1.2.2. Oligonucleotide Studies. The above model studies have been extended to single- and doublestranded oligonucleotides by independently generating the C4′-radicals at defined sites within biopolymers from photochemical precursors. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry and HPLC have provided qualitative and quantitative product analysis during these experiments (100, 101). Comparable observations were made utilizing oligonucleotides containing either a phenyl selenide (63) or tert-butyl ketone (64) as precursors for the C4′-radical (102). Irradiation of 63 under anaerobic conditions gave

rise to 68 and 70, presumably via 65 and 67 (Scheme 16). Two additional products (66 and 69), which could be attributed to trapping 65 and 67, respectively, were observed when 63 was irradiated in the presence of glutathione. When independently generated C4′-radicals (from 63 or 64) were studied under aerobic conditions, as expected, 3′-phosphoglycolate termini (45, eq 11) were formed (102, 103). Trapping of 71 by O2 yielded hydroperoxide 72, which by analogy to bleomycin (4)-mediated DNA damage was suggested to undergo a Criegeé rearrangement en route to the respective 3′-phosphoglycolate product. However, a second hydroperoxide (74) observed was at-

tributed to hydration of 73 (Scheme 17). 3′-Phosphoglycolate (45) and base propenal (46) products are believed to result from 74 via a Grob fragmentation (103). The plausibility of the Grob fragmentation was supported by examination of the decomposition of model compound 75 under mild aqueous conditions, and is consistent with 18O labeling experiments, in which it was shown that one of the glycolate oxygens was derived from O2, and neither can be attributed to H2O (102, 103).

The viability of the Grob fragmentation pathway under biologically relevant conditions is dependent upon the competition between (formal) formation of the olefin cation radical and trapping of the C4′-nucleotide radical by O2. When 44 was produced within biopolymers containing 63 or 64, competition studies involving glutathione as a trap for the initially generated nucleoside radical yielded approximate values for the elimination process of 1 × 103 and 1 × 102 s-1 in single-stranded and double-stranded DNA, respectively (101). These firstorder processes are too slow to compete with trapping of the C4′-nucleotide radical by O2. However, Giese recently observed that O2 trapping of the C4′-nucleotide radical is reversible. Hence, due to this unfavorable equilibrium, DNA damage may be funneled through the olefin cation pathway at low thiol concentrations (104). The olefin cation radical/Grob fragmentation is a viable mechanism for the formation of direct strand breaks in the absence of physiological levels of thiols or bleomycin. However, there is good reason to believe that cleavage by bleomycin (4) follows a mechanism different than that proposed by Schulte-Frohlinde and documented by Giese. Chief among these differences was the observation that DNA cleavage by bleomycin proceeds with release of a proton from the 2′-pro-R position of the deoxyribose undergoing oxidation prior to strand scission (83). In contrast, the Grob fragmentation pathway to 3′-phosphoglycolate produces strand breaks prior to loss of a proton from the C2′-position. In addition, cleavage induced via olefin cation radical formation is independent of O2, whereas bleomycin-mediated strand scission requires O2. Consequently, the presence of bleomycin in the minor groove of DNA upon C4′-hydrogen atom abstraction may result in a bifurcation of the reactivity of the incipient radical. Under aerobic conditions, this bifurcation could involve the proposed Criegeé rearrangement. While the involvement of metal ions in this rearrangement remains unproven, their role in DNA chemistry has precedent. For instance, oxidation of the C1′-nucleotide

Invited Review


Scheme 18

radical by Cu(OP)2 is consistent with the incorporation of 18O from water in lactone-derived products (68). The C4′-nucleotide radical should also be a good reducing radical, and recently, Mn(OAc)3 was shown to oxidize this type of radical in a model compound (105). 4.2. Formation of Alkaline Labile Lesions. Metalmediated oxidation of the C4′-nucleotide radical is relevant to the formation of alkaline labile lesions by molecules such as bleomycin. Isotopic labeling studies involving bleomycin (4) revealed that the source of the oxygen atom is consistent with a major pathway involving trapping of a C4′-carbocation (76) by water (Scheme 18) (82). Minor amounts of the alkaline labile lesion (47) are formed containing 18O in a manner that is consistent with trapping of the C1′-carbocation (77). The C1′carbocation is proposed to result from β-cleavage of the C4′-radical (44), followed by oxidation. Recent studies on independently generated C4′-nucleoside radicals provide support for the initial radical β-fragmentation step (eq 14) (106). However, one cannot rule out reversing the

order of oxidation and ring opening, which would give rise to 77 from 76. 4.3. Using Independently Generated Reactive Intermediates To Probe Electron Transfer in DNA. The studies outlined above address fundamental questions regarding the reactivity of reactive intermediates that are produced in oligonucleotides by DNA-damaging agents. Independent generation of reactive intermediates

at defined sites in oligonucleotides can also be used as mechanistic probes for other physical phenomena in biopolymers. The rate of electron transfer through DNA is an important and contentious issue. In contrast to proteins, where electron transfer rates show an approximate 1 order of magnitude dependence for every 1.5-2.5 Å, the degree of dependence of electron transfer rates on distance in DNA is highly dependent upon the system studied (107-109). In some cases, little or no dependence is observed. This has been attributed to the π-stack in duplex DNA acting as a conduit for electron transfer. However, other systems have been prepared in which the distance dependence of electron transfer rates comparable to those in proteins is observed. All of these studies have utilized metal complexes or incorporation of other nonnative components in DNA to initiate photoinduced electron transfer. Recently, Giese probed the distance dependence of electron transfer rates in nucleic acids by taking advantage of chemistry developed in his laboratories for generating C4′-nucleotide radicals in oligonucleotides (and ultimately olefin cation radicals) from 64 at defined sites in oligonucleotides (110). The olefin cation radicals produced by phosphate elimination from these radicals are strong enough one-electron oxidants to oxidize guanosine bases. Using the reduced enol ether (78, eq 15) as an indicator for electron transfer, the relative rates of electron transfer versus chemical trapping of the olefin cation radical were determined in a series of oligonucleotides. The oligonucleotide sequences differed with re-

spect to the distance between the olefin cation radical and the nearest deoxyguanosine. No distance dependence on electron transfer rates was detected in single-stranded oligonucleotides. This was interpreted to mean that the single-stranded biopolymer was sufficiently flexible so as to enable deoxyguanosines and olefin cation radicals that are separated by varying numbers of nucleotides to interact with one another. In contrast, the natural log of the electron transfer rate showed a linear dependence on distance in more rigid duplex DNA. The slope of the line delineating this dependence (β) was comparable to that observed in proteins (∼1.0), and was interpreted to mean that electron transfer through DNA to the nucleotide olefin cation radical does not benefit from π-stacking.

5. Conclusions The study of reaction mechanisms by independently generating reactive intermediates is an approach traditionally pursued by organic chemists, which only recently has been applied to problems associated with nucleic acid


damage. However, in the less than 10 years since the first report of such a study, this scientific approach has elucidated the reaction mechanisms of some nucleic acid damage processes, revealed others to be even more complicated than originally thought, and shown itself to be useful for probing indirectly related processes. This approach will undoubtedly continue to be a useful tool for elucidating previously observed and uncovering unknown nucleic acid damage processes.

Acknowledgment. Financial support of this work from the National Institutes of Health (GM-54996) and the Alfred P. Sloan Foundation is greatly appreciated. I am grateful to Professors Bernd Giese and Martin Newcomb for sharing results from their laboratories prior to publication and for helpful discussions. I especially thank Keri Tallman for carefully reading the manuscript.

References (1) von Sonntag, C. (1987) The chemical basis of radiation biology, Taylor & Francis, London. (2) Pogozelski, W. K., and Tullius, T. D. (1998) Oxidative strand scission of nucleic acids: routes initiated by hydrogen abstraction from the sugar moiety. Chem. Rev. 98, 1089-1107. (3) Burrows, C. J., and Muller, J. G. (1998) Oxidative nucleobase modifications leading to strand scission. Chem. Rev. 98, 11091151. (4) Morgan, W. F., Day, J. P., Kaplan, M. I., McGhee, E. M., and Limoli, C. L. (1996) Genomic instability induced by ionizing radiation. Radiat. Res. 146, 246-258. (5) von Sonntag, C. (1994) Radiation chemistry in the 1990s: pressing questions relating to the areas of radiation biology and environmental research. Int. J. Radiat. Biol. 65, 19-26. (6) von Sonntag, C. (1994) Topics in free radical-mediated DNA damage: purines and damage amplification-superoxic reactionsbleomycin, the incomplete radiomimetic. Int. J. Radiat. Biol. 66, 485-490. (7) Budzinski, E. E., Dawidzik, J. B., Rajecki, M. J., Wallace, J. C., Schroder, E. A., and Box, H. C. (1997) DNA damage: tandem lesions. Int. J. Radiat. Biol. 71, 327-336. (8) Wang, W., Razskazovskii, Y., and Sevilla, M. D. (1997) Secondary radical attack on DNA nucleotides: reaction by addition to DNA bases and abstraction from sugars. Int. J. Radiat. Biol. 71, 387399. (9) Frankenberg-Schwager, M., and Frankenberg, D. (1990) DNA double-strand breaks: their repair and relationship to cell killing in yeast. Int. J. Radat. Biol. 58, 569-575. (10) Sigman, D. S., Mazumder, A., and Perrin, D. M. (1993) Chemical nucleases. Chem. Rev. 93, 2295-2316. (11) Dervan, P. B. (1992) Characterization of Protein-DNA Complexes by Affinity Cleaving. Methods Enzymol. 208, 497-516. (12) Goldberg, I. H. (1991) Mechanism of neocarzinostatin action: role of DNA microstructure in determination of chemistry of bistranded oxidative damage. Acc. Chem. Res. 24, 191-198. (13) Stubbe, J., and Kozarich, J. W. (1987) Mechanisms of bleomycininduced DNA degradation. Chem. Rev. 87, 1107-1136. (14) Stubbe, J., Kozarich, J. W., Wu, W., and Vandewall, D. E. (1996) Bleomycins: a structural model for specificity, binding, and double strand cleavage. Acc. Chem. Res. 29, 322-330. (15) Dixon, W. J., Hayes, J. J., Levin, J. R., Weidner, M. F., Dombroski, B. A., and Tullius, T. D. (1992) Hydroxyl radical footprinting. Methods Enzymol. 208, 380-414. (16) Huang, H., Zhu, L., Reid, B. R., Drobny, G. P., and Hopkins, P. B. (1995) Solution structure of a cisplatin-induced DNA interstrand cross-link. Science 270, 1842-1845. (17) Tomasz, M., Lipman, R., Chowdary, D., Pawlak, J., Verdine, G. L., and Nakanishi, K. (1987) Isolation and structure of a covalent cross-link adduct between mitomycin C and DNA. Science 235, 1204-1208. (18) Boger, D. L. (1995) The duocarmycins: synthetic and mechanistic studies. Acc. Chem. Res. 28, 20-29. (19) Gajewski, E., Aruoma, O. I., Dizdaroglu, M., and Halliwell, B. (1991) Bleomycin-dependent damage to the bases in DNA is a minor side reaction. Biochemistry 30, 2444-2448. (20) Dizdaroglu, M., Aruoma, O. I., and Halliwell, B. (1990) Modification of bases in DNA by copper ion-1,10-phenanthroline complexes. Biochemistry 29, 8447-8451.

Greenberg (21) Greenberg, M. M., and Matray, T. J. (1997) Inhibition of klenow fragment (exo-) catalyzed DNA polymerization by (5R)-5,6dihydro-5-hydroxythymidine and structural analogue 5,6-dihydro5-methylthymidine. Biochemistry 36, 14071-14079. (22) Hayes, R. C., Petrullo, L. A., Huang, H., Wallace, S. S., and LeClerc, J. E. (1988) Oxidative damage in DNA. Lack of mutagenicity by thymine glycol lesions. J. Mol. Biol. 201, 239-246. (23) Clark, J. M., and Beardsley, P. (1987) Functional effects of cisthymine glycol lesions on DNA synthesis in vitro. Biochemistry 26, 5398-5403. (24) Maccabee, M., Evans, J. S., Glackin, M. P., Hatahet, Z., and Wallace, S. S. (1994) Pyrimidine ring fragmentation products. Effects of lesion structure and sequence context on mutagenesis. J. Mol. Biol. 236, 514-530. (25) Karam, L. R., Dizdaroglu, M., and Simic, M. G. (1988) Intramolecular H atom abstraction from the sugar moiety by thymine radicals in oligo- and polydeoxynucleotides. Radiat. Res. 116, 210-216. (26) Lemaire, D. G. E., Bothe, E., and Schulte-Frohlinde, D. (1984) Yields of radiation-induced main chain scission of poly(U) in aqueous solution: strand break formation via base radicals. Int. J. Radiat. Biol. 45, 351-358. (27) Deeble, D. J., and von Sonntag, C. (1984) γ-Radiolysis of poly(U) in aqueous solution. The role of primary sugar and base radicals in the release of undamaged uracil. Int. J. Radiat. Biol. 46, 247260. (28) Deeble, D. J., Schulz, D., and von Sonntag, C. (1986) Reactions of OH radicals with poly(U) in deoxygenated solutions: sites of OH radical attack and the kinetics of base release. Int. J. Radiat. Biol. 49, 915-926. (29) Hildenbrand, K., and Schulte-Frohlinde, D. (1989) ESR studies on the mechanism of hydroxyl radical-induced strand breakage of polyuridylic acid. Int. J. Radiat. Biol. 55, 725-738. (30) Jones, G. D. D., and O’Neill, P. (1991) Kinetics of radiationinduced strand break formation in single-stranded pyrimidine polynucleotides in the presence and absence of oxygen; a timeresolved light-scattering study. Int. J. Radiat. Biol. 59, 11271145. (31) Hildenbrand, K., Behrens, G., and Schulte-Frohlinde, D. (1989) Comparison of the reaction of •OH and of SO4- radicals with pyrimidine nucleosides. An electron spin resonance study in aqueous solution. J. Chem. Soc., Perkin Trans. 2, 283-289. (32) Barvian, M. R., and Greenberg, M. M. (1992) Independent generation of the major adduct of hydroxyl radical and thymidine. Examination of intramolecular hydrogen atom transfer in competition with thiol trapping. Tetrahedron Lett. 33, 6057-6060. (33) Barvian, M. R., Barkley, R. M., and Greenberg, M. M. (1995) Reactivity of 5,6-dihydro-5-hydroxythymid-6-yl generated via photoinduced single electron transfer and the role of cyclohexa1,4-diene in the photodeoxygenation process. J. Am. Chem. Soc. 117, 4894-4904. (34) Dizdaroglu, M. (1990) Gas chromatography-mass spectrometry of free radical-induced products of pyrimidines and purines. Methods Enzymol. 193, 842-857. (35) Colson, A.-O., and Sevilla, M. D. (1995) Structure and relative stability of deoxyribose radicals in a model DNA backbone: ab initio molecular orbital calculations. J. Phys. Chem. 99, 38673874. (36) Miaskiewicz, K., and Osman, R. (1994) Theoretical study on the deoxyribose radicals formed by hydrogen abstraction. J. Am. Chem. Soc. 116, 232-238. (37) Schulte-Frohlinde, D., Opitz, J., Gorner, H., and Bothe, E. (1985) Model studies for the direct effect of high-energy irradiation on DNA. Mechanism of strand-break formation induced by laser photoionization of poly(U) in aqueous solution. Int. J. Radiat. Biol. 48, 397-408. (38) Wolf, P., Jones, G. D. D., Candeias, L. P., and O’Neill, P. (1993) Induction of strand breaks in polyribonucleotides and DNA by the sulphate radical anion: role of electron loss centres as precursors of strand breakage. Int. J. Radiat. Biol. 64, 7-18. (39) Schulte-Frohlinde, D., and Hildenbrand, K. (1989) Electron spin resonance studies of the reactions of •OH and SO4•- radicals with DNA, polynucleotides and single base model compounds. Free Radicals Synth. Biol., 1989, 335-359. (40) Osman, R., Pardo, L., Banfelder, J., Mazurek, A. P., Shvartzman, L., Strauss, R., and Weinstein, H. (1991) Molecular mechanisms of radiation induced DNA damage: H-addition to bases, direct ionization and double strand break. Free Radical Res. Commun. 12-13, 465-467. (41) Symons, M. C. R. (1987) Application of electron spin resonance spectroscopy to the study of the effects of ionising radiation on DNA and DNA complexes. J. Chem. Soc., Faraday Trans. 1 83, 1-11.

Invited Review (42) Cullis, P. M., Langman, S., Podmore, I. D., and Symons, M. C. R. (1990) Effects of ionising radiation on deoxyribonubleic acid. J. Chem. Soc., Faraday Trans. 1 86, 3267-3271. (43) Barvian, M. R., and Greenberg, M. M. (1995) Independent generation of 5,6-dihydrothymid-5-yl and investigation of its ability to effect nucleic acid strand scission via hydrogen atom abstraction. J. Org. Chem. 60, 1916-1917. (44) Barvian, M. R., and Greenberg, M. M. (1995) Independent generation of of 5,6-dihydrothymid-5-yl in single-stranded polythymidylate. O2 is necessary for strand scission. J. Am. Chem. Soc. 117, 8291-8292. (45) Greenberg, M. M., Barvian, M. R., Cook, G. P., Goodman, B. K., Matray, T. J., Tronche, C., and Venkatesan, H. (1997) DNA damage induced via 5,6-dihydrothymid-5-yl in single-stranded oligonucleotides. J. Am. Chem. Soc. 119, 1828-1839. (46) Matray, T. J., and Greenberg, M. M. (1994) Site-specific incorporation of the alkaline labile, oxidative stress product (5R)-5,6dihydro-5-hydroxythymidine in an oligonucleotide. J. Am. Chem. Soc. 116, 6931-6932. (47) Venkatesan, H., and Greenberg, M. M. (1996) Improved utility of photolabile solid phase synthesis supports for the synthesis of oligonucleotides containing 3′-hydroxyl termini. J. Org. Chem. 61, 525-529. (48) Schulte-Frohlinde, D., and Bothe, E. (1983) Indentification of a major pathway of strand break formation in poly U induced by OH radicals in the presence of oxygen. Z. Naturforsch. 39c, 315319. (49) Bothe, E., Behrens, G., Bohm, E., Sethuram, B., and SchulteFrohlinde, D. (1986) Hydroxyl radical-induced strand break formation of poly(U) in the presence of oxygen: comparison of the rates as determined by conductivity, ESR and rapid-mix experiments with a thiol. Int. J. Radiat. Biol. 49, 57-66. (50) Jones, G. D. D., and O’Neill, P. (1990) The kinetics of radiationinduced strand breakage in polynucleotides in the presence of oxygen: a time-resolved light-scattering study. Int. J. Radiat. Biol. 57, 1123-1139. (51) Schulte-Frohlinde, D., Behrens, G., and Onal, A. (1986) Lifetime of peroxyl radicals of poly(U), poly(A) and single-and doublestranded DNA and the rate of their reaction with thiols. Int. J. Radiat. Biol. 50, 103-110. (52) Adinarayana, M., Bothe, E., and Schulte-Frohlinde, D. (1988) Hydroxyl radical-induced strand break formation in singlestranded polynucleotides and single-stranded DNA in aqueous solution as measured by light scattering and by conductivity. Int. J. Radiat. Biol. 54, 723-737. (53) Deeble, D. J., and von Sonntag, C. (1986) Radiolysis of poly(U) in oxygenated solution. Int. J. Radiat. Biol. 49, 927-936. (54) Tronche, C., Goodman, B. K., and Greenberg, M. M. (1998) DNA damage induced via independent generation of the radical resulting from formal hydrogen atom abstraction from the C1′-position of a nucleotide. Chem. Biol. 5, 263-271. (55) Tallman, K. A., Tronche, C., Yoo, D. J., and Greenberg, M. M. (1998) Release of superoxide from nucleoside peroxyl radicals, a double-edged sword? J. Am. Chem. Soc. 120, 4903-4909. (56) Sugiyama, H., Tsutsumi, Y., and Saito, I. (1990) Highly sequence selective photoreaction of 5-bromouracil-containing deoxyhexanucleotides. J. Am. Chem. Soc. 112, 6720-6721. (57) Ide, H., Yamaoka, T., and Kimura, Y. (1994) Replication of DNA templates containing the R-Anomer of deoxyadenosine, a major adenine lesion produced by hydroxyl radicals. Biochemistry 33, 7127-7133. (58) Lesiak, K. B., and Wheeler, K. T. (1990) Formation of R-deoxyadenosine in polydeoxynucleotides exposed to ionizing radiation under anoxic conditions. Radiat. Res. 121, 328-337. (59) Goodman, B. K., and Greenberg, M. M. (1996) Independent generation and reactivity of 2′-deoxyurid-1′-yl. J. Org. Chem. 61, 2-3. (60) Gimisis, T., Ialongo, G., and Chatgilialoglu, C. (1998) Generation of C-1′ radicals through a β-(acyloxy)alkyl rearrangement in modified purine and pyrimidine nucleosides. Tetrahedron 54, 573-592. (61) Chatgilialoglu, C., Gimisis, T., Guerra, M., Ferreri, C., Emanuel, C. J., Horner, J. W., Newcomb, M., Lucarini, M., and Pedulli, G. F. (1998) Spectra and structure of the 2′-deoxyuridin-1′-yl radical. Tetrahedron Lett. 39, 3947-3950. (62) Weiland, B., Huttermann, J., Malone, M. E., and Cullis, P. M. (1996) Formation of C1′ located sugar radicals from X-irradiated cytosine nucleosides and -tides in BeF2 glasses and frozen aqueous solutions. Int. J. Radiat. Biol. 70, 327-336. (63) Decarroz, C., Wagner, J. R., and Cadet, J. (1987) Specific deprotonation reactions of the pyrimidine radical cation resulting from the menadione mediated photosensitization of 2′-deoxycytidine. Free Radical Res. Commun. 2, 295-301.

Chem. Res. Toxicol., Vol. 11, No. 11, 1998 1247 (64) Hildenbrand, K., and Schulte-Frohlinde, D. (1997) Time-resolved EPR studies on the reaction rates of peroxyl radicals of poly(acrylic acid) and of calf thymus DNA with glutathione. Reexamination of a rate constant for DNA. Int. J. Radiat. Biol. 71, 377-385. (65) Neyhart, G. A., Cheng, C.-C., and Thorp, H. H. (1995) Kinetics and mechanism of the oxidation of sugars and nucleotides by oxoruthenium(IV): model studies for predicting cleavage patterns in polymeric DNA and RNA. J. Am. Chem. Soc. 117, 1463-1471. (66) Cheng, C.-C., Goll, J. G., Neyhart, G. A., Welch, T. W., Singh, P., and Thorp, H. H. (1995) Relative rates and potentials of competing redox processes during DNA cleavage: oxidation mechanisms and sequence-specific catalysis of the self-inactivation of oxometal oxidants by DNA. J. Am. Chem. Soc. 117, 2970-2980. (67) Wayner, D. D. M., and Griller, D. (1989) Electrochemistry of transient free radicals. Mol. Struct. Energ. 11, 109-128. (68) Meijler, M. M., Zelenko, O., and Sigman, D. S. (1997) Chemical mechanism of DNA scission by (1,10-phenanthroline)copper. Carbonyl oxygen of 5-methylenefuranone is derived from water. J. Am. Chem. Soc. 119, 1135-1136. (69) Pitie, M., Bernadou, J., and Meunier, B. (1995) Oxidation at Carbon-1′ of DNA deoxyriboses by the Mn-TMPyP/KHSO5 System results from a cytochrome P-450-type hydroxylation reaction. J. Am. Chem. Soc. 117, 2935-2936. (70) von Sonntag, C., and Schuchmann, H.-P. (1991) The elucidation of peroxyl radical reactions in aqueous solution with the help of radiation-chemical methods. Angew. Chem., Int. Ed. 30, 12291253. (71) Schuchmann, M. N., Schuchmann, H.-P., and von Sonntag, C. (1990) Hydroxyl radical induced oxidation of acetaldehyde dimethyl acetal in oxygenated aqueous solution. Rapid O2•- release from the CH3C(OCH3)2O2• radical. J. Am. Chem. Soc. 112, 403407. (72) Tamir, S., Burney, S., and Tannenbaum, S. R. (1996) DNA Damage by Nitric Oxide. Chem. Res. Toxicol. 9, 821-827. (73) Zelenko, O., Gallagher, J., and Sigman, D. S. (1997) Scission of DNA with bis(1,10-phenanthroline)copper without intramolecular hydrogen migration. Angew. Chem., Int. Ed. 36, 2776-2778. (74) Chen, T., and Greenberg, M. M. (1998) Model studies indicate that copper phenanthroline induces direct strand breaks via β-elimination of the 2′-deoxyribonolactone intermediate observed in enediyne mediated DNA damage. J. Am. Chem. Soc. 120, 3815-3816. (75) McHugh, P. J., and Knowland, J. (1995) Novel reagents for chemical cleavage at abasic sites and UV photoproducts in DNA. Nucleic Acids Res. 23, 1664-1670. (76) Matsumoto, Y., and Kim, K. (1995) Excision of deoxyribose phosphate residues by DNA polymerase β during DNA repair. Science 269, 699-702. (77) Mazumder, A., Gerlt, J. A., Rabow, L., Absalon, M. J., Stubbe, J., and Bolton, P. H. (1989) UV endonuclease V from bacteriophage T4 catalyzes DNA strand cleavage at aldehydic abasic sites by a syn β-elimination reaction. J. Am. Chem. Soc. 111, 80298030. (78) Mazumder, A., Gerlt, J. A., Absalon, M. J., Stubbe, J., Cunningham, R. P., Withka, J., and Bolton, P. H. (1991) Stereochemical studies of the β-elimination reactions at aldehydic abasic sites in DNA: endonuclease III from Escherichia coli, sodium hydroxide and lys-trp-lys. Biochemistry 30, 1119-1126. (79) Sugiyama, H., Xu, C., Murugesan, N., and Hecht, S. M. (1985) Structure of the Alkali-labile product formed during iron(II)bleomycin-mediated DNA strand scisson. J. Am. Chem. Soc. 107, 4104-4105. (80) Kozarich, J. W., Worth, L., Jr., Frank, B. L., Christner, D. F., Vanderwall, D. E., and Stubbe, J. (1989) Sequence-specific isotope effects on the cleavage of DNA by bleomycin. Science 245, 13961399. (81) Rabow, L. E., Stubbe, J., and Kozarich, J. W. (1990) Identification and quantitation of the lesion accompanying base release in bleomycin-mediated DNA degradation. J. Am. Chem. Soc. 112, 3196-3203. (82) Rabos, L. E., McGall, G. H., Stubbe, J., and Kozarich, J. W. (1990) Identification of the source of oxygen in the alkaline-labile product accompanying cytosine release during bleomycin-mediated oxidative degradation of d(CGCGCG). J. Am. Chem. Soc. 112, 32033208. (83) McGall, G. H., Rabow, L. E., Ashley, G. W., Wu, S. H., Kozarich, J. W., and Stubbe, J. (1992) New insight into the mechanism of base propenal formation during bleomycin-mediated DNA degradation. J. Am. Chem. Soc. 114, 4958-4967. (84) Absalon, M. J., Kozarich, J. W., and Stubbe, J. (1995) Sequencespecific double-strand cleavage of DNA by Fe-bleomycin. 1. The


(85) (86) (87)

(88) (89)

(90) (91) (92)

(93) (94) (95) (96) (97)

(98)

detection of sequence-specific double-strand breaks using hairpin oligonucleotides. Biochemistry 34, 2065-2075. Absalon, M. J., Wu, W., Kozarich, J. W., and Stubbe, J. (1995) Sequence-specific double-strand cleavage of DNA by Fe-bleomycin. 2. Mechanism and Dynamics. Biochemistry 34, 2076-2086. Henner, W. D., Rodriguez, L. O., Hecht, S. M., and Haseltine, W. A. (1983) γ Ray induced deoxyribonucleic acid strand breaks. J. Biol. Chem. 258, 711-713. Janicek, M. F., Haseltine, W. A., and Henner, W. D. (1985) Malondialdehyde precursors in gamma-irradiated DNA, deoxynucleotides and deoxynucleosides. Nucleic Acids Res. 13, 90119029. Dizdaroglu, M., von Sonntag, C., and Schulte-Frohlinde, D. (1975) Strand breaks and sugar release by γ-irradiation of DNA in aqueous solution. J. Am. Chem. Soc. 97, 2277-2278. Behrens, G., Koltzenburg, G., and Schulte-Frohlinde, D. (1982) Model reactions for the degradation of DNA-4′ radicals in aqueous solution. Fast hydrolysis of R-alkoxyalkyl radicals with a leaving group in β-position followed by radical rearrangement and elimination reactions. Z. Naturforsch. 37c, 1205-1227. Giese, B., Burger, J., Kang, T. W., Kesselheim, C., and Wittmer, T. (1992) J. Am. Chem. Soc. 114, 7322-7324. Giese, B., Beyrich-Graf, X., Burger, J., Kesselheim, C., Senn, M., and Schafer, T. (1993) Angew. Chem., Int. Ed. 32, 1742-1743. Tronche, C., Martinez, F. N., Horner, J. H., Newcomb, M., Senn, M., and Giese, B. (1996) Polar substituent and solvent effects on the kinetics of radical reactions with thiols. Tetrahedron Lett. 37, 5845-5848. Crich, D., and Mo, X.-S. (1997) Nucleotide C4′ radical fragmentation is base-dependent. Tetrahedron Lett. 38, 8169-8172. Crich, D., and Mo, X.-S. (1997) Nucleotide C3′,4′-radical cations and the effect of a 2′-oxygen substituent. The DNA/RNA paradox. J. Am. Chem. Soc. 119, 249-250. Zipse, H. (1994) The SRN2 pathway: a mechanistic alternative for radicals in polar media. Angew. Chem., Int. Ed. 33, 19851988. Peukert, S., and Giese, B. (1996) Radical-induced SN1 substitution reactions. Tetrahedron Lett. 37, 4365-4368. Choi, S.-Y., Crich, D., Horner, J. H., Huang, X., Martinez, F. N., Newcomb, M., Wink, D. J., and Yao, Q. (1998) Absence of diffusively free radical cation intermediates in reactions of β-(phosphatoxy)alkyl. J. Am. Chem. Soc. 120, 211-212. Cozens, F. L., O’Neill, M., Bogdanova, R., and Schepp, N. (1997) Dynamics of ionization reactions of β-substituted radicals. Substituent and solvent effects. J. Am. Chem. Soc. 119, 10652-10659.

Greenberg (99) Gugger, A., Batra, R., Rzadek, P., Rist, G., and Giese, B. (1997) Spectroscopic evidence for a radical cation as intermediate in a model reaction of the 4′-DNA radical strand cleavage. J. Am. Chem. Soc. 119, 8740-8741. (100) Giese, B., Dussy, A., Elie, C., Erdmann, P., and Schwitter, U. (1994) Synthesis and selective radical cleavage of C-4′-modified oligonucleotides. Angew. Chem., Int. Ed. 33, 1861-1863. (101) Giese, B., Dussy, A., Meggers, E., Petretta, M., and Schwitter, U. (1997) Conformation, lifetime, and repair of 4′-DNA radicals. J. Am. Chem. Soc. 119, 11130-11131. (102) Giese, B., Beyrich-Graf, X., Erdmann, P., Petretta, M., and Schwitter, U. (1995) The chemistry of single-stranded 4′-DNA radicals: influence of the radical precursor on anaerobic and aerobic strand cleavage. Chem. Biol. 2, 367-375. (103) Giese, B., Beyrich-Graf, X., Erdmann, P., Giraud, L., Imwinkelried, P., Muller, S. N., and Schwitter, U. (1995) Cleavage of single-stranded 4′-oligonucleotide radicals in the presence of O2. J. Am. Chem. Soc. 117, 6146-6147. (104) Dussy, A., Meggers, E., and Giese, B. (1998). Spontaneous cleavage of 4′-DNA radicals under aerobic conditions: apparent discrepancy between trapping rates and cleavage products. J. Am. Chem. Soc. 120, 7399-7403. (105) Beyrich-Graf, X., Muller, S. N., and Giese, B. (1998) Oxidation of 4′-deoxyribonucleoside radicals to 4′-deoxyribonucleoside cations. A model for the function of bleomycin. Tetrahedron Lett. 39, 1553-1556. (106) Crich, D., and Yao, Q. (1998) Free radical chemistry of nucleosides and nucleotides. Ring opening of C4′-radicals. Tetrahedron 54, 305-318. (107) Murphy, C. J., Arkin, M. R., Jenkins, Y., Ghatlia, N. D., Bossmann, S. H., Turro, N. J., and Barton, J. K. (1993) Longrange photoinduced electron transfer through a DNA helix. Science 262, 1025-1029. (108) Lewis, F. D., Wu, T., Zhang, Y., Letsinger, R. L., Greenfield, S. R., and Wasielewski, M. R. (1997) Distance-dependent electron transfer in DNA hairpins. Science 277, 673-676. (109) Holmlin, R. E., Danliker, P. J., and Barton, J. K. (1997) Charge Transfer through the DNA base stack. Angew. Chem., Int. Ed. 36, 2715-2730. (110) Meggers, E., Kusch, D., Spichty, M., Wille, U., and Giese, B. (1998) Electron transfer through DNA in the course of radicalinduced strand cleavage. Angew. Chem., Int. Ed. 37, 460-462.

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