Syntheses of prodrug-type 2′-O-methyldithiomethyl oligonucleotides modified at natural four nucleoside residues and their conversions into natural 2′-hydroxy oligonucleotides under reducing condition

Article abstract of DOI:10.1016/j.bmc.2018.10.025

We previously reported that reducing-environment-responsive prodrug-type small interfering RNA (siRNA) bearing 2′-O-methyldithiomethyl (2′-O-MDTM) uridine exhibits efficient knockdown activity and nuclease resistance. In this report, we describe the prepa

Full text of DOI:10.1016/j.bmc.2018.10.025

Accepted Manuscript
Syntheses of prodrug-type 2’-O-methyldithiomethyl oligonucleotides modified
at natural four nucleoside residues and their conversions into natural 2’-hydroxy
oligonucleotides under reducing condition
Junsuke Hayashi, Yosuke Ochi, Yasuyuki Morita, Katsuma Soubou, Yuhei
Ohtomo, Misa Nishigaki, Yuko Tochiyama, Osamu Nakagawa, Shun-ichi
Wada, Hidehito Urata
PII:
S0968-0896(18)31693-6
https://doi.org/10.1016/j.bmc.2018.10.025
BMC 14584
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
1 October 2018
24 October 2018
26 October 2018
Please cite this article as: Hayashi, J., Ochi, Y., Morita, Y., Soubou, K., Ohtomo, Y., Nishigaki, M., Tochiyama,
Y., Nakagawa, O., Wada, S-i., Urata, H., Syntheses of prodrug-type 2’-O-methyldithiomethyl oligonucleotides
modified at natural four nucleoside residues and their conversions into natural 2’-hydroxy oligonucleotides under
reducing condition, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.10.025
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Syntheses of prodrug-type 2’-O-methyldithiomethyl oligonucleotides modified at natural four
nucleoside residues and their conversions into natural 2’-hydroxy oligonucleotides under
reducing condition
Junsuke Hayashi, Yosuke Ochi, Yasuyuki Morita, Katsuma Soubou, Yuhei Ohtomo, Misa Nishigaki,
Yuko Tochiyama, Osamu Nakagawa†, Shun-ichi Wada and Hidehito Urata*
Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan
†Present address: Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka,
Suita, Osaka 565-0871, Japan.
*Corresponding author. TEL & FAX: +81-72-690-1089, E-mail: urata@gly.oups.ac.jp
Keywords: Prodrug-type RNA; reducing environment; 2’-O-modified RNA; post-synthetic
modification
1 / 26

Abstract
We previously reported that reducing-environment-responsive prodrug-type small interfering
RNA (siRNA) bearing 2’-O-methyldithiomethyl (2’-O-MDTM) uridine exhibits efficient
knockdown activity and nuclease resistance. In this report, we describe the preparation of
2’-O-MDTM oligonucleotides modified not only at uridine but also at adenosine, guanosine and
cytidine residues by post-synthetic modification. Precursor oligonucleotides bearing
2’-O-(2,4,6-trimethoxybenzylthiomethyl) (2’-O-TMBTM) adenosine, guanosine, and cytidine were
reacted with dimethyl(methylthio)sulfonium tetrafluoroborate to form 2’-O-MDTM oligonucleotides
in the same manner as the oligonucleotide bearing 2’-O-TMBTM uridine. Furthermore, the
oligonucleotides bearing 2’-O-MDTM adenosine, guanosine, and cytidine were efficiently converted
into corresponding natural 2’-hydroxy oligonucleotides under the cytosol-mimetic reducing
condition.
2 / 26

Graphical abstract
3 / 26

1
2
1. Introduction
Selective gene silencing induced by chemically synthesized oligonucleotides, such as
3
antisense oligonucleotide (AON) and small interfering RNA (siRNA), has shown promise for the
treatment of several intractable diseases.1 In the past 30 years, a small number of AON-type drugs
were approved2 and a novel AON-type drug for the treatment of the hereditary transthyretin
amyloidosis was approved quite recently.3 Recently, the clinical use of siRNA has received
considerable attention because effective knockdown activity can be induced with lower
concentrations of siRNAs than AONs.4 However, the instability of RNA molecules in serum has
hindered the clinical success of RNA-type drugs and only one candidate was approved as the first
siRNA-type drug quite recently.5,6
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
2’-O-Modified RNAs, including 2’-O-alkyl,7 2’-fluoro (2’-F),8,9 2’-O-(2-methoxyethyl)
(2’-O-MOE),10 and bridged nucleic acids (BNAs)11,12 are well used in pharmaceutical research for
enhancement of the nuclease resistance. However, the knockdown activities of 2’-O-modified
siRNAs are often decreased by 2’-O-modification because the modification inhibits the formation of
an RNA-induced silencing complex (RISC) in the RNA interference (RNAi) pathway.13,14 In
particular, the 5’-end side of the antisense strand of siRNA is essential for the formation of
RISC.15-17 Thus, the 2’-O-modifications are mainly introduced at the sense strand or the 3’-end side
of the antisense strand of siRNA.
The prodrug concept has been well-received in oligonucleotide chemistry.18-25 In the prodrug
concept, oligonucleotides are protected by bioreversible functional groups to introduce certain
properties beneficial for pharmaceutical use, such as enhancement of the nuclease resistance and the
cell membrane permeability. These protective groups are cleaved by a trigger reaction to induce the
conversion of the prodrugs into natural active oligonucleotides in the target organ. Thus,
4 / 26

24
25
26
27
28
29
30
31
32
33
34
35
36
37
prodrug-type oligonucleotides would exhibit both the above-mentioned beneficial properties due to
the modification and the gene silencing activity. We originally developed prodrug-type
2’-O-modified RNA containing a 2’-O-methyldithiomethyl (2’-O-MDTM) group, the cleavage of
which is triggered by an intracellular reducing environment, and named it
“Reducing-Environment-Dependent Uncatalyzed Chemical Transforming (REDUCT)-RNA”
(Figure 1).26-28 We reported that the 2’-O-MDTM siRNA modified at the uridine residue of
anti-luciferase siRNAs exhibits stronger knockdown activity than natural 2’-OH siRNA.27
Furthermore, the knockdown activity was basically independent of the position and extent of
modification.28 From this basic research, 2’-O-MDTM siRNA is expected to exhibit sufficient
knockdown activity not only in vitro but also in vivo. However, the 2’-O-MDTM group was
introduced only at the uridine residue in our previous reports. In this report, we describe the
preparation of 2’-O-MDTM oligonucleotides modified at adenosine, guanosine, and cytidine
residues by post-synthetic modification and the conversions into natural 2’-OH oligonucleotides
under the cytosol-mimetic condition.
38
39
40
41
42
43
Figure 1. Conversion of Reducing-Environment-Dependent Uncatalyzed Chemical Transforming
(REDUCT)-RNA modified at uridine residue.
5 / 26

44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
2. Results and discussion
2.1 Synthesis of 2’-O-(2,4,6-trimethoxybenzylthiomethyl)phosphoramidite units
In general, oligonucleotides are synthesized by the phosphoramidite method in which
nucleoside phosphoramidites are employed as monomer units. We tried to prepare the
phosphoramidite unit bearing a 2’-O-MDTM group. However, the 2’-O-MDTM phosphoramidite
unit was not obtained because an intramolecular reaction occurred between reductive P(III) atom and
the disulfide bond. Then, we developed a post-synthetic approach to obtain a 2’-O-MDTM
oligonucleotide modified at the uridine residue (Figure 2).26 In the post-synthetic approach, the
2,4,6-trimethoxybenzylthiomethyl (TMBTM) group was used as the promoiety, which could be
converted into the 2’-O-MDTM group by treatment with dimethyl(methylthio)sulfonium
tetrafluoroborate (DMTSF). In hopes of applying the method to the preparation of 2’-O-MDTM
oligonucleotides modified at other nucleoside residues, such as adenosine, guanosine, and cytidine,
we synthesized 2’-O-TMBTM phosphoramidite units 6a–c (Scheme 1). 2’-O-Methylthiomethyl
(MTM) derivatives 2a–d were obtained from corresponding 2’-OH derivatives 1a–d in good yields.
In the next step, we attempted to synthesize 2’-O-TMBTM derivatives 3a–c according to a
previously reported synthetic procedure for uridine derivatives.26 In the synthesis of uridine
derivative 3d, the solvent and excess SO2Cl2 were removed completely under reduced pressure after
the treatment with SO2Cl2 to afford the 2’-O-chloromethyl intermediate. However, the
2’-O-chloromethyl intermediates of adenosine, guanosine, and cytidine degraded during the removal
of the solvent and excess SO2Cl2. Therefore, the syntheses of 2’-O-TMBTM derivatives 3a–c from
corresponding 2a–c were performed in one pot. 2’-O-MTM nucleosides 2a–c were treated with
SO2Cl2 to afford 2’-O-chloromethyl intermediate. Subsequently, 2,4,6-trimethoxybenzylmercaptan
and excess N,N-diisopropylethylamine (DIEA) were added to this reaction mixture to afford
adenosine, guanosine, and cytidine derivatives 3a–c. In the synthesis of guanosine derivative 3b,
6 / 26

68
69
70
71
72
73
74
75
cyclohexene was added to scavenge chlorine generated from the excess SO2Cl2. The 3’,5’-O-silyl
protecting group of 2’-O-TMBTM derivatives 3a–d was cleaved off by 3HF-Et3N. The 5’-hydroxy
group was protected with the 4,4’-dimethoxytrityl (DMTr) group to afford 5a–d. Finally, the
3’-hydroxy group of 5a–d was phosphitylated with chlorophosphoramidite reagent under the basic
condition to generate the 2’-O-TMBTM phosphoramidite units 6a–d in good yields.
76
77
Figure 2. Post-synthetic approach for the synthesis of 2’-O-MDTM oligonucleotide modified at
78
79
uridine residue.
80
81
82
Scheme 1. Syntheses of phosphoramidite units bearing 2’-O-TMBTM group. (i) DMSO, Ac2O,
7 / 26

83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
AcOH; (ii) SO2Cl2, CH2Cl2; (iii) (MeO)3BnSH, DIEA, CH2Cl2 (for 3a and c); (iv) (MeO)3BnSH,
DIEA, cyclohexene, CH2Cl2 (for 3b); (v) (MeO)3BnSH, NaH, DMF (for 3d); (vi) 3HF-Et3N, THF;
(vii) DMTr-Cl, pyridine; (viii) 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite, DIEA,
DMAP, CH2Cl2.
2.2 Synthesis of 2’-O-MDTM oligonucleotides by post-synthetic modification
2’-O-TMBTM oligonucleotides 7a–d were synthesized by the solid-phase phosphoramidite
method (Table 1). Coupling reactions were conducted with 5-ethylthio-1H-tetrazole (ETT) as the
activator and the coupling time for 2’-O-TMBTM phosphoramidites 6a–d was extended to 600 sec.
Furthermore, the oxidation of P(III) to P(V) was conducted with 0.02 M iodine solution. After
cleavage from the support and deprotection, 2’-O-TMBTM oligonucleotides 7a–d were purified by
reversed-phase HPLC (RP-HPLC) and the structures were characterized by MALDI-TOF mass
spectrometry (Table 1).
Post-synthetic modification for the preparation of 2’-O-MDTM oligonucleotides 8a–d from
2’-O-TMBTM oligonucleotides 7a–d was conducted by treatment with DMTSF.26 An aqueous
DMTSF suspension was added to 0.1 mM solutions of 2’-O-TMBTM oligonucleotides 7a–d in 200
99
mM sodium acetate buffer (pH 4) at 37 °C and the reactions were monitored by RP-HPLC (Figure
100
101
102
103
104
105
106
3). Unexpectedly, the conversion reactions of 7a–d to 8a–d were so fast and almost completed
within 1 min without any detectable side reactions. As it is known that DMTSF easily reacts with
H2O,29 the conversion reactions were conducted by using freshly prepared aqueous DMTSF
suspension. After the reactions, the 2’-O-MDTM oligonucleotides were purified by RP-HPLC. The
disulfide bonds of 2’-O-MDTM oligonucleotides might be cleaved in highly concentrated
triethylammonium acetate (TEAA) buffer. Then, the solutions of 2’-O-MDTM oligonucleotides
obtained from the RP-HPLC purification were desalted by a gel filtration column before
8 / 26

107
108
109
110
111
112
113
114
115
concentration. Finally, the structures of the 2’-O-MDTM oligonucleotides were confirmed by
MALDI-TOF mass spectrometry (Table 1). The results indicate that our post-synthetic approach
would be applicable to the preparation of oligonucleotides bearing 2’-O-MDTM adenosine,
guanosine, cytidine as well as uridine.
Table 1. Sequences and characterization of synthesized oligonucleotides (ONs)
MALDI-TOF Mass
ONs
Sequence (5’ to 3’) a)
Calcd. (M-H)-
3883.7
3899.7
3859.7
3860.6
3749.6
3765.6
3725.5
3726.5
3657.4
3673.4
3633.4
3634.4
Found
3884.2
3899.0
3859.2
3859.1
3750.5
3765.6
3726.1
3727.9
3658.0
3672.5
3632.6
3633.7
d (GCG TTA TTT GCT)
7a
7b
7c
7d
8a
8b
8c
8d
9a
9b
9c
9d
d (GCG TTG TTT GCT)
d (GCG TTC TTT GCT)
d (GCG TTU TTT GCT)
d (GCG TTA TTT GCT)
d (GCG TTG TTT GCT)
d (GCG TTC TTT GCT)
d (GCG TTU TTT GCT)
d (GCG TTA TTT GCT)
d (GCG TTG TTT GCT)
d (GCG TTC TTT GCT)
d (GCG TTU TTT GCT)
116
117
118
119
a) 2’-O-modified positions are underlined. Italic (7a–d), bold (8a–d), and italic bold (9a–d) letters
indicate 2’-O-TMBTM, 2’-O-MDTM, and 2’-OH nucleosides, respectively.
9 / 26

120
121
Figure 3. HPLC charts of the conversions of 2’-O-TMBTM (7a–d) into 2’-O-MDTM (8a–d)
122
123
124
125
126
127
128
129
130
131
132
133
oligonucleotides by treatment with DMTSF in 200 mM sodium acetate buffer (pH 4) at 37 °C.
2.3 Conversion of 2’-O-MDTM oligonucleotides into 2’-OH oligonucleotides under reducing
condition.
We already showed that oligonucleotides bearing 2’-O-MDTM uridine are efficiently
converted into natural oligonucleotides under the cytosol-mimetic reducing condition (10 mM
glutathione, GSH). Then, we carried out the conversion of oligonucleotides bearing 2’-O-MDTM
adenosine, guanosine, cytidine, and uridine residues into corresponding natural 2’-OH
oligonucleotides in the reducing condition. 2’-O-MDTM oligonucleotides (8a–d) were treated with
10 mM GSH in 50 mM phosphate buffer (pH 7.0) at 37 °C and the reactions were analyzed by
RP-HPLC (Figure 4). The 2’-O-MDTM groups of oligonucleotides (8a–d) were cleaved off and the
10 / 26

134
135
136
137
138
139
140
141
corresponding 2’-thiohemiacetal intermediates were generated after reaction for 1.0 h. Subsequently,
the conversion into 2’-OH oligonucleotides (9a–d) was almost completed within 6.0 h. The reactions
were desalted and the structures were characterized by MALDI-TOF mass spectrometry (Table 1).
The results indicate that prodrug-type oligonucleotides bearing 2’-O-MDTM adenosine, guanosine,
and cytidine would be efficiently converted into the corresponding 2’-OH oligonucleotides under the
cytosol-mimetic reducing condition in the same manner as prodrug-type oligonucleotides bearing
2’-O-MDTM uridine.
142
143
Figure 4. HPLC charts of the conversions of 2’-O-MDTM (8a–d) into 2’-OH (9a–d)
oligonucleotides under cytosol-mimetic reducing condition (10 mM GSH in 50 mM phosphate
buffer, pH 7.0 at 37 °C).
144
145
146
147
11 / 26

148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
166
167
168
169
170
171
3. Conclusions
We showed the preparation method of prodrug-type 2’-O-MDTM oligonucleotides modified at
four natural nucleoside residues by post-synthetic modification. The 2’-O-TMBTM amidites of four
natural bases (6a–d) were obtained in 33–49% yield in 5 steps from protected nucleosides (1a–d).
The 2’-O-TMBTM oligonucleotides were synthesized from 2’-O-TMBTM amidites (6a–d) and the
oligonucleotides were efficiently reacted with DMTSF to afford 2’-O-MDTM oligonucleotides.
Furthermore, we confirmed that prodrug-type oligonucleotides bearing 2’-O-MDTM adenosine,
guanosine, cytidine, and uridine were efficiently converted into the corresponding 2’-OH
oligonucleotides under the cytosol-mimetic reducing condition. In our previous report, we
successfully obtained the 2’-O-MDTM RNA modified at the multiple uridine residues by the
post-synthetic modification.26 Hence, the 2’-O-MDTM modifications could be introduced not only at
uridine residues but also at multiple natural four nucleoside residues of RNA sequence by using our
post-synthetic modification method. The evaluation of the biological properties of siRNAs bearing
2’-O-MDTM adenosine, guanosine, cytidine, and uridine residues is ongoing in our laboratory.
4. Experiments
4.1 General methods
All reagents and solvents except 2,4,6-trimethoxybenzylmercaptan were obtained from
commercial sources and used without purification. Silica gel chromatography was performed using
Wakogel C-400HG or Wakosil C-200 (FUJIFILM Wako Pure Chemical Industries, Japan). TLC was
performed on Merck silica gel 60 F254 and compounds were visualized under UV light (254 nm). 1H,
13C, and 31P NMR spectra were measured on an Agilent NMR System 600-DD2 NMR spectrometer.
Reversed-phase HPLC was performed on Nacalai COSMOSIL 5C18-MS-II  4.6 × 250 mm
12 / 26

172
173
174
175
176
177
178
179
180
181
182
183
184
185
186
187
188
189
190
191
192
193
194
195
(analytical column) and  10.0 × 250 mm (purification column) with a linear gradient of acetonitrile
in 50 mM triethylammonium acetate (TEAA) (pH 7). Oligonucleotides were synthesized on an
Applied Biosystems Model 392 DNA/RNA Synthesizer (Applied Biosystems). The mass spectra of
the nucleosides were measured on a JMS-700 mass spectrometer (JEOL, Japan) in the positive-ion
mode. The mass spectra of the oligonucleotides were measured on a Voyager-DE STR MALDI-TOF
mass spectrometer (AB SCIEX) or a Bruker Microflex MALDI-TOF mass spectrometer (Bruker) in
the negative-ion mode. UV quantifications were performed on an Eppendorf BioSpectrometer basic
(Eppendorf) by measuring absorbance at 260 nm.
4.2 Syntheses of 2’-O-(2,4,6-trimethoxybenzylthiomethyl)phosphoramidites
4.2.1 Preparation of N-protected-3',5'-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-2’-O-
[(methylthio)methyl]nucleosides (2a–d)
To
a
solution of each of N-protected-3',5'-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-
diyl)-nucleosides (1a–d) in DMSO (45 eq) were added glacial AcOH (52 eq) and Ac2O (24 eq). The
reaction mixture was stirred at room temperature for 18–24 h. After the reaction, the resulting
solution was poured into sat. NaHCO3 aqueous solution and the mixture was extracted with ethyl
acetate. The organic layer was washed with distilled water and brine, and then dried over anhydrous
sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column
chromatography to afford compounds 2a–d.
4.2.1.1
N-Benzoyl-3',5'-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-
diyl)-2’-O-[(methylthio)methyl]-adenosine (2a).
1
White solid, 73% yield from 1a. H NMR (600 MHz, CDCl3) br s), 8.78 (1H, s),
8.34 (1H, s), 8.04 (2H, d, J = 7.7 Hz), 7.63-7.60 (1H, m), 7.55-7.52 (2H, m), 6.12 (1H, s), 5.07 (1H,
13 / 26

196
197
198
199
200
201
202
203
204
205
206
207
208
209
210
211
212
213
214
215
216
217
218
219
d, J = 11.5 Hz), 5.01 (1H, d, J = 11.7 Hz), 4.73-4.68 (2H, m), 4.26 (1H, br. d, J = 13.2 Hz), 4.20-4.18
(1H, m), 4.04 (1H, dd, J = 13.4, 2.4 Hz), 2.21 (3H, s), m). 13C NMR (151 MHz,
CDCl3): 164.6, 152.5, 150.8, 149.4, 140.9, 133.6, 132.8, 128.9, 127.9, 123.3, 88.7, 81.9, 77.6, 74.8,
68.9, 59.6, 17.44, 17.38, 17.33, 17.29, 17.1, 17.03, 17.01, 16.9, 13.45, 13.42, 12.9, 12.8, 12.6.
HRMS (FAB): m/z calculated for C31H48N5O6SSi2 674.2863 [M+H]+, found 674.2868.
4.2.1.2 N-Isobutyryl-3',5'-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-2’-O-[(methylthio)methyl]-
guanosine (2b).
1
White to yellow solid, 78% yield from 1b; H NMR (600 MHz, CDCl3) 12.0 (1H, s), 8.51
(1H, s), 8.00 (1H, s), 5.86 (1H, s), 5.02 (1H, d, J = 11.4 Hz), 4.96 (1H, d, J = 11.4 Hz), 4.50-4.47 (2H,
m), 4.24 (1H, br d, J = 3.2 Hz), 4.15-4.13 (1H, m), 4.01 (1H, dd, J = 14.4, 2.5 Hz), 2.67 (1H, sep, J =
6.9 Hz), 2.17 (3H, s), 1.29-1.24 (6H, m), 1.14-0.97 (28H, m). 13C NMR (151 MHz, CDCl3) : 178.2,
155.5, 147.4, 146.9, 136.3, 121.9, 87.6, 81.9, 77.8, 74.3, 68.4, 59.6, 36.5, 19.0, 18.9, 17.4, 17.28,
17.23, 17.1, 16.99, 16.97, 16.84, 16.76, 16.71, 13.45, 13.43, 12.9, 12.5. HRMS (FAB): m/z
calculated for C28H50N5O7SSi2 656.2969 [M+H]+, found 656.2969.
4.2.1.3
N-Benzoyl-3',5'-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-
diyl)-2’-O-[(methylthio)methyl]-cytidine (2c).
White solid, 80% yield from 1c; 1H NMR (600 MHz, CDCl3)  8.80 (1H, br s), 8.37 (1H, br
d, J = 7.1 Hz), 7.90 (1H, br s), 7.63-7.51 (4H, m), 5.85 (1H, s), 5.14 (1H. d, J = 11.4 Hz), 5.01 (1H, d,
J = 11.4 Hz), 4.40 (1H, br s), 4.31 (1H, d, J= 13.2 Hz), 4.22 (2H, dd, J = 11.8, 9.9 Hz), 4.01 (1H, d, J
= 13.5 Hz), 2.21 (3H, s), 28H, m). 13C NMR (151 MHz, CDCl3) : 167.7, 162.2, 161.6,
144.8, 133.4, 129.1, 127.6, 98.6, 95.8, 89.9, 82.2, 77.6, 74.4, 67.5, 66.1, 59.3, 17.5, 17.4, 17.3, 17.00,
16.97, 16.9, 16.8, 13.5, 13.3, 13.1, 12.9, 12.5. HRMS (FAB): m/z calculated for C30H48N3O7SSi2
14 / 26

220
221
222
223
224
225
226
227
228
229
230
231
232
233
234
235
236
237
238
239
240
241
242
243
650.2751 [M+H]+, found 650.2748.
4.2.1.4 3',5'-O-(1,1,3,3-Tetraisopropyldisiloxane-1,3-diyl)-2’-O-[(methylthio)methyl]uridine (2d).
White solid, 87% yield from 1d; 1H NMR (600 MHz, CDCl3) 9.02 (1H, s), 7.90 (1H, d, J
= 8.2 Hz), 5.73 (1H, s), 5.69 (1H, dd, J = 8.2, 2.0 Hz), 4.99 (1H, d, J = 11.4 Hz), 4.97 (1H, d, J =
11.4 Hz), 4.36 (1H, d, J = 4.7 Hz), 4.28-4.23 (1H, m), 4.22 (1H, d, J = 4.7 Hz), 4.15-4.13 (1H, m),
3.98 (1H, dd, J = 13.5, 2.4 Hz), 2.19 (1H, s), 1.14-0.94 (28H, m). 13C NMR (151 MHz, CDCl3) :
163.3, 149.8, 139.3, 101.6. 88.8, 82.0, 77.5, 74.2, 67.8, 59.3, 17.5, 17.4, 17.29, 17.25, 17.24, 17.21,
17.00, 16.97, 16.93, 16.8, 13.4, 13.1, 13.0, 12.8, 12.5. HRMS (FAB): m/z calculated for
C23H43N2O7SSi2 547.2329 [M+H]+, found 547.2325.
4.2.2
Preparation
of
N-protected
-3',5'-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-
diyl)-2’-O-(2,4,6-trimethoxybenzylthiomethyl)nucleosides (3a–d)
To a solution of each of N-protected -3',5'-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-
2’-O-[(methylthio)methyl]nucleosides (2a–c) in anhydrous dichloromethane was added dropwise a
solution of sulfuryl chloride (1.3 eq) in anhydrous dichloromethane for 1 min, and the reaction
mixture was stirred at room temperature for 30 min. A mixture of 2,4,6-trimethoxybenzylmercaptan
(2.5 eq) and N,N-diisopropylethylamine (5.0 eq) in anhydrous dichloromethane was added to the
sulfuryl-chloride-treated solution at 0 °C. For the synthesis of guanosine derivative (3b),
cyclohexene was further added to the solution. The reaction mixture was stirred for 1.5–3.0 h at
room temperature and then poured into 0.5 M KH2PO4 aqueous solution. The mixture was extracted
with ethyl acetate. The organic layer was washed with 0.5 M KH2PO4 aqueous solution, distilled
water, and brine, and then dried over anhydrous sodium sulfate, filtered, and concentrated. The crude
15 / 26

244
245
246
247
248
249
250
251
252
253
254
255
256
257
258
259
260
261
262
263
264
265
266
267
mixture was purified by silica gel column chromatography to afford compounds 3a–c.
3',5'-O-(1,1,3,3-Tetraisopropyldisiloxane-1,
3-diyl)-2’-O-(2,4,6-trimethoxybenzylthiomethyl)-uridine (3d) was synthesized according to a
previously reported procedure.26
4.2.2.1
N-Benzoyl-3',5'-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-
diyl)-2’-O-(2,4,6-trimethoxybenzyl-thiomethyl)adenosine (3a).
White solid, 65% yield from 2a; 1H NMR (600 MHz, CDCl3) br s8.74 (1H, s),
8.17 (1H, s), 8.04 (2H, d, J = 7.1 Hz), 7.63-7.60 (1H, m, 1H), 7.53 (2H, t, J = 7.9 Hz), 6.10 (2H, s),
6.01 (1H, s), 5.21 (1H, d, J = 11.8 Hz), 4.97-4.95 (1H, m), 4.95 (1H, d, J = 11.8 Hz), 4.78 (1H, d, J =
5.0 Hz), 4.19 (1H, dd, J = 13.1, 1.9 Hz), 4.16-4.14 (1H, m), 4.03 (1H, dd, J = 13.2, 2.8 Hz), 3.95 (1H,
d, J = 12.7 Hz), 3.81 (1H, d, J = 12.7 Hz), 3.80 (3H, s), 3.77 (6H, s), 28H, m). 13C NMR
(151 MHz, CDCl3) : 164.5, 160.3, 158.8, 152.3, 150.9, 149.3, 142.0, 133.6, 132.8, 130.1, 128.9,
127.9, 108.1, 90.6, 89.2, 81.8, 74.7, 69.7, 60.0, 55.8, 55.3, 22.9, 17.5, 17.4, 17.33, 17.30, 17.2, 17.1,
17.0, 16.9, 13.4, 13.0, 12.8, 12.7. HRMS (FAB): m/z calculated for C40H58N5O9SSi2 840.3493
[M+H]+, found 840.3505.
4.2.2.2
N-Isobutyryl-3',5'-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-
diyl)-2’-O-(2,4,6-trimethoxy-benzylthiomethyl)guanosine (3b).
1
White solid, 62 % yield from 2b, H NMR (600 MHz, CDCl3) 11.9 (1H, s), 8.22 (1H, s),
7.95 (1H,s), 6.11 (2H, s), 5.83 (1H, s), 5.15 (1H, d, J = 11.7 Hz), 5.01 (1H, d, J = 11.5 Hz), 4.59 (1H,
d, J = 4.7 Hz), 4.51 (1H, dd, J = 9.4, 4.7 Hz), 4.21 (1H, br d, J = 13.2 Hz), 4.16-4.13 (1H, m), 4.01
(1H, dd, J = 13.2, 2.7 Hz), 3.93 (1H, d, J = 12.3 Hz), 3.87 (1H, d, J = 12.3 Hz), 3.80 (3H, s), 3.78
16 / 26

268
269
270
271
272
273
274
275
276
277
278
279
280
281
282
283
284
285
286
287
288
289
290
291
(6H, s), 2.41 (1H, sep, J = 7.0 Hz), 1.19 (3H, d, J = 7.0 Hz), 1.15 (3H, d, J = 7.0 Hz), 1.12-0.95 (28H,
m). 13C NMR (151 MHz, CDCl3) : 178.0, 160.5, 158.8, 155.4, 147.2, 147.0, 136.3, 121.9, 107.8,
91.0, 88.1, 81.8, 77.9, 74.1, 69.3, 59.7, 56.0, 55.4, 36.5, 22.9, 18.8, 17.4, 17.29, 17.25, 17.2, 17.05,
16.99, 16.87, 13.4, 12.94, 12.90, 12.6. HRMS (FAB): m/z calculated for C37H59N5O10SSi2Na
844.3418 [M+Na]+, found 844.3422.
4.2.2.3
N-Benzoyl-3',5'-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-
diyl)-2’-O-(2,4,6-trimethoxybenzyl-thiomethyl)cytidine (3c).
White solid, 58% yield from 2c, 1H NMR (600 MHz, CDCl3) 8.69 (1H, br s), 8.35 (1H, br
d, J = 5.0 Hz), 7.89 (2H, br s), 7.62-7.50 (4H, m), 6.13 (2H,s), 5.90 (1H, s), 5.10 (1H, d, J = 11.5 Hz),
5.06 (1H, d, J = 11.4 Hz), 4.44 (1H, d, J = 3.6 Hz), 4.32-4.23 (3H, m), 4.04 (1H, d, J = 12.9 Hz),
4.04-4.02 (1H, m), 3.81 (6H, s), 3.80 (3H, s), 3.76 (1H, d, J = 12.9 Hz), 1.14-0.94 (28H, m). 13C
NMR (151 MHz, CDCl3) : 166.1, 162.3, 160.1, 158.9, 144.6, 133.1, 129.0, 127.5, 108.9, 95.7, 92.3,
90.7, 90.0, 82.1, 78.1, 73.3, 70.2, 67.8, 59.5, 55.8, 55.3, 30.3, 22.0, 17.5, 17.4, 17.33, 17.30, 17.1,
16.99, 16.98, 16.87, 13.5, 13.3, 13.1, 12.9, 12.8, 12.6. HRMS (FAB): m/z calculated for
C39H57N3O10SSi2Na 838.3200 [M+Na]+, found 838.3199.
4.2.2.4
3',5'-O-(1,1,3,3-Tetraisopropyldisiloxane-1,3-
diyl)-2’-O-(2,4,6-trimethoxybenzylthiomethyl)-uridine (3d).
1
White solid, 65% yield from 2d, H NMR (600 MHz, CDCl3) 8.31 (1H, s), 7.85 (1H, d),
6.12 (2H, s), 5.76 (1H,s), 5.66 (1H, d, J = 8.2 Hz), 4.99 (2H, s), 4.44 (1H, d, J = 4.6 Hz), 4.29-4.24
(2H, m), 4.15 (1H, dd, J = 9.7, 1.8 Hz), 3.99 (1H, dd, J = 13.5, 2.4 Hz), 3.95 (1H, d, J = 13.2 Hz),
3.81 (6H, s), 3.80, (3H, s), 3.77 (1H, d, J = 12.9 Hz), 1.11-0.96 (28H, m). 13C NMR (151 MHz,
CDCl3) : 163.0, 160.2, 158.9, 149.5, 139.7, 108.4, 101.4, 90.6, 89.2, 82.0, 77.6, 73.3, 68.3, 59.5,
17 / 26

292
293
294
295
296
297
298
299
300
301
302
303
304
305
306
307
308
309
310
311
312
313
314
315
55.8, 55.3, 22.0, 17.5, 17.4, 17.3, 17.2, 17.1, 16.99, 16.95, 16.8, 13.5, 13.0, 12.9, 12.6. HRMS
(FAB): m/z calculated for C39H57N3O10SSi2Na 735.2778 [M+Na]+, found 735.2772.
4.2.3
Preparation
of
N-protected-2’-O-(2,4,6-trimethoxybenzylthiomethyl)-5’-O
–
(4,4’-dimethoxy-trityl)nucleosides (5a–d)
To a solution of each of N-protected-3’,5’-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-
diyl)-2’-O-(2,4,6-trimethoxybenzylthiomethyl)nucleosides (3a–d) in THF were added Et3N·3HF (3
eq) and Et3N (3 eq). The reaction mixture was stirred at room temperature for 1.5–3.0 h. The mixture
was added to sat. NaHCO3 aqueous solution and extracted with CHCl3. The organic layer was
washed with distilled water and brine, and then dried over anhydrous sodium sulfate, filtered, and
concentrated. The crude mixture was purified by silica gel column chromatography to afford
N-protected-2’-O-(2,4,6-trimethoxybenzylthiomethyl)nucleosides 4a–d. Each of compounds 4a–d
was dissolved in anhydrous pyridine and 4,4’-dimethoxytrityl chloride (1.5 eq) was added to the
solution. The reaction mixture was stirred at room temperature for 1.5–3.0 h. The mixture was added
to sat. NaHCO3 aqueous solution and extracted with ethyl acetate. The organic layer was washed
with distilled water and brine, and then dried over anhydrous sodium sulfate, filtered, and
concentrated. The crude mixture was purified by silica gel column chromatography to afford
compounds 5a–d.
4.2.3.1 N-Benzoyl-2’-O-(2,4,6-trimethoxybenzylthiomethyl)-5’-O – (4,4’-dimethoxytrityl)adenosine
(5a).
1
White solid, 95% yield from 3a; H NMR (600 MHz, CDCl3) br s), 8.71 (1H,
18 / 26

316
317
318
319
320
321
322
323
324
325
326
327
328
329
330
331
332
333
334
335
336
337
338
339
s), 8.18 (1H, s), 8.02 (2H, d, J = 8.2 Hz), 7.63-7.60 (1H, m), 7.55-7.51 (2H, m), 7.44-7.16 (9H, m),
6.84-6.79 (4H, m), 6.21 (1H, d, J = 5.3 Hz), 6.12 (2H, s), 4.94 (1H, d, J = 12.0 Hz), 4.91 (1H, t, J =
5.2 Hz), 4.73 (1H, d, J = 12.0 Hz), 4.57 (1H, dd, J = 9.1, 4.4 Hz), 4.27 (1H, dd, J = 7.3, 3.8 Hz), 3.85
(2H, d, J = 4.1 Hz), 3.81 (6H, s), 3.80 (3H, s), 3.78 (6H, s), 3.50 (1H, dd, J = 10.6, 3.2 Hz), 3.41 (1H,
dd, J = 9.6, 4.1 Hz), 3.02 (1H, d, J = 4.7 Hz). 13C NMR (151 MHz, CDCl3) 160.8, 158.8, 158.6,
152.1, 150.6, 150.2, 147.3, 143.3, 139.4, 133.4, 133.0, 130.1, 129.1, 129.0, 128.2, 127.9, 127.8,
127.6, 127.1, 124.6, 113.2, 106.2, 90.7, 89.6, 88.0, 82.3, 81.4, 75.7, 71.0, 63.3, 55.8, 55.4, 55.2, 24.1.
HRMS (FAB): m/z calculated for C49H50N5O10S 900.3277 [M+H]+, found 900.3282.
4.2.3.2 N-Isobutyryl-2’-O-(2,4,6-trimethoxybenzylthiomethyl)-5’-O–(4,4’-dimethoxytrityl)guanosine
(5b).
White solid, 86% yield from 3b; 1H NMR (600 MHz, CDCl3) : 11.9 (1H, s), 7.78 (1H,
s), 7.56-7.53 (3H, m), 7.41-7.16 (7H, m), 6.84-6.77 (4H, m), 6.11 (2H, s), 5.83 (1H, d, J = 7.1 Hz),
5.20 (1H, dd, J = 7.0, 5.0 Hz), 4.95 (1H, d, J = 12.0 Hz), 4.63 (1H, d, J = 12.0 Hz), 4.52-4.50 (1H,
m), 4.19 (1H, dd, J = 5.3, 2.9 Hz), 3.86 (1H, d, J = 12.3 Hz), 3.84 (1H, d, J = 12.4 Hz), 3.80 (3H,s),
3.78 (6H, s), 3.77 (3H, s), 3.76 (3H, s), 3.51 (1H, dd, J = 10.7, 1.9 Hz), 3.12 (1H, dd, J = 10.7, 3.4
Hz), 2.95 (1H, d, J = 2.9 Hz), 1.59 (1H, sep, J = 6.8 Hz), 0.92 (3H, d, J = 6.8 Hz), 0.70 (3H, d, J =
6.7 Hz). 13C NMR (151 MHz, CDCl3)  178.1, 160.7, 158.8, 158.7, 155.4, 148.1, 147.0, 144.9,
139.4, 139.1, 136.1, 135.7, 130.04, 130.02, 129.1, 128.1, 128.0, 127.8, 127.7, 127.1, 127.0, 122.6,
113.24, 113.22, 113.1, 106.6, 90.6, 86.4, 86.2, 84.4, 78.7, 74.9, 69.9, 63.7, 55.8, 55.4, 55.2, 36.0,
23.7, 18.5, 18.5. HRMS (FAB): m/z calculated for C46H52N5O11S 882.3383 [M+H]+, found 882.3389.
4.2.3.3 N-Benzoyl-2’-O-(2,4,6-trimethoxybenzylthiomethyl)-5’-O–(4,4’-dimethoxytrityl)cytidine (5c).
White solid, 97% yield from 3c; 1H NMR (600 MHz, CDCl3) : 8.59 (1H, br s), 8.51 (1H,
19 / 26

340
341
342
343
344
345
346
347
348
349
350
351
352
353
354
355
356
357
358
359
360
361
362
363
br s), 7.87 (2H, br s), 7.61 (1H, t, J = 7.6 Hz), 7.52 (2H, t, J = 7.6 Hz), 7.45-7.43 (2H, m), 7.35-7.26
(7H, m), 6.90-6.87 (4H, m), 6.12 (2H, s), 5.97 (1H, s), 5.09-5.02(2H, m), 4.47-4.43 (1H, m), 4.29
(1H, d, J = 5.3 Hz ), 4.05-4.02 (1H, m), 3.91 (1H, d, J = 12.7 Hz), 3.832 (3H, s), 3.830 (3H, s), 3.82
(6H, s), 3.81 (1H, d, J = 12.9 Hz), 3.79 (3H, s), 3.59-3.54 (2H, m), 2.99 (1H, d, J = 8.8 Hz). 13C
NMR (151 MHz, CDCl3) 160.6, 160.5, 158.85, 158.82, 158.7, 158.6, 147.3, 144.1, 139.4, 135.7,
135.3, 133.4, 133.2, 130.2, 130.1, 129.1, 128.3, 128.0, 127.84, 127.75, 127.5, 127.2, 127.1, 113.3,
113.2, 106.9, 106.8, 90.7, 89.2, 87.0, 85.6, 83.6, 81.4, 80.2, 80.0, 74.6, 73.8, 68.4, 67.7, 61.4, 61.2,
55.9, 55.38, 55.37, 55.2, 23.5, 22.9. HRMS (FAB): m/z calculated for C48H50N3O11S 876.3165
[M+H]+, found 876.3160.
4.2.3.4 2’-O-(2,4,6-Trimethoxybenzylthio-methyl)-5’-O–(4,4’-dimethoxytrityl)uridine (5d)
White solid, 98% yield from 3d; spectral data are available in a previous report.26
4.2.4 Preparation of N-protected-2’-O-(2,4,6-trimethoxybenzylthiomethyl)-3’-O-[(2-cyanoethyl)-
(N,N-diisopropylamino)phosphoramidyl]-5’-O–(4,4’-dimethoxytrityl)nucleosides (6a–d)
T
o
a
s
o
l
u
t
i
o
n
o
f
e
a
c
h
o
f
N-protected-2’-O-(2,4,6-trimethoxybenzylthiomethyl)-5’-O–(4,4’-dimethoxytrityl)nucleosides
( 5 a – d ) i n a n h y d r o u s d i c h l o r o m e t h a n e w e r e a d d e d
2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (1.5 eq), N,N-diisopropylethylamine (2.0 eq),
and 4-dimethylaminopyridine (0.5 eq) at 0°C. After the reaction mixture was stirred for 0.5–1.5 h at
room temperature, sat. NaHCO3 aqueous solution was added and the mixture was extracted with
ethyl acetate. The organic layer was washed with distilled water and brine and then dried over
20 / 26

364
365
366
367
368
369
370
371
372
373
374
375
376
377
378
379
380
381
382
383
384
385
386
387
anhydrous sodium sulfate, filtered, and concentrated. The crude mixture was purified by silica gel
column chromatography to afford compounds 6a–d.
4.2.4.1 N-Benzoyl-2’-O-(2,4,6-trimethoxybenzylthio-methyl)-3’-O-[(2-cyanoethyl)-(N,N-
diisopropylamino)phosphoramidyl]-5’-O–(4,4’-dimethoxytrityl)adenosine (6a).
White solid, 79% yield from 5a; 31P NMR (243 MHz, CDCl3) : 151.4, 150.7. HRMS (FAB):
m/z calculated for C58H67N7O11PS 1100.4356 [M+H]+, found 1100.4360.
4.2.4.2 N-Isobutyryl-2’-O-(2,4,6-trimethoxybenzylthio-methyl)-3’-O-[(2-cyanoethyl)-(N,N-
diisopropylamino)phosphoramidyl]-5’-O–(4,4’-dimethoxytrityl)guanosine (6b).
White solid, 79% yield from 5b; 31P NMR (243 MHz, CDCl3) : 150.8, 150.4. HRMS
(FAB): m/z calculated for C55H69N7O12PS 1082.4461 [M+H]+, found 1082.4459.
4.2.4.3 N-Benzoyl-2’-O-(2,4,6-trimethoxybenzylthio-methyl)-3’-O-[(2-cyanoethyl)-(N,N-
diisopropylamino)phosphoramidyl]-5’-O–(4,4’-dimethoxytrityl)cytidine (6c)
White solid, 83% yield from 5c; 31P NMR (243 MHz, CDCl3) : 150.9, 150.7. HRMS
(FAB):m/z calculated for C57H67N5O12PS 1076.4244 [M+H]+, found 1076.4241.
4.2.4.4 2’-O-(2,4,6-Trimethoxybenzylthio-methyl)-3’-O-[(2-cyanoethyl)-(N,N-diisopropylamino)-
phosphoramidyl]-5’-O–(4,4’-dimethoxytrityl)uridine (6d)
White solid, 88% yield from 5d; spectral data are available in a previous report.26
21 / 26

388
389
390
391
392
393
4.3 Oligonucleotide synthesis
Oligonucleotides were synthesized on a 1 µmol scale on an ABI Model 392 DNA/RNA
synthesizer in the trityl-on mode. The standard ABI CE DNA synthesis protocol was employed
except for the following points. The coupling wait time was extended to 600 sec for the coupling of
compounds 6a–d with 5-ethylthio-1H-tetrazole (ETT) as the activator. 0.02 M iodine solution was
used for phosphite triester oxidation. Synthesized oligonucleotides were treated with concentrated
394
aqueous ammonia at 55 °C for 8.0 h. After the removal of ammonia in the suspensions, controlled
395
396
397
398
399
400
401
402
403
404
405
406
407
pore glasses (CPGs) were removed by filtration with Millex-LG 0.20 µm (Millipore). The crude
oligonucleotides were analyzed and purified by RP-HPLC. The obtained oligonucleotide solutions
were concentrated in a centrifugal evaporator (EYELA, Japan). After the addition of 2.0 M TEAA,
the oligonucleotides were applied to a Sep-Pak C18 plus (Waters) and then washed with 100 mM
TEAA. Subsequently, the 5’-DMTr group of the oligonucleotides was removed by adding 2% TFA
aqueous solution and desalted with water. Finally, the oligonucleotides were eluted with 50%
acetonitrile/water and concentrated in a centrifugal evaporator. The oligonucleotide solutions were
quantified on a UV spectrometer and lyophilized. The structures were characterized by MALDI-TOF
mass spectrometry.
4.4 Conversion of 2’-O-TMBTM oligonucleotides into 2’-O-MDTM oligonucleotides
To a solution of oligonucleotides (7a–d) (0.1 mM each) in 200 mM sodium acetate buffer
(pH 4) was added freshly prepared DMTSF aqueous suspension (ca. 300 eq) and the reaction was
408
left to stand at 37 °C. After the reaction was monitored by RP-HPLC, excess DMTSF and buffer
409
410
were removed with a gel filtration column (GE Healthcare NAP-25). Oligonucleotides 8a–d were
purified by RP-HPLC and the solutions were desalted by a gel filtration column (GE Healthcare,
22 / 26

411
412
413
414
415
416
417
418
419
420
NAP-25). The oligonucleotide solutions were concentrated and quantified on a UV spectrometer.
The structures were characterized by MALDI-TOF mass spectrometry.
4.5 Conversion of 2’-O-MDTM oligonucleotides into 2’-OH oligonucleotides under glutathione-
based reducing condition
Each of oligonucleotides 8a–d (2 nmol) was dissolved in 36 µL of 50 mM sodium
phosphate buffer (pH 7). To the solution was added 4 µL of 100 mM GSH aqueous solution to adjust
the final concentrations of each oligonucleotide and GSH to 50 µM and 10 mM, respectively. The
421
solution was incubated at 37 °C and the reaction was monitored by RP-HPLC. The samples were
422
423
424
425
426
427
428
429
430
431
432
433
434
desalted by ZipTip C18 (Millipore) and the structures were characterized by MALDI-TOF mass
spectrometry.
Acknowledgments
This work was supported by Grants-in-Aid for Young Scientists (B), No. 15K21517 (to J.H.)
and No. 24790121 (to O.N.), from the Ministry of Education, Culture, Sports, Science, and
Technology, Japan.
23 / 26

435
436
437
438
439
440
441
442
443
References
(1)
(2)
(3)
(4)
Lundin, K. E.; Gissberg, O.; Smith, C. I. E. Hum. Gene Ther. 2015, 26, 475.
444
445
446
447
448
449
450
451
452
453
454
455
456
457
458
Stein, C. A.; Castanotto, D. Mol. Ther. 2017, 25, 1069.
Keam, S. J. Drugs 2018, 78, 1371.
Grünweller, A.; Wyszko, E.; Bieber, B.; Jahnel, R.; Erdmann, V. A.; Kurreck, J. Nucleic Acids
Res. 2003, 31, 3185.
(5)
(6)
(7)
Morrison, C. Nat. Rev. Drug Discov. 2018, 17, 156.
Ledford, H. Nature 2018, 560, 291.
Cummins, L. L.; Owens, S. R.; Risen, L. M.; Lesnik, E. A.; Freier, S. M.; McGee, D.; Guinosso,
C. J.; Cook, P. D. Nucleic Acids Res. 1995, 23, 2019.
(8)
Allerson, C. R.; Sioufi, N.; Jarres, R.; Prakash, T. P.; Naik, N.; Berdeja, A.; Wanders, L.; Griffey,
R. H.; Swayze, E. E.; Bhat, B. J. Med. Chem. 2005, 48, 901.
Layzer, J. M.; McCaffrey, A. P.; Tanner, A. K.; Huang, Z.; Kay, M. A.; Sullenger, B. A. RNA 2004,
10, 766.
(9)
(10)
(11)
Prakash, T. P.; Allerson, C. R.; Dande, P.; Vickers, T. A.; Sioufi, N.; Jarres, R.; Baker, B. F.;
Swayze, E. E.; Griffey, R. H.; Bhat, B. J. Med. Chem. 2005, 48, 4247.
Obika, S.; Nanbu, D.; Hari, Y.; Andoh, J.-i.; Morio, K.-i.; Doi, T.; Imanishi, T. Tetrahedron Lett.
24 / 26

459
460
461
462
463
464
465
466
467
468
469
470
471
472
473
474
475
476
477
478
479
480
481
482
1998, 39, 5401.
(12)
Koshkin, A. A.; Singh, S. K.; Nielsen, P.; Rajwanshi, V. K.; Kumar, R.; Meldgaard, M.; Olsen, C.
E.; Wengel, J. Tetrahedron 1998, 54, 3607.
(13)
(14)
Shukla, S.; Sumaria, C. S.; Pradeepkumar, P. I. ChemMedChem 2010, 5, 328.
Lima, W. F.; Wu, H.; Nichols, J. G.; Sun, H.; Murray, H. M.; Crooke, S. T. J. Biol. Chem. 2009,
284, 26017.
(15)
(16)
(17)
(18)
(19)
Parker, J. S.; Roe, S. M.; Barford, D. Nature 2005, 434, 663.
Ma, J.-B.; Yuan, Y.-R.; Meister, G.; Pei, Y.; Tuschl, T.; Patel, D. J. Nature 2005, 434, 666.
Wang, Y.; Juranek, S.; Li, H.; Sheng, G.; Tuschl, T.; Patel, D. J. Nature 2008, 456, 921.
Debart, F.; Dupouy, C.; Vasseur, J.-J. Beilstein J. Org. Chem. 2018, 14, 436.
Meade, B. R.; Gogoi, K.; Hamil, A. S.; Palm-Apergi, C.; van den Berg, A.; Hagopian, J. C.;
Springer, A. D.; Eguchi, A.; Kacsinta, A. D.; Dowdy, C. F.; Presente, A.; Lonn, P.; Kaulich, M.;
Yoshioka, N.; Gros, E.; Cui, X. S.; Dowdy, S. F. Nat. Biotechnol. 2014, 32, 1256.
Vivès, E.; Dell'Aquila, C.; Bologna, J. C.; Morvan, F.; Rayner, B.; Imbach, J. L. Nucleic Acids
Res. 1999, 27, 4071.
(20)
(21)
Saneyoshi, H.; Iketani, K.; Kondo, K.; Saneyoshi, T.; Okamoto, I.; Ono, A. Bioconjug. Chem.
2016, 27, 2149.
(22)
(23)
Zhang, N.; Tan, C.; Cai, P.; Zhang, P.; Zhao, Y.; Jiang, Y. Chem. Commun. 2009, 3216.
Gauthier, F.; Claveau, S.; Bertrand, J.-R.; Vasseur, J.-J.; Dupouy, C.; Debart, F. Biorg. Med. Chem.
2018, 26, 4635.
(24)
(25)
Lavergne, T.; Baraguey, C.; Dupouy, C.; Parey, N.; Wuensche, W.; Sczakiel, G.; Vasseur, J. J.;
Debart, F. J. Org. Chem. 2011, 76, 5719.
Gauthier, F.; Claveau, S.; Bertrand, J.-R.; Vasseur, J.-J.; Dupouy, C.; Debart, F. Biorg. Med. Chem.
2018, 26, 4635.
25 / 26

483
484
485
486
487
488
489
490
(26)
(27)
Ochi, Y.; Nakagawa, O.; Sakaguchi, K.; Wada, S.; Urata, H. Chem. Commun. 2013, 49, 7620.
Ochi, Y.; Imai, M.; Nakagawa, O.; Hayashi, J.; Wada, S.-i.; Urata, H. Bioorg. Med. Chem. Lett.
2016, 26, 845.
(28)
(29)
Hayashi, J.; Nishigaki, M.; Ochi, Y.; Wada, S.-i.; Wada, F.; Nakagawa, O.; Obika, S.;
Harada-Shiba, M.; Urata, H. Bioorg. Med. Chem. Lett. 2018, 28, 2171.
Adams, E. J. and Oscarson, S. (2005). Dimethyl(methylthio)sulfonium Tetrafluoroborate
(DMTSF).
In
Encyclopedia
of
Reagents
for
Organic
Synthesis.
doi:10.1002/047084289X.rd349.pub2.
491
492
26 / 26