Hydroxyl-radical-induced decomposition of 2'-deoxycytidine in aerated aqueous solutions

Article abstract of DOI:10.1021/ja982461e

The stable products resulting from γ-irradiation of 2'-deoxycytidine in aerated aqueous solutions have been purified to homogeneity and characterized by MS and NMR analyses. The main group of products, which are similar in structure to those that arise from the ·OH-mediated decomposition of thymidine, included four diastereomers of 5,6-dihydroxy-5,6-dihydro-2'- deoxyuridine (2-5), 5-hydroxy-2'-deoxycytidine (6), two diastereomers of N₁- (2-deoxy-β-D-erythro-pentofuranosyl)-5-hydroxyhydantoin (7 and 8), and N₁- (2-deoxy-β-D-erythro-pentofuranosyl)formamide (9). These products are likely formed by way of either intermediate 5(6)-hydroxy-6(5)-peroxyl radicals or the corresponding hydroperoxides. In labeling experiments, ¹⁸O₂ was incorporated into products 2-9 with about 30% label at C₅ and 60% at C₆. Several other products were observed in the γ-irradiation of 2'- deoxycytidine in aerated aqueous solutions. They included four isomeric nucleosides of biuret (10-13), two diastereomers of N₁-(2-deoxy-β-D- erythro-pentofuranosyl)-1-carbamoyl-2-oxo-4,5-dihydroxyimidazoline (14, 15), and aminocarbonyl[2-(2-deoxy-β-D-erythro-pentofuranosyl)amino]-2-oxo- ethylcarbamic acid (16). The first step in the formation of these products is probably intramolecular addition of the 5-hydroxy-6-hydroperoxide to C₄ of the pyrimidine ring, resulting in a 4,6-endoperoxide which decomposes into an open-chain α-hydroxyaldehyde by C₄-C₅ cleavage. The formation of products 10-13 can be explained by subsequent fragmentation of the intermediate α- hydroxyaldehyde, whereas cyclization gives rise to products 14 and 15. In the case of products 16, a series of reactions have been proposed, starting with the rearrangement of the α-hydroxyaldehyde. The above pathways are supported by ¹⁸O₂-labeling experiments. Finally, we characterized two diastereomers of 5',6-cyclo-5-hydroxy-5,6-dihydro-2'-deoxyuridine(17,81) in the γ- irradiation of 2'-deoxycytidine in aerated aqueous solutions. These products likely arise from initial ·OH abstraction of a proton from C(5') of the sugar moiety, followed by intramolecular addition of the resulting radical to C₆ of the cytosine moiety. The ·OH-mediated decomposition of 2'- deoxycytidine in DNA is discussed.

Full text of DOI:10.1021/ja982461e

J. Am. Chem. Soc. 1999, 121, 4101-4110
4101
Hydroxyl-Radical-Induced Decomposition of 2′-Deoxycytidine in
Aerated Aqueous Solutions
,†
‡
‡
,‡
J. Richard Wagner,* Chantal Decarroz, Maurice Berger, and Jean Cadet*
Contribution from the Department of Nuclear Medicine and Radiobiology, Faculty of Medicine,
UniVersity of Sherbrooke, Sherbrooke, Quebec, Canada J1H 5N4, and D e´ partement de Recherche
Fondamentale sur la Mati e` re Condens e´ e, SCIB/LAN, CEA/Grenoble, F-38054 Grenoble, Cedex 9, France
ReceiVed July 13, 1998
Abstract: The stable products resulting from γ-irradiation of 2′-deoxycytidine in aerated aqueous solutions
have been purified to homogeneity and characterized by MS and NMR analyses. The main group of products,
•
which are similar in structure to those that arise from the OH-mediated decomposition of thymidine, included
four diastereomers of 5,6-dihydroxy-5,6-dihydro-2′-deoxyuridine (2-5), 5-hydroxy-2′-deoxycytidine (6), two
diastereomers of N1-(2-deoxy-â-D-erythro-pentofuranosyl)-5-hydroxyhydantoin (7 and 8), and N1-(2-deoxy-
â-D-erythro-pentofuranosyl)formamide (9). These products are likely formed by way of either intermediate
1
8
5
(6)-hydroxy-6(5)-peroxyl radicals or the corresponding hydroperoxides. In labeling experiments, O2 was
incorporated into products 2-9 with about 30% label at C5 and 60% at C6. Several other products were observed
in the γ-irradiation of 2′-deoxycytidine in aerated aqueous solutions. They included four isomeric nucleosides
of biuret (10-13), two diastereomers of N1-(2-deoxy-â-D-erythro-pentofuranosyl)-1-carbamoyl-2-oxo-4,5-
dihydroxyimidazolidine (14, 15), and aminocarbonyl[2-(2-deoxy-â-D-erythro-pentofuranosyl)amino]-2-oxo-
ethylcarbamic acid (16). The first step in the formation of these products is probably intramolecular addition
of the 5-hydroxy-6-hydroperoxide to C4 of the pyrimidine ring, resulting in a 4,6-endoperoxide which
decomposes into an open-chain R-hydroxyaldehyde by C4-C5 cleavage. The formation of products 10-13
can be explained by subsequent fragmentation of the intermediate R-hydroxyaldehyde, whereas cyclization
gives rise to products 14 and 15. In the case of product 16, a series of reactions have been proposed, starting
1
8
with the rearrangement of the R-hydroxyaldehyde. The above pathways are supported by O2-labeling
experiments. Finally, we characterized two diastereomers of 5′,6-cyclo-5-hydroxy-5,6-dihydro-2′-deoxyuridine
(
17, 18) in the γ-irradiation of 2′-deoxycytidine in aerated aqueous solutions. These products likely arise from
•
initial OH abstraction of a proton from C5′ of the sugar moiety, followed by intramolecular addition of the
resulting radical to C6 of the cytosine moiety. The OH-mediated decomposition of 2′-deoxycytidine in DNA
•
is discussed.
Introduction
studies have focused on 8-oxo-7,8-dihydro-2′-deoxyguanosine,
a major oxidation product of 2′-deoxyguanosine in DNA. The
level of 5-hydroxy-2′-deoxycytidine, an oxidation product of
5
The deleterious effects of ionizing radiation may largely be
attributed to DNA damage induced by both the reaction of
hydroxyl radicals ( OH) and direct ionization of DNA. In some
respects, ionizing radiation is similar to oxidative metabolism.
The latter generates copious amounts of superoxide ions and
2
′-deoxycytidine, in cellular DNA was found to be comparable
•
1,2
6
to that of 8-oxo-7,8-dihydro-2′-deoxyguanosine. In addition,
several oxidative modifications of cytosine within DNA have
been shown to be substrates for Escherichia coli endonuclease
III, an enzyme involved in the repair of oxidative DNA damage
•
H2O2, which can produce OH upon reaction with transition
metal ions such as Fe2 and Cu . Oxidative DNA damage
resulting from oxidative metabolism contributes to the natural
processes of aging and cancer.3
+
+
7
by base excision. Using synthetic oligonucleotides, the ef-
ficiency of strand cleavage by endonuclease III was determined
,4
for three oxidative lesions of cytosine: 5,6-glycols > 5-hy-
Hydroxyl-radical-induced damage to DNA leads to a multi-
tude of sugar and base modifications. The majority of biological
8
droxycytosine > 5-hydroxyuracil. It should also be noted that
there is evidence for similar repair activity in human cells.9
Both 5-hydroxy-2′-deoxycytidine and 5-hydroxy-2′-deoxy-
uridine lesions appear to be mutagenic. They have been shown
to miscode during DNA synthesis in vitro such that C is
incorporated opposite 5-hydroxy-2′-deoxycytidine, while A goes
(5) Kasai, H. Mutat. Res. 1997, 387, 147-163.
(6) Wagner, J. R.; Hu, C. C.; Ames, B. N. Proc. Natl. Acad. Sci. U.S.A.
1992, 89, 3380-3384.
(7) (a) Hatahet, Z.; Kow, Y.; Purmal, A. A.; Cunningham, R. P.; Wallace,
S. S. J. Biol. Chem. 1994, 269, 18814-18820. (b) Wagner, J. R.; Blount,
B. C.; Weinfeld, M. Anal. Biochem. 1996, 233, 76-86.
(8) Wang, D.; Essigmann, J. M. Biochemistry 1997, 36, 8628-8633.
(9) Collins, A. R.; Ai-guo, M., Duthie, S. T. Mutat. Res. 1995, 336, 69-
77.
*
Corresponding authors.
University of Sherbrooke.
SCIB/LAN.
†
‡
(
1) von Sonntag, C. The Chemical Basis of Radiation Biology; Taylor
Francis: London, 1987.
2) (a) Cadet, J. DNA Adducts: Identification Biological Significance;
(
Hemminki, K., Dipple, A., Shuker, D. E. G., Kadlubar, F. F., Segerback,
D., Bartsch, H., Eds.; IARC Scientific Publications: Lyon, France, 1994;
Vol. 125, pp 245-276. (b) Cadet, J.; Berger, M.; Douki, T.; Ravanat, J.-L.
ReV. Physiol. Biochem. Pharmacol. 1997, 131, 1-87.
(
3) Sies, H. OxidatiVe Stress: Oxidants and Antioxidants; Academic: New
York, 1991.
(4) Ames, B. N.; Shigenaga. M. K.; Hagen, T. M. Proc. Natl. Acad. Sci.
U.S.A. 1993, 90, 7915-7922.
1
0.1021/ja982461e CCC: $18.00 © 1999 American Chemical Society
Published on Web 04/20/1999

4102 J. Am. Chem. Soc., Vol. 121, No. 17, 1999
Wagner et al.
opposite 5-hydroxy-2′-deoxyuridine.10 This suggests that the
formation of 5-hydroxy-2′-deoxycytidine and 5-hydroxy-2′-
deoxyuridine in DNA may lead to C-to-G transversions and
C-to-T transitions, respectively. Recently, the specific incorpora-
tion of 5-hydroxy-2′-deoxycytidine, 5-hydroxy-2′-deoxyuridine,
and the 5,6-glycol of 2′-deoxyuridine into E. coli was found to
cause C-to-T transitions with a frequency of 0.05%, 83%, and
d-CpC, d-CpCpC, dCMP, and DNA was examined.23 Eleven
modified bases along with an equal number of modified
nucleosides were tentatively identified by FAB-MS. However,
it is difficult to draw any definite conclusions about the
mechanism of decomposition of cytosine derivatives in this
study because of the high concentration of Fenton reagents, i.e.,
2
+
3+
1 mM of Fe or Fe and 2 mM of H2O2, which may interfere
with the fate of intermediate species. For example, transition
metal ions have been shown to induce the reduction or oxidation
1
1
8
0% respectively. In general, we expect that the oxidation
products of 2′-deoxycytidine have a good chance of giving rise
to mutations in cellular DNA because oxidation induces
deamination that directly affects base pairing. This may explain
the bias toward C-to-T transitions in the spectrum of mutations
induced by ionizing radiation, as well as exogenous and
•
24
of initial OH adducts of cytosine derivatives.
In the present work, the separation of dCyd oxidation products
was achieved by a combination of normal- and reversed-phase
HPLC. Photosensitization with menadione and UVA light was
utilized as a means to prepare large quantities of the products
for chemical analyses. Together, the above procedures have
permitted the separation and identification of 17 nucleoside
products that arise from γ-irradiation and photosensitization of
dCyd in aerated aqueous solutions. This work provides the
mechanistic and analytical aspects necessary to explore the
12
endogenous reactive oxygen species.
•
The main products that arise from the OH-mediated decom-
position of uracil, thymine, and cytosine under aerated conditions
have been characterized.1
3-15
These studies have been extended
to pyrimidine nucleosides. For example, 22 modified nucleosides
have been characterized after γ-irradiation of thymidine in
aerated aqueous solutions.16 In addition, the decomposition of
the individual thymidine 5(6)-hydroxy-6(5)-hydroperoxides,
including eight diastereomers, has been studied in detail by
kinetics and product analysis.17 These studies have been
extended to 5-methyl-2′-deoxycytidine, and recently the corre-
sponding 5,6-glycols and methyl oxidation products were
reported.1 In contrast, it has been difficult to study the stable
•
consequences of OH-mediated oxidation products of dCyd in
more complex biological systems.
Experimental Section
2
′-Deoxycytidine (dCyd; 1) was obtained from Sigma (St. Louis,
MO). High-performance liquid chromatography (HPLC) was performed
with dual piston pumps (model M6000, Millipore, Mildford, MA),
equipped with flow cell spectrophotometers (model 441, Millipore) and
refractive index detectors (model 401, Millipore), using either home-
packed silica gel columns with CH Cl/MeOH/H O (4:2:1, 5% CH OH
8,19
•
products from the OH-mediated decomposition of dCyd because
of problems associated with their preparation and purification
from product mixtures. Further complications arise from the
involvement of labile products including the 5,6-glycols and
3
2
3
was added to the organic phase; solvent A), CH
3
2
CH COCH
3
/CH
3
2
CH -
5
(6)-hydroxy-6(5)-hydroperoxides of dCyd.20
OHCH
3
/H
2
O (75:16:9; solvent B), or octadecylsilyl silica gel columns
O without or with CH OH as
(Ultrasphere ODS 2) using bidistilled H
2
3
A number of recent studies has focused on the decomposition
1
13
the mobile phases. H NMR and C NMR were recorded on a Bruker
AM400 spectrometer (Wissembourg, France) in Fourier transform
mode, equipped with an Aspect 3000 computer. Chemical shifts were
of 2′-deoxycytidine (dCyd) induced by various free radical- or
oxidant-generating systems. The γ-irradiation of dCyd in frozen
aqueous solution was investigated and shown to produce 5,6-
dihydro-2′-deoxyuridine and 5′,6-cyclo-5,6-dihydro-2′-deoxy-
uridine, presumably by way of intermediate dCyd radical anions
calibrated in D
2
O with reference to 3-(trimethylsilyl)propionate-2,2,3,3-
1
d
4
as internal standard. H NMR spectra were simulated for best fit by
LAOCOON III software with a precision of 0.05 Hz. Mass spectrometry
analyses were performed on a Kratos MS 50 instrument equipped with
both electron impact (EI) and fast atom bombardement (FAB) attach-
ments. Gycerol was used as the matrix for FAB analysis. Direct analysis
of modified nucleosides was done by desorption chemical ionization
2
1
and cations. The exposure of dCyd to ozone in aqueous
solutions was recently shown to result in the formation of several
radiation-like nucleoside decomposition products.22 In another
study, the Fenton-reaction-mediated decomposition of dCyd,
3 3 3 3
(DCI) with CH CH(CH )CH and NH using a NERMAG apparatus.
(
10) Purmal, A. A.; Kow, Y.-W.; Wallace, S. S. Nucleic Acids Res. 1994,
2, 72-78.
11) Kreutzer, D. A.; Essigmann, J. M. Proc. Natl. Acad. Sci. U.S.A.
998, 95, 3578-3582.
12) (a) Waters, L. C.; Sikpi, M. O.; Preston, R. J.; Mitra, S.;
High-resolution mass spectrometry (HRMS) was carried out on a
ZabSpec-T instrument (Micromass, Manchester, UK) with nanospray
ionization in H O/CH OH/CH COOH (50:50:1).
2 3 3
2
(
1
(
Preparation of dCyd Oxidation Products. The preparation of dCyd
oxidation products in sufficient quantities for spectroscopic analyses
was carried out by menadione photosensitization. The procedure
involved photolysis of aqueous unbuffered solution (2 L) containing
menadione (0.8 mM) and dCyd (10 mM) using a Rayonet photochemi-
cal reactor (Southern New England Ultraviolet Co., Hawden, CT) fitted
Jaberaboansari, A. Radiat. Res. 1991, 127, 190-201. (b) Moraes, E. C.;
Keyse, S. M.; Tyrrell, R. M. Carcinogenesis 1990, 11, 283-293.
(
13) Schuchmann, M. N.; von Sonntag, C. J. Chem Soc., Perkin Trans.
2
1983, 1525-1531.
14) (a) Cadet, J.; T e´ oule, R. Biochim. Biophys. Acta 1971, 238, 8-26.
b) T e´ oule, R.; Cadet, J. J. Chem. Soc., Chem. Commun. 1971, 1269.
15) (a) Polverelli, M.; T e´ oule, R. Z. Naturforsch. 1974, 29c, 12-15.
(
(
(
16
-2
(
with 16 RPR-3500A lamps emitting a total of 1.5 × 10 photons cm
, as determined by ferrioxalate actinometry of UVA light (λmax
50 nm). During photolysis, the solutions were bubbled with O
b) Polverelli, M.; T e´ oule R. Z. Naturforsch. 1974, 29c, 16-18. (c)
-1
s
)
, and
Polverelli, M. Modifications radio-induites de la cytosine en solution aqueuse
a e´ r e´ e et apr e` s irradiation gamma du DNA d’Escherichia coli. Th e` se de
l’Universit e´ Joseph Fourier, Grenoble, France, 1983.
3
2
their temperature was maintained at 10 °C by circulation of cold tap
water. The mixture of dCyd oxidation products was evaporated to
dryness, first by rotary evaporation and then by extensive lyophilization.
Subsequently, the products were fractionated by HPLC using a normal-
phase semipreparative column and solvent A. Each of the fractions
was subsequently purified to homogeneity by reversed-phase HPLC
(
16) Cadet, J.; T e´ oule, R. Bull. Soc. Chim. Fr. 1975, 3-4, 879-884.
Cadet, J.; T e´ oule, R. Bull. Soc. Chim. Fr. 1975, 3-4, 885-890. Cadet, J.;
T e´ oule, R. Bull. Soc. Chim. Fr. 1975, 3-4, 891-895. T e´ oule, R.; Cadet, J.
Mol. Biol. Biochem. Biophys. 1978, 27, 171-203.
(
17) Wagner, J. R.; van Lier, J.; Berger, M.; Cadet, J. J. Am. Chem.
Soc. 1994, 116, 2235-2242.
2
with H O as the mobile phase. Hydroxyl-radical-induced oxidation
(
(
18) Bienvenu, C.; Cadet, J. J. Org. Chem. 1996, 61, 2632-2637.
19) Bienvenu, C.; Wagner, J. R.; Cadet, J. J. Am. Chem. Soc. 1996,
60
products were prepared by γ-irradiation ( Co) of continuously aerated
1
18, 11406-11411.
aqueous solutions of dCyd (1 mM). The solutions were irradiated for
(
20) Wagner, J. R.; van Lier, J. E.; Decarroz, C.; Berger, M.; Cadet, J.
Methods Enzymol. 1990, 186, 502-511.
(23) Luo, Y.; Henle, E. S.; Linn, S. J. Biol. Chem. 1996, 271, 21167-
21176.
(24) Chabita, K.; Saha, A.; Mandal, P. C.; Bhattacharyya, S. N.; Rath,
M. C.; Mukherjee, T. Radiat. Res. 1996, 146, 514-524.
(
21) Shaw, A. A.; Cadet, J. Int. J. Radiat. Biol. 1996, 70, 1-6.
(22) Girault, I.; Fort, S.; Molko, D.; Cadet, J. Free Radical Res. 1997,
2
6, 257-266.

Hydroxyl-Radical-Induced Decomposition of 2′-Deoxycytidine
J. Am. Chem. Soc., Vol. 121, No. 17, 1999 4103
Chart 1. Stuctures of Stable Radiation-Induced Products of
dCyd (1)
5-Hydroxy-2′-deoxycytidine (6). HPLC (silica gel, solvent A): k′
) 15.5 (octadecylsilyl silica gel, H
2
O), k′ ) 6.33. Yield (photolysis):
mg. UV (λmax): 289 nm. H NMR (250 MHz, D O, TSP): δ 7.37 (s,
H, H ), 6.29 (t, 1H, H1′), 4.44 (m, 1H, H3′), 4.04 (m, 1H, H4′), 3.85
1
9
1
2
6
(
dd, 1H, H5′), 3.76 (dd, 1H, H5′′), 2.39 (m, 1H, H2′), 2.27 (m, 1H, H2′′).
1
H NMR (250 MHz, DMSO-d
1
6
,TSP): δ 7.30 (s, 1H, NH), 6.74 (s,
O, TMS): δ 161.3 (C ), 155.6
H, NH). 13C NMR (100.62 MHz, D
2
2
(
3
1
C
4
), 128.1 (C
5
), 123.1 (C
6
), 86.6 (C1′), 86.0 (C4′), 70.6 (C3′), 61.3 (C5′),
+
9.2 (C2′). LC-MS: m/z (relative intensity) 244 (24, MH ), 177 (20),
28 (100), 117 (44, deoxyribose ). HRMS (ES, positive mode): calcd
+
+
for C
5S or 5R)-N
hydantoin (7). HPLC (silica gel, solvent A): k′ ) 3.62 (octadecylsilyl
H
9 13
3
N O
5
([M + H] ) 244.0934, found 244.0934.
(
1
-(2-Deoxy-â-D-erythro-pentofuranosyl)-5-hydroxy-
1
silica gel, H
MHz, D O, TSP): δ 5.99 (dd, 1H, H1′, J1′,2′ ) 8.1 Hz, J 1′,2′′ ) 6.4 Hz),
.54 (s, 1H, H ), 4.39 (m, 1H, H3′, J2′,3′ ) 6.1 Hz, J2′′,3′ ) 3.5 Hz), 3.92
m, 1H, H4′, J3′,4′ ) 3.1 Hz, J4′,5′ ) 4.1 Hz, J4′,5′′ ) 5.5 Hz), 3.73 (m,
2
O), k′ ) 1.83. Yield (photolysis): 9 mg. H NMR (400.13
2
5
(
1
)
6
H, H5′, J5′,5′′ ) -12.3 Hz), 3.65 (m, 1H, H5′′), 2.57 (m, 1H, H2′, J2′,2′′
20 min at a dose rate of 80 Gy/min, as determined by Fricke dosimetry
-13.8 Hz), 2.20 (m, 1H, H2′′). DCI-MS (CH
3
+
CH(CH
), 233 (30, MH ),
3
)CH
3
and
(
total dose ) 1.6 kGy). The structures of 1 and its radiation-induced
+
NH
3
): m/z (relative intensity) 250 (30, M + NH
4
products (2-18) are shown in Chart 1.
+
+
1
34 (60, base NH
mode): calcd for C
5R or 5S)-N
hydantoin (8). HPLC (silica gel, solvent A): k′ ) 4.37 (octadecylsilyl
4
), 117 (50, deoxyribose ). HRMS (ES, positive
cis-(5R,6S)-5,6-Dihydroxy-5,6-dihydro-2′-deoxyuridine (2). HPLC
+
H
8 12
N
2
O
6
(M ) 232.0696, found 232.0695.
(
1
silica gel, solvent A): k′ ) 13.6 (octadecylsilyl silica gel, H
2
O), k′ )
(
1
-(2-Deoxy-â-D-erythro-pentofuranosyl)-5-hydroxy-
.08, where k′ ) (retention volume - column void volume)/column
1
void volume. Yield (photolysis) of 2 and 3: 20 mg. H NMR (400.13
MHz, D
5
1
silica gel, H
MHz, D O, TSP): δ 5.98 (dd, 1H, H1′, J1′,2′ ) 8.4 Hz, J1′,2′′ ) 6.3 Hz),
.43 (s, 1H, H ), 4.43 (m, 1H, H3′, J2′,3′ ) 6.4 Hz, J2′′,3′ ) 3.1 Hz), 3.92
m, 1H, H4′, J3′,4′ ) 2.6 Hz, J4′,5′ ) 4.4 Hz, J4′,5′′ ) 5.6 Hz), 3.69 (m,
2
O), k′ ) 1.50. Yield (photolysis): 9 mg. H NMR (400.13
2
O, TSP): δ 6.17 (dd, 1H, H1′, J1′,2′ ) 8.5 Hz, J1′,2′′ ) 6.2 Hz),
.35 (d, 1H, H , J5,6 ) 3.8 Hz), 4.60 (d, 1H, H ), 4.41 (m, 1H, H3′, J2′,3′
6.4 Hz, J2′′,3′ ) 3.0 Hz), 3.94 (m, 1H, H4′, J3′,4′ ) 3.1 Hz, J4′,5′ ) 3.8
2
6
5
5
(
6
)
Hz, J4′,5′′ ) 5.2 Hz), 3.77 (m, 1H, H5′, J5′,5′′ ) -12.3 Hz), 3.70 (m, 1H,
1
3
1H, H , J
)
NH
) -12.1 Hz), 3.64 (m, 1H, H ), 2.53 (m, 1H, H , J
H
5′′), 2.40 (m, 1H, H2′, J2′,2′′ ) -14.0 Hz), 2.20 (m, 1H, H2′′). C NMR
100.62 MHz, D O, TMS): δ 171.0 (C ), 152.1 (C ), 85.5 (C4′), 84.3
1′), 75.4 (C ), 71 (C3′), 69.0 (C
5′
5′,5′′
5′′
2′
2′,2′′
-13.8 Hz), 2.16 (m, 1H, H2′′). DCI-MS (CH
3
+
CH(CH
), 233 (20, MH ),
3
)CH
3
and
(
(
(
2
4
2
+
): m/z (relative intensity) 250 (30, M + NH
4
C
6
5
), 61.8 (C5′), 36.8 (C2′). FAB-MS
3
+
8 12 2 6
117 (70), 160 (25). HRMS (ES, positive mode): calcd for C H N O
(M ) 232.0696, found 232.0695.
positive mode, glycerol mull): m/z (relative intensity) 377 (25, M +
+
+
+
glycerol + Na), 355 (25, M + glycerol), 285 (45, MNa ), 263 (30,
+
+
N -(2-Deoxy-â-D-erythro-pentofuranosyl)formamide (9). HPLC
MH ), 245 (50, MH - H
mull): m/z (relative intensity) 283 (10, M - H + Na), 261 (40, M
H). DCI-MS (CH CH(CH )CH and NH ): m/z (relative intensity)
), 263 (20, MH ), 245 (60, MH - H
2
O). FAB-MS (negative mode, glycerol
1
-
-
(silica gel, solvent A): k′ ) 9.60 (octadecylsilyl silica gel, H O), k′ )
2
1
-
2
(
3
3
3
3
2
1.08. Yield (photolysis): 9 mg. H NMR (400.13 MHz, D O, TSP): δ
+
+
+
(major) 8.12 (s, 1H, H , J ) 0.9 Hz), 5.89 (t, 1H, H , J ) 7.2 Hz,
80 (15, M + NH
4
2
O), 177
2
2,1′
1′ 1′,2′
+
J1′,2′′ ) 6.0 Hz), 4.39 (m, 1H, H , J
2′,3′ ) 6.2 Hz, J2′′,3′ ) 3.2 Hz, J3′,4′
100), 117 (100, 2-deoxyribose ). HRMS (ES, positive mode): calcd
3′
+
) 3.0 Hz), 3.96 (m, 1H, H , J
) 4.2 Hz, J4′,5′′ ) 5.3 Hz), 3.65 (m,
for C
9
H N O
14 2 7
([M + Na] ) 285.0699, found 285.0699.
4′
4′,5′
cis-(5S,6R)-5,6-Dihydroxy-5,6-dihydro-2′-deoxyuridine (3). HPLC
silica gel, solvent A): k′ ) 13.6 (octadecylsilyl silica gel, H O), k′ )
.08. Yield (photolysis) of 2 and 3: 20 mg. H NMR (400.13 MHz,
O, TSP): δ 6.15 (pst, 1H, H1′, J1′,2′ ) 7.3 Hz, J1′,2′′ ) 6.7 Hz), 5.34
d, 1H, H , J5,6 ) 3.8 Hz), 4.63 (d, 1H, H ), 4.42 (m, 1H, H3′, J2′,3′
.9 Hz, J2′′,3′ ) 4.2 Hz), 3.92 (m, 1H, H4′, J3′,4′ ) 4.3 Hz, J4′,5′ ) 3.6
Hz, J4′,5′′ ) 4.9 Hz), 3.79 (m, 1H, H5′, J5′,5′′ ) -12.3 Hz), 3.73 (m, 1,
1H, H5′, J5′,5′′ ) -12.2 Hz), 3.62 (m, 1H, H5′′), 2.27 (m, 1H, H2′′, J2′,2′′
) -14.0 Hz), 2.14 (m, 1H, H2′); δ (minor) 8.22 (s, 1H, H , J2,1′ ) 0.2
Hz), 5.62 (spt, 1H, H , J ) 7.7 Hz, J1′,2′′ ) 3.0 Hz), 4.38 (m, 1H,
(
1
D
(
2
2
1
1′
1′,2′
H
3′, J2′,3′ ) 6.2 Hz, J2′′,3′ ) 3.2 Hz, J3′,4′ ) 3.0 Hz), 3.95 (m, 1H, H4′
4′,5′ ) 4.2 Hz, J4′,5′′ ) 5.3 Hz), 3.65 (m, 1H, H5′, J5′,5′′ ) -12.2 Hz),
3.62 (m, 1H, H5′′), 2.31 (m, 1H, H2′′, J2′,2′′ ) -14.0 Hz), 2.16 (m, 1H,
,
2
6
5
)
J
6
13
H
2′). C NMR (100.62 MHz, D
2
2
O, TMS): δ (major) 165 (C ), 86.5
13
(C ), 79.2 (C ), 71.9 (C ), 62.3 (C ), 39.1 (C ); δ (minor) 168.1 (C ),
H
5′′), 2.34 (m, 1H, H2′, J2′,2′′ ) -14.1 Hz), 2.24 (m, 1H, H2′′). C NMR
100.62 MHz, D O, TMS): δ 172.3 (C dO), 153.0 (C dO), 85.4 (C4′),
4.9 (C1′), 75.8 (C ), 70.4 (C3′), 69.0 (C ), 61.2 (C5′), 37.3 (C2′). DCI-
4′
1′
3′
5′
2′
2
(
8
2
4
2
86.5 (C4′), 84.7 (C1′), 71.9 (C3′), 62.3 (C5′), 39.4 (C2′). FAB-MS (positive
+
ion, glycerol): m/z (relative intensity) 162 (30, MH ), 184 (15, M +
6
5
+
+
MS (CH
3
CH(CH
3
)CH
3
+
and NH
3
): m/z (relative intensity) 280 (M +
Na ), 254 (20), 117 (40, 2-deoxyribose ). HRMS (ES, positive
+
+
mode): calcd for the diacetylated derivative C H NO ([M + H] )
+
NH
4
, 13), 263 (MH , 17), 245 (MH - H
2
O, 60), 177 (100), 117
6
11
4
+
246.0973, found 246.0993.
-(2-Deoxy-â-D-erythro-pentofuranosyl)biuret (10). HPLC (silica
gel, solvent A): k′ ) 6.87 (octadecylsilyl silica gel, H O), k′ ) 3.66.
O, TSP): δ 5.86
(pst, 1H, H1′, J1′,2′ ) 6.2 Hz, J1′,2′′ ) 7.5 Hz), 4.45 (m, 1H, H3′, J2′,3′
(100). HRMS (ES, positive mode): calcd for C
H N
9 14 2
O
7
([M + Na] )
2
85.0699, found 285.0699.
trans-(5S,6S)-5,6-Dihydroxy-5,6-dihydro-2′-deoxyuridine (4). HPLC
silica gel, solvent A): k′ ) 7.75 (octadecylsilyl silica gel, H O), k′ )
O, TSP):
N
1
2
1
(
0
2
Yield (photolysis): 6 mg. H NMR (400 MHz, D
2
1
.80. Yield (photolysis): 17 mg. H NMR (400.13 MHz, D
2
)
δ 6.26 (dd, 1H, H1′, J1′,2′ ) 7.7 Hz, J1′,2′′ ) 6.2 Hz), 5.36 (d, 1H, H
5,6 ) 2.8 Hz), 4.47 (m, 1H, H3′, J2′,3′ ) 7.2 Hz, J2′′,3′ ) 3.4 Hz, J3′,4′
), 3.99 (m, 1H, H4′, J4′,5′ ) 4.3 Hz, J4′,5′′ ) 5.0
6
,
)
3.2 Hz, J2′′,3′ ) 6.0 Hz), 4.00 (m, 1H, H4′, J3′,4′ ) 2.6 Hz, J4′,5′ ) 4.3
Hz, J4′,5′′ ) 4.9 Hz), 3.70 (m, 2H, H5′, H5′′, J5′,5′′ ) -12.1 Hz), 2.32 (m,
J
2
1
.9 Hz), 4.24 (d, 1H, H
5
1H, H2′, J2′,2′′ ) -14.0 Hz), 2.20 (m, 1H, H2′′). H NMR (250 MHz,
Hz,), 3.80 (m, 2H, H5′,5′′, J5′,5′′ ) -11.5 Hz), 2.45 (m, 1H, H2′, J2′,2′′
)
6
DMSO-d , TMS): δ 8.80 (s, 1H, NH), 8.04 (d, 1H, NH, JNH,1′ ) 9.6
-
14.0 Hz), 2.30 (s, 1H, H2′′). DCI-MS (CH
3
CH(CH
3
+
)CH
3
and NH
3
):
Hz), 6.97 (s, 2H, NH). FAB-MS (positive mode, glycerol): m/e (relative
+
+
+
+
m/z (relative intensity) 280 (M + NH
O, 60), 177 (100), 117 (100).
trans-(5R,6R)-5,6-Dihydroxy-5,6-dihydro-2′-deoxyuridine (5). HPLC
silica gel, solvent A): k′ ) 5.25 (octadecylsilyl silica gel, H O), k′ )
O, TSP):
, J5,6 ) 3.0 Hz), 4.48 (m, 1H, H3′),
), 3.98 (m, 1H, H4′), 3.82 (m, 2H, H5′,5′′), 2.42 (m, 2H,
4
, 13), 263 (MH , 17), 245 (MH
intensity) 220 (10, MH ), 242 (20, MH + Na ), 207 (90), 185 (100).
FAB-MS (negative mode, glycerol mull): m/z (relative intensity) 218
-
H
2
-
(10, M - H ), 191 (60), 183 (70).
(
2
N
1
-(2-Deoxy-r-D-erythro-pentofuranosyl)biuret (11). HPLC (silica
gel, solvent A): k′ ) 6.87 (octadecylsilyl silica gel, H O), k′ ) 6.16.
O, TSP): δ 5.96
1
0
.92. Yield (photolysis): 16 mg. H NMR (400.13 MHz, D
), 5.34 (d, 1H, H
2
2
1
δ 6.30 (pst, 1H, H
.25 (d, 1H, H
1
6
Yield (photolysis): 8 mg. H NMR (400.13 MHz, D
2
4
5
(dd, 1H, H1′, J1′,2′ ) 7.0 Hz, J1′,2′′ ) 2.5 Hz), 4.49 (m, 1H, H3′, J2′,3′
5.6 Hz, J2′′,3′ ) 2.5 Hz), 4.22 (m, 1H, H4′, J3′,4′ ) 3.5 Hz, J4′,5′ ) 4.0
Hz, J4′,5′′ ) 5.3 Hz), 3.66 (m, 2H, H5′, H5′′), 2.48 (m, 1H, H2′, J2′,2′′
3
-14.5 Hz), 2.10 (m, 1H, H2′′). DCI-MS (CH CH(CH )CH and NH ):
)
H
2′,2′′). DCI-MS (CH
3
CH(CH
3
)CH
3
and NH
3
): m/z (relative intensity)
+
+
+
2
80 (M + NH
4
, 13), 263 (MH , 17), 245 (MH - H
2
O, 60), 177
)
(100), 117 (100).
3
3
3

4
104 J. Am. Chem. Soc., Vol. 121, No. 17, 1999
Wagner et al.
CO), 251 (10,
+
+
+
+
m/e (relative intensity) 220 (25, MH ), 237 (10, M + NH
MH - CONH), 160 (70, MH - urea), 117 (50, 2-deoxyribose ).
4
), 117 (100,
310 (10, MH - CH
3
COOH), 268 (20, MH - CH
2
+
+
+
+
+
MH - COOCH
3
), 208 (MH - COCH
3
).
N
1
-(2-Deoxy-â-D-erythro-pyranosyl)biuret (12). HPLC (silica gel,
O), k′) 2.77. Yield
O, TSP): δ 5.41 (dd,
(5′R,5R,6R)-5′,6-Cyclo-5-hydroxy-5,6-dihydro-2′-deoxyuridine (18).
1
solvent A): k′ ) 6.87 (octadecylsilyl silica gel, H
photolysis): 6 mg. H NMR (400.13 MHz, D
H, H1′, J1′,2′ ) 8.0 Hz, J1′,2′′ ) 2.9 Hz), 4.20 (m, 1H, H3′, J2′,3′ ) 2.8
2
Yield (γ-irradiation): 2 mg. H NMR (400.13 MHz, D
6.30 (dd, 1H, H , J
2
O, TSP): δ
1
1′,2′ ) 0.4 Hz, J1′,2′′ ) 6.1 Hz), 4.63 (m, 1H, H3′,
(
1
2
1′
J
2′,3′ ) 7.2 Hz, J2′′,3′ ) 2.8 Hz, J3′,4′ ) 0.4 Hz), 4.51 (dd, 1H, H4′, J4′,5′
) 2.7 Hz, J3′,4′ ) 0.4 Hz), 4.45 (d, 1H, H , J5,6 ) -11.5 Hz), 3.88 (dd,
1H, H5′, J5′,6 ) 3.2 Hz, J4′5′ ) 2.7), 3.55 (dd, 1H, H , J5,6 ) -11.5 Hz,
5′,6 ) 3.2 Hz), 2.50 (m, 1H, H2′, J2′,2′′ ) -15.0 Hz, J2′,3′ ) 7.2 Hz),
2.12 (m, 1H, H , J ) 6.1 Hz, J2′,2′′ ) -15.0 Hz, J2′′,3′ ) 2.8 Hz).
Hz, J2′′,3′ ) 3.5 Hz), 3.90 (m, 1H, H4′, J3′,4′ ) 6.3 Hz, J4′,5′ ) 4.3 Hz,
4′,5′′ ) 8.0 Hz), 3.82 (m, 2H, H5′, H5′′, J5′,5′′ ) -11.3 Hz), 2.14 (m,
H, H2′, J2′,2′′ ) -13.4 Hz), 1.95 (m, 1H, H2′′). FAB-MS (positive mode,
5
J
1
6
J
+
glycerol mull): m/e (relative intensity) 220 (20, MH ), 242 (15, M +
2′′
1′,2′′
+
+
EI-MS (triacetylated derivative, positive mode): m/z (relative intensity)
Na ), 312 (10, M + glycerol).
-(2-Deoxy-r-D-erythro-pyranosyl)biuret (13). HPLC (silica gel,
solvent A): k′ ) 6.87 (octadecylsilyl silica gel, H O), k′ ) 4.55. Yield
O, TSP): δ 5.12 (dd,
+
+
+
3
-
10 (10, MH - CH
3
COOH), 268 (20, MH - CH
2
CO) 251 (10, MH
N
1
+
COOCH
3
) 208 (MH - COCH
3
).
2
1
2-Deoxy-1,4-ribonolactone. HPLC (silica gel, solvent A): k′ ) 1.87.
(
1
photolysis): 6 mg. H NMR (400.13 MHz, D
2
1
Yield (photolysis): 9 mg. H NMR (400.13 MHz, D
m, 1H, H ), 4.55 (m, 1H, H , J2,3 ) 3 Hz, J2′,3 ) 6 Hz), 3.88 (dd, 1H,
), 3.79 (dd, 1H, H5′), 2.78 (dd, 1H, H , J2′,2′ ) 9 Hz), 2.59 (dd, 1H,
O, TMS): δ 181.7 (CdO), 91.2 (C ),
). IR (KBr pellet): λmax 1770 (CdO),
1070 cm (COH). MS (EI, positive mode): m/z (relative intensity)
2
O, TSP): δ 4.58
H, H1′, J1′,2′ ) 2.5 Hz, J1′,2′′ ) 10.0 Hz), 4.06 (m, 1H, H3′, J2′,3′ ) 4.5
Hz, J2′′,3′ ) 10.0 Hz), 3.95 (dd, 1H, H5′, J5′,5′′ ) -12.0 Hz, J4′,5′ ) 1.8
Hz), 3.87 (m, 1H, H4′, J3′,4′ ) 3.0 Hz, J4′,5′′ ) 3.5 Hz), 3.71 (dd, 1H,
5′′), 2.03 (m, 1H, H2′, J2′,2′′ ) -13.0 Hz), 1.90 (m, 1H, H2′′). FAB-
MS (negative mode, glycerol mull): m/e (relative intensity) 218 (25,
(
H
H
4
3
5
2
1
3
2′). C NMR (100.62 MHz, D
), 63.2 (C ), 39.9 (C
2
4
H
7
0.5 (C
3
5
2
-
1
-
-
M - H ), 310 (10, M - H + glycerol), 184 (20), 183 (100).
trans-(4S*,5S*)-N -(2-Deoxy-â-D-erythro-pentofuranosyl)-1-car-
bamoyl-3,4-dihydroxy-2-oxoimidazolidine (14). HPLC (silica gel,
solvent A): k′ ) 6.12 (octadecylsilyl silica gel, H O), k′ ) 2.25. Yield
O, TSP): δ 5.95 (dd,
H, H1′, J1′,2′ ) 8.3 Hz, J1′,2′′ ) 6.1 Hz), 5.54 (s, 1H, H ), 5.23 (s, 1H,
+
+
+
1
M
33 (15, MH ), 114 (20, M - H
2
O), 104 (25, M - CO), 101 (30,
1
+
2
- CH OH).
Synthesis of trans-5-Bromo-6-hydroxy-5,6-dihydro-2′-deoxyuri-
2
dine. The cis diastereomers of the 5,6-glycols of 2′-deoxyuridine (2
and 3) cannot be separated by either normal-phase or reversed-phase
HPLC, and thus they were prepared in two steps from the corresponding
trans diastereomers of the bromohydrins of 2′-deoxyuridine. The latter
were prepared by the addition of bromine (dropwise until coloration)
1
(
1
H
J
J
H
2
2
photolysis): 10 mg. H NMR (400.13 MHz, D
2
5
4
), 4.47 (m, 1H, H3′, J2′,3′ ) 6.0 Hz, J2′′,3′ ) 3.1 Hz), 4.01 (m, 1H, H4′
,
,
3′,4′ ) 3.0 Hz, J4′,5′ ) 4.2 Hz, J4′,5′′ ) 5.5 Hz), 3.75 (m, 2H, H5′, H5′′
5′,5′′ ) -12.2 Hz), 2.59 (m, 1H, H2′, J2′,2′′ ) -14.0 Hz), 2.31 (m, 1H,
to a solution of 2′-deoxyuridine (500 mg/5 mL of H
the reaction, excess bromine was eliminated by bubbling with N
2
O) at 4 °C. After
. The
2′′). DCI-MS (CH
95 (5, M + NH
-deoxyribose ), 98 (100, 2-deoxyribose - H
3
+
CH(CH
3
)CH
3
and NH
3
): m/z (relative intensity)
+
2
4
), 278 (15, MH ), 177 (70), 160 (30), 117 (25,
solution was neutralized by the addition of sodium acetate and taken
to dryness on a rotary evaporator. Two products were obtained after
purification by HPLC using an octadecylsilyl silica gel semipreparative
column and 10% methanol in water as the mobile phase. The first
+
+
2
O). HRMS (ES, positive
+
mode): calcd for C
trans-(4R*,5R*)-N
bamoyl-3,4-dihydroxy-2-oxoimidazolidine (15). HPLC (silica gel,
solvent A): k′ ) 5.25 (octadecylsilyl silica gel, H O), k′ ) 2.42. Yield
O, TSP): δ 5.94 (dd,
H, H1′, J1′,2′ ) 8.2 Hz, J1′,2′′ ) 6.3 Hz), 5.54 (s, 1H, H ), 5.25 (s, 1H,
9
H
15
N O
3 7
([M + Na] ) 300.0808, found 300.0808.
1
-(2-Deoxy-â-D-erythro-pentofuranosyl)-1-car-
fraction (k′ ) 1.1) gave 100 mg (yield 25%) of trans-(5S,6S)-5-bromo-
2
1
6
-hydroxy-5,6-dihydro-2′-deoxyuridine. H NMR (400.13 MHz, D
2
O,
1
(
1
H
J
J
H
8
(
photolysis): 10 mg. H NMR (400.13 MHz, D
2
TSP): δ 6.32 (dd, 1H, H1′, J1′,2′ ) 8.6 Hz, J1′,2′′ ) 6.1 Hz), 5.62 (d, 1H,
, J5,6 ) 2.4 Hz), 4.63 (d, 1H, H ), 4.52 (m, 1H, H3′, J2′,3′ ) 5.7 Hz,
2′′,3′ ) 3.1 Hz), 4.04 (m, 1H, H4′, J3′,4′ ) 3.2 Hz, J4′,5′ ) 4.3 Hz, J4′,5′′
5
H
J
)
J
6
5
4
), 4.52 (m, 1H, H3′, J2′,3′ ) 6.3 Hz, J2′′,3′ ) 3.2 Hz), 4.02 (m, 1H, H4′
,
,
3′,4′ ) 3.2 Hz, J4′,5′ ) 4.0 Hz, J4′,5′′ ) 4.8 Hz), 3.76 (m, 2H, H5′, H5′′
5.0 Hz), 3.84 (m, 2H, H5′5′′, J5′,5′′ ) -12.6 Hz), 2.46 (m, 1H, H2′,
5′,5′′ ) -12.2 Hz), 2.57 (m, 1H, H2′, J2′,2′′ ) -13.9 Hz), 2.30 (m, 1H,
2′,2′′ ) -14.5 Hz), 2.30 (m, 1H, H2′′). FAB-MS (negative mode,
1
3
2′′). C NMR (100.62 MHz, D
2
O, TMS): δ 155 (CO), 154.5 (CO),
+
glycerol mull): m/z (relative intensity) 325 (10, MH ), 243 (10). The
5.8 (C4′), 83.1 (C1′), 81.5 (CHOH), 81.2 (CHOH), 73.3 (C3′), 61.7
5′), 35.4 (C2′). DCI-MS (CH
M + NH
positive mode): calcd for C
second fraction (k′ ) 1.75) gave 200 mg (yield 50%) of trans-(5R,6R)-
C
3
CH(CH
3
)CH
3
and NH
3
): m/z 295 (10,
1
5
-bromo-6-hydroxy-5,6-dihydro-2′-deoxyuridine. H NMR (400.13
+
+
+
4
), 278 (10, MH ), 260 (10, MH - H
2
O). HRMS (ES,
MHz, D
2
O, TSP): δ 6.32 (pst, 1H, H1′, J1′,2′ ) 7.1 Hz, J1′,2′′ ) 6.5 Hz),
.61 (d, 1H, H , J5,6 ) 3.2 Hz), 4.64 (d, 1H, H ), 4.50 (m, 1H, H3′, J2′,3′
6.4 Hz, J2′′,3′ ) 4.2 Hz), 4.03 (m, 1H, H4′, J3′,4′ ) 4.0 Hz, J4′,5′ ) 2.4
+
9
H N
15 3
O
7
([M + Na] ) 300.0808, found
5
)
6
5
3
00.0808.
Aminocarbonyl-[2-(2-deoxy-â-D-erythro-pentofuranosyl)amino]-
-oxomethylcarbamic Acid (16). HPLC (silica gel, solvent A): k′ )
Hz, J4′,5′′ ) 5.2 Hz), 3.86 (m, 2H, H5′5′′, J5′,5′′ ) -12.2 Hz), 2.43 (m,
2
1
1H, H , J
) -14.5 Hz), 2.30 (m, 1H, H ). FAB-MS (negative
2
′
2′,2′′
2′′
+
0.3 (octadecylsilyl silica gel, H
2
O), k′ ) 6.0. Yield (photolysis): 15
O, TSP): δ 5.96 (dd, 1H, H1′), 4.80 (s,
2
), 4.50 (m, 1H, H3′), 4.06 (m, 1H, H4′), 3.75 (m, 2H, H5′, H5′′),
mode, glycerol mull): m/z (relative intensity) 325 (15, MH ), 307 (10,
1
mg. H NMR (400.13 MHz, D
2
-H O), 243 (10, -BrH), 111 (15, uracil).
2
2
2
d
H, CH
Synthesis of 5,6-Dihydroxy-5,6-dihydro-2′-deoxyuridine. The
purified bromohydrins (trans-(5S,6S)- or trans-(5R,6R)-5-bromo-6-
hydroxy-5,6-dihydro-2′-deoxyuridine; 45 mg) were dissolved in 4 mL
of H O and 4 mL of 30% H O , and 100 mg of Ag O was added to the
2 2 2 2
mixture, maintained for 2 min at 4 °C. Hydroperoxides formed in the
1
.32 (m, 1H, H2′), 2.31 (m, 1H, H2′′). H NMR (400.13 MHz, DMSO-
1
3
6
, TMS): δ 8.40 (d, 1H, NH), JNH,1′ ) 9 Hz), 7.2 (s, 2H, NH
O, TMS): δ 171.1 (CONH), 154.8 (COOH),
), 87.0 (C4′), 81.0 (C1′), 72.3 (C3′), 64.4 (CH ), 62.9 (C5′),
2
). C
NMR (100.62 MHz, D
2
1
3
42.7 (CONH
9.4 (C2′). DCI-MS (CH
2
2
3
CH(CH
3
)CH
3
and NH
3
): m/z (relative
reaction were reduced to the alcohol by bubbling with H S. The solution
2
+
+
+
intensity) 295 (10, M + NH
CONH), 192 (95). FAB-MS (positive mode, glycerol mull): m/z
relative intensity) 370 (20, M + glycerol), 300 (20, M + Na ), 278
10, MH ), 162 (20, base H ); diacetylated derivative (positive mode)
4
), 278 (20, MH ), 235 (95, MH
-
was filtered to eliminate the black precipitate, neutralized with sodium
carbonate, and evaporated to dryness. The product mixtures were
purified by HPLC with silica gel and solvent B (k′ ) 13.4), giving 25
mg of each of the corresponding 5,6-glycols (50% yield): cis-(5R,6S)-
5,6-dihydroxy-5,6-dihydro-2′-deoxyuridine (2) and cis-(5S,6R)-5,6-
dihydroxy-5,6-dihydro-2′-deoxyuridine (3).
+
+
(
(
+
+
+
-
m/z 362 (MH ), (negative mode) m/z 360 (M - H).
(5′S,5S,6S)-5′,6-Cyclo-5-hydroxy-5,6-dihydro-2′-deoxyuridine (17).
1
Yield (γ-irradiation): 2 mg. H NMR (400.13 MHz, D
.30 (dd, 1H, H1′, J1′,2′ ) 0.4 Hz, J1′,2′′ ) 6.3 Hz), 4.70 (m, 1H, H3′
2′,3′ ) 7.4 Hz, J2′′,3′ ) 2.9 Hz, J3′,4′ ) 0.4 Hz), 4.35 (dd, 1H, H4′, J4′,5′
5.2 Hz, J3′,4′ ) 0.4 Hz), 4.26 (d, 1H, H , J5,6 ) -10.8 Hz), 4.04 (dd,
H, H5′, J5′,6 ) 8.8 Hz, J4′5′ ) 5.2), 3.39 (dd, 1H, H , J5,6 ) 10.8 Hz,
5′,6 ) 8.8 Hz), 2.50 (m, 1H, H2′, J2′,2′′ ) -14.6 Hz, J2′3′ ) 7.4 Hz),
.16 (m, 1H, H2′′, J1′,2′′ ) 6.3 Hz, J2′,2′′ ) -14.6 Hz, J2′′,3′ ) 2.9 Hz).
2
O, TSP): δ
Quantitation of dCyd Oxidation Products. dCyd (10 mM) and
14
6
J
)
1
J
2
,
2
0.1 mCi of [ C ]-labeled dCyd (New England Nuclear, Boston, MA)
were subjected to either γ-irradiation or menadione photosensitization
using a procedure similar to that described for the preparation of
oxidation products, except that the reactions were done on a smaller
scale (2 mL). The resulting mixture was analyzed by two-dimensional
thin-layer chromatography (2D-TLC) with silica gel and solvent A,
followed at right angles with solvent B. The products which contained
5
6
EI-MS (triacetylated derivative, positive mode): m/z (relative intensity)

Hydroxyl-Radical-Induced Decomposition of 2′-Deoxycytidine
J. Am. Chem. Soc., Vol. 121, No. 17, 1999 4105
Table 1. Yield of Stable Products from γ-Irradiation and Photosensitization of dCyd in Aerated Aqueous Solutionsa
product (dR ) 2-deoxy-â-D-erythro-pentofuranosyl)
γ-irradiationb
UVA/menadione photosensitizationc
5
5
,6-dihydroxy-5,6-dihydro-2′-deoxyuridine (2-5)
-hydroxy-2′-deoxycytidine (6)
28
12
3
27
7
N
N
N
N
1
1
1
1
-(dR)-5-hydroxyhydantoin (7,8)
-(dR)-formamide (9)
2
d
d
12
2
-(dR)-biuret (10-13)
3
17
3
2
6
2
-(dR)-1-carbamoyl-3,4-dihydroxy-2-oxoimidazolidine (14, 15)
aminocarbonyl[2-(dR)-amino]-2-oxomethylcarbamic acid (16)
e
′,6-cyclo-5-hydroxy-5,6-dihydro-2′-deoxyuridine (17, 18)
′-deoxyuridine (19)
∼2
nd
e
nd
36
4
12
ta
f
g
4
total
92
92
a
Yields from normal-phase 2D-TLC and HPLC analyses with 14
-labeled dCyd. b Total loss of substrate (15%); G ) -85 × 10 J mol-1
-9
C
2
.
c
d
e
f
g
Total loss of substrate (22%). Estimated by comparison of refractive index signal with that of 16. Not detected. Trace amounts. Produced in
equal yield with cytosine.
radioactivity were localized by autoradiography and later quantitated
by liquid scintillation counting. Most of the products were well resolved
by 2D-TLC and were identified on the basis of comigration with
authentic standards.20 Since dCyd (1), 5-hydroxy-2′-deoxycytidine (6),
and cytosine are not well separated by 2D-TLC, they were quantitated
by HPLC using an octadecylsilyl silica gel column with water as the
seven protons may be assigned to an intact 2-deoxyribose
moiety. H2′ and H2′′ protons were distinguished on the basis of
25
proton-proton coupling constant arguments. The lower field
component of the H5′ and H5′′ pattern was attributed to H5′, in
line with studies of specifically deuterated nucleosides.26 The
stereospecific synthesis of 2′-deoxyuridine bromohydrins, fol-
lowed by their conversion into 5,6-glycols, confirmed the
absolute configuration of cis diastereomers (2 and 3). The
absolute configuration of trans-2′-deoxyuridine bromohydrins
is based on the difference between 6R and 6S diastereomers of
mobile phase. Finally, the yield of N -(2-deoxy-â-D-erythro-pentofura-
1
nosy)formamide (9), which does not retain radioactivity from the parent
molecule, was estimated by HPLC (silica gel and solvent A) on the
basis of its refractive index signal compared with that of product 16.
1
8
Labeling Experiments with
2
O : γ-Irradiation. dCyd (1 mM)
was irradiated in unbuffered aqueous solutions (100 mL) for 20 min
6
-hydroxy-substituted 5,6-dihydrothymine derivatives with re-
(
total dose 1.6 kGy). Prior to irradiation, the solution was saturated
spect to the conformation of the sugar moiety. As shown for
thymine derivatives, the conversion of pyrimidine bromohydrins
into pyrimidine 5,6-glycols involves a shift of the hydroxyl
group from C6 to C5, leading to inversion of the configuration
1
8
with 99.9%
2
O (CEA, Saclay, France) by two cycles of degassing
1
8
(rotary evaporation) and bubbling with
2
O for 10 min. The last cycle
was completed in the irradiation vessel (1 L), and the vessel was closed
1
8
under
O
2
. After 10 min of irradiation, the solution was resaturated
2
7
with 8O
1
at C5. The conversion preferentially leads to cis-5,6-glycol
products because H2O preferentially attacks the same side of
the zwitterionic intermediate due to anchimeric assistance.28
Thus, trans-(5S,6S)-5-bromo-6-hydroxy-5,6-dihydro-2′-deoxy-
uridine transforms into cis-(5R,6S)-5,6-dihydroxy-5,6-dihydro-
2
. The resulting mixtures were evaporated to dryness, and the
products were initially separated by HPLC using silica gel phase with
solvent A and then with octadecylsilyl silica gel phase with water. The
1
8
percentage of O label in dCyd oxidation products was determined by
DCI-MS.
2
′-deoxyuridine (2), and similarly trans-(5R,6R)-5-bromo-6-
Results
hydroxy-5,6-dihydro-2′-deoxyuridine transforms into cis-(5S,6R)-
5,6-dihydroxy-5,6-dihydro-2′-deoxyuridine (3). The absolute
configuration of products 4 and 5 was established by the
observation that they transform into cis diastereomers by ring-
chain tautomerism, leading to epimerization of C6 (4 into 3 and
5 into 2). This is analogous to the isomerization of thymidine
•
The OH-mediated decomposition of 2′-deoxycytidine (dCyd;
1
) in aerated aqueous solutions gave a complex mixture of
products. It was possible to separate 17 nucleoside products from
this mixture using a combination of normal- and reversed-phase
HPLC. The radiation yields of individual decomposition prod-
ucts were determined by 2D-TLC and/or HPLC analyses of
reaction mixtures containing [ C2]-labeled dCyd (Table 1). The
products may be separated into three groups: (1) nucleosides
which are similar in structure to products that arise from the
2
9
5,6-glycols in aqueous solution.
14
A number of other products were also characterized, including
5-hydroxy-2′-deoxycytidine (6; 12%), two diastereomers of N1-
(2-deoxy-â-D-erythro-pentofuranosyl)-5-hydroxyhydantoin (7
•
OH-mediated decomposition of thymidine (2-9; 55% of the
and 8; 3%), and 1-(2-deoxy-â-D-erythro-pentofuranosyl)forma-
mide (9; 12%). Identification of 7 and 8 was based on a
comparison of their spectroscopic features with those of
analogous compounds obtained by the oxidation of thymidine:
N1-(2-deoxy-â-D-erythro-pentofuranosyl)-5-hydroxy-5-methyl-
total decomposition of dCyd); (2) nucleosides which are unique
to OH-mediated decomposition of dCyd derivatives (10-16;
2
•
3%); and (3) novel cyclic products (17, 18; ∼2%), cytosine
(
12%), and 2-deoxy-1,4-ribonolactone (trace amounts). Taken
together, the above products account for 92% of the overall
30
hydantoin. The absolute configuration of the two diastereomers
was recently addressed by detailed H NMR and molecular
•
OH-induced decomposition of dCyd.
1
Common Pyrimidine Base Modifications. The major prod-
uct of the OH-mediated decomposition of dCyd was 5,6-
dihydroxy-5,6-dihydro-2′-deoxyuridine including four diaster-
eomers (2-5; 28%). The FAB-MS analyses of 2-5 exhibited
a molecular ion at 263 m/z (MH ) with a major ion fragment
•
(25) Altona, C.; Sundaralingam, M. J. Am. Chem. Soc. 1973, 95, 2333.
(26) Ritchie, S.; Perlin, A. S. Carbohydr. Res. 1977, 55, 121-128.
(27) Ryang, H. S.; Wang, S. Y. J. Org. Chem. 1979, 44, 1191-1192.
(28) Harayama, T.; Yanada, R.; Taga, T.; Machida, K.; Cadet, J.; Yoneda,
+
F. J. Chem. Soc., Chem. Commun. 1986, 1469-1471.
at 245 m/z, indicating the loss of H2O from the molecular ion.
(29) (a) Cadet, J.; Ducolomb, R.; Hruska, F. E. Biochim. Biophys. Acta
1979, 563, 206-215. (b) Cadet, J.; Ulrich, J.; T e´ oule, R. Tetrahedron 1975,
1
The H NMR spectra of 2-5 displayed two midrange resonances
3
1, 2057-2061. (c) Lutsig, M. J.; Cadet, J.; Boorstein, R. J.; Teebor, G.
at 4.60 ppm for H5 and at 5.35 ppm for H6 with proton-proton
coupling constants in the range of 2.8-3.8 Hz. The remaining
W. Nucleic Acids Res. 1992, 20, 4839-4845.
(30) Cadet, J.; T e´ oule R. J. Chem Res. (M) 1977, 3215-3232.

4106 J. Am. Chem. Soc., Vol. 121, No. 17, 1999
Wagner et al.
modeling studies.31 Similarly, 5-hydroxy-2′-deoxycytidine (6)
and N1-(2-deoxy-â-D-erythro-pentofuranosyl)formamide (9) were
Chart 2. Possible Stuctures of Aminocarbonyl[2-(2-deoxy-
â-D-erythro-pentofuranosyl)amino]-2-oxomethylcarbamic
Acid (16)
3
2,33
identified by comparison with authentic compounds.
As a
diagnostic feature of product 9, the H NMR and 13C NMR
1
measurements depict the presence of minor (40%) and major
(
60%) species because of hindered rotation about the amide
bond.
•
Modifications Unique to dCyd. The OH-mediated decom-
position products of dCyd led to several products that are not
produced in the related reaction of thymidine. These included
four isomeric nucleosides of biuret (10-13; 3%), two diaster-
eomers of N1-(2-â-D-erythro-pentofuranosyl)-1-carbamoyl-2-
oxo-4,5-dihydroxyimidazolidine (14 and 15; 17%) and ami-
nocarbonyl[2-(2-deoxy-â-D-erythro-pentofuranosyl)amino]-2-
oxoethylcarbamic acid (16; 3%). Together, these products
the presence of an intact 2-deoxyribose moiety as well as two
singlet resonance at 5.5 and 5.2 ppm, which may be assigned
to H4 and H5, respectively. The weak coupling between H4 and
H5 (0.25 Hz) implies that these protons are diequatorial, and
thus the C4 and C5 hydroxyl substituents must be diaxial. The
conformation of the imidazolidine ring may be described as a
half-chair in which C5 is puckered with respect to a plane formed
by N1-C2-N3-C4 atoms of the imidazolidine ring.36 It was
not possible to determine the absolute configuration of these
•
represented about 23% of the overall OH-mediated decomposi-
tion of dCyd.
1
MS and H NMR analyses of products 10-13 revealed that
they were modified nucleosides with biuret attached to R-fura-
nose, â-furanose, R-pyranose, or â-pyranose moieties. The
molecular ions of all four products appeared at 220 m/z, as
observed by FAB- and DCI-MS. Each of the products contained
a biuret moiety, indicated by the presence of two intense
1
diastereomers on the basis of H NMR analyses since the
coupling constants of sugar protons were similar for both
diastereomers (in contrast, the stereochemistry of C6 for 5,6-
saturated pyrimidine derivatives leads to significant changes in
the conformation of the sugar moiety). Last, 14 was observed
to transform into 15 over a period of several days in D2O. For
this to occur, 14 must undergo two ring-chain tautomerism
cycles with epimerization at C4 and C5 positions of the
imidazolidine ring.
+
fragments, one at 177 m/z (MH - CONH) and the other at
+
1
60 m/z (MH - urea). To determine the exact structure of the
sugar moiety required close examination of the proton-proton
coupling constants. The most stable conformations of the
2
-deoxy-â-D-erythro-pentofuranose moiety are 2′-exo-3′-endo
and 2′-endo-3′-exo, which restrict the orientation of sugar
protons such that J1′,2′ + J3′,4′ ≈ 10 Hz, J2′′,3′ ≈ J3′,4′ and J1′,2′ ≈
J2′,3′. In contrast, the pyranose ring system of nucleosides is
Structural elucidation of product 16 required extensive
1
spectroscopic measurements. The H NMR spectrum in D2O
revealed an isolated methylene resonance in midrange field (δ
) 6 ppm) as well as the characteristic protons of an intact
more flexible, leading to unusually large or small coupling for
J1′,2′ or J1′,2′′ and J4′,5′′.34 Thus, products 10 and 11 contain a
2
-deoxyribose moiety. The spectrum in DMSO displayed two
furanose moiety, whereas products 12 and 13 contain a pyranose
moiety. The distinction between R and â furanose anomers was
nonexchangeable protons at low field which may be assigned
to the terminal NH2 group (2H, singlet, 8.4 ppm) and the central
NH group (1H, doublet, 7.2 ppm). The NH proton signal and
its coupling to the anomeric C1′ proton confirmed the presence
based on the proton-proton coupling between H1′ and H2′′, H3′
and H2′′, and H3′ and H4′.35 In the case of the pyranose anomers
(
R or â), the differences in their structure lead to changes in
1
3
of an open-ring structure. The C NMR spectrum displayed
nine signals: one secondary carbon signal (64.4 ppm), three
quartenary carbon signals (142.7, 154.8, and 171.1 ppm), and
five signals which may be assigned to an unmodified 2-deoxy-
ribose moiety (39.4 (C2′), 62.9 (C5′), 72.3 (C3′), 81.0 (C1′), and
the coupling between H1′ and H2′ and H1′ and H2′′. Since the
R-pyranose anomer preferentially assumes a C4 conformation,
1
the coupling between diaxial H1′ and H2′ is strong (J1′,2′′ ) 10
Hz). On the other hand, the â-pyranose anomer preferentially
4
assumes a C1 conformation, leading to strong coupling between
8
7.0 (C4′)). Product 16 was subjected to various mass spec-
H1′ and H2′ (J1′,2′ ) 8 Hz). It should be noted that, in both cases,
the biuret moiety likely adopts a preferential pseudoequatoral
orientation.
trometry analyses. Although the compound was not directly
suitable for analysis by electron impact ionization, the derivative
with two acetyl groups on the 2-deoxyribose moiety (plus 84
Products 14 and 15 were identified as the trans diastereomers
of N1-(2-deoxy-â-D-erythro-pentofuranosyl)-1-carbamoyl-3,4-
dihydroxy-2-oxoimidazolidine. This product is analogous to
trans-1-carbamoyl-3,4-dihydroxy-2-oxoimidazolidine, which is
+
m/z) gave a molecular ion peak at 362 m/z (MH ) and at 360
m/z (M-H) in the positive and negative modes, respectively.
In addition, FAB-MS in the positive mode depicted a molecular
+
•
ion at 278 m/z (MH , 10%) accompanied by characteristic
the major OH-mediated decomposition product of cytosine in
+
+
satellite peaks at 300 m/z (MH + Na, 20%) and 370 m/z (MH
glycerol, 20%), and DCI-MS analyses showed a molecular
aerated aqueous solutions and which has been well characterized
by various measurements, including X-ray crystallography
+
+
+
analysis.1
5,36,37
ion at 278 m/z (MH , 20%) with the satellite peak (M + NH4 ).
The NMR and MS analyses of product 16 were consistent
with two structures (Chart 2). At first, we were reluctant to
assign structure A because it comprises a carbamic acid group
which is not usually stable. For example, the lifetime of
N-carboxyimidazolidone is approximately 2 min in neutral
Thus, identification of the related modified
1
nucleosides was straightforward. The H NMR in D2O indicated
(
31) Ammalahti, E.; Bardet, M.; Cadet, J.; Molko, D. Magn. Reson.
Chem. 1998 36, 363-370.
32) Eaton, M. A.; Hutchinson, D. W. Biochim. Biophys. Acta 1973, 319,
81-287.
33) Cadet, J.; Nardin, R.; Voituriez, L.; Remin, M.; Hruska, F. E. Can.
J. Chem. 1981, 59, 3313-3318.
34) Mariaggi, N.; T e´ oule, R.; Cadet, J.; Dickie, H.; Hughes, E. Radiat.
Res. 1979, 79, 431-438.
35) Cadet, J.; Taieb, C.; Remin, M.; Niemczura, W. P.; Hruska, F. E.
Biochim. Biophys. Acta 1980, 608, 435.
36) Hahn, B. S.; Wang, S. Y.; Flippen, J. L.; Karle, I. L. J. Am. Chem.
Soc. 1973, 95, 2711-2712.
(
2
(
38
solution at 25 °C. In contrast, we did not observe any
decomposition of product 16 in aqueous solutions throughout
the entire study. Thus, we looked for alternatives (structure B).
(
(
(37) Leonard, N. L.; Wiemer, D. F. J. Am. Chem. Soc. 1976, 98, 8218-
8221.
(38) Caplow, M.; Yager, M. J. Am. Chem. Soc. 1967, 89, 4513.
(

Hydroxyl-Radical-Induced Decomposition of 2′-Deoxycytidine
J. Am. Chem. Soc., Vol. 121, No. 17, 1999 4107
Table 2. Incorporation of 18O
Products of dCyd (1)
However, it should be noted that structure B was not completely
compatible with the 13C NMR analyses, since the chemical shifts
2
into Stable Radiation-Induced
of the carbonyl signals for the ureid residues were distinctly
different (142.7 and 171.1 ppm). The assignment of the
carboxylic acid group at 154.8 ppm is consistent with shielding
of this group within carbamic acid derivatives. Nevertheless,
•
ongoing studies of an analogous product obtained from the OH-
mediated decomposition of 5-methyl-2′-deoxycytidine support
the carbamic acid structure.39 The structure of the corresponding
1
13
methyl-substituted derivative has been proven by H- C
hetereonuclear NMR analyses, which indicated long-range
coupling between the downfield carbonyl group of CONH and
the CHCH3 protons of this derivative. Thus, product 16 is likely
a carbamic acid derivative, as depicted in structure A. The
stability of this carbamic acid derivative is unprecedented but
may be a result of strong intramolecular H-bonding with the
terminal amide group.
Novel Cyclic Products. A group of novel 2′-deoxycytidine
products was discovered, including two diastereomers of 5′,6-
cyclo-5-hydroxy-5,6-dihydro-2′-deoxyuridine (17, 18). These
1
products were characterized by extensive MS and H NMR
1
analyses. The salient features of the H NMR spectra were the
lack of detectable coupling between H1′ and H2′ and between
H3′ and H4′, implying strong structural constraints within the
deoxyribose moiety. The stereochemistry of both diastereomers
was determined by comparison of the coupling constants with
those of similar products, i.e., 5′,6-cyclo-5,6-dihydrothymidine.
Thus, the values of J4′,5′ (3.2 Hz) and J5′,6 (2.7 Hz) for 17 were
consistent with the 5′S,6S,5S configuration and those of J4′,5′
thymidine. Taken together, the results indicate that 18O was
2
incorporated at C and C of products 2-9 with a percentage
5
6
of 30%-40% and 60%-70%, respectively.
40
When dCyd (1) was exposed to γ-irradiation in 18O2-saturated
solutions, the majority of trans N1-(2-deoxy-â-D-erythro-pento-
furanosyl)-1-carbamoyl-2-oxo-4,5-dihydroxyimidazolidine (14)
was doubly labeled (65%). In addition, a small fraction of other
molecular ions contained no label (10%), one label (15%), and
two labels (10%). The presence of singly labeled molecules may
(3.2 Hz) and J5′,6 (2.7 Hz) for 18 with the 5′R,6R,5R configu-
ration. It should be noted that only two of the eight possible
diastereomers were detected.
1
8O2-Labeling Experiments. The OH-induced decomposition
18
be explained by exchange of OH with water by a process
of dCyd (1) was carried out in 18O2-saturated solutions in order
involving ring-chain tautomerism. Thus, the initial yield of
doubly labeled molecules may be assumed to contain both singly
and doubly labeled molecules for a total of 80% (15% + 65%).
In addition, the distribution of label within the imidazolidine
ring (C4 and C5) may be estimated upon examination of the
secondary ions resulting from dehydration of the molecular ions.
Upon dehydration, the percentage of doubly labeled molecules
decreased from 65% to 15% (25% less the percentage of triply
labeled molecules (10%)), while the percentage of singly labeled
molecules increased from 10% (15% less the percentage of
singly labeled molecules (5%)) to 65%. These findings indicate
that the majority of label was eliminated upon dehydration. The
C5-OH group is most likely to undergo dehydration since it
sticks out of the plane of the imidazolidine ring. Thus, the
majority of label resides at the C5 position of product 14.
to probe the reaction mechanisms. To study the incorporation
18
of O2, the products were purified by HPLC and then analyzed
by DCI-MS. This method gave intense molecular ions ac-
companied with characteristic satellite peaks occurring at M +
1
7 m/z. The results are summarized in Table 2.
γ-irradiation of dCyd (1) in 18O2-saturated solutions gave
unlabeled and singly labeled trans-(5S,6S)-2′-deoxyuridine 5,6-
glycol (4) in equal amounts (45%), together with a minor amount
of doubly labeled molecules. The presence of unlabeled product
18
may be attributed to the exchange of OH with H2O at C6 by
way of ring-chain tautomerism. The percentage of label in the
+
18
primary MS fragment of 5,6-glycols (MH - H2O or H2 O)
allows one to distinguish the position of label (C5 or C6) since
dehydration takes place with loss of the OH group at C6 (â-
elimination). Thereby, the percentage of label incorporated at
C5 and C6 in product 5 may be estimated to be about 35% (C5)
and 65% (C6). The analysis of label in the other 5,6-gylcols of
Discussion
2
′-deoxyuridine gave similar results. In addition, this was
•
The first steps of the OH-mediated decomposition of dCyd
consistent with the percentage of unlabeled (70%) and singly
labeled (30%) molecules of 5-hydroxy-2′-deoxycytidine (6),
which is formed by dehydration of intermediate 5,6-glycols of
dCyd. Finally, the proportion of unlabeled and singly labeled
molecules was 40% and 60% for 2-deoxy-â-D-erythro-pento-
furanosyl)formamide (9). This suggests that most of the label
are similar to those for the decomposition thymidine and related
pyrimidines. Hydroxyl radicals predominantly react with the 5,6-
double bond of the base moiety of pyrimidines, giving neutral
OH adduct radicals (Scheme 1). On the basis of pulse radiolysis
•
studies, the addition of OH to the base moiety of dCyd leads
predominantly to reducing 5-hydroxyl radical adducts (I). In
view of the rapid rate of reaction of oxygen with pyrimidine
OH radical adducts, the latter species (I) probably quantita-
tively react with O2 to give 5-hydroxy-6-hydroperoxyl radicals
II). Thus, the formation of stable modifications of the base
moiety of dCyd can largely be attributed to subsequent reactions
of intermediate 5-hydroxy-6-hydroperoxyl radicals (Scheme 1).
(60%) was incorporated at C6 since the carbonyl group of the
formamide product is derived from C6 rather than C2 of
pyrimidine, as inferred by studies with radioactive [ C2]-
4
1
1
4
(
39) Bienvenu, C. Oxidations Radicalaires de la 5-methyl-2′-d e´ soxycy-
tidine. Th e` se de l’Universit e´ Joseph Fourier, Grenoble, France, 1996.
40) Shaw, A. A.; Cadet, J.; Voituriez, L.; Gregoli, G.; Symons, M. C.
R. J. Chem. Soc., Perkin Trans. 1988, 11, 1303-1307.
(
(

4108 J. Am. Chem. Soc., Vol. 121, No. 17, 1999
Wagner et al.
Scheme 1. Initial Steps of •OH-Mediated Decomposition of
dCyd (1) in Aerated Aqueous Solutions
In addition to the tetraoxide pathway, the formation of
products 2-9 may involve intermediate 5(6)-hydroxy-6(5)-
hydroperoxides. It should be noted that hydroperoxides are very
sensitive to the presence of trace metal ions in the solution.
For example, the thermal decomposition of thymidine hydro-
peroxides results in the formation of 5,6-glycols even in
1
7
quintuply distilled water. The most sensitive thymidine hy-
droperoxides were the trans diastereomers of 5-hydroxy-6-
hydroperoxides, which decomposed into 5,6-glycols with yields
in the range of 30%-40%. Similarly, metal-catalyzed reduction
of the 5(6)-hydroxy-6(5)-hydroperoxides of dCyd would result
in 5,6-glycol products (2-5; 28%). This can occur by reduction
of the hydroperoxides of dCyd into the corresponding 5,6-
glycols, followed by their deamination. However, since the 5,6-
glycols of dCyd preferentially undergo dehydration as opposed
45
to deamination in aqueous solutions, it is reasonable to propose
an alternative pathway. This would involve initial deamination
of the 5(6)-hydroxy-6(5)-hydroperoxides of dCyd, giving the
5(6)-hydroxy-6(5)-hydroperoxides of 2′-deoxyuridine, followed
by reduction into 5,6-glycols (2-5). The formation of 5-hy-
droxy-2′-deoxycytidine (6; 12%) can be explained either by loss
of superoxide radical ion from intermediate 5-hydroxy-6-hydro-
peroxyl radicals or by loss of H2O2 from 5-hydroxy-6-hydro-
peroxides. In addition, product 6 can arise from dehydration of
labile dCyd 5,6-glycols in aqueous solution. Likewise, the
nucleosides of 5-hydroxyhydantoin (7 and 8; 3%) can be
explained by decomposition of 6-hydroxy-5-hydroxperoxides
•
Initial addition of OH at C6 and the formation of 6-hydroxy-
5
-hydroperoxyl radicals may also contribute to products (not
shown).
In general, peroxyl radicals have a relatively long life. They
decay by way of a bimolecular reaction involving the formation
of transient tetraoxides which rapidly decompose into stable
products. Alternatively, peroxyl radicals may be susceptible to
reduction by superoxide ion radicals or other reducing agents
•
of 2′-deoxyuridine, whose yield is minor in the OH-mediated
oxidation of dCyd. As for N1-(2-deoxy-â-D-erythro-pentofura-
nosyl)formamide (9), this is a minor product (trace amounts)
in the thermal decomposition of thymidine hydroperoxides. In
conclusion, dCyd oxidation products, which are analogous to
thymidine oxidation products, may arise from the decomposition
of either transient tetraoxides or labile 5(6)-hydroxy-6(5)-
42
giving diamagnetic hydroperoxides. The two pathways appear
to be of equal importance for the OH-mediated decomposition
•
of thymidine in aerated aqueous solutions since the initial yield
of hydroperoxides is approximately equal to that of stable
nonperoxide products.43 In contrast, γ-irradiation of dCyd in
aerated aqueous solutions does not give detectable levels of
unstable dCyd hydroperoxides.44 Thus, it is not possible to make
a clear-cut division between tetraoxide- and hydroperoxide-
yielding pathways.
hydroperoxides.
The extent of incorporation of 18O into products 2-9 was
3
5% at C5 and 65% at C6 (Table 2). This suggests that the initial
•
addition of OH radicals to the 5,6-double bond takes place with
The bimolecular decay of peroxyl radicals results in transient
tetraoxides which can decompose either by a concerted (Russell)
pathway into diamagnetic alcohol and ketone fragments or by
cleavage into oxyl radicals (III). In the case of dCyd, a concerted
pathway of decomposition would result in equal amounts of
a percentage of 65% at C . In contrast, pulse radiolysis studies
5
•
indicate that the addition of OH to cytosine derivatives takes
4
6
place at C with a strong bias (87%). Thus, the incorporation
5
1
8
of O at C in products 2-9 is greater (about 20%) than
2
5
expected from the distribution of C -OH and C -OH radicals
5
6
5
,6-glycol and dialuric acid nucleoside products. However,
indicated by pulse radiolysis studies. This suggests that the
neither the nucleoside of dialuric acid nor the product resulting
from its hydrolysis were observed in product analysis, suggesting
that this pathway is negligible. On the other hand, the
decomposition of transient tetraoxides by way of oxyl radicals
may be an important pathway in the formation of products 2-9.
The formation of the 5,6-glycol products (2-5; 28%) from oxyl
radicals can be explained by H atom or electron abstraction. In
addition, the most likely pathway to account for the formation
of N1-(2-deoxy-â-D-erythro-pentofuranosy)formamide (9; 12%)
involves â-cleavage of oxyl radicals, giving rise to an open-
ring dicarbonyl intermediate which then undergoes fragmenta-
tion between N1 and C2. Last, it has been proposed that the
latter ureid intermediate may recyclize to yield ring contraction
formation of products 2-9 from initial C -OH and C -OH
5
6
radicals is not the same. Since both of these radicals likely
convert quantitatively into the corresponding peroxyl radicals
in oxygenated solutions, the difference in the formation of
products 2-9 can be attributed to the chemistry of 5-hydroxy-
6-hydroperoxyl radicals (II) and 6-hydroxy-5-hydroperoxyl
radicals. In particular, if we assume that half of the 5-hydroxy-
6-hydroperoxyl radicals (II) are diverted to the endoperoxide
1
8
pathway (Scheme 2), the percentage of O at C of products
2
5
2-9 would be compatible with pulse radiolysis studies.
The formation of products 10-16 constitutes an unique
•
pathway of OH-mediated decomposition of pyrimidine nucleo-
sides which does not occur for thymidine derivatives (Scheme
2). The primary sequence of steps to these products likely
involves rearrangement of the hydroperoxide to an endoperox-
ide, followed by C4-C5 cleavage into an intermediate R-hy-
droxyketone (IV to V to VI). The effect of pH on the formation
of trans-1-carbamoyl-2-oxo-4,5-dihydroxyimidazolidine (14 or
16
products, i.e., 5-hydroxyhydantoin (7, 8; 3%).
(
41) Willson, R. L. Int. J. Radiat. Biol. 1970, 17, 349-358.
(42) Wagner, J. R.; van Lier, J. E.; Johnston, L. J. Photochem. Photobiol.
1
990, 52, 333-343.
(
43) Wagner, J. R.; van Lier, J. E.; Decarroz, C.; Cadet, J. Bioelectro-
chem. Bioenerg. 1987, 18, 155-162. (c) Wagner, J. R.; Berger, M.; Cadet,
J.; van Lier, J. E. J. Chromatogr. 1990, 504, 191-196.
(45) Wagner, J. R., unpublished results.
(46) Hazra, D. K.; Steenken, S. J. Am. Chem. Soc. 1983, 105, 4380-
4386.
(
44) Daniels, M.; Schweibert, M. C. Biochim. Biophys. Acta 1967, 134,
4
81-483.

Hydroxyl-Radical-Induced Decomposition of 2′-Deoxycytidine
Scheme 2. Proposed Mechanism of Formation of Products
J. Am. Chem. Soc., Vol. 121, No. 17, 1999 4109
Scheme 4. Proposed Mechanism of Formation of
5′,6-Cyclic Products (17, 18)
1
0-16
rearrangement of the intermediate R-hydroxyketone followed
by cyclization and hydrolysis (VI to VII to VIII) explains the
formation of aminocarbonyl[2-(2-deoxy-â-D-erythro-pentofura-
nosyl)amino]-2-oxomethylcarbamic acid (16). In agreement with
18
this pathway, O2-labeling experiments indicated the incorpora-
1
8
Scheme 3. Isomerization of N1-(2-Deoxy-â-D-erythro-
pentofuranosyl)biuret in Aqueous Solution
tion of two atoms of O (Table 2). The presence of singly
labeled molecules implies that the hydroxyl group at C6 of
structure VI may exchange with bulk water.
Finally, the mechanism for the formation of the cyclic
products (17, 18) probably involves initial abstraction of C5′-H
•
by OH radicals, followed by intramolecular addition of the C5′-
centered radical (X) to the C6 position of the cytosine moiety
(Scheme 4). The 5′,6-cyclo base sugar C5 radicals of cytidine
and related nucleosides have previously been characterized by
trapping with 2-methyl-2-nitrosopropane and electron spin
resonance studies on the X irradiation of the substrates in N2O-
48
saturated aqueous solutions. The formation of products 17 and
8 from intermediate XI requires several steps (XI to XII). It
1
is likely that oxygen is incorporated at C5 to give an intermediate
peroxyl radical, which in turn converts into intermediate XII.
In contrast, the C5′ radical of dCyd leads to the formation of
5
′,6-cyclo-5,6-dihydro-2′-deoxycytidine in frozen aqueous solu-
1
5 without the sugar moiety) and N1-glycolylbiuret (VII without
tions in which competing reactions with oxygen cannot occur.
Thus, the identification of products 17 and 18 from γ-irradiation
of dCyd in aerated aqueous solutions suggests that intramo-
lecular addition of the C5′ radical of the sugar moiety to the C6
position of the base moiety competes well with the fortuitous
addition of oxygen to the C5′ radical.
Oxidation of dCyd in DNA. The decay of intermediate 5(6)-
hydroxy-6(5)-hydroperoxyl radicals by a bimolecular route will
be largely suppressed in DNA, giving way to the formation of
hydroperoxides. In fact, the initial yield of DNA hydroperoxides
appears to be about 50% of the total base damage immediately
following γ-irradiation in aqueous aerated solutions.49 Of these
hydroperoxides, it is estimated that one-third arises from dCyd
and two-thirds from thymidine in DNA. Therefore, one can
expect that a good part of the stable products of dCyd in DNA
derive from initial hydroperoxides. A number of stable products
of either cytosine or 2′-deoxycytidine have been detected from
DNA exposed to ionizing radiation in aerated aqueous solutions.
The radiochemical yields of 5-hydroxy-2′-deoxycytidine, 5-hy-
droxy-2′-deoxycytidine, and the 5,6-glycols of 2′-deoxyuridine
•
the sugar) from the OH-mediated decomposition of cytosine
indicates that both products arise from the same intermediate,
4
7
namely an R-hydroxyketone (VI without the sugar). In
contrast, the nucleoside derivative of N1-glycolylbiuret was not
detected in the product mixture, suggesting that it is not stable
in aqueous solution. The formation of 10-16 is explained by
subsequent reactions of an intermediate, R-hydroxyketone (VI).
The formation of 10-13 likely involves initial loss of glyoxal
(CHO-CHO) from VI. The presence of four isomers implies
that the â-furanoid isomerizes into the R-furanoid form and that
the furanose derivatives also isomerize into the pyranose
derivatives (Scheme 3). The acyclic structure of biuret renders
the molecule susceptible to protonation at N1, leading to
isomerization of the sugar moiety and/or anomerization. Fur-
thermore, it should be noted that trace quantities of 2-deoxy-
D-erythro-pentose and biuret were observed in the reaction
mixture. This is strongly indicative of hydrolysis of the
nucleoside presumably via a Schiff base intermediate (IX). The
formation of N1-(2-deoxy-â-D-erythro-pentofuranosyl)-1-car-
bamoyl-2-oxo-4,5-dihydroxyimidazolidine (14, 15) can be
explained by cyclization of the intermediate R-hydroxyketone
-
9
in γ-irradiated DNA under aerated conditions are 2.1 × 10 ,
(48) Hiraoka, W.; Kuwabara, M.; Sato, F.; Matsuda, A.; Ueda, T. Nucleic
Acids Res. 1990, 18, 1217-1223.
18
(VI). This is supported by O2-labeling experiments which show
18
the incorporation of two atoms of O (Table 2). Finally,
(49) (a) Schweibert, M. C.; Daniels, M. Int. J. Radiat. Phys. Chem. 1971,
(
7.
47) Polverelli, M.; Ulrich, J.; T e´ oule, R. Z. Naturforsch. 1983, 39c, 64-
3, 353-366. (b) Michaels, H. B.; Hunt, J. W. Anal. Biochem. 1978, 87,
135-140.
6

4
2
110 J. Am. Chem. Soc., Vol. 121, No. 17, 1999
Wagner et al.
-9
-9
-1
.5 × 10 , and 3.6 × 10 J mol , respectively, as determined
This suggests that the C3-C4 cleavage pathway may be
important in DNA. However, trans-1-carbamoyl-2-oxo-4,5-
dihydroxyimidazolidine is hardly formed in DNA exposed to
ionizing radiation in aerated aqueous solutions (<20-fold lower
by either enzymatic digestion of DNA and HPLC coupled to
electrochemical detection or acid hydrolysis of DNA followed
by purification of the hydrosylate and then gas chromatography/
mass spectrometry analysis with isotopic dilution.6 In addition,
the quantitation of some other cytosine modifications in
γ-irradiated DNA has also been reported, including 5-hydroxy-
,50
•
than the formation of 5-hydroxyhydantoin). The analysis of -
OH-mediated products of dCyd in DNA should be a priority in
future studies.
-
9
-1
51,52
hydantoin (6.0 × 10 J mol ) and isodialuric acid.
Acknowledgment. This paper is dedicated to the memory
of Michel Polverelli, who passed away on September 8, 1997.
We would like to thank the Fonds de Recherche en Sant e´ du
Qu e´ bec for salary support and the North Atlantic Treaty
Organization for providing funds for collaborative research.
Together, the yield of known cytosine products in DNA accounts
for only about 25% of the total damage to this base in DNA.
(50) Douki, T.; Delatour, T.; Paganon, F.; Cadet, J. Chem. Res. Toxicol.
1
996, 9, 1145-1151.
(51) Wagner, J. R. J. Chim. Phys. 1994, 91, 705-716.
(52) Dizdaroglu, M. Free Radical Biol. Med. 1991, 10, 225-242.
JA982461E