Direct and practical synthesis of 2′-O,4′-C-aminomethylene-bridged nucleic acid purine derivatives by transglycosylation

Article abstract of DOI:10.1016/j.tet.2017.01.010

Purine nucleosides of 2′-O,4′-C-aminomethylene-bridged nucleic acid (BNA^NC) have been directly synthesized from pyrimidine BNA^NCusing transglycosylation reaction with high stereoselectivity and excellent yield. The BNA^NCpurine nucleosides (adenosine, guanosine, and inosine) prepared by the direct and practical procedure were derivatized to the corresponding phosphoramidites and were incorporated into DNA. A thermal denaturation study showed that BNA^NC-containing oligonucleotides hybridized to RNA selectively with excellent mismatch discrimination.

Full text of DOI:10.1016/j.tet.2017.01.010

Accepted Manuscript
Direct and practical synthesis of 2′-O,4′-C-aminomethylene-bridged nucleic acid
purine derivatives by transglycosylation
Tadashi Umemoto, Shinichi Masada, Kenichi Miyata, Mari Ogasawara-Shimizu,
Shumpei Murata, Kazunori Nishi, Kazuhiro Ogi, Yoji Hayase, Nobuo Cho
PII:
S0040-4020(17)30023-6
10.1016/j.tet.2017.01.010
TET 28380
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 8 November 2016
Revised Date: 26 December 2016
Accepted Date: 6 January 2017
Please cite this article as: Umemoto T, Masada S, Miyata K, Ogasawara-Shimizu M, Murata S, Nishi K,
Ogi K, Hayase Y, Cho N, Direct and practical synthesis of 2′-O,4′-C-aminomethylene-bridged nucleic
acid purine derivatives by transglycosylation, Tetrahedron (2017), doi: 10.1016/j.tet.2017.01.010.
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Direct and practical synthesis of 2′-O,4′-C-
aminomethylene-bridged nucleic acid purine
derivatives by transglycosylation
Leave this area blank for abstract info.
Tadashi Umemoto*, Shinichi Masada, Kenichi Miyata, Mari Ogasawara-Shimizu, Shumpei Murata, Kazunori
Nishi, Kazuhiro Ogi, Yoji Hayase, and Nobuo Cho
Research, Takeda Pharmaceutical Co. Ltd.

1
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Tetrahedron
journal homepage: www.elsevier.com
Direct and practical synthesis of 2′-O,4′-C-aminomethylene-bridged nucleic acid
purine derivatives by transglycosylation
Tadashi Umemoto*, Shinichi Masada, Kenichi Miyata, Mari Ogasawara-Shimizu, Shumpei Murata,
Kazunori Nishi, Kazuhiro Ogi, Yoji Hayase, and Nobuo Cho
Research, Takeda Pharmaceutical Co., Ltd., Shonan Research Center, 26-1, Muraoka-Higashi 2-Chome, Fujisawa, Kanagawa 251-8555, Japan
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Purine nucleosides of 2′-O,4′-C-aminomethylene-bridged nucleic acid (BNANC) have been
directly synthesized from pyrimidine BNANC using transglycosylation reaction with high
stereoselectivity and excellent yield. The BNANC purine nucleosides (adenosine, guanosine, and
inosine) prepared by the direct and practical procedure were derivatized to the corresponding
phosphoramidites and were incorporated into DNA. A thermal denaturation study showed that
BNANC-containing oligonucleotides hybridized to RNA selectively with excellent mismatch
discrimination.
Available online
Keywords:
Modified nucleic acids
LNA/2′,4′-BNA
Oligonucleotides
Transglycosylation
2009 Elsevier Ltd. All rights reserved
.
aminomethylene-bridged nucleic acid (BNANC) has reported by
Imanishi et al. as a novel BNA derivative containing N-C linkage
in the six membered bridged strugure.9 BNANC has excellent
properties suitable for nucleic acid medicines, such as RNA-
specific stable hybridization, nuclease resistance, and capability
of chemical conjugation with the nitrogen atom in the bridge
structure. However, no practical synthetic method has been
reported for BNANC purine nucleosides.
The synthesis of BNANC pyrimidine nucleosides has been
achieved using a 2,2′-anhydropyrimidine intermediates well as of
other 2′-substituted nucleosides (Scheme 1), such as 2′-amino-2′-
deoxynucleoside,11 2′-deoxy-2′-mercaptonucleoside,12 2′-methyl
(2′-OMe),13 2′-methoxyethylnucleoside (2′-MOE),14 α-L-LNA,6
and 2′-amino/thio-LNAs.5,15 However, the synthetic route is not
applicable for purine nucleoside synthesis because the exocyclic
oxygen atom of pyrimidine is essential for the key intermediate
formation. For the epimerization of the 2′-hydroxyl group of
purine nucleosides, two strategies have been reported as
1. Introduction
Modified nucleic acid derivatives have been widely
investigated and have potential use in nucleic acid-based
medicines, such as antisense oligonucleotides,1 aptamers,2 and
siRNAs3. The modified nucleic acid confers thermal stability on
duplexes and/or triplexes, resistance against nucleases, reduced
immune stimulation, and other properties. Synthesis of modified
nucleic acids often requires a multistep reaction to obtain
monomer units for a solid-phase synthesizer. In particular,
monomer units bearing purine nucleobases are always a source of
concern for the synthesis of chemically modified nucleosides.
One of the most important classes of modified nucleotides is
the LNA/2′,4′-BNA4 family, which shows
a
restricted
conformation of sugar puckering resulting from the addition of a
bridge structure to the skeleton. Originally LNA/2′,4′-BNA was
independently synthesized by the Imanishi et al. and Wengel et
al.4. Since the publication of their articles, many research groups
have reported novel derivatives of the family.5–10 2′-O,4′-C-
alternatives: the one is nucleophilic substitution with
a
carboxylate anion, followed by hydrolysis;6b,16 and the other is
oxidation followed by reduction from the less hindered α-face of
the furanose ring.17 However, both of the two strategies require
multistep reactions, and the total yield is low. To avoid the
epimerization of purine nucleosides for 2′-modified nucleoside
synthesis, a transglycosylation reaction has been reported for 2′-
trifluoroacetamido-2′-deoxyribonucleosides18
and
2′-O-
methylribonucleoside synthesis.19 However, the reactions have
not been applied to nucleosides possessing a bridged sugar
Scheme 1. Inversion of 2′-hydroxyl group of pyrimidine
nucleosides

2
Tetrahedron
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Scheme 2. Synthesis of substrates for the transglycosylation reactions
skeleton as a transglycosyl donor. Therefore, we set out to
establish an efficient synthetic route for purine nucleosides
having a bridged sugar skeleton. Herein, we wish to describe the
direct and practical synthesis of BNANC purine nucleosides from
the corresponding pyrimidine analogs by transglycosylation.
reaction temperature is applicable owing to the relatively high
boiling point, and the other is that the use of chlorinated solvents
should be reduced for the purpose of green chemistry.
Table 2. Synthesis of BNANC purine derivatives by
transglycosylation
2. Results and Discussion
Nucleosides bearing uracilyl, thyminyl, cytosinyl, and their
acylated derivatives have been used for transglycosylation
reactions from pyrimidines to purines.18–20 N4-acetyl cytidine
derivatives have been reported as for β-anomer selective
reactions.19,20 To achieve the direct synthesis of BNANC purine
nucleosides by transglycosylation, thymin-1-yl (1), 4-(1,2,4-
triazol-1-yl)pyrimidin-2-on-1yl (2), cytosin-1-yl (3), and N4-
acetylcytosin-1-yl (4) BNANC analogs were examined as glycosyl
donors (Scheme 2). BNANC thymine (1) was synthesized by the
previously reported procedure.9b BNANC cytidine (3) was
synthesized by a conventional procedure via triazole intermediate
(2) using 1,2,4-triazole and phosphorus oxychloride, followed by
ammonia treatment. The nucleoside (3) was reacted with acetic
anhydride to afford N4-acetylated BNANC cytidine (4) in excellent
yield.
Entry
1
Nucleobase (B-H)
Product
Yield %
-
Description
6
N7:N9=1:1a
(not isolated)
2
7
23
N2-deacylated
(7′, 20%)
3
4
5
6
8
9
99
50
74
Table 1. Transglycosylation reaction for the synthesis of
BNANC adenosine
Cl
10
11
N
N
N
N
H
77
N7-isomerc
Entry
Substrate
Reaction Time
40 min
4 h
Yield %
29
(crude)
(~10%)
1
2
3
4
5
1
1
4
3
2
0a
aIsomer ratio was determined by LC-MS analysis. bdpc:
diphenylcarbamoyl cIsomer ratio was determined by the integrated area of
anomer peak from 1H NMR analysis.
30 min
1 h
70
27
30 min
84
aN6-Acetyl adenosine was collected.
The transglycosylation reaction was optimized with N6-
benzoyladenine as a glycosyl acceptor (Table 1). The reaction
was carried out with BSA and TMSOTf in toluene at 100 °C. At
first, compound 1 was used as a glycosyl donor. The reaction
after 40 min, BNANC N6-benzoyladenosine (5) was obtained in
29% yield with unreacted starting material (entry 1). When the
reaction time was extended to 4 h, the desired product 5 was not
detected (entry 2). LC-MS analysis of the reaction mixture
showed a new peak (rt = 1.68 min, m/z = 593.3). 1H NMR
Facile conditions for the transglycosylation reaction to
modified or unmodified nucleosides have been reported using
N,O-bis-trimethylsilylacetamide
trifluoromethanesulfonate (TMSOTf), and corresponding
nucleobases in 1,2-dichloroethane and/or acetonitrile.18–20 We
used the reported conditions except for solvent. We decided to
use toluene as an alternative solvent for all transglycosylation
reactions for the following two reasons: the one is that a higher
(BSA),
trimethylsilyl

3
analysis indicated that an N6-acetyladenosine analog and
benzamide were present (see supporting information). These
results suggested that the longer reaction time caused amido-
exchange with acetamide derived from BSA. N4-Acetylated
cytidine nucleoside (4) as a glycosyl donor, providing 5 in 70%
yield (entry 3). On the other hand, the reaction using N4-
unprotected cytidine nucleoside (3) proceeded more slowly than
that using 4 (entry 4). Interestingly, transglycosylation reaction
with 2 was completed within 30 min, affording 5 in 84% yield
(entry 5). These results indicated that N4-acetylcytosine and 4-
(1,2,4-triazol-1-yl)primidin-2-one are appropriate nucleobases for
the transglycosylation of BNANC. In all the cases, the reactions
selectively afforded β-nucleosides. We speculate that the
stereoselectivity results from not only steric hindrance by the 2′-
O-4′-C-bridge on the α face of the furanose ring but also
participation of the lone pair of the N–O bond, which regulates
the direction of nucleophilic attack of the incoming nucleobases.
To our knowledge, successful examples of transglycosylation
ACCEPTED MANUSCRIPT
or glycosylation using the unprotected guanine as a glycosyl
acceptor have not been reported. We speculate that the difficulty
of the glycosylation with unprotected guanine is caused from low
conversion to persilylated guanine, due to its poor solubility in
the early step of the reaction. We found that addition of a
catalytic amount of TMSOTf along with BSA accelerated
formation of the persilylated nucleobases before an addition of
transglycosyldonor 4. O6-trimethylsilylguanine, supposed to be a
major intermediate, has a 6 π-system in the purine ring. The
intermediate acted similarly to the 2-amino-6-chloropurine (entry
3), leading to facilitation of the reaction as well as induction of
N9-selectivity. The reaction conditions were thus applied to
BNANC inosine synthesis (entry 6). Finally, we applied the
reaction conditions used in entry 4 to the synthesis of the BNANC
guanosine monomer unit 12. The practical synthesis of N2-
protected BNANC guanosine is described in Scheme 3. The
transglycosylation reaction mixture was quenched and directly
used for the subsequent acetylation to afford N2-acetylated
guanosine 12 in moderate yield (65%, 2 steps). Isolated yield was
improved due to enhanced solubility of desire compound by N2-
acetylation.
Each of the silyl-protected BNANC purine nucleosides (5, 12,
11) was converted to the corresponding phosphoramidites by
conventional deprotection of the silyl group, 4, 4′-dimethoxytrityl
(DMTr) protection of the 5′-hydroxy group, and final 3′-
phosphorylation (Scheme 4). Oligonucleotides were synthesized
by the phosphite triester method using the standard
phosphoramidite protocol. The coupling time for BNANC
phosphoramidites was 20 min with 5-benzylthio-1H-tetrazole
(BTT) as an activator.
The optimized reaction conditions were applied to other
purine nucleoside syntheses (Table 2). Compound 4 has been
selected as a starting material to optimize other purine nucleoside
synthesis since we had developed a procedure for large scale
synthesis of 4 as an important intermediate for cytidine BNANC
.
Coupling N2-isobutylprotected guanine with
4
resulted in
inseparable mixtures of N7/N9 isomers of BNANC guanosine
analogs (entry 1, see the supporting information). N2-acetyl-O6-
diphenylcarbamoyl guanine, which has been reported for N9-
selective glycosylation in guanosine synthesis,21 was used for the
reaction
(entry
2).
BNANC
N2-acetyl-O6-
diphenylcarbamoylguanosine (7) was obtained in 23% yield with
the N2-deacylated by-product (7′, 20%). Reactions with 2-amino-
6-chloropurine (entry 3), guanine (entry 4), 6-chloropurine (entry
5), and hypoxanthine (entry 6) proceeded without marked side
reactions. In each case, the corresponding purine BNANC
nucleosides were obtained in moderate or high yield. The isolated
yield of BNANC guanosine (9) was relatively low due to the poor
solubility to purify by silica-gel column chromatography.
Additionally, the purification of BNANC inosine (11) was
unsuccessful in this step due to the poor solubility of products.
The purification BNANC inosine nucleoside was achieved after
We assessed the effect of BNANC purine incorporation by
measuring melting temperatures of the duplexes with target
DNAs and RNAs using two different sequences for adenosine
and guanosine derivatives (Table 3). Single BNANC incorporated
oligonucleotides (ON2 and ON5) formed stable duplexes with
target RNAs (ON2. ∆Tm = +3.5 °C/mod.; ON5: ∆Tm = +7.3
°C/mod.). In contrast, the oligonucleotides didn't show marked
duplex stabilizing effects against target DNAs. However, triple
BNANC incorporation (ON3 and ON6) contributed to increased
duplex stability with both DNA and RNA targets. The stabilizing
effects against target RNAs were higher than those against target
silyl
deprotection
to
give
2′-O,4′-C-(N-
methylaminomethylene)inosine (13). Inosine with BNANC
skeleton would be useful as an universal base for the therapeutic
use when the target RNA has single nucleotide polymorphism.
Scheme 3. practical synthesis of N2-acetyl BNANC guanosine
Scheme 4. BNANC purine phosphoramidites

4
Tetrahedron
ACCEPTED MANUSCRIPT
DNAs. These results are consistent with those of the previous
study using BNANC pyrimidines.9
transglycosylation. In the reaction, N4-acetylcytosine was used as
an excellent leaving group, as in the case of the previous
examples19,20. We also found that the 4-(1,2,4-triazol-1-yl)-
pyrimidin-2-one nucleoside, which is commonly used as an
intermediate of cytidine synthesis, can be used as a transglycosyl
donor. The transglycosylation could be applied to introduce not
only natural nucleobases but also unnatural nucleobases to the
BNANC skeleton. Further, the transglycosylation can be a
workable solution for synthesis of stereorestricted purine
nucleosides.
Mismatch discrimination properties were measured for ON2
and ON5 (Table 4). We found that BNANC purines could
discriminate the mismatches similarly to the parent
oligonucleotides (ON1 and ON4) in the duplexes with target
DNAs and RNAs. In addition, ON5 could make a wobble base
pairing between BNANC G and DNA-T (X = T, +4.0 °C/mod.)
probably owing to the local A-type structure induced by the sugar
conformation of BNANC
.
3. Conclusion
We have successfully established the efficient and practical
synthetic route for BNANC adenosine, guanosine, and inosine
directly from the corresponding pyrimidines by
Table 3. Melting temperature studies of BNANC-adenosine/guanosine incorporated oligonucleotides with complementary
DNA and RNA
DNA
RNA
Sequence
Tm (°C)b
∆Tm (°C)
∆Tm/mod.
Tm (°C)b
∆Tm (°C)
∆Tm/mod.
(°C)
(°C)
ON1
ON2
ON3
ON4
ON5
ON6
d(GCA TAT CAC T)
d(GCA TABT CAC T)
d(GCAB TABT CABC T)
d(ATG CGC TGT T)
d(ATG CGBC TGT T)
d(ATGB CGBC TGBT T)
38.8
38.4
44.6
48.7
49.6
56.5
-
-
36.5
40.0
53.7
47.3
54.6
67.7
-
-
-0.4
+6.1
-
-0.4
+2.0
-
+3.5
+17.2
-
+3.5
+5.7
-
+0.9
+7.8
+0.9
+2.6
+7.3
+20.4
+7.3
+6.8
b
aAB and GB are adenin-9-yl and guanin-9-yl BNANC (N-Me) monomers respectively. Thermal-denaturation temperature were measured as the maximum of
the first derivative of the melting curve (A260 versus temperature; all curves appeared to be sigmoidal) observed for an equimolar mixture of oligonucleotides (1.0
µM) in 10 mM sodium phosphate buffer (pH 7.0) containing 100 mM NaCl and 0.1 mM EDTA. Complementary DNAs: d(AGT GAT ATG C) for ON1~3,
d(AAC AGC GCA T) for ON4~6; Complementary RNAs: r(AGU GAU AUG C) for ON1~3, r(AAC AGC GCA U) for ON4~6.
Table 4. Mismatch discrimination properties of BNANC adenosine/guanosine-incorporated oligonucleotides
Tm (°C)a
DNAb
RNAc
Sequence
X = A
ntd
X = C
22.3
21.3
48.7
49.6
X = G
29.4
20.7
33.4
30.4
X = T
38.8
38.4
35.5
39.5
Y = A
22.2
22.8
29.4
31.3
Y = C
21.2
25.3
46.0
54.6
Y = G
25.8
25.8
33.4
35.4
Y = U
36.5
40.0
35.4
41.4
ON1
ON2
ON4
ON5
d(GCA TAT CAC T)
d(GCA TABT CAC T)
d(ATG CGC TGT T)
d(ATG CGBC TGT T)
ntd
30.3
30.9
aThermal-denaturation temperature were measured as the maximum of the first derivative of the melting curve (A260 versus temperature; all curves appeared
to be sigmoidal) observed for an equimolar mixture of oligonucleotides (1.0 µM) in 10 mM sodium phosphate buffer (pH 7.0) containing 100 mM NaCl and 0.1
b
c
mM EDTA. Target DNAs: d(AGT GAX ATG C) for ON1~3, d(AAC AGX GCA T) for ON4~6; Target RNAs: r(AGU GAY AUG C) for ON1~3, r(AAC
AGY GCA U) for ON4~6. dnt: No clear transition curve was observed.
pyridine:H2O (7:3, v/v)). The synthesized ONs were purified in
the DMT-ON mode by reversed-phase HPLC (0.1
M
4. Experimental section
triethylamine acetate–acetonitrile system), and their composition
and purity were verified by ion-pair reversed-phase HPLC on an
ACQUITY UPLC OST C18 Column (1.7 µm, 2.1 × 50 mm; A,
400 mM 1,1,1,3,3,3-hexafluoroisopropylalcohol(HFIP), 16 mM
triethylamine(TEA) with 5% MeOH; B, 400 mM HFIP, 16 mM
TEA in MeOH, gradient of B from 10% to 80% in 6 min, flow
rate 0.2 mL/min). Identity of the synthesized ONs was verified
by MALDI-TOF-MS recorded on an Applied Biosystems
Voyager-DE™ spectrometer. Melting temperature studies:
thermal-denaturation temperatures were measured as the
Oligonucleotide (ON) synthesis: A MerMade192 DNA/RNA
synthesizer was used for automated DNA synthesis. The
BNANC(N-Me) purine-incorporated ONs were synthesized by the
conventional solid-phase phosphite triester method using the
corresponding phosphoramidites (activator: 0.3 M 5-benzylthio-
1H-tetrazole in MeCN; coupling time 20 min; detritylation by
3% trichloroacetic acid in toluene (w/v); CapA, 20% 1-
methylimidazole in MeCN (vol%); CapB, 20%Ac2O, 30% 2,4,6-
collidine in MeCN (vol%); oxidation by 0.1 M iodine in

5
maximum of the first derivative of the melting curve (A260
versus temperature) observed for an equimolar mixture of
oligonucleotides (1.0 µM) in 10 mM sodium phosphate buffer
(pH 7.0) containing 100 mM NaCl and 0.1 mM EDTA. The
melting curve measurement was performed on a Shimadzu UV-
2450 (UV–vis spectrophotometer) with TMSPC-8 (TM analysis
system).
17.05, 16.99, 16.89, 16.87, 16.74, 13.55, 12.69, 12.25, 12.16,
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11.83; HRMS (MALDI-TOF) calc. for C24H45N4O6Si2 [M + H]+
m/z=541.2872, found 541.2888.
4.3. N4-Acetyl-3′,5′-O-(tetraisopropyldisiloxane)-5-methyl-2′-
O,4′-C-(N-methylaminomethylene)cytidine (4)
Compound 3 (60.1 g, 111.1 mmol) was dissolved in DMF (1
L) and acetic anhydride (13.6 g, 133.4 mmol) was added. The
mixture was stirred at room temperature for 20 h and water (20
mL) was added. After stirring for 10 min, the mixture was diluted
with ethyl acetate (2 L) and washed with a mixture of 1/4-
saturated sodium hydrogen carbonate solution (2 L), 1/2-
saturated sodium hydrogen carbonate (1 L), and water (1 L). The
resulting organic layer was dried over magnesium sulfate and
evaporated. The residue (85.1 g) was recrystallized with ethyl
acetate–hexane (300 mL, 1:1, v/v) to afford 4 as a white powder
4.1. 1-[3′,5′-O-(Tetraisopropyldisiloxane)-2′-O,4′-C-(N-
methylaminomethylene)-β-D-ribofuranosyl]-5-methyl-4-(1,2,3-
triazol-1-yl)pyrimidin-2-one(2)
Compound 1 (3.2 mmol, 1.8 g) was dissolved in acetonitrile
(20 mL), triethylamine (38 mmol, 5.4 mL), 1,2,4-triazole (26
mmol, 1.8 g), and phosphorus oxychloride (6.4 mmol, 0.59 mL)
were added, and the reaction mixture was stirred at room
temperature for 2 h. Ice was added to the reaction mixture to
quench the reaction, and the mixture was partitioned between
ethyl acetate and water. The separated organic layer was washed
with saturated brine and dried over sodium sulfate. The sodium
sulfate was filtered off, and the organic layer was concentrated.
The residue was purified by silica gel column chromatography
(hexane–ethyl acetate, 30:70 to 0:100, v/v) to yield 2 (1.8 g,
1
(59.2 g, 91%); H NMR (300 MHz, DMSO-d6) δ 9.92 (br. s.,
1H), 7.82 (s, 1H), 6.09 (s, 1H), 4.32 (d, J=3.0 Hz, 1H), 4.07 (d,
J=13.2 Hz, 1H), 3.87 (d, J=3.4 Hz, 1H), 3.67 (d, J=13.6 Hz, 1H),
2.77 (s, 2H), 2.66 (s, 3H), 2.26 (s, 3H), 1.95 (s, 3H), 1.12 – 0.87
(m, 28H); 13C NMR(75 MHz, DMSO-d6) δ 162.03, 153.29,
140.49, 105.18, 86, 83.26, 79.45, 63.53, 59.7, 57.05, 45.33,
25.06, 17.28, 17.25, 17.12, 17.04, 16.94, 16.92, 16.74, 14.12,
12.65, 12.19, 11.83; HRMS (MALDI-TOF) calc. for
C26H47N4O7Si2 [M + H]+ m/z=583.2978, found 583.2977.
1
94%) as a white foam; H NMR (300 MHz, CDCL3) δ 9.31 (s,
1H), 8.35 (s, 1H), 8.12 (s, 1H), 6.39 (s, 1H), 4.51 (d, J=3.0 Hz,
1H), 4.09 (d, J=13.2 Hz, 1H), 3.92 (d, J=3.0 Hz, 1H), 3.71 (d,
J=13.2 Hz, 1H), 2.96 (d, J=11.3 Hz, 1H), 2.79 (s, 3H), 2.65 (d,
J=10.9 Hz, 1H), 2.47 (s, 3H), 1.15 – 0.92 (m, 28H); 13C NMR(75
MHz, CDCL3) δ 158.36, 153.57, 153.43, 146.53, 145.21, 105.48,
87.52, 84.35, 79.92, 77.42, 76.99, 76.56, 63.92, 60.13, 57.53,
45.56, 17.47, 17.3, 17.23, 17.19, 17.13, 17.02, 16.95, 13.42, 12.9,
12.83, 12.41; HRMS (MALDI-TOF) calc. for C26H45N6O6Si2+
[M + H]+ m/z=593.2934, found 593.2932.
4.4. N6-Benzoyl-3′,5′-O-(tetraisopropyldisiloxane)-2′-O,4′-C-(N-
methylaminomethylene)adenosine (5)
Compound 4 (680 mg, 1.17 mmol) and N6-benzoyladenine
(1.4 g, 5.9 mmol) were suspended in toluene (11 mL). To the
suspension was added N,O-bis-trimethylsilylacetamide (BSA, 2.9
mL, 11.7 mmol), and the mixture was heated to 100 °C and
stirred for 1 h until transparency. To the reaction mixture,
trimethylsilyl trifluoromethanesulfonate (TMSOTf, 318 µL, 1.76
mmol) was added, and the mixture was stirred for an additional
30 min. The reaction mixture was cooled, diluted with ethyl
acetate (200 mL), and washed with saturated aqueous sodium
hydrogen carbonate solution, water, and saturated brine (100 mL
each). The resulting organic layer was dried over sodium sulfate
and concentrated. The mixture was purified by silica gel column
chromatography (hexane–ethyl acetate) to afford compound 5
4.2. 3′,5′-O-(Tetraisopropyldisiloxane)-5-methyl-2′-O,4′-C-(N-
methylaminomethylene)cytidine (3)
Compound 1 (75.3g, 139 mmol), 1,2,4-triazole (76.8 g, 1.11
mol) were suspended in acetonitrile (1.3 L) and triethylamine
(168.8 g, 1.67 mol) was added. After stirring for 10 min at room
temperature, the mixture was cooled to −10 °C and phosphorus
oxychloride (42.6 g, 278 mmol) was added dropwise. The
mixture was maintained at room temperature and then stirred for
1 h. Water (102 mL) was added to the reaction mixture to quench
the reaction, and saturated NaCl (aq.) (1 L) and sodium hydrogen
carbonate (aq.) (1 L) were added, followed by extraction with
ethyl acetate (1.5 L). The organic layer was washed twice with
the salt mixture (800 mL, 1:1 (v/v)) and then with brine (800
mL). The organic layer was concentrated to obtain a crude
residue of 2 as pale yellow foam. The intermediate was dissolved
in pyridine (820 mL) and 25% ammonia solution (410 mL) was
added. The mixture was stirred for 4 h and evaporated. Saturated
NaCl (800 mL), THF (550 mL), and ethyl acetate (1.1 L) were
added to the residue. The resulting organic layer was dried over
magnesium sulfate and evaporated. The residue was
recrystallized by the addition of diisopropyl ether (100 mL) and
cooled on ice. The crystals were washed with diisopropyl ether
(twice of 50 mL) and dried in vacuo to afford 3 as white crystals
(50.3 g). The filtrate was concentrated and recrystallized by
diisopropyl ether treatment to afford 3 as white crystals (9.8 g).
1
(533 mg, 0.813 mmol, 70%) as a white foam; H NMR (600
MHz, CDCL3) δ 9.17 (br. s., 1H), 8.81 (s, 1H), 8.35 (s, 1H), 8.03
(d, J=7.7 Hz, 2H), 7.65 – 7.47 (m, 3H), 6.79 (s, 1H), 4.77 (d,
J=2.6 Hz, 1H), 4.50 (d, J=2.9 Hz, 1H), 4.04 (d, J=12.8 Hz, 1H),
3.73 (d, J=12.8 Hz, 1H), 3.01 (d, J=11.0 Hz, 1H), 2.82 (s, 3H),
2.68 (d, J=11.0 Hz, 1H), 1.15 – 0.93 (m, 28H); 13C NMR (150
MHz, CDCL3) δ 164.61, 152.74, 150.56, 149.48, 141.49, 133.69,
132.71, 128.8, 127.81, 123.35, 85.21, 83.32, 80.49, 64.96, 60.26,
57.76, 45.63, 17.39, 17.25, 17.23, 17.2, 17.18, 17.16, 17.04,
16.98, 13.35, 12.84, 12.74, 12.39; HRMS (MALDI-TOF) calc.
for C31H46N6NaO6Si2 [M + H]+ m/z=677.2910, found 677.2907.
4.5. N2-Acetyl-3′,5′-O-(tetraisopropyldisiloxane)-2′-O,4′-C-(N-
methylaminomethylene)-O6-diphenylcarbamoylguanosine(7)
Compound
4
(1.0 g, 1.7 mmol) and N2-acetyl-O6-
diphenylcarbamoylguanine21a (3.6 g, 8.6 mmol) were suspended
in toluene (10 mL). BSA (4.2 mL, 17.2 mmol) was added to the
suspension, and the mixture was stirred at 100 °C for 30 min.
TMSOTf (470 µL, 2.6 mmol) was added to the reaction mixture,
and the mixture was stirred for an additional 30 min. The reaction
mixture was cooled, diluted with ethyl acetate (50 mL), and
washed with saturated aqueous sodium hydrogen carbonate
solution, water, and saturated brine (50 mL each). The resulting
organic layer was dried over sodium sulfate. The insoluble
1
In total, 60.1 g of 3 was obtained (80%); H NMR (300 MHz,
DMSO-d6) δ 8.28 (s, 2H for tautomer A), 7.51 (s, 1H for
tautomer B), 7.32 (br. s., 1H for tautomer B), 6.83 (br. s., 1H),
6.03 (s, 1H), 4.17 (d, J=3.4 Hz, 1H), 4.04 (d, J=13.2 Hz, 1H),
3.86 (d, J=3.4 Hz, 1H), 3.65 (d, J=13.2 Hz, 1H), 2.74 (s, 2H),
2.63 (s, 3H), 1.82 (s, 3H), 1.14 – 0.87 (m, 28H); 13C NMR (75
MHz, DMSO-d6) δ 165.41, 154.64, 146.88, 136.36, 100.52,
85.39, 82.68, 79.93, 63.75, 59.8, 57.16, 45.35, 17.3, 17.25, 17.1,

6
Tetrahedron
ACCEPTED MANUSCRIPT
material was removed by filtration and the filtrate was
concentrated. The mixture was purified by silica gel column
chromatography (hexane–ethyl acetate) to afford compound 7
for 1.5 h and quenched with sodium hydrogen carbonate. Silica
gel was added to the mixture, followed by evaporation. The
resulting residue was purified by silica gel column
chromatography (ethyl acetate–methanol 0%–30%) to afford
1
(325 mg, 0.4 mmol, 23%) as a pale yellow foam; H NMR (300
1
MHz, DMSO-d6) δ 10.77 (s, 1H), 8.30 (s, 1H), 7.56 – 7.26 (m,
10H), 6.57 (s, 1H), 4.93 (d, J=3.0 Hz, 1H), 4.24 (d, J=3.0 Hz,
1H), 4.06 (d, J=13.2 Hz, 1H), 3.69 (d, J=13.2 Hz, 1H), 2.89 –
2.75 (m, 2H), 2.69 (s, 3H), 2.17 (s, 3H), 1.08 – 0.90 (m, 28H);
13C NMR(75 MHz, DMSO-d6) δ 168.52, 155.15, 153.64, 152.09,
149.97, 142.16, 141.59, 129.35, 127.27, 126.94, 120.16, 84.14,
82.38, 79.32, 65.03, 60.17, 57.25, 45.41, 24.37, 17.2, 17.07,
17.04, 16.99, 16.93, 16.8, 12.6, 12.24, 12.01, 11.93; HRMS
compound 9 (484 mg, 50%) as a white powder; H NMR (300
MHz, DMSO-d6) δ 10.67 (br. s., 1H), 7.63 (s, 1H), 6.62 (br. s.,
2H), 6.35 (s, 1H), 4.49 (d, J=3.0 Hz, 1H), 4.20 (d, J=3.0 Hz, 1H),
4.02 (d, J=13.2 Hz, 1H), 3.67 (d, J=13.2 Hz, 1H), 2.78 (s, 2H),
2.66 (s, 3H), 1.10 – 0.91 (m, 28H); 13C NMR (75 MHz, DMSO-
d6) δ 156.68, 153.98, 150.23, 133.17, 116.85, 83.24, 82.09,
79.96, 64.66, 59.99, 57.29, 45.4, 17.23, 17.14, 17.08, 17.07,
16.99, 16.96, 16.88, 16.77, 12.65, 12.23, 12.2, 11.9; HRMS
(MALDI-TOF) calc. for C39H53N7NaO8Si2 [M
+
H]+
(MALDI-TOF) calc. for C24H42N6NaO6Si2 [M
m/z=589.2597, found 589.2586.
+
Na]+
m/z=826.3386, found 826.3410. N2-deacetylated compound 3,5′-
O-(tetraisopropyldisiloxane)-2′-O,4′-C-(N-
4.8. 6-Chloro-9-[3′,5′-O-(tetraisopropyldisiloxane)-2′-O,4′-C-(N-
methylaminomethylene)-β-D-ribofuranosyl]purine (10)
methylaminomethylene)-O6-diphenylcarbamoylguanosine
(7′)
1
was obtained (275 mg, 0.36 mmol, 20%); H NMR (300 MHz,
CDCL3) δ 8.05 (s, 1H), 7.53 – 7.17 (m, 10H), 6.57 (s, 1H), 5.41
(s, 2H), 4.54 (d, J=2.8 Hz, 1H), 4.30 (d, J=3.0 Hz, 1H), 4.03 (d,
J=13.0 Hz, 1H), 3.70 (d, J=13.0 Hz, 1H), 2.98 (d, J=11.0 Hz,
1H), 2.81 (s, 3H), 2.65 (d, J=11.1 Hz, 1H), 1.14 – 0.93 (m, 28H);
13C NMR (75 MHz, DMSO-d6) δ 159.59, 156.35, 154.62, 150.6,
141.86, 138.97, 128.93, 126.62, 118.38, 84.62, 82.87, 80.49,
64.75, 60.17, 57.78, 45.65, 17.35, 17.23, 17.16, 17, 16.94, 13.29,
12.78, 12.76, 12.31; HRMS (MALDI-TOF) calc. for
C37H51N7NaO7Si2 [M + Na]+ m/z=784.3286, found 784.3283.
A mixture of 4 (1.50 mmol, 874 mg), 6-chloropurine (1.95
mmol, 301 mg), and BSA (5.85 mmol, 1.43 mL) in toluene (15
mL) was heated at 100 °C. After the mixture became clear,
TMSOTf (1.95 mmol, 0.35 mL) was added, and the reaction
mixture was stirred at 100 °C for a further 30 min. The reaction
mixture was cooled, diluted with ethyl acetate (50 mL), and
washed with saturated aqueous sodium hydrogen carbonate
solution, water, and saturated brine (50 mL each). The combined
aqueous phases were back-extracted with ethyl acetate. The
resulting organic layer was dried over sodium sulfate, filtered,
and concentrated. The residue was purified by column
chromatography (0 to 30% AcOEt in hexane) to afford 10 (631
4.6. 2-Amino-6-chloro-9-[3′,5′-O-(tetraisopropyldisiloxane)-2′-
O,4′-C-(N-methylaminomethylene)-β-D-ribofuranosyl]purine (8)
1
2-Amino-6-chloropurine (254 mg, 1.5 mmol) and 4 (290 mg,
0.5 mmol) were suspended in toluene (5 mL). To the resulting
suspension, BSA (1.1 mL, 4.5 mmol) was added, and the
suspension was stirred at 100 °C for 30 min. To the solution after
stirring, TMSOTf (186 µL, 0.65 mmol) was added, and the
resulting solution was stirred at 100 °C for 30 min. The solution
after stirring was poured into saturated aqueous sodium hydrogen
carbonate solution (30 mL). Ethyl acetate (40 mL) was added to
the obtained solution, the resulting solution was filtered through
Celite®, and the filtrate was recovered. The filtrate was separated
to yield an organic layer. The resulting organic layer was washed
with saturated aqueous sodium hydrogen carbonate solution (30
mL), water (30 mL), and 1 M hydrochloric acid (30 mL). Sodium
sulfate was added to the organic layer after washing. The
resulting solution was concentrated to dryness under reduced
pressure to afford compound 8 (290 mg, 99%) as a pale yellow
mg, 74%) as a white foam; H NMR (300 MHz, DMSO-d6) δ
8.75 (s, 1H), 8.63 (s, 1H), 6.70 (s, 1H), 4.90 (d, J=3.4 Hz, 1H),
4.51 (d, J=3.4 Hz, 1H), 4.02 (d, J=13.2 Hz, 1H), 3.69 (d, J=13.2
Hz, 1H), 2.90 – 2.77 (m, 2H), 2.71 (s, 3H), 1.10 – 0.90 (m, 28H);
13C NMR(75 MHz, DMSO-d6) δ 151.46, 150.7, 149.22, 145.16,
131.55, 84.54, 82.59, 79.35, 64.92, 60.07, 57.24, 45.35, 17.17,
17.08, 17.01, 16.95, 16.86, 16.81, 12.67, 12.21, 12.01, 11.85;
HRMS (MALDI-TOF) calc. for C24H40ClN5NaO5Si2 [M + Na]+
m/z=592.2154, found 592.2152.
4.9. 3′,5′-O-(Tetraisopropyldisiloxane)-2′-O,4′-C-(N-
methylaminomethylene)inosine (11)
A mixture of hypoxanthine (3.50 g, 25.74 mmol), BSA (12.59
mL, 51.47 mmol), and TMSOTf (0.934 mL, 5.15 mmol) in
toluene (50.0 mL) was stirred at 80 °C for 40 min under an argon
atmosphere. 4 (10.00 g, 17.16 mmol) and TMSOTf (3.11 mL,
17.16 mmol) were added to the solution, and the mixture was
stirred at 80 °C for 3 h. After cooling (~25 °C), the mixture was
diluted with ethyl acetate (50 mL) and quenched with saturated
sodium hydrogen carbonate solution (50 mL, temperature was
~35 °C). After stirring for 10 min, the precipitate was collected
by filtration, washed with water and ethyl acetate, and dried in
vacuo to afford 11 (7.30 g, 13.23 mmol, 77%) as a colorless
solid. This solid contains ~10% of N7-isomer of compound 11;
HRMS (MALDI-TOF) calc. for C24H41N5NaO6Si2 [M + Na]+
m/z=574.2488, found 574.2483.
1
solid; H NMR (300 MHz, DMSO-d6) δ 7.98 (s, 1H), 7.07 (s,
2H), 6.45 (s, 1H), 4.65 (d, J=3.4 Hz, 1H), 4.20 (d, J=3.4 Hz, 1H),
4.04 (d, J=13.2 Hz, 1H), 3.69 (d, J=13.2 Hz, 1H), 2.80 (s, 2H),
2.67 (s, 3H), 1.10 – 0.92 (m, 28H); 13C NMR (75 MHz, DMSO-
d6) δ 159.89, 152.94, 149.48, 138.82, 123.65, 83.66, 82.33,
79.56, 64.67, 59.98, 57.24, 45.4, 17.23, 17.12, 17.06, 17.04, 17,
16.94, 16.79, 12.61, 12.2, 11.88; HRMS (MALDI-TOF) calc. for
C24H42ClN6O5Si2 [M + Na]+ m/z=585.2438, found 585.2443.
4.7. 3′,5′-O-(Tetraisopropyldisiloxane)-2′-O,4′-C-(N-
methylaminomethylene)guanosine(9)
4.10. N2-Acetyl-2′-O,4′-C-(N-
methylaminomethylene)guanosine(12)
Guanine (775 mg, 5.13 mmol) was suspended in BSA (10 mL,
40.9 mmol) and toluene (10 mL), and the resulting suspension
was refluxed for 1 h. To the solution after refluxing, TMSOTf
(60 µL, 0.33 mmol) was added, and the resulting suspension was
refluxed until it became transparent. The solution after refluxing
was cooled to 100 °C, and 4 (1.0 g, 1.71 mmol) and TMSOTf
(300 µL, 1.65 mmol) were added to it. The mixture was stirred
Guanine (775 mg, 5.13 mmol) was suspended in BSA (10 mL,
40.9 mmol) and toluene (10 mL), and the resulting suspension
was heated to 140 °C and refluxed for 1 h. TMSOTf (60 µL, 0.33
mmol) was added to the aforementioned suspension, and the
resulting suspension was refluxed until it became transparent.

7
The solution after refluxing was cooled to 100 °C (the
temperature of oil bath), 4 (1.0 g, 1.71 mmol) and TMSOTf (300
µL, 1.65 mmol) were added, and the resulting solution was
reacted for 30 min. The resulting reaction mixture was poured
into a mixed solution (130 mL) of pyridine-methanol-water-
triethylamine (100:10:20:1, v/v/v/v). The resulting suspension
was stirred at room temperature for 30 min. The suspension after
stirring was concentrated and dried by co-evaporation with dry
pyridine (three times) to yield a residue. Dry pyridine (30 mL),
acetic anhydride (20 mL), and 1-methylimidazole (50 µL) were
added to the obtained residue, and the reaction mixture was
stirred with heating at 70 °C for 4 h. The reaction mixture was
cooled to room temperature, water (100 mL) was added, and the
mixture was stirred for 30 min. To the solution after stirring, 25%
aqueous ammonia (30 mL) was added, and the mixture was
stirred for 15 min. The resulting solution was concentrated under
reduced pressure to yield a residue. Ethyl acetate (200 mL) was
added to the residue, and the resulting solution was washed with
saturated brine–water (1:1, v/v). The resulting solution was
separated to yield an organic layer. The resulting organic layer
was concentrated, and the residue was purified by silica gel
column chromatography (hexane–ethyl acetate=85:15, v/v) to
and washed with sodium hydrogen carbonate solution (80 mL,
ACCEPTED MANUSCRIPT
twice) and brine (80 mL). The resulting organic layer was dried
over anhydrous magnesium sulfate and concentrated. The residue
was purified by silica gel column chromatography (hexane–ethyl
acetate, 40–80%, (v/v)) to afford compound 14 (4.3 g, 92% (2
1
steps)) as a white foam; H NMR (300 MHz, CDCL3) δ 9.19 (s,
1H), 8.80 (s, 1H), 8.33 (s, 1H), 8.01 (d, J=7.5 Hz, 2H), 7.62 –
7.16 (m, 12H), 6.89 – 6.77 (m, 5H), 4.68 (d, J=3.0 Hz, 1H), 4.41
(d, J=4.1 Hz, 1H), 3.78 (s, 6H), 3.39 (s, 2H), 3.07 – 2.83 (m, 3H),
2.78 (s, 3H); 13C NMR(75 MHz, CDCL3) δ 164.59, 158.6,
158.58, 152.75, 150.76, 149.52, 144.31, 141.17, 135.54, 135.32,
133.62, 132.72, 130.03, 129.97, 128.8, 128.05, 127.94, 127.84,
126.99, 123.52, 113.25, 86.5, 84.84, 82.83, 80.93, 66.73, 62.63,
58.5, 55.19, 45.63; HRMS (MALDI-TOF) calc. for C40H39N6O7
[M + H]+ m/z=715.2875, found 715.2880.
4.13. N2-Acetyl-5′-O-(4,4′-dimethoxytriphenyl)methyl-2′-O,4′-C-
(N-methylaminomethylene)guanosine(15)
Compound 12 (1.5 g, 2.5 mmol) was dissolved in THF (25
mL), triethylamine (700 µL,
5
mmol) and TEA·3HF
(triethylamine trihydrofluoride, 410 µL, 2.5 mmol) were added,
and the mixture was stirred at 50 °C for 2 h. After stirring, THF
was evaporated under reduced pressure, and diisopropyl ether (50
mL) was added to form a precipitate. The precipitate was
collected by filtration. The resulting compound 15 was dried by
co-evaporation with dry pyridine (three times), and dissolved in
pyridine (10 mL). DMTr-Cl (1.3 g, 3.75 mmol) was then added,
and the mixture was stirred overnight at room temperature. Water
(20 mL) was added to the reaction mixture to quench the
reaction. The target substance was extracted with ethyl acetate
(200 mL). The ethyl acetate layer was successively washed with
water (100 mL), saturated sodium hydrogen carbonate (100 mL),
and saturated brine (100 mL), and concentrated. The residue was
purified by diol-silica gel column chromatography (hexane–ethyl
acetate=1:9, v/v) to afford 15 (1 g, yield 60%, 2 steps) as white
1
afford 12 (680 mg, 65% (2 steps)) as a white foam; H NMR
(600 MHz, DMSO-d6) δ 12.10 (s, 1H), 11.88 (s, 1H), 7.90 (s,
1H), 6.44 (s, 1H), 4.59 (d, J=3.3 Hz, 1H), 4.20 (d, J=2.9 Hz, 1H),
4.05 (d, J=12.8 Hz, 1H), 3.69 (d, J=12.8 Hz, 1H), 2.80 (s, 2H),
2.67 (s, 3H), 2.18 (s, 3H), 1.09 – 0.91 (m, 28H); 13C NMR(150
MHz, DMSO-d6) δ 173.67, 154.8, 148.18, 147.74, 135.53,
120.59, 83.72, 82.45, 79.95, 64.55, 59.99, 57.27, 45.47, 23.75,
17.29, 17.2, 17.15, 17.12, 17.07, 17.03, 16.97, 16.84, 12.66,
12.26, 12.24, 11.92; HRMS (MALDI-TOF) calc. for
C26H44N6NaO7Si2 [M + Na]+ m/z=631.2702, found 631.2702.
4.11. 2′-O,4′-C-(N-methylaminomethylene)inosine (13)
To a suspension of 11 in DMF(dry) (9.31 mL), triethylamine
(0.113 mL, 0.81 mmol) and triethylamine trihydrofluoride (0.197
mL, 1.21 mmol) were added. The mixture was stirred at room
temperature for 16 h and concentrated under reduced pressure.
The residue was crystallized from 2-propanol and the solvent was
removed under reduced pressure to yield a crude solid. The solid
was recrystallized with 9% aq 2-PrOH (4.5 mL) at 60 °C to yield
2′-O,4′-C-(N-methylaminomethylene)inosine (13, 300 mg, 0.974
1
foam; H NMR (300 MHz, DMSO-d6) δ 12.20 – 11.67 (m, 2H),
8.03 (s, 1H), 7.49 – 7.13 (m, 9H), 6.97 – 6.75 (m, 4H), 6.48 (s,
1H), 5.51 (d, J=5.3 Hz, 1H), 4.43 (d, J=3.0 Hz, 1H), 4.15 (br. s.,
1H), 3.74 (s, 6H), 3.32 – 3.27 (m, 1H), 3.16 (d, J=10.5 Hz, 1H),
2.98 (d, J=11.3 Hz, 1H), 2.78 (d, J=11.3 Hz, 1H), 2.67 (s, 3H),
2.19 (s, 3H); 13C NMR(75 MHz, DMSO-d6) δ 173.6, 158.1,
154.8, 148.06, 147.85, 144.7, 136.47, 135.42, 135.15, 129.77,
129.71, 127.82, 127.68, 126.74, 120.48, 113.17, 85.45, 83.6,
82.06, 80.37, 65.37, 62.57, 57.13, 55, 45.2, 23.71; HRMS
1
mmol, 80%) as an off-white solid. H NMR (300 MHz, DMSO-
d6) δ 12.73 – 11.95 (m, 1H), 8.23 (s, 1H), 8.07 (s, 1H), 6.50 (s,
1H), 5.59 – 5.28 (m, 1H), 5.24 – 4.90 (m, 1H), 4.35 (d, J=3.0 Hz,
1H), 4.06 (d, J=3.0 Hz, 1H), 3.60 (d, J=3.4 Hz, 2H), 2.82 (s, 2H),
2.69 (s, 3H); 13C NMR(75 MHz, DMSO-d6) δ 156.57, 147.29,
145.92, 137.6, 124.44, 83.88, 83.03, 80.81, 64.24, 60.03, 56.97,
45.25; HRMS (MALDI-TOF) calc. for C12H15N5NaO5 [M + Na]+
m/z=332.0971, found 332.0965.
(MALDI-TOF) calc. for C35H36N6NaO8 [M
m/z=691.2487, found 691.2488.
+
Na]+
4.14. 5′-O-(4,4′-Dimethoxytriphenyl)methyl-2′-O,4′-C-(N-
methylaminomethylene)inosine(16)
Compound 13 was co-evaporated twice with dry pyridine and
dissolved in pyridine (dry) (4.9 mL). To the solution, DMTr-Cl
(362 mg, 1.07 mmol) was added, and the mixture was stirred at
room temperature for 20 h. The solvent was removed under
reduced pressure, and the residue was mixed with ethyl acetate
and sodium hydrogen carbonate solution. The organic layer was
purified by column chromatography (silica gel, eluted with 0%–
10% methanol in ethyl acetate) to afford 16 (514 mg, 0.840
4.12. N6-Benzoyl-5′-O-(4,4′-dimethoxytriphenyl)methyl-2′-O,4′-
C-(N-methylaminomethylene)adenosine (14)
Compound 5 (4.3 g, 6.57 mmol) was dissolved in THF (40
mL), triethylamine (1.61 mL, 9.85 mmol) and triethylamine
trihydrofluoride (1.83 mL, 13.1 mmol) were added, and the
mixture was stirred for 2 h at 50 °C. Diisopropylether (40 mL)
was added to the reaction mixture, followed by stirring for 0.5 h
at 0 °C to form a precipitate. The precipitate was collected by
filtration and dried by co-evaporation with dry pyridine. The
residue was dissolved in pyridine (60 mL). To the mixture, 4,4′-
dimethoxytrityl chloride (DMTr-Cl, 2.56 g, 7.57 mmol) was
added, and the mixture was stirred at room temperature
overnight.The mixture was concentrated to 1/3 of its volume. The
residue was diluted with ethyl acetate–THF (80 mL, 1:1 (v/v))
1
mmol, 87% (70%, 2 steps from 11)) as a colorless foam; H
NMR (300 MHz, DMSO-d6) δ 12.40 (br. s., 1H), 8.13 (s, 1H),
8.09 (s, 1H), 7.45 – 7.18 (m, 9H), 6.92 – 6.81 (m, 4H), 6.58 (s,
1H), 5.49 (d, J=3.4 Hz, 1H), 4.45 (d, J=3.0 Hz, 1H), 4.23 (br. s.,
1H), 3.74 (s, 6H), 3.29 – 3.09 (m, J=10.9 Hz, 2H), 2.99 (d,
J=11.3 Hz, 1H), 2.78 (d, J=11.3 Hz, 1H), 2.69 (s, 3H); 13C
NMR(75 MHz, DMSO-d6) δ 158.09, 156.57, 147.4, 145.95,
144.72, 137.53, 135.45, 135.2, 129.75, 129.69, 127.8, 127.67,
126.71, 124.52, 113.16, 85.39, 83.96, 82.12, 80.48, 65.44, 62.58,

8
Tetrahedron
ACCEPTED MANUSCRIPT
57.08, 55, 45.14; HRMS (MALDI-TOF) calc. for C33H33N5NaO7
[M + Na]+ m/z=634.2272, found 634.2271.
4.15. N6-Benzoyl-3′-O-[2-
cyanoethoxy(diisopropylamino)phosphino]-5′-O-(4,4′-
dimethoxytriphenyl)methyl-2′-O,4′-C-(N-
methylaminomethylene)adenosine (17)
added. After stirring for 4 h, additional 1-methylimidazole (0.016
mL, 0.20 mmol), 1H-tetrazole (6.99 mg, 0.10 mmol), and
N,N,N′,N′-tetraisopropyl-2-cyanoethylphosphorodiamidite (0.127
mL, 0.40 mmol) were added to the solution. The mixture was
stirred at room temperature under a dry atmosphere (CaCl2 tube)
for overnight. The mixture was poured into saturated sodium
hydrogen carbonate aq. and extracted with ethyl acetate. The
organic layer was separated, washed with brine, dried over
magnesium sulfate, and concentrated in vacuo. The residue was
purified by column chromatography (silica gel, eluted with 0%–
50% ethyl acetate (+0.5% triethylamine) in hexane (+0.5%
triethylamine)) and (diol silica gel. eluted with 0%–30% acetone
(0.5% triethylamine) in hexane (+0.5% triethylamine)) to afford
19 (1.2 g, 1.5 mmol, 74%) as a colorless oil. 19 was precipitated
from ethyl acetate (0.6 mL)–hexane (25 mL) to afford a white
Compound 14 (410 mg, 0.57 mmol) was dried by co-
evaporation (twice) with anhydrous acetonitrile and dissolved in
anhydrous acetonitrile (2.5 mL). N,N,N′,N′-Tetraisopropyl-2-
cyanoethylphosphorodiamidite (200 µL, 0.63 mmol) and 4,5-
dicyanoimidazole (DCI, 71 mg, 0.6 mmol) were successively
added. The mixture was stirred at room temperature for 2 h, and
the reaction mixture was diluted with ethyl acetate (20 mL).
Saturated aqueous sodium hydrogen carbonate solution (10 mL)
was added to quench the reaction. The organic layer was washed
with saturated aqueous sodium hydrogen carbonate solution (10
mL), dried over anhydrous sodium sulfate, and concentrated. The
residue was purified by silica gel column chromatography (diol-
silica gel, hexane-acetone) to afford 17 (480 mg, 0.52 mmol,
1
solid; H NMR (300 MHz, DMSO-d6) δ 12.61 – 12.15 (m, 1H),
8.26 – 7.90 (m, 2H), 7.44 – 7.34 (m, 2H), 7.34 – 7.15 (m, 7H),
6.86 (dt, J=1.7, 8.8 Hz, 4H), 6.68 – 6.60 (m, J=5.7 Hz, 1H), 4.74
– 4.66 (m, J=7.0 Hz, 1H), 4.63 – 4.49 (m, 1H), 3.73 (d, J=3.2 Hz,
7H), 3.59 – 3.45 (m, 3H), 3.30 – 3.20 (m, 2H), 3.11 – 2.92 (m,
1H), 2.74 – 2.62 (m, 5H), 2.58 – 2.53 (m, 1H), 1.13 – 0.90 (m,
12H); 31P NMR(121 MHz, DMSO-d6) δ 148.36, 147.47; HRMS
1
91%) as a white foam; H NMR (300 MHz, DMSO-d6) δ 11.24
(br. s., 1H), 8.78 – 8.70 (m, 1H), 8.60 – 8.49 (m, 1H), 8.09 – 7.98
(m, 2H), 7.69 – 7.50 (m, 3H), 7.42 – 7.11 (m, 10H), 6.90 – 6.76
(m, 5H), 4.97 – 4.82 (m, 2H), 3.76 – 3.67 (m, 6H), 3.62 – 3.47
(m, 3H), 3.31 – 3.20 (m, 3H), 3.14 – 2.94 (m, 1H), 2.77 – 2.66
(m, 4H), 2.56 (dt, J=2.3, 5.9 Hz, 1H), 1.15 – 0.93 (m, 12H); 31P
NMR(121 MHz, DMSO-d6) δ 148.32, 147.63; HRMS (MALDI-
TOF) calc. for C49H56N8O8P [M + H]+ m/z=915.3953, found
915.3948.
(MALDI-TOF) calc. for C42H50N7NaO8P [M
m/z=834.3351, found 834.3347.
+
Na]+
Acknowledgments
We thank Prof. Takeshi Imanishi for the scientific discussion,
Hirohiko Nishiyama (Hamari Chemicals, Ltd.) for synthesis
support.
4.16. N2-Acetyl-3′-O-[2-
cyanoethoxy(diisopropylamino)phosphino]-5′-O-(4,4′-
dimethoxytriphenyl)methyl-2′-O,4′-C-(N-
methylaminomethylene)guanosine (18)
References and notes
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Compound 15 (870 mg, 1.3 mmol) was dried by co-
evaporation (three times) with dry acetonitrile, and dissolved in
DMF
(9
mL).
N,N,N′,N′-tetraisopropyl-2-
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tetrazole (91 mg, 1.3 mmol) was added. The mixture was stirred
overnight at room temperature. The reaction mixture was diluted
with ethyl acetate (40 mL), and washed twice with saturated
brine (40 mL). The organic layer was dried over anhydrous
sodium sulfate and concentrated. The residue was purified by
diol-silica gel column chromatography (0.5% triethylamine
containing hexane-ethyl acetate) to afford 18 (1.0 g, 1.15 mmol,
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1
88%) as a white foam; H NMR (300 MHz, DMSO-d6) δ 11.93
(br. s., 2H), 8.13 – 7.93 (m, 1H), 7.49 – 7.19 (m, 9H), 6.96 – 6.78
(m, 4H), 6.59 – 6.50 (m, 1H), 4.69 – 4.59 (m, 1H), 4.44 – 4.23
(m, 1H), 3.79 – 3.41 (m, 11H), 3.30 – 3.28 (m, 1H), 3.13 – 2.92
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(MALDI-TOF) calc. for C44H53N8NaO9P [M
m/z=891.3571, found 891.3574.
+
Na]+
4.17. 3′-O-[2-Cyanoethoxy(diisopropylamino)phosphino]-5′-O-
(4,4′-dimethoxytriphenyl)methyl-2′-O,4′-C-(N-
methylaminomethylene)inosine (19)
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solution, 1-methylimidazole (0.159 mL, 1.99 mmol), 1H-
tetrazole (0.070 g, 1.00 mmol), and N,N,N′,N′-tetraisopropyl-2-
cyanoethylphosphorodiamidite (0.127 mL, 0.40 mmol) were
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Supporting information; 1H and 13C NMR spectra of new
compounds, LC-MS analysis data for the reaction mixture entry 2
of Table 1 and MALDI-TOF MS data for ODNs.