Synthesis and evaluation of artificial nucleic acid bearing an oxanorbornane scaffold

Article abstract of DOI:10.3390/molecules25071732

Natural oligonucleotides have many rotatable single bonds, and thus their structures are inherently flexible. Structural flexibility leads to an entropic loss when unwound oligonucleotides form a duplex with single-stranded DNA or RNA. An effective approa

Full text of DOI:10.3390/molecules25071732

molecules
Article
Synthesis and Evaluation of Artificial Nucleic Acid
Bearing an Oxanorbornane Scaffold
Hibiki Komine 1 , Shohei Mori , Kunihiko Morihiro
*
1,2, ,†
Takumi Okuda
,‡
1,‡
, Kenta Ishida 1,
1
, Yuuya Kasahara , Hiroshi Aoyama 1 , Takao Yamaguchi * and
1,2
1,
Satoshi Obika 1,2,*
1
Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan;
flyfly113@gmail.com (H.K.); s7592mori@gmail.com (S.M.); kenta.ishida0918@gmail.com (K.I.);
taku0903taku@gmail.com (T.O.); y-kasahara@nibiohn.go.jp (Y.K.); haoyama@phs.osaka-u.ac.jp (H.A.)
National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN), 7-6-8 Saito-Asagi, Ibaraki,
Osaka 567-0085, Japan
2
*
†
‡
Correspondence: morihiro@bioorg.rcast.u-tokyo.ac.jp (K.M.); yamaguchi-ta@phs.osaka-u.ac.jp (T.Y.);
obika@phs.osaka-u.ac.jp (S.O.); Tel.: +81-6-6879-8200 (S.O.)
Current address: Department of Chemistry and Biotechnology, Graduate School of Engineering,
The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
These authors contributed equally to this work.
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Yoshiyuki Hari
Received: 30 March 2020; Accepted: 7 April 2020; Published: 9 April 2020
ꢀ
ꢁꢂꢃꢄꢅꢆ
Abstract: Natural oligonucleotides have many rotatable single bonds, and thus their structures are
inherently flexible. Structural flexibility leads to an entropic loss when unwound oligonucleotides form
a duplex with single-stranded DNA or RNA. An effective approach to reduce such entropic loss in the
duplex-formation is the conformational restriction of the flexible phosphodiester linkage and/or sugar
moiety. We here report the synthesis and biophysical properties of a novel artificial nucleic acid bearing
an oxanorbornane scaffold (OxNorNA), where the adamant oxanorbornane was expected to rigidify the
structures of both the linkage and sugar parts of nucleic acid. OxNorNA phosphoramidite with a uracil
(U) nucleobase was successfully synthesized over 15 steps from a known sugar-derived cyclopentene.
Thereafter, the given phosphoramidite was incorporated into the designed oligonucleotides. Thermal
denaturation experiments revealed that oligonucleotides modified with the conformationally restricted
OxNorNA-U properly form a duplex with the complementally DNA or RNA strands, although the T
m
values of OxNorNA-U-modified oligonucleotides were lower than those of the corresponding natural
oligonucleotides. As we had designed, entropic loss during the duplex-formation was reduced by
the OxNorNA modification. Moreover, the OxNorNA-U-modified oligonucleotide was confirmed
0
to have extremely high stability against 3 -exonuclease activity, and its stability was even higher
than those of the phosphorothioate-modified counterparts (Sp and Rp). With the overall biophysical
properties of OxNorNA-U, we expect that OxNorNA could be used for specialized applications, such as
conformational fixation and/or bio-stability enhancement of therapeutic oligonucleotides (e.g., aptamers).
Keywords: modified nucleic acid; modified oligonucleotide; oxanorbornane; conformational
restriction; entropy; duplex-forming ability; mismatch discrimination; nuclease resistance
1
. Introduction
The structural flexibility of natural oligonucleotides contributes to the formation of various
higher-order structures. However, when unwound oligonucleotides form a specific structure (e.g.,
duplex), a large entropic loss generally arises. Thus, chemical modifications that restrict the structures
of flexible phosphodiester linkages and/or sugar moieties to those seen in A-form DNA RNA duplexes
·
Molecules 2020, 25, 1732; doi:10.3390/molecules25071732
www.mdpi.com/journal/molecules

Molecules 2020, 25, 1732
2 of 15
have a useful role in producing high-affinity (i.e., potent) antisense oligonucleotides [
1–
3
]. In our
laboratory, a number of conformationally restricted artificial nucleic acids, for which a representative
0
0
0
0
example is 2 ,4 -bridged nucleic acid (2 ,4 -BNA, commonly called locked nucleic acid (LNA)) (Figure 1),
0
0
have been developed [
4–
8
]. 2 ,4 -BNA/LNA has an N-type sugar pucker commonly seen in the A-form
0
0
duplex, and thus the 2 ,4 -BNA/LNA-modified oligonucleotides exhibit a high duplex-forming
ability toward single-stranded RNA (ssRNA). Leumann and co-workers have demonstrated that
oligonucleotides modified with tricyclo-DNA, in which a fused ring system rigidifies the torsion angles
γ
and
Henessian and co-workers have successfully achieved restrictions of both the sugar ring and the torsion
angle by designing -l-triNA 1 [13]. Oligonucleotides containing -l-triNA 1 are known to have a
high binding affinity toward ssRNA.
δ, exhibit increased ssRNA affinity relative to the natural oligonucleotides [9–12]. More recently,
γ
α
α
Throughout our research on conformational restrictions of nucleic acids, we newly designed
an artificial nucleic acid bearing an oxanorbornane scaffold (OxNorNA) (Figure 2). Oxanorbornane,
2-oxabicyclo[2,2,1]heptane, has a rigid structure, and thus the relative position of the phosphodiester
linkage and nucleobase parts of OxNorNA can be strictly fixed. The linkage part of OxNorNA is
0
0
considered a 4 –2 system based on the position of nucleobase. This type of linkage system is rarely
0
0
0
0
seen in the other artificial nucleic acids, although the effects of 5 –2 and 3 –2 linkage systems (isoDNA
and TNA, respectively) on the duplex-forming ability and antisense potency have been intensely
investigated [14–17]. Herein, we report a robust synthetic route for OxNorNA phosphoramite with a
uracil (U) nucleobase and the biophysical properties (duplex-forming ability, mismatch discrimination,
and nuclease resistance) of OxNorNA-U-modified oligonucleotides.
Figure 1. Conformational restrictions of natural oligonucleotides.
Figure 2. Structures of artificial nucleic acid bearing an oxanorbornane scaffold (OxNorNA), isoDNA,
and TNA.
2
. Results and Discussion
2
.1. Synthesis of OxNorNA-U Phosphoramidite
Synthesis of OxNorNA-U phosphoramidite was started from cyclopentene
1
, derived over eight
were
was subjected to a hydroboration reaction to
in a regio- and diastereoselective manner. Silylation was followed
by hydrogenation using Pd/C afforded diol . The primary alcohol of compound was then selectively
protected, and the remaining secondary alcohol was converted into a leaving group (OTf). Subsequent
treatment of sodium azide afforded compound at a 71% yield over two steps. The reduction of the
steps from commercially available d-ribose [18
protected by a benzyl group, and the given compound
produce the desired cyclopentanol
–20] (Scheme 1). At first, two hydroxyl groups of 1
2
3
5
5
7

Molecules 2020, 25, 1732
3 of 15
azide group, using ammonium formate with Pd/C, underwent the removal of the TES group. A uracil
ring was successively formed by a reaction with 3-methoxyacryloyl isocyanate, following the reported
procedure [21
was unsuccessful. The hydroxyl group of
were removed under mildly acidic conditions (CeCl ·7H O, oxalic acid, MeCN, and rt) to afford triol
–
23]. The direct incorporation of the uracil nucleobase to the triflated compound of
6
9
was then mesylated, and the acetonide and TBS group
3
2
1
1
. Intramolecular cyclization of 11 by aqueous (aq.) NaOH regio-selectively afforded the desired
oxanorbornane 12 at a 72% yield. The regioselectivity is explained by the difference in the uracil
nucleobase steric repulsion of the two possible products. That is, 12 obtained from the S 2 reaction of
N
0
the 3 -hydroxyl group of 11 has the uracil nucleobase at a less hindered position than the other product
that can be produced from the S 2 reaction of the 2 -hydroxyl group. The structure of 12 was confirmed
by NMR (For H, C, H– H COSY, DEPT, HMQC, and HMBC NMR spectra, see Supplementary
Material) and X-ray crystal structure (Figure 3). The less hindered 2 -hydroxyl group of 12 was then
0
N
1
13
1
1
0
protected by the dimethoxytrityl (DMTr) group, and the remaining alcohol was phosphytilated to
0
0
afford 14, a suitable building block for the reverse (3 -to-5 ) oligonucleotide synthesis. We note that
tritylation of 12 by using DMTrCl in pyridine resulted in no reaction, but DMTrOTf generated from a
reaction of DMTrCl and AgOTf in CH Cl successfully afforded compound 13 in 93% [16,24].
2
2
Scheme 1. Synthesis of OxNorNA-uracil (U) phosphoramidite 14
.
Reagents and conditions:
◦
◦
◦
(i) NaH, BnBr, DMF, 0 C, 92%; (ii) thexylborane, THF, 0 C to rt; (iii) NaBO
·
H O, H O, 0 C
3
2
2
to rt, 90% over 2 steps; (iv) emphtert-butyldimethylchlorosilane (TBSCl), imidazole, DMF, rt, 74%;
◦
v) HCOONH , Pd/C, EtOH, reflux, 91%; (vi) triethylchlorosilane (TESCl), 2,6-lutidine, CH Cl , –78 C,
4 2 2
(
◦
6%; (vii) trifluoromethanesulfonic anhydride (Tf O), pyridine, CH Cl , 0 C; (viii) NaN , DMF, rt,
2 2 2 3
8
7
◦
1% over 2 steps; (ix) HCOONH , Pd/C, THF, rt, 98%; (x) 3-methoxyacryloyl isocyanate, THF, –40 C to
4
◦
rt; (xi) NH OH, EtOH, 120 C (sealed tube), 48% over 2 steps; (xii) methanesulfonyl chloride (MsCl),
Et N, CH Cl , 0 C, 77%; (xiii) CeCl
rt, 72%; (xv) 4,4 -dimethoxytrityl trifluoromethanesulfonate (DMTrOTf), CH Cl , pyridine, 2,6-lutidine,
0
4
◦
·
7H O, oxalic acid, MeCN, rt, 66%; (xiv) aq. NaOH, 1,4-dioxane,
3
2
2
3
2
0
2
2
◦
C to rt, 93%; (xvi) 2-cyanoethyl N,N-diisopropylchlorophosphoramidite, N,N-diisopropylethylamine
◦
(DIPEA), 1-methylimidazole, MeCN, 0 C to rt, 56%.

Molecules 2020, 25, 1732
4 of 15
Figure 3. ORTEP drawing of the X-ray crystal structure of 12 (thermal ellipsoids at the 50%
probability level).
2
.2. Oligonucleotide Synthesis
Following the conventional phosphoramidite protocol, the given amidite 14 was successfully
incorporated into the designed sequences (Table 1). The coupling time for the incorporation of the
synthesized phosphoramidite 14 was prolonged to 12.5 min. In addition, the detritylation time of all
reverse phosphoramidites was performed in 2 min. The above conditions afforded OxNorNA-U-modified
oligonucleotides ON1–ON3 in 22–47% yields (for high-performance liquid chromatography (HPLC)
charts of purified oligonucleotides, see Supplementary Material).
Table 1. Isolated yields of OxNorNA-U-modified oligonucleotides, together with matrix-assisted laser
desorption/ionization-time of flight mass spectra (MALDI-TOF MS) data.
[M − H]−
ID
Sequence a
Yield (%)
Calcd
Found
0
0
ON1
ON2
ON3
5 -d(GCG TTX TTT GCT)-3
22
31
47
3630.4
3626.3
2977.0
3630.3
3626.3
2976.6
0
0
5 -d(GCG XTX TXT GCT)-3
0
0
5 -d(TTT TTT TTT X)-3
a
X = OxNorNA-U.
2
.3. Duplex-forming Ability and Thermodynamic Parameters
The thermal stabilities of the duplexes formed by the OxNorNA-U-modified oligonucleotides
ON1 and ON2) with the complementary single-stranded DNA (ssDNA) or ssRNA were then
evaluated by ultraviolet (UV) melting experiments. The given melting temperatures (T values) and
thermodynamic parameters are summarized in Tables 2 and 3 (for van’t Hoff plots, see Figures S1 and S2
(
m
0
0
in Supplementary Material). As for the ssDNA complement (sequence: 5 -d(AGCAAAAAACGC)-3 )
(
Table 2), ON1 with a single OxNorNA modification was found to properly form a duplex. However,
◦
◦
the ON1·ssDNA duplex furnished a T
m
value of 45.4 C, which was 4.5 C lower T
m
than that obtained
◦
for the corresponding ON4·ssDNA duplex (T
m
= 49.9 C). Further modification to ON1 with OxNorNA
◦
resulted in a very low affinity for ssDNA (21.0 C for ON2) as compared to the natural oligonucleotide
◦
0
0
(
48.4 C for ON5). Toward the ssRNA complement (sequence: 5 -r(AGCAAAAAACGC)-3 ) (Table 3),
OxNorNA-modified ON1 and ON2 denoted similar tends, but they were found to prefer ssRNA rather
than ssDNA (see their ∆Tm/mod. values).
m
The low T values of the OxNorNA-modified oligonucleotides might be explained by an unusual
–2 linkage system that was changed from the general 5 –3 linkage system found in the natural
0
0
0
0
4
oligonucleotides. The linkage shift may lead the nucleobase of OxNorNA to an unfavorable direction
0
0
for the duplex formations (for the structure comparison between OxNorNA-U and 2 ,4 -BNA/LNA-U
nucleosides, see reference [25]). Thermodynamic data obtained by van’t Hoff plots indicated that
duplex formations by the OxNorNA-modified oligonucleotides were enthalpically unfavored (e.g.,

Molecules 2020, 25, 1732
5 of 15
base stacking), and entropy factors (e.g., structural restriction) had no compensatory effects on the
duplex formations.
Table 2.
T
m
values and thermodynamic parameters of duplexes formed between oligonucleotides and
a
the complementary single-stranded DNA (ssDNA) .
◦
◦
◦
∆G
Tm (∆Tm/mod.)
∆H
∆S
Sequence b
ID
◦
[
C]
[kcal/mol] [kcal/mol] [kcal/mol]
0
0
ON4
ON1
ON5
ON2
5 -d(GCG TTU TTT GCT)-3
49.9
−108.6
−76.4
−81.4
−42.0
−310.1
−214.8
−227.2
−117.2
−12.4
−9.8
0
0
5 -d(GCG TTX TTT GCT)-3
45.4 (−4.5)
0
0
5 -d(GCG UTU TUT GCT)-3
48.4
−11.0
−5.6
0
0
5 -d(GCG XTX TXT GCT)-3
21.0 (−9.1)
a
Conditions: 10 mM sodium phosphate buffer (pH 7.2) containing 100 mM NaCl and 4
values are averages of at least three measurements. The sequence of ssDNA is 5 -d(AGCAAAAAACGC)-3 .
µ
M of each oligonucleotide.
0
0
T
∆
m
◦
T
m
/mod.: the change in T
m
value (
∆
T
m
) per modification compared to the unmodified oligonucleotide.
∆
G
◦
values at 37 C are shown. For thermodynamic parameters and van’t Hoff plots, see Table S1 and Figure S1 in
Supplementary Material. X = OxNorNA-U.
b
Table 3.
T
m
values and thermodynamic parameters of duplexes formed between oligonucleotides and
a
the complementary single-stranded RNA (ssRNA) .
◦
◦
◦
∆G
Tm (∆Tm/mod.)
∆H
∆S
Sequence b
ID
◦
[
C]
[kcal/mol] [kcal/mol] [kcal/mol]
0
0
ON4
ON1
ON5
ON2
5 -d(GCG TTU TTT GCT)-3
46.5
−94.2
−92.0
−86.8
−56.3
−268.3
−265.9
−246.9
−163.6
−11.0
−9.5
0
0
5 -d(GCG TTX TTT GCT)-3
42.4 (−4.1)
0
0
5 -d(GCG UTU TUT GCT)-3
44.7
−10.2
−5.5
0
0
5 -d(GCG XTX TXT GCT)-3
24.7 (−6.7)
a
Conditions: 10 mM sodium phosphate buffer (pH 7.2) containing 100 mM NaCl and 4
values are averages of at least three measurements. The sequence of ssRNA is 5 -r(AGCAAAAAACGC)-3 .
µ
M of each oligonucleotide.
0
0
T
∆
m
◦
T
m
/mod.: the change in T
m
value (
∆
T
m
) per modification compared to the unmodified oligonucleotide.
∆
G
◦
values at 37 C are shown. For thermodynamic parameters and van’t Hoff plots, see Table S2 and Figure S2 in
Supplementary Material. X = OxNorNA-U.
b
2
.4. Mismatch Discrimination
We also evaluated mismatch discrimination of the OxNorNA-U-modified oligonucleotide. As shown
in Tables 4 and 5, OxNorNA-U-modified ON1 exhibited much lower T
one-base-mismatched sequences ( = G, C, T, and U) than toward the fully matched sequence (
A). The base discrimination patterns of OxNorNA-U-modified ON1 were similar to the natural
m
values toward the
N
N
=
counterpart (ON4). This result indicated that a single modification with the adamant OxNorNA does not
interfere with the common base-pairing rules, even the linkage moiety shifts from the natural nucleic acid.
Table 4. Mismatch discrimination of natural and OxNorNA-modified oligonucleotides toward ssDNA a.
Sequence b
◦
ID
Tm (∆Tm = Tm [mismatch] − Tm [match]) [ C]
N = A
N = G
N = C
N = T
0
0
ON4
ON1
5 -d(GCG TTU TTT GCT)-3
49.9
45.4
35.5 (−14.4)
38.5 (−11.4)
36.0 (−9.4)
36.5 (−13.4)
0
0
5 -d(GCG TTX TTT GCT)-3
33.2 (−12.2)
32.5 (−12.9)
a
Conditions: 10 mM sodium phosphate buffer (pH 7.2) containing 100 mM NaCl and 4
values are averages of at least three measurements. The sequence of ssDNA is 5 -d(AGCAAA
µ
M of each oligonucleotide.
0
0
T
m
NAACGC)-3 .
b
X = OxNorNA-U.

Molecules 2020, 25, 1732
6 of 15
Table 5. Mismatch discrimination of natural and OxNorNA-modified oligonucleotides toward ssRNA a.
Sequence b
◦
ID
Tm (∆Tm = Tm [mismatch] − Tm [match]) [ C]
N = A
N = G
N = C
N = U
0
0
ON4
ON1
5 -d(GCG TTU TTT GCT)-3
46.5
42.4
31.9 (−14.6)
41.4 (−5.1)
35.1 (−7.3)
32.6 (−13.9)
0
0
5 -d(GCG TTX TTT GCT)-3
27.8 (−14.6)
27.6 (−14.8)
a
Conditions: 10 mM sodium phosphate buffer (pH 7.2) containing 100 mM NaCl and 4
values are averages of at least three measurements. The sequence of ssRNA is 5 -r(AGCAAA
µ
M of each oligonucleotide.
0
0
T
m
NAACGC)-3 .
b
X = OxNorNA-U.
2
.5. Circular Dichroism Analysis
To analyze the structure of OxNorNA-U-modified oligonucleotide in a single-stranded or duplex
◦
form, circular dichroism (CD) spectra were recorded at 10 C in the same buffer as that used for the UV
melting experiments (Figure 4). As a result, ON1 with one OxNorNA modification showed similar
CD spectra to the natural ON4 (Figure 4a,b); thus, ON1 and ON4 have similar secondary structures
in the presence or absence of complementary strands (ssDNA and ssRNA). From the CD spectra in
Figure 4b, ON1·ssRNA and ON4·ssRNA duplexes were found to form a typical A-form duplex (positive
cotton band at 260 nm and negative cotton band at 210 nm). In contrast to the results in Figure 4a,b,
ON2·ssDNA and ON2·ssRNA duplexes with multiple OxNorNA modifications displayed much
weaker cotton effects than the corresponding ON5·ssDNA and ON5·ssRNA duplexes (Figure 4c,d).
These results probably indicated disrupted base stacking in ON2·ssDNA and ON2·ssRNA duplexes
and thus resulted in duplex destabilizations (low Tm values).
Figure 4. Circular dichroism (CD) spectra of OxNorNA-modified oligonucleotides (ON1 and ON2
and their natural counterparts (ON4 and ON5) in the presence or absence of a complementary strand
ssDNA or ssRNA). ( ON1 and ON4 in the presence or absence of ssDNA; ( ON1 and ON4 in
the presence or absence of ssRNA; ( ON2 and ON5 in the presence or absence of ssDNA; ( ON2
and ON5 in the presence or absence of ssRNA. Conditions: 10 mM sodium phosphate buffer (pH 7.2)
)
(
a)
b)
c)
d)
◦
containing 100 mM NaCl and 4 µM of each oligonucleotide at 10 C. The sequences of the target ssDNA
0
0
0
0
and ssRNA are 5 -d(AGCAAAAAACGC)-3 and 5 -r(AGCAAAAAACGC)-3 , respectively.
2
.6. Stability Against Nuclease Digestion
We next investigated the effect of OxNorNA modification on the stability of oligonucleotides
0
0
against enzymatic degradation. Oligonucleotides with 3 -terminal modifications (sequence: 5 -d(TTT

Molecules 2020, 25, 1732
7 of 15
0
0
TTT TTT
X
X)-3 ,
X
= OxNorNA-U (ON3);
X
= 5 -(R)-phosphorothioate (PS)-modified thymidine (ON6);
0
= 5 -(S)-PS-modified thymidine (ON7);
X
= locked nucleic acid (LNA) (ON8)) were prepared,
0
◦
incubated with 0.133 ug/mL 3 -exonuclease (snake venom phosphodiesterase, svPDE) at 37 C, and
the percentage of the remaining intact oligonucleotides was monitored by HPLC (Figure 5). Under
the conditions we tested, the oligonucleotide modified with LNA (ON8) was digested within 20 min.
Commonly used phosphorothioate (PS) modifications were found to be effective for improving the
oligonucleotide stability against nuclease, but more than 30% of ON6 (Sp) and ON7 (Rp) were cleaved
at 80 min. Conversely, over 80% of OxNorNA-modified ON3 was still intact after 80 min. It was
expected that OxNorNA had successfully escaped from the nuclease recognition since it had an altered
0
0
scaffold from the natural nucleoside and an unusual 4 –2 linkage system.
Figure 5. Enzymatic stability of the OxNorNA-modified oligonucleotide. Conditions: 0.133 ug/mL
snake venom phosphodiesterase (svPDE), 10 mM MgCl , 50 mM Tris-HCl (pH 8.0), and 2 µM each
2
◦
0
0
oligonucleotide at 37 C. The sequence of the oligonucleotides used was 5 -d(TTT TTT TTT
X
)-3 .
= 5 -(S)-phosphorothioate (PS)-modified thymidine (orange
= 5 -(R)-PS-modified thymidine (gray triangle, ON7), and = locked nucleic acid
0
X
= OxNorNA-U (red diamond, ON3), X
0
square, ON6),
X
X
(LNA)-T (blue cross, ON 8).
3
. Materials and Methods
3
.1. General Information
Reagents and solvents were purchased from commercial suppliers and used without purification
unless otherwise specified. All experiments involving air and/or moisture-sensitive compounds
were carried out under an Ar atmosphere. All reactions were monitored with analytical thin-layer
chromatography (TLC; silica gel coated with fluorescent indicator F254; Merck, Darmstadt, Germany).
Flash column chromatography was carried out using EPCLC–W–Prep 2XY (Yamazen, Osaka, Japan).
NMR spectra were recorded on JNM-ECA-500, JNM-ECS-400, and JNM-ECS-300 spectrometers (JEOL,
Akishima, Japan) using CDCl or DMSO–d as the solvent with tetramethylsilane (TMS) as an internal
3
6
standard. For 31P NMR, 5% H PO in D O (0.00 ppm) was used as an external standard. Infrared
3
4
2
(IR) spectra were recorded on an FT/IR–4200 spectrophotometer (JASCO, Hachioji, Japan). Optical
rotations were recorded on a P–2200 instrument (JASCO). Mass spectra of all new compounds were
measured on a SpiralTOF JMS-S3000 (for electrospray ionization, ESI) or a JMS-700 instrument
(JEOL) (for fast atom bombardment, FAB). Solid-phase oligonucleotide synthesis was performed
using an nS–8 Oligonucleotide Synthesizer (GeneDesign, Ibaraki, Japan). Matrix-assisted laser
desorption/ionization-time of flight (MALDI-TOF) mass spectra were recorded on an ultrafleXtreme
or an Autoflex II mass spectrometer (Bruker Daltonics, Billerica, MA, USA). For HPLC, Shimadzu
DGU-20A , LC-20AD, CBM-20A, CTO-20AC, SPD-20A, and FRC-10A were utilized. For UV
3R
absorbance measurements, a UV-1800 spectrometer (Shimadzu, Kyoto, Japan) was utilized. UV melting
experiments were performed using a UV–1650 or UV-1800 UV–Vis spectrophotometer equipped with
a TMSPC–8 Tm analysis accessory (Shimadzu).

Molecules 2020, 25, 1732
8 of 15
3
.2. Synthesis of Compound 2
Compound (3.0 g, 16.1 mmol) was dissolved in dry DMF (160 mL), and the solution was cooled
1
in an ice bath. Sodium hydride (50% w/w in mineral oil, 2.30 g) was carefully added to the solution,
and the resulting mixture was stirred for 30 min. Benzylbromide (5.7 mL, 48.3 mmol) was then
◦
added dropwise, and the mixture was further stirred for 2 h at 0 C. After the reaction was finished,
the mixture was partitioned between CH Cl and H O. The separated organic layer was washed with
2
2
2
brine, dried over Na SO , and concentrated in vacuo. The residue was purified by silica gel column
2
4
chromatography, eluted with hexane/AcOEt (7:1), to give compound
2
as a colorless oil (5.4 g, 92%).
−1
23
1
IR (KBr): νmax 3031, 2985, 2933, 2858, 1605, 1496, 1454 cm ; [
500 MHz, CDCl3):
J = 12.0 Hz), 4.77 (1H, dd, J = 5.7, 5.2 Hz), 4.59 (1H, d, J = 12.0 Hz), 4.54 (1H, d, J = 11.5 Hz), 4.48 (1H,
α
]D
−
98.3 (c 1.00, MeOH); H-NMR
(
δ 7.40–7.25 (10H, m), 5.88–5.86 (1H, m), 4.94 (1H, dd, J = 5.7, 1.2 Hz), 4.85 (1H, d,
13
d, J = 11.5 Hz), 4.33–4.35 (1H, m), 4.15 (2H, s), 1.46 (3H, s), 1.40 (3H, s); C-NMR (125 MHz, CDCl3):
δ
1
2
44.4, 138.4, 138.0, 128.3, 128.2, 127.9, 127.8, 127.6, 127.6, 127.5, 112.1, 82.3, 79.7, 77.6, 72.8, 71.9, 66.5,
+
+
7.7, 26.7; MS (FAB) m/z 389 [M + Na] ; HRMS (FAB) Calcd. for C H O Na [M + Na] : 389.1729;
23 26 4
found: 389.1731.
3
.3. Synthesis of Compound 3
Compound (6.9 g, 18.9 mmol) was dissolved in dry THF (189 mL), and the solution was cooled
2
in an ice bath. Thexylborane (0.5 M in THF, 120 mL) was added dropwise to the solution, and the
resulting mixture was stirred for 3 h at room temperature. After TLC indicated the consumption of
the starting material, the mixture was cooled in an ice bath and was added to NaBO3
·
4H O (14.6 g,
2
9
5 mmol) and H O (60 mL). The mixture was stirred for 1 h at room temperature, diluted with AcOEt,
2
and washed with H O. The organic layer was further washed with brine, dried over Na SO , and
2
2
4
concentrated in vacuo. The residue was purified by silica gel column chromatography, eluted with
hexane/AcOEt (3:1), to give compound as a colorless oil (6.5 g, 90%). IR (KBr): νmax 3449, 3063, 3031,
3
−
1
25
1
2
7
984, 2928, 2917, 2867, 1497, 1454 cm ; [
.35–7.25 (10H, m), 4.78 (1H, d, J = 12.6 Hz), 4.64 (1H, dd, J = 7.5, 4.6 Hz), 4.46–4.53 (3H, m), 4.44 (1H,
α
]D
−
65.5 (c 1.00, MeOH); H-NMR (500 MHz, CDCl ): δ
3
dd, J = 6.9, 2.3 Hz), 4.26–4.30 (1H, m), 3.98 (1H, dd, J = 5.2, 4.6 Hz), 3.73–3.76 (2H, m), 2.43 (1H, d,
J = 2.3 Hz), 2.24–2.32 (1H, m), 1.43 (3H, s), 1.31 (3H, s); 13C-NMR (125 MHz, CDCl3):
138.4, 138.0,
28.4, 128.2, 127.8, 127.7, 127.6, 127.5, 113.0, 86.1, 80.0, 78.0, 77.7, 73.4, 72.8, 68.4, 50.1, 25.9, 24.5; MS
δ
1
+
+
(FAB) m/z 385 [M + H] ; HRMS (FAB) Calcd. for C H O [M + H] : 385.2015; found: 385.2016.
23 29 5
3
.4. Synthesis of Compound 4
Compound (3.00 g, 7.81 mmol) was dissolved in dry DMF (80 mL), and imidazole (1.60 g,
3.4 mmol) and tert-butyldimethylchlorosilane (2.36 g, 15.6 mmol) were added. The resulting mixture
3
2
was stirred for 1 h at room temperature. After the reaction was finished, the mixture was diluted with
AcOEt and washed with saturated aq. NaHCO . The organic layer was washed with brine, dried over
3
Na SO , and concentrated in vacuo. The residue was purified by silica gel column chromatography,
2
4
eluted with hexane/AcOEt (8:1), to give compound
4 as a colorless oil (2.9 g, 74%). IR (KBr): νmax
−
1
25
3
088, 3064, 3031, 2979, 2951, 2928, 2856, 1633, 1605, 1471, 1462, 1532 cm ; [
α
]D
−37.2 (c 1.00, MeOH);
1
H-NMR (500 MHz, CDCl3):
δ 7.23–7.36 (10H, m), 4.68–4.72 (1H, m), 4.51–4.64 (2H, m), 4.52 (1H,
d, J = 12.1 Hz), 4.44 (1H, d, J = 12.1 Hz), 4.29–4.31 (1H, m), 4.11–4.13 (1H, m), 4.05–4.09 (1H, m), 3.77
(
(
1
(
1H, dd, J = 9.2, 6.3 Hz), 3.62 (1H, dd, J = 9.2, 8.6 Hz), 2.30–2.38 (1H, m), 1.41 (3H, s), 1.28 (3H, s), 0.85
9H, s), 0.08 (3H, s), 0.06 (3H, s); 13C-NMR (125 MHz, CDCl3):
138.7, 138.4, 128.3, 127.9, 127.6, 127.6,
27.4, 112.0, 86.4, 79.5, 77.8, 76.9, 76.6, 73.0, 72.6, 67.2, 50.6, 25.8, 25.7, 25.6, 24.0, 17.9, 4.8, 5.0; MS
FAB) m/z 499 [M + H] ; HRMS (FAB) Calcd. for C H O Si [M + H] : 499.2880; found: 499.2884.
δ
−
−
+
+
29
43
5

Molecules 2020, 25, 1732
9 of 15
3
.5. Synthesis of Compound 5
Compound (2.87 g, 5.75 mmol) was dissolved in EtOH (60 mL), and ammonium formate (1.60 g,
5.4 mmol) and 20% Pd(OH) /C (500 mg) were added. The resulting suspension was refluxed for 3 h.
4
2
2
The reaction mixture was cooled to room temperature and filtrated through a Celite pad. The filtrate
was concentrated in vacuo, and the resulting residue was purified by silica gel column chromatography,
eluted with hexane/AcOEt (1:4), to give compound
5
−
as a colorless oil (1.7 g, 91%). IR (KBr): νmax 3427,
−
1
26
1
2
954, 2932, 2858, 1633, 1605, 1472, 1463 cm ; [
α
]D
44.0 (c 1.00, MeOH); H-NMR (500 MHz, CDCl ):
3
δ
4.56 (1H, dd, J = 7.5, 5.2 Hz), 4.41 (1H, dd, J = 7.5, 3.4 Hz), 4.28 (1H, dd, J = 9.2, 3.5 Hz), 4.18–4.23 (1H,
m), 3.81–3.93 (2H, m), 2.71–2.75 (1H, m), 1.97–2.03 (1H, m), 1.51 (3H, s), 1.34 (3H, s), 0.89 (9H, s), 0.11
(
1
3H, s), 0.10 (3H, s); 13C-NMR (125 MHz, CDCl ):
δ
113.6, 87.1, 78.3, 71.4, 60.3, 53.0, 26.0, 25.7, 24.6,
3
+
+
7.9,
−
4.7,
−
5.2; MS (FAB) m/z 319 [M + H] ; HRMS (FAB) Calcd. for C H O Si [M + H] : 319.1941;
15
31
5
found: 319.1950.
3
.6. Synthesis of Compound 6
Compound (2.90 g, 9.09 mmol) was dissolved in dry CH Cl (90 mL), and 2,6-lutidine (3.2 mL,
7.3 mmol) was added. The mixture was cooled to
5
2
2
◦
2
−
78 C, and then chlorotriethylsilane (1.68 mL,
◦
10.0 mmol) was added dropwise. The resulting mixture was stirred for 1 h at
−
78 C. The reaction
was quenched with an addition of MeOH and diluted with CH Cl . The mixture was partitioned
2
2
between CH Cl and saturated aq. NaHCO . The organic layer was concentrated in vacuo, and the
2
2
3
resulting residue was purified by silica gel column chromatography, eluted with hexane/AcOEt (8:1),
−
1
22
to give compound
6
as a colorless oil (3.39 g, 86%). IR (KBr): νmax 3530, 2954, 2877, 2856 cm ; [
α
]D
1
−
32.6 (c 1.00, MeOH); H-NMR (500 MHz, CDCl ):
δ
4.57 (1H, dd, J = 7.5, 5.2 Hz), 4.36 (1H, dd, J = 6.9,
.3 Hz), 4.22 (1H, dd, J = 8.6, 4.6 Hz), 3.92–4.00 (2H, m), 3.69 (1H, dd, J = 9.8, 5.7 Hz), 2.66–2.68 (1H, m),
.08–2.16 (1H, m), 1.50 (3H, s), 1.32 (3H, s), 0.96 (9H, t, J = 8.0 Hz), 0.88 (9H, s), 0.60 (6H, q, J = 8.0 Hz),
3
2
2
0
.08 (3H, s), 0.05 (3H, s); 13C-NMR (125 MHz, CDCl ):
δ
112.9, 86.9, 79.0, 69.8, 59.7, 54.2, 25.9, 25.7, 24.3,
5.1; MS (FAB) m/z 433 [M + H] ; HRMS (FAB) Calcd. for C H O Si [M + H] :
3
+
+
1
4
7.9, 6.7, 4.3,
33.2806; found: 433.2804.
−
4.7,
−
21
44
5
2
3
.7. Synthesis of Compound 7
Compound
6 (3.20 g, 7.37 mmol) was dissolved in dry pyridine (74 mL), and the solution was
cooled in an ice bath. Trifluoromethanesulfonic anhydride (1.5 mL) in dry CH Cl (20 mL) was added
2
2
◦
dropwise, and the reaction mixture was stirred for 1 h at 0 C. After the addition of water, the resulting
mixture was extracted with CH Cl . The organic layer was washed with brine, dried over Na SO ,
2
2
2
4
and concentrated in vacuo. The crude product was co-evaporated with toluene three times and then
used immediately for the next reaction without further purification. The triflate was dissolved in dry
DMF (74 mL), and sodium azide (1.44 g, 22.1 mmol) was added. The resulting mixture was stirred
for 8 h at room temperature. After the addition of water, the resulting mixture was extracted with
AcOEt. The organic layer was washed with brine, dried over Na SO , and concentrated in vacuo.
2
4
The resulting residue was purified by silica gel column chromatography, eluted with hexane/AcOEt
−
1
(
[
12:1), to give compound
7
as a colorless oil (2.40 g, 71% in 2 steps). IR (KBr): νmax 2879, 2103 cm ;
24
1
α
]D
−
27.1 (c 1.00, MeOH); H-NMR (500 MHz, CDCl ):
δ
4.38 (1H, dd, J = 8.0, 5.2 Hz), 4.27 (1H, dd,
3
J = 7.5, 3.4 Hz), 3.95 (1H, dd, J = 9.2, 4.0 Hz), 3.63 (1H, dd, J = 5.2, 10.3 Hz), 3.61–3.59 (2H, m), 1.87–1.81
1H, m), 1.38 (3H, s), 1.21 (3H, s), 0.89 (9H, t, J = 8.0 Hz), 0.81 (9H, s), 0.53 (6H, q, J = 8.0 Hz), 0.03 (3H,
s), 0.00 (3H, s); 13C-NMR (125 MHz, CDCl3):
112.9, 85.9, 83.1, 75.3, 64.6, 57.7, 53.8, 27.1, 25.7, 25.2,
(
δ
+
1
[
7.9, 6.75, 4.33,
M + Na] : 480.2690; found: 480.2701.
−
4.59, 5.19; MS (FAB) m/z 480 [M + Na] ; HRMS (FAB) Calcd. for C H N O Si Na
−
21 43 3 4 2
+

Molecules 2020, 25, 1732
10 of 15
3
.8. Synthesis of Compound 8
Compound (10.1 g, 22.1 mmol) was dissolved in EtOH (220 mL), and ammonium formate (6.97 g,
11 mmol) and 20% Pd(OH) /C (2.00 g) were added. The suspension was vigorously stirred for 3 h at
7
1
2
room temperature and then filtrated through a Celite pad. The filtrate was concentrated in vacuo, and
the resulting residue was purified by silica gel column chromatography, eluted with CHCl /MeOH
3
−1
(
[
8:2), to give compound
8
as a white solid (6.9 g, 98%). IR (KBr): νmax 3184, 2926, 2858, 1596 cm ;
8.33 (1H, s), 5.80 (2H, brs), 4.41 (1H, dd,
J = 4.6, 7.5 Hz), 4.30 (1H, dd, J = 4.6, 7.5 Hz), 3.88 (1H, dd, J = 5.2, 9.7 Hz), 3.60 (1H, dd, J = 3.5, 11.5 Hz),
23
1
α
]D
−
13.4 (c 1.00, MeOH); H-NMR (500 MHz, DMSO-d ):
δ
6
3
.38 (1H, dd, J = 4.0, 11.5 Hz), 3.09 (1H, dd, J = 4.6, 10.3 Hz), 1.86–1.78 (1H, m), 1.38 (3H, s), 1.21 (3H, s);
1
3
C-NMR (125 MHz, DMSO-d6):
5.0; MS (FAB) m/z 318 [M + H] ; HRMS (FAB) Calcd. for C H NO Si [M + H] : 318.2101; found:
δ
165.7, 112.0, 85.3, 82.7, 75.8, 56.1, 54.3, 53.4, 27.0, 25.7, 25.0, 17.7,
−
4.6,
+
+
−
15
32
4
3
18.2104.
3
.9. Synthesis of Compound 9
An amount of 3-methoxyacryloyl chloride (8.52 g, 70.8 mmol) was added to a suspension of
silver cyanate (10.6 g, 70.8 mmol) in dry benzene (300 mL), and the mixture was refluxed for 30 min
and cooled to room temperature. The resulting supernatant was slowly added over 15 min to a
◦
solution of compound
8
(7.5 g, 23.6 mmol) in dry THF (300 mL) at
−
40 C. The mixture was allowed to
gradually warm to room temperature and was stirred for 16 h. After the solvents were removed in
vacuo, the residue was purified by silica gel column chromatography, eluted with hexane/AcOEt (1:2),
to give a white solid. The given solid was dissolved in EtOH (60 mL) and treated with ammonium
◦
hydride (60 mL, 28% in water) in a sealed tube at 120 C for 4 h. The resulting mixture was cooled
at room temperature. After concentrated in vacuo, the residue was purified by silica gel column
chromatography, eluted with hexane/AcOEt (1:2), to give compound
9
−
as a white solid (4.6 g, 48% in 2
16.2 (c 1.00, MeOH); H-NMR
−1
23
1
steps). IR (KBr): νmax 3156, 2931, 2890, 2858, 1715, 1472 cm ; [
α
]D
(
5
3
500 MHz, CDCl3):
δ
9.60 (1H, brs), 7.32 (1H, d, J = 8.0 Hz), 5.72 (1H, d, J = 8.0 Hz), 4.82 (1H, dd, J = 7.4
.2 Hz), 4.53 (1H, dd, J = 10.3, 4.6 Hz), 4.47 (1H, dd, J = 7.5, 4.1 Hz), 4.05 (1H, dd, J = 8.6, 4.0 Hz),
.57–3.72 (2H, m), 2.64 (1H, brs), 2.34–2.44 (1H, m), 1.51 (3H, s), 1.28 (3H, s), 0.89 (9H, s), 0.14 (3H,
,
13
s), 0.10 (3H, s); C-NMR (125 MHz, CDCl3):
8.8, 52.6, 27.1, 25.7, 25.6, 25.0, 17.9, 4.6, 5.2; MS (FAB) m/z 413 [M + H] ; HRMS (FAB) Calcd. for
C H N O Si [M + H] : 413.2108; found: 413.2102.
δ 163.4, 151.3, 142.9, 113.2, 113.2, 102.8, 85.6, 80.7, 62.9,
+
5
−
−
+
19
33
2
6
3
.10. Synthesis of Compound 10
Compound (4.60 g, 11.2 mmol) was dissolved in dry CH Cl (110 mL), and Et N (4.68 mL,
9
2
2
3
◦
3
3.6 mmol) was added. The mixture was cooled to 0 C, and methanesulfonyl chloride (952
µL,
◦
12.3 mmol) was added dropwise. The resulting mixture was stirred for 1 h at 0 C. The reaction was
quenched with an addition of MeOH and diluted with CH Cl . The mixture was then partitioned
2
2
between CH Cl and saturated aq. NaHCO . The organic layer was concentrated in vacuo and the
2
2
3
resulting residue was purified by silica gel column chromatography, eluted with hexane/AcOEt (1:2),
to give compound 10 as a white solid (4.2 g, 77%). IR (KBr): νmax 3173, 2989, 2931, 2886, 2858, 1714,
−
1
23
1
1
472 cm ; [
α
]D
−
24.1 (c 1.00, MeOH); H-NMR (500 MHz, DMSO-d ): δ 11.34 (1H, brs), 7.81 (1H, d,
6
J = 8.0 Hz), 5.61 (1H, dd, J = 8.0, 2.3 Hz), 4.75 (1H, dd, J = 7.5, 5.2 Hz), 4.49–4.57 (1H, m), 4.47 (1H,
dd, J = 8.1, 5.2 Hz), 4.12–4.22 (2H, m), 3.88 (1H, dd, J = 10.4, 5.8 Hz), 3.12 (3H, s), 2.67–2.79 (1H, m),
.43 (3H, s), 1.22 (3H, s), 0.87 (9H, s), 0.11 (3H, s), 0.08 (3H, s); C-NMR (125 MHz, DMSO-d6):
13
1
δ 163.2,
1
51.1, 112.7, 102.1, 84.1,79.6, 75.6, 66.3, 48.1, 36.2, 27.1, 25.6, 25.1, 17.6, 17.6, 4.6, 5.2; MS (FAB) m/z 491
−
−
+
+
[M + H] ; HRMS (FAB) Calcd. for C H N O Si [M + Na] : 491.1883; found: 491.1881.
20 34 2 8

Molecules 2020, 25, 1732
11 of 15
3
.11. Synthesis of Compound 11
Compound 10 (4.20 g, 8.57 mmol) was dissolved in MeCN (86 mL), and oxalic acid (77 mg,
.86 mmol) and CeCl3 7H O (9.58 g, 25.7 mmol) were added. The resulting mixture was stirred for 3 d
0
·
2
at room temperature and then filtrated through a Celite pad. The filtrate was concentrated in vacuo,
and the resulting residue was purified by silica gel column chromatography, eluted with CHCl /MeOH
3
−
1
(
7:3), to give compound 11 as a white solid (1.9 g, 66%). IR (KBr): νmax 3343, 2931, 1690, 1349, 1173 cm
;
25
1
[
α
]D
−
75.5 (c 1.00, MeOH); H-NMR (500 MHz, DMSO-d ): δ 11.2 (1H, s), 7.60 (1H, d, J = 8.1 Hz),
6
.62 (1H, dd, J = 8.1, 2.3 Hz), 5.34 (1H, brs), 4.93 (1H, brs), 4.47–4.42 (1H, m), 4.26–4.13 (3H, m), 3.72
5
13
(1H, dd, J = 5.2, 2.9 Hz), 3.58 (1H, dd, J = 5.2, 3.5 Hz), 3.12 (3H, s), 2.19–2.11 (1H, m); C-NMR (125
MHz, DMSO-d ):
δ
163.3, 151.4, 143.0, 101.8, 75.5, 74.5, 72.6, 69.8, 62.2, 46.6, 36.4; MS (FAB) m/z 337 [M +
6
+
+
H] ; HRMS (FAB) Calcd. for C H N O BS [M + H] : 337.0706; found: 337.0700.
11
17
2
8
3
.12. Synthesis of Compound 12
Compound 11 (1.90 g, 5.65mmol) was dissolved in 1,4-dioxane (57 mL), and 2N aq. NaOH
12 mL) was added. The resulting mixture was stirred for 2 h at room temperature. The mixture was
concentrated in vacuo, and the resulting residue was purified by silica gel column chromatography,
eluted with CHCl /MeOH (8:2), and by crystallization with AcOEt/MeOH (1:1) to give compound 12
(
3
−1
22
as colorless crystals (980 mg, 72%). IR (KBr): νmax 3387, 1685 cm ; [
α
]D
−
170.2 (c 1.00, MeOH);
1
H-NMR (500 MHz, DMSO-d6):
δ 11.28 (1H, brs), 8.05 (1H, d, J = 13.8 Hz), 5.87 (1H, s), 5.54 (1H, d,
J = 13.0 Hz), 5.02 (1H, d, J = 10.7 Hz), 4.54–4.43 (1H, m), 4.32–4.19 (2H, m), 3.87 (1H, dd, J = 3.8, 12.2 Hz),
3
7
.76 (1H, s), 3.64–3.53 (1H, m), 2.33 (1H, s); 13C-NMR (125 MHz, DMSO-d ):
δ
163.2, 151.9, 143.7, 100.0,
6
+
9.1, 78.2, 75.6, 71.4, 65.5, 45.8; MS (FAB) m/z 241 [M + H] ; HRMS (FAB) Calcd. for C H N O
10
13
2
5
+
[M + H] : 241.0824; found: 241.0826.
3
.13. Synthesis of Compound 13
Silver triflate (241 mg, 0.938 mmol) was added portion wise to a solution of 4,4 -dimethoxytrityl
chloride (337 mg, 0.995 mmol) in dry CH Cl (1.5 mL). After stirring for 2 h at room temperature, the
0
2
2
mixture left to stand for 1 h to precipitate AgCl. The supernatant (375 µL) containing DMTrOTf was
added to a solution of compound 12 (30.0 mg, 0.125 mmol) in dry pyridine (370
µ
L) and dry 2,6-lutidine
◦
(370
µL) at 0 C, and the resulting mixture was stirred for 4 h at room temperature. The reaction
was quenched with additions of saturated aq. NaHCO and saturated aq. CuSO . The mixture was
3
4
then filtrated through a Celite pad, and the filtrate was partitioned between AcOEt and saturated aq.
NaHCO . The organic layer was then concentrated in vacuo, and the resulting residue was purified by
3
silica gel column chromatography, eluted with CHCl /MeOH (9:1), to give compound 13 as a white
3
−1
28
solid (63 mg, 93%). IR (KBr): νmax 3405, 3063, 2941, 2843, 1687, 1507, 1613, 1466 cm ; [
α
]D
−1.97
1
(
c 1.00, MeOH); H-NMR (300 MHz, DMSO-d ):
δ
11.3 (1H, d, J = 1.8 Hz), 7.82 (1H, d, J = 8.3 Hz),
6
5.44–5.48 (2H, dd, J = 9.6, 4.8 Hz), 7.26–7.34 (7H, m), 6.85–6.92 (4H,m), 5.96 (1H, d, J = 3.2 Hz), 5.46
(
1H, dd, J = 3.2, 1.6 Hz), 4.86 (1H, d, J = 4.1Hz), 4.50 (1H,d, J = 4.6 Hz), 4.11 (1H, s), 3.89 (1H, dd,
13
J = 4.6, 2.3 Hz), 3.78 (6H, d, J = 4.1 Hz), 3.73–3.78 (1H, m), 2.69 (1H, s), 2.22 (1H, s); C-NMR (75 MHz,
DMSO-d6): 163.5, 168.6, 152.2, 145.9, 143.8, 136.4, 130.2, 128.3, 128.0, 127.2, 113.7, 101.1, 86.5, 81.8,
8.1, 75.8, 72.5, 63.8, 55.5, 46.1, 31.4; MS (FAB) m/z 543 [M + H] ; HRMS (FAB) Calcd. for C H N O
δ
+
7
[
31
31
2
7
+
M + H] : 543.2131; found: 543.2129.
3
.14. Synthesis of Compound 14
N,N-Diisopropylethylamine (40
µL, 0.231 mmol), 1-methylimidazole (9.2
µL, 0.116 mmol), and
2
-cyanoethyl N,N-diisopropylchlorophosphoramidite (26 µL, 0.116 mmol) were added to a degassed
◦
solution of compound 13 (21 mg, 0.0387 mmol) in dry MeCN (385
µL) at 0 C. The resulting mixture
was stirred for 14 h at room temperature. The reaction was quenched with an addition of saturated aq.
NaHCO . The mixture was concentrated in vacuo, and the resulting residue was purified by silica gel
3

Molecules 2020, 25, 1732
12 of 15
column chromatography, eluted with hexane/AcOEt (7:1), and reprecipitation to give compound 14 as
−1 1
a white solid (16 mg, 56%). IR (KBr): νmax 3469, 3064, 2964, 2925, 1690, 1507, 1615, 1460 cm ; H-NMR
400 MHz, CDCl3): 8.53 (1H, s), 7.28–7.50 (8H, m), 7.17–7.26 (2H, m), 6.76–6.82 (4H, m), 5.46 (1/2H, d,
(
δ
J = 8.2 Hz), 5.40 (1/2H, d, J = 8.2 Hz), 4.88 (1/2H, d, J = 3.7 Hz), 4.80 (1/2H, d, J = 3.7 Hz), 4.59 (1/2H, d,
J = 4.1 Hz), 4.54 (1/2H, d, J = 4.1 Hz), 4.29 (1H, d, J = 8.7 Hz), 3.99–4.00 (1H, m), 3.77 (3H, d, J = 3.2 Hz),
3
.76 (3H, d, J = 2.3 Hz), 3.60–3.68 (2H, m), 3.42–3.50 (2H, m), 3.32 (1/2H, s), 3.08 (1/2H, s), 2.40–2.55 (1H,
m), 2.29–2.30 (1H, m), 1.28–1.29 (2H, m), 1.16 (3H, s), 1.14 (3H, s), 1.04 (3H, d, J = 3.7 Hz), 1.03 (3H, d,
13
J = 3.7 Hz); C-NMR (100 MHz, CDCl3):
δ
158.6, 145.1, 143.2, 136.1, 136.0, 130.1, 130.0, 128.0, 127.9,
1
2
26.9, 117.1, 113.3 100.7, 87.0, 79.3, 72.0, 71.8, 64.9, 58.4, 58.2, 55.2, 46.0, 45.9, 43.3, 43.1, 43.0, 31.9, 31.6,
3
1
9.7, 29.6, 24.6, 24.5, 22.6, 20.0, 19.9, 14.1; P-NMR (162 MHz, CDCl ):
δ 150.3, 149.2; MS (FAB) m/z 743
3
+
+
[M + H] ; HRMS (FAB) Calcd. for C H N O P [M + H] : 743.3210; found: 743.3217.
40 48 4 8
3
.15. Energy Minimized Structure (Spartan 16)
The molecular structure run out multi-step stabilized. At first, stable conformers were calculated by
using conformer distribution (MMFF). The given conformers were further calculated using HF/3-21G
equilibrium geometry) and then B97X-D/6-31G* to eliminate high-energy and duplicate conformers.
Finally, the most stable structures were searched by B97X-V/6-3111+G(2df, 2p) density functional model.
(
ω
ω
3
.16. X-ray Crystal Structure
A suitable crystal of compound 12 was carefully selected under an optical microscope and glued
to thin glass fibers and mounted on the goniometer in a liquid nitrogen flow. X-ray diffraction data
were collected on a Rigaku R-AXIS RAPID diffractometer employing graphite-monochromated CuK
α
radiation. The structure was solved by the direct method with the SIR-88 program [26] and refined
with the SHELXL program [27]. The structural model was drawn with the ORTEP-3 program [28].
Further information on the crystal structure determinations has been deposited with the Cambridge
Crystallographic Data Center (1989958). The data can be obtained free of charge via http://www.ccdc.cam.
ac.uk/conts/retrieving.html (or from the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK; Fax: +44 1223
336033; E-mail: deposit@ccdc.cam.ac.uk).
3
.17. Oligonucleotide Synthesis
Oligonucleotides modified with OxNorNA-U (ON1–ON3) were synthesized by the reverse DNA
synthesis using an nS–8 Oligonucleotide Synthesizer (GeneDesign) and a universal CPG solid support
0.2 mol scale, Glen Research). The coupling time for OxNorNA phosphoramidite was extended to
2.5 min, and 5-[3,5-bis(trifluoromethyl)phenyl]-1H-tetrazole was used as an activator. The detritylation
(
µ
1
time of all reverse phosphoramidites was prolonged to 2 min (from 20 s), and 3 w/v% trichloroacetic
acid/dichloromethane solution was used for the detritylation. The other synthetic procedures involved
the standard phosphoramidite protocols. Cleavage from the solid support and removal of protecting
groups was accomplished by mild conditions (tert-butylamine/water (1:3 v/v), 24 h at room temperature,
◦
and then 12 h at 60 C). The resulting oligonucleotides were briefly purified using reverse-phase HPLC
®
(
(
Waters XTerra MS C18 2.5
TEAA) in water), eluent B (MeCN), gradient: 5–9% or 6–15% of eluent B, flow rate = 5.0 mL/min). The
resulting oligonucleotides were further purified with Sep-Pak Plus C18 cartridges, where the DMTr
µm, 10
×
50 mm column, eluent A (0.1 M triethylammonium acetate
®
group was removed by 2% TFA in water, and by reverse-phase HPLC (Waters XTerra® MS C18 2.5
50 mm column, eluent A (0.1 M triethylammonium acetate (TEAA) in water), eluent B (MeCN),
gradient: 5–9% or 6–15% of eluent B, flow rate = 5.0 mL/min). The purified oligonucleotides were
analyzed using reverse-phase HPLC (Waters XTerra® MS C18 2.5
m, 4.6 50 mm column), and their
µm,
10
×
µ
×
compositions were confirmed by MALDI-TOF MS analysis (matrix: 3-hydroxypicorinic acid, additive:
diammonium hydrogen citrate).

Molecules 2020, 25, 1732
13 of 15
3
.18. UV Melting Experiment
The UV melting experiments were performed on Shimadzu UV-1650B and UV-1800 spectrometers
equipped with a T
m
analysis accessory. Samples containing oligonucleotide (4
µ
M), the target DNA
◦
or RNA (4
µM), and 100 mM NaCl in a 10 mM phosphate buffer (pH 7.2) were annealed at 100 C
◦
and then cooled slowly to room temperature. The melting profile was recorded from 5 to 90 C at a
◦
scan rate of 0.5 C/min with detection at 260 nm. The T
m
value was obtained from the temperature for
half-dissociation of the formed duplexes based on the first derivative of the melting curve. For van’t
Hoff plots, T values were determined at several oligonucleotide concentrations (0.90, 1.48, 2.44, 4.00,
.52, and 13.6
m
◦
◦
◦
∆G
6
µ
M) (see Figures S1 and S2 in Supplementary Material). The values of
∆
H ,
∆
S , and
were calculated according to the equations shown below (R is an ideal gas constant, and C
oligonucleotide concentration):
t
indicates
◦
◦
◦
1
/Tm = (R/∆H )·ln(Ct/4) + ∆S /∆H
(1)
(2)
◦
◦
◦
∆
G = ∆H − T∆S
3
.19. CD Spectral Analysis
CD spectra were measured at 10 C in a quartz cuvette with a 1-cm optical path length by using
a J-720W spectrophotometer (JASCO). The sample solutions (360 L) were prepared by dissolving
oligonucleotide (4 M) and NaCl (100 mM) in the phosphate buffer (10 mM, pH 7.2). The molar
ellipticity was calculated from the equation [ ] = /cl, where is the relative intensity, c is the sample
concentration, and l is the cell path length in centimeters.
◦
µ
µ
θ
θ
θ
3
.20. Enzymatic Stability Analysis
Samples (each sample volume: 130
µ
L) containing MgCl (10 mM), oligonucleotide (2
µM),
2
and snake venom phosphodiesterase (svPDE, 0.133
were incubated at 37 C. Portions of the samples (20
svPDE was immediately deactivated by heating the sample at 90 C for 2.5 min. The percentage of the
µg/mL) in the Tris-HCl buffer (50 mM, pH 8.0)
◦
µL) were taken at respective time points, and
◦
remaining intact oligonucleotides was determined by reverse-phase HPLC (2.5
plotted against their reaction time.
µm, 4.6
×
50 mm) and
4
. Conclusions
We synthesized and evaluated structurally restricted OxNorNA-U-modified oligonucleotides.
Although OxNorNA-U-modified oligonucleotides showed a lower duplex-forming ability as compared
to the natural counterparts, the base discrimination ability of those was similar to that obtained for the
natural oligonucleotides. Since OxNorNA has a rigid structure and exhibits extremely high enzymatic
stability, we expect that OxNorNA could be useful for the point modifications of aptamers.
Supplementary Materials: The following are available online, Figure S1: van’t Hoff plots of the duplexes formed
between oligonucleotides (ON1
the duplexes formed between oligonucleotides (ON1
values of duplexes formed between oligonucleotides (ON1
ssDNA, Table S2: T values of duplexes formed between oligonucleotides (ON1
, ON2, ON4, and ON5) and complementary ssDNA, Figure S2: van’t Hoff plots of
,
ON2
,
ON4, and ON5) and complementary ssRNA, Table S1:
ON2 ON4, and ON5) and complementary
ON2 ON4, and ON5) and
T
m
,
,
m
,
,
complementary ssRNA. Copies of the NMR spectra of all new compounds. Copies of the HPLC and MALDI-TOF
MS charts of the synthesized oligonucleotides.
Author Contributions: K.M., Y.K., T.Y., and S.O. designed the experiments; H.K., S.M., K.M., K.I., and T.O.
conducted the synthesis of OxNorNA-U phosphoramidite and OxNorNA-U-modified oligonucleotides, and the
evaluation of the biophysical properties of OxNorNA-U-modified oligonucleotides; H.A. performed the X-ray
crystal structure analysis; and T.Y. wrote the manuscript. All authors have read and agreed to the published
version of the manuscript.
Funding: This work was supported in part by Japan Agency for Medical Research and Development (AMED)
under grant number JP19am0101084, JP18am0301004, and JP19am0401003.

Molecules 2020, 25, 1732
14 of 15
Conflicts of Interest: The authors declare no conflict of interest.
References
1
.
Papargyri, N.; Pontoppidan, M.; Andersen, M.R.; Koch, T.; Hagedorn, P.H. Chemical diversity of locked
nucleic acid-modified antisense oligonucleotides allows optimization of pharmaceutical properties. Mol. Ther.
Nucleic Acids 2020, 19, 706–717. [CrossRef]
2.
3.
4.
5.
Yamamoto, T.; Nakatani, M.; Narukawa, K.; Obika, S. Antisense drug discovery and development.
Future Med. Chem. 2011, 3, 339–365. [CrossRef]
Veedu, R.N.; Wengel, J. Locked nucleic acids: Promising nucleic acid analogs for therapeutic applications.
Chem. Biodivers. 2010, 7, 536–542. [CrossRef]
Morihiro, K.; Kasahara, Y.; Obika, S. Biological applications of xeno nucleic acids. Mol. BioSyst. 2017, 13,
2
35–245. [CrossRef] [PubMed]
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. [CrossRef] [PubMed]
6
.
.
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. [CrossRef]
0
0
7
Yamaguchi, T.; Horiba, M.; Obika, S. Synthesis and properties of 2 -O,4 -C-spirocyclopropylene bridged
0
0
nucleic acid (scpBNA), an analogue of 2 ,4 -BNA/LNA bearing a cyclopropane ring. Chem. Commun. 2015
,
5
1, 9737–9740. [CrossRef]
8
9
1
1
1
1
.
.
Morihiro, K.; Okamoto, A. A highly constrained nucleic acid analog based on
Nucleos. Nucleot. Nucl. 2020, in press. [CrossRef] [PubMed]
Steffens, R.; Leumann, C.J. Tricyclo-DNA: A phosphodiester-backbone based DNA analog exhibiting strong
complementary base-pairing properties. J. Am. Chem. Soc. 1997, 119, 11548–11549. [CrossRef]
α
-L-threosamine.
0. Steffens, R.; Leumann, C.J. Synthesis and thermodynamic and biophysical properties of tricyclo-DNA. J. Am.
Chem. Soc. 1999, 121, 3249–3255. [CrossRef]
1. Renneberg, D.; Leumann, C.J. Watson-Crick base-pairing properties of tricyclo-DNA. J. Am. Chem. Soc. 2002
24, 5993–6002. [CrossRef] [PubMed]
2. Renneberg, D.; Bouliong, E.; Reber, U.; Schümperli, D.; Leumann, C.J. Antisense properties of tricyclo-DNA.
Nucleic Acids Res. 2002, 30, 2751–2757. [CrossRef]
3. Hanessian, S.; Schroeder, B.R.; Giacometti, R.D.; Merner, B.L.; Østergaard, M.E.; Swayze, E.E.; Seth, P.P.
Structure-based design of a highly constrained nucleic acid analogue: Improved duplex stabilization by
,
1
restricting sugar pucker and torsion angle
PubMed]
4. Sheppard, T.L.; Breslow, R.C. Selective Binding of RNA, but Not DNA, by Complementary 2 ,5 -Linked
γ. Angew. Chem. Int. Ed. 2012, 51, 11242–11245. [CrossRef]
[
0
0
1
1
DNA. J. Am. Chem. Soc. 1996, 118, 9810–9811. [CrossRef]
5. Bhan, P.; Bhan, A.; Hong, M.; Hartwell, J.G.; Saunders, J.M.; Hoke, G.D. 2 ,5 -Linked oligo-3 -deoxyribonucleoside
0
0
0
phosphorothioate chimeras: Thermal stability and antisense inhibition of gene expression. Nucleic Acids Res.
1997, 25, 3310–3317. [CrossRef]
1
1
6. Schöning, K.-U.; Scholz, P.; Guntha, S.; Wu, X.; Krishnamurthy, R.; Eschenmoser, A. Chemical etiology of
0 0
α-threofuranosyl-(3 –>2 ) oligonucleotide system. Science 2000, 290, 1347–1351.
nucleic acid structure: The
CrossRef]
7. Liu, L.S.; Leung, H.M.; Tam, D.Y.; Lo, T.W.; Wong, S.W.; Lo, P.K.
antisense oligonucleotides for suppressing gene expression in living cells. ACS Appl. Mater. Interfaces 2018,
0, 9736–9743. [CrossRef]
[
α
-l-Threose nucleic acids as biocompatible
1
1
8. van Boggelen, M.P.; van Dommelen, B.F.G.A.; Jiang, S.; Singh, G. Methyl iodide mediated cleavage of the
nitrogen-oxygen bond of isoxazolidines. Tetrahedron 1997, 53, 16897–16910. [CrossRef]
1
9. Choi, W.J.; Park, J.G.; Yoo, S.J.; Kim, H.O.; Moon, H.R.; Chun, M.W.; Jung, Y.H.; Jeong, L.S. Syntheses of d-
and l-cyclopentenone derivatives using ring-closing metathesis: Versatile intermediates for the synthesis of
d- and l-carbocyclic nucleosides. J. Org. Chem. 2001, 66, 6490–6494. [CrossRef]

Molecules 2020, 25, 1732
15 of 15
2
0. Comin, M.J.; Vu, B.C.; Boyer, P.L.; Liao, C.; Hughes, S.H.; Marquez, V.E. D-(+)-iso-Methanocarbathymidine:
A high affinity substrate for herpes simplex virus 1 thymidine kinase. ChemMedChem 2008, 3, 1129–1134.
CrossRef]
[
2
1. Umemiya, H.; Kagechika, H.; Hashimoto, Y.; Shudo, K. Synthesis of oligopeptides as polynucleotide analogs.
Nucleoside Nucleotides 1996, 15, 465–475. [CrossRef]
2
2. Rao, J.R.; Jha, A.K.; Rawal, R.K.; Sharon, A.; Day, C.W.; Barnard, D.L.; Smee, D.F.; Chu, C.K. (–)-Carbodine:
Enantiomeric synthesis and in vitro antiviral activity against various strains of influenza virus including
H5N1 (avian influenza) and novel 2009 H1N1 (swine flu). Bioorg. Med. Chem. Lett. 2010, 20, 2601–2604.
[CrossRef] [PubMed]
2
3. Akabane-Nakata, M.; Kumar, P.; Das, R.S.; Erande, N.D.; Matsuda, S.; Egli, M.; Manoharan, M. Synthesis and
0
biophysical characterization of RNAs containing 2 -fluorinated northern methanocarbacyclic nucleotides.
Org. Lett. 2019, 21, 1963–1967. [CrossRef] [PubMed]
2
4. Evéquoz, D.; Leumann, C.J. Probing the backbone topology of DNA: Synthesis and properties of
0
0
7
,5 -bicyclo-DNA. Chem. Eur. J. 2017, 23, 7953–7968. [CrossRef]
0
0
2
5. Obika, S.; Nanbu, D.; Hari, Y.; Morio, K.; In, Y.; Ishida, T.; Imanishi, T. Synthesis of 2 -O,4 -C-methyleneuridine
0
and -cytidine. Novel bicyclic nucleosides having a fixed C3 -endo sugar puckering. Tetrahedron Lett. 1997
8, 8735–8738. [CrossRef]
,
3
2
6. Burla, M.C.; Camalli, M.; Cascarano, G.; Giacovazzo, C.; Polidori, G.; Spagna, R.; Viterbo, D. SIR88—A
direct-methods program for the automatic solution of crystal structures. J. Appl. Cryst. 1989, 22, 389–394.
[CrossRef]
2
2
7. Sheldrick, G.M. A Short History of SHELX. Acta Cryst. 2008, A64, 112–122. [CrossRef] [PubMed]
8. Farrugia, L.J. ORTEP-3 for Windows - a version of ORTEP-III with a Graphical User Interface (GUI).
J. Appl. Cryst. 1997, 30, 565. [CrossRef]
Sample Availability: Not available.
©
2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).