2596
M. Endo et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2593–2596
Ministry of Education, Culture, Sports, Science, and
Technology.
A A
L1
L4
A
G
15
16
C
G
U
C
G
C
A
G
C
G
References and notes
C
U
1. Benner, S. A.; Burgstaller, P.; Battersby, T. R.; Jurczyk, S.
In The RNA World; Gesteland, R. F., Cech, T. R., Atkins,
J. F., Eds.; Cold Spring Harbor Laboratory: Cold Spring
Harbor, New York, 1999; pp 163–181.
2. Tor, Y.; Dervan, P. B. J. Am. Chem. Soc. 1993, 115, 4461.
3. Ohtsuki, T.; Kimoto, M.; Ishikawa, M.; Mitsui, T.; Hirao,
I.; Yokoyama, S. Proc. Natl. Acad. Sci. U.S.A. 2001, 98,
4992.
4. Hirao, I.; Ohtsuki, T.; Fujiwara, T.; Mitsui, T.; Yokog-
awa, T.; Okuni, T.; Nakayama, H.; Takio, K.; Yabuki, T.;
Kigawa, T.; Kodama, K.; Yokogawa, T.; Nishikawa, K.;
Yokoyama, S. Nat. Biotechnol. 2002, 20, 177.
5. Kimoto, M.; Endo, M.; Mitsui, T.; Okuni, T.; Hirao, I.;
Yokoyama, S. Chem. Biol. 2003, 11, 47.
C
U
24
10
A
U24: Tm = 63.3 C
y24: Tm = 63.3 C
Ph-y24: Tm = 67.0 C
C
C
G
G
A
C
6
28
A
U
A
G
G
C
G
A
G
G
G
C
G
C
U
C
33
1
6. McMinn, D. L.; Ogawa, A. K.; Wu, Y.; Liu, J.; Schultz, P.
G.; Romesberg, F. E. J. Am. Chem. Soc. 1999, 121, 11585.
7. Mitsui, T.; Kimoto, M.; Sato, A.; Yokoyama, S.; Hirao, I.
Bioorg. Med. Chem. Lett. 2003, 13, 4515.
Figure 3. Site-specific incorporation of Ph-y into a theophylline-
binding RNA aptamer. Unnatural components, y and Ph-y, were
introduced at position 24, in place of the uridine of the original apt-
amer (U24). The numbering of the residues corresponds to the original
duplex aptamer.16;17
8. Nakano, S.; Uotani, Y.; Nakashima, S.; Anno, Y.; Fujii,
M.; Sugimoto, N. J. Am. Chem. Soc. 2003, 125, 8086.
9. Mattaj, I. W.; Nagai, K. Nat. Struct. Biol. 1995, 2, 518.
10. Brown, L. J.; May, J. P.; Brown, T. Tetrahedron Lett.
2001, 42, 2587.
11. 5-Phenylethynyl-3-(b-D-ribofuranosyl)pyridin-2-one (3):
1H NMR (270 MHz, DMSO-d6) d 3.50 (m, 1H), 3.65 (m,
1H), 3.83 (m, 3H), 4.68 (d, 1H, J ¼ 4:1 Hz), 4.73 (d, 1H,
J ¼ 5:1 Hz), 4.96 (t, 1H, J ¼ 6:1 Hz), 5.11 (br s, 1H), 7.39
(m, 3H), 7.48 (m, 2H), 7.70 (s, 1H), 7.72 (s, 1H), 12.11 (s,
1H); 13C NMR (68 MHz, DMSO-d6) d 61.17, 70.57, 74.57,
80.37, 83.47, 86.10, 88.71, 100.17, 122.32, 128.30, 128.54,
130.89, 131.16, 137.68, 138.49, 160.24. HR-MS (FAB, 3-
NBA matrix) calcd for C18H18NO5 (Mþ1) 328.1185,
found: 328.1188.
By the specific transcription involving the unnatural s–y
pair, the hydrophobic Ph-y can be incorporated into
large RNA fragments at desired positions. The DNA
templates containing s are easily prepared by PCR
amplification with a 30-primer containing s.5 Thus, this
specific transcription mediated by unnatural base pairs
is more useful than chemical RNA synthesis especially
for the preparation of long RNA fragments. Efficient
sites in RNA molecules for the incorporation of
unnatural components can be predicted from the 3D
structures of the RNA molecules. Uridine residues in
single-stranded regions, G–U pairs, and certain base
triplets18 in the RNA molecules might be suitable Ph-y
incorporation sites for the thermal stabilization of RNA
molecules.
12. 5-Phenylethynyl-3-(b-D-ribofuranosyl)pyridin-2-one 5-tri-
1
phosphate (4): H NMR (270 MHz, D2O) d 1.11 (t, 27H,
J ¼ 7:3 Hz), 3.03 (q, 18H, J ¼ 7:3 Hz), 4.09 (m, 5H), 4.86
(d, 1H, J ¼ 3:7 Hz), 7.27 (m, 3H), 7.43 (m, 2H), 7.60 (s,
1H), 7.81 (s, 1H). 31P NMR (109 MHz, D2O) d ꢀ22.83 (t,
1H, J ¼ 19:5 Hz, 21.4 Hz), ꢀ10.72 (d, 1H, J ¼ 19:5 Hz),
ꢀ10.15 (d, 1H, J ¼ 21:4 Hz). ESI-MS calcd for
C18H19NO14P3 (Mꢀ1) 566.00, found: 565.50.
€
ꢀ
€
13. Kovacs, T.; Otvos, L. Tetrahedron Lett. 1988, 29, 4525.
14. Mitsui, T.; Kitamura, A.; Kimoto, M.; To, T.; Sato, A.;
Hirao, I.; Yokoyama, S. J. Am. Chem. Soc. 2003, 125,
5298.
Acknowledgements
15. Jenison, R. D.; Gill, S. C.; Pardi, A.; Polinsky, B. Science
1994, 263, 1425.
16. Zimmermann, G. R.; Shields, T. P.; Jenison, R. D.; Wick,
C. L.; Pardi, A. Biochemistry 1998, 37, 9186.
17. Zimmermann, G. R.; Wick, C. L.; Shields, T. P.; Jenison,
R. D.; Pardi, A. RNA 2000, 6, 659.
This work was supported by the RIKEN Structural
Genomics/Proteomics Initiative (RSGI), the National
Project on Protein Structural and Functional Analyses,
Ministry of Education, Culture, Sports, Science and
Technology of Japan, and by a Grant-in-Aid for
Scientific Research (KAKENHI 15350097) from the
18. Walberer, B. J.; Cheng, A. C.; Frankel, A. D. J. Mol. Biol.
2003, 327, 767.