triflate as a promoter for the activation of deoxynucleoside
3′-phosphoramidite building blocks.4 Moreover, we recently
reported the first development of an almost completely
O-selective phosphorylation procedure, i.e., “the activated
phosphite method”, by use of reactive phosphite intermedi-
ates generated from N-unprotected phosphoramidites.5 Our
new strategy proved to be advantageous over the previous
ones because the cleavage step of the N-P (III) bonds which
could be generated by the phosphitylation of the amino
groups of the nucleobases could be omitted.
midite units by treatment of commercially available N-
protected deoxynucleoside 3′-phosphoramidite units with
ammonia or methylamine.
First, we studied the selective removal of the acetyl group
from the 4-N-acetyldeoxycytidine 3′-phosphoramidite unit
to prepare N-free deoxycytidine 3′-phosphoramidite (Scheme
1 and Table 1). As a result, it was found that the 4-N-acetyl
Scheme 1. Deacylation of N-Acylated Phosphoramidite
The activated phosphite method must be very attractive
for the high-throughput synthesis of DNA molecules and the
synthesis of alkali-labile modified DNAs such as oligonucle-
otides containing DNA lesions.6 To demonstrate the useful-
ness in the latter application, we have already reported the
synthesis of oligonucleotides incorporating alkali-labile
modified bases such as 4-N-acetylcytosine by the combined
use of this activated phosphite method and new silyl-type
linkers that could be cleaved under neutral conditions.5a
To expand the activated phosphite method in commercial
custom DNA synthesis, convenient protocols for the syn-
thesis of N-unprotected deoxynucleoside 3′-phosphoramidite
units should be developed. Jones and co-workers reported a
general method for the synthesis of 5′-O-dimethoxytri-
tyldeoxyribonucleosides, which were prepared by dimethoxy-
tritylation of N-dimethylaminoamidinodeoxynucleosides fol-
lowed by removal of the N-protecting groups.7 More
straightfoward methods for the synthesis of 5′-O-tritylated
deoxyribonucleosides directly from deoxynucleosides have
been reported by several research groups.8 In the case of
2′-deoxycytidine and 2′-deoxyadenosine, the 5′-O-DMTr-
N-free deoxynucleoside derivatives can be obtained in
approximately 70% yields by the dimethoxytritylation of
deoxynucleosides in the presence of dichroloacetic acid and
triethylamine.4 However, the isolated yield was very low in
the tritylation of N-free deoxyguanosine because the O-
selectivity of the tritylation was very poor. The O-selective
tritylation of deoxyguanosine in the presence of imidazole,
methanesulfonic acid, and triethylamine reported by Kataoka8b
gave unsatisfactory results in our large-scale synthesis of 5′-
O-DMTr-deoxyguanosine. For these reasons, most of the
previous methods in the synthesis of N-unprotected deoxy-
nucleoside 3′-phosphoramidite units are unsuitable for the
large-scale synthesis of DNA.
Derivatives
Table 1. Preparation of N-Unprotected Phosphoramidite
Derivatives
protecting
group
time yielda
entry
condition
nucleobase
(h)
(%)
1
2
3
2 M NH3/ MeOH cytosine
2 M NH3/ MeOH adenine
2 M NH3/ MeOH guanine
acetyl
2
1
2
94
98
88
phenoxyacetyl
4-isopropyl-
phenoxyacetyl
benzoyl
4
5
2 M NH3/ MeOH cytosine
2 M NH3/ MeOH adenine
12
50
68
56
benzoyl
6
2 M NH3/ MeOH guanine
isobutyryl
22
72
7
8
9
7 M NH3/ MeOH guanine
2 M MeNH2/ THF adenine
2 M MeNH2/ THF cytosine
isobutyryl
benzoyl
6
2
12
14
2
1
2
62
89
85
75
96
97
90
benzoyl
10 2 M MeNH2/ THF guanine
11 2 M MeNH2/ THF cytosine
12 2 M MeNH2/ THF adenine
13 2 M MeNH2/ THF guanine
isobutyryl
acetyl
phenoxyacetyl
4-isopropyl-
phenoxyacetyl
a
Isolated yields of N-free phosphoramidite compounds.
group of the cytosine base could be cleaved completely by
treatment with 2 M NH3/MeOH for 2 h. Although there was
some concern about â-elimination of the 2-cyanoethyl group
of the phosphoramidite unit, this functional group was found
to be stable in 2 M NH3/MeOH for 2 h because the
phosphorus atom of the phosphoramidite unit is trivalent so
that it has poor leaving group ability. The N-free deoxycy-
tidine 3′-phosphoramidite unit was obtained in 94% yield,
as shown in entry 1 of Table 1. Similarly, the N-free
deoxyadenosine 3′-phosphoramidite derivative was obtained
in 98% yield by treatment of the 6-N-phenoxyacetyldeoxa-
denosine 3′-phosphoramidite derivative with 2 M NH3/
MeOH for 1 h (entry 2). The N-free deoxyguanosine
3′-phosphoramidite derivative was also obtained in 88% yield
by using deprotection of 2-N-(4-isopropylphenoxyacetyl)-
deoxyguanosine 3′-phosphoramidite derivative (entry 3). In
contrast, the N-benzoyl group of N-benzoyldeoxycytidine was
more stable under the same conditions using NH3-MeOH.
Since the cleavage reaction required 12 h, side reactions such
In this paper, we report a convenient method for the
synthesis of N-unprotected deoxynucleoside 3′-phosphora-
(4) Hayakawa Y.; Kataoka, M. J. Am. Chem. Soc. 1998, 120, 12395-
12401.
(5) (a) Ohkubo, A.; Ezawa, Y.; Seio, K.; Sekine, M. J. Am. Chem. Soc.
2004, 126, 10884-10896. (b) Ohkubo, A.; Seio, K.; Sekine, M. Tetrahedron
Lett. 2004, 45, 363-366.
(6) (a) Roupioz, Y.; Lhomme, J.; Kotera, M. J. Am. Chem. Soc. 2002,
124, 9129-9135. (b) Kim, J.; Gil, J. M.; Greenberg, M. M. Angew. Chem.,
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(7) Kung, P.-P.; Jones, R. A. Tetrahedron Lett. 1992, 33, 5869-5872.
(8) (a) Adamiak, R. W.; Biala, E.; Grzeskowiak, K.; Kierzek, R.;
Kraszewski, A.; Markiewicz, W. T.; Okupniak, J. Stawinski, J. Wiewiorows-
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Y. J. Org. Chem. 1999, 64, 6087-6089. (c) Nishino, S.; Nagato, Y.;
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