Synthesis of DNA templates containing the fifth base, 2-amino-6-(dimethylamino)purine, for specific transcription involving unnatural base pairs

Article abstract of DOI:10.1246/cl.2001.914

An unnatural nucleobase, pyridin-2-one (denoted as y), can be specifically incorporated into RNA opposite 2-amino-6-(dimethylamino)purine (denoted as x) in DNA templates by T7 RNA polymerase. For the easy preparation of DNA templates containing x, protect

Full text of DOI:10.1246/cl.2001.914

914
Chemistry Letters 2001
Synthesis of DNA Templates Containing the Fifth Base, 2-Amino-6-(dimethylamino)purine, for
Specific Transcription Involving Unnatural Base Pairs
Ichiro Hirao,*† Takahiko Nojima,† Tsuneo Mitsui,† and Shigeyuki Yokoyama*†,††
†Yokoyama CytoLogic Project, ERATO, JST, c/o RIKEN, Hirosawa, Wako, Saitama 351-0198
††Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo,
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033
(Received June 14, 2001; CL-010563)
An unnatural nucleobase, pyridin-2-one (denoted as y), can
be specifically incorporated into RNA opposite 2-amino-6-
(dimethylamino)purine (denoted as x) in DNA templates by T7
RNA polymerase. For the easy preparation of DNA templates
containing x, protecting groups for the 2-amino group of x were
examined. Although the general N2 protecting groups, such as
isobutyryl and acetyl, resisted removal by the usual treatment
with concentrated ammonia at 55 °C for 6 h, the N2-phenoxy-
acetyl group was successfully removed from the protected
oligomers. This DNA synthesis would be a useful method for
preparation of DNA templates toward enzymatic incorporation
of unnatural nucleosides into RNA at desired positions.
tection of x. Among these protecting groups, the N2-
chloroacetyl group of x was unstable, and the dimethy-
laminomethylene was not efficiently introduced to the 2-amino
group. Here, we show the phenoxyacetyl group as the suitable
N2 protecting group of x and the convenient synthesis of DNA
templates containing x.
The phenoxyacetyl group was successfully introduced to
the 2-amino group of 2-amino-6-(dimethylamino)-9-(β-D-ribo-
furanosyl)purine (1), in which all of the hydroxy groups were
temporarily covered with trimethylsilyl groups, using phenoxy-
acetyl chloride and hydroxybenzotriazole (Figure 2). The
ribonucleoside (2) of N2-phenoxyacetylated x was converted to
the 2'-deoxynucleoside (4)10 by 3',5'-di-O-siloxylation, 2'-
deoxygenation via the 2'-O-imidazolethiocarbonyl derivative
(3), and de-silylation, in a 52% overall yield (Figure 2, from 2
to 4). After 5'-O-dimethoxytritylation of 4, the amidite of x (5)
was obtained by phosphitylation. The amidite 5 was character-
ized by 1H NMR and 31P NMR.11
Expansion of the genetic alphabet would allow nucleic
acids to increase their chemical, physical, and biological
abilities.1,2 We have created a novel base pair, pyridin-2-one
(y) and 2-amino-6-(dimethylamino)purine (x) (Figure 1).3
The ribonucleoside triphosphate of y can be site-specifically
incorporated into RNA opposite x in DNA templates.4 Thus,
this base pair would be useful toward a general bioengineering
method for the site-specific incorporation of modified nucleo-
sides into RNA. To synthesize the DNA templates containing
x, we initially employed an isobutyryl, benzoyl or acetyl group
for the protection of the exocyclic 2-amino residue of the
amidite of x. However, even the N2-isobutyryl, which is gen-
erally used for the N2 protecting group of guanosine in DNA
synthesis, and acetyl groups were too stable for removal from
the oligomers by the usual treatment with concentrated ammo-
nia at 55 °C for 6 h (Figure 3a). The complete deprotection of
these groups required drastic conditions involving treatment
with concentrated ammonia at 80 °C for 10 h. To develop an
easy method for the preparation of DNA templates containing
several x’s with a long chain length (up to 100-mer), we exam-
ined base-labile groups, such as phenoxyacetyl,5–7
chloroacetyl,8 and dimethylaminomethylene,9 for the N2 pro-
Copyright © 2001 The Chemical Society of Japan

Chemistry Letters 2001
915
tion of TxAcT was also confirmed by mass spectrometry. Thus,
in order to avoid the undesirable replacement, the capping
reagent was changed to a solution containing 63 mM 4-t-
butylphenoxyacetic anhydride and 1 M 1-methylimidazole in
THF. Using this capping reagent, the undesirable product was
eliminated from the products (Figure 3e), and TxT was
obtained after ammonia treatment at 55 °C for 6 h (Figure 3f).
In addition, as the capping reagent, chloroacetic anhydride
could be used instead of 4-t-butylphenoxyacetic anhydride (data
not shown), although the chloroacetyl group was unstable for
the N2 protection of x.
Oligonucleotides (35-mer and 109-mer) containing one or
two x’s were synthesized by this method. Further enzymatic
reactions using these DNA templates and modified y bases are
currently in progress.
References and Notes
1
S. A. Benner, P. Burgstaller, T. R. Battersby, and S.
Jurczyk, in “The RNA World,” ed. by R. F. Gesteland, T.
R. Cech, J. F. Atkins, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, New York (1999), p. 163.
E. T. Kool, Curr. Opin. Chem. Biol., 4, 602 (2000).
M. Ishikawa, I. Hirao, and S. Yokoyama, Tetrahedron
Lett., 41, 3931 (2000).
2
3
4
T. Ohtsuki, M. Kimoto, M. Ishikawa, T. Mitsui, I. Hirao,
and S. Yokoyama, Proc. Natl. Acad. Sci. U.S.A., 41, 3931
(2000).
The synthesis of a trinucleotide (TxT) was examined using
an automated DNA synthesizer (PE Biosystems, model 392).
The coupling efficiency of amidite 5 was similar to that of the
commercially available phosphoramidites. After the synthesis,
the protected trimer was treated with ammonia for 1 h at room
temperature for the removal of 2-cyanoethyl groups and the
cleavage from the CPG-support. The solution was analyzed by
reverse phase HPLC, and two peaks appeared corresponding to
the phenoxyacetyl-protected trimer (TxPacT) and TxT, which
was produced by partial deprotection of TxPacT (Figure 3b).
While the N2-phenoxyacetyl group of guanosine was easily
removed under the same conditions, the N2-protecting group of
x in the oligomer was rather stable. Thus, the ammonia solu-
tion was heated at 55 °C for 6 h. By this treatment, the phe-
noxyacetyl group was completely removed and TxT was
obtained (Figure 3c).
It has been suggested that the N2-phenoxyacetyl groups of
guanosine in oligomers are partially replaced by acetyl groups
during the capping step using acetic anhydride in DNA solid-
phase synthesis.5 We thus examined the replacement of the
phenoxyacetyl group of x with the acetyl group by the capping
reagent. After DNA synthesis, the TxPacT-bound CPG was
treated with a capping solution (PE Biosystems’reagents: acetic
anhydride, 1-methylimidazole, and pyridine in THF) for 30 min
at room temperature. After ammonia treatment for 1 h at room
temperature, the products were analyzed by HPLC. Even
though the N2-phenoxyacetyl group of x is much more stable
than that of guanine, a peak corresponding to the acetyl-substi-
tuted product (TxAcT) was observed (Figure 3d). The produc-
5
6
7
8
9
J. C. Schuihof, D. Molko, and R. Téoule, Nucleic Acids
Res., 15, 397 (1987).
C. Chaix, D. Molko, and R. Téoule, Tetrahedron Lett., 30,
71 (1989).
C. Chaix. A. M. Duplaa, D. Molko, and R. Téoule, Nucleic
Acids Res., 17, 7381 (1989).
B. E. Ledford and E. M. Carreira, J. Am. Chem. Soc., 117,
11800 (1995).
J. Zemlicka and A. Holy, Collect. Czech. Chem. Commun.,
32, 3159 (1967).
10 Compound 4: 1H NMR (270 MHz, DMSO-d6) δ 2.26, 2.65
(2H, m, H2',2''), 3.18–3.55 (8H, m, bs, -N(CH3)2, H5',5''),
3.83 (1H, m, H4'), 4.38 (1H, m, H3'), 4.89 (1H, t, 5'-OH,
D2O exchange, J = 5.1, 5.4 Hz), 5.05 (2H, s, -COCH2O-),
5.27 (1H, d, 3'-OH, D2O exchange, J = 3.6 Hz), 6.29 (1H,
t, H1', J = 6.6, 6.8 Hz), 6.93 (3H, m, phenoxy), 7.28 (2H,
m, phenoxy), 8.24 (1H, s, H8), 10.08 (1H, s, -NHCO-, D2O
exchange).
1
11 Compound 5: H NMR (270 MHz, CDCl3) δ 1.08–1.19
(12H, m, 2Me2CH), 2.41–2.75 (4H, m, -OCH2CH2CN,
H2',2''), 3.32–3.88 (12H, m, -N(CH3)2, -OCH2CH2CN,
2Me2CH, H5',5''), 3.77 (6H, s, 2CH3O-), 4.26 (1H, m, H4'),
4.70 (1H, m, H3'), 4.87 (2H, s, -COCH2O-), 6.40 (1H, t,
H1', J = 6.3, 6.4 Hz), 6.78, 7.01 (7H, m, phenoxy, DMT-),
7.21–7.41 (11H, m, DMT-), 7.83 (1H, s, s, H8); 31P NMR
(109 MHz, CDCl3) δ 149.38, 149.53 (diastereoisomers).