Deoxyguanosine Adduct Yields a Reactive Catechol - American

Article pubs.acs.org/crt

Hydroxyl Radical-Induced Oxidation of a Phenolic C-Linked 2′Deoxyguanosine Adduct Yields a Reactive Catechol Aaron A. Witham, Daniel G. Beach, Wojciech Gabryelski, and Richard A. Manderville* Departments of Chemistry and Toxicology, University of Guelph, Guelph, Ontario, Canada N1G 2W1 S Supporting Information *

ABSTRACT: Phenolic toxins stimulate oxidative stress and generate Clinked adducts at the C8-site of 2′-deoxyguanosine (dG). We previously reported that the C-linked adduct 8-(4″-hydroxyphenyl)-dG (p-PhOHdG) undergoes oxidation in the presence of Na2IrCl6 or horseradish peroxidase (HRP)/H2O2 to generate polymeric adducts through phenoxyl radical production [Weishar et al. (2008) Org. Lett. 10, 1839−1842]. We now report on reaction of p-PhOH-dG with two radical-generating systems, CuII/H2O2 or FeII-EDTA/H2O2, which were utilized to study the fate of the C-linked adduct in the presence of hydroxyl radical (HO•). The radical-generating systems facilitate (i) hydroxylation of the phenolic ring to afford the catechol adduct 8-(3″,4″-dihydroxyphenyl)-dG (3″,4″-DHPh-dG) and (ii) Hatom abstraction from the sugar moiety to generate the deglycosylated base p-PhOH-G. The ratios of 3″,4″-DHPh-dG to pPhOH-G were ∼1 for CuII/H2O2 and ∼0.13 for FeII-EDTA/H2O2. The formation of 3″,4″-DHPh-dG was found to have important consequences in terms of reactivity. The catechol adduct has a lower oxidation potential than p-PhOH-dG and is sensitive to aqueous basic media, undergoing decomposition to generate a dicarboxylic acid derivative. In the presence of excess N-acetylcysteine (NAC), oxidation of 3″,4″-DHPh-dG produced mono-NAC and di-NAC conjugates. Our results imply that secondary oxidative pathways of phenolic-dG lesions are likely to contribute to toxicity.

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INTRODUCTION Phenols are a class of structurally diverse molecules found abundantly in nature that exhibit a range of biological properties including toxicity.1−5 Phenol toxicity may be dependent on bioactivation by P450 and peroxidase enzymes. P450 catalyzes hydroxylation of phenols to afford catechols,6 which redox cycle to generate semiquinone radicals and quinone electrophiles with generation of superoxide radical anion (O2•−).7 Quinones can cause cellular damage by reacting covalently with nucleophiles to produce adducts and crosslinked structures in DNA and proteins.8−10 Peroxidase enzymes catalyze the one-electron (e−) oxidation of phenols, producing phenoxyl radicals that can generate toxicity by a mechanism described as futile thiol pumping.11 In this mechanism, the enzymatically generated phenoxyl radicals are reduced by thiols to generate thiyl radicals that stimulate the generation of new reactive oxygen species (ROS) such as O2•− and hydroxyl radical (HO•).1,11 Hydroxyl radical causes oxidation of 2′deoxyguanosine (dG) to afford 8-oxo-7,8-dihydro-2′-deoxyguanosine (dOG) as a major product that is commonly used as a biomarker for oxidative damage.12−14 An interesting feature of dOG is its decreased oxidation potential (E° = 0.74 V vs NHE15) relative to dG (E° = 1.29 V vs NHE16), making certain phenoxyl radicals (E° ∼ 1 V vs NHE17) capable of facilitating dOG oxidation. This produces spiroiminohydantoin (Sp) and guanidinohydantoin (Gh) lesions (Scheme 1)18 that are more mutagenic than dOG.19,20 The Burrows laboratory has shown that phenolates can also react covalently with oxidized dOG to generate the tricyclic 4,5PhO-dOG product shown in Scheme 1.21,22 © 2011 American Chemical Society

Scheme 1. Oxidation of dOG

Phenoxyl radicals can also react directly with DNA by addition to the C8-site of dG.23−25 Because of the ambident reactivity of phenoxyl radicals, both oxygen (O)-linked and carbon (C)-linked adducts are produced (Scheme 2). The Clinked adducts shown in Scheme 2 are also generated by mutagenic diazoquinones26 and are structurally related to a family of 8-aryl(Ar)-dG adducts produced by aryl hydrazines,27 polycyclic aromatic hydrocarbons (PAHs),28 estrogens,29 and nitroaromatics.30 Our group has been studying the properties of phenolic Clinked nucleoside adducts because of their expected role in phenol-mediated toxicity.31−34 Electrochemical measurements of the C-linked para-isomer [8-(4″-hydroxyphenyl)-dG (pReceived: August 25, 2011 Published: December 16, 2011 315

dx.doi.org/10.1021/tx200365r | Chem. Res. Toxicol. 2012, 25, 315−325

Chemical Research in Toxicology

Article

more reactive than p-PhOH-dG and decomposes in basic aqueous media and reacts covalently with thiol nucleophiles upon oxidation.

Scheme 2. Ambident Reactivity of Phenoxyl Radical Toward dG

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EXPERIMENTAL PROCEDURES

General Methods. 1H and 13C NMR spectra were recorded at room temperature on a Bruker Avance 300 DPX at 300.1 and 75.5 MHz, respectively. NMR spectra were referenced to the solvent signal of the deuterated solvent. Ammonium acetate, HPLC grade water, acetonitrile, and methanol were purchased from Fisher Scientific (Nepean, ON). Any water used for buffers or spectroscopic solutions was obtained from a Milli-Q filtration system (18.2 MΩ). pH measurements were taken at room temperature with an Accumet 910 pH meter and an Accumet pH Combination Electrode with stirring. Product isolations were carried out using an Agilent 1200 series HPLC equipped with an autosampler, autocollector, fluorescence, and diode array detector. Separations were performed using an Agilent C-18 5 μ 150 mm × 4.6 mm column with a flow rate of 0.5 mL/min for analytical separation and an Apollo C18 5 μ 250 mm × 10 mm column with a flow rate of 2.5 mL/min for semipreparative separation. Mass spectra were obtained at the Biological Mass Spectrometry Facility (BMSF) at the University of Guelph using either a Micromass/Waters Global Ultima quadrupole TOF or a Finnigan LCQ Deca quadrupole ion trap. Sample solutions for analysis were typically prepared in 0.1 mM ammonium acetate in 90:10 (v/v) HPLC grade methanol/water. Each sample was introduced, through electrospray, to the MS instrument using a syringe pump at flow rates of 3−5 μL/min for the ion trap and 0.5 μL/min for the Q-TOF instrument. The nitrogen gas (electrospray desolvation gas) and helium gas (ion trap buffer gas) were obtained from Linde (Guelph, ON). UV−vis and fluorescence emission spectra were recorded on a Cary 300-Bio UV−visible Spectrophotometer and Cary Eclipse Fluorescence Spectrophotometer with built-in Peltier temperature controllers. Standard 10 mm light path quartz glass cells from Hellma GmbH & Co. (Concord, ON) were used for both UV−vis and fluorescence measurements. All UV− vis and fluorescence emission spectra were recorded with baseline correction and stirring. Cyclic voltammetry (CV) and electrolysis experiments were conducted using an Autolab PGSTAT30 potentiostat instrument with glassy carbon working electrodes polished with LECO Microid diamond using Spectrum System 1000. Unless otherwise noted, commercial compounds were used as received. 4-Hydroxyphenyl boronic acid was purchased from Frontier Scientific (Logan, UT), Pd(OAc)2, piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES), EDTA, and N-acetylcysteine (NAC) were from Sigma-Aldrich (Oakville, ON), dG was from ChemGenes (Willmington, MA), 3,3′,3″-phosphinidynetris(benzenesulfonic acid) trisodium salt (TPPTS) was from Alfa Aesar (Ward Hill, MA), and Fe(NH4)2(SO4)2·6H2O and Cu(OAc)2 were from Fisher Scientific (Nepean, ON). The synthesis of 8-bromo-2′-deoxyguanosine (8-BrdG) was performed according to literature procedures by treating dG with N-bromosuccinimide in water−acetonitrile.41 Suzuki−Miyaura cross-coupling reaction between 8-Br-dG and 4-hydroxyphenyl boronic acid to afford p-PhOH-dG was carried out as described previously.31−33 NMR and MS spectra for p-PhOH-dG were identical to those previously published.32 Synthesis of 3″,4″-DHPh-dG. The synthesis of 3″,4″-DHPh-dG was carried out according to the steps outlined in Scheme 3. The

PhOH-dG), Scheme 2] showed that attachment of the phenolic moiety to the C8 site of dG considerably lowers the oxidation potential of the base, making it susceptible to further oxidation.32 Thus, similar to dOG,35 p-PhOH-dG undergoes oxidation by treatment with the transition metal oxidant Na2IrCl636 (E° = 0.9 V vs NHE37). However, unlike dOG that undergoes oxidation to form mutagenic Sp and Gh lesions through H2O attachment to the purine radical cation,18,35 treatment of p-PhOH-dG with Na2IrCl6 or with the horseradish peroxidase (HRP)/H2O2 oxidant system generated C−Clinked polymeric adducts through the intermediacy of the phenoxyl radical.36 This difference in reactivity is caused from conjugation of the phenolic ring in p-PhOH-dG with the purine radical cation, leading to deprotonation of the phenol moiety with generation of the neutral phenolic radical to afford ortho− ortho (o−o) C−C oligomeric cross-linked adducts.36 In the present study, two radical-generating systems, CuII/ H2O238 and FeII-EDTA/H2O2,39 were utilized to study the oxidative fate of p-PhOH-dG. The radical systems differ somewhat in that FeII-EDTA/H2O2 generates freely diffusible HO•,39 while the CuII/H2O2 system can generate a copper-oxo species in which the O-centered radical is attached to the copper ion.40 For the oxidation of dG by CuII/H2O2, coordination of copper to N7 of dG is also thought to direct the oxidation.38 Our goal was to use these systems to study the fate of p-PhOH-dG in the presence of HO•, given that phenolic toxins stimulate ROS production in biological systems,1,11 and form C-linked adducts.25,26,34 Our studies show that both systems facilitate hydroxylation of p-PhOH-dG to afford 3″,4″dihydroxyphenyl-dG (3″,4″-DHPh-dG) and show no propensity for mediating p-PhOH-dG polymerization through phenoxyl radical generation.36 The product 3″,4″-DHPh-dG is Scheme 3. Synthesis of 3″,4″-DHPh-dG

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known 3,4-bis-tert-butyl-dimethylsiloxy(TBDMS)phenyl boronic acid 142 was prepared from catechol by bromination43 followed by protection of the phenolic OH groups and conversion into the boronic acid.42 Suzuki−Miyaura cross-coupling of the TBDMSprotected boronic acid 1 with 8-Br-dG was then performed according to the literature44 with slight modification due to solubility issues with the boronic acid. The TBDMS-protected boronic acid 1 (420 mg, 1.11 mmol) was dissolved in 350 μL of CHCl3, and the solution was purged with argon for 30 min. This solution was then transferred to a 50 mL round-bottom flask containing 8-Br-dG (129 mg, 0.375 mmol), TPPTS (14.8 mg, 0.025 mmol), sodium carbonate (80 mg, 0.75 mmol), and Pd(OAc)2 (2.2 mg, 0.01 mmol) in a solution of 1:1 water:methanol under argon. The reaction was heated to 95 °C and left to stir for 8 h. The reaction was diluted with ca. 20 mL of water, and the pH was adjusted to 6−7 with 10% HCl(aq). The mixture was allowed to cool to 0 °C for several hours before the product was recovered by vacuum filtration. Under the basic conditions of the Suzuki−Miyaura cross-coupling conditions, the TBDMS protecting groups are removed.45,46 The final product 3″,4″-DHPh-dG was obtained as a pale gray solid (0.083 g, 59%). UV−vis (DMSO) λmax 282 nm. 1H NMR (DMSO-d6) (300 MHz) δ: 10.67 (bs, 1H), 9.33 (bs, 1H), 9.26 (bs, 1H), 6.99 (s, 1H), 6.85 (m, 2H), 6.29 (bs, 2H), 6.05 (m, 1H), 5.11 (bs, 1H), 4.97 (bs, 1H), 4.31 (m, 1H), 3.76 (m, 1H), 3.63 (m, 1H), 3.52 (m, 1H), 3.13 (m, 1H), 1.96 (m, 1H). 13C NMR (DMSO-d6) (75.5 MHz) δ: 156.6, 152.7, 151.6, 147.5, 146.8, 145.2, 121.2, 120.6, 116.8, 116.5, 115.4, 87.7, 84.3, 71.1, 62.1, 36.6. HRMS calcd for C16H17N5O6 [M + H]+, 376.1173; found, 376.1146. Deglycosylation of p-PhOH-dG and 3″,4″-DHPh-dG. Deglycosylation reactions of p-PhOH-dG and 3″,4″-DHPh-dG were achieved by placing the nucleosides (18 mg) in ca. 1 mL of 10% formic acid under heat (70 °C), as outlined previously.31,33,47 Authentic samples of p-PhOH-G and 3″,4″-DHPh-G were obtained following HPLC purification using 40 mM ammonium acetate, pH 7.2 (solvent A), and acetonitrile (solvent B) on a C-18 reverse-phase column with UV (280 nm) and fluorescence emission (390 nm) detection. The elution profile was a follows: 95/5 solvent A/B for 3 min followed by 7 min linear gradient to 90/10 A/B, followed by 20 min linear gradient to 70/30 A/B, and back to starting conditions (95/5 A/B) in 4 min. The product peaks were combined, lyophilized to dryness, and characterized by ESI-MS. These deglycosylated products along with the sample of 3″,4″-DHPh-dG were used to construct five-point HPLC calibration curves with UV detection to quantify levels produced from oxidation of p-PhOH-dG. Oxidation of p-PhOH-dG with CuII/H2O2. A stock solution of pPhOH-dG (0.01 M) was prepared in N,N-dimethylformamide (DMF). A solution of Cu(OAc)2 (0.1 M) was prepared in 0.5 M aqueous PIPES buffer, pH 7.3. The reaction mixture consisted of 100 μL of p-PhOH-dG stock to 1 mL of PIPES buffer (0.5 M, pH 7.3) containing 10 μL of the Cu(OAc)2 stock followed by 50 μL of H2O2 (30%). The resulting solution was stirred at 37 °C for various lengths of time and then quenched by the addition of 0.01 M EDTA. HPLC analysis of the reaction mixture was carried out using the protocol outlined for analysis of the deglycosylation reactions. Oxidation of p-PhOH-dG with FeII-EDTA/H2O2. Stock solutions of Fe(NH4)2(SO4)2 (0.01 M) and EDTA (0.02 M) were prepared in H2O. A reaction mixture was prepared by adding 100 μL of the pPhOH-dG DMF stock solution (0.01 M) to 1 mL of PIPES buffer (0.5 M, pH 7.3) containing 10 μL of both the Fe and the EDTA stock solutions. To this solution was added 50 μL of H2O2 (30%), and the reaction mixture was stirred at 37 °C for various lengths of time. Aliquots of the mixture were then analyzed by HPLC. Basic Decomposition of 3″,4″-DHPh-dG in the Absence and Presence of NAC. The basic aqueous decomposition of 3″,4″-DHPhdG was followed spectrophotometrically by UV−vis and fluorescence emission (λext = 290 nm) spectroscopy at ambient temperature and by reverse-phase HPLC with UV detection. Reactions were initiated by adding 150 μL of a DMF stock solution of 3″,4″-DHPh-dG (0.01 M) to a 3 mL cuvette containing 1850 μL of 30% NH4OH. Spectroscopic measurements were taken immediately after the addition of 3″,4″DHPh-dG and following 30 s time intervals until no further change in

the absorbance or emission spectra was observed (∼10 min). The solution was then concentrated under diminished pressure using a ThermoSavant DNA 120 SpeedVac at medium drying rate to remove NH4OH. The resulting neutral aqueous solution was characterized by ESI-MS. In separate experiments, the decomposition of 3″,4″-DHPh-dG in aqueous NH4OH in the absence and presence of 50 equiv of NAC were monitored by reverse-phase HPLC. For these reactions, two solutions of 3″,4″-DHPh-dG were prepared by adding 100 μL of a stock solution in DMF (0.1 M) to 900 μL of water. To one solution was added NAC (81.6 mg, 0.050 mmol, 50 equiv) by a spatula, and the reaction mixture was stirred for ∼2 min to dissolve NAC. To both solutions was added 1 mL of aqueous NH4OH (10%) to initiate the reaction. Following various lengths of incubation time at ambient temperature, 100 μL aliquots were removed and neutralized with 0.1 M HCl to quench the reaction. The resulting neutral solutions were then analyzed by reverse-phase HPLC using an Agilent C-18 5 μ 150 mm × 4.6 mm column with a flow rate of 0.5 mL/min. For solutions lacking NAC, employed were the following gradient conditions: solvent A, aqueous 0.1% formic acid; solvent B, 70:30 ACN:aqueous 0.1% formic acid. The elution profile was as follows: 95/5 solvent A/B for 5 min followed by 5 min linear gradient to 90/10 A/B, followed by 15 min linear gradient to 70/30 A/B, and back to starting conditions (95/5 A/B) in 5 min. For solutions containing NAC, separation was carried out using buffer A [95:5 aqueous 50 mM triethylammonium acetate (TEAA), pH 7.2:ACN] and buffer B (70:30 ACN:50 mM TEAA). The elution profile was a follows: 95/5 solvent A/B for 5 min followed by 5 min linear gradient to 90/10 A/B, followed by 15 min linear gradient to 70/30 A/B, and back to starting conditions (95/5 A/ B) in 5 min. Quantum Yield Measurement. A quantum yield value for 3″,4″DHPh-dG was determined in 10 mM MOPS buffer using the comparative method.48 Quinine bisulfate (ϕfl = 0.546) in 0.5 M H2SO4 was used as the fluorescence quantum yield standard.49 The equation used to calculate the quantum yield was the following; ϕfl(x) = (As/ Af)(Fx/Fs)(ηx/ηs)2ϕfl(s) where s is the standard, x is the unknown, A is the absorbance at the excitation wavelength, F is the integrated area under the emission curve, η is the refractive index of the solvent, and ϕfl is the quantum yield. The corrective term for refractive index was not included due to the similarities in refractive indices between H2O and H2SO4. Absorbance readings were maintained below 0.06, avoiding inner-filter and self-absorbance phenomena. CV. Electrochemical oxidation measurements were conducted in a three-electrode glass cell under N2. Measurements were carried out in a solution of 0.1 M DMF/tetrabutylammonium hexafluorophosphate (TBAF). The working electrode was a glassy carbon electrode, 2 mm in diameter, polished and ultrasonically rinsed with ethanol. The reference electrode was a Ag wire placed in the same solution and separated from the main solution by a fine porosity frit. The reference electrode potential was calibrated in situ against 1 mol equiv of 9,10anthraquinone [−0.800 V vs saturated calomel electrode (SCE)]. The counterelectrode used was Pt wire wrapped in foil that was cleaned by repeated heating with a butane torch with cooling in pure Milli-Q water. For the CV, the starting potential was 0 V vs SCE, and the potential was first scanned 1.6 V toward positive potentials, and then scanned 1.6 V toward negative potentials. The scanning rate used was 0.2 V/s. Peak picking was achieved by manually shifting the CV values to correlate with the 9,10-anthraquinone internal standard and then using the Autolab software Gpes ver. 4.9.005 with data analysis method, peak picking feature, to determine peak potentials and halfpeak potentials. Controlled Potential Electrolysis of p-PhOH-dG and 3″,4″DHPh-dG. Electrochemical oxidation of p-PhOH-dG and 3″,4″DHPh-dG in the absence and presence of 25 equiv of NAC were carried out in a three-electrode glass cell under N2 on a semipreparative scale using controlled electrolysis. For the control experiments, stock solutions were made up with 0.01 mmol of the respective nucleoside (3.75 mg of 3″,4″-DHPh-dG and 3.60 mg of pPhOH-dG in 1 mL of DMF). The entire stock solution was added to 14 mL of a 150 mM phosphate buffer (pH 7.2), and the electrolysis 317



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In the presence of H2O2, CuII has been proposed to generate hydroxyl radical-like species through Fenton type reactions,38 suggesting that the two products were produced by competing processes: (i) hydroxylation of the phenolic ring to generate 3″,4″-DHPh-dG and (ii) H-atom abstraction from the sugar moiety to afford the deglycosylated base p-PhOH-G. Figure 1B shows HPLC elution profiles from incubation of pPhOH-dG in the presence of FeII-EDTA/H2O2. Following 10 min of incubation time (Figure 1Bi), p-PhOH-G and 3″,4″DHPh-dG were present, as noted in the CuII/H2O2 reaction. However, after 30 min of incubation (Figure 1Bii), the peak for p-PhOH-G was the dominant product peak. Also, a new peak, not observed in the CuII/H2O2 reaction, was noted following the 30 min incubation time that eluted at ∼23 min and exhibited an absorbance maximum of 319 nm and coeluted with an authentic sample of the deglycosylated 3″,4″-DHPh-G species. The oxidation reactions were also quenched with acid to deglycosylate all species in solution. The acidic solution was filtered, the precipitate was removed, redissolved in DMSO, and analyzed by HPLC. Following an initial incubation of pPhOH-dG for 30 min in the presence of CuII/H2O2 or FeIIEDTA/H2O2, acid treatment produced the traces labeled (iii) in Figure 1. Both oxidation systems generated clean chromatograms featuring two major peaks that were identified as 3″,4″DHPh-G and p-PhOH-G on the basis of elution times, absorbance maxima, and mass. Spectral data for oxidation of p-PhOH-dG by the two radicalgenerating systems (CuII/H2O2 and FeII-EDTA/H2O2) are summarized in Table 1. Yields of 3″,4″-DHPh-dG, p-PhOH-G

was initiated by applying the appropriate voltage (0.800 V for 3″,4″DHPh-dG) and (1.100 V for p-PhOH-dG) for 90 min. The resulting solutions were analyzed by the HPLC method outlined below. For the experiments using NAC as a trapping agent, stock solutions of the modified bases were prepared in 1.0 mL of DMF/14 mL of phosphate buffer (pH 7.2), as outlined above, but 25 equiv of NAC (40.8 mg, 0.25 mmol) was added to the solutions prior to electrolysis. The potentials and electrolysis time were the same as the control experiments. The working electrode was a glassy carbon electrode, 15 mm in diameter, polished and ultrasonically rinsed with ethanol. The counterelectrode was a Pt wire cleaned by repeated heating with a butane torch. The reference electrode was a Ag wire and was separated from the main solution by a fine porosity frit. The reactions were analyzed by HPLC using 40 mM ammonium acetate, pH 7.2 (solvent A), and 2:1 acetonitrile:40 mM ammonium acetate (solvent B) with a semipreparative C18 column with UV (280 nm) and fluorescence emission (390 nm) detection. The elution profile was a follows: 95/5 solvent A/B for 3 min followed by 7 min linear gradient to 90/10 A/B, followed by 17 min linear gradient to 70/30 A/B, and back to starting conditions (95/5 A/B) in 4 min. The NAC−conjugate peaks were combined, lyophilized, and characterized by UV−vis and ESI-MS.

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RESULTS Hydroxy Radical-Mediated Oxidation of p-PhOH-dG. Incubation of the nucleoside p-PhOH-dG in the presence of CuII/H2O2 for 10 min generated a pair of peaks eluting at ∼25 min (Figure 1Ai). After 30 min of incubation time (Figure

Table 1. Spectral Data for Oxidation of p-PhOH-dGa adduct

tRb

λmaxc

[M − H]−

% yieldd

p-PhOH-dG 3″,4″-DHPh-dG p-PhOH-G 3,4-DHPh-G

26.7 24.9 24.8 22.8

278 280 309 319

358.3 374.1 242.3 258.2

20/3 19/23 −/11

a

A 10 mM concentration of p-PhOH-dG in 0.43 M PIPES buffer, pH 7.3, incubated at 37 °C for 30 min in the presence of CuII/H2O2 or FeII-EDTA/H2O2. bHPLC conditions: 95/5 solvent A (40 mM ammonium acetate pH 7.2)/B (acetonitrile) for 3 min followed by 7 min linear gradient to 90/10 A/B, followed by 20 min linear gradient to 70/30 A/B, and back to starting conditions (95/5 A/B) in 4 min. c In nm. dYield for CuII/FeII reactions.

and 3″,4″-DHPh-G were obtained from the 30 min incubation reaction that generated the highest yields due to further oxidative degradation of p-PhOH-dG upon longer incubation times. The yields were obtained through comparison to HPLC calibration curves using authentic adduct standards. Oxidation of p-PhOH-dG with CuII/H2O2 generated more hydroxylated product (3″,4″-DHPh-dG) than the FeII-EDTA/H2O2 system, while Fe II -EDTA/H 2 O 2 yielded more deglycosylated p-PhOH-G product and generated 3″,4″-DHPh-G, which was not observed in the CuII/H2O2 system until treatment with acid (Figure 1Aiii) . The relatively low yields of product are due to the presence of other product peaks that eluted earlier in the HPLC chromatogram and could not be identified due to the lack of authentic standards for comparison. Properties and Reactivity of 3″,4″-DHPh-dG. Having established that treatment of the parent p-PhOH-dG adduct with CuII/H2O2 or FeII-EDTA/H2O2 yields the hydoxylated

Figure 1. HPLC elution profiles from reaction of p-PhOH-dG with (A) CuII/H2O2 and (B) FeII-EDTA/H2O2 at 37 °C, pH 7.3, following (i) 10 min of incubation, (ii) 30 min of incubation, and (iii) 30 min of incubation followed by treatment with acid (HCO2H).

1Aii), the product peaks were more prominent and showed distinct absorbances at 309 and 282 nm, respectively. The retention time and spectroscopic properties of the product peaks suggested the presence of the hydroxylated species 3″,4″DHPh-dG (indicated by arrow in Figure 1Ai) and the deglycosylated product p-PhOH-G that absorbs at 309 nm. 318



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adduct 3″,4″-DHPh-dG, its properties and reactivity were determined. The optical properties of 3″,4″-DHPh-dG in aqueous media at pH 7 and its electrochemical redox properties in DMF were characterized to draw comparison to the corresponding properties of p-PhOH-dG.32,34,47 Given that other catechols are unstable in aqueous basic media, undergoing conversion to semiquinone radicals and quinones by an autoxidative process,9,50 the reactivity of 3″,4″-DHPh-dG in aqueous NH4OH was studied in the absence and presence of the thiol nucleophile NAC, which was added as a trapping agent. These experiments were then compared to the reactivity of 3″,4″-DHPh-dG in aqueous buffered media, pH 7.2, following electrochemical oxidation in the absence and presence of excess NAC. Optical and Redox Properties of 3″,4″-DHPh-dG. The optical and redox properties of 3″,4″-DHPh-dG are listed in Table 2. Absorption and emission spectra were recorded in Table 2. Photophysical and Redox Parameters for Phenolic C-Linked Adducts adduct

λmax, log εb

λemb (Φflc)

Ep/2 (V)d

p-PhOH-dG 3″,4″-DHPh-dG dGa

278, 4.26 282, 4.37 253, 4.14

390 (0.47) 392 (0.15) 334 (9.7 × 10−5)

0.85 0.71 1.14

a

Optical data for dG taken from ref 51. bDetermined in aqueous 10 mM MOPS buffer, pH 7.0, μ = 0.1 M NaCl. cDetermined using the comparative method with quinine bisulfate in 0.5 M H2SO4 (Φfl = 0.55). dHalf-peak potentials (Ep/2) in volts (V) vs SCE using CV in 0.1 M TBAF in anhydrous DMF with a glassy carbon working electrode.

Figure 2. (A) UV−vis (insert is expansion of 325−450 nm region) spectral changes during the aqueous NH4OH (30%) decomposition of 3″,4″-DHPh-dG at ambient temperature. Absorption measurements were taken immediately after addition of 3″,4″-DHPh-dG to 30% NH4OH and following 30 s time intervals until no further change in absorbance was observed (∼10 min). (B) HPLC elution profile of the aqueous NH4OH (10%) decomposition of 3″,4″-DHPh-dG: (i) before NH4OH treatment and following treatment for (ii) 10, (iii) 30, and (iv) 120 min at ambient temperature.

aqueous MOPS buffer, pH 7.0. Optical data for unmodified dG51 and p-PhOH-dG32,34,47 in aqueous media are included for comparison. As discussed previously,32,47 attachment of the phenolic moiety to the C8 site of dG to afford the nucleoside pPhOH-dG causes a red shift in absorption maxima (278 vs 253 nm) and generates a highly fluorescent nucleobase (λem = 390 nm, Φfl = 0.47) that acts as a pH-sensitive fluorescent probe.32 Attachment of the OH auxochrome to generate 3″,4″-DHPhdG causes a slight red shift in absorption maxima and an increase in extinction coefficient (Table 2). However, the fluorescense intensity of the nucleobase is decreased as compared to p-PhOH-dG. The electron-donor properties of 3″,4″-DHPh-dG were determined using CV in anhydrous DMF, as outlined previously for other nucleoside adducts.32,34,36 Under these experimental conditions, the catechol nucleoside 3″,4″-DHPhdG showed an irreversible 1e− oxidation peak with a half-peak potential (Ep/2) of 0.71 V vs SCE, which is 0.14 V lower than the corresponding value for p-PhOH-dG (0.85 V/SCE) and 0.43 V lower than the value obtained for dG (Ep/2 = 1.14 V/ SCE). Basic Decomposition of 3″,4″-DHPh-dG in the Absence and Presence of NAC. Spectral changes (UV−vis and fluorescence emission) of 3″,4″-DHPh-dG in aqueous NH 4 OH (30%) at ambient temperature were initially monitored to provide information on the stability of 3″,4″DHPh-dG in basic aqueous solution. The neutral species absorbs at ∼282 nm and shows emission at ∼392 nm (Table 2). However, in 30% NH4OH, the initial absorption spectrum (Figure 2A) showed a broad absorption band at ∼435 nm (see insert) that decreased in intensity and was no longer visible after 5 min. After 10 min, no further changes in the collected

spectra were noted, and a product with a maximum peak at 280 nm and a prominent shoulder at 330 nm was present. Over the same time period, the fluorescence emission intensity (λext = 290 nm) of the basic solution increased and shifted from 400 to 390 nm (Figure S1 in the Supporting Information). Under analogous conditions, p-PhOH-dG showed an absorption band at ∼305 nm that did not change over a 2 h time period (Figure S2 in the Supporting Information). The absorption at 305 nm was ascribed to the dianion of p-PhOH-dG from deprotonation of the phenolic OH group and N1 of the dG component, which have pKa values ∼9.32 These results suggested that the dianion of p-PhOH-dG is stable in basic aqueous solution, while the anionic species of 3″,4″-DHPh-dG decomposes. Further information on the aqueous decomposition of 3″,4″DHPh-dG was obtained by HPLC analysis of the reaction mixture (Figure 2B). For HPLC analysis, the decomposition of 3″,4″-DHPh-dG was carried out in 10% NH4OH. Following 2 h of incubation at ambient temperature, 3″,4″-DHPh-dG had completely decomposed (Figure 2Biv), and a single major product (96% yield, based on HPLC peak integration assuming similar ε280 of the product and starting material) eluting at ∼5 min was observed. This product peak had λmax at 280 nm and a prominent shoulder at 330 nm (from diode array analysis), as observed in the UV spectra recorded for the decomposition of 319



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derivatives. In the absence of added H2O2 to the basic solution, electron transfer from the phenolate to O2 generates O2•− and the semiquinone radical.54,55 This process initiates in situ H2O2 production (HOO− in base), and the C−C cleavage in the basecatalyzed autoxidation of catechols also proceeds via the acyclic mechanism involving an o-quinone intermediate.53 The basic decomposition of 3″,4″-DHPh-dG was also carried out in the presence of 50 equiv of NAC in an effort to trap the expected o-quinone intermediate, since o-quinones are wellknown to react with thiols by conjugate addition reactions.9,56−58 HPLC analysis of the reaction mixture (Figure

3″,4″-DHPh-dG in 30% NH4OH following 10 min of incubation at ambient temperature (Figure 2A). The product from the basic aqueous decomposition of 3″,4″DHPh-dG was characterized by ESI-MS. The ESI−-MS analysis showed a product with [M − H]− = 406 (Figure S3 in the Supporting Information) for a neutral mass m/z 407, 32 mass units heavier than 3″,4″-DHPh-dG. A fragment ion at m/z 290 for loss of the deoxyribose (−116) moiety was observed, and this species showed fragment ions at m/z 273 and 247 (Figure S3 in the Supporting Information) for nominal mass losses of 17 and 43, respectively. These losses are also observed for deprotonated p-PhOH-G52 and suggest loss of NH3 (17) and isocyanic acid (HNCO, 43) from the guanine component of the product. The ESI+-MS analysis of the neutral solution (Figure 3) turned out to be more diagnostic of the product

Figure 3. ESI+-MS analysis of proposed diacid product from the aqueous NH4OH (30%) decomposition of 3″,4″-DHPh-dG. (A) ESI+MS spectrum with [M + H]+ = 408 and major fragment at m/z 292 for loss of deoxyribose (−116). (B) MS3 spectrum taken at m/z 292, (C) MS4 spectrum taken at m/z 275, (D) MS5 spectrum taken at m/z 258, and (E) MS6 spectrum taken at m/z 230.

generated from the aqueous decomposition of 3″,4″-DHPh-dG. Here, both the MS3 (Figure 3B) and the MS4 (Figure 3C) spectra of the decomposition product showed single fragments at m/z 275 and 258 for losses of 17 mass units. The MS5 (Figure 3D) and MS6 (Figure 3E) spectra gave fragment ions at m/z 230 and 202, respectively, for losses of 28 mass units. These fragment ions [loss of 17 mass units (OH) followed by loss of 28 mass units (CO)] are indicative of carboxylic acid groups and suggested that 3″,4″-DHPh-dG decomposed into the diacid shown in Figure 3 that has a neutral mass of m/z 407. As described by Sawaki and Foote,53 o-quinones undergo C−C cleavages through reaction with HOO− via an acyclic Baeyer−Villiger type mechanism to afford dicarboxylic acid

Figure 4. (A) HPLC elution profile of the aqueous NH4OH (10%) decomposition of 3″,4″-DHPh-dG in the presence of 50 equiv NAC: (i) initial 3″,4″-DHPh-dG and following treatment for (ii) 30 min, (iii) 4 h, and (iv) 24 h at ambient temperature. (B) ESI−-MS spectrum of peak labeled * for the mono-NAC conjugate (NAC-3″,4″-DHPh-dG); MS3 taken at m/z 406 is shown in the insert.

4A) indicated that NAC decreased the rate of 3″,4″-DHPh-dG decomposition considerably. Following 24 h of incubation (Figure 4Aiv) at ambient temperature, ∼10% 3″,4″-DHPh-dG was still present (following 2 h of incubation in the absence of 320



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NAC, 3″,4″-DHPh-dG had totally decomposed, Figure 2Biv). The major product was the diacid (∼78%), and a new species was present (∼11%) eluting just before the starting material (labeled with the asterisk). ESI−-MS analysis of this product gave an [M − H]− ion at 535 (Figure 4B) for attachment of NAC to 3″,4″-DHPh-dG followed by the loss of 2H [i.e., 375 (3″,4″-DHPh-dG) + 163 (NAC) − 2H = 536]. The fragment ion at m/z 406 is 32 mass units heavier than 3″,4″-DHPh-dG and results from β-elimination of benzoquinol-SH, which is an established fragmentation of S-peptide-benzoquinone adducts.59 The insert in Figure 4B shows the MS3 spectrum taken at m/z 406 showing loss of the deoxyribose moiety (−116 mass units). These results provided further support for the notion that the aqueous decomposition of 3″,4″-DHPh-dG generates an electrophilic o-quinone intermediate. Controlled Potential Electrolysis. To compare the reactivity of p-PhOH-dG to 3″,4″-DHPh-dG in aqueous media at neutral pH in the absence of O2/H2O2, their electrochemical oxidation in the absence and presence of 25 equiv of NAC was examined. These experiments were carried out in aqueous phosphate buffer (pH 7.2) under N2 on a semipreparative scale using controlled electrolysis. With no NAC trapping agent, the pPhOH-dG solution turned a faint yellow color upon electrolysis, and a thick black shiny coating developed on the working electrode surface that could only be removed by vigorous polishing methods. HPLC analysis of the electrolysis mixture showed no new peaks when compared to the initial chromatogram (not shown). In the presence of 25 equiv of NAC, the solution again turned a faint yellow color upon electrolysis, and the thick coating was observed on the electrode surface. HPLC analysis did not show any new peaks from the electrolysis procedure. In both electrolysis experiments, the starting material was diminished after electrolysis, although no new peaks were observed by HPLC. These results suggested polymerization of p-PhOH-dG through phenoxyl radical production36 to generate an insoluble polymer that coated the electrode surface.60 Electrolysis of 3″,4″-DHPh-dG in the absence of NAC generated a yellow solution and a dark coating on the electrode surface. Analysis of the electrolysis solution by HPLC indicated that the starting material had diminished, although no new peaks were observed, as noted for electrolysis of p-PhOH-dG. In the presence of 25 equiv of NAC, the solution again turned yellow, and the dark coating was observed on the electrode surface. However, two new peaks were observed by HPLC eluting faster than the starting material (Figure 5). The UV−vis spectrum of the species giving rise to the peak labeled +1 NAC (Figure 5A) had λmax 261 nm and a broad shoulder peaking at 283 nm (Figure S4A in the Supporting Information) that matched the diode array spectrum for NAC-3″,4″-DHPh-dG generated from the aqueous 10% NH4OH decomposition of 3″,4″-DHPh-dG in the presence of 50 equiv of NAC. ESI−-MS analysis confirmed the presence of the mono-NAC conjugate with an [M − H]− ion at 535 (Figure 4B). Isolation of the species giving rise to the peak labeled +2 NAC (Figure 5A) generated a UV−vis spectrum with absorbances at 320 and 272 nm (Figure S4B in the Supporting Information). ESI−-MS analysis of the product showed an [M − H]− ion at 696 with fragment ions at m/z 567 and 438 for attachment and subsequent cleavage of the S−C bond59 of two NAC groups (Figure 5B). The insert in Figure 5B shows the MS4 spectrum taken at m/z 438 that shows a dominant fragment at m/z 322 for loss of the deoxyribose moiety. These

Figure 5. (A) HPLC elution profile from electrochemical oxidation of 3″,4″-DHPh-dG in the presence of 25 equiv of NAC. (B) ESI−-MS spectrum of peak +2 NAC; MS4 taken at m/z 438 is shown in the insert.

observations were consistent with the formation of a di-NAC conjugate [(NAC)2-3″,4″-DHPh-dG]. The formation of diGSH conjugates is common in reactions of o-quinones with GSH,56−58 indicating that the monoconjugates are very good substrates for further conjugation.57 Unfortunately, efforts to unambiguously characterize the structures of the NAC conjugates using NMR spectroscopy failed to provide meaningful results. The lyophilized NAC conjugate samples proved to be insoluble in most commonly used NMR solvents and decomposed in DMSO-d6.

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DISCUSSION Phenols can undergo oxidative reactions to afford phenoxyl radicals, semiquinone radicals, and quinone electrophiles that can react covalently with DNA to generate DNA adducts. Because dG is the most electron rich DNA base,16 it is particularly susceptible to covalent attachment by phenolic electrophiles1 and is prone to oxidation to generate electrophilic species that can react with phenolate nucleophiles.21,22 This provides a diverse range of phenolic-dG adducts that may retain the dG and phenolic ring systems and are thus prone to further oxidative reactions. In the present study, we have further determined the products generated through the oxidation of the nucleoside adduct p-PhOH-dG (Scheme 2). This adduct is generated by mutagenic diazoquinones26 that break down to furnish aryl 321



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Scheme 4. Proposed Pathway for the Aqueous Decomposition of 3″,4″-DHPh-dG

∼0.13 for FeII-EDTA/H2O2 (Table 1). The ratio determined for the FeII-EDTA/H2O2 system does not take into account the production of 3″,4″-DHPh-G, because its mode of formation is uncertain (deglycosylation of 3″,4″-DHPh-dG or hydroxylation of p-PhOH-G). That the FeII-EDTA/H2O2 system generated more deglycosylated product p-PhOH-G correlated somewhat with findings by the Dedon laboratory on levels of dOG and strand breaks produced by CuII/H2O2 vs FeII-EDTA/H2O2 in reaction with duplex DNA.38 Ratios of dOG to strand breaks were ∼0.4 for CuII/H2O2 and ∼0.03 for FeII-EDTA/H2O2, demonstrating that the FeII system is more efficient at generating strand breaks through H-atom abstraction from the sugar moiety.38 The properties (Table 2) and reactivity of the catechol adduct 3″,4″-DHPh-dG were also determined. The addition of the OH group to afford the catechol adduct lowered the oxidation potential in DMF (Table 2), indicating that 3″,4″DHPh-dG is a better electron donor than p-PhOH-dG. This was found to have important consequences in terms of reactivity. Treatment of p-PhOH-dG with aqueous NH4OH will favor dianion formation, as p-PhOH-dG has two acidic protons (phenolic OH and N1) with pKa values ∼9.32 In NH4OH, the adduct showed an absorption band at ∼305 nm that remained stable over a 2 h period at ambient temperature (Figure S2 in the Supporting Information). In contrast, 3″,4″DHPh-dG was sensitive to aqueous NH4OH and decomposed to a stable product (96% yield) following incubation at ambient temperature (Figure 2). Product analysis by ESI+-MS (Figure 3) was consistent with the formation of a dicarboxylic acid derivative. Diacids are known to be generated from o-quinones by reaction with HOO− that initiates C−C cleavage via an acyclic Baeyer−Villiger type mechanism.53 Outlined in Scheme 4 is a proposal for diacid formation that highlights o-quinone formation and HOO− production from treatment of 3″,4″DHPh-dG with NH4OH in the presence of molecular oxygen (O2). Deprotonation of 3″,4″-DHPh-dG by NH4OH will generate anionic species and the dianion from deprotonation of N1, and the phenolic OH para to the dG component is shown in equilibrium with the trianion resulting from deprotonation of N1 and both phenolic OH groups. The initiating step in the decomposition process involves transfer of

radical intermediates that undergo direct radical addition reactions at the C8-site of dG.61 Structurally similar C-linked phenolic adducts are also derived from sterically hindered phenoxyl radical intermediates.25 In our initial study on the oxidative fate of p-PhOH-dG,36 we employed the transition metal oxidant Na2IrCl6 that was previously utilized by the Burrows laboratory to study the oxidation of dOG.35 In the presence of Na2IrCl6, p-PhOH-dG was converted into a neutral phenoxyl radical that underwent coupling reactions to afford o−o C−C-linked polymeric adducts. HPLC-MS analysis showed the presence of three polymeric adducts (dimer, trimer, and tetramer) that eluted after the starting material p-PhOH-dG.36 Treatment of pPhOH-dG with HRP/H2O2 also generated the C−C-coupled polyphenols through the intermediacy of the phenoxyl radical. In the present study, two radical-generating systems, CuII/ H2O238 and FeII-EDTA/H2O2,39 were utilized to study the oxidative fate of p-PhOH-dG. Treatment of p-PhOH-dG with the radical-generating systems produced products that eluted prior to the starting material (Figure 1). No evidence for C−Clinked polymeric adducts was found indicating that these systems did not facilitate phenoxyl radical production. Following 30 min of incubation of p-PhOH-dG with CuII/ H2O2 or FeII-EDTA/H2O2 in aqueous buffer, the catechol adduct 3″,4″-DHPh-dG was produced. Hydroxylation of the phenolic ring system of p-PhOH-dG by CuII/H2O2 or FeIIEDTA/H2O2 is consistent with the known tendency of HO• to react with substituted benzenes by addition to the ring and not by interaction with the substituent.62 Rate constants for hydroxylation of phenol by HO• are large (107−109 M−1 s−1) with the electrophilic HO• showing a strong preference for attack at the ring positions activated by the phenolic HO group (o and p).62 Given that the p-position of p-PhOH-dG is blocked by the dG moiety, o-hydroxylation to afford 3″,4″-DHPh-dG was observed. Both systems also caused deglycosylation of p-PhOH-dG to generate p-PhOH-G; the FeII-EDTA/H2O2 system also produced 3″,4″-DHPh-G. These findings suggested that Hatom abstraction from the sugar moiety to afford the deglycosylated base p-PhOH-G competes with hydroxylation of the phenolic ring to generate 3″,4″-DHPh-dG. The ratios of 3″,4″-DHPh-dG to p-PhOH-G were ∼1 for CuII/H2O2 and 322



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Scheme 5. Oxidation of BQ-dG

one electron from the anionic species to O2 to form O2•− and the semiquinone radical anion;54,55,57 a possible structure is depicted in Scheme 4. In turn, O2•− reacts with the anion species to generate another equivalent of the semiquinone radical anion and H2O2 (HOO− in base).57 Disproportionation of the transient semiquinone radical anion generates the transient o-quinone anion and anionic species of 3″,4″-DHPhdG.57,63 Reaction of the o-quinone species with HOO− generates the neutral diacid product following acidification and isolation. The aqueous NH4OH decomposition of 3″,4″-DHPh-dG was also carried out in the presence of 50 equiv of NAC (Figure 4). This led to the production of a mono-NAC conjugate (NAC3″,4″-DHPh-dG, ∼11% yield) that was characterized by ESI−MS spectroscopy. The mono-NAC conjugate was also obtained by controlled potential electrolysis of the catechol adduct in neutral pH, under N2, in the presence of excess NAC (Figure 5). For these experiments, both p-PhOH-dG and 3″,4″-DHPhdG were subjected to electrolysis in the absence and presence of 25 equiv of NAC. The potentials employed were slightly above the 1e− oxidation potentials (Table 2) of the nucleoside adducts. For p-PhOH-dG, this will generate the neutral phenolic radical,36 while 3″,4″-DHPh-dG will be converted into the semiquinone radical. Under these electrolysis conditions, p-PhOH-dG appeared to form a polymer on the electrode surface, as polymerization of phenols is known to generate an insulating film that sticks to the electrode60 and failed to react with NAC. 3″,4″-DHPh-dG also appeared to form a polymer on the electrode surface but reacted with NAC to form mono-NAC and di-NAC conjugates (Figure 5). These observations are consistent with the fact the phenolic radicals are known to undergo polymerization reactions,36,60 while semiquinone radicals can also undergo disproportionation to afford o-quinones 57,63 that react covalently with thiol nucleophiles.9,56−58 That electrolysis of 3″,4″-DHPh-dG in the presence of excess NAC generated the di-NAC conjugate (NAC)2-3″,4″-DHPh-dG (Figure 5) is consistent with the fact that o-quinone electrophiles commonly react with GSH to generate di-GSH conjugates.56−58 The results of our studies suggest that secondary oxidative pathways of dG lesions bearing phenolic ring systems are likely to contribute to toxicity. The electrophile p-benzoquinone (BQ) is known to react with dG to afford a benzetheno adduct that contains the phenolic and dG ring systems.64 Gaskell and co-workers have reported that the BQ−dG adduct undergoes hydroxylation of the phenolic ring to afford a catechol adduct.65 More recent results by Linhart et al. show that benzetheno adducts are extensively metabolized in vivo.66 Thus, as outlined in Scheme 5, BQ−dG undergoes hydroxylation at the phenolic ring, which competes with hydroxylation at the C8 site of BQ− dG to afford BQ−dOG in vivo.66 Further oxidative pathways of these hydroxylated adducts may further stimulate mutagenicity, as noted for oxidation of dOG.19,20

A goal of our laboratory is to determine the fate of p-PhOHdG and 3″,4″-DHPh-dG oxidation within a duplex DNA environment. We plan to utilize our recently published postsynthetic palladium-catalyzed cross-coupling strategy67 for site-specific incorporation of these adducts into oligonucleotides.34 It should be interesting to determine how the DNA environment influences the reactivity of the phenoxyl radical and the o-quinone electrophile, given that o-quinones typically undergo conjugate addition by N7 of dG,68 while the phenoxyl radical undergoes C−C coupling,36 which may be impossible within duplex DNA.

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ASSOCIATED CONTENT

S Supporting Information *

Spectral properties of 3″,4″-DHPh-dG and Figures S1−S4 described in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Funding

Support for this research was provided by the Natural Sciences and Engineering Research Council (NSERC) of Canada, the Canada Foundation for Innovation, and the Ontario Innovation Trust Fund.

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ACKNOWLEDGMENTS R.A.M. thanks Prof. Abelaziz Houmam (UOG) for assistance with the electrochemical experiments and the reviewers of this manuscript for helpful comments.

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ABBREVIATIONS dG, 2′-deoxyguanosine; p-PhOH-dG, 8-(4″-hydroxyphenyl)dG; 3″,4″-DHPh-dG, 8-(3″,4″-dihydroxyphenyl)-dG; dOG, 8oxo-7,8-dihydro-dG; Sp, spiroiminohydantoin; Gh, guanidinohydantoin; HRP, horseradish peroxidase; NAC, N-acetylcysteine; DMF, N,N-dimethylformamide; CV, cyclic voltammetry; TBAF, tetrabutylammonium hexafluoro-phosphate; BQ, pbenzoquinone

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REFERENCES

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Deoxyguanosine Adduct Yields a Reactive Catechol - American

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