Synthesis of 3-(2-deoxy-β-D-ribofuranosyl)pyridin-2-one and 2-amino-6- (N,N-dimethylamino)-9-(2-deoxy-β-D-ribofuranosyl)purine derivatives for an unnatural base pair

Article abstract of DOI:10.1016/S0040-4039(00)00520-7

An unnatural base pair, 2-amino-6-(N,N-dimethylamino)purine (denoted x) and pyridin-2-one (denoted y), was designed to prove the structural requirements for base pair formation involving shape complementarity. It was expected that y might satisfy the stru

Full text of DOI:10.1016/S0040-4039(00)00520-7

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 41 (2000) 3931–3934
Synthesis of 3-(2-deoxy-β-D-ribofuranosyl)pyridin-2-one and
2-amino-6-(N,N-dimethylamino)-9-(2-deoxy-β-D-
ribofuranosyl)purine derivatives for an unnatural base pair
Masahide Ishikawa,a Ichiro Hirao a and Shigeyuki Yokoyama a,b,∗
aYokoyama CytoLogic Project, ERATO, JST, c/o The RIKEN Institute, 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
Received 1 March 2000; revised 22 March 2000; accepted 24 March 2000
Abstract
An unnatural base pair, 2-amino-6-(N,N-dimethylamino)purine (denoted x) and pyridin-2-one (denoted y), was
designed to prove the structural requirements for base pair formation involving shape complementarity. It was
expected that y might satisfy the structural requirements for pairing with x, in which the bulky 6-dimethylamino
group may eliminate base pairing with the natural bases. As chemical or biological substrates for DNA synthesis,
the phosphoramidite of x and the 20-deoxy-C3-ribonucleoside triphosphate of y (dyTP) were synthesized, and the
incorporation experiment was demonstrated by using the Klenow fragment of Escherichia coli DNA polymerase
I. © 2000 Elsevier Science Ltd. All rights reserved.
DNA replication is a highly accurate biological event catalyzed by DNA polymerases. To unders-
tand the mechanism of the fidelity, enzymatic incorporation experiments using synthetic unnatural
nucleotides have been employed.1–4 Recently, Kool and co-workers have created non-hydrogen bonded
base pairs, such as difluorotoluene:benzimidazole5–7 and pyrene:abasic site.8 These non-polar bases
can be inserted by a 30-exonuclease-defective Klenow fragment (exo−) opposite bases that have shape
complementarity.5,9 This approach demonstrates the importance of steric matching for specific base pair
formation in replication.3,10,11
On the basis of the steric matching for base pairing, we designed 2-amino-6-(N,N-
dimethylamino)purine (x) as a purine analog and pyridin-2-one (y) as a pyrimidine analog [Fig.
1(a)]. Through enzymatic incorporation experiments for this base pair, which has different shape
complementarity from those of the natural base pairs, the selectivity of the base pairing and the
importance of the steric matching were assessed. These bases would be expected to form the x:y
pair favorably and to avoid non-cognate pairs with natural bases, due to the steric hindrance of the
∗
Corresponding author. Tel: +81 3 5841 4413; fax: +81 3 5841 8057; e-mail: yokoyama@y-sun.biochem.s.u-tokyo.ac.jp (S.
Yokoyama)
0040-4039/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)00520-7
tetl 16799

3932
6-dimethylamino group of x. The formations of x:T or x:C pairs may have disadvantages since the bulky
6-dimethylamino group of x clashes with the 4-keto group of T [Fig. 1(b)] and the 4-amino group of C,
but not with the 6-hydrogen of y. We prepared the 20-deoxy-C3-ribonucleoside triphosphate of y (dyTP)
and DNA templates containing x, and examined the enzymatic incorporation of dyTP into DNA using
the Klenow fragment.
Fig. 1. Cognate and non-cognate base pairs involving unnatural x and y bases
The triphosphate dyTP was synthesized from the corresponding ribonucleosides. Syntheses of both
the ribonucleoside12 and the 20-deoxyribonucleoside13,14 of y have been reported. To prepare both
dyTP and the ribonucleoside triphosphate, ryTP, for replication and transcription experiments, we first
synthesized the ribonucleoside of y, and then converted it into the 20-deoxyribonucleoside. 3-(2-Deoxy-
β-D-ribofuranosyl)-pyridin-2-one was obtained in an overall yield of 13% from the ribonucleoside. The
deoxynucleoside was converted to dyTP15 with phosphorus oxychloride and pyrophosphate. The molar
absorption coefficient of dyTP (7.6×103 at 298 nm) at pH 7.0 was determined by a quantitative analysis
of the phosphorus in the compound. No degradation product of dyTP was observed by HPLC analysis
after standing at room temperature for seven days in a buffer containing 10 mM tris-HCl, pH 7.5, 7 mM
MgCl2, and 0.1 mM DTT. However, heating dyTP at 95°C in the solution gradually yielded an unknown
decomposition product.
The synthesis of the protected 20-deoxynucleoside phosphoramidites (6a and 6b) of x is illustrated in
Scheme 1. Throughout the synthesis, the 2-amino group of the base moiety was protected with a benzoyl
or an acetyl group. The substitutions of the 6-chloro groups of 1a and 1b with dimethylamino groups was
achieved with dimethylamine hydrochloride in 65–75% yields. This approach is more convenient than the
previously reported method using trimethylamine.16 The N2-protected ribonucleosides (3a and 3b) were
converted to 20-deoxyribonucleosides (5a and 5b) via the corresponding 30,50-O-disiloxane derivatives,
followed by dimethoxytritylation and phosphitylation to give the phosphoramidite derivatives.17,18
Before preparing the templates, the trinucleotide [d(TxT)] synthesis was examined by using the x-
amidites, 6a and 6b. The coupling efficiencies of both amidites were similar to those of the commercially
available phosphoramidites. After ammonia treatment for removing the protecting groups and releasing
the trimer from the CPG-resin, the products were analyzed by reverse-phase HPLC and mass spectrome-
try. The deprotection with concentrated ammonia at 60°C for 6 h gave two major products, corresponding
to d(TxT) and the remaining N2-acyl-protected trimers. Due to the incomplete removal of the N2-acetyl
group of x, deprotection with ammonia was carried out at 80°C for 10 h. Both the acetyl and benzoyl
protecting groups were then removed completely, and d(TxT) was obtained without any decomposition
of the trimer. Thus, these deprotection conditions were used to prepare the templates containing x.
We examined the selectivity of the single-nucleotide insertion of dyTP opposite x by the Klenow
fragment (exo+), using a primer-template duplex in which x was adjacent to the 30 end of the primer
(Fig. 2). The insertion products were analyzed by gel electrophoresis after an incubation at 17°C for 30
min with each dN0TP (where N0=A, G, C, T, or y), 50-labeled primer (15-mer), templates containing x

3933
Scheme 1. Synthesis of the amidites of x
or natural bases at a specific position, and the Klenow fragment. As compared with the natural dN0TPs
(where N0=A, G, C, or T), only dyTP was selectively incorporated opposite x in the template (N=x),
and the insertion product was observed as a band corresponding to a 16-mer on the gel (Fig. 2, lanes
7–11). The C-nucleotide, dyTP, was also incorporated opposite A and G (Fig. 2, lanes 3 and 4); the
unnatural base y is a shape analog of T and pairs with A, and wobble pairing between y and G may lead
to the incorporation. However, in the presence of dTTP and dCTP, the insertion of dyTP opposite A or
G was completely prevented (data not shown). Thus, dyTP was selectively incorporated opposite x by
the Klenow fragment (exo+). In the other insertion experiments using the Klenow fragment (exo−), the
incorporation of dTTP opposite x was observed (data not shown). Thus, the 30-exonuclease activity of
the polymerase may be involved in the specificity of the unnatural base pair formation.
Fig. 2. Single-nucleotide insertion of dyTP opposite bases containing x in templates. The insertion was carried out at 17°C for
30 min using the Klenow fragment (2 units, 250 nM), 2 µM template, 1 µM 50-32P-labeled primer, and 0.15 mM dN0TP
We show here that the unnatural x:y pair fits into the shape complementarity theory and non-cognate
base pair formations can be eliminated by introducing steric hindered groups into unnatural bases. To test
the selectivity of the x:y pair formation in replication and transcription by DNA and RNA polymerases,
more precise kinetic and thermodynamic analyses are in progress.

3934
References
1. Verma, S.; Eckstein, F. Annu. Rev. Biochem. 1998, 67, 99–134.
2. Goodman, M. F.; Fygenson, D. K. Genetics 1998, 248, 1475–1482.
3. Kool, E. T. Biopolymers 1998, 48, 3–17.
4. Goodman, M. F.; Creighton, S.; Bloom, L. B.; Petruska, J. Crit. Rev. Biochem. Mol. Biol. 1993, 28, 83–126.
5. Morales, J. C.; Kool, E. T. Nat. Struct. Biol. 1998, 5, 950–954.
6. Moran, S.; Ren, R. X.-F.; Rummey IV, S.; Kool, E. T. J. Am. Chem. Soc. 1997, 119, 2056–2057.
7. Liu, D.; Moran, S.; Kool, E. T. Chem. Biol. 1997, 4, 919–926.
8. Matray, T. J.; Kool, E. T. Nature 1999, 399, 704–708.
9. Morales, J. C.; Kool., E. T. J. Am. Chem. Soc. 1999, 121, 2323–2324.
10. Guckian, K. M.; Krugh, T. R.; Kool, E. T. Nat. Struct. Biol. 1998, 5, 954–959.
11. Barsky, D.; Kool, E. T.; Colvin, M. E. J. Biomol. Struct. Dyn. 1999, 16, 1119–1134.
12. Matulic-Adamic, J.; Beigelman, L. Tetrahedron Lett. 1997, 38, 203–206.
13. Solomon, M. S.; Hopkins, P. B. Tetrahedron Lett. 1991, 32, 3297–3300.
14. Solomon, M. S.; Hopkins, P. B. J. Org. Chem. 1993, 58, 2232–2243.
1
15. H NMR (270.16 MHz, D2O) of dyTP; δ: 7.83 (d, 1H, H4, J=4.9 Hz), 7.35 (d, 1H, H6, J=4.9 Hz), 6.51 (t, 1H, H5, J=4.9
Hz), 5.17 (t, 1H, H10, J=5.0 Hz), 4.56 (br, 1H, H30), 4.06 (br, 1H, H40), 3.99 (br, 2H, H50 and H500), 2.19–2.33 (m, 1H,
H20), 1.81–1.98 (m, 1H, H200). 31P NMR (109.36 MHz, D2O) δ: −10.3 (m, 2P, P1, P3), −22.7 (m, 1P, P2); UV (10 mM
phosphate buffer pH 7.0): λmax=298 nm (ε=7.6×103), 226 nm (ε=7.0×103), λmin=247 nm, 211 nm; MS (ESI−): (M−H−)
449.9.
16. Robins, M. J.; Uznañski, B. Can. J. Chem. 1981, 59, 2601–2607.
1
17. H NMR (270.16 MHz, CDCl3) of 6a; δ: 8.14, 8.13 (s, 1H, H8, diastereomer), 7.72 (s, 1H, BzNH), 7.65–7.70 (m, 2H,
Bz-m), 7.16–7.47 (m, 12H, Bz-p,o, DMTr), 6.70–6.75 (m, 4H, DMTr), 6.27–6.41 (m, 1H, H10), 4.63–4.80 (m, 1H, H30),
4.20–4.27 (m, 1H, H40), 3.74 (s, 6H, OCH3), 3.24–3.72 (m, 10H, H50, H500, NCH (CH3)2, N-CH3), 2.83–3.00 (m, 1H,
H20), 2.40–2.64 (m, 5H, H200, OCH2CH2CN), 1.06–1.19 (m, 12H, NCH (CH3)2); 31P NMR (109.36 MHz, CDCl3) δ:
149.25.
1
18. H NMR (270.16 MHz, CDCl3) of 6b; δ: 7.81, 7.78 (s, 1H, H8, diastereomer), 7.70 (s, 1H, AcNH), 7.18–7.41 (m, 9H,
DMTr), 6.75–6.81 (m, 4H, DMTr), 6.33 (t, 1H, H10, J=6.5 Hz), 4.63–4.70 (m, 1H, H30), 4.27–4.31 (m, 1H, H40), 3.78
(s, 6H, OCH3), 3.30–3.70 (m, 10H, H50, H500, NCH (CH3) 2, N-CH3), 2.72–2.80 (m, 1H, H20), 2.34–2.63 (m, 5H, H200,
OCH2CH2CN), 1.09–1.20 (m, 12H, NCH (CH3) 2); 31P NMR (109.36 MHz, CDCl3) δ: 149.28, 149.61.