Synthesis of selenomethylene-locked nucleic acids (SeLNA) nucleoside unit bearing an adenine base

Article abstract of DOI:10.1080/15257770.2019.1675169

Synthesis of selenomethylene-locked nucleic acids nucleoside bearing an adenine base (SeLNA-A) was investigated. We first examined the stereoinversion reaction at 2′-positions of a 5′,3′-O-TIPDS-protected 4′-C-(hydroxymethyl)ribosyladenine derivative to give the corresponding arabinosyladenine. After triflation, treatment of the arabinosyladenine derivative with a mixture of selenium and sodium borohydride in ethanol managed to construct the desired SeLNA skeleton. Finally, removal of TIPDS by treating with fluoride gave the SeLNA-A nucleoside. In this study, we found the heat-labile property of SeLNA-A. It is necessary to know more precise characteristics of SeLNA to achieve its oligonucleotides synthesis.

Full text of DOI:10.1080/15257770.2019.1675169

Nucleosides, Nucleotides and Nucleic Acids
ISSN: 1525-7770 (Print) 1532-2335 (Online) Journal homepage: https://www.tandfonline.com/loi/lncn20
Synthesis of selenomethylene-locked nucleic acids
(SeLNA) nucleoside unit bearing an adenine base
Yoshihiro Moai, Hidekazu Hiroaki, Saoshi Obika & Tetsuya Kodama
To cite this article: Yoshihiro Moai, Hidekazu Hiroaki, Saoshi Obika & Tetsuya Kodama (2019):
Synthesis of selenomethylene-locked nucleic acids (SeLNA) nucleoside unit bearing an adenine
base, Nucleosides, Nucleotides and Nucleic Acids, DOI: 10.1080/15257770.2019.1675169
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NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
https://doi.org/10.1080/15257770.2019.1675169
Synthesis of selenomethylene-locked nucleic acids
(SeLNA) nucleoside unit bearing an adenine base
Yoshihiro Moaia, Hidekazu Hiroakia , Saoshi Obikab , and Tetsuya
Kodamaa
aGraduate School of Pharmaceutical Sciences, Nagoya University, Nagoya, Aichi, Japan;
bGraduate School of Pharmaceutical Sciences, Osaka University, Suita, Japan
ABSTRACT
ARTICLE HISTORY
Received 30 August 2019
Accepted 26 September 2019
Synthesis of selenomethylene-locked nucleic acids nucleoside
bearing an adenine base (SeLNA-A) was investigated. We first
examined the stereoinversion reaction at 20-positions of a
KEYWORDS
Nucleoside; oligonucleotide;
LNA; seleno-compound
50,30-O-TIPDS-protected
40-C-(hydroxymethyl)ribosyladenine
derivative to give the corresponding arabinosyladenine. After
triflation, treatment of the arabinosyladenine derivative with a
mixture of selenium and sodium borohydride in ethanol man-
aged to construct the desired SeLNA skeleton. Finally, removal
of TIPDS by treating with fluoride gave the SeLNA-A nucleo-
side. In this study, we found the heat-labile property of
SeLNA-A. It is necessary to know more precise characteristics
of SeLNA to achieve its oligonucleotides synthesis.
Introduction
Chemically-modified nucleosides/nucleotides have become important com-
ponents in oligonucleotide-based drug design and sensor technology
because of its nuclease resistance and highly and selectively binding ability
to its target nucleic acids.[1–5] Locked nucleic acid (LNA)[6,7]/20,40-bridged
nucleic acid (20,40-BNA)[8] is one of the most impressive nucleoside compo-
nents that has made recent oligonucleotide-based therapeutic drug develop-
ment attractive. LNA has a methylene bridge between the 20-oxygen and
40-carbon atoms of RNA, which locks its own sugar conformation into the
C30-endo conformation (Figure 1). Since this LNA oligonucleotides show
extremely strong binding affinity against complementary DNA and RNA,[9]
a number of LNA analogs have been developed.[10–17]
We previously reported selenomethylene-locked nucleic acids (SeLNA)
bearing a thymine base (T) (Figure 2).[18] SeLNA-T can be reversibly con-
verted into its oxidized-form (SeOLNA-T), and the hybridization ability
and nuclease resistance of oligonucleotides modified with SeLNA were
CONTACT T. Kodama
kodama@ps.nagoya-u.ac.jp
Graduate School of Pharmaceutical Sciences, Nagoya
University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8601, Japan.
ß 2019 Taylor & Francis Group, LLC

2
Y. MOAI ET AL.
Figure 1. Chemical structures of RNA and LNA/20,40-BNA.
Figure 2. Chemical structures and the properties of SeLNA.
shown to be much higher than those of oligonucleotides modified with
SeOLNA. Moreover, it was also found that KOD XL DNA polymerase was an
efficient enzyme to extend oligonucleotide strands by reading SeLNA-T
nucleotides of the template strand and also by incorporating SeLNA-T triphos-
phate using DNA templates.[19] These findings suggested that SeLNA-modified
oligonucleotides can be developed not only as a sensor module for changes in
the redox environment but also as a material for aptamer technology.
Because selenium-modified nucleic acids are important not only for medi-
cinal use but also for structural analysis,[20–23] selenium-modified nucleoside
at various positions have been developed so far.[24–26] To enable a wide range
of applied research on SeLNA, we also decided to synthesize SeLNA contain-
ing a nucleobase other than thymine base, SeLNA bearing adenine (SeLNA-
A) in this study, and to elucidate the basic properties thereof.
Herein, the synthesis of SeLNA-A from a branched ribose derivative is
described. Seleno bridging reaction between 20- and 40-position as a key step
was performed by subjecting a bis-sulfonated compound to a selenide
solution. Finally, removal of protecting group gave the SeLNA-A nucleoside.
Results and discussion
We investigated the synthesis of selenomethylene-locked nucleic acids
(SeLNA) nucleoside unit bearing an adenine base. Compound 1[27] was
used as a starting material in this study. After tosylation of the hydroxy
group of compound 1, the obtained 2 was treated with acetic anhydride
and acetic acid under sulfuric acid catalytic conditions to afford 3. Further,
acetate group on 1-position of 3 was substituted with 6-N-benzoyladenine
[28]
€
by modified Vorbruggen’s method,
whereby compound 4 was obtained

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
3
over three steps in 60% yield. Acetyl group of 4 was removed by ammonia
treatment to give compound 5 in 84% yield (Scheme 1).
Next, the stereo-inversion reaction at C20 position of 5 was carried out
(Scheme 2 and Table 1). We first attempted to invert the stereochemistry by
performing oxidation and reduction in succession, but only a complex mix-
ture was obtained (Table 1, entry 1). So then, SN2 reaction at 20-position was
examined. Compound 5 was triflated and was subsequently treated with sev-
eral acetoxylation conditions without purification (entries 2–4). When alkali
acetate was used, SN2 reaction was not efficient because the elimination reac-
tion proceeded (entries 2 and 3). Finally, compound 6 was obtained in mod-
erate yield by treating 5 with acetic acid in the presence of DBU (entry 4).
All protecting groups of 6 was removed by continuously performing hydro-
genolysis and aminolysis to afford compound 7 (Scheme 3). Incidentally, it
has been confirmed that the benzoyl group was removed prior to the benzyl
group under the hydrogenolysis conditions. Compound 7 was subjected to
1,1,3,3-tetraisopropyldisiloxane (TIPDS) protection to obtain compound 8 in
94% yield. This compound 8 was triflated again and treated with sodium
hydrogen selenide that was generated by powder selenium and sodium boro-
hydride to afford the TIPDS-protected selenomethylene bridged compound 9.
Subsequently, the silyl group was removed by TBAF treatment, and the desired
SeLNA-A nucleoside (10) was finally obtained in 16% yield over three steps.
As described, we successfully synthesized SeLNA-A nucleoside from a
ribose derivative. In addition, it was found that SeLNA-A nucleoside (10)
was easily decomposed by heating, and b-methyl glycoside of SeLNA was
generated, for example, when recrystallization was carried out in methanol
at around its boiling point (data not shown). The reason why SeLNA-A
nucleoside (10) was obtained only in a low yield is considered to be due to
this heat sensitivity. This suggests that in order to synthesize SeLNA-A-
containing oligodeoxynucleotides, it is necessary to examine in detail the
selection of protecting groups in consideration of its heat-labile property.
Scheme 1. Synthesis of intermediate 5.

4
Y. MOAI ET AL.
Scheme 2. Stereo-inversion reaction at 2’-position.
Table 1. Stereo-inversion reaction at C20 position of compound 5.
Entry
Conditions A
Conditions B
Yield of 6
1
2
3
4
IBX, EtOAc, reflux
NaBH4, MeOH
0%
32%
42%
56%
Tf2O, pyridine, CH2Cl2, ꢁ30 ꢀC
Tf2O, pyridine, CH2Cl2, ꢁ30 ꢀC
Tf2O, pyridine, CH2Cl2, ꢁ30 ꢀC
KOAc, 18-crown-6, toluene
CsOAc, DMSO
DBU, AcOH, toluene, 80 ꢀC
Scheme 3. Synthesis of SeLNA-A nucleoside.
Experimental section
All moisture-sensitive reactions were carried out in well-dried glassware under
an argon or nitrogen atmosphere. 1H NMR spectra were recorded at 400 MHz.
13C NMR spectra were recorded at 100 MHz. 31P NMR spectra were recorded
at 162MHz. Chemical shifts (d) are expressed in ppm relative to internal tetra-
methylsilane (0 ppm), residual CHCl3 (7.26 ppm) or residual DMSO
1
(2.49 ppm) in the H NMR spectra, internal chloroform-d (77.0 ppm) or
dimethyl sulfoxide-d6 (39.5 ppm) in the 13C NMR spectra. FAB mass spectra
of all new compounds were obtained on a LMS-700 instrument. For column
chromatography, FL-60B silica gel was used. The progress of the reactions was
monitored by analytical thin layer chromatography (TLC) on glass plates
(TLC silica gel 60 F254) and the products were visualized by UV light.
6-N-benzoyl-9-[2-O-acetyl-3,5-di-O-benzyl-4-C-(p-toluenesulfonyloxymethyl)-
a-D-ribofuranosyl]adenine (4). Compound 1 (10.4 g, 26.0 mmol) in dichloro-
methane solution (130 mL) was cooled to 0 ꢀC. Triethylamine (21.7 mL,

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
5
156 mmol), 4-dimethylaminopyridine (479 mg, 3.9 mmol) and p-
toluenesulfonyl chloride (7.42 g, 39.0 mmol) was added and stirred at room
temperature for 12 hours. After cold water was added to the reaction solu-
tion to stop the reaction, the organic layer was washed with 1 M HCl, sat.
NaHCO3 and brine. The organic layer was dried over Na2SO4 and evapo-
rated. To a solution of the crude product 2 (15.2 g) in acetic acid (29 mL)
were added acetic anhydride (15.2 mL, 156 mmol) and concentrated sulfuric
acid (0.29 mL, 5.2 mmol) at room temperature. The mixture was stirred at
the same temperature for 3 hours. After the reaction solution was cooled to
0 ꢀC, and ice-cooled sat. NaHCO3 was added to the mixture, and was
stirred for 5 min. The contents were extracted with ethyl acetate, and the
organic layer was washed with saturated sat. NaHCO3 and brine, and dried
over Na2SO4 to give crude acetate 3 (17.0 g). To a suspension of crude acet-
ate 3 (6.54 g, 10.0 mmol) and N-6-benzoyladenine (4.79 g, 20.0 mmol) in
1,2-dichloroethane (100 mL) was added N,O-bis(trimethylsilyl)acetamide
(9.9 mL, 40.0 mmol), and the resulting mixture was refluxed for 2 hours.
After cooled to room temperature, and trimethylsilyl trifluoromethanesulfo-
nate (2.7 mL, 15.0 mmol) was added. The mixture was refluxed again for
8 hours. The reaction mixture was diluted with dichloromethane at room
temperature, and sat. NaHCO3 was added to the stirring mixture. The
organic layer was washed with sat. NaHCO3, water and brine, and dried
over Na2SO4. The obtained crude product was purified by column chromatog-
raphy (SiO2, dichloromethane: acetone ¼ 15:1) to give compound 4 (4.69 g,
6.03 mmol, 60%) as colorless amorphous solid. IR ꢀmax (neat): 3064, 3030,
1
3007, 2925, 2871, 1748, 1699 cmꢁ1. H NMR (400 MHz, CDCl3) d: 9.05 (s,
1H, 6-NH), 8.71 (s, 1H, H2), 8.14 (s, 1H, H8), 8.01 (d, J ¼ 7.3 Hz, 2H), 7.74
(d, J¼ 8.2 Hz, 2H), 7.60 (t, J¼ 7.3 Hz, 1H), 7.51 (t, J ¼ 7.3 Hz, 2H), 7.35–7.18
(m, 12H), 6.12 (d, J¼ 4.6 Hz, 1H, H10), 5.92 (dd, J ¼ 4.6, 5.5 Hz, 1H, H20),
4.73 (d, J ¼ 5.5 Hz, 1H, H30), 4.57 (d, J ¼ 11.2 Hz, 1H), 4.49 (d, J ¼ 11.2 Hz,
1H), 4.45 (d, J¼ 11.9 Hz, 1H), 4.41 (d, J¼ 11.9 Hz, 1H), 4.32 (d, J ¼ 10.5 Hz,
1H), 4.23 (d, J¼ 10.5 Hz, 1H), 3.67 (d, J¼ 10.1 Hz, 1H), 3.56 (d, J ¼ 10.1 Hz,
1H), 2.40 (s, 3H, Ts), 2.03 (s, 3H, Ac). 13C NMR (100 MHz, CD3OD) d:
169.6, 164.5, 152.7, 151.4, 149.5, 145.0, 141.8, 136.9, 136.9, 133.5, 132.8,
132.5, 129.8, 128.8, 128.5, 128.5, 128.2, 128.1,128.0, 127.8, 127.7, 123.1, 86.4,
85.9, 77.8, 74.7, 74.6, 73.6, 70.1, 68.9, 21.6, 20.5. LRMS (FAB) m/z 778
[M þ H]þ. HRMS (FAB): Calcd for C41H40N5O9S [Mþ H]þ: 778.2547.
Found: 778.2505.
6-N-benzoyl-9-[3,5-bi-O-benzyl-4-C-(p-toluenesulfonyloxymethyl)-a-D-
ribofuranosyl]adenine (5). Saturated ammonia methanol solution (55 mL)
was added to a solution of compound 4 (4.25 g, 5.46 mmol) in methanol
(110 mL) at 0 ꢀC and stirred for 19 hours at 0 ꢀC. The reaction mixture
was evaporated, and the obtained crude product is purified by column

6
Y. MOAI ET AL.
chromatography (SiO2, hexane: ethyl acetate ¼ 1:3) to give compound 5
(3.38 g, 4.59 mmol, 84%) as colorless amorphous solid. IR ꢀmax (neat): 3287,
1
3064, 3031, 2925, 2871, 1699 cmꢁ1. H NMR (400 MHz, CDCl3) d: 8.99 (s,
1H), 8.68 (s, 1H), 8.08 (s, 1H), 8.02 (d, J ¼ 7.3 Hz, 2H), 7.76 (d, J ¼ 8.2 Hz,
2H), 7.61 (t, J ¼ 7.3 Hz, 1H), 7.53 (t, J ¼ 7.3 Hz, 2H), 7.37–7.27 (m, 10 H),
7.20–7.18 (m, 2H), 5.89 (d, J ¼ 5.0 Hz, 1H), 4.82 (dd, J ¼ 6.0, 11.0 Hz, 1H),
4.71 (d, J ¼ 11.5 Hz, 1H), 4.68 (d, J ¼ 11.5 Hz, 1H), 4.55 (d, J ¼ 6.0 Hz, 1H),
4.46 (s, 1H), 4.33 (d, J ¼ 10.5 Hz, 1H), 4.30 (d, J ¼ 10.5 Hz, 1H), 3.62 (d,
J ¼ 10.1 Hz, 1H), 3.59 (d, J ¼ 10.1 Hz, 1H), 3.46 (d, J ¼ 6.9 Hz, 1H), 2.41 (s,
3H).13C NMR (100 MHz, CD3OD) d: 164.6, 152.3, 151.1, 149.3, 145.0,
141.9, 137.0, 136.8, 133.5, 132.8, 132.4, 129.8, 128.8, 128.6, 128.5, 128.4,
128.2,128.0, 127.8, 127.7, 122.9, 89.3, 85.9, 79.3, 74.7, 74.6, 73.6, 70.6, 69.1,
21.6. LRMS (FAB) m/z 736 [M þ H]þ. HRMS (FAB): Calcd for
C39H38N5O8S [M þ H]þ: 736.2441. Found: 736.2444.
6-N-benzoyl-9-[2-O-acetyl-3,5-Di-O-benzyl-4-C-(p-toluenesulfonyloxymethyl)-
b-D-arabinofuranosyl]-adenine (6). To a solution of compound 5 (3.14 g,
4.27 mmol) in dichloromethane and pyridine (5:1, 50 mL) was added tri-
fluoromethanesulfonic anhydride (1.4 mL, 8.54 mmol) under at –30 ꢀC and
stirred for 3 hours at the same temperature. The reaction mixture was
diluted with dichloromethane, and was washed with 1 M HCl, sat.
NaHCO3 and brine, and dried over anhydrous Na2SO4. The obtained tri-
flated crude (3.49 g, 4.02 mmol) was used for the next reaction without
purification. In another flask, to a solution of DBU (2.40 mL, 16.0 mmol) in
toluene (40 mL) was added acetic acid (1.84 mL, 32.0 mmol), and the mix-
ture was stirred at room temperature for 1 hour. This mixed solution
(20 mL) was added to a toluene solution (20 mL) of the triflated crude
product (3.49 g, 4.02 mmol) and stirred at 80 ꢀC for 7 hours. After cooling
to room temperature, reaction mixture was diluted with ethyl acetate, and
the organic layer was washed with 1 M HCl, sat. NaHCO3 and brine, and
dried over anhydrous Na2SO4. The obtained crude product was purified by
column chromatography (SiO2, hexane: ethyl acetate ¼ 1:1) to give com-
pound 6 (2.02 g, 2.60 mmol, 61%) as colorless amorphous solid. IR ꢀmax
1
(neat): 3064, 3030, 3008, 2926, 2871, 1749, 1698 cmꢁ1. H NMR (400 MHz,
CDCl3) d: 8.98 (s, 1H), 8.71 (s, 1H), 8.03–8.01 (m, 3H), 7.80 (d, J ¼ 8.2 Hz,
2H), 7.62 (t, J ¼ 7.3 Hz, 1H), 7.53 (t, J ¼ 7.3 Hz, 2H), 7.37–7.24 (m, 10 H),
7.18–7.15 (m, 2H), 6.46 (d, J ¼ 5.7 Hz, 1H), 5.49 (t, J ¼ 5.4 Hz, 1H),
4.70–4.67 (m, 2H), 4.57 (d, J ¼ 12.1 Hz, 1H), 4.50 (d, J ¼ 12.1 Hz, 1H), 4.40
(d, J ¼ 12.1 Hz, 1H), 4.35 (d, J ¼ 10.8 Hz, 1H), 4.32 (d, J ¼ 10.8 Hz, 1H),
4.23 (d, J ¼ 10.5 Hz, 1H), 3.68 (d, J ¼ 9.6 Hz, 1H), 3.55 (d, J ¼ 9.6 Hz, 1H),
2.36 (s, 3H), 1.56 (s, 3H). 13C NMR (100 MHz, CD3OD) d: 169.1, 164.5,
152.6, 151.3, 149.4, 144.9, 142.6, 137.1, 136.8, 133.5, 132.8, 132.7, 129.7,
128.8, 128.4, 128.1, 128.0, 127.9,127.8, 122.6, 83.8, 82.6, 81.4, 76.6, 73.4,

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
7
73.1, 69.3, 69.0, 21.5, 20.0. LRMS (FAB) m/z 778 [M þ H]þ. HRMS (FAB):
Calcd for C41H40N5O9S [M þ H]þ: 778.2547. Found: 778.2543.
9-[4-C-(p-toluenesulfonyloxymethyl)-b-D-arabinofuranosyl]adenine (7).
To a solution of compound 6 (3.00 g, 3.86 mmol) in ethanol (40 mL) was
added 20% Pd(OH)2/C (2.92 g) and cyclohexene (23.5 mL), and the mixture
was refluxed for 15 hours. Since the smarting material did not disappear on
TLC, Pd(OH)2/C was filtered off and the solvent was once removed. The
reaction crude was diluted in ethanol (40 mL), 20% Pd(OH)2/C (2.06 g) and
cyclohexene (7.8 mL) were added to the mixture again, and the mixture
was refluxed for further 13 hours. The Pd(OH)2/C was filtered off and the
solvent was evaporated to obtain a crude product (1.82 g). Saturated ammo-
nia methanol solution (40 mL) was added to the obtained crude product
(1.82 g) at 0 ꢀC, and stirred for 1.5 hours. The crude product removed the
solvent was purified by column chromatography (SiO2, chloroform: metha-
nol ¼ 7:1) to obtain compound 7 (1.20 g, 2.66 mmol, 71%). IR ꢀmax (neat):
3338, 3211, 2746, 1645, 1600, 1356, 1175 cmꢁ1. 1H NMR (400 MHz,
CD3OD) d: 8.40 (s, 1H), 8.34 (s, 1H), 7.81 (d, J ¼ 8.2 Hz, 2H), 7.37 (d,
J ¼ 8.2 Hz, 2H), 6.27 (d, J ¼ 5.0 Hz, 1H), 4.45–4.39 (m, 2H), 4.29 (d,
J ¼ 10.5 Hz, 1H), 4.18 (d, J ¼ 10.5 Hz, 1H), 3.72 (s, 2H), 2.37 (s, 3H). 13C
NMR (100 MHz, CD3OD) d: 157.1, 153.7, 150.4, 146.4, 142.3, 134.2, 130.8,
129.2, 119.5, 86.1, 85.5, 77.8, 77.7, 70.4, 62.6, 21.5. LRMS (FAB) m/z 452
[M þ H]þ. HRMS (FAB): Calcd for C18H22N5O7S [M þ H]þ: 452.1240.
Found: 452.1210.
9-[4-C-(p-toluenesulfonyloxymethyl)-3,5-O-(1,1,3,3,-tetraisopropyldisiloxane-1,3-
diyl)-b-D-arabinofuranosyl]adenine
(8).
1,3-Dichloro-1,1,3,3-
tetraisopropyldisiloxane (0.97 mL, 3.12 mmol) was added to a pyridine
solution (26 mL) of compound 7 (1.17 g, 2.60 mmol) and the mixture was
stirred at room temperature for 6.5 hours. The mixture was diluted with
ethyl acetate, and washed with 1 M HCl, sat. NaHCO3 and brine, and dried
over anhydrous Na2SO4. The obtained crude product was purified by col-
umn chromatography (SiO2, chloroform: methanol ¼ 50:1) to give com-
pound 8 (1.70 g, 2.44 mmol, 94%). IR ꢀmax (neat): 3325, 3175, 2946, 2868,
1
1645, 1599, 1470, 1362, 1177 cmꢁ1. H NMR (400 MHz, CDCl3) d: 8.40 (s,
1H), 8.22 (s, 1H), 7.84–7.82 (m, 3H), 7.35 (d, J ¼ 8.2 Hz, 2H), 6.03 (d,
J ¼ 7.3 Hz, 1H), 5.68 (brs, 2H), 5.23 (d, J ¼ 8.2 Hz, 1H), 4.88–4.82 (m, 1H),
4.38 (d, J ¼ 10.5 Hz, 1H), 4.18 (d, J ¼ 10.5 Hz, 1H), 3.78 (d, J ¼ 11.5 Hz,
1H), 3.70 (d, J ¼ 11.5 Hz, 1H), 3.72 (s, 2H), 2.45 (s, 3H), 1.16–0.80 (m,
28 H). 13C NMR (100 MHz, CDCl3) d: 155.8, 152.5, 149.1, 144.9, 141.4,
132.8, 129.9, 128.0, 120.3, 83.8, 81.7, 79.6, 78.0, 70.1, 66.4, 21.6, 17.3, 17.3,
17.1, 17.1, 17.0, 17.0, 16.9, 13.3, 12.8, 12.5, 12.4. LRMS (FAB) m/z 694
[M þ H]þ. HRMS (FAB): Calcd for C30H48N5O8SSi2 [M þ H]þ: 694.2762.
Found: 694.2764.

8
Y. MOAI ET AL.
9-[2-Se,4-C-methylene-2-seleno-b-D-ribofuranosyl]adenine (10). To a
solution of compound 8 (32 mg, 0.046 mmol) in dichloromethane (0.5 mL) at
0 ꢀC was added 4-dimethylaminopyridine (17 mg, 0.138 mmol) and trifluoro-
methanesulfonyl chloride (8.8 lL, 0.069 mmol), and the mixture was stirred
for 30 min. The reaction mixture was partitioned between ice-cooled CH2Cl2
and water. The organic layer was washed with ice-cooled 1% aqueous HCl,
ice-cooled water, ice-cooled saturated aqueous NaHCO3, and ice-cooled satu-
rated aqueous NaCl, dried over anhydrous MgSO4, filtered, and evaporated to
obtain a crude product of triflate. In another flask, under an argon atmos-
phere, sodium borohydride (10.9 mg, 0.3 mmol) was added to a stirring sus-
pension of selenium powder (7.9 mg, 0.1 mmol) in absolute ethanol (0.3 mL);
black suspension turned to colorless solution. To this solution was added a
solution of crude product of triflate in tetrahydrofuran (0.3 mL), and the mix-
ture was stirred at 40 ꢀC for 17 hours. After cooled to room temperature, the
mixture was diluted with ethyl acetate, mixed with appropriate amount of
SiO2, and evaporated. The obtained crude product on SiO2 was roughly puri-
fied by column chromatography (SiO2, chloroform: methanol ¼ 30:1, 20:1,
10:1, 5:1) to give a mixture of 9 and its partially deprotected nucleosides. To
the mixture in tetrahydrofuran (0.2 mL) was added tetra-n-butylammonium
fluoride (1 M in THF, 15 lL, 15 lmol) and stirred at room temperature for
4 hours. The mixture was directly purified by column chromatography (SiO2,
chloroform: methanol ¼ 15:1, 10:1, 7:1, 5:1) to give compound 10 (2.5 mg,
1
7.3 lmol, 16%) as white solid. H NMR (400 MHz, DMSO-d6) d: 8.38 (s, 1H),
8.13 (s, 1H), 7.28 (s, 2H), 6.34 (s, 1H), 5.73 (d, J ¼ 4.1 Hz, 1H), 5.43 (t,
J ¼ 5.5 Hz, 1H), 4.51–4.48 (m, 1H), 3.91 (d, J ¼ 2.8 Hz, 1H), 3.75 (d,
J ¼ 5.5 Hz, 2H), 2.91 (d, J¼ 9.6 Hz, 1H), 2.86 (d, J ¼ 9.6 Hz, 1H). 13C NMR
(100 MHz, DMSO-d6) d: 156.0, 152.5, 148.3, 137.8, 119.1, 90.7, 89.4, 71.5,
58.8, 48.2, 39.5, 28.2. LRMS (FAB) m/z 344 [M þ H]þ. HRMS (FAB): Calcd
for C11H14N5O3Se [M þ H]þ: 344.0262. Found: 344.0247.
Funding
This work was partially supported by JSPS KAKENHI Numbers
JP17K08364 and by platform project for supporting drug discovery and life
science research (Basis for Supporting Innovative Drug Discovery and Life
Science Research (BINDS)) from AMED under Grant Number
JP19am0101099.
ORCID
Hidekazu Hiroaki
Saoshi Obika
Tetsuya Kodama
http://orcid.org/0000-0003-1621-5893
http://orcid.org/0000-0002-6842-6812
http://orcid.org/0000-0002-5470-5896

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
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References
[1] Khvorova, A.; Watts, J. K. The Chemical Evolution of Oligonucleotide Therapies of
Clinical Utility. Nat. Biotechnol. 2017, 35, 238–248. DOI: 10.1038/nbt.3765.
[2] Deleavey, G. F.; Damha, M. J. Designing Chemically Modified Oligonucleotides for
Targeted Gene Silencing. Chem. Biol. 2012, 19, 937–954. DOI: 10.1016/j.chembiol.
2012.07.011.
[3] Prakash, P. P. An Overview of Sugar-Modified Oligonucleotides for Antisense
Therapeutics. Chem. Biodivers. 2011, 8, 1616–1641. DOI: 10.1002/cbdv.201100081.
[4] Morihiro, K.; Kasahara, Y.; Obika, S. Biological Applications of Xeno Nucleic Acids.
Mol. Biosyst. 2017, 13, 235–245. DOI: 10.1039/C6MB00538A.
€
[5] Rothlisberger, P.; Hollenstein, M. Aptamer Chemistry. Adv. Drug Deliv. Rev. 2018,
134, 3–21. DOI: 10.1016/j.addr.2018.04.007.
[6] Singh, S. K.; Koshkin, A. A.; Wengel, J.; Nielsen, P. LNA (Locked Nucleic Acids):
Synthesis and High-Affinity Nucleic Acid Recognition. Chem. Commun. 1998,
455–456. DOI: 10.1039/a708608c.
[7] Hagedorn, P. H.; Persson, R.; Funder, E. D.; Albaek, N.; Diemer, S. L.; Hansen,
D. J.; Møller, M. R.; Papargyri, N.; Christiansen, H.; Hansen, B. R.; et al. Locked
Nucleic Acid: Modality, Diversity, and Drug Discovery. Drug Discov. Today 2018,
23, 101–114. DOI: 10.1016/j.drudis.2017.09.018.
[8] Obika, S.; Nanbu, D.; Hari, Y.; Morio, K.; In, Y.; Ishida, T.; Imanishi, T. Synthesis
of 20-O,404C-Methyleneuridine and -Cytidine. Novel Bicyclic Nucleosides Having a
Fixed C3’-Endo Sugar Puckering. Tetrahedron Lett. 1997, 38, 8735–8738.
DOI: 10.1016/S0040-4039(97)10322-7.
[9] Vester, B.; Wengel, J. LNA (Locked Nucleic Acid): High-Affinity Targeting of
Complementary RNA and DNA. Biochemistry 2004, 43, 13233–13241.
DOI: 10.1021/bi0485732.
[10] Obika, S.; Imanishi, T.; Kawada, Y.; Baba, T.; Fujisaka, A. Bridged Nucleic Acids:
Development, Synthesis and Properties. Heterocycles 2010, 81, 1347–1392.
DOI: 10.3987/REV-10-667.
[11] Yahara, A.; Shrestha, A. R.; Yamamoto, T.; Hari, Y.; Osawa, T.; Yamaguchi, M.;
Nishida, M.; Kodama, T.; Obika, S. Amido-Bridged Nucleic Acids (AmNAs):
Synthesis, Duplex Stability, Nuclease Resistance, and In Vitro Antisense Potency.
ChemBioChem 2012, 13, 2513–2516. DOI: 10.1002/cbic.201200506.
[12] Hari, Y.; Morikawa, T.; Osawa, T.; Obika, S. Synthesis and Properties of 20-
O,40-C-Ethyleneoxy Bridged 5-Methyluridine. Org. Lett. 2013, 15, 3702–3705. DOI:
10.1021/ol401566r.
[13] Shrestha, A. R.; Kotobuki, Y.; Hari, Y.; Obika, S. Guanidine Bridged Nucleic Acid
(GuNA): An Effect of a Cationic Bridged Nucleic Acid on DNA Binding Affinity.
Chem. Commun. 2014, 50, 575–577. DOI: 10.1039/C3CC46017G.
[14] Mitsuoka, Y.; Fujimura, Y.; Waki, R.; Kugimiya, A.; Yamamoto, T.; Hari, Y.; Obika,
S. Sulfonamide-Bridged Nucleic Acid: Synthesis, High RNA Selective Hybridization,
and High Nuclease Resistance. Org. Lett. 2014, 16, 5640–5643. DOI: 10.1021/
ol503029v.
[15] Barman, L.; Gurav, D.; Oommen, O. P.; Varghese, O. P. 2 N-Guanidino,430C-
Ethylene Bridged Thymidine (GENA-T) Modified Oligonucleotide Exhibits Triplex
Formation with Excellent Enzymatic Stability. RSC Adv. 2015, 5, 12257–12260. DOI:
10.1039/C4RA14721A.

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Y. MOAI ET AL.
[16] Yamaguchi, T.; Horiba, M.; Obika, S. Synthesis and Properties of 20-O,4peC-
Spirocyclopropylene Bridged Nucleic Acid (scpBNA), an Analogue of 20-,40-BNA/
LNA Bearing a Cyclopropane Ring. Chem. Commun. 2015, 51, 9737–9740. DOI: 10.
1039/C5CC02024G.
[17] Ito, Y.; Tsutsui, N.; Osawa, T.; Hari, Y. Synthesis of the Methyl Analog of 20-O,40-C-
Ehylene-Bridged 5-Methyluridine via Intramolecular Radical Cyclization and
Properties of Modified Oligonucleotides. J. Org. Chem. 2019, 84, 9093–9100. DOI:
10.1021/acs.joc.9b01035.
[18] Morihiro, K.; Kodama, T.; Kentefu; Moai, Y.; Veedu, R. N.; Obika, S.
Selenomethylene Locked Nucleic Acid Enables Reversible Hybridization in Response
to Redox Changes. Angew. Chem. Int. Ed. Engl. 2013, 52, 5074–5078.
[19] Wheeler, M.; Chardon, A.; Goubet, A.; Morihiro, K.; Tsan, S. Y.; Edwards, S. L.;
Kodama, T.; Obika, S.; Veedu, R. N. Synthesis of Selenomethylene-Locked Nucleic
Acid (SeLNA)-Modified Oligonucleotides by Polymerases. Chem. Commun. 2012,
48, 11020–11022. DOI: 10.1039/c2cc36464f.
[20] Zhang, W.; Szostak, J. W.; Huang, Z. Nucleic Acid Crystallization and X-Ray
Crystallography Facilitated by Single Selenium Atom. Front. Chem. Sci. Eng. 2016,
10, 196–202. DOI: 10.1007/s11705-016-1565-3.
[21] Salon, J.; Sheng, J.; Jiang, J.; Chen, G.; Caton-Williams, J.; Huang, Z. Oxygen
Replacement with Selenium at the Thymidine 4-Position for the Se Base Pairing and
Crystal Structure Studies. J. Am. Chem. Soc. 2007, 129, 4862–4863. DOI: 10.1021/
ja0680919.
€
[22] Hobartner, C.; Micura, R. Chemical Synthesis of Selenium-Modified
Oligoribonucleotides and Their Enzymatic Ligation Leading to an U6 snRNA Stem-
Loop Segment. J. Am. Chem. Soc. 2004, 126, 1141–1149. DOI: 10.1021/ja038481k.
[23] Wilds, C. J.; Pattanayek, R.; Pan, C.; Wawrzak, Z.; Egli, M. Selenium-Assisted
Nucleic Acid Crystallography: Use of Phosphoroselenoates for MAD Phasing of a
DNA Structure. J. Am. Chem. Soc. 2002, 124, 14910–14916. DOI: 10.1021/
ja021058b.
[24] Sahu, P. K.; Naik, S. D.; Yu, J.; Jeong, L. S. 4. Selenonucleosides as Next-Generation
Nucleosides. Eur. J. Org. Chem. 2015, 2015, 6115–6124. DOI: 10.1002/ejoc.
201500429.
[25] Wanger, M. S.; Schultze, E.; Oliveira, T. L.; Leon, P. M. M.; Thurow, H. S.; Campos,
V. F.; Oliveira, I.; Souza, D.; Rodrigues, O. E. D.; Collares, T.; Seixas, F. K.
Revitalizing the AZT through of the Selenium: An Approach in Human Triple
Negative Breast Cancer Cell Line. Front. Oncol. 2018, 8, 1–10. DOI: 10.3389/fonc.
2018.00525.
[26] Hong, T.; Yuan, Y.; Chen, Z.; Xi, K.; Wang, T.; Xie, Y.; He, Z.; Su, H.; Zhou, Y.;
Tan, Z.; et al. Precise Antibody-Independent m6A Identification via 4SedTTP-
Involved and FTO-Assisted Strategy at Single-Nucleotide Resolution. J. Am. Chem.
Soc. 2018, 140, 5886–5889. DOI: 10.1021/jacs.7b13633.
[27] Rahman, S. M. A.; Seki, S.; Obika, S.; Yoshikawa, H.; Miyashita, K.; Imanishi, T.
Design, Synthesis, and Properties of 20,40-BNA(NC): A Bridged Nucleic Acid
Analogue. J. Am. Chem. Soc. 2008, 130, 4886–4896. DOI: 10.1021/ja710342q.
[28] Vorbru€ggen, H.; Krolikiewicz, K.; Bennua, B. Nucleoside Synthesis with
Trimethylsilyl Triflate and Perchlorate as Catalysts. Chem. Ber. 1981, 114,
1234–1255. DOI: 10.1002/cber.19811140404.