Efficient synthesis of functionalized oligodeoxyribonucleotides with base-labile groups using a new silyl linker

Article abstract of DOI:10.1016/j.bmc.2008.02.076

In this study, we developed new 3′-terminal deoxyribonucleoside-loading reagents 1 with a new silyl-type linker. These reagents could increase the efficiency of introduction of 3′-terminal deoxyribonucleoside components into polymer supports to a level of

Full text of DOI:10.1016/j.bmc.2008.02.076

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 5345–5351
Efficient synthesis of functionalized oligodeoxyribonucleotides
with base-labile groups using a new silyl linker
Akihiro Ohkubo, Rintaro Kasuya, Katsufumi Aoki, Akio Kobori,
Haruhiko Taguchi, Kohji Seio and Mitsuo Sekine*
Department of Life Science, Tokyo Institute of Technology and CREST, JST (Japan Science and Technology Agency), Japan
Received 24 January 2008; revised 22 February 2008; accepted 23 February 2008
Available online 29 February 2008
Abstract—In this study, we developed new 30-terminal deoxyribonucleoside-loading reagents 1 with a new silyl-type linker. These
reagents could increase the efficiency of introduction of 30-terminal deoxyribonucleoside components into polymer supports to a
level of 17–29 lmol/g. The efficiency was higher than that of previous T-loading reagents because reagents 1 contain a 4-aminobu-
tyryl residue as a spacer. Moreover, we could synthesize not only unmodified DNA oligomers but also a base-labile modified DNA
oligomer using resins 9a–d in the activated phosphite method without base protection.
ꢀ 2008 Published by Elsevier Ltd.
1. Introduction
chemistry to efficiently synthesize oligonucleotides con-
taining base-labile functional groups.30,31 In the acti-
vated phosphite method, we can omit the deprotection
step for the removal of the protecting groups of nucleo-
bases. If the oligonucleotides are released from polymer
supports under neutral conditions without using concd
NH3 aq in the final step of the activated phosphite meth-
od, we should overcome the above-mentioned problem.
DNA and RNA oligonucleotides have been used for the
suppression of specific genes,1–5 the exhaustive analysis
of a gene expression,6,7 and the detection of single nucle-
otide polymorphisms (SNPs)8–14 because they can accu-
rately recognize complementary oligonucleotides. A
number of modified oligonucleotides containing func-
tional groups have been developed to enhance their ef-
fects. For example, 20-O-modified RNA oligomers15–21
acquired nuclease resistance compared to unmodified
ones, and oligomers with modified nucleobases22–25 have
demonstrated increased accuracy of base recognition.
Among them, some modified oligomers with base-labile
functional groups,26–28 such as acyl groups. However,
such oligomers cannot be obtained if the standard phos-
phoramidite approach29 is employed in solid-phase syn-
thesis, because base-labile functional groups are easily
decomposed by treatment with concd NH3 aq for the re-
moval of base protecting groups and for their release
from polymer supports.
Kobori et al. previously proposed a loading reagent of a
silyl linker32 with a thymidine instead of a widely useful
linker such as a succinyl linker. This linker can be rap-
idly cleaved by treatment with tetrabutylammonium
fluoride (TBAF)–AcOH in THF under neutral condi-
tions. However, the loading reaction using the proposed
reagent requires a long time because the reactivity of the
reagent is much lower than that of a succinyl linker. The
loading amount of thymidine introduced into highly
cross-linked polystyrene (HCP) resins,33 which could in-
crease the coupling efficiency compared to controlled
pore glass (CPG) resins in the activated phosphite meth-
od was only 6–8 lmol/g for 1 day. This low reactivity
might result from the skeleton of the benzoic acid.
We have recently developed the ‘activated phosphite
method’ without base protection in phosphoramidite
In this paper, we report new loading reagents 1a–d
(Fig. 1) with a silyl linker and four kinds of 20-deoxynu-
cleosides to increase the reactivity of the reagent and the
synthesis of DNA oligomers using this silyl linker in the
activated phosphite method.
Keywords: DNA synthesis; Silyl linker; N-unprotected phosphorami-
dite approach; Solid-phase synthesis.
*
Corresponding author. Tel.: +81 45 924 5706; fax: +81 45 924
5772; e-mail: msekine@bio.titech.ac.jp
0968-0896/$ - see front matter ꢀ 2008 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2008.02.076

5346
A. Ohkubo et al. / Bioorg. Med. Chem. 16 (2008) 5345–5351
fication using silica-gel column chromatography was
pro
pro
B
80%. Compound 7 was converted to a silyl chloride
derivative by treatment with 1,3-dichloro-5,5-dimethyl-
hydantoin. The in situ generated silyl chloride was al-
lowed to react with 50-O-DMTr nucleosides (T, A, G
and C) in the presence of imidazole at room temperature
for 30 min. The amino groups of these 50-O-DMTr
nucleosides were protected with N,N-dibutylamino-
methylene (dbf), N,N-dimethylaminomethylene (dmf),
or acetyl groups to avoid side reactions with nucleobases
during the introduction of the silyl linker into the HCP
resin. For the synthesis of the loading reagent 1, the
selective deprotection of the Fm groups was carried
out by treatment with DBU in CH3CN at room temper-
ature for 10 min. The reagents 1a–d were obtained from
compound 7 via two steps using silica-gel column chro-
matography with 1% Et3N in 65%, 60%, 53%, and 68%
yields, respectively. Reagents 1a–d were condensed with
the amino groups of the HCP resin using DCC in
CH2Cl2 for 12 h. Finally, the capping reactions of these
resins by treatment with Ac2O and DMAP in pyridine
were carried out with the desired T-, dA-, dG-, and
dC-loaded HCP resins 8. The loading amounts of
nucleosides were estimated by DMTr cation assay as
27, 26, 31, and 21 lmol/g, respectively. For the first time,
we could introduce four kinds of 20-deoxyribonucleo-
sides bridged by a silyl linker to polymer supports.
B = T
(1a)
DMTrO
dbf6A (1b)
dmf2G (1c)
ac4C (1d)
O
O
O
Si
OH
O
N
H
1
Figure 1. Skeleton of loading reagents containing a silyl linker.
2. Result and discussion
To increase the loading amount of a nucleoside into
HCP resins, we designed new loading reagents 1 con-
taining a silyl linker that has a 4-aminobutyric acid res-
idue as a spacer. Scheme 1 shows an efficient synthetic
route for T-, A-, G-, or C-loaded HCP resins containing
a silyl linker. 1,4-dibromobenzene 2 was carried out
using stepwise halogen–metal exchange reactions to ob-
tain compound 4 according to the previous method.34
Compound 4 was converted to benzoyl chloride deriva-
tive 5 by treatment with SOCl2 in 77% yield. Subse-
quently, the reaction of compound
5
with 4-
aminobutylic acid in the presence of NaOH gave com-
pound 6 in 65% yield. The carboxyl group of compound
6 was protected by the 9-fluorenylmethyl (Fm) group
using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride (EDC) as a condensing agent to give
compound 7. The isolated yield of compound 7 by puri-
The protecting group should be removed at the first step
of DNA synthesis to avoid decomposition of a base-la-
bile functional group at the deprotection step when the
HSi(
i
Pr)2Cl
CO2
n
BuLi
nBuLi
HSi
COOH
HSi
Br
Br
Br
THF
rt
THF
rt
-78 oC
-78 oC
2
3
4
9-fluorenylmethanol
EDC DMAP
SOCl2
rt
4-aminobutyric acid
O
HSi
HSi
COCl
OH
N
H
1.0 M NaOH aq
rt
1.0 M NaOH aq
rt
O
5
6
Bpro
DMTrO
O
1,3-dichloro-5,5-
dimethylhydantoin
5'-DMTr nucleoside
Imidazole
O
HSi
OFm
N
H
O
CH2Cl2
rt
O
rt
O
7
Si
OFm
N
H
O
pro
O
Bpro
B
DMTrO
DMTrO
O
O
amino-loaded HCP
EDC DMAP
DBU
O
O
CH3CN
rt
O
-
+
H
N
CH2Cl2
Si
Si
O
HNEt3
N
H
N
H
rt
O
O
pro
pro
B = T
(1a)
B = T
(8a)
highly cross-linked
polystyrene (HCP)
dbf6A (1b)
dmf2G (1c)
ac4C (1d)
dbf6A (8b)
dmf2G (8c)
ac4C (8d)
Scheme 1. Synthesis of the nucleoside-loaded HCP resins 8 containing the silyl linker.

A. Ohkubo et al. / Bioorg. Med. Chem. 16 (2008) 5345–5351
5347
nucleoside-loaded HCP resins 8 were used in the acti-
vated phosphite method without base protection. There-
fore, we selected a non-aqueous treatment, MeNH2/
THF,34 to remove the protecting groups of nucleobases
without eliminating the nucleoside from the HCP resin.
First, the T-loaded HCP was treated with MeNH2/THF
for 6 h to estimate the stability of the Si–O bond. As a
result, the ratio of elimination of the nucleoside residue
from the resin was found to be less than 3%, estimated
by the DMTr cation assay. This result indicates that
the Si–O bond in the T-loaded HCP is stable under
the deprotection conditions. Subsequently, the deprotec-
tion of dbf6A-loaded HCP was carried out by treatment
with MeNH2/THF, as shown in Figure 2. The HPLC
analysis shows that the dbf group of the adenine was
completely cleaved in over 3 h. Unexpectedly, the elimi-
nation of the nucleoside residue from the resin increased
to 8%. These results indicate that the stability of the Si–
O bond depends on the kind of nucleobase present.35 In
the case of guanine, 6% elimination of the nucleoside
from the resin was observed during the deprotection
when the dmf group could be rapidly cleaved by treat-
ment with MeNH2/THF. The elimination of cytosine
surprisingly increased to a level of 25% in 3.5 h under
the same conditions. To suppress the elimination of
dC from the resin, the deprotection of the acetyl group
was carried out by treatment with 2 M NH3/MeOH in-
stead of MeNH2/THF. Because the nucleophilicity of
NH3 is lower than that of MeNH2, we could decrease
the elimination to 18% though the reaction rate of the
deprotection was slower than that by treatment with
2 M MeNH2/THF. Consequently, for the first time, we
could synthesize dA-, dG-, and dC-loaded HCP resins
9 containing a silyl linker without base protection. The
loading amounts of nucleosides were finally estimated
by the DMTr cation assay as 24, 29, and 17 lmol/g,
respectively.
paper, the first synthesis of oligodeoxynucleotides using
A-, G-, or C-loaded HCP resins (9b–d) containing the si-
lyl linker is reported. We demonstrated the synthesis of
three unmodified DNA oligomers using the activated
phosphite method and a silyl linker, as shown in Scheme
2. Each chain elongation was carried out using 6-nitro-
1-hydroxybenzotiazole36 in the presence of benzimi-
dazolium triflate (BIT)37 as an activator on the A-, G-,
or C-loaded HCP resins. After chain elongation, the
selective removal of the cyanoethyl groups of the inter-
nucleotidic phosphates was carried out by treatment
with 1 M DBU in CH3CN for 1 min and the successive
release of the 50-terminal DMTr group was carried out
by treatment with 3% trichloroacetic acid in CH2Cl2. Fi-
nally, the unmodified DNA oligomers 10–12 were re-
leased from the resin by treatment with 0.2 M
Et3NÆ3HF in THF at room temperature for 4 h. The
crude products were checked by anion-exchange HPLC,
as shown in Figure 3. Purification gave the oligomers
10–12 in 38%, 33%, and 41% yields, respectively. These
oligomers were characterized by MALDI-TOF mass
spectroscopy. These results show that the A-, G-, and
C-loaded HCP resins containing a silyl linker are very
useful for the synthesis of unmodified DNA oligomers
with the activated phosphite method. Furthermore, we
tried to synthesize a base-labile modified DNA 10mer
d[CCACGA*GTGG] 13 containing 6-acetyl-8-aza-7-
deaza-20-deoxyadenosine (A* = ac6az8c7A).38 In our
recent study, it turned out that introduction of the
modified base, ac6az8c7A, into DNA oligomers in-
creased the hybridization affinity for complementary
DNA oligomers and their base recognition ability com-
pared to unmodified adenine base. However, significant
elimination of the acetyl group occurred by treatment
with concd NH3 aq for deprotection and release of the
DNA oligomer in a standard procedure of DNA synthe-
sis. Therefore, we carried out the synthesis of the modi-
fied DNA 13 with the activated phosphite method using
the G-loaded HCP resin 9c with a silyl linker. As a re-
sult, the modified DNA 13 could be synthesized without
their decomposition and oligomer 13 was obtained in
22% yield, as shown in Figure 3d. These oligomers were
characterized by MALDI-TOF mass spectroscopy.
Previously, we showed the synthesis of oligo DNAs with
base-labile functional groups using T-loaded HCP resin
8a. The oligo DNAs could quantitatively be released
without their decomposition by treatment with TBAF–
AcOH or Et3NÆ3HF under neutral conditions. In this
Bpro
BNH
2
DMTrO
O
DMTrO
O
deprotection
O
O
O
O
H
N
H
N
Si
Si
N
H
N
H
O
O
8b, 8c, 8d
9b 9c, 9d
highly cross-linked
polystyrene (HCP)
elimination of nucleoside
from the HCP resinb
a
entry
nucleobase
conditions
Tcomp
dbf6A
dmf2G
ac4C
2 M MeNH2/THF
2 M MeNH2/THF
2 M MeNH2/THF
2 M NH3/MeOH
1
2
3
4
3 h
0.5 h
3.5 h
15 h
8%
6%
25%
18%
ac4C
a Tcomp was analyzed by using HPLC.
b Elimination of nucleoside was analyzed by using DMTr cation assay.
Figure 2. Removal of the protecting groups on the amino groups of nucleobases on HCP resins.

5348
A. Ohkubo et al. / Bioorg. Med. Chem. 16 (2008) 5345–5351
BNH
2
O
O
BNH
2
DMTrO
O
BNH
O
2
activated phosphite method
ABI 392 DNA synthesizer
DBU
= A, G, C
BNH
O
2
O
P
CH3CN
rt
O
O
OCE
O
H
N
Si
N
H
O
O
H
N
O
Si
9
N
H
O
CH3
NH
unmodified DNA 10-12
modified DNA 13
3% CCl3COOH
Et3N-3HF
O
N
5'-d[TTTTTTTTTTX] 5'-d[CCACGA*GTGG]
13: A* = ac6az8c7A
CH2Cl2
rt
THF
rt
10: X = A
11: X = G
12: X = C
N
N
N
ac6az8c7A
Scheme 2. Synthesis of unmodified DNA 10–12 and modified DNA 13.
oxidatively damaged DNAs or, in the near future, func-
tional DNAs and RNAs with various base-labile modi-
fied structures. Further studies in this direction are
underway.
a
b
d[TTTTTTTTTTA]
d[TTTTTTTTTTG]
4. Experimental
0
10
20
30
40
min
0
10
20
30
40 min
4.1. General remarks
c
d
1H, 13C, and 31P NMR spectra were recorded at 270, 68
and 109 MHz, respectively. The chemical shifts were
1
measured from tetramethylsilane for H NMR spectra,
d[TTTTTTTTTTC]
d[CCACGA*GTGG]
CDCl3 (77 ppm) for 13C NMR spectra and 85% phos-
phoric acid (0 ppm) for 31P NMR spectra. UV spectra
were recorded on a U-2000 spectrometer. Column chro-
matography was performed with silica-gel C-200 pur-
chased from Wako Co. Ltd, and a minipump for a
goldfish bowl was conveniently used to attain sufficient
pressure for rapid chromatographic separation. Anion-
exchange HPLC was done on a Waters Aliance system
with a Waters 3D UV detector and a Gen-PakTM
FAX column (Waters, 4.6 · 100 mm). A linear gradient
(10–60%) of solvent I (1 M NaCl in 25 mM phosphate
buffer (pH 6.0)) in solvent II (25 mM phosphate buffer
(pH 6.0)) was used at 50 ꢁC at a flow rate of 1.0 mL/
min for 40 min. ESI mass was performed by use of Mar-
inerTM (PerSeptive Biosystems Inc.). MALDI-TOF mass
was performed by use of Bruker Daltonics [Matrix: 3-
hydoroxypicolinic acid (100 mg/ml) in H2O–diammo-
nium hydrogen citrate (100 mg/ml) in H2O (10:1, v/v)].
Highly cross-linked polystyrene was purchased from
ABI.
0
10
20
30
40
0
10
20
30
40
min
min
Figure 3. Anion-exchange HPLC profiles of the crude mixtures
obtained using the activated phosphite method under the conditions
used for ABI 392 DNA synthesizer. (a) d[TTTTTTTTTTA], (b)
d[TTTTTTTTTTG], (c) d[TTTTTTTTTTC], and (d) d[CCAC-
GA*GTGG], A* = ac6az8c7A.
3. Conclusions
For the first time, we have synthesized the loading re-
agents 1a–d with a silyl-type of linker and carried out
efficient introduction of them into HCP resins 8a–d.
Subsequently, the deprotection of nucleobase on their
resins was removed by treatment with MeNH2/THF or
NH3/MeOH to prepare resins 9b–d. Moreover, we could
synthesize not only unmodified DNA oligomers but also
a base-labile modified DNA oligomer using resins 9b–d
with the activated phosphite method. The use of a silyl
linker in the activated phosphite method has prompted
us to study the synthesis of base-labile DNAs such as
4.1.1. Synthesis of compound 5. Compound 4 (6.7 g,
28.4 mmol) was added to thionyl chloride (3.2 mL,
42.6 mmol). After being refluxed with stirring for 2 h,
the mixture was distilled under reduced pressure to give

A. Ohkubo et al. / Bioorg. Med. Chem. 16 (2008) 5345–5351
5349
5 (1.7 g, 91%). Bp 102–104 ꢁC (1 mmHg). 1H NMR
(CDCl3) d 0.96–1.07 (m, 12H), 1.18–1.23 (m, 2H), 3.92
(t, 1H, J = 3.1 Hz), 7.36 (d, 2H, J = 6.2 Hz), 7.48 (d,
2H, J = 6.2 Hz). 13C NMR (CDCl3) d 10.7, 18.5, 18.7,
123.9, 130.8, 132.9, 136.9. Anal. Calcd for C13H19ClOSi:
C, 61.27; H, 7.52; Cl, 13.91. Found: C, 61.17; H, 7.48;
Cl, 14.14.
due was chromatographed on a column of silica gel
(30 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 the fractions containing
the desired nucleoside 9 having a Si–O bond. The frac-
tions were collected and evaporated under reduced pres-
sure. 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 dry CH3CN (20 mL). To the
mixture was added 1,8-diazabicyclo[5.4.0]-7-undecene
(670 lL, 4.5 mmol). After the mixture was stirred at
room temperature for 10 min, a 0.2 M solution of trieth-
ylamine hydrogen carbonte (50 mL) was added to the
mixture. After being stirred at room temperature for
5 min, the mixture was partitioned between CHCl3
(100 mL) and brine (50 mL). The organic phase was col-
lected, dried over Na2SO4, filtered, and evaporated un-
4.1.2. Synthesis of compound 6. 4-Aminobutyric acid
(910 mg, 8.9 mmol) was dissolved in 1 M NaOH aq
(9 mL). To the mixture was added compound 5 (1.7 g,
6.7 mmol). After the mixture was stirred at room tem-
perature for 8 h, 12 M HCl aq was added to the mixture
for pH 2. Then, the mixture was partitioned between
CH2Cl2 (400 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 (30 g) with
CHCl3–MeOH (100:0–97:3, v/v) to give 6 (1.4 g, 65%).
1H NMR (CDCl3) d 0.97–1.08 (m, 12H), 1.25–1.30 (m,
2H), 3.98 (t, 1H, J = 3.1 Hz), 7.63 (d, 2H, J = 7.8 Hz),
7.48 (d, 2H, J = 7.8 Hz). 13C NMR (CDCl3) d 10.7,
18.5, 18.7, 128.8, 129.7, 135.4, 142.0, 172.4. Anal. Calcd
for C17H27NO3Si: C, 63.51; H, 8.47; N, 4.36. Found: C,
63.34; H, 8.43; N, 4.31.
der
reduced
pressure.
The
residue
was
chromatographed on a column of silica gel (30 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 1. The frac-
tions were collected and evaporated under reduced pres-
sure. The residue was finally evaporated by repeated
coevaporation three times each with toluene and CHCl3
to remove the last traces of Et3N to give 1.
4.1.3. Synthesis of compound 7. Compound 6 (12.5 g,
38.9 mmol) was dissolved in dry CH2Cl2 (200 mL). To
the mixture were added 9-fluorenylmethanol (11.4 g,
58.4 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodi-
imide hydrochloride (14.8 g, 79.8 mmol), and 4-dimeth-
ylaminopyridine (195 mg, 1.6 mmol). After being stirred
at room temperature for 3 h, the mixture was parti-
tioned between CHCl3 (300 mL) and brine (500 mL).
The organic phase was collected, dried over Na2SO4, fil-
tered, and evaporated under reduced pressure. The resi-
due was chromatographed on a column of silica gel
(200 g) with hexane–ethyl acetate (100:0–50:50, v/v) to
give 7 (15.5 g, 80%). 1H NMR (CDCl3) d 0.91–1.02
(m, 12H), 1.14–1.22 (m, 2H), 1.86 (t, 2H, J = 6.8 Hz),
2.41 (t, 2H, J = 7.0 Hz), 3.36 (dd, 2H, J = 6.5 Hz,
J = 12.7 Hz), 3.92 (t, 1H, J = 2.7 Hz), 4.36 (d, 2H,
J = 7.0 Hz), 6.67 (br s, 1H), 7.21–7.36 (m, 4H), 7.50
(d, 2H, J = 7.0 Hz), 7.68 (d, 2H, J = 7.2 Hz). 13C
NMR (CDCl3) d 10.4, 18.2, 18.4, 24.3, 31.96, 39.2,
46.6, 66.1, 119.8, 124.7, 125.7, 127.0, 127.6, 134.8,
135.4, 141.1, 143.5, 167.5, 173.4, Anal. Calcd for
C31H37NO3Si: C, 74.51; H, 7.46; N, 2.80. Found: C,
74.29; H, 7.62; N, 2.86.
1
4.1.5. Compound 1a. H NMR (CDCl3) d 0.83–0.95 (m,
12H), 1.04–1.20 (m, 11H), 1.41 (s, 3H), 1.86–1.93 (m,
2H), 2.10–2.17 (m, 1H), 2.28–2.37 (m, 3H), 2.96 (dd,
6H, J = 7.3 Hz, J = 14.6 Hz), 3.21 (d, 1H, J = 8.6 Hz),
3.38–3.41 (m, 3H), 3.70 (s, 6H), 4.07 (s, 1H), 4.57 (s,
1H), 6.37 (t, 1H, J = 5.4 Hz), 6.73 (d, J = 6.8 Hz),
7.17–7.30 (m, 9H), 7.44 (d, 2H, J = 7.6 Hz), 7.57 (s,
1H) 7.74 (d, 2H, J = 7.8 Hz), 7.89 (br s, 1H). 13C
NMR (CDCl3) d 8.3, 11.8, 17.1, 17.2, 17.3, 24.4, 34.2,
40.5, 41.5, 45.1, 55.1, 63.3, 73.6, 77.2, 84.8, 86.9, 87.1,
111.1, 113.1, 126.2, 127.0, 127.9, 129.9, 134.2, 135.11,
135.14, 135.5, 137.4, 144.1, 150.5, 158.6, 164.2, 167.4,
179.2. HRMS (ESI) calcd for [C48H57N3O10Si+Na]+
886.3711. Found: 886.37130.
1
4.1.6. Compound 1b. H NMR (CDCl3) d 0.92–1.02 (m,
18H), 1.12–1.28 (m, 8H), 1.37 (t, 4H, J = 7.3 Hz), 1.65
(dd, 4H, J = 7.2 Hz, J = 14.6 Hz), 1.96 (br s, 2H),
2.43–2.56 (m, 3H), 2.75–2.83 (m, 1H), 2.93 (dd, 4H,
J = 7.2 Hz, J = 14.4 Hz), 3.27–3.51 (m, 6H), 3.67–3.76
(m, 8H), 4.24 (d, 1H, J = 2.7 Hz), 4.76 (s, 1H), 6.53 (t,
1H, J = 6.3 Hz), 6.76 (d, 4H, J = 8.4 Hz), 7.20–7.37
(m, 9H), 7.53 (d, 2H, J = 7.8 Hz), 7.82 (d, 2H,
J = 7.8 Hz), 8.06 (s, 1H), 8.10 (br s, 1H), 8.50 (s, 1H),
8.96 (s, 1H). 13C NMR (CDCl3) d 8.5, 11.9, 12.0, 13.6,
13.8, 17.3, 19.7, 20.1, 24.0, 29.1, 30.9, 34.8, 40.6, 41.1,
44.7, 45.1, 51.8, 55.1, 63.2, 73.3, 84.0, 86.4, 86.7, 113.0,
126.2, 126.7, 127.7, 128.0, 129.9, 134.3, 135.6, 135.9,
137.2, 139.8, 144.4, 151.2, 152.6, 158.0, 158.4, 159.9,
167.2, 179.5. HRMS (ESI) m/z calcd for [C57H73N7
O8Si+H]+, 1012.5368. Found: 1012.5371.
4.1.4. A general procedure for the synthesis of compound
1a–d. Compound 7 (830 mg, 1.7 mmol) was rendered
anhydrous by repeated coevaporation with dry CH3CN
(3· 3 mL) and finally dissolved in dry CH2Cl2 (20 mL).
To the mixture was added 1,3-dichloro-5,5-dimethylhy-
dantoin (655 mg, 3.3 mmol). After being stirred at room
temperature for 30 min, imidazole (510 mg, 7.5 mmol)
and 50-O-(4,40-dimethoxytrityl)-20-deoxynucleoside
8
(1.5 mmol) were added. After the mixture was stirred
at room temperature for 30 min, the mixture was parti-
tioned between CHCl3 (100 mL) and brine (100 mL).
The organic phase was collected, dried over Na2SO4, fil-
tered, and evaporated under reduced pressure. The resi-
1
4.1.7. Compound 1c. H NMR (CDCl3) d 0.88–1.03 (m,
12H), 1.08–1.20 (m, 11H), 1.90–2.01 (m, 2H), 2.45 (dd,
4H, J = 6.5 Hz, J = 11.6 Hz), 2.93 (dd, 4H, J = 7.3 Hz,

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A. Ohkubo et al. / Bioorg. Med. Chem. 16 (2008) 5345–5351
J = 14.0 Hz), 3.09 (2s, 6H), 3.30 (d, J = 4.1 Hz), 3.49 (d,
J = 5.7 Hz), 3.77 (s, 6H), 4.23 (s, 1H), 4.62 (s, 1H), 6.42
(t, 1H, J = 6.7 Hz), 6.79 (d, 4H, J = 8.6 Hz), 7.16–7.29
(m, 7H), 7.37 (d, 2H, J = 7.0 Hz), 7.52 (d, 2H,
J = 7.6 Hz), 7.71 (s, 1H), 7.83 (d, 2H, J = 7.6 Hz), 8.41
(br s, 1H), 8.55 (s, 1H). 13C NMR (CDCl3) d 8.8, 11.8,
11.9, 17.17, 17.22, 24.3, 35.1, 35.3, 41.0, 41.3, 41.4,
44.9, 55.1, 63.4, 73.4, 77.2, 83.1, 86.5, 86.8, 113.1,
120.2, 126.8, 127.8, 127.9, 129.9, 134.2, 135.4, 135.6,
135.9, 137.1, 144.3, 150.1, 156.9, 158.0, 158.2, 158.4,
167.1, 180.2. HRMS (ESI) calcd for [C51H61N7O9Si+
Na]+966.4197. Found: 966.4195.
phosphite method.31 The 2-cyanoethyl groups of oligo-
mer obtained after chain elongation was deprotected
by treatment with a 10% DBU solution in CH3CN
(500 lL) at room temperature for 1 min. Then, the mix-
ture containing oligo DNAs was released from the resin
by treatment with a solution of Et3NÆ3HF (0.2 M) and
Et3N (0.4 M) in THF (500 lL) at room temperature
for 4 h. The polymer support was removed by filtration
and washed with 0.1 M ammonium acetate buffer (3·
1 mL). The filtrate was purified by anion-exchange
HPLC to give oligonucleotides 10–13.
Oligonucleotide 10. TTTTTTTTTTA MALDI-TOF
Mass calcd for [C110H143N25O73P10+H]+ 3294.18.
Found: 3293.53.
1
4.1.8. Compound 1d. H NMR (CDCl3) d 0.84–0.91 (m,
12H), 1.05–1.15 (m, 5H), 1.87–2.04 (m, 3H), 2.15 (s,
3H), 2.38 (t, 2H, J = 6.3 Hz), 2.50–2.63 (m, 1H), 2.93
(dd, 4H, J = 7.2 Hz, J = 14.4 Hz), 3.23–3.47 (m, 4H),
3.71 (s, 6H), 4.13 (d, 1H, J = 2.7 Hz), 4.45 (d, 1H,
J = 4.1 Hz), 6.19 (t, 1H, J = 5.7 Hz), 6.73 (d, 4H,
J = 8.1 Hz), 7.08–7.27 (m, 9H), 7.42 (d, 2H,
J = 7.8 Hz), 7.54 (br s, 1H), 7.70 (d, 2H, J = 7.8 Hz),
8.18 (d, 1H, J = 7.3 Hz); 13C NMR (CDCl3) d 7.8, 8.2,
11.7, 17.0, 17.1, 24.3, 24.5, 33.5, 40.1, 42.3, 44.8, 55.0,
57.8, 62.2, 71.9, 77.2, 86.7, 87.0, 96.4, 113.0, 126.0,
126.9, 127.7, 127.8, 129.8, 134.2, 134.8, 135.4, 137.2,
143.8, 144.3, 154.7, 158.4, 162.5, 167.3, 170.8, 178.7.
HRMS (ESI) calcd for [C49H58N4O10Si+Na]+
913.3820. Found: 913.3822.
Oligonucleotide 11. TTTTTTTTTTG MALDI-TOF
Mass calcd for [C110H143N25O74P10+H]+ 3310.18.
Found: 3311.64.
Oligonucleotide 12. TTTTTTTTTTC MALDI-TOF
Mass calcd for [C109H143N23O74P10+H]+ 3270.15.
Found: 3270.72.
Oligonucleotide 13. CCACGA GTGG (A*: ac6az8c7A)
*
MALDI-TOF Mass calcd for [C99H124N41O58P9+H]+
3096.05. Found: 3094.51.
4.1.9. A general procedure for the synthesis of 8. A highly
cross-linked polystyrene (HCP) resin (1 g) having a ben-
zylamino group was washed with dry CH3CN (3· 1 mL)
and dried under reduced pressure. The HCP resin was
added in dry CH2Cl2 (3 mL) in a round flask. To the
mixture were added compound 1 (168 lmol) and DCC
(174 mg, 842 lmol). After a round flask having the mix-
ture was attached to a rotary evaporator and gently ro-
tated for 12 h, the solvent was removed by filtration.
The residual CPG was washed by use of CH2Cl2 (3·
1 mL) 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 lmol). After a
round flask having the mixture was attached to a rotary
evaporator and gently rotated for 2 h, the solvent was
removed by filtration. The residual HCP resin 8 was
washed with CH3CN (3· 1 mL) and dried under reduced
pressure. The amount of the nucleoside introduced into
the resin was estimated by use of the DMTr cation
assay.
Acknowledgments
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 Edu-
cation, Culture, Sports, Science and Technology, Japan
and by the global COE project.
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