F or m a tion of a Meth id e Der iva tive u p on P h otolysis of Th ym id in e
Br om oh yd r in s
Thierry Douki,* Guillaume Vadesne-Bauer, and J ean Cadet*
Laboratoire Le´sions des Acides Nucle´iques, Service de Chimie Inorganique et Biologique, UMR 5046,
CEA/ DSM/ De´partement de Recherche Fondamentale sur la Matie`re Condense´e, CEA-Grenoble,
38054 Grenoble Cedex 9, France
tdouki@cea.fr; jcadet@cea.fr
Received October 25, 2002
Reaction of bromine with thymidine in aqueous solution produces, in high yield, the corresponding
5-bromo-6-hydroxy-5,6-dihydroderivative (thymidine bromohydrins). UVC photolysis of thymidine
bromohydrins gives rise to a reactive intermediate that is converted into 5-(hydroxymethyl)-2′-
deoxyuridine upon incubation in water. When the former compound is left in methanol, ethanol,
or propanol, the corresponding 5-alkoxymethyl derivatives are produced. The proposed structure
for the primary photolysis product of thymidine bromohydrins is a methide derivative of the thymine
ring. This compound could be an interesting intermediate in the synthesis of methyl-substituted
thymidine.
In tr od u ction
Resu lts
Id en tifica tion of th e F in a l Sta ble P r od u cts of
Modification of the chemical structure of DNA bases
is associated with deleterious cellular processes including
lethality and mutagenicity. In that respect, radical-
induced degradation of nucleobases has been extensively
investigated on model systems such as nucleosides and
short oligonucleotides.1 Photochemical generation of nu-
cleobase radicals is a powerful tool for the understanding
of the degradation pathways.2 Recently, we used thymine
bromohydrins, previously used as synthetic precursors
of pyrimidine hydroperoxides,3 to photochemically trigger
the formation of tandem lesions in a dinucleoside mono-
phosphate carrying a vicinal guanine moiety.4 In the
latter experiment, products only modified on their thy-
mine moiety were also obtained in high yield.
P h otolysis of Th ym id in e Br om oh yd r in s. (5R,6S)-
and (5S,6R)-5-Bromo-6-hydroxy-5,6-dihydrothymidine
(trans bromohydrins of thymidine, 2) were synthesized
in high yield as previously reported by addition of
bromine to an aqueous solution of thymidine (1).5 The
reaction mixture was injected on a reverse-phase HPLC
column, and a fraction containing the two bromohydrins
was collected. The resulting solution was then exposed
to the UVC light emitted by a germicidal lamp. Thymi-
dine bromohydrins are very reactive under these condi-
tions, since, after 30 min of irradiation, HPLC analysis
shows that 2 is completely converted. Instead, the
HPLC-UV chromatogram exhibits two main peaks cor-
responding to closely eluting photoproducts 3 and 3′ (yield
ca. 95%) at shorter retention times than those obtained
for 2 (Figure 1). Thymidine glycols that may arise from
either the hydrolytic conversion of bromohydrins or the
fate of the 6-hydroxy-5-yl radical are also obtained in low
yield (<5%). When irradiated bromohydrins mixtures are
left in aqueous solution at room temperature, two new
compounds, 4 and 4′, are produced at the expense of 3
and 3′, as shown by HPLC-MS/MS analyses (vide infra).
The same observations are made when the crude bromi-
nation mixture was irradiated instead of HPLC-purified
bromohydrins.
We presently report a more thorough study of the
photolysis of thymine bromohydrins at the nucleoside
level. The final stable products were characterized, on
1
the basis of H NMR and mass spectrometry analyses,
as 5-(hydroxymethyl)-uracil nucleosides. Their formation
involved an intermediate that was partially identified by
1H NMR and mass spectrometry. A methide structure,
in agreement with the reactivity in water and alcohols,
was proposed for this transient bromohydrins photolysis
product.
(1) (a) Breen, A. P.; Murphy, J . A. Free Radical Biol. Med. 1995,
18, 1033-1077. (b) Cadet, J .; Berger, M.; Douki, T.; Ravanat, J .-L. Rev.
Physiol. Biochem. Pharmacol. 1997, 131, 1-87.
(2) (a) Greenberg, M. M. Chem. Res. Toxicol. 1998, 11, 1235-1248.
(b) Romieu, A.; Bellon, S.; Gasparutto, D.; Cadet, J . Org. Lett. 2000, 2,
1085-1088.
(3) (a) Cadet, J .; Te´oule, R. Biochim. Biophys. Acta 1971, 238, 8-26.
(b) Cadet, J .; Te´oule, R. C. R. Acad. Sci. Paris 1973, 276, 1743-1746.
(c) Wagner, R. J .; van Lier, J . E.; Berger, M.; Cadet, J . J . Am. Chem.
Soc. 1994, 116, 2235-2242. (d) Tremblay, S.; Douki, T.; Cadet, J .;
Wagner, R. J . Biol. Chem. 1999, 274, 20833-20838.
(4) Douki, T.; Rivie`re, J .; Cadet, J . Chem. Res. Toxicol. 2002, 15,
445-454.
4 and 4′ were isolated separately by reverse phase
HPLC. Compound 4 was identified as 5-(hydroxymethyl)-
2′-deoxyuridine (5-HMdUrd) on the basis of its chroma-
1
tography, mass spectrometry (Figure 2),6 and H NMR
(5) Ulrich, J .; Cadet, J .; Te´oule, R. Org. Mass Spectrom. 1973, 7,
543-554.
(6) Frelon, S.; Douki, T.; Ravanat, J .-L.; Tornabene, C.; Cadet, J .
Chem. Res. Toxicol. 2000, 13, 1002-1010.
10.1021/jo026606i CCC: $25.00 © 2003 American Chemical Society
Published on Web 12/10/2002
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J . Org. Chem. 2003, 68, 478-482