Synthesis and UV photolysis of oligodeoxynucleotides that contain 5-(phenylthiomethyl)-2'-deoxyuridine: a specific photolabile precursor of 5-(2'-deoxyuridilyl)methyl radical.

Article abstract of DOI:10.1021/ol005643y

[formula: see text] The title exocyclic radical (2) is generated via photochemical cleavage of 5-(phenylthiomethyl)-2'-deoxyuridine (8). The latter thionucleoside (8) was successfully incorporated into DNA oligomers by automated DNA synthesis using phosphoramidite chemistry. UV exposure of 8 containing oligonucleotides under (an)aerobic conditions gives rise to specific base lesions. The photoproducts have been isolated and further characterized on the basis of detailed NMR and mass spectrometric analyses.

Full text of DOI:10.1021/ol005643y

ORGANIC
LETTERS
2000
Vol. 2, No. 8
1085-1088
Synthesis and UV Photolysis of
Oligodeoxynucleotides That Contain
5-(Phenylthiomethyl)-2′-deoxyuridine:
A Specific Photolabile Precursor of
5-(2′-Deoxyuridilyl)methyl Radical
Anthony Romieu,† Sophie Bellon, Didier Gasparutto, and Jean Cadet*
Laboratoire des Le´sions des Acides Nucle´iques, SerVice de Chimie Inorganique et
Biologique, UMR 5046, De´partement de Recherche Fondamentale sur la Matie`re
Condense´e, CEA-Grenoble, F-38054 Grenoble Cedex 9, France
cadet@drfmc.ceng.cea.fr
Received February 10, 2000
ABSTRACT
The title exocyclic radical (2) is generated via photochemical cleavage of 5-(phenylthiomethyl)-2′-deoxyuridine (8). The latter thionucleoside (8)
was successfully incorporated into DNA oligomers by automated DNA synthesis using phosphoramidite chemistry. UV exposure of 8 containing
oligonucleotides under (an)aerobic conditions gives rise to specific base lesions. The photoproducts have been isolated and further characterized
on the basis of detailed NMR and mass spectrometric analyses.
During the past five years, many efforts have been devoted
to the synthesis of oligodeoxynucleotides (ODNs) that
contained photoreactive precursors of radical intermediates
involved in the radiation-induced decomposition of purine
and pyrimidine nucleic acid components.1 These modified
DNA fragments are powerful tools for mechanistic studies
aimed at elucidating the role of individual nucleobase and
osidic reactive intermediates in the formation of nucleic acid
lesions. These include oxidized bases, abasic sites, and DNA
strand breaks. Indeed, the UV-mediated independent genera-
tion of a specific radical at a defined site in an ODN enables
one to investigate the chemistry of this radical under
conditions in which other radicals derived from nucleosides
are not formed. This approach offers a distinct advantage
over the use of ionizing radiation that gives rise to multiple
reactive intermediates. Interestingly, the chemistry of the
osidic radicals resulting from C-1′- and C-4′-hydrogen atom
abstraction has been studied in detail by using such modified
ODNs. The latter mechanistic investigations have involved
extensive product analysis, together with kinetics and EPR
measurements.1,2
To investigate the reactivity of the 5-(2′-deoxyuridilyl)-
methyl radical 2 in DNA oligomers, we have developed a
similar methodolgy by synthesizing 8, a specific photopre-
† Current address: Unite´ de Pharmacochimie Mole´culaire et Structurale,
U266 INSERM - UMR 8600 CNRS, Universite´ Rene´ Descartes, UFR des
Sciences Pharmaceutiques et Biologiques, 4, Avenue de l’Observatoire,
F-75270 Paris Cedex 06, France.
(1) For reviews, see: Greenberg, M. M. Chem. Res. Toxicol. 1998, 11,
1235-1248. Beckwith, A. L. J.; Crich, D.; Duggan, P. J.; Yao, Q. Chem.
ReV. 1997, 97, 3273-3312.
(2) (a) Chatgilialoglu, C.; Gimisis, T.; Guerra, M.; Ferreri, C.; Emanuel,
C. J.; Horner, J. H.; Newcomb, M.; Lucarini, M.; Pedulli, G.; Tetrahedron
Lett. 1998, 39, 3947-3950. (b) Emanuel, C. J.; Newcomb, M.; Ferreri, C.;
Chatgilialoglu, C. J. Am. Chem. Soc. 1999, 121, 2927-2928. (c) Giese,
B.; Wessely, S.; Spormann, M.; Lindemann, U.; Meggers, E.; Michel-
Beyerle, M. E. Angew. Chem., Int. Ed. 1999, 38, 996-998. (d) Hwang,
J.-T.; Greenberg, M. M. J. Am. Chem. Soc. 1999, 121, 4311-4315.
10.1021/ol005643y CCC: $19.00 © 2000 American Chemical Society
Published on Web 03/28/2000

cursor to radical 2 derived from thymidine. Radical 2 is one
of the main reactive radiation-induced and type I photosen-
sitized decomposition products of thymidine 1.3,4 It results
interact with neighboring guanines, resulting in the formation
of DNA lesions with two adjacent modified bases (“tandem
DNA lesions”).5,6 With the aim to further substantiate the
latter mechanistic pathways, thionucleoside 8 was site-
specifically incorporated into synthesized ODNs via phos-
phoramidite 9. Then, radical 2 was independently generated
by UV-C irradiation (λmax ) 254 nm) under both aerobic
and anaerobic conditions.
•
from either OH-mediated hydrogen atom abstraction from
the 5-methyl group or deprotonation of the radical cation
intermediate produced by photosensitizers operating through
a type I mechanism such as benzophenone or menadione
(Scheme 1). Subsequent reaction with oxygen leads to
We report the synthesis of these modified ODNs together
with the isolation and the characterization of DNA damage
induced via the 5-(2′-deoxyuridilyl)methyl radical. It is
assumed that 2 is generated via photochemical homolytic
cleavage of the C-SPh bond of 5-(phenylthiomethyl)-2′-
deoxyuridine (dPhSU) 8. This is supported by the formation
of cyclonucleosides from several thionucleosides possessing
this photoreactive group via the generation of similar alkyl
radicals.7-9 The synthesis of 8 and 9 is outlined in Scheme
2. Hydroxymethylation of 2′-deoxyuridine 6 with paraform-
Scheme 1a
Scheme 2. Synthetic Reactions Used for the Preparation of
the Phosphoramidite Synthon of dPhSUa
a (a) (CH2O)n, TEA, H2O, 60 °C, 66 h; (b) (CH3CO)2O, pyridine,
15 h; (c) PhSH, TEA, DMF, 70 °C, 24 h; (d) NH4OH 32% aqueous,
CH3OH, 15 h, 13% (a + b + c + d); (e) DMTrCl, pyridine, 23 h,
64%; (f) NCCH2CH2OP(Cl)NPri2, DIEA, CH2Cl2, argon, 30 min,
96%.
aldehyde in alkaline medium followed by acetylation and
selective substitution of the benzylic acetate group by
(5) Delatour, T.; Douki, T.; Gasparutto, D.; Brochier, M.-C.; Cadet, J.
Chem. Res. Toxicol. 1998, 11, 1005-1013.
(6) (a) Box, H. C.; Budzinski, E. E.; Dawidzik, J. D.; Wallace, J. C.;
Evans, M. S.; Gobey, J. S. Radiat. Res. 1996, 145, 641-643. (b) Budzinski,
E. E.; Dawidzik, J. D.; Rajecki, M. J.; Wallace, J. C.; Schroder, E. A.;
Box, H. C. Int. J. Radiat. Biol. 1997, 71, 327-336. (c) Box, H. C.;
Budzinski, E. E.; Dawidzik, J. D.; Gobey, J. S.; Freund, H. G. Free Radical
Biol. Med. 1997, 23, 1021-1030. (d) Box, H. C.; Budzinski, E. E.;
Dawidzik, J. D.; Wallace, J. C.; Iijima, H. Radiat. Res. 1998, 149, 433-
439.
(7) (a) Romieu, A.; Gasparutto, D. Molko, D.; Cadet, J. J. Org. Chem.
1998, 63, 5245-5249. (b) Romieu, A.; Gasparutto, D.; Cadet, J. Chem.
Res. Toxicol. 1999, 12, 412-421.
(8) (a) Usui, H.; Ueda, T. Nucleic Acids Res. 1984, (S), 61-63. (b)
Matsuda, A.; Muneyama, K.; Nishida, T.; Sato, T.; Ueda, T. Nucleic Acids
Res. 1976, 3, 3349-3357.
a (a) The 5-(2′-deoxyuridilyl)methyl radical 2 reaction manifold.
(b) Generation of 2 by UV-C photolysis of the thionucleoside 8.
hydroperoxide 3 which decomposes into 5-(hydroxymethyl)-
2′-deoxyuridine (dHMU) 4 and 5-formyl-2′-deoxyuridine
(dFU) 5. Furthermore, the exocyclic radical is suspected to
(3) Cadet, J.; Delatour, T.; Douki, T.; Gasparutto, D.; Pouget, J.-P.;
Ravanat, J.-L.; Sauvaigo, S. Mutat. Res. 1999, 424, 9-21.
(4) (a) Decarroz, C.; Wagner, J. R.; van Lier, J. E.; Muri-Krishna, C.;
Riesz, P.; Cadet, J. Int. J. Radiat. Biol. 1986, 50, 491-505. (b) Wagner, J.
R.; van Lier, J. E.; Decarroz, C.; Berger, M.; Cadet, J. Methods Enzymol.
1990, 186, 502-511. (c) Delatour, T.; Douki, T.; D’Ham, C.; Cadet, J. J.
Photochem. Photobiol. B 1998, 44, 191-198.
(9) Recently, Mehl and Begley reported the synthesis of a bispyrimidine
model system containing the PhS group as a precursor to the C-6 spore
photoproduct radical: Mehl, R. A.; Begley, T. P. Org. Lett. 1999, 1, 1065-
1066.
1086
Org. Lett., Vol. 2, No. 8, 2000

thiophenol produced the fully protected thionucleoside 7.10
Deprotection of 7 afforded dPhSU 8, which was further
converted into phosphoramidite 9 via the protection of the
primary alcohol with dimethoxytrityl chloride (DMTrCl)11
followed by treatment with 2-cyanoethyl N,N-diisopropy-
lphosphoramidochloridite.12 It was found that 8 is stable in
the presence of the reagents (trichloroacetic acid, tetrazole,
iodine/pyridine/THF/H2O, and acetic anhydride/N-meth-
ylimidazole) used in automated chemical DNA synthesis.
However, 8 was fully decomposed under standard base-
deprotecting conditions (30% ammonium hydroxide, 55 °C,
12 h).13 This was circumvented by using the highly alkali-
labile amino-protecting groups developed by Schulhof et al.,14
which allow for a complete deprotection of synthetic
oligonucleotides in 30% aqueous ammonia at room temper-
ature for 4 h. In the latter conditions, no degradation of the
modified nucleoside 8 was detected.
analysis of the nucleoside mixture obtained by enzymatic
digestion of 9-mer 12 by nuclease P1 followed by alkaline
phosphatase provided dC, dG, dT, dA, and dPhSU in a
2:1:3:2:1 ratio, confirming the structure (Figure 1).
Figure 1. HPLC elution profile of the enzymatic digestion mixture
of 9-mer d(ATCPhSUGTACT) 12.
The modified ODNs 10-12 (Table 1) were synthesized
according to the solid-phase phosphoramidite method (1 and
When 2-mer [5′-d(PhSUG)-3′] 10 is UV-irradiated under
aerobic conditions, high yields of [5′-d(HMUG)-3′] 13 and
[5′-d(FUG)-3′] 14 are obtained (Figure 2a). Structural insights
Table 1. Sequences and Relative Molecular Masses (Da) of
the Modified ODNs
mass
massb
name
sequencesa
XG
CXGA
ATCXGTACT
HMUG
FUG
CHMUGA
CFUGA
length (calcd) (Da)
(found) (Da)
10
11
12
13
14
15
16
17
18
19
20
21
2
4
9
2
2
4
4
9
9
2
2
2
679.40
1281.80
2796.80
587.40
679.40 ( 0.10
1281.51 ( 0.31
2797.23 ( 1.74
587.50 ( 0.10
585.40 ( 0.10
1190.00 ( 0.10
1187.90 ( 0.10
2704.50 ( 0.27
2701.80 ( 1.50
571.40 ( 0.10
569.40 ( 0.10
1140.90 ( 0.10
585.40
1189.80
1187.80
2704.80
2702.80
571.40
ATCHMUGTACT
ATCFUGTACT
TG
TMeC8
G
569.40
1140.90
dimeric TG
a X is PhSU. b All the ODN masses were obtained by ESI-MS in the
negative mode.
Figure 2. HPLC elution profiles of UV-irradiated d(PhSUG) 10
(a) under atmospheric O2 and (b) N2-purged solution.
10 µmol scales) on an automated DNA synthesizer using 9
and commercially available phenoxyacetyl-dA-, phenoxy-
acetyl-dG-, and isobutyryl-dC-cyanoethyl phosphoramidites.
After cleavage of the support and removal of the alkali-labile
groups with 30% ammonium hydroxide at room temperature
for 4 h, the 5′-DMTr-oligomers were purified and deprotected
on line by reversed-phase HPLC.7a Electrospray ionization
mass spectrometry (ESI-MS) measurements of the modified
ODNs (Table 1) confirmed the incorporation of dPhSU (8)
and the purity of oligomers 10-12. Furthermore, HPLC
into the modified dinucleoside monophosphates 13 and 14
were gained from ESI-MS measurements (Table 1). In
addition, both HPLC peaks were identified by comigration
with authentic samples of 13 and 14 which were prepared
by using synthetic methodologies described in the litera-
ture.15,16 Similar results were obtained with the other dPhSU-
containing ODNs 11-12. Altogether these results suggest
that the 5-(2′-deoxyuridilyl)methyl radical 2 is formed in a
high yield and trapped efficiently by oxygen when 8 is UV-
irradiated within these biopolymers.
(10) Suzuki, Y.; Matsuda, A.; Ueda, T. Chem. Pharm. Bull. 1987, 35,
1085-1092.
(11) Agarwal, K. L.; Yamazaki, A.; Cashion, P. J.; Khorana, H. G.
Angew. Chem., Int. Ed. Engl. 1972, 11, 451-459.
(12) Sinha, N. D.; Biernat, J. Ko¨ster, H. Tetrahedron Lett. 1983, 24,
5843-5846.
(13) Complete conversion into 5-(aminomethyl)-2′-deoxyuridine was
observed.
(14) Schulhof, J. C.; Molko, D.; Te´oule, R. Nucleic Acids Res. 1987,
15, 397-415.
(15) For [5′-d(HMUG)-3′], see: Sowers, L. C.; Beardsley, G. P. J. Org.
Chem. 1993, 58, 1664-1665.
(16) For [5′-d(FUG)-3′], see: Berthod, T. Pe´tillot, Y.; Guy, A.; Cadet,
J.; Forest, E.; Molko, D. Nucleosides Nucleotides 1996, 15, 1287-1305.
Org. Lett., Vol. 2, No. 8, 2000
1087

Thereafter, photolysis of 10 in deaerated aqueous solution
resulted in the formation of [5′-d(TG)-3′] 19 as the major
compound (Figure 2b). This product is likely to arise from
trapping of 2 by thiophenol, a hydrogen atom donor,
produced during photoirradiation. Furthermore, two other
products which illustrate the reactivity of the 5-(2′-deoxy-
uridilyl)methyl radical under anaerobic conditions have been
detected in the crude photolysate. The most polar compound
was isolated in 12% yield and subjected to mass spectrometry
measurements. The molecular weight of 569.4 is in good
agreement with the calculated mass of 569.4 for the thymine-
guanine tandem lesion [5′-d(TMeC8G)-3′] 20 which has a
covalent bond between the methyl carbon atom of thymine
and the C-8 carbon atom of guanine. The latter product was
first identified by Box et al. in DNA oligomers, namely, [5′-
d(CGTA)-3′] and [5′-d(ACGT)-3′], exposed to X irradiation
in deoxygenated aqueous solution. This was achieved by 1H
NMR spectroscopy.6a,c The structure of the vicinal DNA
lesion 20 was confirmed by using tandem mass spectrometry
(ESI-MS/MS) which allows the determination of the frag-
mentation pattern of the molecule. Thus, a characteristic
568-470 transition which results from a loss of the sugar
residue at the 5′-end of the dinucleoside monophosphate 20
was observed in the MS/MS spectra in the negative mode.
In the latter fragment, the thymine remains covalently
attached through the methylene bridge. To generate enough
product 20 for more extensive NMR studies, [5′-d(PhSUG)-
3′] was synthesized in large amounts (1 mmol scale) by
solution phosphotriester chemistry and subsequently irradi-
hydrolysis of the phosphodiester bond was observed under
the three enzymatic degradation conditions used as inferred
from RP-HPLC and mass spectrometry analyses of the
reaction mixtures. The last compound isolated from the
HPLC elution profile of the photolysis reaction has a
molecular weight of 1140.9. This suggests a dimeric structure
of [5′-d(TG)-3′] where the two methyl carbon atoms of
thymines are connected by a covalent bond (calculated
1140.9). This structure was confirmed by submitting the
compound to nuclease P1 and alkaline phosphatase digestion.
RP-HPLC analysis of the hydrolysis mixture followed by
tandem mass spectrometry measurements of the collected
peaks revealed the presence of a resistant and characteristic
d(TMeMeT) residue (mass calculated 482.4; mass found 482.2).
Thus, under anaerobic conditions, the 5-(2′-deoxyuridilyl)-
methyl radical can react with an adjacent guanine base to
give [5′-d(TMeC8G)-3′] or undergo a radical pair combination
to generate the dimeric adduct derived from [5′-d(TG)-3′].
In summary, the present studies demonstrated that 5-
(phenylthiomethyl)-2′-deoxyuridine is an efficient photopre-
cursor of the 5-(2′-deoxyuridilyl)methyl radical within DNA
oligomers. Work is in progress to generate 20 in longer
ODNs which will be suitable for studies aimed at determining
both the biochemical (mutagenesis, repair) and conforma-
tional features of the tandem lesion. In addition, availability
of such modified ODNs will facilitate the development and
the optimization of HPLC-MS/MS assay for monitoring the
formation of the latter damage within isolated and cellular
oxidized DNA.
1
ated. The H NMR spectrum of 20 collected from HPLC
Acknowledgment. The contribution of Colette Lebrun
(SCIB/LRI/CEA, Grenoble, France) for the measurement of
the mass spectra is gratefully acknowledged. We thank Drs.
Thierry Douki and Jean Luc Ravanat for fruitful discussions
about tandem mass spectrometry experiments. This work was
supported in part by grants from the French Ministry of
Science and Research (ACC-SV Grant 8-MESR 1995) and
Electricite´ de France (Comite´ de Radioprotection).
runs, exhibits the same features as those previously described
by Box et al. for the analogous product in the [5′-d(CGTA)-
3′] oligomer. As striking characteristics, the thymine methyl
proton resonance and the guanine H-8 resonance are absent.
Moreover, two doublet signals which correspond to the
nonmagnetically equivalent methylenic protons are observed
(chemical shifts at 3.65 and 3.95 ppm, respectively). This
unambiguously confirms the assigned structure of 20. The
resistance of the latter adduct to enzymatic digestion by
several endo- and exonucleases, namely, nuclease P1, calf
spleen phosphodiesterase, and snake venom phosphodi-
esterase, was investigated as previously described.7,17-19 No
Supporting Information Available: Typical experimen-
tal procedures and characterization of compounds 8 and 9
and modified oligonucleotides. This material is available free
of charge via the Internet at http://pubs.acs.org.
OL005643Y
(17) Gasparutto, D.; Ravanat, J.-L.; Ge´rot, O.; Cadet, J. J. Am. Chem.
Soc. 1998, 120, 10283-10286.
(18) Romieu, A.; Gasparutto, D. Molko, D.; Ravanat, J.-L.; Cadet, J.
Eur. J. Org. Chem. 1999, 49-56.
(19) Romieu, A.; Gasparutto, D.; Cadet, J. J. Chem. Soc, Perkin Trans.
1 1999, 1257-1263.
1088
Org. Lett., Vol. 2, No. 8, 2000