Synthesis of 6-(2-thienyl)purine nucleoside derivatives that form unnatural base pairs with pyridin-2-one nucleosides

Article abstract of DOI:10.1016/S0960-894X(01)00415-2

Unnatural bases, 2-amino-6-(2-thienyl)purine and 2-amino-6-(2-furanyl)purine, were newly designed to replace the previously developed purine analogue, 2-amino-6-(N,N-dimethylamino)purine, which specifically pairs with pyridin-2-one. These nucleoside derivatives were synthesized via the 6-substitution of 6-iodopurine nucleosides with tributylstannylthiophene or tributylstannylfuran. As compared with 2-amino-6-(N,N-dimethylamino)purine, 2-amino-6-(2-thienyl)purine reduced the interference in the stacking interactions with the neighboring bases in a DNA duplex and improved the efficiency of the enzymatic incorporation of the nucleoside triphosphate of pyridin-2-one opposite the unnatural base.

Full text of DOI:10.1016/S0960-894X(01)00415-2

Bioorganic & Medicinal Chemistry Letters 11 (2001) 2221–2223
Synthesis of 6-(2-Thienyl)purine Nucleoside Derivatives That
Form Unnatural Base Pairs with Pyridin-2-one Nucleosides
Tsuyoshi Fujiwara,a Michiko Kimoto,b,c Hiroshi Sugiyama,d Ichiro Hiraoa,* and
Shigeyuki Yokoyamaa,b,c,*
aYokoyama CytoLogic Project, ERATO, JST, c/o RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan
bDepartment of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,
Tokyo 113-0033, Japan
cCellular Signaling Laboratory, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan
dDivision of Biofunctional Molecules, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University,
2-3-10 Kanda, Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan
Received 1 May 2001; accepted 13 June 2001
Abstract—Unnatural bases, 2-amino-6-(2-thienyl)purine and 2-amino-6-(2-furanyl)purine, were newly designed to replace the pre-
viously developed purine analogue, 2-amino-6-(N,N-dimethylamino)purine, which specifically pairs with pyridin-2-one. These
nucleoside derivatives were synthesized via the 6-substitution of 6-iodopurine nucleosides with tributylstannylthiophene or tribu-
tylstannylfuran. As compared with 2-amino-6-(N,N-dimethylamino)purine, 2-amino-6-(2-thienyl)purine reduced the interference in
the stacking interactions with the neighboring bases in a DNA duplex and improved the efficiency of the enzymatic incorporation of
the nucleoside triphosphate of pyridin-2-one opposite the unnatural base. # 2001Elsevier Science Ltd. All rights reserved.
The development of unnatural nucleobase pairs would
increase nucleic acid functionality and eventually lead to
an expansion of the genetic alphabet.1ꢀ4 We previously
designed an unnatural base pair, 2-amino-6-(N,N-dime-
thylamino)purine (designated as x) and pyridin-2-one
(designated as y) (Fig. 1a)5,6 on the basis of the concepts
of both the hydrogen-bond donor–acceptor pattern and
the shape complementarity. The bulky 6-dimethylamino
group of x efficiently prevents the non-cognate base
pairing, such as x–T (or x–U) (Fig. 1b) and x–C, and
the ribonucleoside triphosphate of y can be specifically
incorporated into RNA opposite x of a DNA template
by T7 RNA polymerase.6 This transcription enables the
synthesis of functional RNAs containing unnatural
nucleosides at desired positions6 and facilitates the
expansion of the genetic code for the site-specific incor-
poration of unnatural amino acids into proteins. How-
ever, the dimethylamino group interferes with the
stacking interactions between the unnatural and the
neighboring base pairs in a DNA duplex. Accordingly,
further applications, especially replication, of the x–y
pair have remained restricted.
To reduce the steric hindrance between the unnatural
and the neighboring base pairs, we replaced the 6-
dimethylamino group of x with heterocycles, such as
thiophene and furan (Fig. 1c). These heterocycles might
function as bulky groups on the pairing surface as well
as the 6-dimethylamino group of x (Fig. 1d). In addi-
tion, these heterocycles may increase the stacking stabi-
lity with the neighboring base pairs in DNA duplexes as
compared to the dimethylamino group of x, although
there is a possibility that the heterocycle moieties devi-
ate from the surface of the purine base. We synthesized
these unnatural nucleosides and examined the thermal
stability of the DNA duplexes and the incorporation
into DNA involving the unnatural base pair between 2-
amino-6-(2-thienyl)purine (1a) and y.
We first examined the photochemical synthesis of the 6-
(2-thienyl)purine 20-deoxyribonucleosides according to
the literature.7 Photoarylation was carried out with 2-
isobutyrylamino-6-iodo-9-(2-deoxy-3,5-di-O-isobutyryl-
b-d-ribofuranosyl)purine (2)8,9 in nitrogen-purged thio-
phene (Scheme 1, Method A). After irradiation for 24 h,
*Corresponding authors. Tel.: +81-48-468-7187; fax: +81-48-
468-4779;
e-mail: hirao@cytologic.riken.go.jp. Tel.: +81-3-5841-
4413; fax: +81-3-5841-8057; e-mail: yokoyama@y-sun.biochem.s.u-
tokyo.ac.jp
0960-894X/01/$ - see front matter # 2001Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00415-2

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T. Fujiwara et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2221–2223
N2-isobutyrylamino-6-(2-thienyl)-9-(2-deoxy-3,5-di-O-
isobutyryl-b-d-ribofuranosyl)purine (3a) was obtained
in a 78% yield. After deprotection of the isobutyryl
groups of 3a [1M NaOH in pyridine/MeOH/H 2O
(17:6:1) for 4 h at rt], the product was purified by C18-
HPLC. However, the 1H NMR spectrum indicated
contamination with a by-product, which was deter-
mined to be 2-amino-6-(2,20-bithienyl)-9-(2-deoxy-3,5-
di-O-acetyl-b-d-ribofuranosyl)purine (5) (see Scheme 1);
the ratio of 20-deoxyribonucleosides of 4a to 5 was 9:1
(Fig. 2, upper chart). The by-product may result from
the addition of thiophene to the radical intermediate of
3a during irradiation.
bis(triphenylphosphine)palladium(II) chloride (Scheme
1, Method B).10 By this coupling in DMF for 2 h at
100 ꢁC, 3a11 was obtained in a 94% yield, and no by-
product was observed in the products after deprotection
and HPLC purification (Fig. 2, lower chart). In addi-
tion, the 20-deoxyribonucleoside of 1b (3b) was synth-
esized by using tributylstannylfuran in a 98% yield.
Similarly, the corresponding nucleosides without the 2-
amino group, 6-(2-thienyl)-9-(2-deoxy-3,5-di-O-acetyl-
b-d-ribofuranosyl)purine and 6-(2-furanyl)-9-(2-deoxy-
3,5-di-O-acetyl-b-d-ribofuranosyl)purine, were also
synthesized from 6-iodo-9-(2-deoxy-3,5-di-O-acetyl-b-d-
ribofuranosyl)purine in 79 and 87% yields, respectively
(Scheme 1).
Then, we examined the synthesis of 3a by the palladium
catalyzed coupling of 2 with tributylstannylthiophene and
The 20-deoxyribonucleoside of 2-amino-6-(2-thi-
enyl)purine (1a) was converted to the corresponding
amidite (8) for DNA synthesis (Scheme 1). The furanyl
derivatives gradually decomposed under the basic con-
ditions used in DNA synthesis; nucleoside 4d decom-
posed in a concentrated ammonia solution at room
temperature. Thus, we synthesized DNA fragments
containing 1a. For the protection of the 2-amino group
of 1a during DNA synthesis, a phenoxycarbonyl
group12 was used, because the isobutyryl group was too
stable as the protecting group. The deprotection of
fully-protected DNA fragments was carried out by a
conventional mꢁethod (concentrated NH4OH at rt for
1h and then 55 C for 6 h).
To assess the potential of the unnatural 1a–y pair, we
examined the thermal stability of the DNA duplex and
the single-nucleotide insertion13 by the exonuclease-
deficient Klenow fragment of Escherichia coli DNA
polymerase I. As expected, the thermal denaturation
experiments showed the increased stability of the DNA
duplex (12-mer, 50-CGCAT1aGTTACC-30/50-GGTAA-
Figure 1. Unnatural and non-cognate base pairs. Compound 1a: X=S
and R=NH2, 1b: X=O and R=NH2, 1c: X=S and R=H, and 1d:
X=O and R=H.
Scheme 1.

T. Fujiwara et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2221–2223
2223
opposite 1a of the template was also increased and was
higher than those of the non-cognate pairing with nat-
ural substrates. On the other hand, the incorporation of
y opposite x was less efficient than the incorporation of
T opposite x. Thus, our results show that the improve-
ment of the stacking stability of the DNA duplex con-
taining the unnatural base pair concurrently increased
the incorporation efficiency of the unnatural base pair-
ing. Although the incorporation specificity of the 1a–y
pairing is still insufficient for practical use in replication,
the transcription specificity of the 1a–y pairing was
improved as compared to that of the x–y pairing (data
not shown). Transcription–translation experiments
for the incorporation of unnatural amino acids into
proteins using the novel codon–anticodon interaction
involving the 1a–y pair are in progress.
Figure 2. 1H NMR spectrum (DMSO-d6) of 4a synthesized by method
A (upper) and method B (lower). The integration of the aromatic
region is shown. The peaks assigned to by-product 5 are indicated by
the arrows in the upper chart.
References and Notes
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 Har-
bor, New York, 1999; pp 163–181.
2. Piccirilli, J. A.; Krauch, T.; Moroney, S. E.; Benner, S. A.
Nature 1990, 343, 33.
Table 1. Steady-state kinetic parameters for insertion of single
nucleotides into a template-primer duplex by the exonuclease-deficient
Klenow fragment
3. Kool, E. T.; Morales, J. C.; Guckian, K. M. Angew. Chem.,
Int. Ed. 2000, 39, 990.
4. Wu, Y.; Ogawa, A. K.; Berger, M.; McMinn, D. L.;
Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc. 2000, 122,
7621.
5. Ishikawa, M.; Hirao, I.; Yokoyama, S. Tetrahedron Lett.
2000, 41, 3931.
Template
base (N)
Nucleotide
triphosphate (N0)
KM
(mM)
Vmax
(% minꢀ1
Efficiency
(Vmax/KM)
)
6. Ohtsuki, T.; Kimoto, M.; Ishikawa, M.; Mitsui, T.; Hirao,
I.; Yokoyama, S. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 4922.
7. Nair, V.; Richardson, S. G.; Coffmann, R. E. J. Org. Chem.
1982, 47, 4520.
8. Gaffney, B. L.; Markey, L. A.; Jones, R. A. Tetrahedron
1984, 40, 3.
9. Nair, V.; Richardson, S. G. J. Org. Chem. 1980, 45, 3969.
10. Gundersen, L.-L.; Bakkestuen, A. K.; Aasen, A. J.;
Overas, H.; Rise, F. Tetrahedron 1994, 50, 9743.
11. Compound 4a: 1H NMR (270 MHz, DMSO-d6): d 2.24
(ddd, 1H, J=13, 6.1, 3.1 Hz), 2.64 (ddd, 1H, J=13, 7.6,
6.4 Hz), 3.39 (m, 2H), 3.84 (m, 1H), 4.38 (m, 1H), 4.98 (t, 1H,
J=5.4 Hz), 5.30 (d, 1H, J=4.0 Hz), 6.27 (dd, 1H, J=7.6,
6.1Hz), 6.56 (bs, 2H), 7.26 (dd, 1H, J=5.0, 3.7 Hz), 7.80 (dd,
1H, J=5.0, 1.2 Hz), 8.35 (s, 1H), 8.52 (dd, 1H, J=3.7, 1.2 Hz).
Electron spray mass spectrum: M+1(positive)=333.90
(found), 334.099 (calcd), M+HCOOꢀ (negative)=377.76
(found), 378.089 (calcd).
y
1a
1a
1a
1a
1a
x
x
x
x
x
130 (30)
270 (70)
1000 (100)
n.d.
150 (40)
440 (210)
550 (160)
1700 (200)
110 (40)
220 (50)
1.2 (0.1)
9.2ꢂ103
2.4ꢂ103
3.8ꢂ103
2.2ꢂ103
2.7ꢂ103
4.9ꢂ103
6.5ꢂ102
6.4ꢂ102
1.5ꢂ103
T
C
G
A
y
T
C
G
A
0.64 (0.13)
3.8 (0.6)
n.d.
0.33 (0.04)
1.2 (0.3)
2.7 (0.4)
1.1 (0.1)
0.07 (0.01)
0.33 (0.05)
Assay was carried out at 37 ꢁC for 7–20 min using 5 mM template–pri-
mer duplex, 20 or 50 nM enzyme, and 0.03–3 mM dNTP in a solution
(10 mL) containing 50 mM Tris–HCl (pH 7.5), 10 mM MgCl2, 1 mM
DTT, and 0.05 mg/mL bovine serum albmin.
n.d. No inserted products were detected after incubation for 20 min
with 3 mM dNTP. Parenthetic values are standard deviations.
CyATGCG-30) containing the 1a–y pair (Tm=43.4 ꢁC)
as compared with that containing the x–y pair
(Tm=39.5 ꢁC). As shown in Table 1, the enzymatic
incorporation efficiency of triphosphate y into DNA
´
12. Chaix, C.; Molko, D.; Teoule, R. Tetrahedron Lett. 1989,
30, 71 .
13. Boosalis, M. S.; Petruska, J.; Goodman, M. F. J. Biol.
Chem. 1987, 262, 14689.