A novel nitration product formed during the reaction of peroxynitrite with 2',3',5'-tri-O-acetyl-7,8-dihydro-8-oxoguanosine: N-nitro-N'-[1-(2,3,5- tri-O-acetyl-β-D-erythro-pentofuranosyl)-2,4-dioxoimidazolidin-5- ylidene]guanidine

Article abstract of DOI:10.1021/tx0000318

A novel nitration product, formed during the reaction of peroxynitrite with 2',3',5'-tri-O-acetyl-7,8-dihydro-8-oxoguanosine, has been characterized using a combination of UV/vis, CD, and NMR spectroscopy and mass spectrometry. This compound has been identified as N-nitro-N'-[1-(2,3,5-tri- P-O-acetyl-β-D-erythro-pentofuranosyl)-2,4-dioxoimidazolin-5- ylidene]guanidine (IV). Upon base hydrolysis, IV releases nitroguanidine (IVa) and an intermediate, 1-(2,3,5-tri-O-acetyl-β-D-erythro- pentofuranosyl)-5-iminoimidazolidine-2,4-dione (IVb). This intermediate is ultimately hydrolyzed to the stable 3-(2,3,5-tri-O-acetyl-β-D-erythro- pentofuranosyl)oxaluric acid (IVc). IV can be reduced by sodium borohydride to a pair of stable diastereomers (IV(red)). The formation of this product is rationalized in terms of initial oxidation of 2',3',5'-tri-O-acetyl-7,8- dihydro-8-oxoguanosine to a quinonoid diimine intermediate, 3. Nucleophilic attack at C5 of 3 by peroxynitrite leads to formation of a C5-oxyl radical species, 5, which then undergoes a series of rearrangements to yield an ylidene radical, 7. Combination of this radical species with nitrogen dioxide results in the formation of product IV.

Full text of DOI:10.1021/tx0000318

390
Chem. Res. Toxicol. 2000, 13, 390-396
A Novel Nitr a tion P r od u ct F or m ed d u r in g th e Rea ction
of P er oxyn itr ite w ith
2′,3′,5′-Tr i-O-a cetyl-7,8-d ih yd r o-8-oxogu a n osin e:
N-Nitr o-N′-[1-(2,3,5-Tr i-O-a cetyl-â-D-er yth r o-p en tofu r a n osyl)-
2,4-d ioxoim id a zolid in -5-ylid en e]gu a n id in e
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 February 16, 2000
A novel nitration product, formed during the reaction of peroxynitrite with 2′,3′,5′-tri-O-
acetyl-7,8-dihydro-8-oxoguanosine, has been characterized using a combination of UV/vis, CD,
and NMR spectroscopy and mass spectrometry. This compound has been identified as N-nitro-
N′-[1-(2,3,5-tri-O-acetyl-â-D-erythro-pentofuranosyl)-2,4-dioxoimidazolidin-5-ylidene]guani-
dine (IV). Upon base hydrolysis, IV releases nitroguanidine (IVa ) and an intermediate, 1-(2,3,5-
tri-O-acetyl-â-D-erythro-pentofuranosyl)-5-iminoimidazolidine-2,4-dione (IVb). This intermediate
is ultimately hydrolyzed to the stable 3-(2,3,5-tri-O-acetyl-â-D-erythro-pentofuranosyl)oxaluric
acid (IVc). IV can be reduced by sodium borohydride to a pair of stable diastereomers (IVr ed ).
The formation of this product is rationalized in terms of initial oxidation of 2′,3′,5′-tri-O-acetyl-
7,8-dihydro-8-oxoguanosine to a quinonoid diimine intermediate, 3. Nucleophilic attack at C5
of 3 by peroxynitrite leads to formation of a C5-oxyl radical species, 5, which then undergoes
a series of rearrangements to yield an ylidene radical, 7. Combination of this radical species
with nitrogen dioxide results in the formation of product IV.
including the oxidation products, 8-oxo-7,8-dihydro-2′-
deoxyguanosine (8-oxodGuo) (24), 2,2-diamino-4-[(2-deoxy-
â-D-erythro-pentofuranosyl)amino]-5(2H)-oxazolone, and
4-hydroxy-8-oxo-4,8-dihydro-2′-deoxyguanosine (25); the
nitration product, 8-nitroguanine (27); and the addition
product, 4,5-dihydro-5-hydroxy-4-(nitrosooxy)-2′-deoxy-
guanosine (28).
In tr od u ction
Peroxynitrite (ONOO-) is a physiologically relevant
and reactive molecule formed by the diffusion-limited
reaction of nitric oxide (•NO) with superoxide (O2-•) (1).
Peroxynitrite is stable in alkaline solutions, but at
physiologic pH, it is protonated to form peroxynitrous
acid, which rapidly decomposes (2). The reactive species
produced in the absence of carbon dioxide are believed
to be HOONO*, an excited state of peroxynitrous acid
(3), and/or the HO•/•NO2 radical pair (4-6). In the
presence of carbon dioxide, peroxynitrite is catalytically
While dGuo is reactive toward peroxynitrite, its oxida-
tion product, 8-oxodGuo, is at least 1000 times more
reactive (29). The steady-state level of 8-oxodGuo present
in tissues is about 4-11 molecules per 107 bases (30),
reflecting a balance between formation and removal
(enzymatic and nonenzymatic, respectively) of this lesion.
We have hypothesized, therefore, that 8-oxodGuo in
genomic DNA might be an important target of peroxy-
nitrite chemistry, and that this reaction might contribute
to lowering steady-state 8-oxodGuo levels. Consequently,
we have been interested in identifying the products of
the reaction of peroxynitrite with 8-oxodGuo. Four im-
mediate products (I-IV) are formed during this reaction.
Products I-III have been previously characterized as the
3′,5′-di-O-acetyl-â-D-erythro-pentofuranosyl derivatives of
2,4,6-trioxo[1,3,5]triazinane-1-carboxamidine (I), (2-imino-
5-oxoimidazolidin-4-ylidine)urea (II), and parabanic acid
(III) (31). The fourth product, IV, has now been identi-
fied, and its characterization is reported here.
-
decomposed to NO3 (7, 8). The reactive intermediates
-•
formed under these conditions are the carbonate (CO3
)
and nitrogen dioxide (•NO2) radicals (9-11), the former
species having been recently observed directly in EPR
studies (12).
Peroxynitrite reacts with a variety of substrates,
resulting in either oxidation or nitration. These sub-
strates include low-molecular weight antioxidants such
as ascorbate (13, 14), trolox (14), and glutathione (3, 14),
and larger biomolecules such as lipids (15), proteins (16-
21), and DNA. With DNA, sugar damage results in
strand breaks (22-24). Base damage also occurs and has
been studied extensively. Of the DNA nucleosides, 2′-
deoxyguanosine (dGuo) is the most reactive (25, 26).
Several products of this reaction have been characterized,
Exp er im en ta l P r oced u r es
* To whom correspondence should be addressed: 77 Massachusetts
Ave., Room 56-731A, Massachusetts Institute of Technology, Cam-
bridge, MA 02139. Telephone: (617) 253-3729. Fax: (617) 252-1787.
E-mail: srt@mit.edu.
Gen er a l. DMSO-d6 and D2O were obtained from Cambridge
Isotope Laboratories (Andover, MA); Na15NO2 and Na15NO3
were from Isotech (Miamisburg, OH). All solvents were HPLC
grade.
† Division of Bioengineering and Environmental Health.
‡ Department of Chemistry.
10.1021/tx0000318 CCC: $19.00 © 2000 American Chemical Society
Published on Web 04/08/2000

N-Nitration during the Reaction of ONOO- with 8-OxodGuo
Chem. Res. Toxicol., Vol. 13, No. 5, 2000 391
In str u m en ta tion . UV/vis measurements were made using
an HP8452 diode array spectrophotometer (Hewlett-Packard,
Palo Alto, CA) and CD spectra recorded on a 62D Aviv circular
dichrometer. 1H NMR spectra were recorded at 300 or 500 MHz
and 13C NMR spectra (proton-decoupled) at 75 or 125 MHz on
Unity 300 and Inova 500 spectrometers, respectively, using the
standard parameters provided by Varian. δ values are given in
parts per million. COSY, HMQC, and HMBC experiments were
used to aid in the assignment of sugar and base protons and
carbons. High-performance liquid chromatography (HPLC) was
performed using an HP1100 pump equipped with a model 1090
diode array detector (Hewlett-Packard). Electrospray ionization
mass spectrometry (ESI-MS) and tandem mass spectrometry
(ESI-MS/MS) experiments were carried out using an HP 5989B
(Hewlett-Packard) and TSQ 7000 (Finnigan, San J ose, CA) mass
spectrometers, respectively. High-resolution mass spectrometry
(HRMS) experiments were carried out in ESI mode on an APEX
II FTMS instrument (Bruker, Billerica, MA). ESI-MS and ESI-
MS/MS spectra were obtained in negative ion mode using a
spraying solution (78/20/2 water/2-propanol/ammonium hydrox-
ide).
P er oxyn itr ite Syn th esis. O14NOO- was prepared either by
ozonolysis of an alkaline solution of a sodium azide (Fisher)
solution (32) or by the reaction of isoamyl nitrite (Aldrich,
Milwaukee, WI) with hydrogen peroxide (Fisher) at pH 12-14
(33). In the latter case, excess hydrogen peroxide was removed
by passing the peroxynitrite solution through a 7.5 cm × 1.5
cm manganese oxide (Aldrich) column. O15NOO- was prepared
by reacting 0.58 mmol of acidified hydrogen peroxide with 0.58
mmol of Na15NO2 as previously described (34). Excess hydrogen
peroxide was removed by passing the peroxynitrite solution
through a 6 cm × 0.5 cm manganese oxide column. Peroxynitrite
concentrations were determined by making dilutions in 0.1 M
NaOH and measuring the absorbance at λ ) 302 nm (ꢀ ) 1670
M-1 cm-1) (34).
clear yellow solution arose over the course of the reaction. This
mixture was rotary evaporated to apparent dryness and taken
up in 50 mL of doubly distilled water. This solution was loaded
onto an 11 cm × 3 cm gravity column packed with C18, 125 Å,
55-105 µm beads (Waters, Milford, MA) and pre-equilibrated
with water. After washing with 400 mL of doubly distilled water,
the 2′,3′,5′-tri-O-acetyl-8-oxoGuo was eluted with methanol. This
fraction was dried in vacuo to obtain the desired 2′,3′,5′-tri-O-
acetyl-8-oxoGuo. Yield: 1.96 g (4.61 mmol, 88%). 1H NMR
(DMSO-d6, δ): 10.84 (s, 1H, NH), 6.53 (s, 2H, NH2), 6.00 (m,
1H, H1′), 5.72 (m, 1H, H2′), 5.53 (m, 1H, H3′), 4.37 (m, 1H, H4′),
4.19 (m, 1H, H5′), 4.11 (m, 1H, H5′′), 2.08-2.00 (m, 9H, 3 ×
CH3CO). 13C NMR (DMSO-d6, δ): 170.13, 169.46, 169.42,
153.49, 151.30, 151.09, 146.74, 98.73, 83.29, 78.65, 70.54, 70.25,
62.93, 20.54, 20.35, 20.30. ESI-MS (m/z): 424 (M - H)-. UV/
vis (0.1 M HCl): λmax ) 248 and 292 nm.
Rea ction of 2′,3′,5′-Tr i-O-a cetyl-8-oxoGu o w ith P er oxy-
n itr ite. The concentrations of 2′,3′,5′-tri-O-acetyl-8-oxoGuo
stock solutions were determined by diluting in 0.1 M HCl and
measuring the absorbance at λ ) 248 nm (ꢀ ) 13.5 mM-1 cm-1
)
(37). For analytical studies, 2′,3′,5′-tri-O-acetyl-8-oxoGuo (100-
500 nmol) was dissolved in 150 mM KH2PO4, pH 7.2 buffer (1
mL) containing either no sodium bicarbonate or 25 mM sodium
bicarbonate. This solution was prepared in an Eppendorf tube
(1.5 mL). Peroxynitrite (0.1-1 µmol) was added as a bolus on
the underside of the cap. To initiate the reaction, the tube was
gently closed and the peroxynitrite vortexed into the buffered
solution containing the 2′,3′,5′-tri-O-acetyl-8-oxoGuo. Mixing
was continued for 1 min. The mixture was either reduced with
2-5 µL of a 1 M stock of sodium borohydride (American
Bioanalytical, Natick, MA) or analyzed directly by HPLC. For
HPLC analysis, solvent A was either 50 mM ammonium acetate
(Fisher Scientific Co., Fair Lawn, NJ ) or ammonium formate
(Sigma) and solvent B was acetonitrile, and a 250 mm × 4.6
mm, 5 µm Columbus C18 column (Phenomenex) was used.
Elution conditions were as follows: isocratic, 5% B for 10 min;
5 to 40% B over the course of 20 min; isocratic, 40% B for 2
min; 40 to 5% B over the course of 3 min. The flow rate was 1.0
mL/min, and products were monitored simultaneously at 230
and 254 nm.
Syn th esis of 8-Br om ogu a n osin e. 8-Bromoguanosine was
prepared using a previously published procedure (35). Briefly,
3.67 g (13 mmol) of Guo (Sigma, St. Louis, MO) suspended in
22 mL of doubly distilled water was reacted with 100 mL of
saturated bromine (Aldrich) water. The latter was added in 8
mL aliquots with vigorous stirring of the reaction mixture, and
the yellow color was allowed to fade between additions. The
precipitated 8-bromoguanosine was recovered by filtration,
washed extensively with cold water, followed by cold acetone,
and air-dried. Yield: 4.03 g (11.1 mmol, 85%). 1H NMR (DMSO-
d6, δ): 10.81 (s, 1H, NH), 6.49 (s, 2H, NH2), 5.66 (d, 1H, H1′),
5.44 (d, 1H, H2′), 5.09 (d, 1H, 2′-OH), 5.00 (m, 1H, 3′-OH), 4.91
(m, 1H, 5′-OH), 4.11 (m, 1H, H3′), 3.83 (m, 1H, H4′), 3.63 (m,
1H, H5′), 3.49 (m, 1H, H5′′). 13C NMR (DMSO-d6, δ): 155.46,
153.45, 152.08, 121.20, 117.50, 89.67, 85.87, 70.53, 70.29, 62.03.
ESI-MS (m/z): 362 (50%), 360 (50%) (M - H)-. UV/vis (H2O):
λmax ) 260 nm.
On a preparative scale, 2′,3′,5′-tri-O-acetyl-8-oxoGuo (200 mg,
0.47 mmol) dissolved in 6 mL of methanol was added to 300
mL of 150 mM KH2PO4, 25 mM NaHCO3, pH 7.2 buffer. This
mixture was vigorously stirred while peroxynitrite (4.7 mmol,
16 mL) was forcefully dumped in to initiate the reaction. The
mixture was stirred for 5 min before sodium borohydride (5
mmol) was added in portions, with the addition of hydrochloric
acid (1 M) to keep the pH equal to 7.2-7.5. Stirring was
continued for an additional 15 min, after which the solution was
loaded on a 4 cm × 2.5 cm C18 (125 Å, 55-105 µm) gravity
column. The column was washed with 200 mL of water to effect
desalting, and the crude products were eluted in methanol. The
methanol fraction was dried in vacuo and then taken up in 75/
25 water/methanol (10 mL). The desired product, IVr ed , was
isolated by semipreparative HPLC using 50 mM ammonium
acetate (solvent A) and acetonitrile (solvent B) and a 250 mm
× 10 mm, 5 µm Nucleosil C18 column (Alltech). Elution
conditions were as follows: isocratic wash with 5% B for 10 min,
5 to 40% B over the course of 20 min, isocratic wash with 40%
B for 4 min, 40 to 5% B over the course of 6 min. The flow rate
was 3.0 mL/min, and products were monitored simultaneously
at 230 and 254 nm. IVr ed eluted after 29 min under these
conditions. Yield: 11.6 mg (0.025 mmol, 5%). See the Results
Acetylation of 8-Br om ogu an osin e. 8-Bromoguanosine (4.03
g, 11.1 mmol) was dissolved in 10 mL of pyridine (Aldrich) and
7 mL of acetic anhydride (Mallinckrodt, Paris, KY). This mixture
was stirred in an ice bath for 3.5 h after which 2′,3′,5′-tri-O-
acetyl-8-bromoguanosine precipitated. The precipitate was sus-
pended in ∼50 mL of ice-cold water and filtered and the residue
washed with ∼350 mL of ice-cold water and air-dried. Yield:
1
2.55 g (5.23 mmol, 47%). H NMR (DMSO-d6, δ): 10.93 (s, 1H,
NH), 6.61 (s, 2H, NH2), 6.01 (m, 1H, H1′), 5.88 (d, 1H, H2′),
5.65 (m, 1H, H3′), 4.42 (m, 1H, H4′), 4.32 (m, 1H, H5′), 4.21 (m,
1H, H5′′), 2.11-2.00 (m, 9H, 3 × CH3CO). 13C NMR (DMSO-d6,
δ): 170.73, 170.12, 170.04, 156.05, 154.44, 152.54, 120.75,
117.80, 88.26, 79.93, 71.91, 70.55, 63.41, 21.14, 20.95. ESI-MS
(m/z): 486 (50%), 484 (50%) (M - H)-.
for 1H and 13C NMR data. UV/vis (50 mM NH4OAc): λmax
)
224 and 268 nm. HRMS calcd for C15H20N6O11 [M - H]-
459.1111, found 459.1110.
Syn th esis of 2′,3′,5′-Tr i-O-a cetyl-8-oxoGu o. This com-
pound was synthesized as previously described by Ikehara et
al. (36). Briefly, 2.55 g (5.23 mmol) of 2′,3′,5′-tri-O-acetyl-8-
bromoguanosine and 4.3 g (52.3 mmol) of anhydrous sodium
acetate (Mallinckrodt) were suspended in 50 mL of glacial acetic
acid (Mallinckrodt), and the mixture was refluxed for 1.7 h. A
Syn th esis of 3-(2,3,5-Tr i-O-a cetyl-â-D-er yth r o-p en tofu r a -
n osyl)oxa lu r ic a cid (IVc). 2′,3′,5′-Tri-O-acetyl-8-oxoGuo (0.13
mmol, 50 mg) was dissolved in 150 mM KH2PO4, 25 mM
NaHCO3, pH 7.2 buffer (25 mL) and treated with peroxynitrite
(0.64 mmol). The reaction was initiated by forcefully pipetting
the peroxynitrite into the vigorously stirred nucleoside solution.

392 Chem. Res. Toxicol., Vol. 13, No. 5, 2000
Niles et al.
The mixture was left at room temperature for 2 days and
concentrated by rotary evaporation, and the desired product was
obtained using the same semipreparative HPLC conditions
described above. The 3-(2,3,5-tri-O-acetyl-â-D-erythro-pento-
furanosyl)oxaluric acid was desalted on a C18 Mega Bond Elut
column (Varian), eluted with methanol, and dried in vacuo.
Yield: 4.3 mg (0.01 mmol, 8%). 1H NMR (DMSO-d6, δ): 9.81 (s,
1H, COOH), 8.87 (d, J ) 9.3 Hz, 1H, NH), 7.04 (s, 1H, NH),
5.51 (dd, J ) 5.1, 9.3 Hz, 1H, H1′), 5.22 (m, 2H, H2′ and H3′),
4.23 (m, 1H, H4′), 4.11 (m, 2H, H5′ and H5′′), 2.07 (m, 9H, 3 ×
CH3). 13C NMR (DMSO-d6, δ): 170.10, 169.54, 169.45, 165.92,
160.53, 152.20, 82.29, 78.18, 72.93, 70.47, 62.30, 20.56, 20.41,
20.29. UV/vis (50 mM NH4OAc): λmax ) 213 nm. ESI-MS (m/z):
389 [M - H]-. ESI-MS/MS (negative ions, m/z): 317 [M -
C2HO3]-, 274 [M - C3H2NO4]-.
Resu lts a n d Discu ssion
The products formed when peroxynitrite reacts with
8-oxoGuo were very polar and, consequently, were not
well retained or separated by reverse phase HPLC (RP-
HPLC). However, when the acetylated starting material,
2′,3′,5′-tri-O-acetyl-8-oxoGuo was used, the products were
retained longer and could be resolved during RP-HPLC,
with the starting material and four additional products
having capacity factors, κ′,1 between 6.7 and 7.3. Products
I-IV were observed immediately following the reaction
of peroxynitrite with 2′,3′,5′-tri-O-acetyl-8-oxoGuo. I-III
have been identified previously (31) and will not be
discussed here. Instead, this paper will deal specifically
with the characterization of the novel product, IV.
The UV/vis spectrum of IV exhibited two absorbance
maxima at λ ) 238 nm (ꢀ ) 18.6 mM-1 cm-1) and 272
nm (ꢀ ) 19.3 mM-1 cm-1). This compound was hydro-
lytically unstable, and in 150 mM KH2PO4, 25 mM
NaHCO3, pH 7.2 buffer, the half-life of IV was 7.5 h. Two
stable hydrolysis products arose from IV, namely, IVa
and IVc, the latter forming via an intermediate, IVb.
This instability presented a major obstacle to the isola-
tion of sufficient quantities of pure IV for NMR analysis.
Our efforts to RP-HPLC purify IV on a semipreparative
scale followed by lyophilization to remove the HPLC
solvents (50 mM ammonium acetate and acetonitrile)
resulted in a complex mixture of IV and its degradation
products, preventing an informative NMR analysis. To
facilitate NMR spectroscopic studies, we explored ways
to stabilize IV. Reduction of this compound with sodium
borohydride resulted in a stable product, IVr ed . Sufficient
quantities for NMR analysis were then purified without
degradation by semipreparative HPLC.
F igu r e 1. Overlaid UV spectra of authentic nitroguanidine (-)
and IVa (- - -) released from IV by hydrolysis. The absorbance
maximum in both cases occurs at λ ) 264 nm.
oxoGuo. A similar reaction was also carried out using
O14NOO-. IV isolated from the reaction of 2′,3′,5′-tri-O-
acetyl-8-oxoGuo with O14NOO- and O15NOO- produced
[M - H]- ions at m/z 457 and 458, respectively, as
determined by ESI-MS. Nitrite and nitrate, however,
often contaminate peroxynitrite preparations. To rule
these out as sources of the additional nitrogen atom in
IV, reactions were carried out with O14NOO-, as de-
scribed above, in the presence of added Na15NO2 and
Na15NO3 (∼10 mM). No 15N incorporation into IV was
observed. Taken together, these data show that peroxy-
nitrite, and not its degradation products, is the source of
the additional nitrogen atom in IV.
Id en tifica tion of th e Hyd r olysis P r od u cts of IV.
Hydrolysis of IV produced two stable products, IVa and
IVc, the latter forming via an intermediate, IVb. IVa
has a characteristic UV spectrum with a λmax of 264 nm.
IVa produced an [M - H]- ion at m/z 103 during ESI-
MS, indicating that its molecular weight is 104. IVa was
determined to be nitroguanidine since the molecular
weight and UV spectrum were identical to those of
authentic nitroguanidine (Figure 1). Further, nitroguani-
dine (IVa ) released from product IV obtained from the
reaction of 2′,3′,5′-tri-O-acetyl-8-oxoGuo with O15NOO-
produces an [M - H]- ion at m/z 104. This indicates that
a peroxynitrite-derived nitro group has been incorporated
into IV and that this nitro group is adducted to one of
the 2′,3′,5′-tri-O-acetyl-8-oxoGuo-derived guanidino ni-
trogen atoms.
In summary, IV was identified through (i) low- and
high-resolution mass spectrometric studies on IV itself,
(ii) identification of the hydrolysis products, IVa , IVb,
and IVc, and (iii) NMR analysis of the stable reduction
The intermediate, IVb, has C14H17N3O9 as its molec-
ular formula as determined by HRMS (calculated for [M
- H]-, 370.0887; found, 370.0879) and two exchangeable
protons. The latter was determined by using H2O and
D2O in the spraying solvent during ESI-MS experiments
in which the [M - H]- ion of IVb appeared at m/z 370
and 371, respectively. Since one proton (deuteron) is lost
to produce the observed negative ion, the ∆(m/z) of 1
when D2O is used indicates that IVb has two exchange-
able protons. These data, taken together, are consistent
with the identification of IVb as 1-(2,3,5-tri-O-acetyl-â-
D-erythro-pentofuranosyl)-5-iminoimidazolidine-2,4-di-
one. The hydrolytic instability of the intermediate IVb
precluded its characterization by NMR spectroscopy.
product, IVr ed
.
Ma ss Sp ectr om etr ic Ch a r a cter iza tion of IV. The
exact mass of IV, determined by high-resolution mass
spectrometry, was 458.1027, consistent with C15H18N6O11
as its molecular formula (calculated exact mass of
458.1034). This indicated that IV contains one more
nitrogen atom than its parent, 2′,3′,5′-tri-O-acetyl-8-
oxoGuo (C16H19N5O9). Peroxynitrite is the most likely
source of this nitrogen atom. To test this, O15NOO- was
synthesized and reacted with 2′,3′,5′-tri-O-acetyl-8-
1
The capacity factor, κ′, is calculated using the equation κ′ ) (t -
t0)/t0, where t is the retention time of a product and t0 is the time needed
for the injected solvent to elute from the column.

N-Nitration during the Reaction of ONOO- with 8-OxodGuo
Chem. Res. Toxicol., Vol. 13, No. 5, 2000 393
Ta ble 1. Su m m a r y of th e 1H a n d 13C NMR Da ta for th e
â-D-er yth r o-P en tofu r a n osyl Moiety of th e Dia ster eom er s
of IVr ed
1H
(δ, ppm)
H1′
H2′
H3′
H4′
H5′ H5′′ CH3COOa
IVr ed (A) 6.05 5.65 5.48 4.15 4.30 4.25 2.00-2.08
IVr ed (B) 5.51 5.31 5.22 4.07 4.15 4.00
13C
(δ, ppm) C1′
IVr ed (A) 85.48 72.49 69.21 78.06 63.45 169.25- 20.20-
170.20 20.51
C2′
C3′
C4′
C5′ CH3COOa CH3COOa
F igu r e 2. Hydrolysis of IV to nitroguanidine (IVa ), 1-(2,3,5-
tri-O-acetyl-â-D-erythro-pentofuranosyl)-5-iminoimidazolidine-
2,4-dione (IVb), and 3-(2,3,5-tri-O-acetyl-â-D-erythro-pento-
furanosyl)oxaluric acid (IVc) and reduction to N-nitro-N′-[1-
(2,3,5-tri-O-acetyl-â-D-erythro-pentofuranosyl)-2,4-dioxoimida-
zolidin-5-ylidene]guanidine (IVr ed ).
IVr ed (B) 82.63 83.72 69.61 77.78 62.55
a
Significant overlap of the 1H and 13C signals arising from the
acetyl groups was observed in both one- and two-dimensional
experiments, thus preventing the unambiguous assignment of
these resonances to a specific diastereomer.
Ta ble 2. Su m m a r y of th e 1H a n d 13C NMR Da ta for th e
Ba se Moiety of th e Dia ster eom er s of IVr ed
1Η
(δ, ppm)
NH
NH2
8.38
N′H
H5
IVred
11.50
7.75
5.51/5.35
13C
(δ, ppm)
C2
C4a
C5a
C6b
IVr ed (A)
IVr ed (B)
149.04
148.34
155.96
155.12
69.78
69.38
158.09
158.09
a
These assignments might be interchanged. b Signal broadened
by the quadrupolar effect of the 14N nucleus, thereby preventing
resolution of this signal into individual peaks.
During analytical HPLC in which 50 mM NH4OAc was
used as the aqueous buffer, IVr ed eluted as a single peak
(κ′ ) 6.4). However, when 50 mM ammonium formate
was used as the aqueous buffer, IVr ed eluted as two peaks
with κ′ values of 6.6 and 6.7, respectively. The separated
peaks were collected, and their CD spectra, shown in
Figure 3, were recorded. The CD spectrum of the κ′ )
6.6 peak exhibited mirror image symmetry with that of
the κ′ ) 6.7 peak. Therefore, it was concluded that IVr ed
is a mixture of two diastereomeric compounds. Neither
50 mM ammonium acetate nor formate during semi-
preparative HPLC facilitated baseline separation of the
F igu r e 3. CD spectra of the diastereomers of IVr ed . The
spectrum of the earlier eluting peak (κ′ ) 6.6) is denoted by the
dashed line (- - -) and the later eluting peak (κ′ ) 6.7) by the
solid line (-).
However, its stable hydrolysis product, IVc, could be
characterized unambiguously as 3-(2,3,5-tri-O-acetyl-â-
D-erythro-pentofuranosyl)oxaluric acid. For this identifi-
cation, authentic 3-(2,3,5-tri-O-acetyl-â-D-erythro-pento-
furanosyl)oxaluric acid was prepared and structurally
characterized (see Experimental Procedures). IVc was
then shown to be identical to the authentic sample by
LC/ESI-MS and MS/MS experiments (results not shown).
Identification of the hydrolysis products of IV led to some
important insights regarding its structure. Specifically,
IVb and IVa indicate that IV has an imidazolidine-2,4-
dione nucleus with nitroguanidine as a substituent. By
using these facts, together with the determined molecular
formula, IV could be tentatively assigned as N-nitro-N′-
[1-(2,3,5-tri-O-acetyl-â-D-erythro-pentofuranosyl)-2,4-di-
oxoimidazolidin-5-ylidene]guanidine (Figure 2).
P h ysicoch em ica l a n d Str u ctu r a l Ch a r a cter iza -
tion of th e Red u ction P r od u ct IVr ed . The UV/vis
spectrum of IVr ed revealed λmax values at 224 and 268
nm. This hypsochromic shift in the λmax of IVr ed relative
to that of IV is consistent with a decreased level of
conjugation in the former. HRMS indicated that this
compound has C15H20N6O11 as its molecular formula
(calculated exact mass, 460.1190; found, 460.1189), a
finding consistent with the two-electron reduction of IV.
diastereomeric pair, IVr ed
.
Since IVr ed could not be resolved into its individual
diastereomers, NMR studies were carried out on the
diastereomeric mixture. The sugar 1H and 13C NMR
signals could be assigned unambiguously using a com-
bination of COSY and HMQC experiments. While the
1
one-dimensional H and 13C NMR spectra were compli-
cated due to overlapping resonances, the signals arising
from each diastereomer of IVr ed could be distinguished
in the two-dimensional experiments that were carried out
(Table 1). These data together with the 1H and 13C
assignments for the base moiety are consistent with the
identification of IVr ed as N-nitro-N′-[1-(2,3,5-tri-O-acetyl-
â-D-erythro-pentofuranosyl)-2,4-dioxoimidazolidin-5-yl]-
guanidine, the product expected from reduction of the
imino bond in IV (Figure 2).
The base moiety of each diastereomer has one non-
exchanging proton and four exchangeable protons (Table
2). The nonexchanging proton was assigned as the H5
proton since its chemical shift (δ ) 5.35/5.51 ppm) is very
similar to the H5 proton of the model compound allantoin
[δ ) 5.27 ppm (38)]. The downfield shift observed for H5
in IVr ed likely results from the deshielding effect of the
electron-withdrawing nitroguanidine group bound to C5.

394 Chem. Res. Toxicol., Vol. 13, No. 5, 2000
Niles et al.
The downfield proton at 11.50 ppm corresponds to the
3-NH of the imidazolidine-2,4-dione moiety. The two
protons at δ ) 8.38 ppm were assigned as the NH2
protons of the nitroguanidine moiety of IVr ed by com-
parison to authentic nitroguanidine (δ ) 8.85 ppm). The
single proton at δ ) 7.75 ppm corresponds to the
substituted N′H of the nitroguanidine moiety. Only one
set of exchangeable protons could be observed, in contrast
to the observation made for the sugar and H5 protons.
The former signals are inherently broad, however, and
significant overlap of signals arising from diastereomers
is to be expected.
Table 2 also summarizes the assignments of the base
carbons. The 13C spectrum indicated that the base moiety
of each diastereomer of IVr ed contains one sp3 and three
quaternary carbons. The sp3 carbon is most likely the site
of reduction. Indeed, the nonexchanging H5 proton is
attached to the sp3 carbon as determined by HMQC
experiments. Therefore, the signals at 69.78 and 69.38
ppm are assigned to C5. Of the quaternary signals, the
δ ) 158.09 ppm signal is assigned as C6 of the nitro-
guanidine fragment by comparison with authentic ni-
troguanidine (δ ) 160.97 ppm). The signals at 149.04/
148.34 and 155.96/155.12 ppm were assigned to C2 and
C4, respectively. Further, the 149.04 ppm signal arises
from diastereomer A as long-range coupling between the
C2 and H1′ of diastereomer A was observed in HMBC
experiments. The 148.34 ppm signal could, therefore, be
assigned to diastereomer B. Similar assignments could
not be made unambiguously for the C4 and C5 reso-
nances.
F igu r e 4. Proposed mechanism for the formation of N-
nitro-N′-[1-(2,3,5-tri-O-acetyl-â-D-erythro-pentofuranosyl)-2,4-dioxo-
imidazolidin-5-ylidene]guanidine (IV) during the reaction of
peroxynitrite with 2′,3′,5′-tri-O-acetyl-8-oxoGuo.
enzyme-bound intermediate which is subsequently hy-
drolyzed to allantoin, an imidazolidine-2,4-dione deriva-
tive.
Peroxynitrite is a very potent one-electron oxidant,
producing HO• radical (E° ) 1.90 V) (42) and CO3
-•
radical (E° ) 1.5 V) (43) in the absence and presence of
carbon dioxide, respectively, and NO2 (E° ) 1.04 V) (42)
•
in both cases. These species are all thermodynamically
capable of oxidizing 8-oxoGuo (E° ) 0.58 V) (44). A one-
electron oxidation of 8-oxoGuo would result in formation
of the radical cation species which, by analogy to the
guanine radical cation (45, 46), is rapidly deprotonated
to form 2 (Figure 4). This intermediate in a subsequent
one-electron oxidation followed by deprotonation will
yield the same quinonoid diimine intermediate, 3, ob-
served during the electrochemical oxidation of 8-oxoGuo
and the enzymatic oxidation of uric acid.
Su m m a r y. The hydrolytic and reductive transforma-
tions of IV are summarized in Figure 2. The hydrolysis
products have been identified as nitroguanidine (IVa )
and 1-(2,3,5-tri-O-acetyl-â-D-erythro-pentofuranosyl)-5-
iminoimidazolidine-2,4-dione (IVb) which ultimately is
hydrolyzed to 3-(2,3,5-tri-O-acetyl-â-D-erythro-pentofur-
anosyl)oxaluric acid (IVc). The reduction product, IVr ed
,
During the electrochemical oxidation of 8-oxoGuo and
the enzymatic oxidation of uric acid, hydrolysis of 3 leads
to the formation of the observed products. In the latter
case, water has been shown unambiguously to be the
source of the O4 atom (47). We, therefore, reacted 2′,3′,5′-
tri-O-acetyl-8-oxoGuo with peroxynitrite in H218O to test
whether it was the source of the O4 atom in IV. No 18O
was incorporated into IV, arguing against hydrolysis of
3 as a necessary step in the formation of IV. Further-
more, reactions carried out in the presence of 18O2 and
C18O2 resulted in no 18O incorporation into IV. After
ruling out O2, H2O, and CO2 as possible sources of the
exogenous oxygen atom incorporated into IV, we came
to the conclusion that peroxynitrite is most likely the
source of the O4 atom. ONOO- is a very good nucleophile,
capable of reacting with CO2 with a bimolecular rate
constant of 3-5 × 104 M-1 s-1 (7, 8). Given that 3 is more
susceptible to nucleophilic attack than CO2, it is likely
to undergo direct reaction with ONOO- to yield 4. In our
experiments, which were conducted at pH 7.2, 70% of the
added peroxynitrite [pKa ) 6.8 (17)] was present as the
anion. Thus, ONOO- is available for direct nucleophilic
addition to both CO2 and 3.
was determined to be a diastereomeric mixture of N-
nitro-N′-[1-(2,3,5-tri-O-acetyl-â-D-erythro-pentofuranosyl)-
2,4-dioxoimidazolidin-5-yl]guanidine. Taken together, these
results support the identification of IV as N-nitro-N′-
[1-(2,3,5-tri-O-acetyl-â-D-erythro-pentofuranosyl)-2,4-di-
oxoimidazolidin-5-ylidene]guanidine.
A Mech a n istic Ra tion a liza tion for th e F or m a tion
of P r od u ct IV. Formation of IV requires both oxidation
and nitration reactions. Additionally, since IV has an
imidazolidine-2,4-dione nucleus and retains C8 of 2′,3′,5′-
tri-O-acetyl-8-oxoGuo (data not shown), this suggests that
the imidazole ring of the parent compound remains
intact. Therefore, to form the C4-oxo bond in IV, an
oxygen atom must be incorporated from an exogenous
source.
For the oxidation of 8-oxoGuo, important insight can
be gained from the previously reported studies on the
electrochemical oxidation of this compound and the
enzymatic oxidation of its analogue, uric acid. From the
electrochemical model (39, 40), it has been determined
that 8-oxoGuo will undergo a rapid, two-electron oxida-
tion to yield a quinonoid diimine intermediate, 3 (Figure
4). This intermediate is transient since it is extremely
susceptible to nucleophilic attack and is readily hydro-
lyzed to yield several products, including 2-amino-4,5,6-
trioxopyrimidine and 5-guanidinohydantoin. Similarly,
in the enzymatic oxidation of uric acid (41), a species
analogous to 3, namely, dehydrourate, is formed as an
The peroxo bond in HOONO is very weak, with a Gibbs
free energy change for homolysis in aqueous solution
estimated to be about 12.7 ( 3.7 kcal/mol (48). This fact
partially explains the ease of homolytic cleavage of the
•
peroxo bond in HOONO which yields HO• and NO2. Like

N-Nitration during the Reaction of ONOO- with 8-OxodGuo
Chem. Res. Toxicol., Vol. 13, No. 5, 2000 395
HOONO, 4 is expected to undergo peroxo bond cleavage
to give the C5-oxyl radical species, 5, with concomitant
•NO2 release. C5-oxyl radical species have also been
proposed as intermediates in the hydroxyl radical-medi-
ated and type I photosensitization oxidation of guanine
(49). Cleavage of the C5-C6 bond in 5 results in the
formation of 6.2 The latter intermediate subsequently
eliminates CO or CO2 to yield the ylidene radical, 7.
Ack n ow led gm en t. We thank J eff Simpson for his
tremendous assistance in acquiring HMQC and HMBC
data, Gary Kruppa at Bruker for providing access to an
APEX II fourier transform mass spectrometer, Christian
Berg and Paul Speir for their assistance in acquiring
HRMS data, and Robert Kennedy for his assistance in
acquiring CD spectra. NIH Grants 5-F31-HG00144 and
CA 26731 supported this work.
•
Radical-radical combination of 7 with NO2 then results
in the formation of IV.
Su p p or tin g In for m a tion Ava ila ble: The 300 MHz 1H
COSY, 75 MHz 13C NMR, and 500 MHz HMQC spectra for
IVr ed . This material is available free of charge via the Internet
at http://pubs.acs.org.
An alternative to the radical-based mechanism for the
formation of IV involves formation of N-[1-(2,3,5-tri-O-
acetyl-â-D-erythro-pentofuranosyl)-2,4-dioxoimidazolidin-
5-ylidene]guanidine, 8, with subsequent N-nitration oc-
curring via the nitronium ion, NO2+, which has also been
suggested as a nitrating species produced during per-
oxynitrite reactions carried out in the presence of transi-
tion metals and superoxide dismutase (50). However, to
N-nitrate guanidino compounds, extremely harsh condi-
tions, involving the use of mixtures of concentrated
sulfuric and nitric acids, must be employed (51). This fact,
together with previous reports on peroxynitrite-mediated
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•
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•
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Con clu sion
This work identifies N-nitro-N′-[1-(2,3,5-tri-O-acetyl-
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2
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