Preparation of short oligonucleotides via the phosphoramidite method using a tetrazole promoter in a catalytic manner

Article abstract of DOI:10.1021/ja970685b

A facile phosphoramidite method using a tetrazole promoter in a catalytic manner has been developed for the condensation of a nucleoside 3'-phosphoramidite and a nucleoside. This method is particularly useful for a large-scale synthesis of short oligonucl

Full text of DOI:10.1021/ja970685b

11758
J. Am. Chem. Soc. 1997, 119, 11758-11762
Preparation of Short Oligonucleotides via the Phosphoramidite
Method Using a Tetrazole Promoter in a Catalytic Manner
Yoshihiro Hayakawa* and Masanori Kataoka
Contribution from the Laboratory of Bioorganic Chemistry, Graduate School of Human
Informatics, Nagoya UniVersity, Chikusa, Nagoya 464-01, Japan
ReceiVed March 4, 1997X
Abstract: A facile phosphoramidite method using a tetrazole promoter in a catalytic manner has been developed for
the condensation of a nucleoside 3′-phosphoramidite and a nucleoside. This method is particularly useful for a
large-scale synthesis of short oligonucleotides. For example, dinucleoside phosphates are prepared on a multigram
scale in 92-99% yields through the reaction of nucleoside 3′-N,N-diethylphosphoramidites (1.05 equiv) and 5′-O-
free nucleosides (1.00 equiv) with 5-(p-nitrophenyl)-1H-tetrazole (NPT) (0.05 equiv) in the presence of molecular
sieves 13X in acetonitrile (40 °C, 60 min) followed by trimethylsilyl triflate-catalyzed oxidation with bis(trimethylsilyl)
peroxide in dichloromethane (40 °C, 10 min). The NPT-catalytic approach is also effective for the synthesis of
longer deoxyribonucleotides such as d(5′CTACCTGT3′) and 2′-5′- or 3′-5′-linked ribonucleotides.
Introduction
phosphoramidite. Hence, a stoichiometric amount of the
tetrazole is the minimum required for completion of the reaction.
Here, if the generated amine can be removed, the tetrazole would
catalytically promote the condensation as shown in Scheme 1.
This was actually achieved by the use of molecular sieves (MS)
13X (10 Å pore size; ca. 2 µm particle size) as the amine
scavenger.
An efficient large-scale synthesis of short oligonucleotides
in a solution phase has become an important subject in nucleic
acid chemistry. The synthesis is currently achieved by con-
densation of a nucleoside and a nucleoside phosphoramidite with
promotion by a tetrazole such as 1H-tetrazole,1 5-(p-nitrophe-
nyl)-1H-tetrazole (NPT),2 or 5-(ethylthio)-1H-tetrazole3 as a key
step.4 This reaction usually requires 2-4 equiv each of a
promoter and a phosphoramidite to a nucleoside for gaining an
acceptable reaction rate.5 However, the use of an excess of
the tetrazole compound, particularly NPT, poses serious prob-
lems in large-scale synthesis because they are fairly expensive,
harmful to health,6 potentially explosive, and poorly soluble in
a reaction solvent. Thus, methods using a catalytic amount of
the tetrazole activator have been strongly desired, but not yet
viable.5b,c,7 This paper describes the first tetrazole-catalyzed
condensation of a nucleoside and a nucleoside phosphoramidite.
The use of an O-allyl N,N-diethylphosphoramidite as the
amidite, NPT as the promoter, and MS 13X as the amine
scavenger is most effective for the reaction to proceed at a
reasonable rate and with a high yield. For example, the
condensation of the nucleoside 2 (1.00 equiv) and the amidite
Results and Discussion
In the phosphoramidite method, the acidic tetrazole acts not
only as an activator of the amidite but also as a scavenger of
the generated amine, where the scavenger takes precedence over
the activator because the amine is a stronger base than the
X Abstract published in AdVance ACS Abstracts, November 15, 1997.
(1) Beaucage, S. L.; Caruthers, M. H. Tetrahedron Lett. 1981, 22, 1859-
1862.
(2) Froehler, B. C.; Matteucci, M. D. Tetrahedron Lett. 1983, 24, 3171-
3174.
(3) Wright, P.; Lloyd, D.; Rapp, W.; Andrus, A. Tetrahedron Lett. 1993,
34, 3373-3376.
(4) (a) Beaucage, S. L.; Iyer, R. P. Tetrahedron 1992, 48, 2223-2311
and references cited therein. See also: (b) Beaucage, S. L.; Iyer, R. P.
Tetrahedron 1993, 49, 6123-6194.
(5) (a) Stec, W. J.; Zon, G. Tetrahedron Lett. 1984, 25, 5279-5282. (b)
Dahl, B. H.; Nielsen, J.; Dahl, O. Nucleic Acids Res. 1987, 15, 1729-
1743. (c) Dahl, B. H.; Nielsen, J.; Dahl, O. Nucleosides Nucleotides 1987,
6, 457-460.
(6) Sigma-Aldrich Library of Chemical Safety 2; Sigma-Aldrich: Mil-
waukee, 1990; p 3313D (Sigma-Aldrich Material Safety Data Sheets;
Product No. 33644-0).
(7) A few phosphoramidite methods using a catalytic promoter have been
reported in the preparation of simple phosphites and their derivatives. (a)
Nifant’ev, E. E.; Ivanova, N. L. Vestn. Mosk. UniV. Khim. 1968, 23, 104-
106; Moskow UniV. Chem. Bull. (Engl. Transl. 1968, 23, 78-79). (b) Dahl,
O. Phosphorus Sulfur 1983, 18, 201-204. (c) Stec, W. J.; Zon, G.
Tetrahedron Lett. 1984, 25, 4279-4282.
S0002-7863(97)00685-9 CCC: $14.00 © 1997 American Chemical Society

Preparation of Short Oligonucleotides
J. Am. Chem. Soc., Vol. 119, No. 49, 1997 11759
Scheme 1
Table 1. Synthesis of Dinucleoside Phosphatesa
Figure 1. Profile of the condensation of 2 and 7. The reaction was
conducted using 5 mol % of a promoter in the presence or absence of
an amine scavenger in acetonitrile at 40 °C. The yield of the phosphite
was estimated by the 31P NMR analysis with triphenylphosphine oxide
as a standard; O, NPT/MS 13X; b, NPT/DIAION WK-40; 0,
benzimidazolium triflate/MS 13X; 9, 1H-tetrazole/MS 13X, 4, NPT/
without scavenger; 2, NPT/DOWEX 50W-X8.
nucleoside
amidite
product
yield,b %
1
1
1
2
2
2
2
2
4
5
6
7
4
5
6
7
11
12
13
14
15
16
17
18
97
96
92
98
97
97
95
99
weak cation exchanger such as DIAION WK-40 (H-form) was
less effective, requiring 72 h longer for completion of the
condensation. Mineral acids such as p-toluenesulfonic acid or
strong acid resins such as Dowex 50W-8X and Nafion brought
about decomposition of the phosphoramidite, detritylation, and,
in the cases with deoxyadenosine and deoxyguanosine deriva-
tives, depurination. The use of 1H-tetrazole, 5-(ethylthio)-1H-
tetrazole, benzimidazolium triflate,12 pyridinium chloride,13 or
N-methylanilinium trifluoroacetate14 as a catalytic promoter gave
little of the coupling products. Figure 1 shows some of these
results including that obtained by the use of MS 13X and NPT.
The utility of several phosphoramidites was also examined. Allyl
N,N-dimethylphosphoramidites serve as alternative phosphora-
midites. The NPT-catalyzed reaction of 2 and the freshly
prepared 8 was finished in 15 min to afford after the TM-
SOOTMS oxidation 18 in 93% yield. The dimethylphosphora-
midites, however, are not absolutely useful because they lack
stability and therefore require strict conditions for storage.15
Allyl N,N-diisopropylphosphoramidites may be used, but these
substrates require longer reaction time or harder reaction
conditions. That is, the condensation was generally ac-
complished in 24 h at 25 °C or in 1 h at reflux temperature.
When less reactive allyl phosphoromorpholidites were used, the
reaction was incomplete even after 12 h at reflux temperature.
Amidites with the electron-withdrawing alkyl substituent such
as 2-cyanoethyl or o-chlorophenyl in place of the allyl on the
ester part decelerate the reaction. For instance, the condensation
of 3 and the 2-cyanoethylated N,N-diethylphosphoramidite 9
with a catalytic amount of NPT in acetonitrile at 40 °C needed
ca. 8 h for completion. The reaction using the O-o-chlorophenyl
analog 10 was not finished even after 12 h; the yield of the
desired product was ca. 20%.
a Condensation was carried out using amidite, nucleoside, and NPT
in a 1.05:1.00:0.05 molar ratio in acetonitrile at 40 °C for 60 min. The
resulting phosphite was directly oxidized by bis(trimethylsilyl) peroxide/
trimethylsilyl triflate in acetonitrile-CH2Cl2 (40 °C, 5 min) to the
phosphate. b Isolated yield. The product was obtained as a ca. 1:1
mixture of diastereomers.
78 (1.05 equiv) using a catalytic amount of NPT (0.05 equiv)
in the presence of MS 13X in acetonitrile (40 °C, 60 min)9
followed by oxidation with bis(trimethylsilyl) peroxide (TM-
SOOTMS) (2.0 equiv) assisted by trimethylsilyl triflate (TM-
SOTf) (0.05 equiv) in dichloromethane10 (40 °C, 10 min)
afforded 18 as a ca. 1:1 mixture of diastereomers (31P NMR:
-1.4 and -1.1 ppm, H3PO4 standard) in 99% yield. The
phosphitylation was also achieved at 25 °C but required longer
time (80-120 min). In this reaction, the dimethoxytrityl,
(allyloxy)carbonyl (AOC), and allyl protectors were left intact.
MS 13X is recyclable as the amine scavenger after drying at
200 °C for 12 h. Table 1 shows several examples of the
dinucleoside phosphotriesters obtained by the new method. The
products could be converted generally in >95% yield to the
unprotected derivatives through detritylation with dichloroacetic
acid and removal of the allyl and AOC protectors with
Pd[P(C6H5)3]4/P(C6H5)38,11 in the presence of diethylammonium
hydrogen carbonate.11
We investigated the efficiency of other amine scavengers and
promoters in the reaction of 2 and 7. MS 3A and 4A with
smaller pore sizes were not suitable as the amine scavenger. A
(8) (a) Purrung, M. C.; Fallon, L.; Lever, D. C.; Shuey, S. W. J. Org.
Chem. 1996, 61, 2129-2136. See also: (b) Hayakawa Y.; Uchiyama, M.;
Kato, H.; Noyori, R. Tetrahedron Lett. 1985, 26, 6505-6508. (c) Hayakawa,
Y.; Kato, H.; Uchiyama, M.; Kajino, H.; Noyori, R. J. Org. Chem. 1986,
51, 2400-2402. (d) Hayakawa, Y.; Wakabayashi, S.; Kato, H.; Noyori, R.
J. Am. Chem. Soc. 1990, 112, 1691-1696.
(9) The controlled experiment without NPT at 40 °C did not give 18
even after 5 h. This result indicates that NPT serves as a catalyst in the
reaction.
(10) Hayakawa, Y.; Uchiyama, M.; Noyori, R. Tetrahedron Lett. 1986,
27, 4191-4194.
(11) (a) Hayakawa, Y.; Hirose, M.; Noyori, R. Nucleosides Nucleotides
1989, 8, 867-870. (b) Hayakawa, Y.; Hirose, M.; Noyori, R. J. Org. Chem.
1993, 58, 5551-5555. (c) Hayakawa, Y.; Hirose, M.; Hayakawa, M.;
Noyori, R. ibid. 1995, 60, 925-930. (d) Hayakawa, Y.; Hirose, M.; Noyori,
R. Tetrahedron 1995, 36, 9899-9916.
This NPT-catalyzed approach is also useful for the preparation
of longer deoxyribonucleotides. Synthesis of the octamer d(5′-
(12) Hayakawa, Y.; Kataoka, M.; Noyori, R. J. Org. Chem. 1996, 61,
7996-7997.
(13) (a) Nelson, K. A.; Sopchik, A. E.; Bentrude, W. G. J. Am. Chem.
Soc. 1983, 105, 7752-7754. (b) Gryaznov, S. M.; Letsinger, R. L. Nucleic
Acids Res. 1992, 20, 1879-1882.
(14) Fourrey, J. L.; Varenne, J. Tetrahedron Lett. 1984, 25, 4511-4514.
See also: Fourrey, J. L.; Varenne, J.; Fontaine, C.; Guittet, E.; Yang, Z.
W. Tetrahedron Lett. 1987, 28, 1769-1772.
(15) In the storage of 8 under argon at -40 or +25 °C for 24 h, ca. 2 or
15% of decomposition, respectively, was observed. Cf. Sinha, N. D.; Biernat,
J.; McManus, J.; Ko¨ster, H. Nucleic Acids Res. 1984, 12, 4539-4557.

11760 J. Am. Chem. Soc., Vol. 119, No. 49, 1997
Hayakawa and Kataoka
CTACCTGT3′) (21) was representatively demonstrated. The
Figure 2. HPLC and 31P NMR profile of the crude octamer d(5′-
CTACCTGT3′) (21): (A) the HPLC chart obtained using an ODS-5µm
column (4.6 × 250 mm) eluted with a 0.1 M triethylammonium acetate
buffer containing 5-15% acetonitrile (v/v) (linear gradient in 30 min);
(b) the 31P NMR spectrum in D2O with 85% H3PO4 as an external
standard.
condensation of 2 and 6 with NPT (40 °C, 60 min) and the
subsequent TMSOOTMS/TMSOTf oxidation (40 °C, 10 min)
gave a diasteromeric mixture of the dinucleoside phosphate 17,
which was detritylated by dichloroacetic acid in dichloromethane
(25 °C, 10 min) to afford 19 in 95% overall yield. Repetition
of (1) the phosphitylation with a suitable nucleoside 3′-
phosphoramidite among 4-7 (40 °C, 1-2 h), (2) the TM-
SOOTMS oxidation, and (3) the detrityaltion converted the
dimer 19 to the protected octamer 20 in 57% overall yield.16 In
this preparation, the phosphitylation proceeded with 91-95%
yield. The yield is comparable with that in the existing typical
approach using 2 equiv each of the phosphoramidite and NPT.
The allyl and AOC protectors of 20 were removed by treatment
with catalytic amounts of Pd[P(C6H5)3]4 and P(C6H5)3 in the
presence of diethylammonium hydrogen carbonate in aqueous
tetrahydrofuran to give 21 in ca. 91% yield. The HPLC and
31P NMR analysis (Figure 2) indicated that the purity of crude
21 is >98%. HPLC of the product obtained by digestion of 21
with snake venom phosphodiesterase and bacterial alkaline
phosphatase17 showed four peaks due to dA, dC, dG, and T in
a ratio of 0.9:3.0:0.9:3.1. The experimentally derived base
composition agreed well with the ratio calculated for 21 to
confirm the structure.
Conclusion
We have realized for the first time a facile method for the
preparation of nucleotides via the tetrazole-catalyzed condensa-
tion of a nucleoside and a nucleoside phosphoramidite using a
trapping agent of the amine generated in the reaction, where
the use of NPT as the promoter, the allyl N,N-diethylphos-
phoramidite in deoxyribonucleotide synthesis or the allyl N,N-
diisopropylphosphoramidite in ribonucleotide synthesis as the
phosphoramidite, and MS 13X as the amine scavenger is most
effective. In the new approach, another remarkable result was
obtained; that is, the condensation can be performed with an
excellent yield by use of nearly equimolar amounts of a
nucleoside phosphoramidite and a nucleoside. The use of the
expensive promoter or nucleoside phosphoramidites in a cata-
lytic or stoichiometric manner, respectively, is strongly de-
manded from an economical point of view in large-scale
synthesis. Thus, the present method meeting these requirements
is highly superior to existing approaches.4,5a
The catalytic method can also be applied to the synthesis of
ribonucleotides bearing 2′-5′- or 3′-5′-internucleotide linkages.
In these cases, since high quality of N,N-diethylphosphoramid-
ites is not easily accessible, N,N-diisopropylphosphoramidites
were employed. Thus, the NPT-catalyzed reaction of the
nucleoside 22 and the phosphoramidite 23 in the presence of
MS 13X (8 h, acetonitrile reflux temperature) followed by the
TMSOOTMS/TMSOTf oxidation gave 25 in 95% yield. Simi-
larly, the 2′-5′-linked nucleotide 26 was produced in 96% yield
from 22 and 24.
Experimental Section
General Procedures and Materials. IR spectra were measured in
KBr on a JASCO FT/IR-5300 spectrometer. UV spectra were taken
in MeOH on a JASCO V-550 spectrometer. NMR spectra were
obtained in CDCl3 on a JEOL R-400 instrument. The 1H and 31P
chemical shifts are described as δ values in parts per million relative
to (CH3)4Si and 85% H3PO4, respectively. High-performance liquid
chromatography (HPLC) was achieved using a COSMOSIL 5C18-MS
(16) Preparation of nonamers from 20 was attempted under several
conditions, but it was not achieved in a satisfactory yield since the solubility
of the 5′-O-free derivative of 20 in acetonitrile is too low to allow
homogeneous reaction of this component and a phosphoramidite.
(17) Ogilvie, K. K.; Thompson, E. A.; Quilliam, M. A.; Westmore, J.
B. Tetrahedron Lett. 1974, 2865-2868.

Preparation of Short Oligonucleotides
J. Am. Chem. Soc., Vol. 119, No. 49, 1997 11761
(nacalai tesque, ODS-5µm) column on a JASCO 88-PU chromatograph
with a JASCO 870-UV detector. Elemental analysis was performed
at the Graduate School of Human Informatics, Nagoya University. The
nucleosides, 1,11c 2,12 3,17 and 22,11d and the nucleoside phosphora-
midites, 4,8a 5,8a 6,8a 7,8a 9,5b 10,5b 14,8a 23,13 and 24,11d were prepared
by the reported methods. The amidites were stored in a refrigerator.
Acetonitrile used as the reaction solvent was distilled from CaH2.
Column chromatography was achieved with E. Merck Kieselgel 60
(70-230 mesh) deactivated by adding 6% water. Solvents for the
chromatography were used after simple distillation of the commercially
supplied ones. 5-(p-Nitrophenyl)-1H-tetrazole (Lancaster) and molec-
ular sieves 13X powder (Aldrich) were used as commercially supplied.
The products, 18,12 25,12 and 26,11d are known the in the literature.
The spectral and physical data of the new compounds, 8, 11-17, 19,
and 21, are as follows.
5′-O-(p,p′-Dimethoxytrityl)thymidine 3′-(Allyl N,N-dimethylphos-
phoramidite) (8). To a suspension of 5′-O-(p,p′-dimethoxytrityl)-
thymidine (5.00 g, 9.19 mmol), (allyloxy)bis(dimethylamino)phosphine
(1.49 g, 9.19 mmol), and molecular sieves 3A (200 mg) in acetonitrile
(50 mL) was added N-methylimidazolium triflate18 (1.07 g, 4.60 mmol)
in five portions. The resulting mixture was stirred at 25 °C for 1.5 h
and then poured into ether (200 mL). The organic solution was washed
with brine (100 mL × 2) and concentrated to give 8 (6.09 g, 98%
yield) as a colorless solid: IR 1690, 1609, 1510, 1464 cm-1; UV λmax
235 (ꢀ 28 300), 268 nm (14 100); 1H NMR δ 1.44 (s, 3H), 2.24-2.35
(m, 1H), 2.41-2.70 (m, 7H), 3.28-3.35 (m, 1H), 3.46-3.51 (m, 1H),
3.79 (s, 6H), 4.03-4.07 (m, 1H), 4.11-4.19 (m, 2H), 4.66-4.71 (m,
1H), 5.08-5.28 (m, 2H), 5.77-5.95 (m, 1H), 6.38-6.44 (m, 1H), 6.83
(d, 4H, J ) 9.3 Hz), 7.21-7.41 (m, 10H), 7.60, 7.61 (2 s, 1H); 31P
NMR δ -148, -147. This compound is too labile to be subjected to
the mass spectral and elemental analyses. The crude product was used
for further reaction immediately after preparation without purification.
5.82-5.97 (m, 4H), 6.19-6.27 (m, 2H), 6.83-6.85 (m, 4H), 7.03-
7.06 (m, 1H), 7.24-7.36 (m, 11H), 7.96-8.22 (m, 2H), 8.44-8.66
(br s, 1H); 31P NMR δ -1.3, -1.1. Anal. Calcd for C54H59N6O18P:
C, 58.38; H, 5.35; N, 7.56 . Found: C, 58.25; H, 5.26; N, 5.77.
Allyl [N2-[(Allyloxy)carbonyl]-O6-allyl-5′-O-(p,p′-dimethoxytrityl)-
2′-deoxyguanylyl]-(3′-5′)-N4,3′-O-bis[(allyloxy)carbonyl]-2′-deoxy-
cytidine (13): a colorless amorphous solid; IR 1752, 1698, 1609, 1510,
1460, 1418 cm-1; UV λmax 237 (ꢀ 59 300), 269 nm (50 200); 1H NMR
δ 2.10-2.22 (m, 1H), 2.69-2.89 (m, 2H), 3.08-3.20 (m, 1H), 3.35-
3.51 (m, 2H), 3.75, 3.76 (2 s, 6H), 4.33-4.39 (m, 4H), 4.50-4.68 (m,
8H), 5.07 (d, 2H, J ) 5.3 Hz), 5.17-5.49 (m, 12H), 5.82-6.01 (m,
4H), 6.10-6.20 (m, 1H), 6.28 (t, 1H, J ) 6.8 Hz), 6.33-6.39 (m, 1H),
6.72-6.77 (m, 4H), 7.16-7.37 (m, 10H), 7.53, 7.66 (2 s, 1H), 7.76-
7.85 (br s, 1H), 7.93, 7.97 (2 s, 1H), 7.93, 7.97 (2 d, 1H, J ) 11.7 Hz);
31P NMR δ -1.1, -1.0. Anal. Calcd for C58H63N8O18P: C, 58.48;
H, 5.33; N, 9.41. Found: C, 58.25; H, 5.33; N, 9.51.
Allyl [5′-O-(p,p′-Dimethoxytrityl)thymidylyl]-(3′-5′)-N4,3′-O-bis-
[(allyloxy)carbonyl]-2′-deoxycytidine (14): a colorless amorphous
solid; IR 1750, 1686, 1561, 1508, 1460 cm-1; UV λmax 237 (ꢀ 33 700),
272 nm (14 400); 1H NMR δ 1.41 (s, 3H), 2.07-2.17 (m, 1H), 2.39-
2.47 (m, 1H), 2.60-2.65 (m, 1H), 2.83 (dt, 1H, J ) 4.9, 15.1 Hz),
3.38 (dd, 1H, J ) 11.2, 11.7 Hz), 3.50-3.55 (m, 1H), 3.79, 3.90 (2 s,
6H), 4.22-4.38 (m, 3H), 4.44-4.66 (m, 7H), 5.12-5.39 (m, 8H), 5.79-
5.97 (m, 3H), 6.26 (t, 1H, J ) 6.5 Hz), 6.41, 6.42 (2 s, 1H, J ) 8.8
Hz), 6.84 (d, 4H, J ) 8.3 Hz), 7.20-7.39 (m, 10H), 7.54, 7.57 (2 s,
1H), 7.98, 8.04 (2 d, 1H, J ) 7.5 Hz), 8.09 (s, 1H), 9.11, 9.16 (2 s,
1H); 31P NMR δ -1.1, -1.0. Anal. Calcd for C51H56N5O17P: C,
58.79; H, 5.42; N, 6.72. Found: C, 58.78; H, 5.44; N, 6.70.
Allyl [N6-[(Allyloxy)carbonyl]-5′-O-(p,p′-dimethoxytrityl)-2′-
deoxyadenylyl]-(3′-5′)-3′-O-[(allyloxy)carbonyl]thymidine (15): a
colorless amorphous solid; IR 1752, 1698, 1609, 1510, 1460, 1418
1
cm-1; UV λmax 237 (ꢀ 39 200), 269 nm (33 700); H NMR δ 1.91,
1.94 (2 s, 3H), 2.20-2.31 (m, 1H), 2.47-2.53 (m, 1H), 2.49, 2.50 (dt,
1H, J ) 5.8, 7.3 Hz), 2.76-2.84 (m, 1H), 3.09-3.18 (m, 1H), 3.37-
3.50 (m, 1H), 3.76, 3.77 (2 s, 6H), 4.21-4.41 (m, 4H), 4.51-4.69 (m,
4H), 4.76 (d, 2H, J ) 4.4 Hz), 5.18-5.45 (m, 8H), 5.85-6.05 (m,
3H), 6.31-6.38 (m, 1H), 6.45-6.52 (m, 1H), 6.77-6.80 (m, 4H), 7.19-
7.41 (m, 10H), 8.65, 8.66 (2 s, 1H), 8.82, 8.83 (2 s, 1H), 8.87-9.91
(br s, 1H), 9.92-10.01 (br s, 1H); 31P NMR δ -1.5, -1.1. Anal. Calcd
for C52H56N7O16P: C, 58.59; H, 5.29; N, 9.20. Found: C, 58.59; H,
5.26; N, 9.25.
Allyl [N4-[(Allyloxy)carbonyl]-5′-O-(p,p′-dimethoxytrityl)-2′-
deoxycytidylyl]-(3′-5′)-3′-O-[(allyloxy)carbonyl]thymidine (16): a
colorless amorphous solid; IR 1748, 1688, 1508, 1466 cm-1; UV λmax
235 (ꢀ 23 600), 267 nm (11 100); 1H NMR 1.87, 1.89 (2 s, 3H), 2.18-
2.52 (m, 3H), 2.82-2.94 (m, 1H), 3.33-3.49 (m, 2H), 3.77, 3.78 (2 s,
6H), 4.15-4.69 (m, 10H), 5.07-5.42 (m, 8H), 5.84-6.01 (m, 3H),
6.17-6.38 (m, 2H), 6.82-6.84 (m, 4H), 6.95 (d, 1H, J ) 5.7 Hz),
7.17-7.41 (m, 10H), 8.02-8.08 (m, 1H), 8.23-8.47 (br s, 1H), 9.55-
9.78 (br s, 1H); 31P NMR δ -1.4, -1.1. Anal. Calcd for
C51H56N5O17P: C, 58.79; H, 5.42; N, 6.72. Found: C, 58.56; H, 5.45;
N, 6.88.
Preparation of Allyl [5′-O-(p,p′-Dimethoxytrityl)thymidylyl]-(3′-
5′)-3′-O-[(allyloxy)carbonyl]thymidine (18). The Typical Procedure
for the Synthesis of Dinucleoside Phosphates. To a solution of the
nucleoside 2 (8.16 g, 25.0 mmol), the amidite 7 (17.9 g, 26.3 mmol),
and NPT (243 mg, 1.25 mmol) in acetonitrile (50 mL) was added
molecular sieves (MS) 13X (20.0 g). The resulting heterogeneous
mixture was vigorously stirred at 40 °C for 60 min. To the reaction
mixture were added a 1.0 M solution of bis(trimethylsilyl) peroxide
(TMSOOTMS) in dichloromethane (30.0 mL, 30.0 mmol) and a 0.25
M solution of trimethylsilyl triflate (TMSOTf) in dichloromethane (6.00
mL, 1.50 mmol),10 and stirring was continued for 10 min. After
removal of the molecular sieves by filtration, the filtrate was concen-
trated to give an oil, which was dissolved in a 9:1 mixture of ethyl
acetate and hexane (60 mL). The solution was passed through a silica
gel pad (20 g) and washed with a 9:1 mixture of ethyl acetate and
hexane (40 mL). Concentration of the combined filtrate and washing
afforded 18 (24.1 g, 99% yield) as a colorless amorphous solid.
The dinucleoside phosphates, 11-17, were prepared in a similar
manner.
Allyl [N6-[(Allyloxy)carbonyl]-5′-O-(p,p′-dimethoxytrityl)-2′-
deoxyadenylyl]-(3′-5′)-N4,3′-O-bis[(allyloxy)carbonyl]-2′-deoxycy-
tidine (11): a colorless amorphous solid; IR 1749, 1671, 1613, 1584,
1508, 1464 cm-1; UV λmax 238 (ꢀ 26 900), 268 nm (18 300); 1H NMR
δ 2.09-2.19 (m, 1H), 2.72-2.30 (m, 2H), 3.12-3.19 (m, 1H), 3.34-
3.45 (m, 2H), 3.72, 3.73 (2 s, 6H), 4.32-4.61 (m, 10H), 4.71 (d, 2H,
J ) 4.6 Hz), 5.15-5.36 (m, 10H), 5.82-5.96 (m, 4H), 6.21-6.26 (m,
1H), 6.43-6.47 (m, 1H), 6.74 (d, 4H, J ) 8.8 Hz), 7.14-7.26 (m,
10H), 7.99-8.02 (m, 1H), 8.17 (d, 1H, J ) 10.7 Hz), 8.60-8.62 (m,
2H), 8.84-9.89 (br s, 1H); 31P NMR δ -1.1, -1.0. Anal. Calcd for
C55H59N8O17P: C, 58.20; H, 5.24; N, 9.87. Found: C, 58.21; H, 5.27;
N, 9.97.
Allyl [N4-[(Allyloxy)carbonyl]-5′-O-(p,p′-dimethoxytrityl)-2′-
deoxycytidylyl]-(3′-5′)-N4,3′-O-bis[(allyloxy)carbonyl]-2′-deoxycy-
tidine (12): a colorless amorphous solid; IR 1751, 1653, 1561, 1503,
1402 cm-1; UV λmax 238 (ꢀ 20 700), 293 nm (6700); 1H NMR δ 2.15-
2.25 (m, 1H), 2.30-2.36 (m, 1H), 2.76-2.91 (m, 2H), 3.37-3.48 (m,
2H), 3.79, 3.80 (2 s, 6H), 4.28-4.67 (m, 12H), 5.07-5.39 (m, 10H),
Allyl [N2-[(Allyloxy)carbonyl]-O6-allyl-5′-O-(p,p′-dimethoxytrityl)-
2′-deoxyguanylyl]-(3′-5′)-3′-O-[(allyloxy)carbonyl]thymidine (17):
a colorless amorphous solid; IR 1752, 1698, 1609, 1510, 1460, 1418
1
cm-1; UV λmax 237 (ꢀ 26 700), 269 nm (25 400); H NMR δ 1.92 (s,
3H), 2.21-2.38 (m, 1H), 2.42-2.56 (m, 1H), 2.69-2.81 (m, 1H), 3.02-
3.12 (m, 1H), 3.32-3.38 (m, 1H), 3.44-3.48 (m, 1H), 3.75, 3.76 (2 s,
6H), 4.20-4.41 (m, 4H), 4.50-4.68 (m, 6H), 5.07 (d, 2H, J ) 4.9
Hz), 5.22-5.49 (m, 10H), 5.81-6.02 (m, 3H), 6.09-6.18 (m, 1H),
6.28-6.32 (m, 1H), 6.39 (t, 1H, J ) 6.3 Hz), 6.72-6.79 (m, 4H), 7.16-
7.39 (m, 10H), 7.60, 7.73 (2 s, 1H), 7.91, 7.92 (2 s, 1H), 8.86-9.02 (2
br s, 1H); 31P NMR δ -1.4, -1.1. Anal. Calcd for C55H60N7O17P:
C, 58.87; H, 5.39; N, 8.74. Found: C, 58.67; H, 5.27; N, 8.94.
Preparation of Allyl [N2-[(Allyloxy)carbonyl]-O6-allyl-2′-deox-
yguanylyl]-(3′-5′)-3′-O-[(allyloxy)carbonyl]thymidine (19). A het-
erogeneous mixture of 2 (978 mg, 3.00 mmol), 6 (2.52 g, 3.15 mmol),
NPT (28.7 mg, 150 µmol), and MS 13X (2.50 g) in acetonitrile (10
mL) was vigorously stirred at 40 °C for 60 min. To this mixture were
added a 1.0 M solution of TMSOOTMS in dichloromethane (4.00 mL,
4.00 mmol) and a 0.25 M solution of trimethylsilyl triflate in
dichloromethane (0.80 mL, 200 µmol), and stirring was continued for
(18) Hostomsky, Z.; Smrt, J.; Arnold, L.; Toc´ık, Z.; Paces, V. Nucleic
Acids Res. 1987, 15, 4849-4856.

11762 J. Am. Chem. Soc., Vol. 119, No. 49, 1997
Hayakawa and Kataoka
10 min. MS 13X was removed by filtration, and the filtrate was
concentrated to give a gummy oil. This material was dissolved in 3%
dichloroacetic acid in a 3:97 mixture of methanol and dichloromethane
(100 mL)19 and stirred for 10 min. The solution was poured into an
aqueous sodium hydrogen carbonate solution (200 mL). The separated
organic layer was washed with brine (100 mL) and concentrated to the
half-volume. The resulting solution was poured into a 1:1 mixture of
diethyl ether and hexane (500 mL) to precipitate 19 (2.34 g, 95%) as
a colorless powder, which was collected by filtration: IR 1751, 1701,
1609, 1534, 1462, 1414 cm-1; UV λmax 215 (ꢀ 30 500), 267 nm
lammonium salts of 21 (4.90 g, 115 800 OD260, 52% overall yield from
19) as a colorless solid: 31P NMR δ -0.77. The HPLC analysis of
the product with a 0.1 M triethylammonium acetate buffer containing
5-15% acetonitrile (v/v) (linear gradient in 30 min) indicated that the
purity of 21 is >98%.
Enzymatic Digestion of the Octamer 21 and HPLC Analysis of
the Resulting Products. A mixture of the purified octamer 21 (0.40
OD260) in H2O (10 µL), snake venom phosphodiesterase (1 µL, 0.1
unit), bacterial alkaline phosphatase (10 µL, 2.5 units), 300 mM Tris-
HCl (5 µL), and 300 mM MgCl2 (2.5 µL) was incubated at 37 °C for
24 h and then heated at 90 °C for 1 h. The aliquot (5 µL) was subjected
directly to HPLC analysis with a 9:1 mixture of H2O-MeOH as eluent
(1 mL/min, 40 °C), which showed four peaks due to dC (tR ) 3.5
min), dG (4.6 min), T (5.8 min), and dA (7.0 min) in a 3.0:0.9:3.1:0.9
ratio. No N-AOC-protected nucleosides were detected.
1
(19 200); H NMR δ 1.94, 1.95 (2 s, 3H), 2.30-2.44 (m, 1H), 2.47-
2.62 (m, 2H), 3.17-3.29 (m, 1H), 3.78-3.99 (m, 3H), 4.27-4.31 (m,
1H), 4.32-4.44 (m, 3H), 4.60-4.66 (m, 4H), 4.69 (d, 2H, J ) 5.4
Hz), 4.83 (ddd, 1H, J ) 26.6, 10.0, 3.4 Hz), 5.09 (d, 2H, J ) 5.8 Hz),
5.23-5.49 (m, 10H), 5.87-6.02 (m, 2H), 6.14 (ddt, 1H, J ) 10.5,
17.4, 5.4 Hz), 6.24-6.33 (m, 2H), 7.37, 7.39 (2 s, 1H), 7.61 (s, 1H),
7.89, 7.90 (2 s, 1H), 8.64-8.79 (2 br s, 1H); 31P NMR δ -1.7, -1.0.
Anal. Calcd for C34H42N7O15P: C, 49.82; H, 5.16, N, 11.96. Found:
C, 49.95; H, 5.08; N, 11.95.
Acknowledgment. This work was partially supported by
Grants-in-Aid for Scientific Research (Nos. 08454200 and
09273228), a Grant-in-Aid for Scientific Research on Priority
Areas (No. 09874152) (Y. H.), and a Grant-in-Aid for JSPS
Fellows (No. 80003104) (M.K.) from the Ministry of Education,
Science, Sports and Culture, Japan, and the Takeda Foundation.
Synthesis of d(5′CTACCTGT3′) (21). Elongation of the 5′-O-free
nucleotide 19 was achieved via a procedure similar to that for the
formation 19 described above to give the protected octamer 20 as a
colorless powder (5.45 g, 57% overall yield). The whole crude product
was dissolved in THF (50 mL) containing degassed H2O (10 mL), and
to this were added Pd[P(C6H5)3]4 (593 mg, 513 µmol), P(C6H5)3 (83.1
mg, 313 µmol), and diethylammonium hydrogen carbonate (5.54 g,
41.0 mmol). The solution was stirred at 25 °C for 3 h and then poured
into dichloromethane (50 mL) with vigorous stirring to give diethy-
Supporting Information Available: Characterization data
including 1H and 31P NMR spectral charts for all new
compounds (20 pages). See any current masthead page for
ordering or Internet access instructions.
(19) Habus, I.; Agrawal, S. Nucleic Acids Res. 1994, 22, 4350-4351.
JA970685B