Evidence for glycosidic bond rotation in a nucleobase peroxyl radical and its effect on tandem lesion formation

Nucleobase peroxyl radicals are the major reactive intermediates formed in DNA when the biopolymer is exposed to gamma-radiolysis under aerobic conditions. The major reaction pathways for the peroxyl radical (1) derived from 5,6-dihydro-2'-deoxyuridin-6-yl involve pi-bond addition to or hydrogen atom abstraction from the adjacent nucleotides to produce tandem lesions. The ability to independently generate 1 at a defined site in DNA enabled us to probe its reactivity by varying the local DNA structure. The effect of DNA structure variation reveals that 1 reacts from its syn- and anti-conformations in competition with trapping by thiol. These experiments also reveal that tandem lesions will be produced as a mixture of diastereomers, which could impact their biological effects.     

Independent generation of the 5-hydroxy-5,6-dihydrothymidin-6-yl radical and its reactivity in dinucleoside monophosphates

Hydroxyl radical is a major reactive oxygen species produced by gamma-radiolysis of water or Fenton reaction. It attacks pyrimidine bases and gives the 5-hydroxy-5,6-dihydropyrimidin-6-yl radical as the major product. Here we report the synthesis of all four stereoisomers of 5-hydroxy-6-phenylthio-5,6-dihydrothymidine (T*), which, upon 254 nm UV irradiation, give rise to the 5-hydroxy-5,6-dihydrothymidin-6-yl radical (I). We also incorporated the photolabile radical precursors into dinucleoside monophosphates d(GT*) and d(TT*) and characterized major products resulting from the 254-nm irradiation of these dinucleoside monophosphates. Our results showed that, under anaerobic conditions, the most abundant product emanating from the 254-nm irradiation of d(GT*) and d(TT*) is an abasic site lesion. Products with the thymine portion being modified to thymine glycol and 5-hydroxy-5,6-dihydrothymine were also observed. In addition, we demonstrated that radical I can attack the C8 carbon atom of its 5' neighboring guanine and give rise to a novel cross-link lesion. Moreover, LC-MS/MS results showed that gamma-radiation of d(GT) under anaerobic condition yielded the same type of cross-link lesions.     

Synthesis and analysis of oligonucleotides containing abasic site analogues

DNA damage results in the formation of abasic sites from the formal hydrolysis of the glycosidic bond (AP) and several oxidized abasic lesions. Previous studies on AP sites revealed that DNA polymerases preferentially incorporated dA opposite them in approximately 80% of the replication events in Escherichia coli. These results were consistent with the hypothesis that the AP sites are noninstructive lesions due to the absence of a Watson-Crick base whose bypass adheres to the "A-rule." Recent replication studies of the oxidized abasic lesion, 2-deoxyribonolactone (L), revealed that DNA polymerase(s) does not apply the A-rule when bypassing it and incorporates large amounts of dG opposite L. These studies suggested that abasic sites such as L do direct polymerases to selectively incorporate nucleotides opposite them. However, it was not possible to determine the structural basis for this molecular recognition from these experiments. A group of oligonucleotides containing analogues of the AP and L lesions were synthesized and characterized as probes to gain insight into the structural basis for the distinct effect of 2-deoxyribonolactone on replication. These molecules will be useful tools for studying replication in cells and in vitro.     

Product and mechanistic analysis of the reactivity of a C6-pyrimidine radical in RNA

Nucleobase radicals are the major reactive intermediates produced when hydroxyl radical reacts with nucleic acids. 5,6-Dihydrouridin-6-yl radical (1) was independently generated from a ketone precursor via Norrish Type I photocleavage in a dinucleotide, single-stranded, and double-stranded RNA. This radical is a model of the major hydroxyl radical adduct of uridine. Tandem lesions resulting from addition of the peroxyl radical derived from 1 to the 5'-adjacent nucleotide are observed by ESI-MS. Radical 1 produces direct strand breaks at the 5'-adjacent nucleotide and at the initial site of generation. The preference for cleavage at these two positions depends upon the secondary structure of the RNA and whether O(2) is present or not. Varying the identity of the 5'-adjacent nucleotide has little effect on strand scission. In general, strand scission is significantly more efficient under anaerobic conditions than when O(2) is present. Strand scission is more than twice as efficient in double-stranded RNA than in a single-stranded oligonucleotide under anaerobic conditions. Internucleotidyl strand scission occurs via β-fragmentation following C2'-hydrogen atom abstraction by 1. The subsequently formed olefin cation radical ultimately yields products containing 3'-phosphate or 3'-deoxy-2'-ketouridine termini. These end groups are proposed to result from competing deprotonation pathways. The dependence of strand scission efficiency from 1 on secondary structure under anaerobic conditions suggests that this reactivity may be useful for extracting additional RNA structural information from hydroxyl radical reactions.     

Tandem lesions are the major products resulting from a pyrimidine nucleobase radical

Nucleobase radicals (e.g., 1) are the major family of reactive intermediates formed when DNA is exposed to gamma-radiolysis. Independent generation of 1 in chemically synthesized oligonucleotides reveals that formation of this nucleobase radical under aerobic conditions results in the formation of tandem lesions approximately 65% of the time. The distribution of lesions formed with the 5'- and 3'-adjacent nucleotides is dependent upon the secondary structure of duplex DNA. Tandem lesions, which are defined as two contiguously, damaged nucleotides in a single DNA strand, are of significant biological interest. The yield of tandem lesions from 1 is much greater than was previously believed. The observations presented could have significant ramifications on how scientists interpret the effects of gamma-radiolysis on DNA.     

Independent generation and study of 5,6-Dihydro-2'-deoxyuridin-6-yl,a member of the major family of reactive intermediates formed in DNA from the effects of gamma-radiolysis

Nucleobase radicals are the major family of reactive intermediates formed when nucleic acids are exposed to gamma-radiolysis. Elucidation of their reactivity is complicated by the formation of multiple species randomly throughout the biopolymers. 5,6-Dihydro-2'-deoxyuridin-6-yl (1) was generated upon photolysis (350 nm) of the respective tert-butyl ketone (2). The radical abstracts hydrogen atoms from beta-mercaptoethanol (k = 8.8 +/- 0.5 x 10(6) M(-)(1) s(-)(1)) and 2,5-dimethyltetrahydrofuran (k = 31 +/- 2.5 M(-)(1) s(-)(1)). The latter was used as a model for the 2-deoxyribose component of DNA. The major product formed in the presence of O(2) was 6-hydroxy-5,6-dihydro-2'-deoxyuridine (11), which is believed to be formed directly from the peroxy precursor and not via elimination of superoxide. Small amounts of 2-deoxyribonolactone (13) were also formed under aerobic conditions. This product is believed to result from intramolecular hydrogen atom abstraction by the C6-peroxyl radical (14) and suggests that gamma-radiolysis may indirectly result in oxidation of the C1'-position of nucleotides, despite the inaccessibility of this hydrogen in duplex DNA.     

Separation and identification of DMPO adducts of oxygen-centered radicals formed from organic hydroperoxides by HPLC-ESR,ESI-MS and MS/MS

Many electron spin resonance (ESR) spectra of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) radical adducts from the reaction of organic hydroperoxides with heme proteins or Fe(2+) were assigned to the adducts of DMPO with peroxyl, alkoxyl, and alkyl radicals. In particular, the controversial assignment of DMPO/peroxyl radical adducts was based on the close similarity of their ESR spectra to that of the DMPO/superoxide radical adduct in conjunction with their insensitivity to superoxide dismutase, which distinguishes the peroxyl adducts from the DMPO/superoxide adduct. Although recent reports assigned the spectra suggested to be DMPO/peroxyl radical adducts to the DMPO/methoxyl adduct based on independent synthesis of the adduct and/or (17)O-labeling, (17)O-labeling is extremely expensive, and both of these assignments were still based on hyperfine coupling constants, which have not been confirmed by independent techniques. In this study, we have used online high performance liquid chromatography (HPLC or LC)/ESR, electrospray ionization-mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS) to separate and directly characterize DMPO oxygen-centered radical adducts formed from the reaction of Fe(2+) with t-butyl or cumene hydroperoxide. In each reaction system, two DMPO oxygen-centered radical adducts were separated and detected by online LC/ESR. The first DMPO radical adduct from both systems showed identical chromatographic retention times (t(R) = 9.6 min) and hyperfine coupling constants (a(N) = 14.51 G, a(H)(beta) = 10.71 G, and a(H)(gamma) = 1.32 G). The ESI-MS and MS/MS spectra demonstrated that this radical was the DMPO/methoxyl radical adduct, not the peroxyl radical adduct as was thought at one time, although its ESR spectrum is nearly identical to that of the DMPO/superoxide radical adduct. Similarly, based on their MS/MS spectra, we verified that the second adducts (a(N) = 14.86 G and a(H)(beta) = 16.06 G in the reaction system containing t-butyl hydroperoxide and a(N) = 14.60 G and a(H)(beta) = 15.61 G in the reaction mixture containing cumene hydroperoxide), previously assigned as DMPO adducts of t-butyloxyl and cumyloxyl radical, were indeed from trapping t-butyloxyl and cumyloxyl radicals, respectively.     

Study of the reaction products of flavonols with 2,2-diphenyl-1-picrylhydrazyl using liquid chromatography coupled with negative electrospray ionization tandem mass spectrometry

The products obtained after the reaction between flavonols and the stable free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH(*)) in both methanol and acetonitrile were characterized using liquid chromatography coupled with negative electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) and NMR spectroscopy. The flavonols studied were quercetin, kaempferol and myricetin. In methanol, two reaction products of oxidized quercetin were identified using LC/ESI-MS/MS and NMR. Quercetin was oxidized through a transfer of two H-atoms to DPPH(*) and subsequently incorporated either two CH(3)OH molecules or one CH(3)OH- and one H(2)O molecule giving the products 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-2,3-dimethoxy-2,3-dihydrochromen-4-one and 2-(3,4-dihydroxyphenyl)-3,3,5,7-tetrahydroxy-2-methoxy-2,3-dihydrochromen-4-one, respectively. LC/ESI-MS/MS analysis revealed that in methanol, kaempferol and myricetin also gave rise to methoxylated oxidation products similar to that identified for quercetin. Kaempferol, in addition, also exhibited products where a kaempferol radical, obtained by a transfer of one H-atom to DPPH(*), reacted with CH(3)OH through the addition of CH(3)O(*), yielding two isomeric products. When the reaction took place in acetonitrile, LC/ESI-MS/MS analysis showed that both quercetin and myricetin formed stable isomeric quinone products obtained by a transfer of two H-atoms to DPPH(*). In contrast, kaempferol formed two isomeric products where a kaempferol radical reacted with H(2)O through the addition of OH(*), i.e. similar to the reaction of kaempferol radicals with CH(3)OH.     

Oxygen-dependent DNA damage amplification involving 5,6-dihydrothymidin-5-yl in a structurally minimal system.

5,6-Dihydrothymidin-5-yl (1) was independently generated in a dinucleotide from a phenyl selenide precursor (4). Under free radical chain propagation conditions, the products resulting from hydrogen atom donation and radical-pair reaction are the major observed products in the absence of O(2). The stereoselectivity of the trapping process is dependent on the structure of the hydrogen atom donor. No evidence for internucleotidyl hydrogen atom abstraction by 1 was detected. The tandem lesion (17) resulting from hydrogen atom abstraction from the C1' position of the adjacent 2'-deoxyuridine by the peroxyl radical derived from 1 (3) is observed under aerobic conditions. The structure of this product is confirmed by independent synthesis and its transformation into a second independently synthesized product (24). Internucleotidyl hydrogen atom abstraction is effected selectively by the 5S-diastereomer of the peroxyl radical. The formation of dinucleotide 17 provides further support for the novel O(2)-dependent DNA damage amplification mechanism involving 1 reported previously (Greenberg, M. M.; et al. J. Am. Chem. Soc. 1997, 119, 1828).     

Dimethylselenide as a probe for reactions of halogenated alkoxyl radicals in aqueous solution. Degradation of dichloro- and dibromomethane

Using pulse radiolysis and steady-state gamma-radiolysis techniques, it has been established that, in air-saturated aqueous solutions, peroxyl radicals CH 2HalOO (*) (Hal = halogen) derived from CH 2Cl 2 and CH 2Br 2 react with dimethyl selenide (Me 2Se), with k on the order of 7 x 10 (7) M (-1) s (-1), to form HCO 2H, CH 2O, CO 2, and CO as final products. An overall two-electron oxidation process leads directly to dimethyl selenoxide (Me 2SeO), along with oxyl radical CH 2HalO (*). The latter subsequently oxidizes another Me 2Se molecule by a much faster one-electron transfer mechanism, leading to the formation of equal yields of CH 2O and the dimer radical cation (Me 2Se) 2 (*+). In absolute terms, these yields amount to 18% and 28% of the CH 2ClO (*) and CH 2BrO (*) yields, respectively, at 1 mM Me 2Se. In competition, CH 2HalO (*) rearranges into (*)CH(OH)Hal. These C-centered radicals react further via two pathways: (a) Addition of an oxygen molecule leads to the corresponding peroxyl radicals, that is, species prone to decomposition into H (+)/O 2 (*-) and formylhalide, HC(O)Hal, which further degrades mostly to H (+)/Hal (-) and CO. (b) Elimination of HHal yields the formyl radical H-C(*)=O with a rate constant of about 6 x 10 (5) s (-1) for Hal = Cl. In an air-saturated solution, the predominant reaction pathway of the H-C(*)=O radical is addition of oxygen. The formylperoxyl radical HC(O)OO (*) thus formed reacts with Me 2Se via an overall two-electron transfer mechanism, giving additional Me 2SeO and formyloxyl radicals HC(O)O(*). The latter rearrange via a 1,2 H-atom shift into (*)C(O)OH, which reacts with O2 to give CO2 and O2(*)(-). The minor fraction of H-C(*)=O undergoes hydration, with an estimated rate constant of k approximately 2 x 10(5) s(-1). The resulting HC(*)(OH)2 radical, upon reaction with O2, yields HCO 2H and H (+)/O2(*-). Some of the conclusions about the reactions of halogenated alkoxyl radicals are supported by quantum chemical calculations [B3LYP/6-31G(d,p)] taking into account the influence of water as a dielectric continuum [by the self-consistent reaction field polarized continuum model (SCRF=PCM) technique]. Based on detailed product studies, mechanisms are proposed for the free-radical degradation of CH 2Cl 2 and CH 2Br 2 in the presence of oxygen and an electron donor (namely, Me 2Se in this study), and properties of the reactive intermediates are discussed.     

Histone-catalyzed cleavage of nucleosomal DNA containing 2-deoxyribonolactone

Oxidized abasic sites such as 2-deoxyribonolactone (L) are produced in DNA by a variety of oxidizing agents, including potent cytotoxic antitumor natural products. 2-Deoxyribonolactone is labile under alkaline conditions, but its half-life in free DNA at pH 7.5 is approximately 1 week. Independent generation of L at defined positions within nucleosomes reveals that the histone proteins catalyze strand scission and increase the rate between 11- and ～43-fold. Mechanistic studies indicate that DNA-protein cross-links are not intermediates en route to strand scission and that C2 deprotonation is the rate-determining step. The use of mutant histone H4 proteins demonstrates that the lysine-rich tail that is often post-translationally modified in cells contributes to the cleavage of L but is not the sole source of the enhanced cleavage rates. Consideration of DNA repair in cells suggests that L formation in nucleosomal DNA as part of bistranded lesions by antitumor antibiotics results in de facto double strand breaks, the most deleterious form of DNA damage.     

Location of abasic sites in oligodeoxynucleotides by tandem mass spectrometry and by a chemical cleavage initiated by an unusual reaction of the ODN with MALDI matrix

We describe two approaches employing electrospray ionization (ESI) tandem mass spectrometry (MS/MS) and matrix assisted laser desorption/ionization (MALDI) post-source decay (PSD) for determining the location of an abasic site in modified oligodeoxynucleotides (ODNs). With MS/MS, we found both complementary fragment ions (a_n′ and w_n′) produced at the abasic site were predominant in the mass spectra and allowed the location to be determined. Under MALDI conditions, most ODNs carrying an abasic site are singly charged, and PSD gives predominately w_n′ ions at the abasic sites, revealing their location. We also describe another approach for identifying and locating abasic sites in model ODNs; namely, an “in situ” derivatization coupled with MALDI mass spectrometry (MS). In general, an ODN n-mer containing an abasic site at the m-th position from the 5′-terminus can react with the matrix component, anthranilic acid, to form a Schiff base. The adduct upon MALDI breaks into 3′ and 5′ fragments (w_n−m, b_m, a_m, d_m−1) at the abasic site, revealing its location. ESI MS methods are also applicable for detecting the hydrazone derivatives of abasic sites, and the fragmentation of hydrazones shows the location of the abasic site.     

Hydrogen bonding contributes to the selectivity of nucleotide incorporation opposite an oxidized abasic lesion

The ability of DNA polymerases to maintain the integrity of the genome even after it has been structurally altered is vital. There is considerable interest in determining the structural properties of the DNA template that polymerases recognize when determining which nucleotide to add to a nascent strand. Mechanistic, synthetic, and structural chemistries have been used to study how DNA polymerase activity is affected by size, shape, pi-stacking, and hydrogen bonds of the template molecules. Herein, we probe the structural aspects of abasic lesions that result in their distinct coding potential in Escherichia coli despite lacking a Watson-Crick base. In particular, we investigate why bypass of 2-deoxyribonolactone (L) results in significant amounts of dG incorporation opposite the lesion, whereas other abasic lesions (e.g., AP) adhere to the "A-rule". Experiments using synthetic analogues reveal that DNA polymerase V bypasses L and increased levels of dG incorporation result from a hydrogen bonding interaction between the carbonyl oxygen and dG. These results show that a DNA polymerase utilizes hydrogen bonding as one structural parameter when decoding an abasic lesion.     

Detection and characterization by mass spectrometry of radical adducts produced by linoleic acid oxidation

The formation of linoleic acid radical species under the oxidative conditions of the Fenton reaction (using hydrogen peroxide and Fe (II)) was monitored by FAB-MS and ES-MS using the spin trap 5,5-dimethyl-1-pyrrolidine-N-oxide, DMPO. Both the FAB and ES mass spectra were very similar and showed the presence of ions corresponding to carbon- and oxygen centered spin adducts (DMPO/L*, DMPO/LO*, and DMPO/LOO*). Cyclic structures, formed between the DMPO oxygen and the neighboring carbon of the fatty acid, were also observed. Electrospray tandem mass spectrometry of these ions was performed to confirm the proposed structure of these adducts. All MS/MS spectra showed an ion at m/z 114, correspondent to the [DMPO + H]+, and a fragment ion due to loss of DMPO (loss of 113 Da), confirming that they are DMPO adducts. ES-MS/MS spectra of alkoxyl radical adducts (DMPO/LO*) showed an additional ion at m/z 130 [DMPO - O + H]+, while ES MS/MS of peroxyl radical adducts (DMPO/LOO*) showed a fragment ion at m/z 146 [DMPO - OO + H]+, confirming both structures. Other fragment ions were observed, such as alkyl acylium radical ions, formed by cleavage of the alkyl chain after loss of water and the DMPO molecule. The identification of fragment ions observed in the MS/MS spectra of the different DMPO adducts suggests the occurrence of structural isomers containing the DMPO moiety both at C9 and C13. The use of ES tandem mass spectrometry, associated with spin trapping experiments, has been shown to be a valuable tool for the structural characterization of carbon and oxygen-centered spin adducts of lipid radicals.     

Self-assembly formation of the magic ion of (H2O)20O+: observation of nanoscale cages of oxygenated water clusters induced from iron nanoparticles

For the first time, we observed a stable and intense ion (m/z 376) of the oxygenated water cluster ion ((H(2)O)(20)O(+)) produced from simply spraying an aqueous solution of iron nanoparticles (Fe NPs) into an electrospray mass spectrometry (ESI-MS) system. Tandem mass spectrometric (MS/MS and MS/MS/MS) results were applied to identify the assignments of the fragment ions of m/z 376 in order to explore the possible structures of this cluster ion. The possible structures of the (H(2)O)(20)O(+) ions are proposed as pentagonal dodecahedron water clathrate cages from the results of tandem mass spectrometry since eliminations of five water molecules were frequently observed in the MS/MS results for many subsequent fragment ions of m/z 376. The formation of this oxygenated water cluster ion ((H(2)O)(20)O(+)) in ESI-MS is attributed to the high surface reactivity and surface energy of Fe NPs during ESI processes (under high temperature and high voltage (5 kV) of ESI spray environment). We believe that the observation of self-assembly formation of oxygenated water clusters is an important issue in nanoscience as well as in the fields of water clusters.     

Photochemical generation and reactivity of the major hydroxyl radical adduct of thymidine

5,6-Dihydro-5-hydroxythymidin-6-yl radical (1), the major reactive intermediate resulting from hydroxyl radical addition to C5 of the pyrimidine, is produced via 350 nm photolysis of a 2,5-dimethoxyphenylsulfide precursor (2). Competition between O(2) and thiol for 1 suggests that the radical reacts relatively slowly with β-mercaptoethanol compared to other alkyl radicals. Overall, aryl sulfide 2 should be an effective precursor for the major hydroxyl radical adduct of thymidine in DNA.     

Liquid chromatography/tandem mass spectrometry analysis of long-chain oxidation products of cardiolipin induced by the hydroxyl radical

The anionic phospholipid cardiolipin (CL) is found almost exclusively in the inner membrane of mitochondria, playing an important role in energy metabolism. Oxidation of CL has been associated with apoptotic events and various pathologies. In this study, electrospray ionization mass spectrometry coupled with liquid chromatography (LC/ESI-MS) was used to identify tetralinoleoyl-cardiolipin (TLCL) modifications induced by the OH(·) radical generated under Fenton reaction conditions (H(2)O(2) and Fe(2+)). The identified oxidation products of TLCL contained 2, 4, 6 and 8 additional oxygen atoms. These long-chain oxidation products were characterized by LC/ESI-MS/MS as doubly [M-2H](2-) and singly charged [M-H](-) ions. A detailed analysis of the fragmentation pathways of these precursor ions allowed the identification of hydroperoxy derivatives of CL. MS/MS analysis indicated that CL oxidation products with 4, 6 and 8 oxygen atoms have one fatty acyl chain bearing 4 oxygen atoms ([RCOO+4O](-)). Even when the TLCL molecule was oxidized by the addition of eight oxygen atoms, one of the acyl chains remained non-modified and one fatty acyl chain contained three or four oxygen atoms. This led us to conclude that under oxidative conditions by the OH(·) radical, the distribution of oxygens/peroxy groups in the CL molecule is not random, even when CL has the same fatty acyl chains in all the positions. Using mass spectrometry, the oxidation products have been unequivocally assigned, which may be useful for their detection in biological samples.     

Negative-ion electrospray ionization tandem mass spectrometry of N-phosphoryl amino acids and dipeptides

The negative-ions of N-phosphoryl amino acids were studied by electrospray ionization tandem mass spectrometry (ESI-MS/MS). The negative-ion ESI-MS/MS of N-phosphoryl amino acids showed characteristic fragmentation patterns different from those observed in the corresponding positive-ion ESI-MS/MS and negative-ion fast-atom bombardment mass spectra. For negative-ion ESI-MS/MS, a unique fragmentation from the N-terminal of N-phosphoryl amino acids or peptides containing a free beta-OH or CO(2)H group was observed to yield the characteristic fragment ion (RO)(2)P(O)O(-). The ease of the rearrangement depended on the position of the hydroxyl group in amino acids or peptides, and the N --> O rearrangement mechanism was proposed to involve the participation of the hydroxyl group. From previous solution-phase experiments and theoretical calculations, it was found that the beta-OH group was more active than gamma-OH, and the corresponding difference in negative-ion ESI-MS/MS was consistent with those previous findings.     

Theoretical calculations of carbon-oxygen bond dissociation enthalpies of peroxyl radicals formed in the autoxidation of lipids

Theoretical calculations were carried out to provide a framework for understanding the free radical oxidation of unsaturated lipids. The carbon[bond]hydrogen bond dissociation enthalpies (BDEs) of organic model compounds and oxidizable lipids (R[bond]H) and the carbon[bond]oxygen bond dissociation enthalpies of peroxyl radical intermediates (R[bond]OO*) have been calculated. The carbon[bond]hydrogen BDEs correlate with the rate constant for propagation of free radical autoxidation, and the carbon[bond]oxygen BDEs of peroxyl radicals correlate with rate constants for beta-fragmentation of these intermediates. Oxygen addition to intermediate carbon radicals apparently occurs preferentially at centers having the highest spin density. The calculated spin distribution therefore provides guidance about the partitioning of oxygen to delocalized carbon radicals. Where the C[bond]H BDEs are a function of the extent of conjugation in the parent lipid and the stability of the carbon radical derived therefrom, C[bond]OO* BDEs are also affected by hyperconjugation. This gives way to different rates of beta-fragmentation of peroxyl radicals formed from oxygen addition at different sites along the same delocalized radical. We have also studied by both theory and experiment the propensity for benzylic radicals to undergo oxygen addition at their ortho and para carbons which, combined, possess an equivalent unpaired electron spin density as the benzylic position itself. We find that the intermediate peroxyl radicals in these cases have negative C[bond]OO* BDEs and, thus, have rate constants for beta-fragmentation that exceed the diffusion-controlled limit for the reaction of a carbon-centered radical with oxygen.     

Direct strand scission in double stranded RNA via a C5-pyrimidine radical

Nucleobase radicals are the major family of reactive intermediates produced when nucleic acids are exposed to γ-radiolysis. The 5,6-dihydrouridin-5-yl radical (1), the formal product of hydrogen atom addition and a model for hydroxyl radical addition, was independently generated from a ketone precursor via Norrish Type I photocleavage in single and double stranded RNA. Radical 1 produces direct strand breaks at the 5'-adjacent nucleotide and only minor amounts of strand scission are observed at the initial site of radical generation. Strand scission occurs preferentially in double stranded RNA and in the absence of O(2). The dependence of strand scission efficiency from the 5,6-dihydrouridin-5-yl radical (1) on secondary structure under anaerobic conditions suggests that this reactivity may be useful for extracting additional RNA structural information from hydroxyl radical reactions. Varying the identity of the 5'-adjacent nucleotide has little effect on strand scission. Internucleotidyl strand scission occurs via β-elimination of the 3'-phosphate following C2'-hydrogen atom abstraction by 1. The subsequently formed olefin cation radical yields RNA fragments containing 3'-phosphate or 3'-deoxy-2'-ketonucleotide termini from competing deprotonation pathways. The ketonucleotide end group is favored in the presence of low concentrations of thiol, presumably by reducing the cation radical to the enol. Competition studies with thiol show that strand scission from the 5,6-dihydrouridin-5-yl radical (1) is significantly faster than from the 5,6-dihydrouridin-6-yl radical (2) and is consistent with computational studies using the G3B3 approach that predict the latter to be more stable than 1 by 2.8 kcal/mol.