Introduction of 3-terminal nucleosides having a silyl-type linker into polymer supports without base protection

Article abstract of DOI:10.1021/jo9000965

New 3 -terminal deoxyribonucleoside-loading reagents having a silyl-type linker were developed. They were effectively introduced into polymer supports under the conditions of Huisgen [3 + 2] cycloaddition without base protection. Moreover, four unmodified

Full text of DOI:10.1021/jo9000965

Introduction of 3′-Terminal Nucleosides Having a Silyl-Type Linker
into Polymer Supports without Base Protection
Akihiro Ohkubo, Yasuhiro Noma, Katsufumi Aoki, Hirosuke Tsunoda, Kohji Seio, and
Mitsuo Sekine*
Department of Life Science, Tokyo Institute of Technology, Yokohama 226-8501, Japan
msekine@bio.titech.ac.jp
ReceiVed January 15, 2009
New 3′-terminal deoxyribonucleoside-loading reagents having a silyl-type linker were developed. They
were effectively introduced into polymer supports under the conditions of Huisgen [3 + 2] cycloaddition
without base protection. Moreover, four unmodified DNA oligomers d[TACCTAAATCCAX] (X ) T,
A, C, and A) and a base-labile modified DNA 12mer d[A*C*T*C*C*GT*C*T*A*C*G] 16 (A* ) 6-N-
acetyl-8-aza-7-deaza-2′-deoxyadenosine, C* ) 4-N-acetyl-2′-deoxyctydine, T* ) 2-thio-T) were suc-
cessfully synthesized by cleavage of the silyl-type linker using Bu4NF under neutral conditions in our
N-unprotected phosphoramidite method. In this paper, we also report a new reaction of chlorination of
cytosine base using 1,3-dichloro-5,5-dimethylhydantoin.
Introduction
but also oligonucleotides having various functional groups2 are
required for current research because the diversification of
studies in these fields has progressed considerably. However,
some useful oligonucleotides having base-labile functional
groups, such as aminoacylated RNAs,3 N-acylated oligonucle-
otides,4 and 2′-O-acyloxymethyl RNAs,5 cannot be synthesized
via solid-phase synthesis using the standard phosphoramidite
procedure.1 This is because the latter method uses ammonium
The fields of chemical biology, biotechnology, and molecular
biology have progressed rapidly following the development of
the phosphoramidite method as a practical procedure for DNA
and RNA synthesis by Caruthers and co-workers,1 largely
because researchers can easily obtain oligonucleotides having
the desired sequences. Not only unmodified oligonucleotides
(1) (a) Beaucage, S. L.; Caruthers, M. H. Tetrahedron Lett. 1981, 22, 1859–
1862. (b) Matteucci, M. D.; Caruthers, M. H. J. Am. Chem. Soc. 1981, 103,
3185–3191. (c) Beaucage, S. L., Bergstrom, D. E., Glick, G. D., Jones, R. A.,
Eds. Current Protocols in Nucleic Acid Chemistry; John Wiley & Sons, Inc.:
New York, 2000.
(2) (a) Stec, W. J.; Zon, G.; Egan, W.; Stec, B. J. Am. Chem. Soc. 1984,
106, 6077–6079. (b) Miller, P. S.; Agris, C. H.; Murakami, A.; Reddy, M. P.;
Spitz, S. A.; Ts’o, P. O. Nucleic Acids Res. 1983, 11, 6225–6242. (c) Nielsen,
P. E.; Egholm, M.; Berg, R. H.; Buchardt, O. Science 1991, 254, 1497–1500.
(d) Obika, S.; Nanbu, D.; Hari, Y.; Morio, K.; In, Y.; Ishida, T.; Imanishi, T.
Tetrahedron Lett. 1997, 39, 5401–5404. (e) Xu, Y.; Kool, E. T. J. Am. Chem.
Soc. 2000, 122, 9040–9041. (f) Gartner, Z. J.; Kanan, M. W.; Liu, D. R. J. Am.
Chem. Soc. 2002, 124, 10304–10306. (g) Saneyoshi, H.; Seio, K.; Sekine, M. J.
Org. Chem. 2005, 70, 10453–10460. (h) Ohkubo, A.; Aoki, K.; Ezawa, Y.; Sato,
Y.; Taguchi, H.; Seio, K.; Sekine, M. Tetrahedron Lett. 2005, 46, 8953–8957.
(3) (a) Robertson, S. A.; Ellman, J. A.; Schultz, P. G. J. Am. Chem. Soc.
1991, 113, 2722–2729. (b) Lodder, M.; Wang, B.; Hecht, S. M. Methods 2005,
36, 245–251.
(4) (a) Wada, T.; Kobori, A.; Kawahara, S.; Sekine, S. Eur. J. Org. Chem.
2001, 4583–4593. (b) Wada, T.; Kobori, A.; Kawahara, S.; Sekine, M.
Tetrahedron Lett. 1998, 39, 6907–6910. (c) Ohkubo, A.; Kasuya, R.; Sakamoto,
K.; Miyata, K.; Nagasawa, H.; Tsukahara, T.; Maki, Y.; Watanobe, T.; Seio,
K.; Sekine, M. Nucleic Acids Res. 2008, 36, 1952–1964.
(5) Parey, N.; Baraguey, C.; Vasseur, J.-J.; Debart, F. Org. Lett. 2006, 8,
3869–3872.
(6) (a) Ohkubo, A.; Seio, K.; Sekine, M. Tetrahedron Lett. 2004, 45, 363–
366. (b) Ohkubo, A.; Seio, K.; Sekine, M. J. Am. Chem. Soc. 2004, 126, 10884–
10896. (c) Ohkubo, A.; Kuwayama, Y.; Kudo, T.; Tsunoda, H.; Seio, K.; Sekine,
M. Org. Lett. 2008, 10, 2793–2796.
10.1021/jo9000965 CCC: $40.75
Published on Web 02/27/2009
2009 American Chemical Society
J. Org. Chem. 2009, 74, 2817–2823 2817

Ohkubo et al.
SCHEME 1. Synthesis of T- and dA-Loading Reagents 1a
and 1b without Base Protection
FIGURE 1. Skeletons of previous N-protected loading reagents and
loading reagents 1a-d containing a silyl linker without base protection.
hydroxide for the removal of the protecting groups and their
release from polymer supports; base-labile functional groups
are decomposed by this treatment. To overcome this serious
problem, we developed a so-called “activated phosphite
method”6 for O-selective condensation using 3′-terminal deoxy-
ribonucleoside-loading reagents having a silyl-type linker6b,7 that
can be cleaved under neutral conditions. Using this approach,
we succeeded in synthesizing oligonucleotides containing base-
labile deoxynucleoside derivatives such as N4-acetyl-2′-deoxy-
cytidine or N6-acetyl-8-aza-7-deaza-2′-deoxyadenosine in good
yields. Nevertheless, there is still a problem: the amino groups
of the previous loading reagents7 must be protected to avoid
side reactions on the nucleobases since the loading reaction
requires a coupling between the amino group on the polymer
supports and the carboxyl group of the silyl linker attached to
the 3′-OH group of the nucleosides (Figure 1). Therefore, in
the synthesis of oligonucleotides incorporating base-labile
functional groups using our N-unprotected phosphoramidite
approach, we needed to treat the resin with methylamine to
remove the base-protecting group from the 3′-terminal N-
protected deoxynucleoside-loaded resin. Unfortunately, we
observed that 6-18% of the once-loaded deoxynucleoside was
partially eliminated from the resins by treatment with methy-
lamine. In addition, various 3′-terminal nucleosides having base-
labile functional groups at the same position cannot be used
because they decompose under the treatment with methylamine,
such as 2′-O-modifided nucleosides or 5-modifided pyrimidines.
If a 3′-terminal nucleoside component can be introduced into
the resin without base protection, the problems just described
can be resolved because then the first deprotection step would
be unnecessary.
SCHEME 2. Silylation of DMT-C 6c Using the Silane
Derivative 5 in the Presence of
1,3-Dichloro-5,5-dimethylhydantoin
Results and Discussion
Scheme 1 shows an efficient synthetic route to the T- and
A-loading reagents 1a and 1b, respectively. Compound 2 was
first esterified to increase the solubility in THF. Subsequently,
the resulting ester 3 was reduced to alcohol 4 using LiAlH4 in
THF. The introduction of an azido group was carried out using
CBr4-Ph3P-NaN312 to obtain compound 5 in 78% yield. This
product was converted to a silyl chloride derivative by treatment
with 2 equiv of 1,3-dichloro-5,5-dimethylhydantoin for 2 h. The
in situ generated silyl chloride derivative was allowed to react
with 5′-O-DMTr-T 6a and 5′-O-DMTr-dA 6b without base
protection in the presence of imidazole to produce compounds
1a and 1b in 93 and 65% yields, respectively. However, when
5′-O-DMTr-dC was used under the same conditions, instead of
the desired 3′-O-silylated product, an unexpected species was
obtained (Scheme 2). Interestingly, the NMR and mass data of
this unexpected product suggested that not only silylation of
the 3′-hydroxyl group but also chlorination of the cytosine base
had occurred. Since the 5- and 6-protons of the cytosine base
were clearly observed in the 1H NMR spectrum (see Supporting
Information), there was a possibility that the chlorination had
occurred on the exo 4-amino group or heterocyclic 3-nitrogen.
Such an unusual chlorination of the cytosine base has not been
reported until now. To determine the location where the chloro
group was introduced, a similar reaction was carried out using
a 15N-labeled 3′,5′-O-bis(TBDMS)-dC derivative 8 (Scheme 3).
We found that the amino group of 8 readily reacted with 1,3-
dichloro-5,5-dimethylhydantoin within 10 min to produce a
similar base-chlorinated product 9 that could be isolated as a
stable material in 93% yield by silica gel column chromatog-
raphy. Since the peak of an imino proton observed at around
Copper-catalyzed Huisgen [3 + 2] cycloadditions between
azido and alkyne groups, which were developed by Meldal8
and Sharpless,9 have been widely used. Recently, some groups
have reported that the reaction could be used to functionalize
alkyne-modified oligonucleotides without base protection in
good yield.10 Those results indicated that the N-unprotected
loading reagents 1a-d having an azido group (Figure 1) could
be efficiently incorporated into alkyne-modified resins without
side reactions. We report the synthesis of the reagents 1a-d
and their introduction into highly cross-linked polystyrene11
(HCP) resins without base protection.
(7) Ohkubo, A.; Kasuya, R.; Aoki, K.; Kobori, A.; Taguchi, H.; Seio, K.;
Sekine, M. Bioorg. Med. Chem. 2008, 16, 5345–5351.
(8) Tornøe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002, 67,
3057–3064.
(9) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew.
Chem., Int. Ed. 2002, 41, 2596–2599.
(10) (a) Burley, G. A.; Gierlich, J.; Mofid, M. R.; Nir, H.; Tal, S.; Eichen,
Y.; Carell, T. J. Am. Chem. Soc. 2006, 128, 1398–1399. (b) Seela, F.; Sirivolu,
V. R. Chem. BiodiV. 2006, 3, 509–514. (c) Kocalka, P.; El-Sagheer, A. H.; Brown,
T. Chembiochem 2008, 9, 1280–1285. (d) Gramlich, P. M. E.; Warncke, S.;
Gierlich, J.; Carell, T. Angew. Chem., Int. Ed. 2008, 47, 3442–3444.
(11) McCollum, C.; Andrus, A. Tetrahedron Lett. 1991, 32, 4069–4072.
(12) (a) Hata, T.; Yamamoto, I.; Sekine, M. Chem. Lett. 1975, 977–980. (b)
Yamamoto, I.; Sekine, M.; Hata, T. J. Chem. Soc., Perkin Trans. 1 1980, 306–
310.
2818 J. Org. Chem. Vol. 74, No. 7, 2009

3′-Terminal Nucleosides HaVing a Silyl-Type Linker
SCHEME 3. Chlorination of 15N-Labeled dC Derivative 8
Using 1,3-Dichloro-5,5-dimethylhydantoin
SCHEME 5. Introduction of 3′-Terminal
Nucleoside-Loading Reagents 1 into HCP Resins by
Copper-Catalyzed Huisgen [3 + 2] Cycloaddition
SCHEME 4. Synthesis of dC- and dG-Loading Reagents 1c
and 1d Using Acetyl or DMF Groups
27, 30, 27, and 34 µmol/g, respectively. For the first time, we
could efficiently introduce an N-unprotected nucleoside into
polymer supports. Moreover, we also found by the DMTr cation
assay that these deoxynucleosides could be completely released
from the resins by treatment with a 1 M THF solution of
TBAF-AcOH for 6 h under neutral conditions, although release
of the thymidine residue from the resin 13a by treatment with
a 0.2 M THF solution of Et3N-3HF was very slow (tcomp
24 h), as shown in Figure 2.
)
Further, we demonstrated the synthesis of four DNA oligo-
mers d[TACCTAAATCCAX] (X ) T, A, C, and A) by the
activated phosphite method (Scheme 6). Each chain elongation
was carried out using 6-nitro-1-hydroxybenzotriazole (HOnBt)
in the presence of benzimidazolium triflate14 (BIT) as an
activator on the resins 13a-d containing a silyl linker. After
chain elongation, selective removal of the 2-cyanoethyl groups
of the internucleotidic phosphates was carried out by treatment
with 1 M DBU in CH3CN for 1 min, and the successive removal
of the 5′-terminal DMTr group was carried out by treatment
with 3% trichloroacetic acid in CH2Cl2. Finally, the desired DNA
oligomers 15a-d were released from the resin by treatment
with 1 M Bu4NF (TBAF)-AcOH in THF at room temperature.
Each complete release of DNA oligomers required a prolonged
time (24 h; Figure 3), compared with that of only deoxynucleo-
sides. This decrease of reactivity in the Si-O bond cleavage
might result from the effect of anionic charges of the phosphate
groups. The crude products were analyzed by anion-exchange
HPLC (Figure 4). The purification produced the oligomers
15a-d in 44, 37, 52, and 33% yields, respectively. These
oligomers were characterized by MALDI-TOF mass spectrom-
etry. These results show that the 3′-terminal 2′-deoxynucleoside-
loaded HCP resins prepared using the loading reagents 1a-d
are very useful for the synthesis of DNA oligomers by the
activated phosphite method.
Finally, we carried out the synthesis of a base-labile modified
DNA 12mer d[A*C*T*C*C*GT*C*T*A*C*G] 16 (A* ) 6-N-
acetyl-8-aza-7-deaza-2′-deoxyadenosine: ac6az8c7A, C* ) 4-N-
acetyl-2′-deoxyctydine: ac4C, T* ) 2-thio-T: s2T).4c In our
recent study, it turned out that introduction of the modified base,
ac6az8c7A, ac4C, and s2T into DNA oligomers increased the
hybridization affinity for the cDNA oligomers and their base
recognition ability compared to the unmodified adenine base.
However, significant elimination of the acetyl group occurred
upon treatment with ammonium hydroxide for deprotection and
release of the DNA oligomer in the standard procedure for DNA
synthesis. Therefore, we carried out the synthesis of the modified
8.5 ppm was a singlet rather than a doublet, we concluded that
the chlorinated compound has a 15N-Cl bond instead of a
15N-1H bond at the 4-position. Although this chlorination was
an undesired side reaction in the present study, the facile
selective N-chlorination of the cytosine moiety might be very
interesting and useful for obtaining synthetic intermediates in
nucleoside and oligonucleotide chemistry.
We had previously carried out efficient silylation of an
N-acetyl dC derivative in the presence of 1,3-dichloro-5,5-
dimethylhydantoin. This result suggested that the protection for
the amino group of the cytosine base using an acetyl group could
completely avoid the undesired chlorination described previ-
ously. Therefore, 5′-O-DMTr-4-N-acetyl-dC 6d was employed
for introducing the silyl residue into its 3′-position (Scheme 4).
This reaction yielded the desired product, which in turn was
treated with 20% tBuNH2/MeOH. As a result, the dC-loading
reagent 1c was obtained in 81% yield via two steps. On the
other hand, for the synthesis of the dG-loading reagent 1d, the
guanine base was protected with an N,N-dimethylaminometh-
ylene (DMF) group to simplify purification of the target
compound. Thus, the 3′-O-silylated product was easily obtained,
and the DMF group was selectively removed by treatment with
2 M methylamine/THF13 to produce compound 1d in 50% yield
via two steps.
Next, we examined the efficiency of the introduction of the
3′-terminal nucleoside residues into polymer supports using the
loading reagents 1a-d under various conditions (Scheme 5).
We found that the loading reagents could be efficiently
introduced into the alkyne-loaded HCP resin 12 (the loading
amount of the terminal amino group: 34 µmol/g) in the presence
of CuI and Et3N under reflux for 12 h. The T-, dA-, dC-, and
dG-loading amounts were estimated by DMTr cation assay as
(13) Ohkubo, A.; Sakamoto, K.; Miyata, K.; Taguchi, H.; Seio, K.; Sekine,
M. Org. Lett. 2005, 8, 3869–3872.
(14) Hayakawa, Y.; Kataoka, M.; Noyori, R. J. Org. Chem. 1996, 61, 7996–
7997.
J. Org. Chem. Vol. 74, No. 7, 2009 2819

Ohkubo et al.
FIGURE 2. Release of nucleosides from the HCP resins 13a-d by treatment with 0.2 M THF solution of Et3N-3HF or 1 M THF solution of
TBAF-AcOH.
FIGURE 3. Release of oligonucleotides 15a-d from the HCP resins by treatment with 1 M THF solution of TBAF-AcOH.
SCHEME 6. Synthesis of DNA Oligomers in the Activated Phosphite Method with 3′-Terminal Nucleoside-Loaded HCP Resins
13 Containing a Silyl Linker
DNA 16 by the activated phosphite method using a G-loaded
HCP resin 13d with a silyl linker. As a result, the modified
DNA 16 could be synthesized without decomposition and the
oligomer 16 was obtained in 41% yield (Figure 4e). This
oligomer was characterized by MALDI-TOF mass spectrometry.
into HCP resins without side reactions under the conditions of
Huisgen [3 + 2] cycloaddition. Moreover, we succeeded in
synthesizing DNA oligomers using the nucleoside-loaded resins
having a silyl linker by the activated phosphite method, without
using a standard procedure such as the addition of ammonium
hydroxide for release of oligomers from resins. These results
have encouraged us to study the synthesis of base-labile
oligonucleotides having functional groups at the 3′-terminal
position, such as aminoacylated RNAs. Further studies are now
underway in this direction.
Conclusion
We report the first synthesis of 3′-terminal N-unprotected
deoxynucleoside-loading reagents 1a-d having a silyl linker.
We also found that these reagents can be effectively introduced
2820 J. Org. Chem. Vol. 74, No. 7, 2009

3′-Terminal Nucleosides HaVing a Silyl-Type Linker
FIGURE 4. Anion-exchange HPLC profiles of the crude mixtures obtained by the activated phosphite method: (a) d[TACCTAAATCCAT] 15a,
(b) d[TACCTAAATCCAA] 15b, (c) d[TACCTAAATCCAC] 15c, (d) d[TACCTAAATCCAG] 15d, and (e) d[A*C*T*C*C*GT*C*T*A*C*G]
16, A* ) 6-N-acetyl-8-aza-7-deaza-A, C* ) 4-N-acetyl-C, T* ) 2-thio-T.
134.4, 135.9, 136.2. Anal. Calcd for C13H21N3Si: C, 63.11; H, 8.45;
N, 16.83. Found: C, 63.42; H, 8.45; N, 16.83.
Experimental Section
Synthesis of Compound 3. Compound 2 (9 g, 38 mmol) was
added to 5% H2SO4 in MeOH (300 mL). After being refluxed with
stirring for 1 h, the mixture was partitioned between CH2Cl2 (300
mL) and brine (300 mL). The organic phase was collected, dried
over Na2SO4, filtered, and evaporated under reduced pressure. The
residue was chromatographed on a column of silica gel (150 g)
Synthesis of Compound 1a. Compound 5 (742 mg, 3 mmol)
was rendered anhydrous by repeated coevaporation with dry CH3CN
(3 mL × 3) and finally dissolved in dry CH2Cl2 (15 mL). To the
mixture was added 1,3-dichloro-5,5-dimethylhydantoin (1.18 g, 6
mmol). After being stirred at room temperature for 2 h, imidazole
(1.02 g, 15 mmol) and 5′-O-(4,4′-dimethoxytrityl)thymidine 6a
(1.68 g, 2.7 mmol) were added. After the mixture was stirred at
room temperature for 2 h, the mixture was partitioned between
CHCl3 (100 mL) and brine (100 mL). The organic phase was
collected, dried over Na2SO4, filtered, and evaporated under reduced
pressure. The residue was chromatographed on a column of silica
gel (40 g) with hexane/CHCl3 (50:50-0:100, v/v) containing 1%
pyridine and then CHCl3/MeOH (100:0-97:3, v/v) containing 1%
pyridine to give 1a (1.98 g, 93%): 1H NMR (CDCl3) δ 0.94-1.07
(m, 12H), 1.17-1.27 (m, 2H), 1.47 (s, 3H), 2.19-2.29 (m, 1H),
2.41-2.48 (m, 1H), 3.25 (dd, 1H, J ) 2.4, 10.5 Hz), 3.47 (dd, 1H,
J ) 2.3, 10.6 Hz), 3.79 (s, 6H), 4.13 (d, 1H, J ) 2.2 Hz), 4.35 (s,
2H), 4.64-4.66 (m, 1H), 6.47 (dd, 1H, J ) 5.8, 7.7 Hz), 6.79 (dd,
4H, J ) 2.2, 8.9 Hz), 7.23-7.37 (m, 11H), 7.48 (d, 2H, J ) 8.1
Hz), 7.65 (s, 1H), 8.32 (br s, 1H); 13C NMR (CDCl3) δ 11.8,
11.9,12.0, 12.5, 17.3, 40.6, 41.8, 54.7, 55.2, 62.4,63.3, 72.9, 73.4,
77.2, 81.4, 84.9, 86.9, 87.2, 87.5, 88.0, 111.1, 113.1, 113.2, 127.0,
127.1, 127.4, 127.5, 127.7, 127.8, 127.9, 128.0, 129.1, 130.0, 133.8,
133.9, 134.9, 135.0, 135.3, 135.6, 136.7, 137.1, 139.4, 144.2, 147.3,
150.2, 150.3, 158.6, 158.7, 163.5, 163.7; HRMS (ESI) calcd for
[C44H51N5O7Si + Na]+ 812.3455, found 812.3461.
Synthesis of Compound 1b. Compound 5 (247 mg, 1 mmol)
was rendered anhydrous by repeated coevaporation with dry CH3CN
(3 mL × 3) and finally dissolved in dry CH2Cl2 (5 mL). To the
mixture was added 1,3-dichloro-5,5-dimethylhydantoin (394 mg,
2 mmol). After being stirred at room temperature for 2 h, imidazole
(340 mg, 5 mmol) and 5′-O-(4,4′-dimethoxytrityl)-2′-deoxyadenos-
ine 6b (498 mg, 0.9 mmol) were added. After the mixture was
stirred at room temperature for 2 h, the mixture was partitioned
between CHCl3 (50 mL) and brine (50 mL). The organic phase
was collected, dried over Na2SO4, filtered, and evaporated under
reduced pressure. The residue was chromatographed on a column
of silica gel (15 g) with hexane/CHCl3 (50:50-0:100, v/v)
containing 1% pyridine and then CHCl3/MeOH (100:0-97:3, v/v)
containing 1% pyridine to give 1b (469 mg, 65%): 1H NMR
1
with hexane/AcOEt (100:0-95:5, v/v) to give 3 (7.2 g, 76%): H
NMR (CDCl3) δ 0.93-1.06 (m, 12H), 1.18-1.27 (m, 2H), 3.90
(s, 3H), 3.96 (t, 1H, J ) 3.2 Hz), 7.58 (d, 2H, J ) 8.1 Hz), 7.98
(d, 2H, J ) 8.1 Hz); 13C NMR (CDCl3) δ 10.6, 18.5, 18.6, 52.2,
128.1, 128.2, 128.3, 130.5, 140.6, 167.1. Anal. Calcd for
C14H22O2Si: C, 67.15; H, 8.86. Found: C, 67.27; H, 8.68.
Synthesis of Compound 4. LiAlH4 (1.76 g, 46.5 mmol) was
dissolved in dry THF (50 mL). To the solution was added dropwise
the dry THF solution (50 mL) of compound 3 (10.6 g, 42.3 mmol)
at 0 °C. After the mixture was stirred at room temperature for 15
min, AcOEt (20 mL) was added dropwise to the mixture. The
mixture was filtered using a Celite bed. The mixture was partitioned
between CH2Cl2 (500 mL) and 0.2 M aqueous HCl (300 mL). The
organic phase was collected, dried over Na2SO4, filtered, and
evaporated under reduced pressure. The residue was chromato-
graphed on a column of silica gel (150 g) with hexane/AcOEt (100:
0-90:10, v/v) to give 4 (9.4 g, 85%): 1H NMR (CDCl3) δ
0.96-1.07 (m, 12H), 1.17-1.23 (m, 2H), 3.09 (br s, 1H), 3.94 (t,
1H, J ) 3.2 Hz), 4.58 (s, 2H), 7.29 (d, 2H, J ) 7.6 Hz), 7.48 (d,
2H, J ) 7.6 Hz); 13C NMR (CDCl3) δ 10.7, 18.4, 18.6, 64.8.2,
126.0, 132.9, 135.4, 141.6. Anal. Calcd for C13H22OSi: C, 70.21;
H, 9.97. Found: C, 69.93; H, 10.00.
Synthesis of Compound 5. Compound 4 (440 mg, 2 mmol) was
rendered anhydrous by repeated coevaporation with dry CH3CN
and finally dissolved in dry DMF (4 mL). To the solution were
added CBr4 (680 m g, 2.1 mmol), PPh3 (540 mg, 2.1 mmol), and
NaN3 (650 mg, 10 mmol). After the mixture was stirred at room
temperature for 12 h, the mixture was partitioned between AcOEt
(50 mL) and brine (50 mL). The organic phase was collected, dried
over Na2SO4, filtered, and evaporated under reduced pressure. The
residue was chromatographed on a column of silica gel (10 g) with
1
hexane/AcOEt (100:0-90:10, v/v) to give 4 (380 mg, 78%): H
NMR (CDCl3) δ 0.97-1.11 (m, 12H), 1.18-1.29 (m, 2H), 3.95 (t,
1H, J ) 3.1 Hz), 4.36 (s, 2H), 7.30 (d, 2H, J ) 7.8 Hz), 7.54 (d,
2H, J ) 8.1 Hz); 13C NMR (CDCl3) δ 10.6, 18.4, 18.6, 54.8, 127.3,
J. Org. Chem. Vol. 74, No. 7, 2009 2821

Ohkubo et al.
(CDCl3) δ 0.97-1.05 (m, 12H), 1.25-1.28 (m, 2H), 2.52-2.58
(m, 1H), 2.75-2.80 (m, 1H), 3.29 (dd, 1H, J ) 4.4, 10.5 Hz), 3.40
(dd, 1H, J ) 4.4, 10.4 Hz), 3.80 (s, 6H), 4.28 (d, 1H, J ) 2.4 Hz),
4.35 (s, 2H), 4.73 (t, 1H, J ) 2.7 Hz), 6.04 (br s, 2H), 6.50 (t, 1H,
J ) 6.7 Hz), 6.77 (d, J ) 8.8 Hz), 7.20-7.28 (m, 9H), 7.35 (d,
2H, J ) 6.8 Hz), 7.51 (d, 2H, J ) 7.6 Hz), 8.02 (s, 1H), 8.28 (s,
1H); 13C NMR (CDCl3) δ 12.1, 12.2, 17.5, 41.0, 54.9, 55.3, 63.7,
73.7, 84.7, 86.7, 87.3, 113.3, 120.3, 127.0, 127.6, 128.3, 130.2,
134.1, 135.1, 135.8, 136.9, 139.3, 144.7, 149.8, 152.9, 155.5, 158.7;
HRMS (ESI) calcd for [C44H50N8O5Si + H]+ 799.3752, found
799.3751.
Synthesis of Compound 7. Compound 5 (65 mg, 0.26 mmol)
was rendered anhydrous by repeated coevaporation with dry CH3CN
(3 mL × 3) and finally dissolved in dry CH2Cl2 (5 mL). To the
mixture was added 1,3-dichloro-5,5-dimethylhydantoin (102 mg,
0.52 mmol). After being stirred at room temperature for 2 h,
imidazole (88 mg, 1.3 mmol) and 5′-O-(4,4′-dimethoxytrityl)-2′-
deoxycytidine 6c (124 mg, 0.23 mmol) were added. After the
mixture was stirred at room temperature for 30 min, the mixture
was partitioned between CHCl3 (50 mL) and brine (50 mL). The
organic phase was collected, dried over Na2SO4, filtered, and
evaporated under reduced pressure The residue was chromato-
graphed on a column of silica gel (10 g) with hexane/CHCl3 (50:
50-0:100, v/v) containing 1% pyridine and then CHCl3/MeOH
(100:0-97:3, v/v) containing 1% pyridine to give 7 (101 mg, 53%):
1H NMR (CDCl3) δ 0.96-1.07 (m, 12H), 1.20-1.26 (m, 2H),
2.14-2.20 (m, 1H), 2.39-2.44 (m, 1H), 3.32 (d, 1H, J ) 7.5 Hz),
3.46 (d, 1H, J ) 7.5 Hz), 3.79 (s, 1H), 4.11 (s, 1H), 4.36 (s, 2H),
4.65 (t, 1H, J ) 6.3 Hz), 5.49 (d, 1H, J ) 8.0 Hz), 6.37 (t, 1H, J
) 6.5 Hz), 6.81-6.84 (m, 4H), 7.22-7.50 (m, 14H), 8.43 (s, 1H);
13C NMR (CDCl3) δ 11.9, 12.0, 12.4, 17.3, 41.5, 54.6, 54.7, 55.1,
55.2, 55.3, 62.8, 62.9, 63.0, 72.7, 72.8, 77.2, 84.7, 84.8, 86.8, 86.9,
99.9, 113.1, 113.2, 127.1, 12.3, 127.4, 127.5, 127.9, 128.0, 128.1,
130.0, 130.1, 133.1, 133.7, 134.6, 134.9, 135.0, 135.2, 136.8, 144.1,
148.3, 156.3, 158.6; HRMS (ESI) calcd for [C43H49ClN6O6Si +
Na]+ 831.3069, found 831.3049.
and evaporated under reduced pressure. The residue was finally
evaporated by repeated coevaporation three times each with toluene
and CHCl3 to remove the last traces of pyridine. Subsequently, the
residue was dissolved in 20% tBuNH2/MeOH (10 mL). After the
mixture was stirred at room temperature for 7.5 h, the mixture was
evaporated under reduced pressure. The residue was chromato-
graphed on a column of silica gel (15 g) with hexane/CHCl3 (50:
50-0:100, v/v) containing 1% pyridine and then CHCl3/MeOH
(100:0-97:3, v/v) containing 1% pyridine to give 1c (564 mg,
1
81%): H NMR (CDCl3) δ 0.90-0.96 (m, 12H), 1.03-1.18 (m,
2H), 2.11-2.19 (m, 1H), 2.56-2.62 (m, 1H), 3.27 (dd, 1H, J )
3.2, 10.5 Hz), 3.52 (dd, 1H, J ) 2.7, 10.8 Hz), 3.77 (s, 6H), 4.10
(d, 1H, J ) 3.8 Hz), 4.32 (s, 2H), 4.55-4.59 (m, 1H), 5.42 (d, 1H,
J ) 7.3 Hz), 6.32 (t, 1H, J ) 5 0.7 Hz), 6.81 (dd, 4H, J ) 2.2, 8.6
Hz), 7.20-7.47 (m, 13H), 7.99 (d, 1H, J ) 7.3 Hz); 13C NMR
(CDCl3) δ 12.2, 17.5, 42.7, 54.9, 55.4, 62.1, 77.2, 86.4, 86.8, 86.9,
94.0, 113.3, 113.4, 127., 127.6, 128.0, 128.3, 128.4, 130.3, 133.3,
134.0, 135.1, 135.5, 136.8, 141.6, 144.4, 155.9, 158.8, 165.5; HRMS
(ESI) calcd for [C43H50N8O6Si + Na]+ 797.3459, found 797.3452.
Synthesis of Compound 1d. Compound 5 (136 mg, 0.55 mmol)
was rendered anhydrous by repeated coevaporation with dry CH3CN
(3 mL × 3) and finally dissolved in dry CH2Cl2 (5 mL). To the
mixture was added 1,3-dichloro-5,5-dimethylhydantoin (217 mg,
1.1 mmol). After being stirred at room temperature for 2 h,
imidazole (170 mg, 2.5 mmol) and 5′-O-(4,4′-dimethoxytrityl)-N2-
dimethylaminomethylene-2′-deoxyguanosine 6e (312 mg, 0.5 mmol)
were added. After the mixture was stirred at room temperature for
2 h, the mixture was partitioned between CHCl3 (50 mL) and brine
(50 mL). The organic phase was collected, dried over Na2SO4,
filtered, and evaporated under reduced pressure. The residue was
chromatographed on a column of silica gel (10 g) with hexane/
CHCl3 (50:50-0:100, v/v) containing 1% Et3N and then CHCl3/
MeOH (100:0-97:3, v/v) containing 1% Et3N to give the fractions
containing the desired nucleoside 11 having a Si-O bond. The
fractions were collected and evaporated under reduced pressure.
The residue was finally evaporated by repeated coevaporation three
times each with toluene and CHCl3 to remove the last traces of
pyridine. Subsequently, the residue was dissolved in 2 M MeNH2/
THF (5 mL). After the mixture was stirred at room temperature
for 7.5 h, the mixture was evaporated under reduced pressure. The
residue was chromatographed on a column of silica gel (10 g) with
hexane/CHCl3 (50:50-0:100, v/v) containing 1% pyridine and then
CHCl3/MeOH (100:0-97:3, v/v) containing 1% pyridine to give
1d (204 mg, 50%): 1H NMR (DMSO-d6) δ 0.90-0.958 (m, 12H),
1.15-1.24 (m, 2H), 2.34-2.50 (m, 1H), 2.69-2.43 (m, 1H),
3.10-3.25 (m, 2H), 3.71 (s, 6H), 3.98 (s, 1H), 4.46 (s, 2H), 4.59
(s, 1H), 6.19 (d, 1H, J ) 5.7 Hz), 6.43 (br s, 2H), 6.81 (d, 4H, J
) 7.0 Hz), 7.17-7.32 (m, 11H), 7.48 (d, 1H, J ) 5.7 Hz), 7.78 (d,
1H, J ) 4.6 Hz), 8.31 (d, 1H, J ) 5.9 Hz), 10.63 (br s, 1H); 13C
NMR (DMSO-d6) δ 11.1, 11.2, 17.1, 17.2, 24.6, 53.5, 55.0, 62.8,
63.5, 72.9, 79.1, 82.1, 85.7, 85.8, 112.2, 113.1, 116.8, 126.6, 127.6,
127.7, 129.6, 133.1, 134.6, 134.7, 134.8, 135.4, 137.0, 144.7, 144.7,
151.1, 153.7, 156.7, 158.0; HRMS (ESI) calcd for [C44H50N8O6Si
+ Na]+ 837.3520, found 837.3529.
Synthesis of Compound 9. Compound 8 (456 mg, 1 mmol) was
rendered anhydrous by repeated coevaporation with dry CH3CN
(3 mL × 3) and finally dissolved in dry CH2Cl2 (5 mL). To the
mixture was added 1,3-dichloro-5,5-dimethylhydantoin (216 mg,
1.1 mmol). After the mixture was stirred at room temperature for
10 min, the mixture was partitioned between CHCl3 (50 mL) and
brine (50 mL). The organic phase was collected, dried over Na2SO4,
filtered, and evaporated under reduced pressure. The residue was
chromatographed on a column of silica gel (15 g) with hexane/
CHCl3 (50:50-0:100, v/v) and then CHCl3/MeOH (100:0-97:3,
1
v/v) to give 9 (455 mg, 93%): H NMR (CDCl3) δ 0.00-0.041
(m, 12H), 0.69-0.82 (m, 18H), 1.89-1.99 (m, 1H), 2.11-2.19 (m,
1H), 3.64 (d, 1H, J ) 9.5 Hz), 3.75-3.80 (m, 2H), 4.28-4.33 (m,
1H), 5.69 (d, 1H, J ) 5.1 Hz), 6.17 (t, 1H, J ) 6.3 Hz), 7.34 (d,
1H, J ) 5.1 Hz), 8.48 (br s, 1H); 13C NMR (CDCl3) δ -5.7, -5.6,
-4.9, -4.7, 17.9, 18.2, 25.6, 25.8, 41.3, 62.4, 71.4, 84.7, 87.6,
99.7, 99.9, 132.9, 133.0, 148.3, 156.3, 156.4; HRMS (ESI) calcd
for [C21H40N215NO4Si + H]+ 491.2295, found 491.2283.
Synthesis of Compound 1c. Compound 5 (136 mg, 1 mmol)
was rendered anhydrous by repeated coevaporation with dry CH3CN
(3 mL × 3) and finally dissolved in dry CH2Cl2 (5 mL). To the
mixture was added 1,3-dichloro-5,5-dimethylhydantoin (394 mg,
2 mmol). After being stirred at room temperature for 2 h, imidazole
(340 mg, 5 mmol) and 5′-O-(4,4′-dimethoxytrityl)-N4-acetyl-2′-
deoxycytidine 6d (514 mg, 0.9 mmol) were added. After the mixture
was stirred at room temperature for 2 h, the mixture was partitioned
between CHCl3 (100 mL) and brine (100 mL). The organic phase
was collected, dried over Na2SO4, filtered, and evaporated under
reduced pressure. The residue was chromatographed on a column
of silica gel (15 g) with hexane/CHCl3 (50:50-0:100, v/v)
containing 1% Et3N and then CHCl3/MeOH (100:0-97:3, v/v)
containing 1% Et3N to give the fractions containing the desired
nucleoside 10 having a Si-O bond. The fractions were collected
Synthesis of Resin 12. A highly cross-linked polystyrene (HCP)
resin (1 g) having a benzylamino group (34 µmol/g) was washed
with dry CH3CN (1 mL × 3) and dried under reduced pressure.
The HCP resin was added in dry CH2Cl2 (10 mL) in a round flask.
To the mixture were added propiolic acid (20 µL, 170 µmol) and
DCC (175 mg, 850 µmol). After a round flask having the mixture
was attached to a rotary evaporator and gently rotated for 3 h, the
solvent was removed by filtration. The introduction of propiolic
acid described above was repeated again. The residual CPG was
washed by use of CH2Cl2 (1 mL × 3) and dried under reduced
pressure. Subsequently, the resin was dissolved in pyridine/Ac2O
(9:1, v/v, 10 mL) in a round flask. To the mixture was added
4-(dimethylamino)pyridine (30 mg, 240 µmol). After a round flask
having the mixture was attached to a rotary evaporator and gently
2822 J. Org. Chem. Vol. 74, No. 7, 2009

3′-Terminal Nucleosides HaVing a Silyl-Type Linker
rotated for 2 h, the solvent was removed by filtration to give the
HCP resin 12.
TOF mass calcd for [C126H161N45O75P12 + H]+ 3876.71, found
3874.87. Oligonucleotide 15b: d[TACCTAAATCCAA] MALDI-
TOF mass calcd for [C126H160N48O73P12 + H]+ 3885.72, found
3882.48. Oligonucleotide 15c: d[TACCTAAATCCAC] MALDI-
TOF mass calcd for [C125H160N46O74P12 + H]+ 3861.71, found
3866.52. Oligonucleotide 15d: d[TACCTAAATCCAG] MALDI-
TOF mass calcd for [C126H160N48O73P12 + H]+ 3901.72, found
3904.40. Oligonucleotide 16: d[A*C*T*C*C*GT*C*T*A*C*G],
A* ) 8-aza-7-deaza-A, C* ) 4-N-acetyl-C, T* ) 2-thio-T,
MALDI-TOF mass calcd for [C129H163N41O75P11S3 + H]+ 3922.64,
found 3921.70.
Synthesis of 3′-Terminal Nucleoside-Loaded Resins 13a-d.
The HCP resin 10 (118 mg) with was washed with dry CH3CN
(1 mL × 3) and dried under reduced pressure. The HCP resin was
added in dry THF (2 mL) in a round flask. To the mixture were
added 3′-terminal nucleoside-loading reagent 1 (40 µmol), Et3N
(5.52 µL, 40 µmol), and CuI (7.6 mg, 40 µmol). After being refluxed
for 12 h, the solvent was removed by filtration. The residual HCP
resin was washed by use of CH3CN (1 mL × 3) and dried under
reduced pressure. The amount of the nucleoside introduced into
the resin was estimated by use of the DMTr cation assay, as shown
in Scheme 5.
Synthesis of Oligonucleotides 15a-d and 16. The synthesis
of oligodeoxynucleotides was carried out on a HCP resin 13 having
a silyl linker in an ABI 392 DNA synthesizer by use of the reaction
cycle in activated phosphite method. The 2-cyanoethyl groups of
oligomer obtained after chain elongation was deprotected by
treatment with a 10% DBU solution in CH3CN (500 µL) at room
temperature for 1 min. Then, the mixture containing oligo DNAs
was released from the resin by treatment with a solution of TBAF
(1 M) and AcOH (1 M) in THF (500 µL) at room temperature for
24 h. The polymer support was removed by filtration and washed
with 0.1 M ammonium acetate buffer (1 mL × 3). The filtrate was
purified by anion-exchange HPLC to give oligonucleotides 15a-d
and 16. Oligonucleotide 15a: d[TACCTAAATCCAT] MALDI-
Acknowledgment. This work was supported by a Grant from
CREST of JST (Japan Science and Technology Agency) and a
Grant-in-Aid for Scientific Research from the Ministry of
Education, Culture, Sports, Science and Technology, Japan. This
work was supported in part by a grant of the Genome Network
Project from the Ministry of Education, Culture, Sports, Science
and Technology, Japan and by the global COE project.
Supporting Information Available: The 1H, 13C data of all
new products. This material is available free of charge via the
Internet at http://pubs.acs.org.
JO9000965
J. Org. Chem. Vol. 74, No. 7, 2009 2823