Peroxynitrite reacts with 8-oitropurines to yield 8-oxopurines

Article abstract of DOI:10.1021/tx010093d

Peroxynitrite reacts with 2′-deoxyguanosine to yield several major products, including 8-oxo2′-deoxyguanosine (8-oxodG) and 8-nitroguanine (8-nitroGua). While the terminal products formed during the reaction of 8-oxodG with peroxynitrite have been previously characterized, those formed from 8-nitroGua have not. To identify these products, 9-ethyl-8-nitroxanthine was used as a model for 8-nitroGua, since the former could be easily synthesized in high yield, and facilitated reversed-phase HPLC separation of the resulting products. Using this model substrate, the products formed during the peroxynitrite reaction were identified as the ethyl derivatives of oxaluric acid, 5-iminoimidazolidin-2,4-dione, III, [N-nitro-N'-[2,4-dioxo-imidazolidine-5-ylidene]-urea, V, dehydroallantoin, parabanic acid, cyanuric acid, and uric acid. Upon the basis of the previous studies with 8-oxodG, these products were recognized as those expected to arise from peroxynitrite-mediated uric acid oxidation. Furthermore, the presence of uric acid in the reaction mixture led us to propose a model in which the 8-nitropurine is first converted to the 8-oxopurine which is further oxidized by peroxynitrite to give the observed final products. We have also provided evidence suggesting that the peroxynitrite anion, acting as a nucleophile, might be responsible for the initial conversion of the 8-nitropurine to the 8-oxopurine and that a hydroxyl radical or oxidative process is less likely to explain this conversion.

Full text of DOI:10.1021/tx010093d

Chem. Res. Toxicol. 2002, 15, 7-14
7
P er oxyn itr ite Rea cts w ith 8-Nitr op u r in es to Yield
8-Oxop u r in es
J oseph M. Lee,† J acquin C. Niles,† J ohn S. Wishnok,† and
Steven R. Tannenbaum*,†,‡
Division of Bioengineering and Environmental Health and Department of Chemistry,
Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
Received May 15, 2001
Peroxynitrite reacts with 2′-deoxyguanosine to yield several major products, including 8-oxo-
2′-deoxyguanosine (8-oxodG) and 8-nitroguanine (8-nitroGua). While the terminal products
formed during the reaction of 8-oxodG with peroxynitrite have been previously characterized,
those formed from 8-nitroGua have not. To identify these products, 9-ethyl-8-nitroxanthine
was used as a model for 8-nitroGua, since the former could be easily synthesized in high yield,
and facilitated reversed-phase HPLC separation of the resulting products. Using this model
substrate, the products formed during the peroxynitrite reaction were identified as the ethyl
derivatives of oxaluric acid, 5-iminoimidazolidin-2,4-dione, III, [N-nitro-N′-[2,4-dioxo-imida-
zolidine-5-ylidene]-urea, V, dehydroallantoin, parabanic acid, cyanuric acid, and uric acid. Upon
the basis of the previous studies with 8-oxodG, these products were recognized as those expected
to arise from peroxynitrite-mediated uric acid oxidation. Furthermore, the presence of uric
acid in the reaction mixture led us to propose a model in which the 8-nitropurine is first
converted to the 8-oxopurine which is further oxidized by peroxynitrite to give the observed
final products. We have also provided evidence suggesting that the peroxynitrite anion, acting
as a nucleophile, might be responsible for the initial conversion of the 8-nitropurine to the
8-oxopurine and that a hydroxyl radical or oxidative process is less likely to explain this
conversion.
acid, and cyanuric acid (21, 22). Three key intermediates,
I, II, and IV, shown in Figure 1, were also identified. I
and II were shown to undergo hydrolysis to yield para-
banic and oxaluric acid, via III, while IV underwent
hydrolysis to cyanuric acid. The results at the nucleoside
level were reproduced in 8-oxoG-containing double
stranded oligonucleotides treated with peroxynitrite, with
the exception that no parabanic acid was detectable (23).
In tr od u ction
During chronic inflammation and infection, activated
immune cells release increased amounts of reactive
oxygen and nitrogen species, which can lead to tissue
damage (1-3). Peroxynitrite, a one- and two-electron
oxidizing and nitrating agent, is one such toxic agent that
can directly modify a wide range of biological targets,
including lipids (4), proteins (5-9), and DNA bases/
sugars (10, 11). Peroxynitrite is formed from the diffu-
sion-limited reaction between nitric oxide and superoxide
(12, 13), and at the nucleoside and DNA levels, it reacts
almost exclusively with Gua and dG, respectively (14, 15).
Several oxidation and nitration products, illustrated in
Figure 1, are formed including 8-nitroguanine (8-ni-
troGua) (11), 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-
oxodG) (16), 4,5-dihydro-5-hydroxy-4-(nitrosooxy)-gua-
nine (nox-dG) (17), and 2,2-diamino-4-[(2′-deoxy-â-D-
erythro-pentofuranosyl)amino-5-(2H)-oxazolone, dX (18-
20).
It was subsequently shown that 8-nitroGua (14) and
8-oxodG (14, 16) were not only major peroxynitrite
products but also several orders of magnitude more
reactive than dG toward peroxynitrite. The reaction of
peroxynitrite with 8-oxodG has been studied, and the
final products have been characterized as the â-D-erythro-
pentofuranosyl deriviatives of oxaluric acid, parabanic
* To whom correspondence should be addressed. Phone (617) 253-
3729. Fax: (617) 252-1787. E-mail: srt@mit.edu.
† Division of Bioengineering and Environmental Health.
‡ Department of Chemistry.
8-NitroGua is formed in a dose-dependent fashion in
vitro when Gua-containing oligonucleotides have been
treated with peroxynitrite (24). In studies by Tretyakova
10.1021/tx010093d CCC: $22.00 © 2002 American Chemical Society
Published on Web 12/22/2001

8
Chem. Res. Toxicol., Vol. 15, No. 1, 2002
Lee et al.
F igu r e 1. Structures of 8-oxodG, 8-nitroGua, nox-dG, dX, and the former two products peroxynitrite products (R ) ethyl except for
8-oxodG, dX, and nox-dG where R′ ) 3′,5′-di-O-Ac-beta-D-erythropentafuranosyl).
(Andover, MA). Peroxynitrite was prepared as described by
Pryor et al. (29), and the concentration was determined by
measuring its UV absorbance (ꢀ302nm ) 1670 M-1 cm-1) (30).
Partisil ODS-40 (C18) packing material and C18 Sep-packs were
acquired from Whatman (Springfield Mill, U.K.) and Varian
(Harbor City, CA), respectively. All solvents were HPLC-grade.
In str u m en ta tion . UV-vis spectra were obtained using an
HP8452 Diode Array Spectrophotometer (Hewlett-Packard). 1H
and 13C NMR (proton decoupled) spectra were obtained using a
Unity 300 spectrometer at 300 and 75 MHz, respectively. HPLC
analyses were performed using an HP 1100 pump equipped with
an 1090 Diode Array Detector (Hewlett-Packard, Palo Alto, CA).
All HPLC and LC-MS runs were carried out using either a 250
× 4.6 mm, 5 µm LC18-DB column (Supelco, Bellefonte, PA) or
a 30 cm × 1 mm, 5 µm LC18-DB column (Supelco). Electrospray
ionization mass spectrometry (ESI-MS), tandem mass spec-
trometry (ESI-MS/MS), and ion trap mass spectrometry experi-
ments were performed using an HP 5989B (Hewlett-Packard),
a TSQ 7000 (Finnigan, San J ose, CA), and an 1100 LC/MSD
Ion Trap (Agilent, Palo Alto, CA), respectively. FT-MS spectra
were obtained using ESI in negative ions mode on an APEX II
FT-MS (Bruker, Billerica, MA). Spectra were obtained in either
positive or negative ions mode using a spraying solution
consisting of a 50:50 water-methanol solution spiked with 0.02%
glacial acetic acid. Hydroxyl radical was generated in situ by
irradiating N2O-flushed aqueous samples with γ-rays emitted
at 2.36 Gy/min by a Gammacell-220 machine equipped with a
60Co source.
For the 250 × 4.6 mm, 5 µm LC18-DB column, two HPLC
methods were employed. Method A: isocratic elution at 1.0 mL/
min, using a mixture consisting of 90% 100 mM triethylammo-
nium acetate (TEAA), pH 7 and 10% methanol. Method B: 100%
20 mM ammonium acetate, pH 7 for 10 min, followed by an
acetonitrile gradient from 0 to 25% over the next 15 min with
a flow rate of 1.0 mL/min. Similarly, for the 30 cm × 1 mm, 5
µm LC18-DB column, two HPLC methods, C and D were used.
In Methods C and D, respectively, the column was isocratically
eluted with water and 20 mM ammonium acetate, at a flow rate
of 0.06 mL/min.
Syn th esis of 8-Nitr ogu a . 8-nitroGua was synthesized by
stirring 8-bromoguanosine (1 g, 2.7 mmol) with five equivalents
of sodium nitrite (0.95 g, 14 mmol) under reflux in DMF at 60
°C overnight to produce both 8-nitroGua and 8-nitroxanthine.
The DMF reaction mixture was next concentrated in vacuo. The
dried orange solid was dissolved in 100 mM TEAA (pH 7) and
loaded onto a 2 cm × 8.5 cm MegaBond Elut C18 sep pack
et al., 8-NitroGua formation reached a maximum at a
peroxynitrite concentration of 400 µM and subsequently
declined with increasing peroxynitrite concentration (25).
When peroxynitrite reacts with DNA, the 8-nitrodG
formed rapidly depurinates [t1/2 ) 1-4 h at neutral pH
and room temperature (25)] to yield the measurable
8-nitroGua. However, since complete depurination of
8-nitrodG was ensured prior to measurement of 8-ni-
troGua, the decline in 8-nitroGua formation could not be
attributed to incomplete depurination. Instead, this
result suggested that both at the nucleobase and DNA
level, 8-nitroGua can undergo further reaction with
peroxynitrite, leading us to hypothesize that similar to
the 8-oxodG/peroxynitrite reaction, peroxynitrite can
induce formation of 8-nitroGua oxidation and nitration
products.
In this paper, we have studied the reactions of 8-ni-
troGua, 8-nitroGuo and 9-ethyl-8-nitroxanthine with
peroxynitrite in an effort to elucidate the final products
and the mechanism by which this chemistry occurs. We
found 9-ethyl-8-nitroxanthine to be a useful model com-
pound in these studies since it is readily synthesized in
high yield, recapitulates 8-nitroGua chemistry almost
identically, and along with its oxidation products, is
amenable to reversed-phase chromatography. In addition,
we have provided in this paper a reproducible method
for synthesizing 8-nitroGua, and have characterized this
compound directly by several spectroscopic methods,
instead of by the indirect methods that have been
previously employed (11, 24, 26-28).
Exp er im en ta l P r oced u r es
Gen er a l. Guanosine, 4-amino-5-imidazole-carboxyamide
(AICA), benzoyl isothiocyanate, copper (II) acetate, bromoet-
hane, sodium nitrite, and 15N nitric acid (70 wt %, 98 atom %
15N) were obtained from Aldrich (Milwaukee, WI). Uric acid,
9-methyluric acid, 8-aminoguanosine, 8-bromoguanosine, and
2′-deoxyguanosine were obtained from Sigma (St. Louis, MO).
Acetic anhydride, pyridine, dimethylformamide (DMF), dimethyl
sulfoxide (DMSO), and hydrazine, were purchased from either
Mallinckrodt (Paris, KY) or Aldrich. NMR solvents, DMSO-d6
and D2O, were obtained from Cambridge Isotope Laboratories

Peroxynitrite Reactions to Yield 8-Oxopurines
Chem. Res. Toxicol., Vol. 15, No. 1, 2002
9
(Varian) that was preequilibrated with 100 mM TEAA. The
desired products were eluted with 100 mM TEAA buffer, while
the unreacted starting material was later eluted with methanol.
The fractions containing the products were concentrated and
further purified on a 3 cm × 18 cm ODS-40 C18 column that
was preequilibrated and eluted under gravity with 100 mM
TEAA. The second eluting yellow band was collected, concen-
trated, and rechromatographed on a 2 cm × 8.5 cm C18 Sep
Pak with ddH2O as the eluent to remove excess salts in and
obtain pure 8-nitroGua. Yield: 0.027 g, (0.14 mmol, 5.2%). 1H
NMR (DMSO-d6, δ) 10.25 ppm (s, 1H, NH), 5.89 ppm (s, 2H,
NH2); 13C NMR (DMSO-d6, δ) 159.42, 158.76, 157.60, 151.77,
and 121.27 ppm; HRMS calculated for C5H3N6O3 [M-H]-
195.0267, observed 195.0269; UV-vis (H2O) λmax ) 400 nm;
HPLC Method A, single peak eluting at 4 min.
Syn th esis of 8-n itr oGu o. Using a similar strategy as above,
8-nitroGuo was prepared by stirring 8-bromoguanosine (31) (1
g, 2.8 mmol) with 1.5 equiv potassium nitrite (0.35 g, 4.2 mmol)
in DMF at room temperature for 2 days. 8-NitroGua and
8-nitroxanthine were also present as side products. Using the
same purification steps outlined above, 8-nitroGuo was purified
and desalted. Yield: 0.045 g, (0.14 mmol, 3.3%). ESI-MS 327,
[M - H]-; UV-vis (H2O): λmax ) 394 nm; HPLC Method A,
single peak eluting at 12.3 min.
results and devised another synthetic route involving the
nitrite-mediated Br- displacement from 8-bromogua-
nosine to give 8-nitroGuo and 8-nitroGua by depurination
1
of the former. UV-vis spectroscopy, FT-MS, H NMR,
and13C NMR were used to characterize the 8-nitroGua.
In agreement with previous reports, 8-nitroGua has a
maximum UV-vis absorbance at 400 nm and is ex-
tremely stable even at pH 1-14 and elevated tempera-
tures (up to 100 °C). An exact mass of 195.0267 calculated
for the formula C5H3N6O3 [M - H]- ion was consistent
with the observed 195.0269. 1H NMR carried out in
DMSO-d6 revealed two resonances at 10.25 ppm (1H) and
5.89 ppm (2H), corresponding to the N1 proton and the
exocyclic N2 amino protons, respectively. Noticeably, the
resonance at 8 ppm corresponding to the C8-H in the
parent Guo is absent, as would be expected for 8-ni-
troGua.
Rea ction s of P er oxyn itr ite w ith 8-Nitr oGu a a n d
8-Nitr oGu o. When 8-nitroGua (100 µM) was treated
with peroxynitrite (0.5-5 mM), there was a dose-depend-
ent decrease in the amount of 8-nitroGua remaining at
the end of the reaction. At 5 mM peroxynitrite, all the
8-nitroGua had been destroyed. The increased destruc-
tion of 8-nitroGua was accompanied by the appearance
of at least four new peaks during HPLC analysis.
Unfortunately, these products were extremely polar and
very poorly resolved by reversed-phase chromatography.
In an effort to improve our chromatography, we synthe-
sized 8-nitroGuo, which itself is well retained on a
reversed-phase column (retention time ) 12.3 min using
HPLC Method A). However, we encountered several
problems with this strategy. First, 8-nitroGuo will un-
dergo depurination in aqueous solution, albeit slowly at
room temperature, at a rate of 0.6%/h. Second, addition
of peroxynitrite leads to rapid and near complete depu-
rination despite there being no change in the pH of the
reaction mixture. Consequently, the peroxynitrite prod-
ucts observed using 8-nitroGuo were the same as those
between 8-nitroGua with peroxynitrite. Hence, 8-ni-
troGuo did not offer any advantage over using 8-ni-
troGua. As a result, we turned our attention toward
finding a suitable substrate that did not undergo depu-
rination, but was amenable to reversed-phase chroma-
tography, readily synthesized and expected to undergo
chemistry similar to that of 8-nitroGua.
Syn th esis of 9-Eth ylgu a n in e. 9-Ethylguanine was synthe-
sized according to a previously published report (32). ESI-MS
180, [M + H]+; UV-vis (H2O): λmax ) 272, 252 nm; HPLC
Method B, single peak eluting at 19 min.
Syn t h esis of 9-E t h yl-8-n it r oxa n t h in e. 9-Ethyl-8-nitro-
xanthine was prepared by refluxing 9-ethylguanine (0.501 g,
2.8 mmol) in glacial acetic acid (10 mL) and nitric acid solutions
(1 mL, 70%) at 132 °C for 50 min, followed by careful rotary
evaporation of the greenish solution to apparent dryness on an
ice bath. The product was taken up in ddH2O and desalted on
an ODS-40 (C18) 3 cm × 18 cm column that was preequilibrated,
washed, and eluted with water. Yield: 0.6 g, (2.8 mmol, 100%).
ESI-MS: 224, [M - H]-; UV-vis (H2O): λmax ) 410 nm; HPLC
Method B, single peak eluting at 23.3 min. Likewise, 9-ethyl-
8-15NO2-xanthine was synthesized using 1 mL of a 40 wt % nitric
acid (98% atom 15N) solution. Yield: 0.6 g, (2.8 mol, 100%). ESI-
MS: 225, [M - H]-; UV-vis (H2O): λmax ) 412 nm; HPLC
Method B, single peak eluting at 23.3 min.
Rea ction of 8-Nitr o a n d 8-Oxop u r in es w ith P er oxyn i-
tr ite. The reaction of peroxynitrite with several modified DNA
bases was analyzed by measuring either the loss of starting
material or by the detection of new products using HPLC and
LC-MS. These reactions were carried out in a 1 mL eppendorf
tube containing 0.5-5 mM peroxynitrite and 100 µM 8-ni-
troGua, 8-nitroGuo, or 9-ethyl-8-nitroxanthine (14NO2 and 15NO2
labeled), in 120 mM potassium phosphate, 25 mM sodium
bicarbonate, pH 7.2 buffer.
Syn th esis of 9-Eth yl-8-n itr oxa n th in e a s a Mod el
Com p ou n d . We chose to use 9-ethyl-8-nitroxanthine to
model the reaction of 8-nitroGua with peroxynitrite.
First, since this compound is alkyl substituted at the N9,
it will not undergo depurination, as would the N9-â-D-
erythro-pentofuranosyl-substituted compound. Second,
the precursor 9-ethylguanine is readily afforded in good
yield by a previously reported synthesis (32). Third, C8-
nitration of 9-ethylguanine using concentrated nitric and
glacial acetic acid gives quantitatively, and in one step,
the 9-ethyl-8-nitroxanthine, in contrast with the low
yields (∼5%) expected for the route to the corresponding
9-ethyl-8-nitroGua via the nitrite-mediated displacement
of Br- from 9-ethyl-8-bromoguanine. Reversible protec-
tion of the N2-exocyclic amino group of 9-ethylguanine
during the reaction with concentrated nitric and glacial
acetic acid was not a viable option, since most of these
protecting moieties contain acyl groups that are readily
hydrolyzed under these harsh nitrating conditions. Fi-
nally, 9-ethyl-8-nitroxanthine is more water-soluble than
R ea ct ion of 9-E t h yl-8-n it r oxa n t h in e w it h H yd r oxyl
Ra d ica l. Samples containing 9-ethyl-8-nitroxanthine (100 µM)
in 100 mM phosphate buffer (pH 7.4), were prepared in 1.5 mL
eppendorf tubes and sealed with rubber septae. The tubes were
flushed with a 9:1 nitrous oxide (N2O):oxygen gas (O2) mixture
for five minutes, prior to irradiation with up to 1000 Gy. HPLC
and off-line ESI-MS were used to analyze the reaction mixture.
R ea ct ion of 9-E t h yl-8-Nit r oxa n t h in e w it h H yd r oxyl-
a m in e. The 9-ethyl-8-nitroxanthine standard (100 µM) was
incubated with hydroxylamine (15 µM to 1.5 M) in 0.1 N KOH
(pH 13) for up to 4 h, to determine whether nucleophilic
displacement of the nitro group occurred. The reaction was
monitored by UV-vis spectroscopy at 412 nm (λmax for 9-ethyl-
8-nitroxanthine).
Resu lts a n d Discu ssion
Ch a r a cter iza tion of 8-Nitr oGu a a n d 8-Nitr oGu o.
The synthesis of 8-nitroGua via the reaction of 8-diazo-
guanine with sodium nitrite has previously been reported
(33-35). However, we were unable to reproduce these

10 Chem. Res. Toxicol., Vol. 15, No. 1, 2002
Lee et al.
account for the presence of an amino group in the 3′,5′-
di-O-Ac-8-oxodG versus a hydroxyl group in 9-ethyl-8-
nitroxanthine.
Ch ar acter ization of th e P r odu cts Elu tin g in P eaks
1-5. Table 1, parts a and b, summarizes the results of
the comparison between the products arising from the
reaction of 3′,5′-di-O-Ac-8-oxodG and 9-ethyl-8-nitro-
xanthine with peroxynitrite. Using HPLC Method B,
peak 1 was not sufficiently resolved from the solvent front
to facilitate ESI-MS analysis. However, when the peak
was collected, dried in vacuo and reanalyzed by LC-MS
using Method D, two new peaks arose and were identified
as 1-ethyl-5-iminoimidazolidin-2,4-dione, III and 1-ethyl-
oxaluric acid. For peak 2, we were also unable to
ascertain its molecular ion weight since it eluted at the
end of the solvent front. However, this compound has a
characteristic UV spectrum shown in Figure 3, with λmax
at 228 and 264 nm, that is essentially identical to that
reported for II (22) as illustrated in Figure 3. Thus, we
conclude that peak 2 corresponds to [N-nitro-N′-[1-ethyl-
2,4-dioxoimidazolidin-5-ylidene]-urea, V. Using Method
D in LC-MS experiments, the product eluting in peak 3
was determined to be 1-ethyldehydroallantoin, which is
analogous to I formed in the reaction of 3′,5′-di-O-Ac-8-
oxodG with peroxynitrite. Peak 4 was analyzed by LC-
MS (using HPLC Method D) and determined to be
1-ethylparabanic acid, and peak 5 as 1-ethylcyanuric
acid. During our LC-MS experiments using a narrow bore
column and lower flow rates (60 µL/min), we were able
to detect an additional compound with λmax ) 245 and
290 nm, and that gave rise to an [M - H]- ion at m/z
195. The UV spectrum was identical to that of authentic
9-methyluric acid, so based upon the UV-vis spectrum
and the molecular weight, this compound was identified
as 9-ethyluric acid. Furthermore, we ascertained that the
C8-nitro group was not retained in any of these products
by carrying out the same experiments using 9-ethyl-8-
[15N]nitroxanthine, and determining that the molecular
weights of the products arising from the 14N- and 15N-
labeled substrates were identical.
F igu r e 2. (a) Five new reaction products arising from the
reaction between 9-ethyl-8-nitroxanthine and 5 mM peroxyni-
trite. Employed Method C and analyzed reaction with λ ) 238
nm (solid line) and λ ) 282 nm (dashed line). (b) Dose-dependent
depletion of 9-ethyl-8-nitroxanthine in the presence of increasing
peroxynitrite concentration (pH 7.2).
8-nitroGua, and is well retained during reversed-phase
chromatography, eluting at 23.3 min using HPLC Method
B.
Rea ction of 9-Eth yl-8-n itr oxa n th in e w ith P er oxy-
n itr ite. When we reacted 9-ethyl-8-nitroxanthine (100
µM) with peroxynitrite (0.5-5 mM), there was a dose-
dependent decline in the amount of starting material
present at the end of the reaction (Figure 2b). Similar to
the reaction with 8-nitroGua, all of the 9-ethyl-8-nitro-
xanthine had reacted at a peroxynitrite concentration of
5 mM. Correspondingly, five new peaks appeared when
the reaction mixture was analyzed by reversed-phase
HPLC (Figure 2a).
Upon the basis of our initial studies of the reaction
products formed during the reaction of 8-nitroGua and
8-nitroGuo with peroxynitrite, we suspected that the
8-nitropurines were giving rise to terminal products
corresponding to those known to arise from the reaction
of the 8-oxopurine with peroxynitrite. Therefore, we
hypothesized that the 8-nitropurine products might be
identical or analogous to the 8-oxopurine products and
set out to compare the identity of the products arising
from these two 8-substituted purines using UV-vis and
ESI-MS. Since the 3′,5′-di-O-Ac-8-oxodG products had
previously been characterized (21, 22), we could calculate
the expected molecular weight of the various products
by (i) subtracting 201 amu for the 3′,5′-di-O-Ac-â-D-
erythro-pentofuranosyl moiety and adding 29 amu to
account for the ethyl substituted derivative used in this
study, and (ii) adding 1 amu to the expected molecular
weight of any product retaining the C2-oxo group, to
P r op osed Mech a n ism for th e Rea ction of 9-Eth yl-
8-n itr oxa n th in e w ith P er oxyn itr ite. The above data
confirm the hypothesis that the terminal products arising
from the 8-nitropurine are identical to those arising from
the 8-oxopurine. The presence of 9-ethyluric acid in the
reaction mixture also suggests that displacement of the
C8-nitro group occurs during the reaction of the 8-nitro-
purine with peroxynitrite. Upon the basis of the data
above, we therefore propose that early in the reaction,
peroxynitrite mediates conversion of the 8-nitropurine to
the 8-oxopurine, and that subsequently, the 8-oxopurine
reacts with peroxynitrite to give the observed terminal
products. In exploring the mechanism, we hypothesized
that either oxidative or nucleophilic processes might be
involved in the initial displacement of the C8-nitro group
and the subsequent oxidation of the 8-oxopurine, since
peroxynitrite is a powerful oxidant (36, 37) and a good
nucleophile (30, 38). It has been shown previously that
peroxynitrite decomposes into potent one-electron oxi-
dants, either by a proton or CO2-catalyzed process. The
former pathway yields the hydroxyl radical (•OH) and
nitrogen dioxide (•NO2) (39-41), while the latter results
•
in formation of the carbonate radical (CO3•-) and NO2
radicals, via homolytic cleavage of peroxynitrosocarbon-
ate (ONOOCO2-) (30, 36, 38, 42, 43).

Peroxynitrite Reactions to Yield 8-Oxopurines
Chem. Res. Toxicol., Vol. 15, No. 1, 2002 11
Ta ble 1. (a ) Com p a r ison betw een P r od u cts Ar isin g fr om th e Rea ction of 3′,5′-Di-O-Ac-8-oxod G a n d
9-Eth yl-8-n itr oxa n th in e w ith P er oxyn itr ite Wh er e th e P r od u cts of th e F or m er Con ta in a C2-oxo Gr ou p ; (b) Com p a r ison
betw een P r od u cts Ar isin g fr om th e Rea ction of 3′,5′-Di-O-Ac-8-oxod G a n d 9-Eth yl-8-n itr oxa n th in e w ith P er oxyn itr ite
Wh er e th e P r od u cts of th e F or m er Reta in th e C2-Am in o Gr ou p
Sch em e 1. P er oxyn itr ite Ad d ition a n d Hyd r oxyl
Ra d ica l In ser tion w ith 9-Eth yl-8-n itr oxa n th in e to
Yield 9-Eth ylu r ic Acid (R ) eth yl)
to explain the formation of dX by C4 addition, and
F igu r e 3. Overlaid UV-vis spectra of peak 2 (s) and II (---).
8-oxodG and formamidopyrimidine (Fapy G) by C8 ad-
Absorbance maximum measured at λ ) 228, 264 nm and λ )
238, 272 nm, respectively, using ddH2O.
dition, during the •OH-mediated dG oxidation (44).
•
Indeed, as shown in Scheme 1, OH addition at C8 can
•
The OH may play a role in the reaction of 9-ethyl-8-
give rise to the N7-centered radical A, followed by
hydrogen abstraction to form B, which then eliminates
nitrous acid to yield the 9-ethyluric acid. However,
nitroxanthine by initial insertion into the purine ring.
Such a mechanism has been previously been proposed

12 Chem. Res. Toxicol., Vol. 15, No. 1, 2002
Lee et al.
Sch em e 2. On e Electr on Oxid a tion of
9-Eth yl-8-n itr oxa n th in e to Yield th e Obser ved
In ter m ed ia tes a n d Ter m in a l P r od u cts (R ) eth yl)
6.6 (50), respectively], and, therefore, will rapidly depro-
tonate to the neutral 8-nitroxanthine radical, F . In fact,
given that the overall oxidation of 8-nitroxanthine by the
•OH and CO3 radicals is likely close to thermoneutral,
•-
proton transfer is likely to accompany electron transfer,
as the former provides additional energy to make the
oxidation step more favorable (53). Hydration of the
neutral radical at the C8 to give G, followed by elimina-
tion of nitrous acid would then yield the neutral uric acid
radical, H. This species could either abstract a H-atom
to give 9-ethyluric acid, or undergo further oxidation to
the quinone diimine species, J , analogous to that previ-
ously reported (54). The latter species may also be an
important intermediate in the reaction of 9-ethyluric acid
with peroxynitrite to give the observed final products.
Another initiating reaction involves the peroxynitrite
anion, ONOO-, acting as a nucleophile, leading to loss
of the C8-nitro group. ONOO- is considered a good
nucleophile, and is the species that reacts with CO2 to
give peroxynitrosocarbonate, ONOOCO2- (42). To assess
whether a nucleophile could displace the C8-nitro group
in 9-ethyl-8-nitroxanthine, we decided to use hydroxy-
lamine, which was expected to give the stable 9-ethyl-8-
hydroxylaminoxanthine for isolation and characteriza-
tion. Therefore, we incubated 9-ethyl-8-nitroxanthine
(100 µM) with hydroxylamine (15 µM to 1.5 M) in 0.1 M
potassium hydroxide solution and monitored the decline
in absorbance at 412 nm with time. At a hydroxylamine
concentration g10 mM, there was a progressive decline
in the absorbance, which indicated that the C8-nitro
group was being displaced. Indeed, when the reaction
mixture was neutralized, and analyzed by HPLC and off-
line ESI-MS, the major product gave rise to an [M - H]-
ion at m/z 210, consistent with that expected for the
9-ethyl-8-hydroxylaminoxanthine. Furthermore, the λmax
) 240 and 285 nm at pH 9 are in close agreement with
those reported for 8-hydroxylaminoxanthosine (λmax ) 246
and 291 nm at pH 11) (55). Additionally, 9-ethyluric acid
was a very minor product, likely arising as a result of
hydroxide-mediated nitro group displacement. In a con-
trol experiment, incubation of 9-ethyl-8-nitroxanthine
(100 µM) in 0.1 M potassium hydroxide resulted in no
significant degradation of this substrate, demonstrating
its stability under these conditions. This finding supports
the hypothesis that nucleophilic displacement of the C8-
nitro group can occur. Since peroxynitrite has a pKa of
∼6.8 (29), at pH 7.2-7.4, between 70 and 80% is present
as ONOO- and thus is available to react as a nucleophile.
The displacement of the C8-nitro group is expected to
occur via an addition-elimination route as shown in
Scheme 1. Under the experimental conditions used (pH
7.2-7.4), 9-ethyl-8-nitroxanthine exists as the neutral
molecule, since pKa values of 4.1 and 13.2 have previously
been reported for the analogous 9-methyl-8-nitroxanthine
(56). Addition of ONOO- at the C8 of 9-ethyl-8-nitrox-
anthine thus leads to formation of C, which, like
ONOOCO2-, contains a weak peroxo bond and undergoes
homolysis to give D. H-atom abstraction by this species
then leads to B, which then eliminates either nitrous acid
or water to yield, respectively, 9-ethyluric acid, or the
starting 9-ethyl-8-nitroxanthine.
•
certain facts argue against a primary role for OH in this
mechanism. First, all our reactions with peroxynitrite
were carried out in 25 mM sodium bicarbonate-contain-
ing buffers (∼1 mM CO2). Under these conditions, per-
oxynitrite is expected to decay predominantly into the
•
CO3•-/•NO2 radical pair, without significant OH forma-
tion (42). Second, we treated 9-ethyl-8-nitroxanthine with
•OH generated in situ by γ-irradiation of N2O/O2 purged
aqueous solutions. At the 1000 Gy dose, over 95% of the
starting material had been consumed, and analysis of the
reaction mixture demonstrated formation of 1-ethyl-
parabanic acid as the major product along with a minor
unknown compound (λmax ) 240 nm). Since 1-ethylpara-
banic acid is the major product with OH radical, and only
a minor product with peroxynitrite, it is unlikely that
the OH plays a significant role in the overall chemistry
with peroxynitrite. However, we cannot fully exclude the
possibility that •OH plays a minor role in the initial
conversion of the 8-nitropurine to the 8-oxopurine, and
that the differences in product profiles between the OH
and peroxynitrite reactions arise due to additional chem-
istry when the latter is present.
Alternatively, since the OH and CO3 radicals are
potent oxidants [E° ) 1.9 V (45) and 1.5 V (46), respec-
tively], while NO2 is a milder oxidant (E° ) 1.04 V) (45),
these species may mediate one-electron oxidation of
9-ethyl-8-nitroxanthine to initiate the reaction. Unfor-
tunately, redox potentials have not been determined for
any of the 8-nitropurines, so meaningful predictions
about the ability of the peroxynitrite-derived species to
effect one-electron oxidation of these substrates cannot
be made. 1-Electron oxidation of 8-nitroxanthine, in a
manner similar to that previously reported for dGuo (47-
50) and 8-oxodG (51, 52) will lead to formation of the
8-nitroxanthine radical cation, E, as shown in Scheme
2. Because of the strong electron-withdrawing C8-nitro
group, this radical is expected to be even more acidic than
the dG and 8-oxodG radical cations [pKa ) 3.9 (49) and
•
•
•
•
•-
•
Con clu sion
The reaction of peroxynitrite with both 8-oxodG and
9-ethyl-8-nitroxanthine (a model for 8-nitroGua) results

Peroxynitrite Reactions to Yield 8-Oxopurines
Chem. Res. Toxicol., Vol. 15, No. 1, 2002 13
(11) Yermilov, V., Rubio, J ., Becchi, M., Friesen, M. D., Pignatelli, B.,
and Oshima, H. (1995) Formation of 8-nitroguanine by the
reaction of guanine with peroxynitrite in vitro. Carcinogenesis 16,
2045-2050.
in the formation of four identical and two analogous
products, the latter arising only because of the presence
of a C2-oxo group in the model compound versus a C2-
NH2 group in 8-oxodG. These findings suggest that the
two reactions converge, likely due to formation of the
8-oxopurine from the 8-nitropurine and subsequent reac-
tion of the 8-oxopurine with peroxynitrite. A primary role
for the hydroxyl radical in the peroxynitrite-mediated
chemistry has been ruled out, since a very distinct
product profile results when this species reacts with
9-ethyl-8-nitroxanthine. Absence of an 15N-label in the
products arising from the reaction of 9-ethyl-8-[15NO2]-
nitroxanthine with peroxynitrite, along with the hy-
droxylamine-mediated displacement of the C8-nitro group,
argue in favor of a mechanism in which ONOO- initially
attacks the C8 of 9-ethyl-8-nitroxanthine, leading to loss
of the C8-nitro group and ultimately to 9-ethyluric acid
formation. This work, therefore, illustrates an interesting
and novel aspect of 8-nitropurine oxidation by peroxyni-
trite as well as suggests an additional mechanism via
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Ack n ow led gm en t. We would like to thank Gary
Kruppa from Bru¨ker for allowing us to run our 8-ni-
troGua sample on the APEX II Fourier Transform Mass
Sspectrometer. We would also like to acknowledge Mo-
hamad Awada and Professor R. P. Marini for their
support in using the Gammacell-220 machine and ac-
quiring N2O, respectively. This work was supported by
NIH Grants T32ES07020 and 5P01CA26731. J .C.N. was
supported by 5F31HG00144.
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