Characterization of Hydantoin Products from One-Electron Oxidation

Burney, S., Niles, J. C., Dedon, P. C., and Tannenbaum, S. R. (1999) DNA damage ...... Yu Ye, James G. Muller, Wenchen Luo, Charles L. Mayne, Anthony ...
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Chem. Res. Toxicol. 2001, 14, 927-938

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Characterization of Hydantoin Products from One-Electron Oxidation of 8-Oxo-7,8-dihydroguanosine in a Nucleoside Model Wenchen Luo, James G. Muller, Elliot M. Rachlin, and Cynthia J. Burrows* Contribution from the Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, Utah 84112-0850 Received April 10, 2001

Use of one-electron oxidants such as Na2IrCl6 to oxidize 8-oxo-7,8-dihydro-2′-deoxyguanosine (OG) residues in oligodeoxynucleotides was previously shown to lead to predominant formation of a base lesion of mass M - 10 compared to starting material [Duarte et al. (1999) Nucleic Acids Res. 27, 596-502]. To thoroughly characterize the structure of this lesion, the oxidation of the nucleoside 9-N-(2′,3′,5′-tri-O-acetyl-β-D-erythro-pentanosyl)-8-oxo-7,8-dihydroguanine with one-electron oxidants at pH 2-4 was used as a model for duplex DNA oxidation of OG residues. 1H NMR and H,H COSY NMR studies in CD OD along with LC-ESI-MS/MS fragmentation 3 analysis are consistent with the assignment of the M - 10 species as a mixture of two pHdependent equilibrating isomers, a guanidinohydantoin (Gh) and an iminoallantoin (Ia) nucleoside, both present as mixtures of epimers at the C5 position of the hydantoin ring, i.e., four total isomers are formed. The Gh/Ia mixture is formed from hydration and decarboxylation of the initially formed intermediate 5-hydroxy-8-oxo-7,8-dihydroguanosine, a species that is also produced by four-electron oxidation (e.g., singlet oxygen) of guanosine. The product mixture can be further oxidized to a species designated Iaox, a hydrolytically unstable material at pH 7 that has been characterized by ESI-MS and 1H NMR. Competition studies with 8-oxo-7,8dihydroadenosine placed the redox potential of Gh/Ia at about 1.0 V vs NHE. These studies provide important information concerning the structures of lesions obtained when OG, a “hot spot” for oxidative damage, serves as a “hole trap” in long-range electron-transfer studies.

Introduction Being the most electron rich of the four DNA bases, guanine (G) is highly susceptible to attack by electrophiles including oxidizing agents. A central player in G oxidation is the 8-oxo-7,8-dihydro-2′-deoxyguanosine (OG, 1) lesion whose role in DNA mutagenesis and repair is under continuing investigation (1-3). While OG is one of the major products formed by ionizing radiation, oneelectron oxidation and singlet oxygen attack at G residues, more than a dozen other lesions have been identified or proposed as minor products of the above pathways or as major products with other oxidants (Scheme 1) (4, 5). These include a 2,5-diaminoimidazolone product (Iz) identified by Cadet et al. (6) and the formamidopyrimidine-Gua (Fapy-G) ring-opened lesion. The preference for one pathway over another depends in part upon pH, on stacking in the duplex, and on the availability of additional oxidants and nucleophiles that may participate at a later stage in the mechanism. While the probability of two sequential oxidation events occurring at the same base in the genome is low, the ability of oxidative damage to migrate over long distances in DNA (7) suggests that certain lesions may act as “sinks” for electron hole trapping. In this scenario, DNA that is subject to a second oxidizing event might accumulate damage at the previously damaged site. This will be particularly true for (a) oxidized lesions, since most oxopurines and hydroxypyrimidines have much lower redox potentials than their parent base, and (b) second oxidations following a one-electron pathway that

allows hole migration in the duplex (8, 9). OG, with a redox potential approximately 0.5 V less than G (1013), is one example of a common oxidative lesion that might be expected to undergo additional oxidative modification. Indeed, OG is highly reactive toward further oxidation, and several in vitro studies now support OG as a “hot spot” for oxidized damage (10, 14-19). Synthesis of OG-containing oligomers necessitates precautionary measures to prevent OG oxidation during the final deprotection steps of most solid-phase synthesis procedures (20). Exposure of OG to singlet oxygen leads to the formation of a variety of products variously proposed to include cyanuric acid, Iz, oxaluric acid, and a sevenmembered ring heterocycle (21-26), while oxidation with peroxynitrite forms oxaluric acid, parabanic acid, and cyanuric acid after hydrolysis of marginally stable intermediates (15-17, 27). Our laboratory has recently focused on the oxidation of OG triggered by one-electron oxidants because this mechanism can allow equilibration of the electron hole to an OG, potentially over long distances, and it should therefore be highly relevant to cellular damage. Our initial studies identified the commercially available iridium(IV) salts Na2IrCl6 and Na2IrBr6 as convenient oneelectron oxidants that, as anions, do not bind to DNA and react cleanly as outer-sphere, one-electron oxidants (19), making them excellent models for the wide variety of oneelectron oxidants that may be present in the cell. The redox potentials of IrCl62- and IrBr62- [0.9 and 0.82 V vs NHE (19)] are appropriate for oxidation of OG with very

10.1021/tx010072j CCC: $20.00 © 2001 American Chemical Society Published on Web 05/23/2001

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Chem. Res. Toxicol., Vol. 14, No. 7, 2001 Scheme 1

Luo et al. Scheme 2

Scheme 3

little, if any, side reaction with G. That IrIV reacts with OG by the transfer of one electron is further substantiated by the observation of the predicted (28) sequence dependence of OG oxidation; an OG residue stacked 5′ to a G in duplex DNA was more readily oxidized than one 5′ to a pyrimidine (18), similar to the tendency of 5′-GG-3′ to be more readily oxidized than 5′-GPy-3′ sequences (29, 30). The products of IrIV-mediated oxidation of OG were initially identified in oligodeoxynucleotides as a mixture of M + 16 and M - 10 species (31), both of which lead to strand scission upon heating with piperidine (5). A detailed analysis of the M + 16 product involving LC-ESI-MS/MS and NMR analysis along with independent synthesis supports the assignment of this species as a spiroiminodihydantoin (Sp) nucleoside (4, Scheme 2) (32). Sp predominates under conditions of high temperature (∼50 °C) in a single-stranded oligonucleotide (33) and is the sole product of oxidation of the nucleoside (pH 7, 25 °C) (32), and consequently, it is likely a relevant product of oxidation of OG in the cellular nucleoside pool or in single-stranded DNA. In addition, Sp was recently identified as the major nucleoside product of both peroxynitrite oxidation of OG in the presence of thiols (an overall two-electron oxidation) and singlet oxygen oxida-

tion of G (34). For the latter reaction, the structure of Sp has been misinterpreted as 4-hydroxy-8-oxoG for more than a decade (24, 35-41). On the other hand, the M - 10 product predominates in oxidation of OG by IrIV in duplex DNA, and it is the sole product from oxidation of single-stranded oligomers with IrIV at low temperature (4 °C) (33). It is not surprising that formation of a bulky heterocycle such as Sp would be highly disfavored in duplex DNA, and thus an altered product distribution may be expected due to the unfavorable or impossible nature of installing the spiro lesion into duplex DNA. Because it is easier to fully characterize a lesion in a monomer rather than in an oligonucleotide, we sought conditions to generate the M - 10 lesion from the OG nucleoside. We originally proposed that the M - 10 product is a guanidinohydantoin (Gh) nucleoside that would likely be present as a mixture of two epimers (31). Data presented herein is consistent with a more complex view in which Gh participates in a slow and reversible isomerization to two epimers of iminoallantoin (Ia, Scheme 3) in a fashion analogous to that found in the oxidation of uric acid and other purine derivatives (42-49). The mixture of Gh + Ia is also subject to further oxidation and to base hydrolysis.

Experimental Procedures Materials. Guanosine hydrate and 4-(dimethylamino)pyridine were purchased from Acros, Br2 and CoCl2‚6H2O from Fisher Scientific, benzyl alcohol and Pd (10% on activated carbon) from Aldrich, Na from Mallinckrodt, KHSO5 (Oxone) from Sigma, Na2IrCl6 from Alfa Aesar, and H218O (>93.5% purity) from Icon. The nucleoside 2′,3′,5′-triacetoxy-8-oxo-7,8dihydroguanosine (1) was synthesized by a modified method (32) of Holmes et al. (50) and Matsuda et al. (51) NiCR was synthesized by the method of Karn and Busch (52). Instrumentation. 1H NMR and 13C NMR spectra were recorded on a Varian VXR-500 MHz spectrometer, FAB mass spectra on a Finnigan MAT 95 spectrometer, and ESI mass spectra on a Micromass Quatro II spectrometer. HPLC analyses were carried out using a Beckman System Gold 126NM solvent module attached to a Beckman 168NM diode arrray detector

Hydantoin Products from Oxidation of 8-OxoG

Figure 1. Reversed-phase HPLC analysis of products of oxidation of OG; see Experimental Section for details. (a) Starting material (OG, 1). (b) Reaction mixture after addition of 1 equiv of Na2IrCl6, pH 4, 40 min. (c) Product mixture after isolation of peaks A + B by silica gel chromatography. (d) Product of oxidation of OG with 4.4 equiv of Na2IrCl6 at pH 4 for 40 min.

Chem. Res. Toxicol., Vol. 14, No. 7, 2001 929 Chromatographic Purification of N1-(2′,3′,5′-tri-O-acetylβ-D-erythro-pentanosyl)-5-guanidinohydantoin (Gh, 2) and N6-(2′,3′,5′-tri-O-acetyl-β-D-erythro-pentanosyl)-2-iminoallantoin (Ia, 3). Compound 1 (425 mg, 1 mmol) was dissolved in 135 mL of water upon stirring. CoCl2‚6H2O (4 mg, 16.8 µmol) and Oxone (338 mg, including KHSO5 1.1 mmol) were added, and the reaction solution was stirred for 40 min at room temperature and then stored in the refrigerator overnight. The solution was concentrated under vacuum at room temperature. The residue was suspended in 4:1 CHCl3:MeOH (v:v) and purified by a short silica gel column using 4:1 CHCl3:MeOH (v:v) as mobile phase. The residue obtained was purified again by a short silica gel column using CHCl3:MeOH/8:1 (v:v) as mobile phase. The purity of Gh and Ia was >95% according to HPLC and NMR, and the ratio (Ia:Gh) was 1.4:1 by 1H NMR. 1H NMR (DMSO-d ) δ 11.77 (b,1H), 11.65 (b, 1H), 8.58 (b, 2H), 6 8.41 (b, 2H), 7.64 (b, 14H), 5.84 (s, 2H), 5.82 (s, 2H), 5.48-5.52 (m, 4H), 5.24-5.30 (m, 6H), 5.14 (m, 2H), 4.06-4.24 (m, 12H), 2.00-2.10 (m, 36H) ppm. 1H NMR (CD3OD) δ 5.76 (s, 1H), 5.75 (s, 1H), 5.739 (s, 1H), 5.736 (s, 1H), 5.68-5.69 (d, 2H), 5.585.61 (dd, 2H), 5.37-5.41 (m, 6H), 5.22-5.24 (dd, 2H), 4.224.36 (m, 12H), 2.08-2.11 (m, 36H) ppm. 13C NMR (CD3OD) δ 172.57, 171.75, 170.74, 170.52, 158.55, 157.23, 156.30, 86.68, 84.20, 80.31, 80.25, 72.34, 71.96, 71.57, 71.54, 66.23, 64.71, 64.37, 20.75, 20.48, 20.47 ppm. IR (KBr) ν 3368, 3159, 2360, 2341, 1745, 1680, 1653, 1614, 1437, 1385, 1241, 1110, 1045 cm-1. UV-vis at pH 7 (50 mM KPi buffer), λmax 225 nm,  ) 4229 L M-1 cm-1. In H2O at pH 3 no λmax was detected >200 nm, 220 nm ) 3260 L M-1 cm-1. FAB-HRMS m/z calcd for C15H22N5O9 416.14022, found 416.14175. Further Oxidation of Gh (2) and Ia (3) with Na2IrCl6. To a final volume of 20 µL of 75 mM potassium phosphate, Gh (2) and Ia (3) (7.5 mM total) were incubated with Na2IrCl6 (37.5 mM) for 40 min at room temperature. The reaction mixture was directly analyzed by HPLC or LC-ESI-MS (10 µL).

Figure 2. Conversion of OG (b) to products Gh + Ia (9) and Iaox (2) as a function of added IrIV as monitored by LC-MS. using an Alltech Alltima C-18 Nuc analytical reverse phase column (5 µm, 250 mm × 4.6 mm) or Waters Xterra MS C-18 analytical reversed-phase column (5 µm, 250 mm × 4.6 mm). All solvents used for HPLC were HPLC-grade and filtered and sonicated before use. All aqueous solutions utilized purified water (Nanopure, Sybron/Barnsted). Oxidation of 1 with Na2IrCl6, NiCR/KHSO5, or CoCl2/ KHSO5. Compound 1 (7.5 mM in H2O) was incubated with CoCl2‚6H2O (0.125 mM) + KHSO5 (7.5 mM) or NiCR (0.125 mM) + KHSO5 (7.5 mM) at room temperature for 40 min. In the case of oxidation with Na2IrCl6, one-fourth of the Na2IrCl6 was added every 10 min to a final concentration of 7.5 mM. The final pH of the three oxidation systems was 3-4. The reaction mixture was directly injected for HPLC or LC-ESI-MS analysis (10 µL). The reaction mixture was analyzed by HPLC with a linear gradient of 10% solvent B to 20% solvent B in 20 min at 1 mL/ min. Solvent A was 0.1% aqueous CF3CO2H (TFA); solvent B was 0.08% TFA in CH3CN, and the flow rate was1 mL/min. The chromatograms were recorded with monitoring at 220 nm. For the product distribution experiments (Figure 2), compound 1 (7.5 mM in 75 mM potassium phosphate buffer at pH 4.5) was incubated with Na2IrCl6 for 30 min. The final concentrations of Na2IrCl6 were 3.75-30 mM. Aliquots (10 µL) of the reaction mixture were analyzed by LC-ESI-MS.

1H NMR Analysis of N6-(2′,3′,5′-tri-O-acetyl-β-D-erythropentanosyl)-1,5-dehydro-2-iminoallantoin (Iaox). OG, 1, (9.4 mg, 2.21 × 10-2 mmol) was dissolved in 2.85 mL water with vortexing. A 200-µL aqueous solutionof Na2IrCl6 (54.2 mg, 9.71 × 10-2 mmol) was added in 10 portions during 2 min. After 30 min, the reaction solution was divided into 10 Eppendorf tubes and freeze-dried under vacuum for 1 h. DMSO-d6 (0.5 mL) was added to dissolve the residue and the mixture was centrifuged for 1 min. The brown solution on top of a solid residue was analyzed by 500 MHz NMR at room temperature. 1H NMR (DMSO-d6) δ 8.53 (b, 2H), 8.46 (b, 2H), 5.82 (dd, 1H), 5.69 (d, 1H), 5.43 (dd, 1H), 4.34 (m, 1H), 4.25 (m, 1H), 4.10 (m, 1H), 2.05 (s, 3H), 2.09 (s, 3H), 2.16 (s, 3H).

H218O Labeling Experiments. After 30 µL of an aqueous solution of 1 (7.5 mM) was lyophilized to dryness, H218O (30 µL) was added. Na2IrCl6 was dissolved in 1.5 µL of H218O and added into the solution over 40 min to make its final concentration 7.5 mM. The reaction mixture was analyzed by LC-ESI-MS. In the preparative reaction, 1 (3.9 mg) was dissolved in 120 µL of H218O. Na2IrCl6 (6.9 mg) in 20 µL of H218O was added in four portions over 80 min. Gh (18O) and Ia (18O) were collected by HPLC using an Alltech Alltima C-18 Nuc analytical reversedphase column (5 µm, 250 mm × 4.6 mm). The fractions were combined and lyophilized. Further acylation of Gh (2) and Ia (3). A mixture of Gh (2) and Ia (3) (1.1 mg, 2.64 µmol) was dissolved in 200 µL of CH3CN. A 5-µL CH3CN solution containing N,N-(dimethylamino)pyridine (24 µg, 0.2 µmol), 0.5 µL of acetic anhydride (5.28 µmol) and 0.8 µL of triethylamine (5.81 µmol) were added. The reaction mixture was shaken for 40 min and quenched by addition of 5 µL of CH3OH. The mixture was analyzed by ESILC-MS. The same reaction was performed with Gh (18O) and Ia (18O). The reaction mixture was analyzed by negative ion ESI-LCMS with a linear gradient of 10% solvent B to 20% solvent B in

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20 min at 0.8 mL/min, isocratic of 20% B for 5 min and then 20% B to 30% B in 10 min. Solvent A was 1 mM NH4OAc buffer (pH 8.3) and solvent B was CH3CN.

Results Synthesis and Purification of Guanidinohydantoin/Iminoallantoin Nucleosides. Since the oxidation process generally leads to more polar species, the trisacetylated ribonucleoside substrate 1 (Scheme 2, R ) 2,3,5-tri-O-acetyl-β-D-erythro-pentanosyl) was chosen to provide a somewhat longer retention time for reversedphase HPLC analysis. As previously reported, the OG nucleoside 1 is cleanly converted to the Sp nucleoside when treated with 2 equiv. Na2IrCl6 at pH 7 (KPi buffer) (32). In contrast, we find that Sp is a minor product when the reaction is carried out at pH 4, and the reaction at low pH thus mimics the oxidation of duplex oligodeoxynucleotides at room temperature and neutral pH. In the experiment, oxidation of a 7.5 mM solution of OG (1) in water at 22 °C by addition of Na2IrCl6 (final concentration 7.5 mM) led to the formation of two major stable new products, A and B, that eluted at shorter retention times, 15.0 and 15.7 min (Figure 1b), respectively, compared to OG (Figure 1a) by reversed-phase HPLC. Essentially identical results were obtained with other oxidants such as KHSO5 (7.5 mM) in the presence of a catalytic amount of CoCl2 (0.13 mM) or NiCR (53) (0.13 mM), both of which are thought to form SO4•- (54). The pH of the reaction solution ranged from 3 to 4, depending upon the oxidant. The area ratio of the two peaks, A:B, was 1.5: 1 (later identified as Gh:Ia) at pH 2 by HPLC (Figure 1b). LCESI-MS analysis of the products indicated that the two peaks represented materials of the same mass (vide infra), which was 10 amu smaller than that of OG. The similar retention times, UV spectra and mass suggested that the two resolved peaks represented either diastereomers or other isomers with very similar structures. The products could be purified from starting material and trace products by silica gel chromatography although they remained a mixture as shown by HPLC (Figure 1c). All attempts to collect one peak in preference to the other resulted in equilibration to the original mixture of two HPLC peaks. At pH 2.5, the materials of the two HPLC peaks had nearly identical UV-vis spectra (see Supporting Information), but at pH >5.8, a shoulder grew in at 226 nm for peak A vs 228 nm for peak B. The highresolution FAB mass spectrum of the mixture indicated a molecular formula of C15H21N5O9, which is consistent with either N1-(2′,3′,5′-tri-O-acetyl-erythro-pentanosyl)5-guanidinohydantoin (Gh, 2) (55) or its isomer (see Scheme 3) N6-(2′,3′,5′-tri-O-acetyl-β-D-erythro-pentanosyl)-2-iminoallantoin (Ia, 3), and clearly different from the seven-membered ring product (5) of OG oxidation with singlet oxygen reported by Sheu and Foote (21, 22), whose molecular formula would be C15H17N3O11 although its mass is the same. The mixture of Gh/Ia was readily further oxidized with excess IrIV to a material with a molecular weight of 2 amu less or -12 compared to OG. Four equivalents of IrIV were sufficient for nearly complete conversion to this species (Figure 2) which is a single broad peak (later assigned as Iaox) in the HPLC at somewhat longer retention time (Figure 1d). This material underwent hydrolysis to further products, but it could be characterized to some extent by ESI-MS/MS and 1H NMR (vide infra).

Figure 3. 500 MHz H, H COSY NMR of Gh + Ia in CD3OD. Insert: expansion of 1D-1H NMR of peaks centered at 5.75 ppm.

Figure 4. Expanded view of 500 MHz 1H NMR spectrum of Gh + Ia in CD3OD.

NMR Characterization of Guanidinohydantoin and Iminoallantoin. Integration of the 1H NMR signals suggested two isomeric species, A and B, in a 1.4:1 ratio, consistent with the1.5:1 ratio observed by HPLC monitored at 220 nm. The 1H NMR spectra in CD3OD and DMSO-d6 were complicated and hard to interpret individually. By combining 1H NMR and H,H-COSY NMR analysis, it was possible to assign all of the CH protons on the ribose (Figures 3 and 4). Use of CD3OD as a less viscous solvent gave consistently higher resolution spectra than DMSO-d6, although only DMSO-d6 permitted observation of NH protons. The spectral data obtained in CD3OD can be interpreted as follows. From the unambiguously assigned doublet 1′B at 5.69 ppm in H,HCOSY NMR (Figure 3), its cross-peak immediately indicates the position of the 2′B multiplet in the region δ 5.37-5.41 ppm. The multiplet at 5.60 ppm could only be the signal from 2′A based on the peak area and dd

Hydantoin Products from Oxidation of 8-OxoG

Chem. Res. Toxicol., Vol. 14, No. 7, 2001 931 Scheme 4

Figure 5. 500 mHz 1H NMR spectrum of Gh + Ia in DMSOd6.

splitting pattern. Starting from this 2′A signal at 5.60 ppm, we were able to determine that the signals of 1′A and 3′A were both in the region δ 5.37-5.41 ppm. This continued analysis allowed assignment of all the CH protons on the ribose (see Figure 3). What remained to be assigned were the peaks centered at 5.75 ppm for which there are no cross-peaks (Figure 3), indicating that they represent hydrogens on the base rather than the sugar. It was also known that these four peaks are not readily D2O exchangeable by 1H NMR in DMSO-d6 containing D2O. It could therefore be assigned that these four singlets represent the hydrogen at C-5 of the hydantoin ring of the epimers of Gh (2) and the hydrogen at C-5 of the iminohydantoin ring of epimers of Ia (3). These four resonances at 5.74-5.76 ppm (CD3OD) are comparable to the epimeric hydrogen atoms at C-5 of the imidazolone ring which appear as two singlets at 5.44 and 5.48 (D2O) in the diastereomers of the proposed N8-2-fluorene adduct of iminoallantoin characterized by Johnson and co-workers (56, 57) although somewhat different from the epimeric hydrogen at C-5 of iminoallantoin diastereomers reported by Tannenbaum and co-workers (5.19 and 5.30 in DMSO-d6) (16). The existence of a carbon-bound proton on the base of the nucleoside again excluded the seven-membered ring structure 5 (21, 22), which has the same mass as Gh and Ia. Moreover, the further splitting of the dd resonance of 2′A at 5.60 ppm and of 3′B at 5.23 ppm clearly showed that the resonances comprised signals of two disastereomers, i.e., a total of four isomers (Figure 4) (58). Five groups of D2O-exchangeable hydrogen resonances were observed in the 1H NMR spectrum in DMSO-d6 (Figure 5). Signals at 11.77 ppm (1H) and 11.65 ppm (1H) could be assigned as the N3-H of Gh diastereomers. Signals at 8.58 ppm (2H) and 8.41 ppm (2H) are consistent with the C2-NH2 groups of Ia diastereomers. A broad peak at 7.64 ppm (∼14H) was collectively assigned as the guanidine protons of Gh (8H for two diastereomers) and 1-NH and 8-NH2 of Ia (6H for two diastereomers). On the basis of the similar ratios of A:B obtained from the HPLC trace compared to the 1H NMR spectrum immediately following the reaction, the faster eluting peak A was assigned as diastereomers of Gh and the slower eluting peak B as those of Ia. Part of the reasoning of this assignment was the expectation that the guani-

dino group of Gh would be protonated in the pH range studied and, therefore, be eluted faster than Ia, whose guanidine moiety is acylated thereby raising its pKa. The ratio of a freshly prepared sample of Gh:Ia was 1.4:1 at pH 2.5. The diastereomeric ratios of both Gh and Ia are approximately 1:1 based on 1H NMR. The 13C NMR spectrum of Gh plus Ia in CD3OD indicated a new resonance at 66.2 ppm (see Supporting Information) that was similar to that for C-5 of allantoin [62.3 ppm in DMSO-d6 (59)]. It should be pointed out that due to the extreme structural similarity between diastereomers of Gh and Ia, some of the resonances were detected as only one signal both in the 1H NMR and 13C NMR spectra. For example, only one signal at 66 ppm was detected for C5 to represent the diastereomers of Gh and Ia (see Supporting Information). Mass Spectrometric Analysis of Guanidinohydantoin and Iminoallantoin. Although NMR spectroscopy provided important structural information, we sought further support for the structures of Gh and Ia from an LC-ESI-MS/MS fragmentation study. Initially, the two HPLC peaks A and B were analyzed by positive ion mode LC-ESI-MS/MS since the peaks showed nearly baseline separation on reversed-phase HPLC (0.1% TFA in H2O + 0.08% TFA in CH3CN gradient). Interestingly, both peaks gave identical fragmentation patterns (data not shown). This result is consistent with two interpretations: (i) the two HPLC peaks represent C5 epimers of a single constitutional isomer, either Gh or Ia, or (ii) the two peaks separately represent Gh and Ia, but they undergo rapid isomerization in the gas-phase MS experiment, thereby giving identical fragmentation. To further investigate the latter possibility, we sought to prevent isomerization via N-acylation (Scheme 4). In the experiment, the A + B purified mixture (Figure 1c) was treated with 2.2 equiv. acetic anhydride to produce mainly diacylated materials of mass ) 499. The acylated products were more conveniently studied by negative ion LC-ESI-MS/MS, and so the ions of m/z 498 were selected in MS-1, further fragmented, and analyzed in MS-2. Two major peaks in the HPLC trace of the acylated materials displayed m/z 498, and their fragmentation data are shown in Figures 6 and 7 (top). For the diacylated product in Figure 6, a structure is proposed in which both acyl groups are on the guanidine group. The fragmentation pattern is consistent with this assignment, since the major one-bond fragmentation products (m/z 83, 142, and 413) correspond to bond cleavages as shown in Figure 6 (dashed lines). For example, loss of the neutral carbodiimide fragment (CH2N2Ac) of mass 85 by homolytic cleavage of the C-N bond of the molecular anion (mass 498) yields the relatively abundant 413 ion.

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Figure 6. Negative ion LC-ESI-MS/MS data for GhAc2. (Top) Oxidation of OG carried out in H216O. (Bottom) Oxidation of OG carried out in H218O for isotopic incorporation (b).

Figure 7. Negative ion LC-ESI-MS/MS data for IaAc2. (Top) Oxidation of OG carried out in H216O. (Bottom) Oxidation of OG carried out in H218O for isotopic incorporation (b).

If an analogous fragmentation occurs heterolytically, the neutral fragment CHN2Ac could be deprotonated to form the observed base peak at 83. In a related fragmentation study of the Sp nucleoside 4, we noted that the new oxygen atom introduced upon oxidation of OG was derived from H2O (32). This allowed preparation of specifically labeled hydantoin products through the use of H218O. When LC-ESI-MS/MS analysis was repeated on an 18O-labeled material in the present study, the 413 peak shifted to 415 as expected (Figure 6, bottom). When the other major peak in the HPLC representing diacylated material was analyzed in the same fashion, the data shown in Figure 7 were obtained. The fragmentation is most consistent with the diacyliminoallantoin structure shown. A simple fragmentation of one C-N bond accounts for the two major fragment ions (138 and 359) observed. Since only the 138 ion shifts by 2 mass units in the H218O experiment, this fragment must be derived from the hydantoin ring. The structures presented in Figures 6 and 7 represent the simplest interpretations of the data. While other possibilities exist, any other structures would require at least two bond fragmentations of the hydantoin ring to explain the isotope patterns observed. pH-Dependent Equilibration and Degradation of Gh and Ia. Oxidation of OG at pH 3-4 followed by immediate reversed-phase HPLC analysis in 50 mM potassium phosphate buffer (pH 2) showed an initial ratio of the peaks A:B of 1.4:1. However, this ratio slowly changed reaching an equilibrium value of 1:1.2 after

storing the mixture for several weeks at -20 °C. In the course of examining HPLC separation conditions at various pHs, it was found that the area ratio of A compared to B decreased as the pH of the mobile phase increased over the range pH 2.5-5.8. This was suggestive of a pH-dependent equilibration between the two isomers in which A reaches its maximum at low pH, although B is generally the major thermodynamic product. At pH 5.8, the A:B ratio was 1:1.6. From pH 5.8 to 6.8, peak B underwent a shift to shorter retention time finally overlapping with peak A (see Supporting Information) (60). The pH-dependent equilibration of A and B was fully reversible. Preliminary experiments were carried out to determine how fast the equilibration occurred between A and B. After raising the pH 10 in a buffered solution for 17 h and then readjusting the pH back to 2 with H3PO4, it was found that 2 h were required to reestablish the stable 1:1.2 ratio of A:B. Overall, the behavior of this system is consistent with the assignment of peak A as guanidinohydantoin (Gh, 2), formed initially to a higher extent than peak B, iminoallantoin (Ia, 3), as expected from the mechanism of its formation. The isomerization of Gh to Ia shown in Scheme 3 is reminiscent of the behavior of allantoin isomerization between the two open structures through a symmetrical bicyclic intermediate 14 (Scheme 5a) (44, 61). It also parallels the product isomerization of the aerial oxidation reaction of 8-arylamine derivatives of guanosine in which the guanidinohydantoin form was favored in acidic conditions while the iminoallantoin analogue was favored under basic conditions (Scheme 5b) (56, 57). The Gh nucleoside would be expected to be protonated in the pH range studied here, and protonation would also reduce the nucleophilicity of the nitrogen undergoing attack at the C4 carbonyl of the hydantoin ring. The abrupt change in retention time of peak B (Ia) in the pH range 5.8-6.8 suggests that a functional group of Ia is undergoing a change in protonation. This could also be monitored by UV spectroscopy. At pH 5.8 (50 mM KPi), Gh shows λmax at 226 nm while Ia has λmax at 224 nm (see Supporting Information). In a titration experiment, a peak grows in rather dramatically at 224 nm as the pH is increased in from 5.6 to 6.8 (see Supporting Information). An increase in  at this wavelength would be consistent with deprotonation of the exocyclic ammonium of Ia near pH 6, providing more extensive conjugation in the β-amino-enone chromophore of Ia.

Hydantoin Products from Oxidation of 8-OxoG

Chem. Res. Toxicol., Vol. 14, No. 7, 2001 933

Scheme 6

Figure 8. 500 mHz 1H NMR spectrum of Iaox in DMSO-d6.

We reported earlier that the products of OG oxidation by IrIV are piperidine-labile lesions leading to deglycosylation and DNA strand scission in oligodeoxynucleotides (18, 19, 31). Treatment of the Gh + Ia mixture at pH 10 indicated half-lives of Ia ) 29 h and Gh ) 23 h at room temperature as analyzed by HPLC. Incubation of the mixture of Gh and Ia in pH 10 phosphate buffer at 90 °C for 5 min led to 87% degradation, while at pH 12, Gh and Ia were decomposed within one min. This is consistent with the observation of piperidine-induced strand scission under Maxam-Gilbert conditions (90 °C, 30 min) for oligodeoxynucleotides containing an M - 10 lesion from OG oxidation (31), although some of the decomposition may be due to hydrolysis of the acetyl groups of the ribose rather than deglycosylation alone. Characterization of Further Oxidation/Hydrolysis Products of Gh and Ia. Vialas et al. proposed that the oxometalloporphyrin system Mn-TMPyP/KHSO5 could further oxidize a guanidinohydantoin intermediate that was formed by oxidation of guanine in an oligonucleotide (62); however, the further oxidized product was not very stable. To compare this process with a stepwise oneelectron oxidation, we examined the reaction of Gh + Ia with additional IrIV. Even in the fresh reaction sample of OG oxidized with only 1 equiv of IrIV, both in water (pH 4) and in pH 7 buffer, a small amount of further oxidized material was detected at 23 min retention time by HPLC (Figure 1b). When the purified mixture of Gh + Ia (Figure 1c, 7.5 mM in 75 mM KPi, pH 7) was treated with 37.5 mM Na2IrCl6 at room temperature for 40 min and analyzed by HPLC, it was found that more than 90% of Gh + Ia was oxidized to form Ghox (17) and/or Iaox (18), having a mass of 413 (Scheme 6). High resolution FABMS was consistent with the molecular formula of these isomers. Only one new peak was observed by HPLC, and all attempts to resolve it into more than one peak failed. The same peak could be obtained by incubating OG directly with 4.4 equiv Na2IrCl6, and in this case, no Gh or Ia was observed by HPLC (Figure 1d), although they were likely intermediates in the formation of the final oxidized product. In competition studies with the 8-oxo-7,8-dihydroadenosine (OA) analogue of 1, it was found that OA was

always more readily oxidized than Gh + Ia at either pH 4.5 or pH 7 by about a factor of 3-7 (see Supporting Information). The redox potential for the OA nucleoside has been reported to be 0.92 V vs NHE (11), which would place the potential for Gh/Ia near 1.0 V. All oxidations were more efficient at pH 7 compared to pH 4.5, consistent with the notion that neutral amine lone pairs will be more readily oxidized than protonated species (see Supporting Information). The oxidized product was relatively unstable in aqueous solution, but the 1H NMR spectrum of the fresh reaction mixture in DMSO-d6 indicated that there was only one major compound as product (Figure 8). Two D2O exchangeable proton resonances were observed. The signals at 8.4 ppm (2H) and 8.5 (2H) could be best described as the two NH2 groups of Iaox (18) (63). We propose that Ia should be somewhat easier to oxidize than Gh (present mainly as GhH+), thereby shifting the Gh a Ia equilibrium toward the formation of Iaox exclusively. Poje et al. found that oxidized allantoin was not able to undergo equilibrating isomerization through a bicyclic intermediate (Scheme 5d) (44). Our observations are inconclusive regarding this hypothesis; either Iaox is the strongly favored species in the equilibrium (Scheme 5d) or Iaox (18) does not isomerize to Ghox (17) at all, although the reverse (Ghox f Iaox) is not excluded. When Iaox (possibly containing a trace of Ghox) was incubated in potassium phosphate buffer (pH 4-7) for 3 h, oxaluric acid riboside, 10 (MH+ ) 391), was observed by LC-MS as the major product, and only a trace of parabanic acid, 8, (MH+ ) 373), possibly formed from hydrolysis of a small amount of Ghox, was observed (Scheme 6). When Iaox was incubated in concentrated ammonia solution, diaminoimidazolone (11, deglycosylated), MH+ ) 113, was observed after 3 min by HPLC and LC-MS. Formation of 11 is further evidence for the assignment of the Iaox rather than the Ghox structure to the major product of Gh/Ia oxidation. Additional Mechanistic Studies. The oxidation of OG conducted under an argon atmosphere gave essentially the same HPLC trace indicating that oxygen is not a significant oxidant in the system. Since the use of 2 equiv of IrIV led to nearly complete conversion of OG to products, it is clear that IrIV is the only oxidant involved in formation of Gh + Ia. Injection of the fresh reaction mixture obtained from oxidation of OG with 1 equiv of Na2IrCl6 after 15-30 min reaction time (75 mM KPi buffer, pH 4) into LC-ESI-MS, revealed two additional transient LC peaks eluting at 25.2 and 26.2 min and having mass M + 16 compared to OG (Figure 1b). Essentially, the same intermediates were observed when oxidation of OG was carried out at pH 7, which would produce Sp as the major product (32). A

934

Chem. Res. Toxicol., Vol. 14, No. 7, 2001 Scheme 7

detailed analysis of these peaks could not be carried out because of their small quantities and instability, but both showed masses of 442 while having significantly longer retention times than Gh and Ia. One possible explanation is that they represent epimers of the common precursor to Gh and Sp, namely 5-OH-OG, 13 (Scheme 2). Use of H218O (>93.5% purity) as solvent in oxidation reactions of OG led to >90% incorporation of 18O into the parent molecular ion (M + H+) of Gh and Ia as well as the proposed intermediate 5-OH-OG (13). This confirmed that water trapped the reactive radical species generated upon oxidation by IrIV, and that O2 is not involved in the reaction. The further oxidized species Iaox also retained one atom of 18O initially incorporated from solvent.

Discussion The iridium(IV) salts Na2IrCl6 and Na2IrBr6 undergo one-electron reduction to the corresponding iridium(III) species with concomitant one-electron oxidation of OG to form OG•+. The stability of the radical cation of OG should be much greater than that of G•+ due to the 0.5 V difference in redox potentials of G vs OG. G•+ with a pKa of 3.9 (64, 65) undergoes rapid deprotonation as a nucleoside, and the subsequent chemistry of G• is then dominated by its reaction with O2, ultimately forming the imidazolone product Iz (6). In contrast, OG•+ has a pKa of 6.6 (10), is slower to deprotonate and does not appear to react rapidly with O2. We propose the pathway shown in the top of Scheme 7 to account for the formation of the intermediate, 5-OH-OG (13), by facile one-electron oxidation and proton loss from the initially formed adduct 20 of water adding to OG•+. However, we cannot rule out the alternative pathway in which two sequential oneelectron oxidations occur using 2 equiv of IrIV prior to attack of a solvent water at C5 of cation 22. In any case, the two-electron oxidized species 5-OH-OG (13) appears to be the initial product in this cascade. The subsequent bifurcation of the pathway from 5-OHOG to two different types of products, a spiroiminodihydantoin product 4 vs ring-opened products Gh and Ia is highly dependent on pH for nucleosides and upon temperature and base stacking in the case of oligodeoxynucleotides (Scheme 8) (33). Both of these pathways are precedented elsewhere, the chief prototype being urate oxidation. A detailed mechanism for the pathway of 5-hydroxyurate conversion to allantoin has been proposed by Tipton and co-workers (48, 49), and the spirodihydantoin side product of urate oxidation was observed by Poje et al. (47). Similarly, the aerobic oxidation of 8-arylamine adducts of G support the general observation of 5-hy-

Luo et al. Scheme 8

droxypurines leading to both acyl migration to a spiro product that is preferred at high temperature compared to ring-opening and decarboxylation to allantoin-type products at lower temperature (56, 57, 66). The more facile hydrolysis of the N1-C6 bond in intermediate 13 compared to G or OG is likely a function of the dearomatization that has occurred as a result of C-5 hydroxylation. Furthermore, the pKa of the acylguanidine moiety of 13 should be higher than that of G or OG, and protonation of N1 or N3 would facilitate hydration to 23 and ring opening to 24 (Scheme 8) (67), accounting for the preference of this pathway leading to Gh and Ia under acidic conditions compared to formation of Sp. In urate oxidation, Tipton and co-workers have also obtained evidence for an acyl migration akin to the 24 f 25 rearrangment (48), but we have no data to support or refute this step. In any case, decarboxylation of either 25 or 24 would lead to 2 or its enol tautomer 2′, respectively. From a mechanistic standpoint, guanidinohydantoin should be the initially formed product of mass OG-10, and indeed the more rapidly eluting peak A in the HPLC chromatogram has been assigned to this structure (Figure 1b). This is based partly on the notion that the guanidino group should have a pKa > 12, and Gh should therefore be protonated at all pHs studied here, making it the more rapidly eluting substance in reversed-phase HPLC. In addition, the NMR spectrum in DMSO-d6 is more consistent with this assignment. Upon standing, Gh equilibrates to Ia, to reach a pH-dependent equilibrium mixture of the two isomers, with Gh achieving its highest concentration at lower pH via conversion to GhH+. Each isomer is furthermore present as an approximately 50:50 mixture of two C-5 epimers. That four total isomers are present in the Gh + Ia reaction mixture is evident from the number of 1H resonances observed in the spectrum obtained under the highest resolution

Hydantoin Products from Oxidation of 8-OxoG

conditions using CD3OD as solvent. The pattern of NH protons in the spectrum obtained in DMSO-d6, the ESIMS/MS fragmentation patterns of the further acylated products GhAc2 + IaAc2, and the oxidation to Iaox are all further evidence for the formation of iminoallantoin in addition to guanidinohydantoin as a product of OG oxidation. Furthermore, Gh and Ia have different UV spectra with different pH-dependent features, which would be a surprising phenomenon if only two epimers of one or the other species were present. Instead, we believe that the predominant tautomer of Ia bearing an endocyclic CdN bond would likely undergo protonation of the exocyclic amino group at pH