A novel nitroimidazole compound formed during the reaction of peroxynitrite with 2′,3′,5′-tri-O-acetyl-guanosine

Article abstract of DOI:10.1021/ja004296k

Peroxynitrite reacts with 2′,3′,5′-tri-O-acetyl-guanosine to yield a novel compound identified as 1-(2,3,5-tri-O-acetyl-β-D-erythro-pentofuranosyl) -5-guanidino-4-nitroimidazole (6). This characterization was achieved using a combination of UV/vis spectroscopy and ESI-MS. Additionally, 1 -(β-D-erythro-pentofuranosyl) 5-guanidino-4-nitroimidazole (6a) was synthesized by an independent route, characterized by UV/vis spectroscopy, ESI-MS, and ¹H- and ¹³C NMR, and shown to be identical to deacetylated 6. This product is extremely stable in aqueous solution at both pH extremes and is formed in significant yields. These characteristics suggest that this lesion may be useful as a specific biomarker of peroxynitrite-induced DNA damage. We also observed formation of 2′,3′,5′-tri-O-acetyl-8-nitroguanosine (2′,3′,5′-tri-O-acetyl-8-NO₂Guo), 2-amino-5-[(2,3,5 tri-O-acetyl-β-D-erythro-pentofuranosyl)amino]-4H-imidazol-4-one (2′,3′,5′-tri-O-acetyl-Iz), and the peroxy-nitrite-induced oxidation products of 2′,3′,5′-tri-O-acetyl-8-oxoGuo. The formation of 6 and 2′,3′,5′-tri-O-acetyl-8-NO₂Guo was rationalized by a mechanism invoking formation of the guanine radical.

Full text of DOI:10.1021/ja004296k

J. Am. Chem. Soc. 2001, 123, 12147-12151
12147
A Novel Nitroimidazole Compound Formed during the Reaction of
Peroxynitrite with 2′,3′,5′-Tri-O-Acetyl-Guanosine
†
†
,†,‡
Jacquin C. Niles, John S. Wishnok, and Steven R. Tannenbaum*
Contribution from the DiVision of Bioengineering and Department of Chemistry, Massachusetts Institute of
Technology, 77 Massachusetts AVenue, 56-731A, Cambridge, Massachusetts 02139
ReceiVed December 19, 2000. ReVised Manuscript ReceiVed August 9, 2001
Abstract: Peroxynitrite reacts with 2′,3′,5′-tri-O-acetyl-guanosine to yield a novel compound identified as
1
-(2,3,5-tri-O-acetyl-â-D-erythro-pentofuranosyl)-5-guanidino-4-nitroimidazole (6). This characterization was
achieved using a combination of UV/vis spectroscopy and ESI-MS. Additionally, 1-(â-D-erythro-pentofuranosyl)-
-guanidino-4-nitroimidazole (6a) was synthesized by an independent route, characterized by UV/vis
5
1
13
spectroscopy, ESI-MS, and H- and C NMR, and shown to be identical to deacetylated 6. This product is
extremely stable in aqueous solution at both pH extremes and is formed in significant yields. These characteristics
suggest that this lesion may be useful as a specific biomarker of peroxynitrite-induced DNA damage. We also
observed formation of 2′,3′,5′-tri-O-acetyl-8-nitroguanosine (2′,3′,5′-tri-O-acetyl-8-NO2Guo), 2-amino-5-[(2,3,5-
tri-O-acetyl-â-D-erythro-pentofuranosyl)amino]-4H-imidazol-4-one (2′,3′,5′-tri-O-acetyl-Iz), and the peroxy-
nitrite-induced oxidation products of 2′,3′,5′-tri-O-acetyl-8-oxoGuo. The formation of 6 and 2′,3′,5′-tri-O-
acetyl-8-NO2Guo was rationalized by a mechanism invoking formation of the guanine radical.
sugar damage results in strand breaks,1
9-21
and several base
Introduction
lesions have been identified. Both in DNA and at the nucleoside
-
Peroxynitrite (ONOO ) is a physiologically relevant and
reactive molecule formed by the diffusion-limited reaction of
22,23
level, guanine is the most reactive nucleobase.
Several
products of the reaction between 2′-deoxyguanosine (dG) and
•
•-
1
-
nitric oxide ( NO) with superoxide (O2 ). While ONOO is
stable in alkaline solutions, its conjugate acid, peroxynitrous
-
ONOO have been characterized (see Figure 1), including the
oxidation products, 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-
2
acid (ONOOH), is very short-lived (t1/2 ≈ 1 s), giving rise to
21
oxodG), 2-amino-5-[(3,5-di-O-acetyl-â-D-erythro-pentofura-
•
•
3-5
the HO / NO2 radical pair in ∼33% yield. The decomposition
nosyl)amino]-4H-imidazol-4-one (dIz), the precursor to 2,2-
diamino-4-[(2-deoxy-â-D-erythro-pentofuranosyl)amino]-5-(2H)-
-
of ONOO is dramatically enhanced in the presence of carbon
dioxide. Through intermediate formation of peroxynitrosocar-
24
oxazolone (dZ) and a spiroiminodihydantoin. The nitration
-
•-
•
bonate (ONOOCO2 ), the CO3 and NO2 free radicals are
25
product, 8-nitroguanine, has also been described, as well as
generated, each in 33% yield.6
-8
an addition product, 4,5-dihydro-5-hydroxy-4-(nitrosooxy)-2′-
2
6
Peroxynitrite causes oxidation and nitration of a variety of
substrates, including low molecular weight antioxidants such
deoxyguanosine (nox-dG).
Despite identification of the various reaction products, a clear
mechanism has not emerged to fully account for their formation.
In this contribution, we report the characterization of the novel
as ascorbate,9 trolox, and glutathione
,10
10
10,11
and larger bio-
and DNA. With DNA,
12
13-18
molecules such as lipids, proteins,
†
Division of Bioengineering and Environmental Health.
Department of Chemistry.
(14) Radi, R.; Beckman, J. S.; Bush, K. M.; Freeman, B. A. J. Biol.
Chem. 1991, 266, 4244-4250.
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‡
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(
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(
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(17) Mohr, S.; Stamler, J. S.; Brune, B. FEBS Lett. 1994, 348, 223-
227.
Chem. Soc. 1998, 120, 7211-7219.
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3544-3552.
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A. Biochem. Biophys. Res. Commun. 1995, 210, 1025-1030.
(20) Yermilov, V.; Yoshie, Y.; Rubio, J.; Ohshima, H. FEBS Lett. 1996,
399, 67-70.
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R.; Dedon, P. C. Chem. Res. Toxicol. 1997, 10, 386-392.
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(23) Burney, S.; Niles, J. C.; Dedon, P. C.; Tannenbaum, S. R. Chem.
Res. Toxicol. 1999, 12, 513-520.
(24) Niles, J. C.; Wishnok, J. S.; Tannenbaum, S. R. Org. Lett. 2001, 3,
963-966.
(
1
(
1
996, 327, 335-343.
(
(
(
7) Lymar, S. V.; Hurst, J. K. Inorg. Chem. 1998, 37, 294-301.
8) Goldstein, S.; Czapski, G. J. Am. Chem. Soc. 1998, 120, 3458-3463.
9) Squadrito, G. L.; Jin, X.; Pryor, W. A. Arch. Biochem. Biophys. 1995,
3
22, 53-59.
10) Lee, J.; Hunt, J. A.; Groves, J. T. J. Am. Chem. Soc. 1998, 120, 0,
053-6061.
11) Koppenol, W. H.; Moreno, J. J.; Pryor, W. A.; Ischiropoulos, H.;
Beckman, J. S. Chem. Res. Toxicol. 1992, 5, 834-842.
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Biochem. Biophys. 1991, 288, 481-487.
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82.
(
6
(
(
(25) Yermilov, V.; Rubio, J.; Becchi, M.; Friesen, M. D.; Pignatelli, B.;
Ohshima, H. Carcinogenesis 1995, 16, 2045-2050.
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3-7.
(
2
1
0.1021/ja004296k CCC: $20.00 © 2001 American Chemical Society
Published on Web 11/10/2001

12148 J. Am. Chem. Soc., Vol. 123, No. 49, 2001
Niles et al.
concentrations were determined by making dilutions in 0.1 M NaOH
-
1
-1 29
and measuring the absorbance at λ ) 302 nm (ꢀ ) 1670 M cm ).
Acetylation of Guo and Synthesis of 2,3,5-Tri-O-acetyl-[C8-D]-
Guo. Guo (10.2 g, 36 mmol) was acetylated by suspending in a 2:1
v/v pyridine/acetic anhydride mixture until all of the starting material
had been converted into the triacetylated product as determined by
HPLC and ESI-MS. Conversion of Guo into [C8-D]-Guo was achieved
30
as previously reported. Exchange was confirmed by negative ion ESI-
1
1
6
MS and H NMR in DMSO-d . From the H NMR, it was determined
that the product was 92% [C8-D]-Guo and 8% [C8-H]-Guo. Acetylation
was carried out as described above.
Synthesis of 1-(â-D-erythro-Pentofuranosyl)-5-cyanamido-4-ni-
troimidazole (5). To prepare this compound, 2,4,5-tribromoimidazole
3
1
(
1) was reduced to 4-bromoimidazole (2) and nitrated to give 5(4)-
32
bromo-4(5)-nitroimidazole (3), which was subsequently fused with
Figure 1. Summary of products we have confirmed to be formed
during the reaction of peroxynitrite with 3′,5′-di-O-acetyl-dG and
tetra-O-acetyl-ribose to give 1-(2,3,5-tri-O-acetyl-â-D-erythro-pento-
furanosyl)-5-bromo-4-nitroimidazole (4). The latter compound (1.0
33
2
′,3′,5′-tri-O-acetyl-Guo.
g, 2.22 mmol) was allowed to react with cyanamide (0.36 g, 6.66 mmol)
and sodium methoxide (0.28 g, 6.66 mmol) in anhydrous methanol
(45 mL) at room temperature for 2 days, at which time the reaction
was complete as determined by UV/vis spectroscopy and HPLC. For
characterization, 10 mL of the reaction mixture was evaporated to
dryness and taken up in ∼15 mL double distilled water and loaded
onto a C18, 125 Å, 55-105 µm beads (Waters, Milford, MA) column
preequilibrated and washed with a 99/1 water/acetonitrile mixture. The
product eluted as a broad band and was collected in 15 mL fractions.
compound, 6, formed during the reaction of 2′,3′,5′-tri-O-acetyl-
Guo with peroxynitrite. This product, together with 2′,3′,5′-tri-
O-acetyl-8-NO2Guo, gives definitive insights into the mechanism-
-
(
s) of 2′,3′,5′-tri-O-acetyl-Guo oxidation by ONOO . Thus, we
have proposed that the guanine radical is a key intermediate
from which 6 and 2′,3′,5′-tri-O-acetyl-8-NO2Guo are derived.
Furthermore, of potential biological significance is the fact
that 6 is a significant and stable product of 2′,3′,5′-tri-O-acetyl-
Guo oxidation even at low peroxynitrite concentrations and is
likely to arise only from the reaction of 2′,3′,5′-tri-O-acetyl-
Guo with peroxynitrite. Therefore, this compound may be
important in understanding peroxynitrite-induced mutagenesis
and also may serve as a specific biomarker of peroxynitrite-
induced DNA damage.
The third fraction was dried in vacuo and used as the analytical sample.
1
H NMR (D O) δ (ppm) 7.45 (s, 1H, H2), 5.58 (d, 1H, H1′), 4.37 (t,
2
1
H, H2′), 4.13 (t, 1H, H3′), 3.96 (m, 1H, H4′), 3.71-3.58 (m, 2H, H5′
13
and H5′′). C NMR (DMSO-d ) δ (ppm) 146.86 (C4), 133.02 (C5),
6
130.26 (C2), 118.96 (C7), 87.11 (C1′), 85.40 (C4′), 74.70 (C2′), 70.83
-
(C3′), 62.01 (C5′). ESI-MS: 284 (M - H) ; UV/vis: λmax ) 230 nm,
-
4
2
10 nm. HRMS calcd for C H N O
9 10 5 6
84.0632.
[M - H] 284.0631, found
Synthesis of Authentic 1-(â-D-erythro-Pentofuranosyl)-5-guani-
dino-4-nitroimidazole, 6a. The crude reaction mixture containing 5
Experimental Section
(
(
20 mL, 0.44 g, 0.99 mmol) was refluxed with ammonium chloride
1.65 g, 30.8 mmol) for 5 days after which the mixture was cooled
6 2
General. DMSO-d and D O were obtained from Cambridge Isotope
15
and evaporated to dryness in vacuo. The residue was taken up in water
and purified by semipreparative HPLC on a 250 mm × 10 mm, 5 µm
Nucleosil C18 column (Alltech). Ammonium acetate (50 mM) and
acetonitrile were used as solvents A and B, respectively. The column
was eluted isocratically with 1% B for 10 min before a gradient from
Laboratories (Andover, MA); Na NO
2
was from Isotech (Miamisburg,
OH). All solvents were HPLC grade.
Instrumentation. UV/vis measurements were made using an
HP8452 diode array spectrophotometer (Hewlett-Packard, Palo Alto,
1
13
CA). H NMR spectra were recorded at 500 MHz, and C NMR spectra
proton decoupled), at 125 MHz on an Inova 500 spectrometer (Varian).
1
to 20% B over 10 min was initiated. After a 2 min isocratic wash
(
with 20% B, the eluent composition was restored to 1% B over 3 min.
A flow rate of 4.0 mL/min was used, and products were monitored
1
H COSY and HETCOR 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 1090 or an 1100 diode array detector (Hewlett-
Packard). Electrospray ionization mass spectrometry (ESI-MS) and
tandem mass spectrometry (ESI-MS/MS) experiments were carried out
using either an HP 5989B (Hewlett-Packard) or TSQ 7000 (Finnigan,
San Jose, CA) mass spectrometer, respectively. Unless stated otherwise,
ESI-MS and ESI-MS/MS spectra were obtained in negative and positive
ion mode using spraying solutions with the composition 78/20 water/
simultaneously at 230, 380, and 410 nm. Approximate yield of 6a was
1
1
4
2
0-15%. H NMR (D
2
O) δ (ppm) 7.59 (s, 1H, H2), 5.56 (d, 1H, H1′),
.37 (t, 1H, H2′), 4.15 (t, 1H, H3′), 3.97 (m, 1H, H4′), 3.72-3.52 (m,
H, H5′ and H5′′). 13C NMR (DMSO-d
6
) δ (ppm) 158.07 (C7), 143.64
(
C4), 133.16 (C5), 129.99 (C2), 87.08 (C1′), 84.77 (C4′), 74.55 (C2′),
+
6
3
3
9.90 (C3′), 60.99 (C5′). ESI-MS: 303 (M + H) . UV/vis: 230 nm,
+
80 nm. HRMS calcd for C H N O
9 15 6 6
03.1059.
[M + H] 303.1053, found
Reaction of 2′,3′,5′-tri-O-Acetyl-guanosine with Peroxynitrite.
′,3′,5′-Tri-O-acetyl-Guo (100 nmol) was reacted with peroxynitrite (5
2
-propanol with 2% ammonium hydroxide or 50/50 water/methanol
2
with 0.05% acetic acid, repsectively.
2 4 3
µmol) in 150 mM KH PO , 25 mM NaHCO , pH 7.2 buffer (1 mL).
1
4
Peroxynitrite Synthesis. N-Peroxynitrite was prepared either by
ozonolysis of an alkaline solution of sodium azide (Fisher) solution27
or by the reaction of isoamyl nitrite (Aldrich, Milwaukee, WI) with
hydrogen peroxide (Fisher) at pH 12-14.28 In the latter case, excess
hydrogen peroxide was removed by passing the peroxynitrite solution
This mixture was purified on a 250 mm × 4.6 mm, 5 µm Columbus
C18 column (Phenomenex) using 50 mM ammonium acetate (solvent
A) and acetonitrile (solvent B) as mobile phases. The column was eluted
isocratically with 5% B for 10 min, followed by a gradient from 5 to
4
0% B in 20 min, another isocratic wash with 40% B for 5 min, and
15
through a 7.5 cm × 1.5 cm manganese oxide (Aldrich) column. N-
Peroxynitrite was prepared by reacting 53 µmol of H in 1 M HCl
as previously described. The reaction was
quenched by rapidly adding 35 µL of 5 M NaOH. Peroxynitrite
finally a gradient from 40 to 5% B over 5 min. The flow rate was 1.0
mL/min, and products were monitored simultaneously at 230, 252, and
2 2
O
1
5
29
with 53 µmol of Na NO
2
(30) Agasimundin, Y. S.; Oakes, F. T.; Kostuba, L. J.; Leonard, N. J. J.
Org. Chem. 1985, 50, 2468-2474.
(
27) Pryor, W. A.; Cueto, R.; Jin, X.; Koppenol, W. H.; Ngu-Schwemlein,
(31) Balaban, I. E.; Pyman, F. L. J. Chem. Soc. 1922, 121, 947-958.
(32) Barrio, M. G.; Scopes, D. I. C.; Holtwick, J. B.; Leonard, N. J.
Proc. Natl. Acad. Sci. U.S.A. 1981, 78, 3986-3988.
(33) Hasan, A.; Lambert, C. R.; Srivastava, P. C. J. Heterocycl. Chem.
1990, 27, 1877-1883.
M.; Squadrito, G. L.; Uppu, P. L.; Uppu, R. M. Free Radical Biol. Med.
1
995, 18, 75-83.
(
(
28) Uppu, R. M.; Pryor, W. A. Anal. Biochem. 1996, 236, 242-249.
29) Hughes, M. N.; Nicklin, H. G. J. Chem. Soc. 1968, 450-452.

NoVel Nitroimidazole Compound
J. Am. Chem. Soc., Vol. 123, No. 49, 2001 12149
Scheme 1. Synthesis of Authentic
1
6
-(â-D-erythro-Pentofuranosyl)-5-guanidino-4-nitroimidazole,
a
a
a
a) NH
4
SO
4
, chlorotrimethylsilane, anhydrous hexamethyldisilazane,
Cl
) 2,3,5-tri-O-acetyl-â-D-
) â-D-pentofuranosyl.
reflux for 16 h; b) 1,2,3,5-tetra-O-acetyl-â-D-ribofuranose, CH
SnCl , stir at rt for 18 h under Ar atm, R
erythro-pentofuranosyl, and R
2
2
,
4
1
2
2
Figure 2. Impact of CO on the relative yields of the various products
the range 250-320 nm. This suggested that the purine ring
of peroxynitrite-mediated 2′,3′,5′-tri-O-acetyl-Guo oxidation. VII (pres-
ently uncharacterized) is the precursor to 3-(2,3,5-tri-O-acetyl-â-D-
erythro-pentofuranosyl)-2,4,6-trioxo-[1,3,5]triazinane-1-carboxami-
dine, I.
system had been destroyed. ESI-MS studies revealed that this
-
compound had a molecular weight of 428 [(M - H) ) 427].
ESI-MS deuterium exchange studies were carried out to
determine the number of exchangeable protons in 6. To do so,
the sample was exchanged into D2O (99.9%) by repeated
lyophilization and resuspension in D2O. In addition, a spraying
solution with composition 80/20 2-propanol/D2O, 2% am-
380 nm. The putative nitroimidazole product, 6, was collected and dried
in vacuo. Deacetylation was effected, as necessary, by dissolving the
residue in 0.5 M NaOH for 30-60 min, and the product was isolated
with a 1% B isocratic wash using the same solvents and column as
above.
Comparison of 6a and Deacetylated 6. 6a and deacetylated 6 were
compared using HPLC, UV/vis, and ESI-MS. For the HPLC studies, a
-
monium hydroxide was used. In these studies, the [M - H]
ion was observed at m/z ) 430. Since one proton (deuteron) is
lost to produce the observed negative ion, the ∆(m/z) ) +3
when D2O is used indicates that 6 has four exchangeable
protons. Furthermore, we were also able to show that when (a)
1
5
50 mm × 2.0 mm, 5 µm Columbus C18 column was used along with
0 mM ammonium acetate (solvent A) and acetonitrile (solvent B) as
1
4
2
′,3′,5′-tri-O-acetyl-[C8-D]-Guo reacted with N-peroxynitrite,
mobile phases. The column was eluted isocratically with 1% B at a
flow rate of 0.2 mL/min, and products were monitored simultaneously
at 230 and 380 nm.
15
and (b) 2′,3′,5′-tri-O-acetyl-Guo reacted with N-peroxynitrite,
the molecular weight of 6 increased by 1 amu (data not shown).
These results indicated, respectively, that (i) the C8-H of the
parent 2′,3′,5′-tri-O-acetyl-Guo was retained, and (ii) in con-
junction with the molecular weight of 6, that a peroxynitrite-
derived nitro group was incorporated into 6. Altogether, the
above findings were consistent with the base moiety of 6 being
Results
When we reacted 2′,3′,5′-tri-O-acetyl-Guo with peroxynitrite,
the previously reported products 2′,3′,5′-tri-O-acetyl-8-NO2Guo
and 2′,3′,5′-tri-O-acetyl-Iz were detected by both HPLC-UV
and ESI-MS (see Figure 2A). No 2′,3′,5′-tri-O-acetyl-8-oxoGuo
5
(4)-guanidino-4(5)-nitroimidazole. To prove this, we synthe-
sized authentic 1-(â-D-erythro-pentofuranosyl)-5-guanidino-4-
nitroimidazole, 6a, by an independent route for comparison with
deacetylated 6.
Synthesis of 6a. The synthesis of 6a is summarized in
Scheme 1. The preparation of compounds 2, 3, and 4 has been
was detected by these methods, but its peroxynitrite-induced
oxidation products I, II and IV3
4-36
were identified (Figures 1
and 2A). Since 8-oxodG is at least 1000 times more readily
oxidized by peroxynitrite than dG,37 this finding indicated that
2
2
′,3′,5′-tri-O-acetyl-8-oxoGuo formed during the oxidation of
′,3′,5′-tri-O-acetyl-Guo was further oxidized. Previously, nox-
3
1
32
reported previously by Balaban et al., Barrio et al., and Hasan
3
3
et al., respectively. In the report by Hasan et al., a mixture of
the structural isomers 1-(2,3,5-tri-O-acetyl-â-D-erythro-pento-
furanosyl)-5-bromo-4-nitroimidazole (4) and 1-(2,3,5-tri-O-
acetyl-â-D-erythro-pentofuranosyl)-4-bromo-5-nitroimidazole was
obtained during the fusion of the ribosyl moiety and the 4(5)-
bromo-5(4)-nitroimidazole. However, in our experiments using
the same conditions, a single product having UV/vis charac-
teristics of the 1-(2,3,5-tri-O-acetyl-â-D-erythro-pentofuranosyl)-
dG was identified as a product arising from the addition of
2
6
peroxynitrite across the C4-C5 double bond of dG. In our
experiments, though, we could not detect any products having
a molecular weight corresponding to this compound. However,
a novel and stable nitroimidazole compound, 6, was isolated
and characterized as detailed below. The impact of CO2 on the
yield of the various products and on the destruction of 2′,3′,5′-
tri-O-acetyl-Guo was also investigated. We found that CO2
enhanced both the oxidation and nitration product yields (Figure
33
3
8
5
-bromo-4-nitroimidazole isomer was isolated. To verify this
observation, we reacted this product (0.30 g, 0.67 mmol) with
sodium cyanide (0.17 g, 3.5 mmol) and potassium iodide (17.66
mg, 0.11 mmol) in DMSO at ambient temperature. Under these
conditions, cyanide is known to displace the bromo group of
2A), and correspondingly, more 2′,3′,5′-tri-O-acetyl-Guo reacted
in the presence of CO2 than in its absence (Figure 2B).
Characterization of 6. The UV/vis spectrum of 6 exhibited
maxima at 230 and 380 nm, with no significant absorbance in
1
-(2,3,5-tri-O-acetyl-â-D-erythro-pentofuranosyl)-5-bromo-4-ni-
3
9,40
(
34) Niles, J. C.; Burney, S.; Singh, S. P.; Wishnok, J. S.; Tannenbaum,
S. R. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 11729-11734.
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36) Tretyakova, N. Y.; Niles, J. C.; Burney, S.; Wishnok, J. S.;
Tannenbaum, S. R. Chem. Res. Toxicol. 1999, 12, 459-466.
37) Uppu, R. M.; Cueto, R.; Squadrito, G. L.; Salgo, M. G.; Pryor, W.
A. Free Radical Biol. Med. 1996, 21, 407-411.
troimidazole but not that of its structural isomer.
Analysis
(
(38) Rousseau, R. J.; Robins, R. K.; Townsend, L. B. J. Am. Chem. Soc.
1968, 90, 2661-2668.
2
(
(39) Baddiley, J.; Buchanan, J. G.; Hardy, F. E.; Stewart, J. J. Chem.
Soc. 1959, 2893-2901.
(
(40) Rousseau, R. J.; Townsend, L. B.; Robins, R. K. Chem. Commun.
1966, 9, 265-266.

12150 J. Am. Chem. Soc., Vol. 123, No. 49, 2001
Niles et al.
Figure 3. Both deacetylated 6 (above) and authentic 1-(â-D-erythro-pentofuranosyl)-5-guanidino-4-nitroimidazole, 6a (below) have identical HPLC
retention times and UV spectra.
of the reaction mixture by HPLC and ESI-MS revealed that
the starting material had been quantitatively converted into the
cyano derivative.41 This finding confirmed, therefore, that
have shown that oxidation and nitration yields are enhanced in
CO -containing versus CO -free buffers. In addition to confirm-
2
2
ing earlier reports of 8-oxoGuo, 8-NO Guo, and Iz formation,
2
1-(2,3,5-tri-O-acetyl-â-D-erythro-pentofuranosyl)-5-bromo-4-ni-
we have identified the novel compound, 5-guanidino-4-nitroimi-
troimidazole is the sole product of the glycosylation reaction.
The synthesis of 5 has not previously been described and was
achieved by displacing the bromo group of 4 with cyanamide
in a reaction analogous to the cyanide displacement reaction.
No displacement of the nitro group was observed, and this
reaction was quantitative (see Experimental Section for char-
acterization). Finally, the cyanamido derivative was converted
to 6a in ∼10-15% yield by refluxing with ammonium chloride,
purified by HPLC and characterized as described in the
Experimental section, confirming its identity as 1-(â-D-erythro-
pentofuranosyl)-5-guanidino-4-nitroimidazole.
dazole, 6. The formation of both 6 and 2′,3′,5′-tri-O-acetyl-8-
NO2Guo provide important insight into the mechanism by which
peroxynitrite oxidizes 2′,3′,5′-tri-O-acetyl-Guo.
Mechanism of Peroxynitrite-Mediated Oxidation of 2′,3′,5′-
Tri-O-acetyl-Guo Leading to Formation of 6 and 2′,3′,5′-
Tri-O-acetyl-8-NO2Guo. We have proposed the mechanism
summarized in Scheme 2 that involves, as a first step, the one-
electron oxidation of 2′,3′,5′-tri-O-acetyl-Guo to produce the
•
+
radical cation, 2′,3′,5′-tri-O-acetyl-Guo . Similar one-electron
•
-
•- 42
43
oxidations of Guo by Br2 and SO4
,
photoionization, and
44
•+
MnTmPyp/KHSO5 have been previously described. The Guo
Comparison of 6a with Deacetylated 6. Deacetylated 6 was
compared with 6a using HPLC-UV and ESI-MS. Each
compound was analyzed separately and found to have the same
retention time (4.3 min) and UV/vis spectrum as shown in Figure
42,45
(pKa ) 3.9
) will rapidly deprotonate at pH 7.2-7.4 to yield
•
the neutral radical, Guo . In fact, the species produced from
•
-
46
•
peroxynitrite, namely CO3 (E° ) 1.5 V ) and HO (E° ) 1.9
V ), are sufficiently potent to oxidize Guo (E° ) 1.29 V ).
Also, the Guo has significant unpaired electron density at the
O6, C5, and C8 positions. These properties are expected to
4
7
48
3. When both compounds were mixed, a single peak eluted after
•
4
.3 min. Additionally, we verified that 6a remained unchanged
4
9
during deacetylation by subjecting it to the same conditions as
followed by HPLC analysis to determine that the retention
6
(
42) Candeias, L. P.; Steenken, S. J. Am. Chem. Soc. 1989, 111, 1094-
099.
(43) Candeias, L. P.; Steenken, S. J. Am. Chem. Soc. 1992, 114, 699-
04.
44) Vialas, C.; Pratviel, G.; Claparols, C.; Meunier, B. J. Am. Chem.
Soc. 1998, 120, 11548-11553.
45) Steenken, S. Chem. ReV. 1989, 89, 503-520.
(46) Huie, R. E.; Clifton, C. L.; Neta, P. Radiat. Phys. Chem. 1991, 38,
477-481.
47) Stanbury, D. M. AdV. Inorg. Chem. 1989, 33, 69-138.
(48) Steenken, S.; Jovanovic, S. V. J. Am. Chem. Soc. 1997, 119, 617-
618.
time and UV spectrum had not changed. Taken together, these
data confirmed the identity of 6 as 1-(2,3,5-tri-O-acetyl-â-D-
erythro-pentofuranosyl)-5-guanidino-4-nitroimidazole.
1
7
(
Discussion
(
In this study, we have examined the impact of CO2 on the
peroxynitrite-mediated oxidation of 2′,3′,5′-tri-O-acetyl-Guo, and
(
(41) During the reaction with cyanide, some diacetylated cyano product
is also formed, and the yield increases with reaction time. This most likely
results from deacetylation of 5 due to trace amounts of water present, which
leads to formation of HBr and acidification of the reaction mixture.
(49) Hole, E. O.; Nelson, W. H.; Close, D. M.; Sagstuen, E. J. Chem.
Phys. 1987, 86, 5218-5219.

NoVel Nitroimidazole Compound
J. Am. Chem. Soc., Vol. 123, No. 49, 2001 12151
Scheme 2. Proposed Mechanism for the Formation of 6 and
It is worth emphasizing that although the reaction of
peroxynitrite with dG has been previously studied, this is the
first report of nitroimidazole product formation during this
reaction. In many of the prior studies, products had been
characterized but no mechanistic rationalization provided. Here,
we have proposed a radical-based mechanism for the formation
of 6, consistent with the extensively studied mechanism of
peroxynitrite decomposition.
2
′,3′,5′-Tri-O-acetyl-8-NO2Guo during the
Peroxynitrite-Induced Oxidation of 2′,3′,5′-Tri-O-acetyl-Guo
Biological Significance
Product 6 is a significant product of peroxynitrite-mediated
2
′,3′,5′-tri-O-acetyl-Guo oxidation. Importantly, this product is
extremely stable and is expected to accumulate in DNA unless
it is efficiently repaired. Therefore, this lesion might contribute
to the mutagenicity of peroxynitrite, which has been shown to
induce predominantly G f T (75%), G f A (14%), and G f
C (12%) mutations in the supF gene treated in vitro and
5
0
transiently transfected into human AD293 cells. The specific
lesion(s) causing these mutations remain(s) unknown, and thus,
to evaluate the contribution of 6, understanding both the
mutagenic potential and kinds of mutations induced by 6
becomes critical. Additionally, the stability of this lesion implies
that it might be useful as a highly specific biomarker of
peroxynitrite-induced DNA damage. This is particularly inter-
esting since it can facilitate a comparison of the extent of
peroxynitrite-induced DNA damage occurring in inflamed
tissues, where peroxynitrite production is expected to be high,
relative to normal tissues.
Summary
•+
remain unchanged for 2′,3′,5′-tri-O-acetyl-Guo . Thus, radical
We have identified the novel compound 6 as a stable and
significant product of the reaction between peroxynitrite and
′,3′,5′-tri-O-acetyl-Guo. From the product identity, we have
proposed a mechanism that implicates the guanosine radical as
a key intermediate in the formation of 6 and 2′,3′,5′-tri-O-acetyl-
-NO2Guo. However, involvement of the guanosine radical
cation cannot be completely excluded. As the product 6 is likely
to be a specific peroxynitrite-induced product, it has the potential
to serve as a biomarker of peroxynitrite-induced DNA damage
and, at the same time, expand our understanding of the toxicity
and mutagenicity of peroxynitrite-induced DNA base lesions.
•
combination between NO2 and the C8 or C5 positions of
•
2
′,3′,5′-tri-O-acetyl-Guo , respectively, gives the intermediates
2
e and f, as shown in Scheme 2. In the case of C8 addition, e
rearomatizes to yield 2′,3′,5′-tri-O-acetyl-8-NO2Guo. In the case
of C5 addition, f can be attacked by water at the electrophilic
C6 to yield g, with subsequent cleavage of the C5-C6 bond to
give the carbamate derivative, h. Decarboxylation of h then leads
to formation of the final product 6. While deprotonation of the
8
•
+
2
′,3′,5′-tri-O-acetyl-Guo is likely to occur concurrently with
or rapidly following its formation, it is possible for the 2′,3′,5′-
•
+
tri-O-acetyl-Guo to serve as a precursor to the observed
products as shown in Scheme 2. The reactions are analogous
•
Acknowledgment. We thank Jinping Gan for preparation
of the C8-[D]-Guo, and Li Li in the Department of Chemistry
Instrument Facility for obtaining HRMS data. The National
Institutes of Health Grants 5-F31-HG00144 and CA26731
supported this work.
to those described for the 2′,3′,5′-tri-O-acetyl-Guo , with forma-
•
tion of a and b resulting from the combination of NO2 with
•
+
the C8 and C5 of the 2′,3′,5′-tri-O-acetyl-Guo , respectively.
Deprotonation of a then leads to 2′,3′,5′-tri-O-acetyl-8-NO2Guo
formation, while b is hydrolyzed to give c, which undergoes
C5-C6 bond cleavage to yield d. This intermediate then
undergoes decarboxylation and deprotonation to yield the final
product 6.
JA004296K
(50) Juedes, M. J.; Wogan, G. N. Mutat. Res. 1996, 349, 51-61.