Photochemistry of the Nonspecific Hydroxyl Radical Generator,<i>N</i>-Hydroxypyridine-2(1<i>H</i>)-thione

Aveline, Béatrice M.; Kochevar, and Irene E.; Redmond, Robert W.

doi:10.1021/ja961988t

Cited by 68 publications

(95 citation statements)

References 43 publications

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“…Redmond and co-workers used this reaction for the detection of • OH radicals that are formed as a result of the laser flash photolysis of N -hydroxy-2(1 H )-pyridone and N -hydroxypyridine-2(1 H )-thione 59. In the photolysis of AAPH, the formation of • OH radicals can be excluded.…”

Section: Resultsmentioning

confidence: 99%

Oxidative Modification of Guanine Bases Initiated by Oxyl Radicals Derived from Photolysis of Azo Compounds

2010

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Oxidative damage to guanine bases initiated by photolysis of the water-soluble radical generator 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) has been investigated by laser kinetic spectroscopy. In the neutral oxygenated aqueous solutions, 355 nm laser flash photolysis of AAPH generates a whole spectrum of free radicals including 2-amidinoprop-2-peroxyl (ROO • ), 2-amidinoprop-2-oxyl (RO • ), and superoxide (O 2 •− ) radicals. These oxyl radicals with negligible absorption in a near UV -visible range were monitored in the reactions leading to the products with characteristic absorption spectra. This approach reveals that RO • radicals induce fast one-electron oxidation of 2′-deoxyguanosine (dG) to form guanine neutral radicals, dG(-H) • . In contrast, ROO • radicals do not react with observable rates with dG. The O 2 •− radicals were detected using a classical test reaction with tetranitromethane to form nitroform. The major pathway for formation of the end products of guanine oxidation is combination of the G(-H) • and O 2 •− radicals to form 2,5-diamino-4H-imidazolone (Iz). This mechanism was confirmed by analysis of the end products produced by oxidation of two substrates: (1) guanosine derivative 2′,3′,5′-tri-O-acetylguanosine (tri-O-Ac-G), and (2) the 5′-d(CCATCGCTACC) sequence. The major products isolated by HPLC and identified by mass spectrometry methods were the tri-O-Ac-Iz and 5′-d(CCATC[Iz]CTACC products.

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Section: Resultsmentioning

confidence: 99%

Oxidative Modification of Guanine Bases Initiated by Oxyl Radicals Derived from Photolysis of Azo Compounds

2010

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“…Redmond and co-workers have reported that laser photolysis of aqueous solutions of N-HPT at 355 nm produced hydroxyl radicals with quantum yields of 12-24% as determined by reaction of the hydroxyl radical with thiocyanate (23). We investigated the steady-state photolysis of dilute (1 mM) solutions of N-HPT and found that 5 min of irradiation with a lamp at 350 nm was sufficient to convert most of the N-HPT to products.…”

Section: Discussionmentioning

confidence: 99%

“…N-HPT generates several reactive intermediates upon photolysis in neutral aqueous solutions where N-HPT is largely ionized (Scheme 1; 23). They include the 2-pyridylthiyl radicals, the N-oxy-2-pyridylthiyl radicals, and a solvated electron (the latter two occurring as a result of photoionization with Φ e-) 0.05 at 355 nm) in addition to hydroxyl radicals (23). It is possible that the pyridylthiyl radicals may initiate RNA cleavage during our reactions.…”

Section: Discussionmentioning

confidence: 99%

“…Examples include alkylperoxides such as 1 (19) that undergo bond homolysis upon photolysis, yielding an alkoxyl radical and a hydroxyl radical (although these reactions may be more complex ; 20), N-oxides such as 2 (21), and N-hydroxypyridines such as N-hydroxypyridine-2(1H)-one (N-HP) (22) and N-hydroxypyridine-2(1H)-thione (N-HPT) (23) that undergo N-O bond homolysis (eq 1). Little is known about either the interactions of these compounds with nucleic acids or the chemistry resulting from their irradiation in the presence of nucleic acids.…”

Section: +mentioning

confidence: 99%

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Studies of RNA Cleavage by Photolysis of N-Hydroxypyridine-2(1H)-thione. A New Photochemical Footprinting Method

2000

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N-Hydroxypyridine-2(1H)-thione (N-HPT) has been studied as a photochemical source of hydroxyl radicals for use in photoinitiated nucleic acid footprinting experiments. Steady-state photolysis of dilute aqueous solutions of N-HPT at 350 nm in the presence of a 385 nucleotide 32 P-labeled RNA, the Tetrahymena L-21 ribozyme, resulted in cleavage of the RNA at nucleotide resolution. No cleavage of the RNA occurred in the absence of light or in the absence of N-HPT. Photolysis of the analogous pyridine lacking the N-hydroxyl group did not result in detectable amounts of RNA cleavage. The addition of RNA to preirradiated solutions of N-HPT gave no apparent RNA cleavage products, suggesting that the photoproducts of N-HPT do not result in RNA modification. Cleavage of RNA, upon photolysis in the presence of N-HPT, occurred in a sequence-independent fashion with double-stranded RNA being cleaved as efficiently as single-stranded RNA. Based on these observations, we conclude that photochemically generated diffusable hydroxyl radicals are responsible for the RNA cleavage. Experiments involving the photolysis of N-HPT in the presence of the Tetrahymena ribozyme and magnesium showed a magnesiumdependent protection from RNA cleavage due to formation of a folded RNA tertiary structure. The locations and amount of protection were identical to those observed in footprinting experiments performed with other hydroxyl radical sources. The presence of N-HPT had no effect on either the rate of folding or the catalytic activity of the folded RNA, indicating that this reagent does not disrupt RNA tertiary structure or otherwise affect activity. Thus, N-HPT is established as a new reagent for use in photoinitiated RNA footprinting experiments.Hydroxyl radicals are important reactive species which have been widely used in studies of higher order nucleic acid structure and complexes formed between small molecules or proteins and nucleic acids (1, 2). Initial hydrogen abstraction from the ribose or the deoxyribose moiety is followed by a reaction cascade which results in cleavage of the phosphodiester backbone. Sequence-independent cleavage of both single-stranded and double-stranded DNA and RNA molecules can be analyzed at nucleotide resolution by denaturing polyacrylamide gel electrophoresis (PAGE) 1 of 32 P-labeled molecules. The formation of either higher order structure or specific complexes can lead to protection from such cleavage. A region of reduced cleavage, usually detected by denaturing PAGE, is referred to as a footprint. Footprinting experiments have typically been used to provide structural information on systems at equilibrium. When it is possible to measure footprints as a function of time, it then becomes possible to make inferences about rates and mechanisms of dynamic processes of which RNA folding is one example.

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“…Some photochemical sources of oxyl radicals, which display DNA oxidizing activity (strand breaks and base modification under relatively mild conditions), have been reported during the last decade (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13). N-hydroxy-and N-alkoxypyridinethiones 1 (Chart 1) have served as convenient oxyl radical sources to assess oxidative damage (6,7); indeed, the molecular mechanism of their photooxidative DNA damage has been studied thoroughly (14)(15)(16)(17). Although pyridinethiones 1 are readily available and serve as efficient oxyl-radical sources, their DNA damage is complicated: not only do the photoreleased oxyl radicals cause strand breaks and base modifications, but the photoproducts of the pyridinethiones display significant photosensitizing DNA damage (14).…”

Section: Introductionmentioning

confidence: 99%

N-Hydroxy-4-(4-chlorophenyl)thiazole-2(3H)-thione as a Photochemical Hydroxyl-radical Source: Photochemistry and Oxidative Damage of DNA (Strand Breaks) and 2′-Deoxyguanosine (8-oxodG Formation)¶

Adam¹,

Hartung²,

Okamoto³

et al. 2007

Photochemistry and Photobiology

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On irradiation of N‐hydroxythiazole‐2(3H)‐thione 3 at 300 nm, the photoproducts disulfide 4, bisthiazole 5 and thiazole 6 are formed. During this photolysis, hydroxyl radicals are released, which have been detected by spin trapping with 5,5‐dimethyl‐1‐pyrroline N‐oxide (DMPO), coupled with electron paramagnetic resonance spectroscopy. In the presence of supercoiled pBR322 DNA, irradiation of thiazolethione 3 induces strand breaks through the photogenerated hydroxyl‐radicals, as confirmed by control experiment with the hydroxyl‐radical scavenger isopropanol. Singlet oxygen appears not to be involved, as attested by the lack of a D2O isotope effect. During the photoreaction of thiazolethione 3 in the presence of 2′‐deoxyguanosine (dG), the latter is photooxidized (ca 10% conversion after 2 h of irradiation) to the 7,8‐dihydro‐8‐oxo‐2′‐deoxyguanosine as the main oxidation product. The dG conversion levels off after complete consumption of thiazolethione 3 and is suppressed by the addition of the hydroxyl‐radical scavenger 2,6‐di‐tert‐butylcresol or DMPO. Since the photoproducts 4–6 are ineffective as sensitizers for the photooxidation of dG and DNA, the hydroxyl radicals released in the photolysis of thiazolethione 3 are the oxidizing species of DNA and dG. These results suggest that the thiazolethione 3 may serve as a novel and effective photochemical hydroxyl‐radical source for photobiological studies.

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Photochemistry of the Nonspecific Hydroxyl Radical Generator,N-Hydroxypyridine-2(1H)-thione

Cited by 68 publications

References 43 publications

Oxidative Modification of Guanine Bases Initiated by Oxyl Radicals Derived from Photolysis of Azo Compounds

Oxidative Modification of Guanine Bases Initiated by Oxyl Radicals Derived from Photolysis of Azo Compounds

Studies of RNA Cleavage by Photolysis of N-Hydroxypyridine-2(1H)-thione. A New Photochemical Footprinting Method

N-Hydroxy-4-(4-chlorophenyl)thiazole-2(3H)-thione as a Photochemical Hydroxyl-radical Source: Photochemistry and Oxidative Damage of DNA (Strand Breaks) and 2′-Deoxyguanosine (8-oxodG Formation)¶

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