Effect of molecular sieves in the liquid-phase synthesis of nucleotides via the phosphoramidite method

Article abstract of DOI:10.1016/S0040-4020(01)00880-8

It is demonstrated that the reaction of a nucleoside phosphoramidite and a nucleoside aided by a suitable promoter in the presence of molecular sieves 3A or 4A in a liquid phase is efficiently performed by the use of stoichiometric amounts of the reactant

Full text of DOI:10.1016/S0040-4020(01)00880-8

TETRAHEDRON
Pergamon
Tetrahedron 57 ꢀ2001) 8823±8826
Effect of molecular sieves in the liquid-phase synthesis of
nucleotides via the phosphoramidite method
p
Yoshihiro Hayakawa, Akiyoshi Hirata, Jun-ichiro Sugimoto, Rie Kawai, Akira Sakakura
and Masanori Kataoka
Laboratory of Bioorganic Chemistry, Graduate School of Human Informatics, Nagoya University, Chikusa, Nagoya 464-8601, Japan
Received 19 July 2001; accepted 31 August 2001
AbstractÐIt is demonstrated that the reaction of a nucleoside phosphoramidite and a nucleoside aided by a suitable promoter in the presence
of molecular sieves 3A or 4A in a liquid phase is ef®ciently performed by the use of stoichiometric amounts of the reactants to give the
desired coupling product in an excellent yield. q 2001 Elsevier Science Ltd. All rights reserved.
1
. Introduction
of a phosphoramidite, a nucleoside, and a promoter, the
yield of the target product is rather lowered. A reason for
the low yield may be that moisture contained in the reaction
solvent and reactants themselves decompose the phosphor-
amidite or/and its activated species. Accordingly, we
examined the reaction of a nucleoside phosphoramidite
and a nucleoside in the presence of molecular sieves ꢀMS)
3A or 4A as the moisture scavenger and realized the high-
yield condensation using stoichiometric amounts of the
In recent years, a variety of small-size nucleotides and their
related compounds with biologically attractive activities
and properties have been discovered. They include
0
0
0
0
adenylylꢀ2 ±5 )adenylylꢀ2 ±5 )adenosine ꢀ2±5A core) and
1
0
cytidine-5 -monophosphono-N-acetylneuraminic acid ꢀCMP-
0
2
related derivatives, cyclic bisꢀ3 !5 )diguanylic acid,
0
Neu5Ac), aminoacylamido-AMP, phosmidosine, agrocin
3
4
5
6
7
4, and dinogunellin. Much research has been devoted
10
8
reactants and a promoter.
to the biological activities of these compounds. In order to
perform wide and detailed investigation, it is essential to
obtain a suf®cient amount of these substrates. In other
words, it is important to develop an ef®cient synthetic
method that provides a large amount of nucleotides and
their related compounds. Large-scale synthesis of short-
length nucleotide derivatives, such as those described
here, are generally carried out in a solution phase. In
large-scale, liquid-phase synthesis, the use of reactants in
excess is a serious economical disadvantage. In addition,
even when the internucleotide-bond formation reaction is
carried out quantitatively, reactants used in excess result
in the formation of by-products that must then be removed
by troublesome operations. It is therefore important that an
unnecessarily excess amount of reactants is avoided.
However, existing methods for the construction of the inter-
nucleotide linkage generally employ excess amounts of
reactants in order to obtain the target product in a high
2
. Results and discussion
First, we carried out the condensation of a deoxyribonucleo-
side phosphoramidite and a nucleoside in the presence of
MS and, as a control experiment, in the absence of MS. The
0
reaction ꢀ30 min) of the nucleoside 3 -ꢀallyl phosphorami-
0
H-tetrazole as the promoter in the presence of MS 3A,
dite) 4 and the 5 -O-unsubstituted nucleoside 11 using
1
where 4, 11, and the promoter are used in 5.0 mmol each
in a 0.1 M acetonitrile solution, and the subsequent oxi-
dation by means of 2-butanone peroxide or tert-butyl
11
12
hydroperoxide ꢀTBHP) gave the dinucleoside phosphate
1
5 in a 99% yield [calculated on the basis of weight and the
31
purity ꢀ P NMR assay) of the isolated product]. In contrast,
the preparation without MS 3A gave 15 in an only 85%
yield. In this case, the undesired formation of the H-phos-
phonate ꢀca. 15% yield), which results from hydrolysis of 4
and/or its activated species, was observed. Cyanoethyl phos-
phoramidites are also usable as the nucleoside phosphor-
amidite. The reaction using the cyanoethyl phosphorami-
dite generally requires a longer time for the completion.
For example, 1H-tetrazole-promoted condensation of 8
and 11 by the use of stoichiometric amounts of reactants
was completed in 60 min to give, after the oxidation, 19 in a
8
yield. For example, the standard phosphoramidite method
normally uses 2±4 equiv. of a phosphoramidite and a
9
promoter toward a nucleoside. When the internucleotide-
bond formation is carried out using stoichiometric amounts
Keywords: nucleotides; molecular sieves; phosphoramidites.
Corresponding author. Tel.: 181-52-789-4848; fax: 181-52-789-5646;
e-mail: yoshi@info.human.nagoya-u.ac.jp
p
0040±4020/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020ꢀ01)00880-8

8824
Y. Hayakawa et al. / Tetrahedron 57 ꢀ2001) 8823±8826
Table 1. Synthesis of dinucleoside phosphates
a
Amidite
Nucleoside
Promoter
Reaction time ꢀmin)
Product
Yield ꢀ%)
b
c
1
2
2
3
3
4
4
4
4
4
5
5
6
7
7
8
8
8
11
9
9
9
9
N-PhIMT
BIT
5
1
1
5
5
1
1
30
1
5
3
3
3
10
10
3
12
13
13
14
14
15
15
15
15
15
16
16
17
18
18
19
19
19
27
27
27
28
29
29
30
30
31
31
100, 99
89
90
91
93
95, 91
99, 96
N-MeBIT
BIT
N-MeBIT
BIT
N-MeBIT
1H-tetrazole
BrDCI
PyTFB
BIT
c
11
11
11
11
11
10
10
11
11
11
11
11
11
24
24
24
24
26
26
25
25
26
26
c
99
94
95
83
80
88
92
90
95
90
93
87
91
90
96
91
88
93
91
93
93
N-MeBIT
N-PhIMT
b
BIT
N-MeBIT
BIT
N-MeBIT
1H-tetrazole
BIT
3
60
15
10
90
15
15
15
15
15
15
15
20
20
20
21
21
21
22
22
23
23
N-MeBIT
ETT
N-PhIMT
b
BIT
N-MeBIT
BIT
N-MeBIT
BIT
N-MeBIT
The preparation was carried out on a 0.1±5.0 mmol scale.
a
b
c
31
Determined by P NMR analysis of the crude product unless otherwise noted.
These results have been reported in Ref. 10.
Yield of the product isolated by chromatography. Purity of the isolated product is generally .98%.
6
3% yield. In the synthesis using the O -unprotected
15
10
9
ꢀETT), and azolium salts such as N-ꢀphenyl)imidazolium
tri¯ate ꢀN-PhIMT), N-ꢀp-acetylphenyl)imidazolium tri¯ate
guanosine derivative 7 or 10 as a building block, there are
cases that the desired product is obtained in a somewhat low
yield. In such cases, the H-phosphonate and the phosphor-
amidate, which arise from hydrolysis and oxidation, respec-
tively, of 7 or 10, were obtained as by-products. While, any
1
6
ꢀN-AcPhIMT), benzimidazolium tri¯ate ꢀBIT),
and
N-ꢀmethyl)benzimidazolium tri¯ate ꢀN-MeBIT). These
promoters have higher reactivity than 1H-tetrazole to
complete the condensation more rapidly. For example,
N-PhIMT accomplished the reaction of 4 and 11 in 1 min
and the subsequent 2-butanone peroxide oxidation provided
15 in a 99% isolated yield. In this case, in the absence of MS
6
derivatives with the guanine-O -modi®ed structure was not
detected. This fact that the guanine-O -modi®cation was not
6
caused is an advantage of the present method using a
stoichiometric amount of reactants. As the promoter, a
variety of compounds can be used, which include
3
1
3A, the yield of the coupling product ꢀ P NMR analysis)
1
3
2
tetra¯uoroborate ꢀPyTFB),
-ꢀbromo)-4,5-ꢀdicyano)imidazole ꢀBrDCI), pyridinium
5-ꢀethylthio)-1H-tetrazole
1
4

Y. Hayakawa et al. / Tetrahedron 57 ꢀ2001) 8823±8826
8825
6
methanesulfonic acid ꢀCentral Glass), N -ꢀbenzoyl)-5 -O-
0
0
N,N-diisopropylphosphoramidite) ꢀ5) ꢀGlen Research),
0
0
ꢀ
p -dimethoxytrityl)-2 -deoxyadenosine 3 -ꢀ2-cyanoethyl
4
N -ꢀacetyl)-5 -O-ꢀp,p -dimethoxytrityl)-2 -deoxycytidine
0
0
0
0
Glen Research), N -ꢀisobutyryl)-5 -O-ꢀp,p -dimethoxy-
3
ꢀ
-ꢀ2-cyanoethyl N,N-diisopropylphosphoramidite) ꢀ6)
2
0
trityl)-2 -deoxyguanosine 3 -ꢀ2-cyanoethyl N,N-diiso-
0
0
propylphosphoramidite) ꢀ7) ꢀGlen Research),5 -O-ꢀp,p -
0
0
dimethoxytrityl)thymidine 3 -ꢀ2-cyanoethyl N,N-diiso-
0
0
propylphosphoramidite) ꢀ8) ꢀGlen Research), 2-butanone
peroxide as a 55% solution in dimethyl phthalate ꢀKishida)
were commercially supplied. The following compounds and
a solution are reported in literature or prepared by reported
1
0
methods. Azolium salts: N-ꢀphenyl)imidazolium tri¯ate,
1
6
benzimidazolium tri¯ate,
zolium tri¯ate; protected 2 -deoxyribonucleoside 3 -phos-
and N-ꢀmethyl)benzimida-
0
and 4; 3 -O-protected 2 -
1
0
0
1
7,18 17,18 17,18
17
0
deoxyribonucleosides: 9, 10, and 11; protected ribo-
0
phoramidites: 1,
2,
93,
1
20
21
0
18
nucleoside 3 -phosphoramidites: 20, 21, 22, and 23;
22
18
10
0
23
,3 -O-protected ribonucleosides: 24, 25, and 26;
.0 M tert-butyl hydroperoxide/toluene solution.
0
23
23
2
1
a
Aceto-
1
2,24
nitrile was distilled from CaH . Powdery molecular sieves
2
3A and 4A were used after drying the commercially
supplied one ꢀNacalai tesque) at 2008C for 12h. Nacalai
tesque silica gel 60 ꢀneutrality, 75 mm) was used for column
chromatography.
4.2. A general procedure for the preparation of
dinucleoside phosphates
A heterogeneous mixture of a nucleoside phosphoramidite
ꢀ5.0 mmol), a nucleoside ꢀ5.0 mmol), a promoter ꢀ5.0
was 79% and the undesired H-phosphonate was formed in a
21% yield. The present procedure is also effective for the
condensation of a ribonucleoside 3 -phosphoramidite and a
mmol), and molecular sieves 3A or 4A ꢀca. 1 g) in aceto-
nitrile ꢀ50 mL) was stirred at 258C. The reaction was
monitored by TLC. After completing the reaction, to the
mixture was added a 6.7% 2-butanone peroxide/dimethyl
phthalate-toluene solution ꢀ10 mL) or a 1.0 M TBHP/
0
-O-free ribonucleoside in stoichiometric use of the reac-
0
5
tants and a promoter. In this case, a longer time, usually
0±15 min, is required for completing the condensation.
1
toluene solution ꢀ10 mL) and stirring was continued for an
additional 5 min. [The `product yield determined by
Several examples of the preparation of dinucleoside phos-
phates via the present method are listed in Table 1. In all
cases described above, the use of MS 4A in place of MS 3A
as the moisture scavenger gave similar results.
31
P
NMR analysis' shown in Table 1 was obtained in this
stage according to the following procedure. To the reaction
mixture was added a suitable amount of triphenylphosphine
oxide. An aliquot of the mixture was taken and subjected to
3
1
the P NMR measurement, in which the irradiation mode
was set to NOE-Eliminated H Complete Decoupling
3. Conclusion
1
Measurement ꢀNNE mode). Comparison of intensity of
signals due to the dinucleoside phosphate due to triphenyl-
phosphine oxide gave the yield of the target product.] The
reaction mixture was diluted with ethyl acetate ꢀ200 mL)
and the molecular sieves were removed by ®ltration. The
We developed a highly ef®cient phosphoramidite method
for the liquid-phase synthesis of short-length nucleotides,
which provides the desired product in a high yield by the
use of stoichiometric amounts of reactants and a promoter in
the presence of MS 3A or 4A. Compared with existing
methods requiring an excess of reactants and the promoter,
the present approach is advantageous in regard to its
relatively low cost and operational simplicity of product
®
NaHCO ꢀ50 mL) and brine ꢀ50 mL). The organic layer was
ltrate was washed with an aqueous solution saturated with
3
dried and concentrated. The resulting crude material
was dissolved in ethyl acetate ꢀ20 mL) and poured to a
vigorously stirred petroleum ether ꢀ500 mL) to precipitate
a target dinucleoside phosphate as colorless power in an
almost quantitative amount.
1
0
isolation. As previously reported, this strategy was applied
to the synthesis of CMP-Neu5Ac and 2±5A core.
4
. Experimental
4.3. Determination of structure and purity of the product
4
.1. Materials
1
0
All dinucleoside phosphates listed in Table 1 are known.
Structure of the product was con®rmed by comparison of its
N-ꢀPhenyl)imidazole ꢀAldrich), benzimidazole ꢀNacalai
Tesque), N-ꢀmethyl)benzimidazole ꢀWako), tri¯uoro-
1
31
H NMR, P NMR, and TOF mass spectral data with those

8826
Y. Hayakawa et al. / Tetrahedron 57 ꢀ2001) 8823±8826
1
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3
1
31
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1
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This work was supported in part by a Grant-in-Aid for
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