Synthesis and Properties of Oligonucleotides Having Ethynylphosphonate Linkages

Article abstract of DOI:10.1021/acs.joc.9b01318

Ethynylphosphonate (EP)-linked thymidine dimers were synthesized via a palladium-catalyzed cross-coupling reaction and successfully incorporated into oligonucleotides. The oligonucleotides containing EP linkages appropriately formed a duplex with their complementary single-stranded RNA (ssRNA) and single-stranded DNA. The oligonucleotides containing both the EP linkages and 2′-O,4′-C-methylene-bridged nucleic acid/locked nucleic acid exhibited strong duplex-forming ability toward the complementary ssRNA. The EP-modified oligonucleotides exhibited higher exonuclease resistances than their natural counterparts. Moreover, one EP modification to a gapmer-type antisense oligonucleotide resulted in a switch of the cleavage site in the target ssRNA. Therefore, the EP modification can be applied for controlling the cleavage site in the RNase H-dependent mechanism.

Full text of DOI:10.1021/acs.joc.9b01318

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Article
Synthesis and properties of oligonucleotides
having ethynylphosphonate (EP) linkages
Masahiko Horiba, Takao Yamaguchi, and Satoshi Obika
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.9b01318 • Publication Date (Web): 23 Dec 2019
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The Journal of Organic Chemistry
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Synthesis and properties of oligonucleotides having ethynylphospho-
nate (EP) linkages
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Masahiko Horiba, Takao Yamaguchi, Satoshi Obika*
Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565–0871, Japan
Supporting Information Placeholder
O
EP-linked oligonucleotides
Me
NH
O
N
O
DNA or 2',4'-BNA/LNA
O
O
O
Me
O
P
O
O
Ethynylphosphonate (EP) linkage
NH
N
O
O
Duplex-forming ability toward ssDNA or ssRNA
Stability against 3'- or 5'-exonuclease
RNase H-Mediated cleavage of target RNA
O
ABSTRACT: Ethynylphosphonate (EP)-linked thymidine dimers were synthesized via a palladium-catalyzed cross-coupling reac-
tion and successfully incorporated into oligonucleotides. The oligonucleotides containing EP linkages appropriately formed a du-
plex with their complementary single-stranded RNA (ssRNA) and single-stranded DNA. The oligonucleotides containing both the
EP linkages and 2’-O,4’-C-methylene-bridged nucleic acid/locked nucleic acid exhibited strong duplex-forming ability toward the
complementary ssRNA. The EP-modified oligonucleotides exhibited higher exonuclease resistances than their natural counterparts.
Moreover, one EP modification to a gapmer-type antisense oligonucleotide resulted in a switch of the cleavage site in the target
ssRNA. Therefore, the EP modification can be applied for controlling the cleavage site in the RNase H-dependent mechanism.
the duplex formation.6b On the basis of this hypothesis, we
proposed the ethynylphosphonate (EP) linkage. The EP link-
INTRODUCTION
Phosphorothioate (PS) linkages have been introduced into a
age has no hydrogen atom that may cause steric hindrance
number of preclinical and clinical antisense oligonucleotides
(ASOs) (Figure 1).1 The PS modification imparts some benefi-
with the nucleobase. Moreover, we supposed that linearly
aligned P–C6’–C5’–C4’ atoms of the EP linkage would mimic
cial properties to ASOs, such as high stability against nuclease
digestions, improved affinity for plasma proteins, and high
tissue/cell distributions. Moreover, the modification exhibits
little or no negative effect on the recruitment of ribonuclease
H (RNase H).2 One matter for concern in the PS modification
is a chiral center at the phosphorus atom, which produces 2n
diastereomers (where n is the number of PS modifications)
having different biophysical properties3 in the more common
oligonucleotide (ON) synthesis. As such, the other types of
linkage modifications are still being developed. For example,
ONs modified with sulfur,4 nitrogen,5 or carbon6 atoms instead
of the 5’- or 3’-oxygen have been synthesized and evaluated.
In the case of the sulfur and nitrogen modifications, some ad-
vantages have been recognized in in vivo experiments.4a,5c In
contrast, carbon-modified analogs (i.e., alkylphosphonate)
have not been thoroughly studied for their biological proper-
ties,6 most likely because many of them show weak duplex-
forming ability. As typical examples, 5’-methylphosphonate
(5’-MP)- and vinylphosphonate (VP)-modified ONs exhibit a
weak ability to form duplexes toward the complementary sin-
gle-stranded DNA (ssDNA).6b,6c It is believed that a steric
repulsion between the hydrogen atom(s) at the C6’-position
and the nucleobase in these analogs causes adverse effects on
Figure 1. Structures of natural and modified (PS, 5’-MP, VP, and
EP) nucleic acids.
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the general torsion angle β (P–O5’–C5’–C4’ ≈ 180º) in the A-
Page 2 of 9
(trimethylsilyl)ethoxymethyl (SEM) group,12 and the di-
methoxytrityl (DMTr) group was removed under acidic condi-
tions. An oxidation of the primary alcohol of 2 using Dess-
Martin periodinane, followed by the conversion of the result-
ing aldehyde into 1,1-dibromoalkene by a Corey-Fuchs reac-
tion, gave 3 in 85% yield over 2 steps. The SEM group was
then removed with tin(IV) tetrachloride,12 and the TBDPS
group was subsequently deprotected by treatment with TBAF.
This step-by-step approach afforded 4 in 97% yield. The given
substrate 4 and H-phosphonate 7, derived from 5’-O-DMTr-
thymidine, were subjected to a cross-coupling reaction under
microwave irradiation [conditions: Pd(OAc)2, 1,1’-
bis(diphenylphosphino)ferrocene (dppf), and propylene oxide
in DMF at 90 ºC]. This resulted in the formation of dimer 5 in
53% yield. In the last step, phosphitylation of the 3’-hydroxyl
group of 5 produced the desired amidite block 6 in 46% yield.
Similar to the above synthetic approach, we also prepared a
dimer amidite containing the EP linkage and 2’,4’-BNA/LNA
(Scheme 2).
form or B-form duplex (Figure S1, Supporting Information).7
Herein, we describe the synthesis of EP-linked thymidine di-
mers and their incorporation into ONs. The ONs having the
EP linkages as well as both the EP linkages and 2’-O,4’-C-
methylene-bridged nucleic acid8/locked nucleic acid9 (2’,4’-
BNA/LNA) structures were evaluated for their duplex-forming
abilities toward single-stranded RNA (ssRNA) and ssDNA.
Furthermore, the nuclease resistance and RNase H-recruiting
ability of the EP-modified ONs were evaluated.
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RESULTS AND DISCUSSION
Synthesis of EP-linked dimers
The hydroxyl group at the 5’-position acts as a nucleophile
during the synthesis of natural ONs using phosphoramidite
chemistry. However, since this general method cannot be ap-
plied for the EP modification, a dimer containing the EP link-
age was prepared and used for the ON synthesis (Scheme 1).
A palladium-catalyzed cross-coupling of H-phosphonate and
dibromoalkene was performed and is a key reaction in the
dimer synthesis.10 Firstly, the imide at the nucleobase of start-
Synthesis of EP-linked ONs
The synthesis of EP-linked ONs was performed on a 1-
micromolar scale by using an automated DNA synthesizer.
The coupling time was set to 2 min for EP-linked dimer
ing
material
111
was
protected
with
a
2-
Scheme 1. Synthesis of EP-linked thymidine dimer phosphoramidite 6. Reagents and conditions: (a) SEMCl, DBU, CH2Cl2, –78 ºC, 1 h;
(b) TFA, MeOH, rt, 20 h, 59% (2 steps); (c) Dess-Martin periodinane, CH2Cl2, rt, 1 h; (d) CBr4, PPh3, CH2Cl2, 0 ºC, 20 min, 85% (2
steps); (e) SnCl4, CH2Cl2, rt, 9 h; (f) TBAF, AcOH, THF, rt, 2 h, 97% (2 steps); (g) 7, Pd(OAc)2, dppf, propylene oxide, DMF, 90 ºC
microwave, 1 h, 53%; (h) iPr2NP(Cl)OCH2CH2CN, DIPEA, MeCN, rt, 1 h, 46%.
Scheme 2. Synthesis of EP-linked thymidine dimer phosphoramidite 11 containing 2’,4’-BNA/LNA. Reagents and conditions: (a) 1H-
tetrazole, MeCN/H2O, rt, 30 min, quant.; (b) 4, Pd(OAc)2, dppf, propylene oxide, DMF, 90 ºC microwave, 30 min, 41%; (c)
iPr2NP(Cl)OCH2CH2CN, DIPEA, MeCN, rt, 2 h, 66%.
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amidites. After the synthesis, the crude ONs were purified by
Table 1. Tm values of the duplexes formed between ON1–ON10
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3
4
5
6
7
8
reverse-phase HPLC, and the designed ONs containing
straight and rigid EP linkage were successfully isolated. How-
ever, we observed a large quantity of short chains that failed to
react with the EP-linked dimer amidites (see Figure S2, Sup-
porting Information), and most of the ON syntheses resulted in
low yields (0.2–2.2%) (Table S1, Supporting Information).
Several attempts at varying the coupling time (between 32 s
and 8 min) and/or activators (ETT and less acidic DCI) were
unsuccessful. Despite the low yields, the EP-linked ONs from
several syntheses were obtained and utilized for the biophysi-
cal evaluations.
and their complementary ssRNA or ssDNA[a]
Tm (ΔTm) (ºC)
ONs
ssRNA
48
ssDNA
52
5’-d(GCG TTTTTT GCT)-3’
5’-d(GCG TeTTTTT GCT)-3’
5’-d(GCG TTTeTTT GCT)-3’
5’-d(GCG TTTTTeT GCT)-3’
5’-d(GCG TTTeTTT GCT)-3’
5’-d(GCG TTTeTTT GCT)-3’
5’-d(GCG TTTeTTT GCT)-3’
5’-d(GCG TTTTTT GCT)-3’
5’-d(GCG TTTTTT GCT)-3’
5’-d(GCG TTTTTT GCT)-3’
ON1
ON2
ON3
ON4
ON5
ON6
ON7
ON8
ON9
ON10
45 (–3)
44 (–4)
45 (–3)
50 (+2)
48 (+0)
50 (+2)
53 (+5)
52 (+4)
52 (+4)
48 (–4)
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47 (–5)
49 (–3)
49 (–3)
49 (–3)
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53 (+1)
53 (+1)
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Duplex-forming ability of the EP-linked ONs
The melting temperature (Tm) of the duplexes formed be-
tween the EP-linked ONs and the complementary ssRNA or
ssDNA were measured by UV-melting experiments (Table 1).
In contrast to our initial speculation, ONs modified with the
rigid EP linkage (ON2–ON4) were found to show a much
lower affinity toward ssRNA (ΔTm = –3 to –4 °C) and ssDNA
(ΔTm = –4 to –5 °C) than the natural counterpart (ON1). The
other EP-linked ONs having a different sequence similarly
showed decreased duplex-forming ability (Table S2, Support-
ing Information). As an exception, the EP linkage at the 5’-end
had a smaller negative affect on the duplex-forming ability of
the ON (see ON13 in Table S2). We have found that, in most
cases, a combination of the EP linkage and 2’,4’-BNA/LNA
results in improved Tm values for both ssRNA and ssDNA
(e.g., ON2–ON4 versus ON5–ON7).
[a] Conditions: 10 mM phosphate buffer (pH 7.2), 100 mM NaCl,
and 4 µM of each ON. The Tm values reflect the average of three
measurements. Target sequences: 5’-r(AGC AAAAAA CGC)-3’
(ssRNA), 5’-d(AGC AAAAAA CGC)-3’ (ssDNA). T = 2’,4’-
BNA/LNA, e = EP. All linkages except for EP are phos-
phodiesters.
Circular dichroism spectra of phosphodiester- and EP-
linked dimers
Circular dichroism (CD) spectra are useful to determine the
higher-order structures of dinucleotides, single strands, du-
plexes, and quadruplexes. In the case of dinucleotides, cotton
bands at 260–280 nm correlate with the base–base interac-
tions.13 Thymidine dimers modified with EP linkages and/or
2’,4’-BNA/LNA were prepared and their CD spectra evaluated.
As depicted in Figure 2, all dimers D1–D4 had maximum
band intensity at 276 to 279 nm and minimum one at 246 to
254 nm. This suggests that these dimers have similar higher-
order structures. However, the band intensity of the EP-linked
dimer (D3) was weaker than that of the natural one (D1).
Therefore, the linearly fixed EP linkages seem to disrupt the
base–base interactions. The strength of base-stacking should
be reflected in the enthalpic aspect of the thermodynamic pa-
rameters. Consistent with the CD data, the duplex formed be-
tween ON3 and ssRNA showed a loss in enthalpy as com-
pared to the ON1/ssRNA duplex (Table S3 and Figure S3,
Supporting Information). In contrast to D3, a dimer containing
2’,4’-BNA/LNA (D2) showed double the intensity at around
280 nm compared to D1, which revealed that 2’,4’-BNA/LNA
positively affects the base-stacking as well as preorganization
for the duplex formation. A dimer containing both the EP
linkage and 2’,4’-BNA/LNA (D4) showed increased band
intensity relative to D3; thus the weakened base-stacking
caused by the EP modification, which probably led to the de-
creased Tm values (Table 1), could be restored by the addition
of 2’,4’-BNA/LNA.
Figure 2. CD spectra of phosphodiester- and EP-linked dimers.
Conditions: 10 mM phosphate buffer (pH 7.2), 100 mM NaCl, and
0.1 µM of each dimer (at 5 ºC). T = 2’,4’-BNA/LNA, e = EP. The
linkages of D1 and D2 are phosphodiesters.
natural ON (ON19) rapidly degraded under the conditions we
tested, whereas the PS-modified ON (ON20) remained intact
more than 80% after 80 min. In the case of the EP-modified
ON (ON21), 7% of the intact ON survived until the last time-
point. Thus, the EP linkage slightly increased the stability
against 3’-exonuclease. It is considered that changes in the
skeletal conformation, based on CD spectral analysis (Figure
2), and the electrophilicity at the phosphorus atom by EP mod-
ification, positively affected the stability towards nucleases.
Against 5’-exonuclease, the ON modified with EP (ON13)
showed high stability similar to the ON modified with PS
(ON22). It has been reported that the 5’-oxygen atom of the
phosphodiester acts as a leaving group in the hydrolysis by
BSPD (5’-exonuclease), whereas the 3’-oxygen atom is a leav-
ing group in the case of SVPD (3’-exonuclease). The greatly
enhanced stability seen in the EP-modified ON13 against the
Stability of the EP-linked ONs against nuclease diges-
tions
The stability of ONs against 3’- or 5’-exonuclease (SVPD
or BSPD) was investigated by monitoring the percentage of
non-degraded ONs (Table 2). In the case of 3’-exonuclease,
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Table 2. Stabilities of the ONs against 3’- or 5’-exonuclease digestion
1
2
3
Intact ONs (%)
3’-Exonuclease[a]
5’-Exonuclease[b]
4
5
6
7
Time (min)
ON19
ON20
88
ON21
23
ON11
ON22
–
ON13
–
20
80
0
0
–
0
83
7
79
88
8
9
[a] Conditions: 50 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 0.2 µg/mL SVPD, and 4 µM each ON (at 37 ºC). Sequences: 5’-d(TTT TTT
TTTT)-3’ (ON19), 5’-d(TTT TTT TTTsT)-3’ (ON20), 5’-d(TTT TTT TTTeT)-3’ (ON21). [b] Conditions: 50 mM phosphate buffer (pH
6.0), 500 µg/mL BSPD, and 4 µM of each ON (at 37 ºC). Sequences: 5’-d(TTT ACG CAG TTT)-3’ (ON11), 5’-d(TsTT ACG CAG TTT)-
3’ (ON22), 5’-d(TeTT ACG CAG TTT)-3’ (ON13); s = PS, e = EP. All linkages except for PS and EP are phosphodiesters.
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5’-exonuclease can be explained by the low reactivity of the
ethynyl substituent.
Table 3. Tm values of the duplexes formed between ON23–ON28
and their complementary ssRNA[a]
RNase H-recruiting ability of the EP-linked ONs
ONs
Tm (ΔTm) (ºC)
55
We also evaluated the RNase H-recruiting ability of the EP-
linked ONs (Table 3 and Figure 3). A gapmer-type 14-mer ON,
an octathymidine flanked by 2’-O-methyl RNA (2’-OMe) was
synthesized. An RNase H-activity assay was performed using
an ON having eight PS modifications (ON24) and ONs con-
taining one EP linkage (ON25–ON28) in the gap region. The
Tm values of these gapmers were confirmed to be higher than
45 ºC as shown in Table 2. After annealing of ON23–ON28 to
the complementary ssRNA labeled by fluorescein at the 5’-end
(Fl-RNA), RNase H was added and the mixture stood at 37 ºC
for 40 min. The resulting mixture was separated on a denatur-
ing urea polyacrylamide gel electrophoresis (urea-PAGE) and
the cleaved 5’-fragments of Fl-RNA were monitored using a
fluorescence imaging system. As shown in Figure 3, the natu-
ral and PS-modified ONs (ON23 and ON24) induced the
5’-d(gcg TTTTTTTT gcu)-3’
ON23
5’-d(gcg sTsTsTsTsTsTsTsT gcu)-3’ ON24
46 (–9)
54 (–1)
53 (–2)
54 (–1)
53 (–2)
5’-d(gcg TeTTTTTTT gcu)-3’
5’-d(gcg TTTeTTTTT gcu)-3’
5’-d(gcg TTTTTeTTT gcu)-3’
5’-d(gcg TTTTTTTeT gcu)-3’
ON25
ON26
ON27
ON28
[a] Conditions: 10 mM phosphate buffer (pH 7.2), 100 mM NaCl,
and 2 µM each ON. The Tm values reflect the average of three
measurements. Target sequence: 5’-r(AGC AAAAAAAA CGC)-
3’. g = 2’-OMe-G, c = 2’-OMe-C, u = 2’-OMe-U, s = PS, e = EP.
All linkages except for PS and EP are phosphodiesters.
Figure 3. (a) Analysis of the 5’-fragments of Fl-RNA degraded by RNase H. (b) Illustrated presentation of the cleaved sites in Fl-RNA.
Conditions: 50 mM Tris-HCl (pH 8.0), 3 mM MgCl2, 75 mM KCl, 10 mM DTT, 0.1 unit/µL RNase H, 6 µM of each ON, and 30 µM Fl-
RNA (at 37 ºC for 40 min). g = 2’-OMe-G, c = 2’-OMe-C, u = 2’-OMe-U, s = PS, e = EP. All linkages except for PS and EP are phos-
phodiesters. Arrows mean the cleaved sites, and the length of the arrows corresponds to the rate of hydrolysis. 5’-r(AGC AAAAAAA)-3’
(10-mer), 5’-r(AGC AAAAA)-3’ (8-mer), and 5’-r(AGC AAA)-3’ (6-mer) were labeled by fluorescein at the 5’-end and used as size mark-
ers.
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cleavage of Fl-RNA mainly at the third position from the 3’-
HCO3 was added at 0 ºC and the resulting mixture was ex-
tracted with EtOAc. The combined organic layer was washed
with water and brine, dried over Na2SO4, and concentrated.
The crude product (9.89 g) was used for the next reaction
without further purification.
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end of Fl-RNA. Under the same conditions, fully 2’-OMe-
modified ON (ON29) did not produce the cleaved fragments.
It was interesting that ONs modified with the EP linkage
(ON25–ON28) drastically changed the cleavage sites in the
target Fl-RNA. RNase H is known to recognize several resi-
dues on ASOs during the enzymatic reaction.14 Inclusion of
the EP linkage to the RNase H recognition region (the region
located several residues away from the cleavage site) led to a
reduced enzymatic hydrolysis at the corresponding site. This
means that the cleavage sites can be controlled by the EP mod-
ification. Recently, Seth and coworkers have successfully de-
veloped ASOs allele-selectively inhibiting mutant huntingtin
gene by an incorporation of artificial nucleic acids into the gap
region.15 Moreover, they have demonstrated that site-specific
replacement of PS with alkyl phosphonate linkages enhances
the therapeutic profile of ASOs.16 The EP linkage would be
used for these kinds of applications.
To a solution of the crude product (9.89 g) in MeOH (2.0
mL) was added trifluoroacetic acid (0.68 mL, 8.9 mmol) at 0
ºC. After stirring at room temperature for 2 h, saturated aq.
NaHCO3 was added and the resulting mixture was extracted
with EtOAc. The combined organic layers were washed with
water and brine, dried over Na2SO4, and concentrated. The
crude product was purified by column chromatography (SiO2,
15–75% EtOAc in n-hexane) to afford 2 (3.22 g, 59% in 2
steps) as a white solid: 1H NMR (300 MHz, CDCl3) δ –0.01 (s,
9H), 0.93–0.99 (m, 2H), 1.09 (s, 9H), 1.84–1.88 (m, 4H), 2.15
(ddd, J = 6.2, 7.9, 13.7 Hz, 1H), 2.30 (ddd, J = 3.1, 5.9, 13.4
Hz, 1H), 3.25 (ddd, J = 3.1, 6.2, 12.0 Hz, 1H), 3.60–3.69 (m,
3H), 3.96–3.99 (m, 1H), 4.41–4.45 (m, 1H), 5.36 (d, J = 9.6
Hz, 1H), 5.39 (d, J = 10.0 Hz, 1H), 6.26 (dd, J = 6.2, 7.6 Hz,
1H), 7.27 (d, J = 1.0 Hz, 1H), 7.37–7.47 (m, 6H), 7.62–7.67
(m, 4H); 13C{1H} NMR (75.5 MHz, CDCl3) δ –1.3, 13.4, 18.2,
19.1, 27.0, 40.5, 62.2, 67.6, 70.3, 73.1, 87.5, 87.7, 110.4,
128.0, 130.2, 130.3, 133.2, 133.4, 135.5, 135.8, 135.9, 151.1,
163.5; IR (KBr): 3481, 2952, 1708, 1670, 1469, 1249, 1099,
836, 703 cm–1; [α]D20 +30.7 (c 1.00, CHCl3); HRMS (MALDI-
TOF) Calcd. for C32H46N2O6NaSi2 [M+Na]+: 633.2787, Found
633.2776.
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CONCLUSIONS
The EP-linked thymidine dimers were successfully synthe-
sized via a palladium-catalyzed cross-coupling reaction of H-
phosphonates and dibromoalkenes. The resulting dimers were
incorporated into the synthesized ON sequences. All the EP-
linked ONs formed appropriate duplexes with ssRNA and
ssDNA. Furthermore, the ONs modified with EP linkage and
2’,4’-BNA/LNA showed a relatively strong tendency to form
duplexes. The EP linkage was also found to enhance their
stability towards nuclease activity. An important feature ob-
served in the EP-linked ONs was the change in cleavage sites
of the target RNA in the RNase H-dependent mechanism.
Therefore, ASOs having EP linkages might be used for allele-
selective gene regulation.
6’-Dibromo-(5’-deoxy-5’-methylidene)-3’-O-(tert-
butyldiphenylsilyl)-3-(2-
trimethylsilylethoxymethyl)thymidine (3)
To a solution of 2 (600 mg, 982 µmol) in dichloromethane
(10 mL) was added Dess-Martin periodinane (500 mg, 1.20
mmol) at 0 ºC. After stirring at room temperature for 1 h, satu-
rated aq. Na2S2O3 and saturated aq. NaHCO3 were added and
the resulting mixture was stirred for 10 min. The mixture was
extracted with EtOAc. The combined organic layer was
washed with water and brine, dried over Na2SO4, and concen-
trated. The crude aldehyde (633 mg) was used for the next step
without further purification.
EXPERIMENTAL SECTION
General
Dry dichloromethane, dry acetonitrile, and deoxygenated
N,N-dimethylformamide were used as purchased. ON1, ON8–
11, ON17–20, ON22–24, ON29 and their complementary
ssRNAs and ssDNAs were purchased from GeneDesign, Inc.
1H NMR spectra were recorded at 300 or 500 MHz. 13C{1H}
and 31P{1H} NMR spectra were recorded at 75 and 200 MHz,
respectively. Chemical shift values are expressed in δ values
(ppm) relative to internal tetramethylsilane (0.00 ppm), resid-
To a solution of carbon tetrabromide (1.31 g, 3.95 mmol) in
dry dichloromethane (10 mL) was added triphenylphosphine
(2.07 g, 7.89 mmol) and the reaction mixture was stirred at 0
ºC for 30 min under N2 atmosphere. To the resulting red solu-
tion was added the crude aldehyde (633 mg) in dry dichloro-
methane (10 mL) at 0 ºC. After stirring at 0 ºC for 20 min,
saturated aq. NH4Cl was added and the resulting mixture was
filtered through Celite. The filtrate was extracted with EtOAc
and the combined organic layers were washed with water and
brine, dried over Na2SO4, and concentrated. To the residue
was added hexane and the resulting mixture was filtered. The
filtrate was concentrated and purified by column chromatog-
raphy (SiO2, 10–25% EtOAc in n-hexane) to afford 3 (639 mg,
1
ual chloroform (7.26 ppm), or CHD2OD (3.31 ppm) for H
NMR and chloroform-d1 (77.16 ppm) or methanol-d4 (49.00
ppm) for 13C{1H} NMR. For 31P{1H} NMR, 5% H3PO4 (0.00
ppm) was used as an external standard. Mass spectra of all
new compounds and ONs were measured on a SpiralTOF
JMS-S300 instrument. PSQ-100B, FL-100D, PSQ-60B, FL-
60D, or Chromatorex-DIOL silica gel was used for column
chromatography. PLC Silica gel 60 F254 was used for prepara-
tive thin layer chromatography.
1
85% in 2 steps) as a white solid: H NMR (300 MHz, CDCl3)
δ –0.01 (s, 9H), 0.93–0.98 (m, 2H), 1.09 (s, 9H), 1.82–1.91 (m,
4H), 2.38 (ddd, J = 4.1, 6.5, 13.7 Hz, 1H), 3.62–3.68 (m, 2H),
4.24 (ddd, J = 3.8, 3.8, 6.2 Hz, 1H), 4.65 (dd, J = 3.8, 8.6 Hz,
1H), 5.35 (d, J = 10.0 Hz, 1H), 5.38 (d, J = 9.6 Hz, 1H), 6.22
(d, J = 8.9 Hz, 1H), 6.24 (dd, J = 6.5, 6.5 Hz, 1H), 6.91 (d, J =
1.1 Hz, 1H), 7.37–7.48 (m, 6H), 7.63–7.66 (m, 4H); 13C{1H}
NMR (75.5 MHz, CDCl3) δ –1.3, 13.5, 18.3, 19.1, 26.9, 40.3,
67.6, 70.3, 75.9, 86.1, 86.5, 96.0, 110.6, 128.1, 128.1, 130.3,
132.7, 133.0, 134.0, 134.9, 135.9, 135.9, 150.8, 163.3; IR
3’-O-(tert-Butyldiphenylsilyl)-3-(2-
trimethylsilylethoxymethyl)thymidine (2)
1,8-Diazabicyclo[5.4.0]-7-undecene (7.8 mL, 51 mmol) and
2-(chloromethoxy)ethyltrimethylsilane (4.5 mL, 26 mmol)
were added in three portions to a solution of 111 (6.96 g, 8.89
mmol) in dry dichloromethane (70 mL) at –78 ºC under a N2
atmosphere. After stirring at –78 ºC for 1 h, saturated aq. Na-
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Page 6 of 9
(KBr): 2953, 1711, 1671, 1465, 1249, 1112, 830, 702 cm–1;
ºC under a N2 atmosphere. After stirring at room temperature
for 1 h, the reaction mixture was concentrated. The residue
was purified by column chromatography (Chromatorex-DIOL
silica, 75% AcOEt in n-hexane) to afford 6 (789 mg, 46%) as
20
1
2
3
4
5
6
7
8
[α]D +17.8 (c 1.00, CHCl3); HRMS (MALDI-TOF) Calcd.
for C33H44N2O5NaSi2Br2 [M+Na]+: 785.1048, Found 785.1044.
6’-Dibromo-(5’-deoxy-5’-methylidene)thymidine (4)
1
a white solid: H NMR (300 MHz, CDCl3) δ 1.18–1.21 (m,
To a solution of 3 (224 mg, 293 µmol) in dry dichloro-
methane (3 mL) was added tin(IV) chloride (1.0 M in di-
chloromethane, 0.33 mL, 0.33 mmol) at 0 ºC under a N2 at-
mosphere. After stirring at room temperature for 9 h, saturated
aq. NaHCO3 was added and the resulting mixture was extract-
ed with EtOAc. The combined organic layers were washed
with water and brine, dried over Na2SO4, and concentrated.
The crude product (264 mg) was used for the next reaction
without further purification.
12H), 1.39 (s, 3/2H), 1.39 (s, 3/2H), 1.93–1.94 (m, 3H), 2.35–
2.80 (m, 8H), 3.39–3.90 (m, 12H), 4.21–4.35 (m, 3H), 4.79–
4.96 (m, 2H), 5.28–5.32 (m, 1H), 6.34–6.39 (m, 1H), 6.47 (dd,
J = 5.2, 8.9 Hz, 1H), 6.84 (d, 4H), 7.23–7.38 (m, 10H), 7.55 (d,
J = 1.4 Hz, 1/2H), 7.57 (s, 1/2H), 8.43 (s, 2H); 31P{1H} NMR
(202.5 MHz, CDCl3) δ –8.7, –8.6, –8.5, 149.4, 149.5, 150.6,
150.6; HRMS (FAB) Calcd. for C54H63N7O14NaP2 [M+Na]+:
1118.3806, Found 1118.3805.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2’,4’-BNA/LNA H-phosphonate 9
To a solution of the crude product (264 mg) and acetic acid
(50 µL, 0.88 mmol) in tetrahydrofuran (3.0 mL) was added
tetrabutylammonium fluoride (1.0 M in tetrahydrofuran, 586
µL, 586 µmol) at room temperature. After stirring for 2 h,
water was added and the resulting mixture was extracted with
EtOAc. The combined organic layer was washed with brine,
dried over Na2SO4, and concentrated. The obtained residue
was purified by column chromatography (SiO2, 75% EtOAc in
n-hexane) to afford 4 (113 mg, 97% in 2 steps) as a white sol-
To a solution of 8 (100 mg, 129 µmol) in acetonitrile/water
(1.6 mL, 7:1) was added 1H-tetrazole (18 mg, 0.26 mmol) at 0
ºC. After stirring at room temperature for 30 min, brine was
added and the resulting mixture was extracted with CH2Cl2.
The combined organic layers were dried over Na2SO4. After
the removal of the organic solvent, 9 (100 mg, quant.) was
1
obtained as a white solid: H NMR (300 MHz, CDCl3) δ 1.55
(s, 3/2H), 1.65 (s, 3/2H), 2.40–2.61 (m, 1H), 2.74 (t, J = 6.0
Hz, 1H), 3.34 (d, J = 11.5 Hz, 1/2H), 3.45 (d, J = 11.0 Hz,
1/2H), 3.69 (d, J = 11.0 Hz, 1/2H), 3.75 (d, J = 11.0 Hz, 1/2H),
3.68–3.91 (m, 8H), 4.01–4.12 (m, 1H), 4.26–4.34 (m, 1H),
4.72 (s, 1/2H), 4.74 (s, 1/2H), 5.00 (d, J = 6.4 Hz), 5.03 (d, J =
6.9 Hz, 1/2H), 5.69 (s, 1/2H), 5.70 (s, 1/2H), 6.72 (d, J = 738
Hz, 1H), 6.86–6.89 (m, 4H), 6.96 (d, J = 739 Hz, 1/2H), 7.26–
7.62 (m, 9H), 7.63 (s, 1/2H), 7.63 (s, 1/2H), 8.65 (s, 1/2H),
8.69 (s, 1/2); 31P{1H} NMR (121.6 MHz, CDCl3) δ 7.09, 7.46;
HRMS (FAB) Calcd. for C35H36N3O10P [M]+: 689.2138,
Found 689.2139.
1
id: H NMR (300 MHz, CD3OD) δ 1.90 (d, J = 1.4 Hz, 3H),
2.23–2.40 (m, 2H), 4.35 (ddd, J = 3.7, 3.7, 5.9 Hz, 1H), 4.53
(dd, J = 3.2, 8.7 Hz, 1H), 6.21 (dd, J = 6.9, 6.9 Hz, 1H), 6.82
(d, J = 8.3 Hz, 1H), 7.44 (d, J = 1.4 Hz, 1H); 13C{1H} NMR
(75.5 MHz, CD3OD) δ 12.4, 39.8, 75.6, 87.4, 87.7, 94.5, 111.9,
137.9, 138.2, 152.2, 166.4; IR (KBr): 3348, 1719, 1655, 1273
23
cm–1; [α]D +45.1 (c 1.04, DMSO); HRMS (MALDI-TOF)
Calcd. for C11H12N2O4NaBr2 [M+Na]+: 416.9056, Found
416.9069.
EP-Linked thymidine dimer 5
EP-linked thymidine dimer 10
A solution of palladium(II) acetate (136 mg, 606 mmol) and
1,1’-bis(diphenylphosphino)ferrocene (672 mg, 1.21 mmol) in
deoxygenated N,N-dimethylformamide (20 mL) was stirred at
room temperature for 1 h. To the resulting yellow solution was
added a solution of 4 (1.20 g, 3.03 mmol), H-phosphonate 7
(3.01 g, 4.55 mmol), and propylene oxide (2.2 mL, 30 mmol)
in deoxygenated N,N-dimethylformamide (3 mL). After stir-
ring at 90 ºC under microwave irradiation for 1 h, the resulting
mixture was concentrated. The residue was purified by column
chromatography (SiO2, 0–10% MeOH in CHCl3), followed by
an additional purification by column chromatography (SiO2,
50–100% EtOAc in n-hexane and then 10% MeOH in EtOAc),
to afford 5 (1.45 g, 53%) as a white solid: 1H NMR (500 MHz,
CDCl3) δ 1.40 (s, 3/2H), 1.43 (d, J = 1.2 Hz, 3/2H), 1.92 (s,
3/2H), 1.94 (s, 3/2H), 2.31–2.35 (m, 1H), 2.45–2.51 (m, 2H),
2.72–2.81 (m, 5/2H), 2.90 (dd, J = 4.6, 13.8 Hz, 1/2H), 3.38–
3.42 (m, 1H), 3.52–3.54 (m, 1H), 3.79 (s, 6H), 4.16 (d, J = 3.5
Hz, 1/2H), 4.26–4.35 (m, 3H), 4.41 (d, J = 3.5 Hz, 1/2H),
4.68–4.78 (m, 2H), 5.23–5.29 (m, 1H), 6.35–6.47 (m, 2H),
6.83–6.85 (m, 4H), 7.23–7.36 (m, 10H), 7.56 (s, 1/2H), 7.61 (s,
1/2H), 9.50 (s, 1/2H), 9.53 (s, 1/2H), 9.70 (s, 1/2H), 9.98 (s,
1/2H); 31P{1H} NMR (202.5 MHz, CDCl3) δ –9.3, –9.0;
HRMS (MALDI-TOF) Calcd. for C45H46N5O13NaP [M+Na]+:
918.2722, Found 918.2726.
A solution of palladium(II) acetate (2 mg, 9 µmol) and 1,1’-
bis(diphenylphosphino)ferrocene (11 mg, 20 µmol) in deoxy-
genated N,N-dimethylformamide (0.5 mL) was stirred at room
temperature for 1 h. To the resulting yellow solution was add-
ed a solution of 4 (20 mg, 51 µmol), H-phosphonate 9 (52 mg,
76 µmol), and propylene oxide (37 µL, 0.51 mmol) in deoxy-
genated N,N-dimethylformamide (0.5 mL). After stirring at 90
ºC under microwave irradiation for 30 min, the resulting mix-
ture was concentrated. The residue was purified by column
chromatography (SiO2, 5–9% MeOH in CHCl3), followed by
an additional purification by column chromatography (SiO2,
5% MeOH in CHCl3) to afford 10 (19 mg, 41%) as a white
1
solid: H NMR (300 MHz, CDCl3) δ 1.60 (s, 3/2H), 1.69 (s,
3/2H), 1.87 (s, 3/2H), 1.87 (s, 3/2H), 2.31–2.34 (m, 1H), 2.50–
2.75 (m, 3H), 3.45–3.66 (m, 2H), 3.78 (s, 6H), 3.83–4.37 (m,
4H), 4.63–4.76 (m, 3H), 4.89–5.02 (m, 2H), 5.64 (s, 1/2H),
5.67 (s, 1/2H), 6.32–6.39 (m, 1H), 6.86 (d, J = 8.7 Hz, 4H),
7.25–7.32 (m, 8H), 7.41–7.43 (m, 2H), 7.60 (s, 1/2H), 7.63 (s,
1/2H), 9.95 (s, 1/2H), 9.98 (s, 1/2H), 10.4 (s, 1/2H), 10.4 (s,
1/2H); 31P{1H} NMR (121.6 MHz, CDCl3) δ –10.0, –8.5;
HRMS (FAB) Calcd. for C46H46N5O14P [M]+: 923.2779,
Found 923.2777.
EP-linked thymidine dimer amidite 11
EP-Linked thymidine dimer amidite 6
To a solution of 10 (20 mg, 22 µmol) in dry acetonitrile (1
mL) was added N,N-diisopropylethylamine (12 µL, 69 µmol)
and 2-cyanoethyl-N,N-diisopropylphosphoramidochloridite (8
µL, 0.04 mmol) at room temperature under N2 atmosphere.
After stirring for 1 h, N,N-diisopropylethylamine (12 µL, 69
To a solution of 5 (1.39 g, 1.55 mmol) in dry acetonitrile
(15 mL) was added N,N-diisopropylethylamine (0.85 mL, 4.9
mmol)
and
2-cyanoethyl-N,N-
diisopropylphosphoramidochloridite (0.52 mL, 2.3 mmol) at 0
µmol)
and
2-cyanoethyl-N,N-
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The Journal of Organic Chemistry
diisopropylphosphoramidochloridite (8 µL, 0.04 mmol) were
Calcd. for [M–H]–: 3654.4, Found 3654.7; ON12: 1.3% yield,
MS (MALDI-TOF) Calcd. for [M–H]–: 3619.4, Found 3620.0;
ON13: 2.2% yield, MS (MALDI-TOF) Calcd. for [M–H]–:
3619.4, Found 3618.9; ON14: 2.1% yield, MS (MALDI-TOF)
Calcd. for [M–H]–: 3647.4, Found 3647.6; ON15: 1.2% yield,
MS (MALDI-TOF) Calcd. for [M–H]–: 3647.4, Found 3647.3;
ON16: 1.6% yield, MS (MALDI-TOF) Calcd. for [M–H]–:
3647.4, Found 3648.2; ON21: 0.9% yield, MS (MALDI-TOF)
Calcd. for [M–H]–: 2973.0, Found 2972.6; ON25: 0.3% yield,
MS (MALDI-TOF) Calcd. for [M–H]–: 4400.9, Found 4400.8;
ON26: 0.2% yield, MS (MALDI-TOF) Calcd. for [M–H]–:
4400.9, Found 4401.2; ON27: 0.5% yield, MS (MALDI-TOF)
Calcd. for [M–H]–: 4400.9, Found 4401.7; ON28: 0.8% yield,
MS (MALDI-TOF) Calcd. for [M–H]–: 4400.9, Found 4401.1.
1
2
3
4
5
6
7
8
added. After further stirring at room temperature for 1 h, the
reaction mixture was concentrated. The residue was briefly
purified by column chromatography (Chromatorex-DIOL sili-
ca, 2% MeOH in CHCl3), and the resulting product was tritu-
rated with n-hexane to afford 11 (16 mg, 66%) as a white sol-
id: 1H NMR (500 MHz, CDCl3) δ 1.18–1.20 (m, 12H), 1.58 (s,
3/4H), 1.59 (s, 3/4H), 1.67 (s, 3/4H), 1.69 (s, 3/4H), 1.88–1.90
(m, 3H), 2.36–2.81 (m, 6H), 3.45–4.36 (m, 16H), 4.76–5.02
(m, 4H), 5.66 (s, 1/4H), 5.67 (s, 1/4H), 5.71 (s, 1/2H), 6.21–
6.37 (m, 1H), 6.85–6.88 (m, 4H), 7.20–7.35 (m, 8H), 7.41–
7.46 (m, 2H), 7.59 (s, 1H), 8.97 (s, 2H); 31P{1H} NMR (121.6
MHz, CDCl3) δ –9.8, –9.2, –8.6, –8.6, 149.1, 149.3, 150.4,
150.6; HRMS (FAB) Calcd. for C55H64N7O15P2 [M+H]+:
1124.3936, Found 1124.3937.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
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59
60
Melting temperature (Tm) measurements
Synthesis, purification and characterization of EP-linked
dimers D3 and D4
The Tm values were measured on UV-1650B and UV-1800
spectrometers (SHIMADZU) equipped with a Tm analysis
accessory. Samples containing NaCl (100 mM), ON (4 µM),
and target ssRNA (4 µM) or ssDNA (4 µM) in the phosphate
buffer (10 mM, pH 7.2) were annealed by heating up to 95 ºC
followed by cooling to room temperature slowly (overnight in
a typical pattern). The absorbance at 260 nm was recorded
from 5 to 90 ºC at a scan rate of 0.5 ºC/min. The Tm values
were defined as the temperature at which 50% of the formed
duplexes dissociated and were calculated based on the first
derivative of the melting curve.
Compounds 5 and 10 (1.0 mg, 1.1 µmol) independently
stood in 28% ammonium hydroxide at room temperature for 2
h. The samples were lyophilized, and each dimethoxytrityl
group was deprotected on Pak® Plus C18 cartridge with 0.5%
trifluoroacetic acid. The fractions containing the dimers were
purified by reverse-phase HPLC (2.5 µm, 10×50 mm) with a
linear gradient [MeCN (3 to 9% over 15 min) in 0.1 M TEAA
buffer (pH 7.0)]. The purities of the dimers were evaluated by
reverse-phase HPLC (2.5 µm, 4.6×50 mm) and the dimers
were identified by liquid chromatography-mass spectrometer.
The concentrations of the dimer solutions were determined
from the absorbance at 260 nm. D3: MS (ESI) Calcd. for
C21H24N4O11P [M–H]–: 539.1, Found 539.2; D4: MS (ESI)
Calcd. for C22H24N2O12P [M–H]–: 567.1, Found 567.1.
Circular dichroism (CD) spectral analyses
CD spectra were measured at 5 ºC in a quartz cuvette having
1.00-cm optical path length by using a JASCO J-720W spec-
trophotometer. The sample solutions (360 µL) were prepared
by dissolving the dimer (0.1 mM) and NaCl (100 mM) in the
phosphate buffer (10 mM, pH 7.2). The molar ellipticity was
calculated from the equation [θ] = θ/cl, where θ is the relative
intensity, c is the sample concentration, and l is the cell path
length in centimeters.
Synthesis, purification, and characterization of ONs
The ON synthesis was performed on a 1-micromolar scale
using an automated DNA synthesizer following the standard
phosphoramidite protocol. 5-(Ethylthio)-1H-tetrazole was
used as an activator, and the coupling time for EP-linked di-
mer amidites was prolonged from 40 s to 2 min. The coupling
time for 2’,4’-BNA/LNA and 2’-OMe phosphoramidites was
10 min. The synthesis was carried out in the trityl-off mode for
ON2–ON7, ON12–ON16, and ON21 and in the trityl-on
Stability against 3’-exonuclease
Samples (each sample volume: 130 µL) containing MgCl2
(10 mM), ON (4 µM), and snake venom phosphodiesterase
(SVPD, 0.2 µg/mL) in the Tris-HCl buffer (50 mM, pH 8.0)
were incubated at 37 ºC. Portions of the samples (20 µL) were
taken at respective time points, and SVPD was immediately
deactivated by heating sample at 90 ºC for 2.5 min. The per-
centage of the remaining intact ONs was determined by re-
verse-phase HPLC (2.5 µm, 4.6×50 mm) and plotted against
their reaction time.
mode for ON25–ON28. As
a capping reagent, tert-
butylphenoxyacetic acid anhydride (TAC2O) was utilized. For
the cleavage of the solid support and the removal of the pro-
tecting groups, ON2–ON7, ON12–ON16, and ON21 were
treated with 28% ammonium hydroxide at room temperature
for 2 h, and ON25–ON28 were treated with AMA (28% am-
monium hydroxide/40% methylamine = 1:1) at room tempera-
ture for 5 min and then at 65 ºC for 1 h. For the deprotection
of the dimethoxytrityl group, ON25–ON28 were subsequently
treated with 0.5% trifluoroacetic acid on a Sep-Pak® Plus C18
cartridge. The crude ONs were purified by reverse-phase
HPLC. The purity of the ONs was evaluated by reverse-phase
HPLC, and the ONs were identified using a MALDI-TOF
mass spectrometer. The yields of the ONs were determined
from their absorbance at 260 nm. ON2: 1.5% yield, MS
(MALDI-TOF) Calcd. for [M–H]–: 3626.4, Found 3626.4;
ON3: 1.3% yield, MS (MALDI-TOF) Calcd. for [M–H]–:
3626.4, Found 3626.4; ON4: 1.0% yield, MS (MALDI-TOF)
Calcd. for [M–H]–: 3626.4, Found 3626.1; ON5: 1.0% yield,
MS (MALDI-TOF) Calcd. for [M–H]–: 3654.4, Found 3654.1;
ON6: 1.7% yield, MS (MALDI-TOF) Calcd. for [M–H]–:
3654.4, Found 3655.4; ON7: 0.7% yield, MS (MALDI-TOF)
Stability against 5’-exonuclease
Samples (each sample volume: 130 µL) containing ON (4
µM), and bovine spleen phosphodiesterase (BSPD, 500
µg/mL) in the phosphate buffer (50 mM, pH 6.0) were incu-
bated at 37 ºC. Portions of the samples (20 µL) were taken at
respective time points, and BSPD was immediately deactivat-
ed by heating sample at 90 ºC for 2.5 min. The percentage of
the remaining intact ONs was determined by reverse-phase
HPLC (2.5 µm, 4.6×50 mm) and plotted against their reaction
time.
RNase H activity evaluations
The samples containing ON (30 µM), target ssRNA labeled
by fluorescein at the 5’-end (300 mM), MgCl2 (100 mM), KCl
(300 mM), and DTT (100 mM) in the Tris-HCl buffer (500
mM, pH 8.0) were annealed by heating up to 60 ºC followed
by slowly cooling to room temperature (e.g., over 30 min). E.
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coli RNase H having the DNA·RNA hybrid-binding domain
Page 8 of 9
2. (a) Stein, C. A.; Subasinghe, C.; Shinozuka, K.; Cohen,
J. S. Physicochemical Properties of Phosphorothioate
Oligodeoxynucleotides. Nucleic Acids Res. 1988, 16,
3209–3221. (b) Srinivasan, S. K.; Tewary, H. K.;
Iversen, P. L. Characterization of Binding Sites, Extent
of Binding, and Drug Interactions of Oligonucleotides
with Albumin. Antisense Res. Dev. 1995, 5, 131–139.
(c) Geary, R. S.; Yu, R. Z.; Watanabe, T.; Henry, S. P.;
Hardee, G. E.; Chappell, A.; Matson, J.; Sasmor, H.;
Cummins, L. E. N.; Levin, A. A. Pharmacokinetics of a
Tumor Necrosis Factor-α Phosphorothioate 2’-O-(2-
Methoxyethyl) Modified Antisense Oligonucleotide:
Comparison Across Species. Drug Metab. Dispos.
2003, 31, 1419–1428. (d) Miller, C. M.; Donner, A. J.;
Blank, E. E.; Egger, A. W.; Kellar, B. M.; Østergaard,
M. E.; Seth, P. P.; Harris, E. N. Stabilin-1 and Stabilin-
2 are Specific Receptors for the Cellular Internalization
of Phosphorothioate-modified Antisense Oligonucleo-
tides (ASOs) in the Liver. Nucleic Acids Res. 2016, 44,
2782–2794.
1
2
3
4
5
6
7
8
(purchased from Takara Bio Inc.) was added to each sample
(0.1 unit/µL), and the resulting mixtures stood at 37 ºC for 40
min. To a portion of each sample (10 µL), formamide (28 µL)
and EDTA (100 mM, 2 µL) were added to deactivate RNase H.
The samples were separated by denaturing urea-PAGE (20%
acrylamide) and analyzed using a fluorescence imaging sys-
tem (ImageQuant LAS 4010).
9
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Supporting Information
The Supporting Information is available free of charge on
the ACS Publications website.
1H, 13C{1H}, and 31P{1H} NMR spectra for all new com-
pounds, HPLC charts and MALDI-TOF MS data for all new
ONs, and supplementary data.
3. (a) Wan, W. B.; Migawa, M. T.; Vasquez, G.; Murray,
H. M.; Nichols, J. G.; Gaus, H.; Berdeja, A.; Lee, S.;
Hart, C. E.; Lima, W. F.; Swayze, E. E.; Seth, P. P.
Synthesis, Biophysical Properties and Biological Activ-
ity of Second Generation Antisense Oligonucleotides
Containing Chiral Phosphorothioate Linkages. Nucleic
Acids Res. 2014, 42, 13456–13468. (b) Li, M.;
Lightfoot, H. L.; Halloy, F.; Malinowska, A. L.; Berk,
C.; Behera, A.; Schümperli, D.; Hall, J. Synthesis and
Cellular Activity of Stereochemically-pure 2’-O-(2-
AUTHOR INFORMATION
Corresponding Author
* E-mail: obika@phs.osaka-u.ac.jp
ORCID
Takao Yamaguchi: 0000-0003-3180-0257
Satoshi Obika: 0000-0002-6842-6812
Notes
The authors declare no competing financial interest.
Methoxyethyl)-phosphorothioate
Oligonucleotides.
Chem. Commun. 2017, 53, 541–544. (c) Iwamoto, N.;
Butler, D. C. D.; Svrzikapa, N.; Mohapatra, S.; Zlatev,
I.; Sah, D. W. Y.; Meena; Standley, S. M.; Lu, G.;
Apponi, L. H.; Frank-Kamenetsky, M.; Zhang, J. J.;
Vargeese, C.; Verdine, G. L. Control of Phosphorothio-
ate Stereochemistry Substantially Increases the Effica-
cy of Antisense Oligonucleotides. Nat. Biotechnol.
2017, 35, 845–851.
ACKNOWLEDGMENT
This work was supported in part by JSPS KAKENHI Grant
Number 16J06367 to M.H. and 16K17930 to T.Y., the Plat-
form 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 JP18am0101084, and the Basic Science and
Platform Technology Program for Innovative Biological Med-
icine from AMED under Grant Number JP18am0301004.
4. (a) Islam, M. A.; Waki, R.; Fujisaka, A.; Ito, K. R.;
Obika, S. In Vitro and in Vivo Biophysical Properties of
Oligonucleotides Containing 5’-Thio Nucleosides.
Drug Discov. Ther. 2016, 10, 263–270. (b) Islam, M.
A.; Fujisaka, A.; Mori, S.; Ito, K. R.; Yamaguchi, T.;
Obika, S. Synthesis and Biophysical Properties of 5’-
Thio-2’,4’-BNA/LNA Oligonucleotide. Bioorg. Med.
Chem. 2018, 26, 3634–3638. (c) Bentley, J.; Brazier, J.
A.; Fisher, J.; Cosstick, R. Duplex Stability of
DNA·DNA and DNA·RNA Duplexes Containing 3’-S-
Phosphorothiolate Linkages. Org. Biomol. Chem. 2007,
5, 3698–3702.
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