A new strategy for the synthesis of oligodeoxynucleotides directed towards perfect O-selective internucleotidic bond formation without base protection

Article abstract of DOI:10.1016/j.tetlet.2003.10.156

Deoxyadenosine and deoxycytidine have nucleophilic amino groups so that the undesired N-phosphitylation of these amino groups occurred in the previous phosphoramidite methods without base protection. We report that the N-phosphitylation could be considerably suppressed in our new HOBt-mediated coupling strategy via phosphite intermediates as reactive species. Thus, 99.7-99.9% O-selective internucleotidic bond formation was achieved.

Full text of DOI:10.1016/j.tetlet.2003.10.156

Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 363–366
A new strategy for the synthesis of oligodeoxynucleotides
directed towards perfect O-selective internucleotidic bond
formation without base protection
a,c b,c
Akihiro Ohkubo, Kohji Seio and Mitsuo Sekine
a,c,
*
a
Department of Life Science, Tokyo Institute of Technology, Nagatsuta, Mirdoriku, Yokohama 226-8501, Japan
b
Division of Collaborative Research for Bioscience and Biotechnology, Frontier Collaborative Research Center,
Nagatsuta, Mirdoriku, Yokohama 226-8501, Japan
CREST, JST (Japan Science and Technology Corporation), Nagatsuta, Mirdoriku, Yokohama 226-8501, Japan
c
Received 17 September 2003;revised 22 October 2003;accepted 24 October 2003
Abstract—Deoxyadenosine and deoxycytidine have nucleophilic amino groups so that the undesired N-phosphitylation of these
amino groups occurred in the previous phosphoramidite methods without base protection. We report that the N-phosphitylation
could be considerably suppressed in our new HOBt-mediated coupling strategy via phosphite intermediates as reactive species. Thus,
9
9.7–99.9% O-selective internucleotidic bond formation was achieved.
Ó 2003 Published by Elsevier Ltd.
The chemical synthesis of oligodeoxynucleotides has
contributed to biological and structural studies of
nucleic acids. Recently, much attention has been paid
on the base residues at every coupling step. In addition,
we have quite recently reported that the latter method
7a;8
was not generally applicable.
1
to the development of more straightforward methods
for the synthesis of DNA fragments in the post-genome
days. Particularly, our interest has been focused on a
synthetic strategy without base protection, since the
procedure prescribed for protection and deprotection of
In an attempt to eliminate this P–N bond cleavage step,
we have recently developed a new phosphoramidite
approach called Ôproton-block methodÕ based on the
7
hitherto simplest principle of acid–base reactions.
2–5
the N-protecting groups can be eliminated.
However, the yield of the product was low in the case
of longer oligodeoxynucleotides because byproducts
exponentially increased. As a matter of fact, completely
O-selective phosphitylation has not yet been achieved in
the phosphoramidite method to date.
6
Letsinger first proposed the phosphoramidite method
without nucleobase protection by use of pyridinium
3
chloride as a promoter in 1991. Later, Hayakawa
changed this hygroscopic reagent to nonhygroscopic
imidazolinium triflate (IMT) and succeeded in syn-
On the other hand, we previously reported O-selective
internucleotidic bond formation by the H-phosphonate
5
thesizing oligodeoxyribonucleotides. However, these
precedents require an additional post-treatment with
9
method. In this method, we observed that phosphite
3
pyridinium hydrochloride/aniline or benzimidazolium
5
intermediates generated from H-phosphonate building
blocks reacted scarcely with the exocyclic amino groups
of the base residues. This high O-selectivity was
explained in terms of two kinds of HOMO–LUMO
zinteractions between the activated phosphite species
triflate (BIT)/MeOH for decomposition of undesired
N-phosphitylated side chains containing P(III)–N bonds
0
and the 5 -hydroxyl function. Based on these previous
results, we considered that, if phosphite-type intermedi-
ates could be generated from deoxynucleoside phos-
phoramidite derivatives by an alcohol-type activator,
they would not react with the amino groups.
Keywords: Chemical synthesis of DNA;HOBt; O-Selective phosphor-
ylation;Activator;Phosphoramidite approach without base protection.
*
Corresponding author. Tel.: +81-45-924-5706;fax: +81-45-924-5772;
e-mail: msekine@bio.titech.ac.jp
0
040-4039/$ - see front matter Ó 2003 Published by Elsevier Ltd.
doi:10.1016/j.tetlet.2003.10.156

364
A. Ohkubo et al. / Tetrahedron Letters 45 (2004) 363–366
Figure 1. The anion-exchange HPLC profiles of the crude mixtures from synthesis with unprotected nucleobases. (a) d[ApT], IMT;(b) d[CpT], IMT;
c) d[ApT], HOBt;(d) d[CpT], HOBt;(e) d[GpT], HOBt;(f) d[TpT], HOBt.
(
Therefore, we extensively searched for suitable activa-
tors to realize this idea. In this paper, we report that
acetic anhydride, which would react readily with the
amino groups of oligodeoxynucleotides. Thus, two
consecutive couplings at each cycle were employed,
and this operation was superior to the one-coupling
mode.
10
1
most effective as an activator in the N-unprotected
-hydroxybenzotriazole [HOBt (pK
a
¼ 5:3)] was the
phosphoramidite approach.
To check the O/N-selectivity of internucleotidic bond
formation of the previously reported methods, several
experiments were conducted. When IMT, previously
reported as the best reagent for the O-selective phos-
The result of the synthesis of d[ApT] is shown in Figure
1c and Table 1. There was almost no peak correspond-
ing to the N-phosphorylated product at around
18.5 min. Hundred-fold expansion of the HPLC chart
exhibited the remarkably high selectivity (the ratio of O/
N-phosphorylation in this condensation) that was esti-
mated to be 99.7:0.3. The condensation in the synthesis
of d[CpT] using HOBt was also almost perfectly O-se-
lective (99.9%), as shown in Figure 1d. In the case of
d[GpT] and d[TpT], similarly, no N-phosphorylated
byproducts were detected at all, as shown in Figure 1e
and f. Thus, these products, d[ApT], d[CpT], d[GpT]
and d[TpT], were isolated in 92%, 94%, 90% and 92%
yields, respectively.
5a
phitylation, was used in the solid-phase synthesis of
d[ApT] and d[CpT] on highly cross-linked polystyrene
HCP) resins using a manual operation, the N-phos-
11
(
phorylated derivatives of deoxyadenosine and deoxy-
cytidine were considerably formed in 23% and 17%
yields, respectively, as shown in Figure 1a and b.
To examine the side reaction of the base parts in our
HOBt-mediated internucleotidic bond formation on
HCP, d[ApT], d[CpT], d[GpT] and d[TpT] were syn-
thesized according to Figure 2.
Furthermore, the condensation of d[Ap(ce)T]-HCP,
d[Cp(ce)T]-HCP and d[Gp(ce)T]-HCP, which were
prepared by the same method as described in Figure 2,
The efficiency of the condensation should be increased
as much as possible to avoid the capping reaction using
10 equiv
B
N
N
DMTrO
1
O
O
N
N
NH
O
O
2
P
OH
N
HO
O
CEO
20 equiv
3% CCl COOH
0.1M I
2
3
O
Py - H O ( 9:1 v/v )
2 2
CH Cl
r.t., 1 min
CH
3
CN - NMP ( 9:1 v/v )
r.t., 1 min
2
r.t., 2 min
HCP
n
highly cross-linked
polystyrene resin
NMP = N-Methyl-2-pyrrolidone
NH NH
conc NH
3
2
2
NH
2
oligodeoxynucleotides
B
= Th, Ad, Cy, Gu,
r.t., 40 min
a
b
c
d
Figure 2. Solid-phase synthesis of oligodeoxynucleotides in the HOBt approach.

A. Ohkubo et al. / Tetrahedron Letters 45 (2004) 363–366
Table 1. The O-selectivity of condensation in the HOBt approach
365
Amidite
Polymer
Final product
O-Selectivity (%)
HOBt
IMT
tf-HOBt
1
1
1
1
1
1
b
d[T]-HCP
d[T]-HCP
d[T]-HCP
d[ApT]
d[CpT]
d[GpT]
77.0
82.9
99.7
99.9
99.3
98.9
c
d
a
a
a
>99.9
90.5
9.7
>99.9
>99.9
>99.9
>99.9
>99.9
99.1
98.7
d[Ap(ce)T]-HCP
d[Cp(ce)T]-HCP
d[Gp(ce)T]-HCP
d[TpApT]
d[TpCpT]
d[TpGpT]
>99.9
>99.9
with 1a was carried out in the presence of HOBt. These
results are summarized in Table 1. The amounts of these
N-phosphorylated species became closer to the noise
level as shown in the expanded HPLC spectra of Figure
Figure 3f and g. These results indicate that N-phos-
phorylation was greatly suppressed even in the consec-
utive sequence of dA or dC. In these cases, the coupling
efficiency was ca. 99% (trityl cation assay). Thus, d[A T]
6
3
c–e so that the O-selectivity in these cases was calcu-
and d[C
spectively. As a dG-rich sequence, we chose d[GGT]
having the same number of dG as the above 7-mers,
since sequences such as d[G T] having consecutive dGs
6
T] were isolated in 48% and 67% yields, re-
lated to be more than 99.9%. Thus, d[TpApT],
d[TpCpT] and d[TpGpT] were isolated in 89%, 83% and
3
8
4% yields, respectively. When IMT was used as the
6
activator, considerable side reactions were detected, as
shown in Figure 3a and b and Table 1. Particularly, the
reaction of d[Cp(ce)T]-HCP with 1a gave extremely
poor selectivity of 9.7%. Surprisingly, the N-phosphory-
lated product was formed as the major product, as
shown in Figure 3b. On the other hand, the use of a
more acidic HOBt derivative, 1-hydroxy-6-(trifluoro-
tend to aggregate giving complex HPLC patterns. In this
case, the average coupling yield was 98.1% so that the
desired 9-mer was formed as the predominant peak with
small amounts of the n ꢀ 1-mers (Fig. 3h). Conse-
quently, d[GGT] was isolated in 48% yield. In a similar
3
manner, a DNA 12-mer d[CAGT] could be isolated in
3
36% yield (Fig. 3i). The n ꢀ 1-mers observed in Figure 3i
methyl)benzotriazole (tf-HOBt, pK
a slight decrease of the O-selectivity compared with
a
¼ 3:94), resulted in
is due mainly to the somewhat lower coupling efficiency
at the dG positions. In the P NMR spectrum of crude
d[CAGT] , there were no significant resonance signals of
31
HOBt, as shown in Table 1.
3
N-phosphorylated byproducts. These isolated oligomers
were well characterized by enzyme digestion and
MALDI-TOF mass spectroscopy.
In the synthesis of d[A T] and d[C T], the desired
6
products were obtained as the main peaks, as shown in
6
Figure 3. The anion-exchange HPLC profiles of the crude mixtures obtained by the solid-phase synthesis. (a) d[TAT], IMT;(b) d[TCT], IMT;
c) d[TAT], HOBt;(d) d[TCT], HOBt;(e) d[TGT], HOBt;(f) d[A T], HOBt;(g) d[C T], HOBt;(h) d[GGT] , HOBt;(i) d[CAGT] , HOBt.
(
6
6
3
3

366
A. Ohkubo et al. / Tetrahedron Letters 45 (2004) 363–366
In conclusion, we have realized the hitherto highest O-
selective phosphorylation and synthesizing oligodeoxy-
ribonucleotides in satisfactory yields without using base
amino protecting groups, capping reaction and post-
treatment for the P–N bond cleavage after condensa-
tion. This new strategy would provide new insight into
the synthesis of base-sensitive modified oligonucleotides
and the high-throughput synthesis of DNA fragments
on DNA chips. Further studies are now under way.
winski, J.;Wiewiorowski, M. Nucleic Acids Res. 1978, 5,
889–1905.
. (a) Gryaznov, S. M.;Letsinger, R. L. J. Am. Chem. Soc.
991, 113, 5876–5877;(b) Gryaznov, S. M.;Letsinger, R.
L. Nucleic Acids Res. 1992, 20, 1879–1882.
. Kung, P.-P.;Jones, R. A. Tetrahedron Lett. 1992, 33,
1
3
1
4
5
5
869–5872.
. (a) Hayakawa, Y.;Kataoka, M. J. Am. Chem. Soc. 1998,
20, 12395–12401;(b) Hayakawa, Y.;Kawai, R.;Kat-
1
aoka, M. Eur. J. Pharm. Sci. 2001, 13, 5–16;(c) Hayak-
awa, Y. Bull. Chem. Soc. Jpn. 2001, 74, 1547–1565.
. (a) Beaucage, S. L.;Caruthers, M. H. Tetrahedron Lett.
6
1
981, 22, 1859–1862;(b) Beaucage, S. L.;Iyer, R. P.
Tetrahedron 1992, 48, 2223–2311, and references cited
therein.
. (a) Sekine, M.;Ohkubo, A.;Seio, K. J. Org. Chem. 2003,
Acknowledgements
7
This work was supported by a Grant-in-Aid for Scien-
tific Research from the Ministry of Education, Culture,
Sports, Science and Technology, Japan. This work was
also supported by CREST of JST (Japan Science and
Technology).
1
4, 5478–5492;(b) Sekine, M.;Ohkubo, A.;Seio, K.
Nucleic Acids Res. Suppl. 2001, 1, 77–78.
8. Similar results could not be obtained when CPG or
5a
HCP resins were used in place of Tenta gel. Instead,
serious internucleotide bond cleavage was observed
when the post-treatment using MeOH–BIT was carried
7
b
out. .
. Wada, T.;Sato, Y.;Honda, F.;Kawahara, S.;Sekine, M.
J. Am. Chem. Soc. 1997, 119, 12710–12721.
9
References and Notes
10. (a) van der Marel, G.;van Boeckel, C. A. A.;Wille, G.;
van Boom, J. H. Tetrahedron Lett. 1981, 22, 3887–3890;
(b) van der Marel, G. A.;van Boeckel, C. A. A.;Wille, G.;
van Boom, J. H. Nucleic Acids Res. 1982, 10, 2337–
2351.
1
2
. Current Protocols in Nucleic Acid Chemistry;Beaucage, S.
L., Bergstrom, D. E., Glick, G. D., Jones, R. A., Eds.;
John Wiley: New York, 2000.
. Adamiak, R. W.;Biala, E.;Grzeskowiak, K.;Kierzek, R.;
Kraszewski, A.;Markiewicz, W. T.;Okupniak, J.;Sta-
11. McCollum, C.;Andrus, A. Tetrahedron Lett. 1991, 32,
4069–4072.