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725
mer was converted to a single major product, and small amounts
of several by-products with slightly longer retention times were
detected. The flow-through peak detected for the reaction mixture
was probably due to contaminants from the reagent or the buffer,
because its UV absorption spectrum was different from that of the
nucleic acids. The major product was purified by HPLC in an isola-
tion yield of 43% (2.3 nmol from 5.4 nmol), and was analyzed by
matrix-assisted laser desorption/ionization time of flight (MALDI-
TOF) mass spectrometry. The observed m/z value of the obtained
product in the negative-ion mode was 3208.5 (calculated m/z,
3208.6), whereas that of the starting material, that is, the thymine
glycol-containing 11-mer, was 3323.8 (calculated m/z, 3323.6).
This result demonstrated that the thymine glycol in the 11-mer oli-
gonucleotide was successfully converted into the formamide le-
sion. To verify that the same conversion can be performed in
longer oligonucleotides, a 30-mer, d(CTCGTCAGCATCTTgCATCATA-
CAGTCAGTG), was treated with NaIO4 in the same manner. HPLC
analysis revealed that a single major product, which could be sep-
arated from the starting material, was obtained, as shown in Figure
3.
In this study, we found that thymine glycol could be converted
into the formamide lesion quantitatively by the NaIO4 oxidation,
and applied this procedure to the lesion-containing oligonucleo-
tides. Since oligonucleotides containing thymine glycol are now
commercially available, by using its building block,2 the post-syn-
thetic conversion method described in this Letter will enable biol-
ogists, who may find chemical synthesis difficult to accomplish, to
obtain oligonucleotides containing the formamide lesion, and thus
will facilitate biochemical studies on this type of DNA damage.
Figure 2. HPLC analysis of the thymine glycol-containing 11-mer before (a) and
after (b) the periodate oxidation. The conditions were the same as those described
in the legend to Figure 1a, except that the acetonitrile gradient was 5–11% for
20 min plus 25% for 5 min.
used in the synthesis of modified oligonucleotides,17 we confirmed
that unmodified oligonucleotides remained intact after an over-
night treatment with NaIO4, prior to the following experiments.
In addition, it should be noted that the aforementioned reaction
with lead tetraacetate cannot be used for oligonucleotides, because
this reagent is promptly hydrolyzed by water. An oligonucleotide
containing 5R-thymine glycol, d(CGTACTgCATGC), in which Tg rep-
resents 5R-thymine glycol, was prepared by using the (5R,6S)-thy-
mine glycol building block, as described previously,2 and this
oligonucleotide was treated with 150 mM NaIO4 in 250 mM so-
dium acetate (pH 6.0) at room temperature for 15 h. After desalting
on a gel-filtration column, the reaction mixture was analyzed by
HPLC. As shown in Figure 2, the thymine glycol-containing 11-
Acknowledgments
This study was supported by a Grant-in-Aid for Scientific Re-
search from the Ministry of Education, Culture, Sports, Science,
and Technology, Japan. J.Y. was supported by a Research Fellow-
ship of the Japan Society for the Promotion of Science for Young
Scientists, and expresses his special thanks to The Global COE Pro-
gram of Osaka University.
Supplementary data
Supplementary data associated with this article can be found, in
References and notes
1. Friedberg, E. C.; Walker, G. C.; Siede, W.; Wood, R. D.; Schultz, R. A.; Ellenberger,
T. In DNA Repair and Mutagenesis, 2nd ed.; ASM Press: Washington DC, 2006; pp
25–29.
2. (a) Iwai, S. Angew. Chem., Int. Ed. 2000, 39, 3874; (b) Iwai, S. Chem. Eur. J. 2001, 7,
4343; (c) Shimizu, T.; Manabe, K.; Yoshikawa, S.; Kawasaki, Y.; Iwai, S. Nucleic
Acids Res. 2006, 34, 313.
3. (a) Katafuchi, A.; Nakano, T.; Masaoka, A.; Terato, H.; Iwai, S.; Hanaoka, F.; Ide,
H. J. Biol. Chem. 2004, 279, 14464; (b) Kusumoto, R.; Masutani, C.; Iwai, S.;
Hanaoka, F. Biochemistry 2002, 41, 6090.
4. Ide, H.; Kow, Y. W.; Wallace, S. S. Nucleic Acids Res. 1985, 13, 8035.
5. Lustig, M. J.; Cadet, J.; Boorstein, R. J.; Teebor, G. W. Nucleic Acids Res. 1992, 20,
4839.
6. (a) Breimer, L. H.; Lindahl, T. Biochemistry 1985, 24, 4018; (b) Olinski, R.;
Zastawny, T.; Budzbon, J.; Skokowski, J.; Zegarski, W.; Dizdaroglu, M. FEBS Lett.
1992, 309, 193; (c) Höss, A.; Jaruga, P.; Zastawny, T. H.; Dizdaroglu, M.; Pääbo,
S. Nucleic Acids Res. 1996, 24, 1304.
7. Cadet, J.; Narin, R.; Voituriez, L.; Remin, M.; Hruska, F. E. Can. J. Chem. 1981, 59,
3313.
8. Claridge, T. D. W. In High-Resolution NMR Techniques in Organic Chemistry;
Elsevier Science: Oxford, 1999. pp 313–328.
9. (a) Paul, C. R.; Wallace, J. C.; Alderfer, J. L.; Box, H. C. Int. J. Radiat. Biol. 1988, 54,
403; (b) Wagner, J. R.; Decarroz, C.; Berger, M.; Cadet, J. J. Am. Chem. Soc. 1999,
121, 4101.
10. (a) Girault, I.; Molko, D.; Cadet, J. Free Radical Res. 1994, 20, 315; (b) Girault, I.;
Fort, S.; Molko, D.; Cadet, J. Free Radical Res. 1997, 26, 257.
11. Luo, Y.; Henle, E. S.; Linn, S. J. Biol. Chem. 1996, 271, 21167.
Figure 3. HPLC analysis of the 30-mer oligonucleotide. (a) The crude sample after
the NaIO4 treatment. (b) Co-injection with the thymine glycol-containing 30-mer.
The conditions were the same as those described in the legend to Figure 1a, except
that the acetonitrile gradient was 7–13% for 20 min.