Solid-phase synthesis of oligodeoxyribonucleotides without base protection utilizing o-selective reaction of oxazaphospholidine derivatives

Article abstract of DOI:10.1080/15257771003612839

A study on the development of a novel method to synthesize oligodeoxyribonucleotides without base protection is described. We found that nucleoside 3′-O-oxazaphospholidine derivatives exclusively react with the hydroxy group of nucleosides in the presence

Full text of DOI:10.1080/15257771003612839

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Solid-Phase Synthesis of
Oligodeoxyribonucleotides without Base
Protection Utilizing O-Selective Reaction
of Oxazaphospholidine Derivatives
Natsuhisa Oka a , Yukihiro Maizuru a , Mamoru Shimizu a & Takeshi
Wada a
a Department of Medical Genome Sciences, Graduate School of
Frontier Sciences , The University of Tokyo , Kashiwa, Chiba, Japan
Published online: 24 Feb 2010.
To cite this article: Natsuhisa Oka , Yukihiro Maizuru , Mamoru Shimizu & Takeshi Wada (2010) Solid-
Phase Synthesis of Oligodeoxyribonucleotides without Base Protection Utilizing O-Selective Reaction
of Oxazaphospholidine Derivatives, Nucleosides, Nucleotides and Nucleic Acids, 29:2, 144-154, DOI:
10.1080/15257771003612839
To link to this article: http://dx.doi.org/10.1080/15257771003612839
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Nucleosides, Nucleotides and Nucleic Acids, 29:144–154, 2010
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257771003612839
SOLID-PHASE SYNTHESIS OF OLIGODEOXYRIBONUCLEOTIDES
WITHOUT BASE PROTECTION UTILIZING O-SELECTIVE REACTION
OF OXAZAPHOSPHOLIDINE DERIVATIVES
Natsuhisa Oka, Yukihiro Maizuru, Mamoru Shimizu, and Takeshi Wada
Department of Medical Genome Sciences, Graduate School of Frontier Sciences,
The University of Tokyo, Kashiwa, Chiba, Japan
A study on the development of a novel method to synthesize oligodeoxyribonucleotides without
2
base protection is described. We found that nucleoside 3 ꢁ-O-oxazaphospholidine derivatives exclu-
sively react with the hydroxy group of nucleosides in the presence of unprotected nucleobase amino
groups. Since the O-chemoselectivity of the oxazaphospholidine derivatives is likely due to their ring
structure, which allows the regeneration of the oxazaphospholidine derivatives from the correspond-
ing base phosphitylation adducts via an intramolecular recyclization, the method is expected to be
compatible with any kinds of acidic activators.
Keywords Oligonucleotide; oxazaphospholidine; base protection; phosphoramidite;
O-selective reaction
INTRODUCTION
Chemically synthesized oligonucleotides and their analogues have been
used for a wide range of applications (e.g., diagnostics, therapeutics, nan-
otechnology),[1] and the growing demands for them in recent years have
created interest in the development of a more efficient method to synthe-
size oligonucleotides and their analogues than the conventional phospho-
ramidite method.[2] The use of monomers without base protection is one of
the most promising approaches to date owing to their improved synthetic
efficiency by eliminating at least several steps for protection and deprotec-
tion.[3,4] The method is also advantageous for the synthesis of base-labile
oligonucleotide analogues because the deprotection of nucleobases, which
is usually carried out under strongly basic conditions, is not required.[3,4]
Received 24 November 2009; accepted 11 January 2010.
We thank Professor Kazuhiko Saigo (University of Tokyo) for helpful suggestions. This research was
supported by grants from MEXT Japan.
Address correspondence to Takeshi Wada, Department of Medical Genome Sciences, Graduate
School of Frontier Sciences, The University of Tokyo, Bioscience Building 702, 5-1-5 Kashiwanoha,
Kashiwa 277-8562, Japan. E-mail: wada@k.u-tokyo.ac.jp
144

Solid-Phase Synthesis of Oligodeoxyribonucleotides
145
For the synthesis of oligonucleotides without base protection, a highly
O-chemoselective condensation between the phosphorus atom of monomer
units and the terminal hydroxy group of oligonucleotides in the presence
of highly reactive unprotected nucleobase amino groups[5] is requisite. The
following two approaches have achieved the O-selective condensation and
the resultant synthesis of oligonucleotides without base protection. The
first one is the H-phosphonate method, in which base unprotected nu-
cleoside 3ꢁ-H-phosphonate monoesters are used as monomer units. The
H-phosphonate monomers exclusively react with hydroxy groups of nucleo-
sides in the presence of nucleobase amino groups. Relatively short oligonu-
cleotides have been synthesized utilizing this chemoselectivity. However,
it is difficult to synthesize long oligomers due to the inherent instability
of the H-phosphonate diester internucleotide linkages.[4] The second ap-
proach uses base unprotected nucleoside 3ꢁ-phosphoramidites as monomer
units. Since the condensation of nucleoside phosphoramidite derivatives
promoted by commonly used acidic activators, such as 1H-tetrazole, does
not have a high O/N -chemoselectivity, various acidic activators have been
developed to achieve a highly chemoselective condensation between the
phosphoramidites and nucleoside hydroxy groups by base protonation or
generating reactive intermediates that have a high O-chemoselectivity. How-
ever, complete O-chemoselectivity has not been achieved in many cases,
which still suffer from the generation of N -phosphitylation byproducts.[3]
On the other hand, during the course of our study on the synthesis of
stereoregulated backbone-modified oligonucleotide analogues by using nu-
cleoside 3ꢁ-O-(1,3,2-oxazaphospholidine) monomer units,[6] we have found
that the oxazaphospholidine monomers do not give any N -phosphitylation
byproducts when mixed with N -unprotected nucleosides and an acidic acti-
vator.[6c] This finding led us to develop a new method to synthesize oligonu-
cleotides without base protection. The results of this study are described
below.
RESULTS AND DISCUSSION
To examine the reactivity of nucleoside 3ꢁ-O-oxazaphospholidine deriva-
tives toward nucleobase amino groups in the presence of 1H-tetrazole,
one of the conventional activators for the phosphoramidite method,[2]
we analyzed the reaction between a 3ꢁ,5ꢁ-protected deoxycytidine deriva-
tive (Scheme 1, 1c) and an oxazaphospholidine derivative (trans-3)[6a–c] in
the presence of 1H-tetrazole by 31P NMR spectroscopy. Since trans-3 was
synthesized from racemic 2-methylamino-1-phenylethanol as a mixture of
two P-diastereomers, the corresponding two 31P NMR signals are observed
(Figure 1B, 136.5, 135.6 ppm). A reaction, in which the oxazaphospholidine
trans-3 was replaced by a regular phosphoramidite monomer (2), was also

146
N. Oka et al.
DMTrO
Th
DMTrO
Th
O
O
O
O
P
O
P
N
HN
CN
CN
N
N
O
O
N
HN
(4 equiv)
N
2
(2 equiv)
TBDMSO
N
O
CH3CN−CD3CN (4:1, v/v)
, etc.
rt, 30 min
OTBDMS
NH2
N
4
DMTrO
Th
O
DMTrO
Th
TBDMSO
N
O
O
O
O
O
P
P
OTBDMS
N
N
HN
O
N
+
HN
1c
O
H2N
N
Ph
trans-3
(2 equiv)
(4 equiv)
Ph
N
1c + trans-3 + cis-3
TBDMSO
N
O
O
CH3CN CD3CN (4:1, v/v)
−
rt, 30 min
OTBDMS
5
SCHEME 1 Phosphitylation of cytosine exocyclic amino group (1c) with conventional nucleoside 3ꢁ-
phosphoramidite (2) and nucleoside 3ꢁ-O-oxazaphospholidine (3).
monitored by 31P NMR. The 31P NMR analysis showed that the phospho-
ramidite 2 gave N -phosphitylation adducts, such as 4,[3d] by the appearance
of several new 31P NMR signals around 125 ppm (Figure 1A). In sharp con-
trast, the reaction between the oxazaphospholidine trans-3 and 1c did not
give any N -phosphitylation adducts, although the epimerization of trans-3
which generated a mixture of cis-3 (139.5, 137.6 ppm) and trans-3 was ob-
served as reported in the previous reports (Figure 1B).[6a–c] We confirmed
that trans-3 also epimerized in the absence of 1c. Therefore, the epimeriza-
tion of trans-3 is probably due to the repetitive attacks of 1H-tetrazole to the
chiral phosphorus atom of 3, although the involvement of the intermediate 5
cannot be excluded. The experiments using the 3ꢁ,5ꢁ-protected deoxyadeno-
sine counterpart (1a) gave virtually the same results (data not shown). Thus,
the experiments demonstrated that the nucleoside 3ꢁ-O-oxazaphospholidine
derivative did not give any N -phosphitylation adducts in the presence of
unprotected nucleobases and 1H-tetrazole in contrast to the conventional
phosphoramidite. It can be attributed to the structure of the oxazaphos-
pholidine ring, which allows the regeneration of the oxazaphospholidine
derivative 3 from the corresponding base phosphitylation adducts via an in-
tramolecular recyclization (Figure 1, 5), although further investigations are
necessary for full clarification of the mechanism.

Solid-Phase Synthesis of Oligodeoxyribonucleotides
147
FIGURE 1 31P NMR analysis. A: 1c + 2 + 1H-tetrazole; B: 1c + trans-3 + 1H-tetrazole.
The results shown above prompted us to develop a new method to
synthesize oligodeoxyribonucleotides by using base unprotected nucleo-
side 3ꢁ-O-oxazaphospholidine monomers. First, the synthesis of nucleo-
side 3ꢁ-O-oxazaphospholidine monomers without protection of the nucle-
obases was investigated. As the first design of the oxazaphospholidine
monomers, we adopted the simplest ring structure derived from 2-(methyl-
amino)ethanol.[7] In general, the nucleoside 3ꢁ-O-oxazaphospholidine
derivatives are synthesized by the reaction between the 3ꢁ-OH of ap-
propriately protected nucleosides and 2-chlorooxazaphospholidine deriva-
tives, which are prepared from the corresponding 1,2-amino alcohols and
PCl3 in the presence of a tertiary amine.[7] However, the 2-chlorooxaza-
phospholidine derivatives prepared by this method suffer from contamina-
tion by the ammonium salt, which is difficult to remove completely even by
filtration and distillation. To circumvent this problem, we chose a new syn-
thetic route, in which N ,O-bis(trimethylsilyl)-2-(methylamino)ethanol 6[8]
was allowed to react with PCl3. Salt-free 7 was obtained after a simple vac-
uum distillation in good yield and used for the following synthesis of the
nucleoside 3ꢁ-O-oxazaphospholidine monomers (Table 1).
The thymidine monomer 8a was synthesized by the reaction between
5ꢁ-O-DMTr-thymidine and 7 in the presence of Et3N at room temperature
according to the procedure reported in the literature (Table 1, entry 1).[7] In

148
N. Oka et al.
TABLE 1 Synthesis of N -unprotected nucleoside 3ꢁ-O-oxazaphospholidine monomers 8a–d
DMTrO
B
O
N
5'-O-DMTr-nucleoside
Cl
P
PCl3
Et3N
TMS
O
N TMS
O
N
O
P
0 °C then rt, 1 h
THF, −78 °C then rt, 30 min (8a)
or −78 °C, 12 h (8b−d)
then vacuum distillation
6
O
7
72%
8a−d
Entry
8
B
Isolated yield (%)
Purity (%)a
1
2
3
4
a
b
c
thymin-1-yl
cytosine-1-yl
adenine-9-yl
guanine-9-yl
72
65
85
84
98
92
94
90
d
aDetermined by 31P NMR.
contrast, the other monomers 8b–d have a nucleobase amino group, which
can react with 7 under the same conditions. In fact, when base unprotected
5ꢁ-O-DMTr-2ꢁ-deoxycytidine, deoxyadenosine, and deoxyguanosine were re-
acted with 7 under the same conditions, serious base-phosphitylations were
observed by 31P NMR (signals at 130–120 ppm). We found that the base-
phosphitylations were minimized when the reactions were carried out at
−78◦C. Thus, the 2-chlorooxazaphospholidine 7 reacted with the 3ꢁ-OH of
5ꢁ-O-DMTr-2ꢁ-deoxycytidine, deoxyadenosine, and deoxyguanosine almost
exclusively in THF −78◦C to afford the desired monomer units 8b–d in
modest to good yields. The monomer units of sufficient purity were ob-
tained by simple precipitation. The monomer units were stable at least for
several months under proper storage conditions (at −30◦C in a glass vial
flushed with an inert gas).
With the base-unprotected nucleoside 3ꢁ-O-oxazaphospholidine mono-
mer units 8 in hand, a d[CAGT] 4mer was manually synthesized on a
controlled-pore glass (CPG). The synthesis cycle is given in Table 2. The
TABLE 2 Procedure for manual solid-phase synthesis (0.5 µmol)
Step
Operation
Reagents and solvents
Time
1
2
detritylation
washing
3% DCA in CH2Cl2
(i) CH2Cl2 (ii) dry CH3CN (iii) drying
in vacuo.
30 seconds
—
3
4
coupling
washing
8 (0.09 M, 40 equiv) and 1H-tetrazole
(0.45 M, 200 equiv) in dry CH3CN
under argon
(i) dry CH3CN (ii) dry NMP (for dG)
(iii) drying in vacuo.
3 minutes
—
5
6
oxidation
washing
t-BuOOH (1 M) in dry toluene
(i) dry CH3CN (ii) CH2Cl2
3 minutes
—

Solid-Phase Synthesis of Oligodeoxyribonucleotides
149
FIGURE 2 A) Anion-exchange HPLC profile: a linear gradient of 0–30% 1 M NaCl in 10 mM phosphate
buffer (pH 6.9) at 50◦C for 30 minutes at a rate of 0.5 mL/min using Gen-Pak FAX column (4.6 × 100
mm) (Waters). B) RP-HPLC profile: a linear gradient of 0–20% acetonitrile in 0.1 M ammonium acetate
buffer (pH 7.0) at 50◦C for 60 minutes at a rate of 0.5 mL/min using a µBondasphere 5 µm C18 column
(3.9 × 150 mm) (Waters, Milford, MA, USA).
average coupling yield estimated by DMTr+ assay was 96%. It should be
noted that the solid-support was washed with N -methylpyrrolidone (NMP)
after the cycle to incorporate a dG to wash away the dG-monomer and/or
its residue, which had rather low solubility to the organic solvents used for
the synthesis. After the chain elongation, the DMTr-on CPG was treated with
conc. NH3 at 55◦C overnight for deprotection and cleavage from the sup-
port. The 5ꢁ-O-DMTr group was then removed by treatment with 80% AcOH,
and the crude d[CAGT] was analyzed by anion-exchange and reserve-phase
HPLC. The analysis showed that the desired 4mer was obtained as a major
product (Figure 2).
Since the deprotection of the internucleotidic phosphates required
somewhat harsh conditions (The RP-HPLC profile indicates that small
amounts of oligos having one or two protected phosphate groups remained
even after the treatment with conc. NH3 at 55◦C for about 12 hours), it
would be necessary to redesign the oxazaphospholidine ring structure for the
synthesis of base-labile oligonucleotide analogues. Fortunately, preliminary
experiments with a dithymidine phosphate derivative showed that the de-
protection of the phosphate was completed under milder conditions (conc
NH3, room temperature, 30 minutes or 0.2 M DBU in MeCN, room tem-
perature, 30 minutes) when the oxazaphospholidine monomer trans-3 was
used in the place of 8, indicating that the method developed in this study is
potentially applicable to the synthesis of base-labile oligonucleotides, such
as oligoribonucleotides.

150
N. Oka et al.
CONCLUSION
In conclusion, we demonstrated that the nucleoside 3ꢁ-O-oxazaphos-
pholidine derivatives had a complete O/N -chemoselectivity and were use-
ful for the synthesis of DNA oligomers without base protection. Since the
chemoselectivity of the oxazaphospholidine derivatives is likely due to their
ring structure, which allows the regeneration of the oxazaphospholidine
derivatives from the corresponding base phosphitylation adducts via an in-
tramolecular recyclization, it is expected to be compatible with any kinds of
acidic activators. Further investigation on the synthesis of oligonucleotides
and their analogues based on this new concept is now in progress.
EXPERIMENTAL
General
Infrared (IR) spectra were recorded on a JASCO (Tokyo, Japan) FT/IR-
480 Plus spectrophotometer. All NMR spectra were recorded on a Varian
1
(Palo Alto, CA, USA) Mercury 300. H NMR spectra were obtained at 300
MHz with tetramethylsilane (TMS; δ 0.0) as an internal standard in CDCl3.
13C NMR spectra were obtained at 75.5 MHz with CDCl3 as an internal
standard (δ 77.0) in CDCl3. 31P NMR spectra were obtained at 121.5 MHz
with 85% H3PO4 (δ 0.0) as an external standard in CDCl3. Dry THF was
prepared by distillation from sodium benzophenone ketyl under argon prior
to use. Other dry organic solvents were prepared by appropriate procedures.
The other organic solvents were reagent grade and used as received. Manual
solid-phase synthesis was performed by using a glass filter (10 mm × 50
mm) with a stopper at the top and a stopcock at the bottom as a reaction
vessel. Controlled-pore glass (CPG) was purchased from 3-Prime (Aston,
PA, USA).
N,O-Bis(trimethylsilyl)-2-(methylamino)ethanol (6). Compound 6 was syn-
thesized according to the procedure described in the literature.[8] 2-
(Methylamino)ethanol (8.03 mL, 100 mmol) was dissolved in dry Et2O
(100 mL) under argon and the mixture was cooled with a dry ice–hexane
bath. A 2.67 M solution of BuLi in hexane (78.7 mL, 210 mmol) was added
dropwise to the mixture over 30 minutes with stirring. The cooling bath was
removed and the mixture was stirred for 30 minutes. The mixture was then
cooled again with a dry ice–hexane bath and TMSCl (26.5 mL, 210 mmol)
was added dropwise over 10 minutes. The mixture was then stirred for 2
hours at room temperature. Any volatile reagents were removed by distilla-
tion at atmospheric pressure under argon (bath temperature = 80◦C) and
the residue was purified by vacuum distillation to afford 6 (15.9 g, 72 mmol,
72%) as a colorless liquid. b.p. 78–79 ◦C/12 mmHg (lit.[8] 86◦C/17 mmHg).

Solid-Phase Synthesis of Oligodeoxyribonucleotides
151
1H NMR (300 MHz, CDCl3) δ 3.54 (t, J = 6.9 Hz, 2H), 2.83 (t, J = 6.9 Hz,
2H), 2.48 (s, 3H), 0.13 (s, 9H), 0.06 (s, 9H).
2-Chloro-3-methyl-1,3,2-oxazaphospholidine (7).[7,9] N ,O-Bis(trimethylsilyl)-
2- (methylamino)ethanol (6) (15.9 g, 72 mmol) was added dropwise to PCl3
(6.28 mL, 72 mmol) at 0◦C under argon, and the mixture was stirred for 1
hour at room temperature. The resultant TMSCl was removed by distillation
at atmospheric pressure under argon (bath temperature = 100◦C) and the
residue was purified by vacuum distillation to afford 7 (9.3 g, 67 mmol, 93%)
as a colorless liquid. b.p. 77–78◦C/15 mmHg (lit.[9] 57–58◦C/2 mmHg). 1H
NMR (300 MHz, CDCl3) δ 4.61 (brs, 1H), 4.33 (brs, 1H), 3.18 (m, 2H), 2.77
(d, J = 13.5 Hz, 3H). 31P NMR (121 MHz, CDCl3) δ 169.5.
5ꢁ-O-DMTr-thymidine 3ꢁ-O-oxazaphospholidine (8a).[7] 5ꢁ-O-DMTr-thymi-
dine (0.545 g, 1.0 mmol) was dried by repeated coevaporations with dry
pyridine and dry toluene under argon and dissolved in freshly distilled THF
(12.0 mL). Et3N (0.67 mL, 5.0 mmol) was added, and the mixture was
cooled to −78◦C. The 2-chlorooxazaphospholidine 7 (0.167 g, 1.2 mmol)
was then added dropwise, and the mixture was stirred for 30 minutes at
room temperature. The mixture was poured into a saturated NaHCO3 aque-
ous solution (100 mL) and extracted with CHCl3 (100 mL). The organic
layer was washed with saturated NaHCO3 aqueous solutions (2 × 100 mL)
and combined aqueous layers were back-extracted with CHCl3 (100 mL).
The organic layers were combined, dried over Na2SO4, filtered, and con-
centrated to dryness under reduced pressure. The residue was precipitated
from dry hexane (100 mL) and dried in vacuo to afford 8a (0.470 g, 0.72
mmol, 72%) as a white powder. 98% purity (31P NMR). 50:50 mixture of
P-diastereomers (1H NMR). IR (KBr) 3422, 3194, 3060, 2931, 2836, 1691,
1608, 1509, 1466, 1274, 1252, 1177, 1065, 1033, 927, 830, 792, 757, 728, 702,
585 cm−1. 1H NMR (300 MHz, CDCl3) δ 8.93 (brs, 1H), 7.60 (d, J = 0.9 Hz,
1H), 7.40–7.22 (m, 9H), 6.82 (d, J = 8.7 Hz, 4H), 6.35 (m, 1H), 4.77 (m,
1H), 4.27 (m, 1H), 4.14 (m, 1H), 4.02 (m, 1H), 3.78 (s, 6H), 3.54–3.44 (m,
1H), 3.32 (ddd, J = 10.4, 6.2, 2.7 Hz, 1H), 3.07 (m, 1H), 2.93 (m, 1H), 2.68
(d, J = 12.3 Hz, 1.5H), 2.62 (d, J = 12.3 Hz, 1.5H), 2.48–2.25 (m, 2H), 1.42
(d, J = 0.9 Hz, 1.5 H), 1.41 (d, J = 0.9 Hz, 1.5H). 13C NMR (75 MHz, CDCl3)
δ 163.5, 158.4, 150.1, 144.0, 143.9, 135.3, 135.1, 135.0, 135.0, 129.9, 127.9,
2
127.8, 126.9, 113.0, 111.1, 86.8, 86.7, 85.5, 85.4, 84.4, 84.3, 72.6 (d, J PC
=
14.7 Hz), 72.3 (d, 2J PC = 15.3 Hz), 68.8 (d, 2J PC = 3.5 Hz), 68.7 (d, 2J PC
=
2
2
3.5 Hz), 62.9, 62.8, 55.2, 49.0 (d, J PC = 4.9 Hz), 49.0 (d, J PC = 4.9 Hz),
40.4 (d, 2J PC = 4.3 Hz), 40.2, 31.5 (d, 2J PC = 21.1 Hz), 31.5 (d, 2J PC = 21.2
Hz), 11.7, 11.7. 31P NMR (121 MHz, CDCl3) δ 139.9, 139.7.
5ꢁ-O-DMTr-2ꢁ-deoxycytidine 3ꢁ-O-oxazaphospholidine (8b). 5ꢁ-O-DMTr-2ꢁ-
deoxycytidine (0.530 g, 1.0 mmol) was dried by repeated coevaporations
with dry pyridine and dry toluene under argon and dissolved in freshly
distilled THF (12.0 mL). Et3N (0.67 mL, 5.0 mmol) was added, and the
mixture was cooled to −78◦C. The 2-chlorooxazaphospholidine 7 (0.210 g,

152
N. Oka et al.
1.5 mmol) was then added dropwise, and the mixture was stirred for 12
hours at −78◦C. The mixture was poured into a saturated NaHCO3 aque-
ous solution (100 mL) and extracted with CHCl3 (100 mL). The organic
layer was washed with saturated NaHCO3 aqueous solutions (2 × 100 mL)
and combined aqueous layers were back-extracted with CHCl3 (100 mL).
The organic layers were combined, dried over Na2SO4, filtered, and con-
centrated to dryness under reduced pressure. The residue was precipitated
from dry Et2O (100 mL) and dried in vacuo to afford 8b (0.413 g, 0.65
mmol, 65%) as a white powder. 92% purity (31P NMR). 50:50 mixture of
P-diastereomers (31P NMR). IR (KBr) 3351, 3194, 2934, 2837, 1652, 1509,
1410, 1362, 1286, 1252, 1178, 1115, 1071, 1033, 927, 829, 791, 757, 727, 702,
1
585 cm−1. H NMR (300 MHz, CDCl3) δ 7.89 (d, J = 6.6 Hz, 0.5H), 7.87
(d, J = 6.6 Hz, 0.5H), 7.43–7.22 (m, 9H), 6.84 (d, J = 8.4 Hz, 4H), 6.25 (t,
J = 5.7 Hz, 1H), 5.49 (d, J = 7.8 Hz, 0.5H), 5.46 (d, J = 7.5 Hz, 0.5H), 4.72
(m, 1H), 4.26 (m, 1H), 4.12 (m, 1H), 4.00 (m, 1H), 3.78 (s, 6H), 3.41 (m,
1H), 3.05 (m, 1H), 2.91 (m, 1H), 2.65 (d, J = 12.3 Hz, 1.5H), 2.59 (d, J =
12.3 Hz, 1.5H), 2.54–2.43 (m, 1H), 2.26–2.16 (m, 1H). 13C NMR (75 MHz,
CDCl3) δ 165.6, 158.5, 155.8, 144.4, 144.3, 140.9, 135.4, 135.4, 135.3, 130.1,
2
128.1, 127.9, 126.9, 113.1, 86.6, 85.6, 85.5, 84.9, 71.2 (d, J PC = 14.6 Hz),
70.8 (d, 2J PC = 14.7 Hz), 68.8, 68.7, 62.1, 61.9, 55.2, 55.2, 49.0 (d, 2J PC = 4.9
2
2
Hz), 49.0 (d, J PC = 5.2 Hz), 41.1, 40.9, 31.5 (d, J PC = 21.4 Hz), 31.4 (d,
2J PC = 21.0 Hz). 31P NMR (121 MHz, CDCl3) δ 140.8, 140.4.
5ꢁ-O-DMTr-2ꢁ-deoxyadenosine 3ꢁ-O-oxazaphospholidine (8c). 5ꢁ-O-DMTr-2ꢁ-
deoxyadenosine (0.554 g, 1.0 mmol) was dried by repeated coevaporations
with dry pyridine and dry toluene under argon and dissolved in freshly dis-
tilled THF (12.0 mL). Et3N (0.67 mL, 5.0 mmol) was added, and the mixture
was cooled to −78◦C. The 2-chlorooxazaphospholidine 7 (0.210 g, 1.5 mmol)
was then added dropwise, and the mixture was stirred for 12 hours at −78◦C.
The mixture was poured into a saturated NaHCO3 aqueous solution (100
mL) and extracted with CHCl3 (100 mL). The organic layer was washed with
saturated NaHCO3 aqueous solutions (2 × 100 mL) and combined aqueous
layers were back-extracted with CHCl3 (100 mL). The organic layers were
combined, dried over Na2SO4, filtered, and concentrated to dryness under
reduced pressure. The residue was precipitated from dry hexane (100 mL)
and dried in vacuo to afford 8c (0.556 g, 0.85 mmol, 85%) as a white pow-
der. 94% purity (31P NMR). 55:45 mixture of P-diastereomers (31P NMR).
IR (KBr) 3328, 3174, 2931, 2836, 1642, 1605, 1509, 1470, 1445, 1418, 1364,
1331, 1301, 1251, 1177, 1073, 1033, 927, 829, 798, 756, 727, 702, 651, 585
1
cm−1. H NMR (300 MHz, CDCl3) δ 8.30 (s, 0.5H), 8.30 (s, 0.5H), 8.00 (s,
0.5H), 7.99 (s, 0.5H), 7.42–7.18 (m, 9H), 6.80 (d, J = 7.8 Hz, 4H), 6.42 (m,
1H), 5.86 (brs, 2H), 4.88 (m, 1H), 4.35 (m, 1H), 4.17 (m, 2H), 3.78 (s, 6H),
3.43–3.31 (m, 2H), 3.12 (m, 1H), 2.96 (m, 1H), 2.84 (m, 1H), 2.69 (d, J =
12.0 Hz, 1.5H), 2.66 (d, J = 12.0 Hz, 1.5H), 2.57–2.47 (m, 1H).13C NMR (75

Solid-Phase Synthesis of Oligodeoxyribonucleotides
153
MHz, CDCl3) δ 158.5, 155.4, 152.9, 149.6, 144.5, 144.4, 138.9, 138.8, 135.7,
135.6, 135.6, 130.0, 128.1, 127.8, 126.9, 120.0, 113.1, 86.5, 85.7, 84.3, 84.2,
72.6 (d, 2J PC = 10.0 Hz), 69.0, 68.8, 63.2, 63.1, 55.2, 55.2, 49.3 (d, 2J PC = 5.4
2
2
Hz), 49.1 (d, J PC = 5.5 Hz), 40.0, 39.8, 31.6 (d, J PC = 21.9 Hz).31P NMR
(121 MHz, CDCl3) δ 139.1, 138.0.
5ꢁ-O-DMTr-2ꢁ-deoxyguanosine 3ꢁ-O-oxazaphospholidine (3d). 5ꢁ-O-DMTr-2ꢁ-
deoxyguanosine (0.285 g, 0.50 mmol) was dried by repeated coevapora-
tions with dry pyridine and dry toluene under argon and dissolved in freshly
distilled THF (6.0 mL). Et3N (0.35 mL, 2.5 mmol) was added, and the mix-
ture was cooled to −78◦C. The 2-chlorooxazaphospholidine 7 (0.105 g, 0.75
mmol) was then added dropwise, and the mixture was stirred for 12 hours at
−78◦C. The mixture was poured into a saturated NaHCO3 aqueous solution
(50 mL) and extracted with CHCl3 (50 mL). The organic layer was washed
with saturated NaHCO3 aqueous solutions (2 × 50 mL) and combined aque-
ous layers were back-extracted with CHCl3 (50 mL). The organic layers were
combined, dried over Na2SO4, filtered, and concentrated to dryness under
reduced pressure. The residue was precipitated from dry Et2O (50 mL) and
dried in vacuo to afford 8d (0.285 g, 0.42 mmol, 84%) as a white powder.
90% purity (31P NMR). 51:49 mixture of P-diastereomers (31P NMR). IR
(KBr) 3343, 3206, 2933, 2837, 1688, 1607, 1532, 1509, 1465, 1445, 1413,
1360, 1302, 1251, 1177, 1073, 1033, 927, 830, 784, 756, 728, 702, 584 cm−1.
1H NMR (300 MHz, CDCl3) δ 10.4, 7.83 (d, J = 3.6 Hz, 1H), 7.36–7.21 (m,
9H), 6.85 (d, J = 8.7 Hz, 2H), 6.85 (d, J = 9.0 Hz, 2H), 6.50 (brs, 2H), 6.11
(m, 1H), 4.68 (m, 1H), 4.25 (m, 1H), 4.07 (m, 1H), 3.88 (m, 1H), 3.73 (s,
6H), 3.24–3.09 (m, 2H), 3.03–2.90 (m, 2H), 2.79–2.70 (m, 1H), 2.56 (d, J =
11.7 Hz, 1.5H), 2.54 (d, J = 12.6 Hz, 1.5H), 2.40–2.28 (m, 1H). 13C NMR (75
MHz, CDCl3) δ 159.0, 157.7, 154.7, 152.0, 145.7, 136.4, 135.8, 130.6, 128.8,
128.6, 127.7, 117.7, 114.1, 86.6, 85.5, 83.1, 72.9 (d, 2J PC = 9.5 Hz), 69.8, 69.7,
64.3, 64.1, 56.0, 55.9, 49.7 (d, 2J PC = 5.5 Hz), 49.4 (d, 2J PC = 5.4 Hz), 39.3,
39.0, 31.9 (d, 2J PC = 22.5 Hz). 31P NMR (121 MHz, CDCl3) δ 136.5, 135.3.
A general procedure for manual solid-phase synthesis of d[CAGT]. 5ꢁ-O-DMTr-
thymidine-loaded CPG (0.5 µmol) via a succinyl linker was used for the syn-
thesis. Chain elongation was performed by repeating the steps in Table 2.
After the chain elongation, the CPG was treated with conc NH3 (5 mL)
in a tightly-sealed flask at 55◦C overnight. The CPG was then filtered off,
and the filtrate was concentrated under reduced pressure. The residue was
treated with an 80% AcOH aqueous solution at room temperature for 1
hour and concentrated under reduced pressure. The residue was dissolved
in H2O (5 mL), washed with Et2O (3 × 5 mL), and concentrated to dry-
ness under reduced pressure. The resultant crude d[CAGT] was analyzed
by anion-exchange and reverse-phase HPLC. The main product was identi-
cal to an authentic sample prepared by the conventional phosphoramidite
method.[2]

154
N. Oka et al.
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