2′,4′-BNA/LNA with 9-(2-Aminoethoxy)-1,3-diaza-2-oxophenoxazine Efficiently Forms Duplexes and Has Enhanced Enzymatic Resistance**

Article abstract of DOI:10.1002/chem.202003982

Artificial nucleic acids are widely used in various technologies, such as nucleic acid therapeutics and DNA nanotechnologies requiring excellent duplex-forming abilities and enhanced nuclease resistance. 2′-O,4′-C-Methylene-bridged nucleic acid/locked nuc

Full text of DOI:10.1002/chem.202003982

Accepted Article
Accepted Article
Title: 2′,4′-BNA/LNA with 9-(2-aminoethoxy)-1,3-diaza-2-
oxophenoxazine efficiently forms duplexes and has enhanced
enzymatic resistance
Authors: Yuki Kishimoto, Osamu Nakagawa, Akane Fujii, Kotaro
Yoshioka, Tetsuya Nagata, Takanori Yokota, Yoshiyuki Hari,
and Satoshi Obika
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.202003982
Link to VoR: https://doi.org/10.1002/chem.202003982
01/2020

10.1002/chem.202003982
Chemistry - A European Journal
FULL PAPER
2′,4′-BNA/LNA with 9-(2-aminoethoxy)-1,3-diaza-2-
oxophenoxazine efficiently forms duplexes and has enhanced
enzymatic resistance
Yuki Kishimoto,[a,b] Osamu Nakagawa,*[a,b,d] Akane Fujii,[a,b] Kotaro Yoshioka,[b,c] Tetsuya Nagata,[b,c]
Takanori Yokota,[b,c] Yoshiyuki Hari,[d] and Satoshi Obika*[a,b]
Abstract: Artificial nucleic acids are widely used in various
technologies such as nucleic acid therapeutics and DNA
nanotechnologies requiring excellent duplex-forming abilities and
enhanced nuclease resistance. We previously reported 2′-O,4′-C-
methylene-bridged nucleic acid (2′,4′-BNA/LNA) with 1,3-diaza-2-
oxophenoxazine (tCO), which was named BNAP (BH). In this study,
acids include nucleobases, sugars, and the phosphate backbone.
Among these, nucleobase modification can dramatically affect
base recognition.[3] The 1,3-diaza-2-oxophenoxazine (known as
tCO or phenoxazine) artificial nucleobase is a tricyclic cytosine
analog that has been extensively studied for its ability to improve
thermal stability in duplex (Figure 1a).[4] 9-(2-Aminoethoxy)-1,3-
diaza-2-oxophenoxazine (G-clamp) has also been developed
(Figure 1b),[5] to improve the - stacking interaction with adjacent
bases and form additional hydrogen bonds with the 6-carbonyl
group of guanine. Therefore, oligodeoxynucleotides (ODNs) that
contain G-clamps could dramatically improve duplex-forming
abilities. Based on the G-clamp design, several artificial
nucleobases with expanded aromatic rings have been developed
for improving - stacking interactions.[6] Recently, we developed
a novel phenoxazine analog (1,3,9-triaza-2-oxophenoxazine [9-
TAP]) with improved triplex-forming abilities[7] and metal-mediated
we designed
a
novel BH analog, 2′,4′-BNA/LNA with 9-(2-
aminoethoxy)-1,3-diaza-2-oxophenoxazine (G-clamp), named BNAP-
AEO (BAEO). The BAEO nucleoside was successfully synthesized and
incorporated into oligodeoxynucleotides (ODNs). ODNs containing
BAEO possessed up to 104-fold, 152-fold, and 11-fold higher binding
affinities for complementary RNA (cRNA) than did ODNs containing
2′-deoxycytidine (C), 2′,4′-BNA/LNA with 5-methylcytosine (L), or 2′-
deoxyribonucleoside with G-clamp (PAEO), respectively. Moreover,
duplexes formed by ODN bearing BAEO with complementary DNA
(cDNA) and cRNA were thermally stable even in molecular crowding
conditions induced by the addition of polyethylene glycol. Furthermore, base pairs[8].
ODN bearing BAEO was more resistant to 3′-exonuclease than ODNs
a)
b)
N
H
with phosphorothioate linkages.
O
H
H
O
N
N
N
O
N
N
N
H
H
N
N
O
O
N
N
Introduction
H
H
N
N
N
N
N
N
H
H
O
O
Artificial nucleic acids are widely used in DNA
nanotechnologies[1] and nucleic acid-based therapies[2]. The high
duplex-forming ability of these artificial nucleic acids improves
H
H
tCO:guanine
G-clamp:guanine
Figure 1. Base pairs of tCO: guanine (a) and G-clamp: guanine (b).
gene expression inhibition-based oligonucleotide (ON)
therapeutics.[2] To maintain their activity within the cell, these ON’s
must be nuclease-resistant.[2] Briefly, the moieties modified for
improving duplex stability and enzymatic resistance of nucleic
We recently developed a 2′-O,4′-C-methylene-bridged nucleic
acid (or locked nucleic acid) (2′,4′-BNA/LNA) with a phenoxazine
(tCO) base (BNAP (BH)) (Figure 2c).[9] 2′,4′-BNA/LNA (Figure 2a),
which has a fixed N-type sugar conformation and improves duplex
stability.[10,11] The original design concept of 2′,4′-BNA/LNA was to
prevent entropy loss by preorganization into an N-type sugar
conformation in A-form DNA duplexes. Another mechanism for
improving duplex stability that improves the - stacking
interaction with adjacent bases has been proposed.[12] We
expected that BH, which combines 2′,4′-BNA/LNA, and a tCO
base, would further improve duplex stability by increasing -
stacking interactions. As expected, BH showed high thermal
stability relative to 2′,4′-BNA/LNA with 5-methylcytosine (L), and
2′-deoxyribonucleoside with tCO (PH).
In this study, we developed a novel artificial nucleic acid that
possesses excellent duplex-forming abilities and enhanced
enzymatic resistance. We believe that such a nucleic acid will be
a promising tool in DNA nanotechnologies and ON therapeutics.
Based on this concept, we designed a 2′,4′-BNA/LNA with a G-
clamp (BNAP-AEO (BAEO); BAEO means BH with 2-aminoethoxy
(AEO) at position 9) (Figure 2e). Duplexes containing BAEO were
very stable.
[a]
[b]
Dr. Y. Kishimoto, Dr. O. Nakagawa*, A. Fujii, Prof. Dr. S. Obika*
Graduate School of Pharmaceutical Sciences
Osaka University
1-6 Yamadaoka Suita, Osaka 565-0871, Japan
E-mail: obika@phs.osaka-u.ac.jp (S.O.)
Dr. Y. Kishimoto, Dr. O. Nakagawa*, A. Fujii, Dr. K. Yoshioka,
Dr. T. Nagata, Prof. Dr. T. Yokota, Prof. Dr. S. Obika*
Core Research for Evolutional Science and Technology (CREST),
Japan Sciences and Technology Agency (JST), 7 Gobancho,
Chiyoda-ku, Tokyo 102-0076, Japan
[c]
[d]
Dr. K. Yoshioka, Dr. T. Nagata, Prof. Dr. T. Yokota
Department of Neurology and Neurological Science, Graduate
School of Medical and Dental Sciences, Tokyo Medical and Dental
University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan
Dr. O. Nakagawa*, Prof. Dr. Y. Hari
Faculty of Pharmaceutical Sciences, Tokushima Bunri University,
180 Nishihamahoji, Yamashiro-cho, Tokushima 770-8514, Japan
E-mail: osamu_nakagawa@ph.bunri-u.ac.jp (O.N.)
Supporting information for this article is given via a link at the end of
the document.
This article is protected by copyright. All rights reserved.

10.1002/chem.202003982
Chemistry - A European Journal
FULL PAPER
Finally, we investigated the detailed properties of ODN-BAEO,
including its enzymatic resistance and fluorescence.
a)
b)
c)
NH2
N
NH
NH
Me
O
O
N
N
ODN
O
ODN
ODN
Results and Discussion
N
O
N
O
N
O
O
O
O
O
O
Synthesis of ODN-containing BAEO
O
O
O
O
P
O
P
BAEO phosphoramidite (9), a precursor for incorporation into
ODNs, was synthesized using 2′,4′-BNA/LNA with 5-bromouracil
(1) as the starting material (Scheme 1, Figure S1). Compound 1
is an intermediate used for the synthesis of BH[9]; the synthesis of
9 could be achieved according to the synthesis of BH[9] and a 2′-
deoxyribonucleoside with a G-clamp (PAEO)[5]. First, 1 was
O
P
O
O
O
O
O
ODN
ODN
ODN
2'-deoxyribonucleotide 2',4'-BNA/LNA with tCO
2',4'-BNA/LNA with
5-methylcytosine ( )
PH
BH
L
with tCO (
)
(BNAP [ ])
NH3
NH3
chlorinated at position
4
by the Appel reaction with
d)
e)
O
O
triphenylphosphine (PPh3) and tetrachlorocarbon (CCl4) in
dichloromethane (DCM), followed by the addition of 2-
aminoresorcinol in the presence of 1,8-diazabicyclo[5.4.0]undec-
7-ene (DBU) at reflux to obtain 2 (73% yield). Then, a
benzyloxycarbonyl (Cbz)-protected aminoethanol unit was
NH
NH
O
O
N
N
ODN
O
ODN
N
O
N
O
O
O
O
introduced to
2 by the Mitsunobu reaction using diethyl
O
O
O
P
azodicarboxylate (DEAD), PPh3 in DCM/tetrahydrofuran (THF) to
obtain 3 (95%). The 3′- and 5′-diacetyl groups of 3 were removed
using an ammonia methanol solution to obtain 4. The
phenoxazine skeleton was constructed from 4 using DBU in
ethanol under reflux (57% in 2 steps). The Cbz group of 2-
aminoethanol at position 9 of 5 was removed to obtain the BAEO
nucleoside (6). In 1H-NMR (DMSO-d6) of 6, two tautomers
between the 10- and 1-positions in 6 were confirmed (ratio: 4/1
[major (NH(10)-C=N(1))/minor (N(10)=C-NH(1))]). Compound 6
O
P
O
O
O
ODN
ODN
2'-deoxyribonucleotide 2',4'-BNA/LNA with G-clamp
PAEO BAEO
with G-clamp (
)
(BNAP-AEO [
])
Figure 2. Structures of artificial nucleic acids used in this study (ODN:
oligodeoxynucleotide).
Scheme 1. Synthesis of ODN containing BAEO
.
Reagents and conditions: (a) PPh3, CCl4, DCM, reflux, 2 h, then 2-aminoresorcinol, DBU, room temperature, 6 h (73%) (b) benzyl-N-(2-hydroxyethyl)carbamate,
DEAD, PPh3, DCM/THF, 0 °C → room temperature, 5 h (95%); (c) 7 M NH3 in MeOH, room temperature, 2 h; (d) DBU, EtOH, reflux, 5 h (2 steps in 57%); (e)
Pd(OH)2/C, cyclohexene, MeOH, reflux 5 h (93%); (f) TFAA, pyridine, 0 °C → room temperature, 2 h; (g) DMTrCl, AgOTf, 2,6-lutidine, DCM, 0 °C → room
temperature, 2 h (2 steps, 47%); (h) i-Pr2NP(Cl)OCH2CH2CN, DIPEA, DCM, room temperature, 2 h (77%); (i) DNA synthesizer.
This article is protected by copyright. All rights reserved.

10.1002/chem.202003982
Chemistry - A European Journal
FULL PAPER
Table 1. Sequences and MALDI-TOF data for ODNs containing BAEO
.
was protected with
a trifluoroacetyl group at the 9-(2-
aminoethoxy) moiety at the phenoxazine base to obtain 7.
Compound 7 was protected with a 4,4′-dimethoxytrityl group to
acquire 8, which was phosphitylated to obtain 9.
MALDI-TOF Mass
Sequences
Calcd. (M-H)–
3794.5
Found
3793.8
3788.4
3763.6
3813.0
3744.2
5′-d(GCGTTBAEOTTTGCT)-3′
5′-d(GCGTCBAEOATTGCT)-3′
5′-d(GCGTCBAEOCTTGCT)-3′
5′-d(GCGTABAEOATTGCT)-3′
5′-d(GCGTGBAEOGTTGCT)-3′
5′-d(GCGTBAEOCBAEOTTGCT)-3′
5′-d(GCGTCBAEOBAEOTTGCT)-3′
5′-d(GCGTBAEOBAEOBAEOTTGCT)-3′
5′-d(TTTTTTTTTBAEO)-3′
Compound 9 was incorporated into an ODN using an automated
DNA/RNA synthesizer, 5-[3,5-Bis(trifluoromethyl)phenyl]-1H-
tetrazole (Activator® 42), as an activator with a prolonged
coupling time of 720 s (natural nucleoside: 30 s). Fast-
deprotected phosphoramidites (N-phenoxyacetyl (Pac)-2'-
deoxyadenosine, N-p-isopropyl-Pac-2'-deoxyguanosine, and N-
acetyl-2'-deoxycytidine) were used as the phosphoramidites of
the natural 2′-deoxynucleosides of A, G, and C. Phosphoramidite
of natural thymidine was used for T. Cleavage from the controlled
pore glass (CPG), and deprotection of the ODN-containing BAEO
were carried out with a 50 mM potassium carbonate (K2CO3)
methanol solution at room temperature for 4 h. We previously
found that the deprotection of ODN bearing BH was effective in
ammonium hydroxide:methylamine (AMA) (v/v, 1:1) at 65 °C for
10 min.[9] However, ODN containing BAEOs decomposed under
such conditions. Furthermore, the ODN bearing BAEOs with
adjacent 5′-and 3′ purine bases (guanine and adenine) of BAEO
tended to show increased decomposition under the AMA
conditions. The ODNs containing BAEOs were purified by high-
performance liquid chromatography (HPLC) using a reverse-
phase octadecylsilyl column (Figure S2). The ODNs containing
3788.5
3764.5
3812.6
3844.6
3941.7
3941.7
4118.8
3141.1
3942.6
3940.4
4117.8
3141.3
Thermal stabilities of duplexes containing BAEO with multiple
adjacent bases
The thermal stabilities of duplexes formed between ODNs
containing BAEO (12-mer) and complementary (c) DNA and RNA
were investigated using ultraviolet (UV) melting experiments
(Table 2, Figure S4). The concept behind the design of BAEO was
to improve its duplex-forming ability by increasing guanine
hydrogen bonding; - stacking interactions with adjacent
nucleobases. Therefore, ODNs containing various 5′- and 3′-
adjacent bases of BAEO were evaluated. Melting temperatures
(Tm) were determined at 260 nm in a 2 mM sodium phosphate
buffer (pH 7.2) containing 20 mM sodium chloride. The
concentration of each ODN was 2 M. ODNs containing 2′,4′-
BNA/LNA with 5-methylcytosine (L), or 2′-deoxyribonucleoside
BAEO
were
characterized
by
matrix-assisted
laser
desorption/ionization time-of-flight (MALDI-TOF) analysis (Table
1 and Figure S3).
with G-clamp (PAEO
) were also investigated as controls.
Table 2. The Tm of duplexes formed by ODNs with complementary (c) DNA and cRNA.
Tm (Tm: ([Tm (L, PAEO or BAEO) -Tm (C)]/modification) (°C)
Target DNA Target RNA
PAEO
ODNs
(5′-NNN-3′)
X:
C
40
44
L
BAEO
53 (+13)
53 (+9)
C
L
PAEO
BAEO
TXT
CXA
44 (+4)
48 (+4)
50 (+10)
55 (+11)
41
46
48 (+7)
53 (+7)
52 (+11)
59 (+13)
57 (+16)
62 (+16)
54
43*
61 (+7)
67 (+13)
56 (+13)*
72 (+18)
61 (+18)*
CXC
45
51 (+6)
61 (+16)
64 (+19)
AXA
41
46
45 (+4)
48 (+2)
43 (+2)
54 (+8)
42 (+1)
51 (+5)
38
46
44 (+6)
51 (+5)
45 (+7)
48 (+2)
47 (+9)
53 (+7)
GXG
54
43*
67 (+6.5)
74 (+10.0)
62 (+9.5)*
n.d.
73 (+15.0)*
XCX
45
55 (+5.0)
69 (+12.0)
75 (+15.0)
54
43*
65 (+5.5)
71 (+5.7)
76 (+11.0)
66 (+11.5)*
80 (+13.0)
68 (+12.5)*
CXX
45
45
53 (+4.0)
58 (+4.3)
65 (+10.0)
77 (+10.7)
63 (+9.0)
70 (+8.3)
54
43*
n.d.
74 (+10.3)*
n.d.
76 (+11.0)*
XXX
UV melting profiles were measured in 2 mM sodium phosphate buffer (pH 7.2) containing 20 mM NaCl at a scan rate of 0.5 °C/min at 260 nm. The concentration
of oligonucleotide was 2 μM for each strand. The error in Tm was ± 0.5 °C. *: These were measured under non-NaCl conditions.
C: 2′-deoxycytidine, L: 2′,4′-BNA/LNA with 5-methylcytosine, PAEO: 2′-deoxyribonucleoside with G-clamp, BAEO: BNAP-AEO.
Sequence: 5′-d(GCGTNNNTTGCT)-3′/3′-CGCAQQQAACGA-5′ (X (in N): C, L, PAEO or BAEO, Q: Corresponding matching base).
This article is protected by copyright. All rights reserved.

10.1002/chem.202003982
Chemistry - A European Journal
FULL PAPER
Thereafter, ODN-X and duplex-X indicate ODN and duplex
ODN-L/cRNA, and ODN-PAEO/cRNA, even with adjacent purine
bases of BAEO. ODN-BAEO/cRNA with 5′-adjacent pyrimidines
possessed an unusually high thermal stability (Tm: +16∼+18 °C).
In the case of two, one interval modifications (XCX) and two or
three continuous modifications (CXX) (XXX), ODN-BAEO/cRNA
maintained the high duplex-forming ability that was observed with
one modification (Tm/modification: +11∼+15 °C). For the
extremely high thermal stabilities observed for multiple BAEO
modifications, Tm was redetermined under non-NaCl conditions.
In the case of continuous modifications, ODN-BAEO/cRNA
containing X, respectively, where X represents C, PAEO, or BAEO
.
To depict the 5′- and 3′-adjacent bases of X in ODN-X, the
notation ODN(NXN) is used (N is one of the four natural
nucleobases, adenine (A), guanine (G), cytosine (C), or thymine
(T) as 5′- and 3′-adjacent bases of X). For example, ODN(TBAEOT)
means ODN containing 5′- and 3′-adjacent TT bases of BAEO
.
ODN(NXN)/cDNA and ODN/(NXN)/cRNA indicate the duplex
formed with complementary DNA and RNA, respectively. Tm is
the difference from Tm of X to Tm of C unless otherwise noted.
First, the thermal stabilities of ODN(TXT)/cDNA were measured. demonstrated an advantage over ODN-BAEO/cDNA. Moreover,
The Tm of ODN(TBAEOT)/cDNA was 53 °C, 13 °C higher than that
of ODN(TCT)/cDNA. Furthermore, the Tm of ODN(TBAEOT)/cDNA
was 9 °C and 3 °C higher than those of ODN(TLT) and
ODN(TPAEOT), respectively. For ODN(TXT)/cDNA, the Tms were
even in the case of adjacent purines, ODN-BAEO/cRNA performed
better than ODN-BAEO/cDNA.
According to Ortega’s report, we also analyzed ODN-BAEO
s
using their sequence and buffer (Table S7) [5b]. In this condition,
in decreasing order: BAEO > PAEO >> L > C. For adjacent CC bases, the Tm of ODN(CBAEOC)/cDNA and Tm of ODN(CPAEOC)/cDNA
the Tm of ODN(CBAEOC)/cDNA was 64 °C. The Tm of
ODN(CBAEOC)/cDNA was 19 °C, extremely higher than that of
ODN(CCC)/cDNA. The Tm of ODN(CBAEOC)/cDNA was 6 °C
higher than that of ODN(TBAEOT)/cDNA. The thermal stability of
ODN(CXC)/cDNA decreased in the order: BAEO > PAEO >> L > C.
The thermal stability of ODN-BAEO/cDNA was effectively improved
when the 5′ and 3′-adjacent base of BAEO was CC rather than TT
(Tm: +19 °C (CBAEOC), +13 °C (TBAEOT)). In contrast, the Tm
ODN(CBAEOA)/cDNA was decreased 10 °C compared with that of
were +22 °C and +17 °C, respectively. Furthermore, the Tm of
ODN(CBAEOC)/cRNA and ODN(CPAEOC)/cRNA was +20 °C and
+15 °C. The high thermal stability of duplex-BAEO was maintained
under these conditions.
Thus, duplex-BAEO showed excellent thermal stability in
comparison to duplexes-C, L, and PAEO. We previously reported
the thermal stabilities of duplexes-PH and BH in the same
sequence and conditions (Table S2).[9] When with 5′-pyrimidine
adjacent bases of BH in duplex, duplex-BH, as well as duplex-BAEO,
showed excellent thermal stability. In summary, the order of
thermal stabilizes of duplex with 5′-pyrimidine adjacent bases was
BAEO > PAEO > BH > L > C.
ODN(CBAEOC)/cDNA and was
ODN(CPAEOA)/cDNA. For adjacent AA bases of BAEO
2 °C lower than that of
,
ODN(ABAEOA)/cDNA did not improve the Tm, which was lower
than those of ODN(ALA)/cDNA and ODN(APAEOA)/cDNA (L >
PAEO > BAEO > C). For adjacent GG BAEO bases, the Tm of
ODN(GBAEOG)/cDNA was 51 °C, which was 5 °C and 3 °C higher
than those of ODN(GCG)/cDNA and ODN(GLG)/cDNA,
respectively. However, the Tm of ODN(GBAEOG)/cDNA was not
improved over that of ODN(GPAEOG) (Tm (BAEO-PAEO): -3 °C).
Thus, BAEO could obtain excellent performance with pyrimidine
(especially C rather than T) bases at both 5′ and 3′-adjacent bases.
In our previous study on BH, the thermal stability of ODN/DNA
duplexes containing PH and BH also improved with adjacent
pyrimidines (C and T) rather than purines (A and G) (Table S2).[9]
Mismatch discrimination ability of BAEO in duplexes
We investigated the mismatch discrimination ability of BAEO in
ODN/DNA and ODN/RNA (Table 3, Figure S6). ODN(CXC) with
5′,3′-adjacent CC bases were used because the sequence
showed high thermal stabilities in the above experiment.
BAEO showed excellent base discrimination (Tm(BAEO): -26 °C
(A-G), -29 °C (T-G), -36 °C (C-G)) in ODN/DNA. The mismatched
Tm of ODN-BAEO/DNA was +6 °C (A), +6 °C (T), and +4 °C (C)
higher than those of ODN-C/DNA. When comparing BAEO and L,
the matched Tm of ODN-BAEO/DNA was high (+13 °C); in contrast,
the mismatched Tm of ODN-BAEO/DNA were similar to those of
ODN-L/DNA (Tm: +1 °C (A), +2 °C (T), +1 °C (C)). The
mismatched Tm of ODN-BAEO/DNA was nearly identical to that of
ODN-PAEO/DNA. Thus, ODN-BAEO/DNA demonstrated high
thermal stability in cases of matched G, but dramatically reduced
thermal stability in the presence of mismatched bases.
For ODN/RNA, BAEO also showed excellent base discrimination
(Tm(BAEO): -25 °C (A-G), -27 °C (U-G), -30 °C (C-G)). However,
unlike in ODN-BAEO/DNA, the Tm of ODN-BAEO/RNA compared
with ODN-C/RNA increased with mismatched bases (Tm: +8 °C
(A), +14 °C (U), +10 °C (C) in RNA). In contrast, the base
discrimination of BAEO was slightly decreased compared with that
of L (Tm(BAEO-L): +2 °C (A), +5 °C (U), +4 °C (C) in RNA).
We previously reported the base discrimination of BH in duplex
(Table S3).[9] The base discrimination ability of BH was lower than
that of BAEO. With mismatched A and T (or U), the Tm of duplex-
BH was higher than that of duplex-BAEO (Tm(BAEO-BH): -1 °C (A),
-4 °C (T), +2 °C (C) with DNA), suggesting that the tCO base
without a 9-AEO unit had low discrimination abilities. The 9-AEO
We investigated ODN-containing two or three continuous
modifications (X) (ODN(CXX) ODN(XXX)), which were expected
to improve - stacking interaction between BAEOs. With such
modifications,
ODN(CBAEOBAEO)/cDNA
and
ODN(BAEOBAEOBAEO)/cDNA increased the Tm, as compared with
ODN(CLL)/cDNA and ODN(LLL)/cDNA, respectively. However,
ODN(CBAEOBAEO)/cDNA and ODN(BAEOBAEOBAEO)/cDNA had a
slightly lower Tms than ODN(CPAEOPAEO)/cDNA and
ODN(PAEOPAEOPAEO)/cDNA. The Tm of ODN-BAEO/cDNA was not
improved by the continuous modifications when compared with
the single modifications such as ODN(TBAEOT)/cDNA and
ODN(CBAEOC)/cDNA. In contrast, ODN(BAEOCBAEO)/cDNA
containing two, one interval modifications of BAEO maintained the
high Tm of the single modifications ODN(TBAEOT)/cDNA and
ODN(CBAEOC)/cDNA.
The thermal stabilities of ODN-BAEOs and cRNA were also
measured. Overall, ODN-BAEO/cRNA had higher thermal stability
than ODN-BAEO/cDNA ((Tm: +7∼+18 °C (cRNA)). The ODN-
BAEO/cRNA showed excellent thermal stability in all ODN-C/cRNA,
This article is protected by copyright. All rights reserved.

10.1002/chem.202003982
Chemistry - A European Journal
FULL PAPER
group may contribute significantly to the high base discrimination
ability of tCO analogs. Thus, BAEO possesses excellent base
discrimination abilities in both ODN/DNA and ODN/RNA.
(a)
80
60
40
Table 3. Comparison of matched versus mismatched hybridization
5′-d(GCGTCXCTTGCT)-3′
3′-d(CGCAGYGAACGA)-5′
20
0
Tm (Tm: Tm[mismatch]-Tm[match]) (°C)
X
Y:
G
A
T
C
-20
-40
-60
C
L
PAEO
BAEO
45
51
61
64
32 (−13)
37 (−14)
38 (−23)
38 (−26)
29 (−16)
33 (−18)
34 (−27)
35 (−29)
24 (−21)
27 (−24)
24 (−37)
28 (−36)
220
240
260
280
300
320
Wavelength (nm)
5′-d(GCGTCXCTTGCT)-3′
3′-r(CGCAGYGAACGA)-5′
(b)
Tm (Tm: Tm[mismatch]-Tm[match]) (°C)
X
60
50
40
30
20
10
0
Y:
G
A
U
C
C
L
PAEO
BAEO
54
61
67
72
39 (−15)
45 (−16)
44 (−23)
47 (−25)
31 (−23)
40 (−21)
41 (−26)
45 (−27)
32 (−22)
38 (−23)
38 (−29)
42 (−30)
Values in parentheses are the difference between the Tm when the ODN was
bound with the guanine-containing target and the Tm when the ODN was bound
with a mismatched base. The buffer conditions were the same as given in Table
2. C: 2′-deoxycytidine, L: 2′,4′-BNA/LNA with 5-methylcytosine, PAEO: 2′-
deoxyribonucleoside with G-clamp, BAEO: BNAP-AEO.
-10
-20
-30
Circular dichroism (CD) analysis of duplexes containing BAEO
220
240
260
280
300
320
The structure of duplex-BAEO was investigated using CD (Figure
3). Measurements were performed using the buffer conditions
used for UV melting experiments.
The formation of the ODN(CBAEOC)/cDNA duplex did not
induce large conformational changes when compared with
ODN(CCC)/cDNA. The ODN(CBAEOC)/cDNA maintained a typical
B-form DNA/DNA structure. Furthermore, ODN(CBAEOC)/cRNA,
like ODN(CCC)/cRNA, maintained an A-form duplex. Thus, the
duplexes formed between adjacent CC bases of BAEO did not
undergo large conformational changes.
Wavelength (nm)
Figure 3. CD spectra of ODN(CXC)/cDNA (panel a) and
ODN(CXC)/cRNA (panel b) duplexes.
Conditions: 2 mM sodium phosphate buffer (pH 7.2), 20 mM NaCl, 2 μM
each strand. C: 2′-deoxycytidine (black), L: 2′,4′-BNA/LNA with 5-
methylcytosine (orange), PAEO: 2′-deoxyribonucleoside with G-clamp
(blue), BAEO: BNAP-AEO (red).
Sequence: 5′-d(GCGTCXCTTGCT)-3′/3′-CGCAGGGAACGA-5′ (X: C,
L, PAEO or BAEO).
We also investigated the duplex structures of ODN(ABAEOA)
using cDNA and cRNA (Figure S7). The structure of
ODN(ABAEOA)/cDNA was slightly different from the typical B-form
structure observed with ODN(ACA)/cDNA. Furthermore,
ODN(ABAEOA)/cRNA possessed small conformational changes in
structure that were not present in the A-form of ODN(ACA)/cRNA.
Thus, the observation that duplexes-BAEO with adjacent CC
results in non-conformational changes reveals that the presence
of BAEO in duplexes may contribute to the maintenance of B- and
A-form structures with high thermal stability. On the contrary,
Molecular dynamics analyses of duplexes containing BAEO
We investigated the structure of duplex-BAEO by molecular
dynamics (MD) calculations using 12-mers of ODN-
(CBAEOC)/cDNA and ODN-(CBAEOC)/cRNA (Figures 4 and S8).
The structure of ODN(CBAEOC)/cDNA was maintained in typical
B-form duplexes without distortion around BAEO (Figure 4a). The
G-clamp base moiety in BAEO was effectively overlapped by the
5'-adjacent cytosine base (Figure 4b). In contrast, the G-clamp
base in BAEO was not sufficiently overlapped by the 3′-adjacent
cytosine. The A-form duplex was maintained with
ODN(CBAEOC)/cRNA without disturbing the structure around BAEO
(Figure S8). The overlap between the BAEO base and the 5’-
adjacent C base tended to be larger in DNA/DNA than in
DNA/RNA. In other words, the overlap between bases was more
apparent in B-form than in A-form duplexes.[9] Therefore, the
adjacent base independence of BAEO might be smaller in B-form
than in A-form duplexes. These simulation MD results were in
good agreement with the experimental CD measurements.
duplexes-BAEO with adjacent AA bases showed
a small
conformational change; therefore, the thermal stabilities of
duplex-BAEO with adjacent AA might be decreased compared with
those of duplex-BAEO with adjacent CC.
This article is protected by copyright. All rights reserved.

10.1002/chem.202003982
Chemistry - A European Journal
FULL PAPER
mol−1 K−1). BAEO and PAEO had similar patterns in ODN/RNA;
(a)
(b)
therefore, G-clamp was expected to predominantly influence
rather than 2′,4′-BNA/LNA in BAEO
.
By comparing L and PH in previous thermodynamic analyses of
BH, we determined that the tCO moiety of BH primarily functions
in DNA/DNA, while 2′,4′-BNA/LNA primarily functions in
DNA/RNA (Table S4).[9] Therefore, the mechanisms by which BH
and BAEO confer duplex stability are different.
Next, the free energy changes of BAEO duplexes were
calculated (G(X-C, cDNA): -3.8 (BAEO), -3.2 (PAEO), -1.1
kcal/mol (L)) (G(X-C, cRNA): -5.7 (BAEO), -4.2 (PAEO), -2.6
kcal/mol (L)). The BAEO in ODN/RNA was stable relative to that in
ODN/DNA.
Figure 4. Snapshot of molecular dynamics (MD) analysis of
ODN(CBAEOC)/cDNA duplex.
The binding constants (Ks) of ODN-BAEO with cDNA and cRNA
were calculated using the above thermodynamic parameters. The
Ks of ODN-BAEO with cDNA was 2.69 x 1010 M-1. This value was
~480 times higher than that of ODN-C. In contrast, the Ks of ODN-
L was only six-fold higher than that of ODN-C. As a corollary, the
Ks of ODN-BAEO was 80-fold higher than that of ODN-L.
Furthermore, the Ks of ODN-BAEO was shown to be only 2.6-fold
Side view (left), expanded top view of part of CBAEOC (right). MD
calculation performed with MacroModel software [Schrçdinger, LLC],
OPLS3 force field (in water at 300 K for 20 ns). Purple: 5′-adjacent cytidine
to BAEO, Green: BAEO
.
Sequence: 5′-d(GCGTCBAEOCTTGCT)-3′/3′-d(CGCAGGGAACGA)-5′
higher than that of ODN-PAEO
.
Thermodynamic parameters of duplexes containing BAEO
With respect to cRNA, ODN-BAEO exhibited an extremely high
binding affinity (Ks = 2.88 x 1013 M-1), approximately 104-fold
higher than that of ODN-C. The binding affinities of ODN-L and
ODN-PAEO were 68 and 913 times higher than that of ODN-C. This
suggests that ODN-BAEO had a Ks 152- and 11-fold higher than
those of ODN-L and ODN-PAEO, respectively.
To verify the molecular mechanism underlying the improved
stability of duplex-BAEO
, thermodynamics parameters were
obtained by van’t Hoff plots based on the Tm results obtained at
multiple concentrations (Table 4, Table S4, Figure S9).
For adjacent CC bases, ODN-BAEO/cDNA showed a favorable
entropy change (H: +14.1 kcal mol−1, S: +57 cal mol−1 K−1);
H, S, or G indicate differences from the corresponding H,
S, or G values obtained for ODN-C/cDNA or cRNA. ODN-
PAEO/cDNA and ODN-L/cDNA also showed favorable changes in
entropy. Therefore, the modifications of L, PAEO, and BAEO into
ODN/DNA might use the same mechanisms for stabilizing
duplexes. Thus, both the G-clamp and 2′,4′-BNA/LNA moiety in
BAEO were associated with duplex stability.
We also calculated the Ks of ODN-BH with cDNA and cRNA
(Table S4). ODN-BAEOs had significantly higher binding affinities
than ODN-BH. Thus, in terms of Ks, the stability conferred by these
modifications was in decreasing order: BAEO > PAEO > BH > PH > L
> C. These results clearly show that the introduction of BAEO into
the duplex dramatically improved the high duplex-forming ability
in ODN/DNA and ODN/RNA.
ODN-BAEO/cRNA had a favorable change in enthalpy that was
different from that of ODN-BAEO/cDNA (H: -5.6 kcal mol−1, S:
+1 cal mol−1 K−1), ODN-PAEO/cRNA also showed this pattern
(H: -4.0 kcal mol−1, S: +1 cal mol−1 K−1), whereas ODN-
L/cRNA had a substantial favorable change in enthalpy and an
unfavorable change in entropy (H: -7.2 kcal mol−1, S: -15 cal
Thermal stabilities of duplexes containing BAEO in molecular
crowding conditions
To evaluate the thermal stabilities of duplexes-BAEO under
molecular crowding conditions,[13] UV melting experiments were
performed in a buffer with or without 40 wt% PEG-200 (Table 5).
Table 4. Thermodynamic properties of duplexes with cDNA and cRNA.
Target DNA
Target RNA
X =
S
(cal/mol・K)
Ks
S
(cal/mol・K)
Ks
H
(kcal/mol)
G(37°C)
(kcal/mol)
H
(kcal/mol)
G(37°C)
(kcal/mol)
(M-1)
(M-1)
C
−93.3
−78.2
−79.8
−79.2
−265
−213
−211
−208
−11.0
−12.1
−14.2
−14.8
5.65 x 107
3.37 x 108
1.02 x 1010
2.69 x 1010
−97.4
−104.6
−101.4
−103.0
−271
−286
−270
−270
−13.4
−16.0
−17.6
−19.1
2.77 x 109
1.89 x 1011
2.53 x 1012
2.88 x 1013
L
PAEO
BAEO
C: 2′-deoxycytidine, L: 2′,4′-BNA/LNA with 5-methylcytosine, PAEO: 2′-deoxyribonucleoside with G-clamp, BAEO: BNAP-AEO.
Sequence: 5′-d(GCGTCXCTTGCT)-3′/3′-CGCAGCGAACGA-5′ (X: C, L, PAEO or BAEO).
This article is protected by copyright. All rights reserved.

10.1002/chem.202003982
Chemistry - A European Journal
FULL PAPER
Table 6. Tm values of BAEO modified duplexes with 5′-and 3′-
adjacent mismatch base pairs.
Table 5. The Tm of duplexes formed by ODNs with complementary
(c) DNA and cRNA in molecular crowding (PEG) conditions.
Tm (Tm: (Tm (L, PAEO or BAEO) -Tm (C))) (°C)
X
Target DNA
PEG (-) PEG (+)
Target RNA
PEG (-) PEG (+)
C
L
55
43
61
49
Tm (ΔTm = Tm [mismatch] - Tm [match]) (oC)
61 (+6)
70 (+15)
73 (+18)
50 (+7)
54 (+11)
59 (+16)
70 (+9)
75 (+14)
80 (+19)
58 (+9)
58 (+9)
66 (+17)
Sequence
PAEO
BAEO
PAEO
BAEO
5’-CXC-3’
X = C
L
3’-YGY-5’
CXC
UV
melting
profiles
were
measured
in
10
mM
45
51
61
64
tris(hydroxymethyl)aminomethane (Tris)-HCl buffer (pH 7.2) containing 40%
polyethylene glycol (PEG)-200 and 100 mM KCl at a scan rate of 0.5 °C/min
at 260 nm. The concentration of oligonucleotide was 2 M for each strand.
The error in Tm values was ±0.5 °C. C: 2′-deoxycytidine, L: 2′,4′-BNA/LNA
GGG
CXC
AGG
CXC
TGG
CXC
CGG
29 (-16)
21 (-24)
20 (-25)
31 (-14)
32 (-13)
29 (-16)
31 (-20)
25 (-26)
17 (-34)
35 (-16)
34 (-17)
34 (-17)
39 (-22)
27 (-34)
24 (-37)
39 (-22)
37 (-24)
37 (-24)
40 (-24)
29 (-35)
25 (-39)
42 (-22)
39 (-25)
39 (-25)
with 5-methylcytosine, PAEO
:
2′-deoxyribonucleoside with 9-AEO-
phenoxazine, BAEO: BNAP-AEO. Sequence: 5′-d(GCGTCXCTTGCT)-3′/3′-
CGCAGGGAACGA-5′ (X: C, L, PAEO or BAEO).
In this section, Tm represents the difference between Tm of ODN-
X/cDNA (or cRNA) and that of ODN-C/cDNA(or cRNA) under the
same buffer conditions. In other words, this Tm refers to the
CXC
GGA
CXC
GGT
CXC
GGC
thermal stabilization of each modification, L, PAEO or BAEO
,
compared with C. First, it was found that the thermal stabilization
of ODN-L/cDNA did not change in the absence or presence of
PEG (Tm: +6 °C and +7°C, respectively); the same was observed
for ODN-L/cRNA (Tm: +9 °C and +9 °C). ODN-PAEO showed high
thermal stabilization in the PEG(-) condition (Tm: +15 °C (cDNA)
and +14°C (cRNA)), which was greater than that of ODN-L.
Notably, the Tms in the PEG(+) condition (+11 °C (cDNA) and
+9 °C (cRNA)) were 4 and 5 °C lower than the Tms in the PEG(-)
condition. On the other hand, ODN-BAEO showed even better
thermal stabilization: the Tms in the PEG(-) condition were
+18 °C (cDNA) and +19 °C (cRNA); in the PEG(+) condition, the
Tms were +16 °C (cDNA) and +17 °C (cRNA). Thus, the
excellent thermal stabilization of ODN-BAEO was maintained even
under the molecular crowding conditions.
The buffer conditions were the same as given in Table 2. C: 2′-
deoxycytidine, L: 2′,4′-BNA/LNA with 5-methylcytosine, PAEO
deoxyribonucleoside with G-clamp, BAEO: BNAP-AEO.
: 2′-
case of 5′-mismatched base pairs (C:A, C:T, and C:C), the Tm was
effectively decreased (Tm(mismatch-match): -24 °C (C:A), -
35 °C (C:T), -39 °C (C:C)). Mismatched 5′-adjacent base pairs of
BAEO:G largely decreased Tm compared with those of C:G. For
mismatched 3′-adjacent base pairs of X:G, ODN-BAEO/DNA
decreased Tm (Tm(mismatch-match): -22 °C (C:A), -25 °C (C:T),
-25 °C (C:C)) (for BAEO). Thermal stabilities of ODN-BAEO/DNA
were effectively decreased with 5′-mismatch base pairs rather
than 3′- mismatch base pairs (Tms: -24∼-39 °C (5′-mismatch), -
22∼-25 °C (3′-mismatch)). G-clamp base in BAEO can overlap
more effectively with 5′-adjacent matched base pairs than 3′-
adjacent matched base pairs (Figure 4). It was expected that G-
clamp base in BAEO was effectively inhibited overlap for -
stacking interaction with 5′-unstabled mismatched base pairs.
Thus, the - stacking ability of BAEO in duplex is destabilized,
even when adjacent base pairs are mismatched. This property is
likely to be useful for technologies that require high sequence
discrimination.
This suggests that artificial nucleobases, which have increased
- stacking interactions, have disadvantages when it comes to
changes in surrounding conditions. The stabilization effects of
duplex-PAEO and duplex-BAEO were different even in between ours
and Ortega’s[5b] buffer conditions (Table S7). In duplexes-BAEO
,
the - stacking function of G-clamp bases could be successfully
overcome by the addition of 2′,4′-BNA/LNA. As such, we expect
ODN-BAEO to be useful in cellular environments, such as for ON-
based therapies.
Thermal stabilities of BAEO modified duplexes with
mismatched adjacent base pairs
We measured how the mismatched 5′- and 3′-adjacent base
pairs of BAEO:G in ODN/DNA affect thermal stability (Table 6 and
Table S5). BAEO was designed to enhance duplex-forming ability
through increased - stacking interactions with adjacent bases
in duplexes. Therefore, we hypothesized that adjacent,
mismatched base pairs of BAEO:G in duplex would decrease -
stacking interactions and, therefore, reduce duplex stability. In the
Fluorescence measurements of ODN containing BAEO
tCO and G-clamp bases are known to fluoresce at 450 nm with
360 nm excitation.[14] We investigated the fluorescence properties
of ODN-BAEO with/without targeted DNA and RNA (Figure 5). The
fluorescence intensity change of BAEO by base discrimination was
This article is protected by copyright. All rights reserved.

10.1002/chem.202003982
Chemistry - A European Journal
FULL PAPER
(a)
4000
(b)
4000
(c)
4000
(d)
4000
3500
3000
2500
2000
1500
1000
500
3500
3000
2500
2000
1500
1000
500
3500
3000
2500
2000
1500
1000
500
3500
3000
2500
2000
1500
1000
500
0
0
0
0
380 430 480 530 580
Wavelength (nm)
380 430 480 530 580
Wavelength (nm)
380 430 480 530 580
Wavelength (nm)
380 430 480 530 580
Wavelength (nm)
Figure 5. Fluorescence spectra of ODN-BAEO/DNA (a), ODN-PAEO/DNA (b), ODN-BAEO/RNA (c) and ODN-PAEO/RNA (d) duplexes.
Conditions: 2 mM sodium phosphate buffer (pH 7.2), 20 mM NaCl, 2 μM each strand.
Sequence: 5′-d(GCGTCXCTTGCT)-3′/3′-CGCAGYGAACGA-5′ (X: PAEO or BAEO). PAEO: 2′-deoxyribonucleoside with G-clamp, BAEO: BNAP-AEO.
Black: single strand ODN. X:Y = X:A (dark blue), X:G (orange), X:C (green), X:T (or X:U) (red).
also measured. Single-stranded ODN-BAEO possessed a similar
fluorescence pattern to ODN-PAEO (maximum emission: 450 nm,
excitation: 360 nm), although the fluorescence intensity of ODN-
BAEO was slightly smaller than that of ODN-PAEO. The fluorescent
base discrimination of BAEO in duplexes was measured with
matched G and mismatched A, T, and C. The fluorescence
intensities of BAEO were, in the following descending order,
Enzymatic resistance of ODN containing BAEO
We investigated the exonuclease resistance of BAEO in ODN.
3′-exonuclease (Crotalus Admanteus venom phosphodiesterase)
was used against a 10-mer ODN (5′-TTTTTTTTTBAEO-3′) (Figure
6). ODNs containing L, PAEO, and two phosphorothioates (PS)
linkages (Sp and Rp isomers) were used as controls. About 80%
of the full-length ODN-BAEO remained intact under these harsh
conditions, in contrast to natural ODN-C, which was completely
degraded within 10 min. In these conditions, the full-length ODN-
PS(Rp), ODN-L, and ODN-PS(Sp) were maintained at 5%, 20%
and 52%, respectively. We confirmed that ODN-PAEO is known to
have resistance against 3′-exonuclease.[15] The resistance of
ODN-BAEO was similar to that of ODN-PAEO. This result suggests
that the high enzymatic resistance of BAEO was conferred by the
G-clamp base moiety rather than the 2′,4′-BNA/LNA sugar moiety.
Thus, the high enzymatic stability of ODN-BAEO may also be
useful in cellular applications such as ON-based therapies.
duplex(BAEO:T) > duplex(BAEO:C) > single strand ODN-BAEO
>
duplex(BAEO:G) > duplex(BAEO:A). This result was similar to that
obtained for PAEO, except that the intensity of duplex(BAEO:G) was
slightly higher than that of duplex(PAEO:G).
The fluorescence properties of BAEO in ODN/RNA were also
evaluated. The fluorescence intensity pattern of ODN-BAEO/RNA
was different from that of ODN-BAEO/DNA and was in the order:
(duplex(BAEO:C) > single strand ODN-BAEO > duplex(BAEO:G) >
duplex(BAEO:U) > duplex(BAEO:A). In contrast, the intensity
differences between duplex(BAEO:U) and duplex(PAEO:U) were
slightly different in ODN/RNA.
The quenching mechanism of fluorescence nucleobase is
generally known to base on Photoinduced electron Transfer
(PeT)[16] and Fluorescence Resonance Energy Transfer
(FRET)[14]. The main mechanism of fluorescence quenching of G-
clamp is expected to be PeT.[16] Quenching ability by PeT is based
on electrochemical oxidation potentials of the nucleobase (in
order: G>A>C≈T).[17] In this study, duplex with A and G in the
opposite base of PAEO and BAEO tended to show effective
fluorescence quenching. Despite PAEO and BAEO having the same
fluorophore as G-clamp base, each duplex containing PAEO and
BAEO showed different fluorescence intensities and patterns. It
was reported that 2′,4′-BNA/LNA can improve - stacking
interaction with adjacent bases in duplex.[12] This difference of
fluorescence between BAEO and PAEO is expected to be caused by
changes in - stacking interactions and the surrounding structure
induced by the 2′,4′-BNA/LNA sugar. Furthermore, the
fluorescence patterns of BAEO and PAEO were different even in
between ODN/DNA (B-form) and ODN/RNA (A-form). This might
be induced by the difference of - stacking interaction against 5′-
and 3′-adjacent bases of BAEO and PAEO in B-form and A-form
duplex.
100
80
60
40
20
0
0
10
20
30
40
50
60
Time (min)
Figure 6. Resistance of ODNs containing BAEO against 3′-
exonuclease.
Conditions: 10 mM MgCl2, 50 mM Tris-HCl pH 8.0, 2 M ODN, Crotalus
Admanteus venom phosphodiesterase 114 g/ml at 37 °C. ODN sequence: 5′-
TTTTTTTTTX-3′. X: C (black), L (blue), phosphorothiate (PS) (Rp) (orange),
PS(Sp) (ocher), PAEO (pink) or BAEO (red).
This article is protected by copyright. All rights reserved.

10.1002/chem.202003982
Chemistry - A European Journal
FULL PAPER
= 97 / 3 to 95 / 5) to give 2 as a yellow solid (3.10 g, 73%); [α]ꢀꢁꢂ
+14.4 (c 1.0, CH3CN); IRmax (KBr): 1025, 1057, 1092, 1129,
1232, 1292, 1346, 1372, 1422, 1447, 1500, 1586, 1650, 1758,
1853, 2359, 2569, 2763, 2895, 2955, 3020, 3088, 3299 cm-1; 1H-
NMR (400 MHz, CD3CN):  2.06 (3H, s), 2.14 (3H, s), 3.93, 3.99
(2H, ABq, J = 8.0 Hz), 4.37, 4.47 (2H, ABq, J = 12.0 Hz), 4.57 (1H,
s), 4.94 (1H, s), 5.59 (1H, s), 6.48 (2H, d, J = 8.0 Hz), 6.97 (1H, t,
J = 8.0 Hz), 8.01 (1H, s), 8.05 (1H, s), 9.28 (1H, s); 13C NMR (101
MHz, CD3CN):  20.9, 21.0, 59.7, 71.7, 72.5, 78.5, 86.8, 88.6,
89.2, 109.7, 115.5, 118.3 128.4, 141.8, 151.1, 153.2, 158.3, 170.7,
171.0; HR-MS (MALDI) calculated for C20H21N3O9Br [M+H]+:
526.0456, found: 526.0450.
Conclusion
We successfully synthesized
a
BAEO nucleoside and
incorporated it into ODNs. BAEO dramatically improved duplex
stability when compared with PAEO, BH. PH, L, or C. The binding
constant (Ks) of ODN-BAEO for cRNA was 104- and 152-fold higher
than those of ODN-C and ODN-L, respectively. BAEO had base
discrimination abilities that were similar to those of PAEO. Thermal
stabilities of duplexes-PAEO were same or slightly higher than
those of duplexes-L in molecular crowding PEG(+) conditions,
although thermal stabilities of duplexes-PAEO were higher than
those of duplexes-L in PEG(-) conditions. On the other hand,
duplexes-BAEO maintained high thermal stability in both PEG(+)
and PEG(-) conditions compared with duplexes-PAEO. The high
enzymatic resistance of BAEO could offer several advantages in
clinical cellular applications. To the best of our knowledge, BAEO
possesses excellent duplex-forming abilities in all artificial nucleic
acids developed to date. Thus, BAEO is likely to be useful in DNA
nanotechnologies and nucleic acid-based therapies that require
high duplex-forming ability and enzymatic stability.
5-Bromo-3′,5′-di-O-acetyl-N4-{2-(2-
(benzyloxycarbonylamino)ethoxy)-6-hydroxyphenyl}-2′-O,4′-
C-methylenecytidine (3)
Under argon atmosphere, to a solution of 2 (3.10 g, 5.89 mmol) in
CH2Cl2/THF (130/65 ml) were added Ph3P (1.72 g, 6.54 mmol)
and benzyl-N-(2-hydroxyethyl)carbamate (1.28 g, 6.57 mmol),
and diethyl azodicarboxylate (DEAD, 2.95 ml, 16.2 mmol). The
mixture was refluxed for 5 h. After addition of saturated aqueous
NaHCO3 at 0 °C, the mixture was extracted with CHCl3. The
organic layer was washed with saturated aqueous NaHCO3
solution and H2O, and dried over Na2SO4, and then concentrated
under reduced pressure. The residue was purified by silica gel
column chromatography ([1] CHCl3/AcOEt = 8/2 to 6/4; [2]
CHCl3/MeOH = 95/5 to 93/7) to give 3 (3.94 g, 95%) as a yellow
foam; [α]ꢁꢀꢃ +5.5 (c 1.0, CHCl3); IR max (KBr): 1058, 1094, 1129,
1238, 1281, 1331, 1369, 1391, 1419, 1508, 1570, 1622, 1677,
1716, 1749, 2359, 2570, 2765, 2890, 2954, 3011, 3086, 3329 cm-
1; 1H-NMR (400 MHz, CDCl3):  2.12 (3H, s), 2.20 (3H, s), 3.66-
3.70 (2H, m), 3.96, 4.01 (2H, ABq, J = 8.0 Hz), 4.10-4.16 (2H, m),
4.39, 4.46 (2H, ABq, J = 12.0 Hz), 4.73 (1H, s), 4.85 (1H, s), 5.12
(2H, s), 5.48-5.51 (1H, m), 5.70 (1H, s), 6.40 (1H, d, J = 8.0 Hz),
6.67 (1H, dd, J = 0.9, 8.4 Hz), 7.00 (1H, t, J = 8.0 Hz), 7.30-7.35
(5H, m),7.92 (1H, s), 8.09 (1H, s), 11.05 (1H, s); 13C-NMR (100.5
MHz, CDCl3):  20.9, 20.9, 40.8, 58.5, 67.0. 67.7, 70.6, 71.7, 77.4,
77.6, 85.8, 85.8, 87.8, 89.0, 102.6, 114.2, 115.6, 127.3, 128.2,
128.3, 128.6, 136.4, 140.1, 150.1, 150.2, 151.8, 156.3, 156.5,
169.7, 170.1; HR-MS (MALDI) calcd. for C30H31N4O11NaBr
[M+Na]+: 725.1065, found: 725.1085.
Experimental Section
General
Dehydrated
acetonitrile,
dichloromethane,
N,N-
dimethylformamide (DMF), pyridine, and tetrahydrofran (THF)
were purchased from Wako Pure Chemical. Fuji Silysia PSQ-60B,
PSQ-100B, or FL-100D were used for silica gel column
chromatography. 1H-NMR, 13C-NMR, 31P-NMR were recorded on
JEOL JNM-ECS300, JNM-ECS400, and JNM-ECA500
spectrometers. Chemical shift values were reported in part per
million (ppm) relative to internal tetramethylsilane (0.00 ppm),
residual CHD2OD (3.31 ppm), CHD2CN (1.94 ppm) or
CHD2S(=O)D3 (2.50 ppm) for H-NMR. For 13C-NMR, chemical
1
shift values are reported in ppm relative to chloroform-d1 (77.2
ppm), methanol-d4 (49.0 ppm), acetonitrile-d3 (1.32 ppm) or
DMSO-d6 (39.5 ppm). Chemical shift values were reported in ppm
relative to 5% H3PO4 (0.00pm) and trifluoroacetic acid (-78.5 ppm)
as the external standard for 31P-NMR and 19F-NMR, respectively.
IR spectra were recorded on a JASCO FT/IR-4200 spectrometers.
Optical rotations were recorded on a JASCO DIP-370 instrument.
MALDI-TOF mass were recorded on JEOL SpiralTOF JMS-
S3000.
3-(2′-O,4′-C-methylene-β-D-ribofuranosyl)-9-{2-
benzyloxycarbonylamino)ethoxy}-1,3-diaza-2-
oxophenoxazine (5)
To 3 (3.50 g, 4.98 mmol) was added 7 M NH3/MeOH solution. The
mixture reacted at room temperature for 3 h under Ar atmosphere
and was concentrated under reduced pressure. The residue 4
(3.00 g) was used without purification into next reaction. Under
argon atmosphere, to a solution of 4 (3.00 g) in EtOH (300 ml)
was added DBU (800 μl, 5.35 mmol), and the mixture was
refluxed for 5 h. The reaction mixture was concentrated under
reduced pressure, and the residue was dissolved in CH3Cl. The
organic solution was washed with saturated aqueous NaHCO3,
dried over with Na2SO4, and concentrated under reduced
pressure. The residue was purified by silica gel column
chromatography (CHCl3 / MeOH = 97/ 3 to 90 / 10) to give 5 (1.53
g, 57% in 2 steps) as an orange foam; [α]ꢁꢀꢃ -7.3 (c 1.0, CHCl3);
IR max (KBr): 1015, 1055, 1075, 1098, 1147, 1233, 1254, 1280,
1333, 1363, 1382, 1418, 1476, 1510, 1559, 1611, 1671, 1699,
5-Bromo-3′,5′-di-O-acetyl-N4-(2,6-dihydroxyphenyl)-2′-O,4′-C-
methylenecytidine (2)
Under argon atmosphere, triphenyl phosphine (Ph3P) (4.30 g,
16.4 mmol) and CCl4 (80 ml) were added to a solution of 1 (3.40
g, 8.11 mmol) in CH2Cl2 (90 ml). The mixture was refluxed for 2 h.
After the reaction mixture was cooled to room temperature, 2-
aminoresorcinol
(2.08
g,
16.7
mmol)
and
1,8-
diazabicyclo[5.4.0]undec-7-ene (DBU) (2.4 ml, 16 mmol) were
added and stirred at room temperature for 6 h. The reaction
mixture was concentrated under reduced pressure, and the
residue was dissolved in CHCl3. This organic solution was
washed with 5% citric acid aqueous solution, dried over with
Na2SO4, and concentrated under reduced pressure. The residue
was purified by silica gel column chromatography (CHCl3 / MeOH
This article is protected by copyright. All rights reserved.

10.1002/chem.202003982
Chemistry - A European Journal
FULL PAPER
2359, 2884, 2947, 3091, 3275 cm-1; 1H-NMR (400 MHz, CD3OD):
 3.48-3.59 (2H, m), 3.75, 3.95 (2H, ABq, J = 6.0 Hz), 3.90 (2H,
s), 4.02 (2H, t, J = 4.0 Hz), 4.08 (1H, s), 4.31 (1H, s), 5.10, 5.14
(2H, ABq, J = 12.0 Hz), 5.49 (1H, s), 6.38 (1H, d, J = 8.0 Hz), 6.54
(1H, d, J = 8.0 Hz), 6.79 (1H, t, J = 10.0 Hz), 7.23-7.33 (5H, m),
7.51 (1H, s); 13C-NMR (100.5 MHz, CD3OD):  41.2, 57.6, 67.5,
69.6, 70.2, 72.4, 80.7, 88.7, 90.4, 108.2, 109.3, 116.8, 123.1,
124.9, 128.6, 128.9, 129.4, 129.6, 138.4, 144.3, 147.8, 155.8,
156.2, 159.1; HR-MS (MALDI) calcd. for C26H26N4O9Na [M+Na]+:
561.1592, found: 561.1595.
3275 cm-1; 1H -NMR (400 MHz, CD3CN):  3.39 (2H, s), 3.66, 3.77
(2H, ABq, J = 8.0 Hz), 3.68-3.72 (2H, m) 3.74 (6H, s), 4.03-4.11
(2H, m), 4.18 (1H, d, J = 4.0 Hz), 4.30 (1H, d, J = 4.0 Hz), 4.39
(1H, s), 5.40 (1H, s), 6.21 (1H, dd, J = 8.3, 0.8 Hz), 6.49 (1H, dd,
J = 8.4, 0.7 Hz), 6.75 (1H, t, J = 8.0 Hz), 6.88 (4H, m), 7.23-7.27
(1H, m), 7.31-7.38 (6H, m), 7.47-7.50 (3H, m), 8.00 (1H, s), 8.24
(1H, t, J = 6.0 Hz); 13C-NMR (100.5 MHz, CD3CN):  39.9, 55.9,
59.1, 68.1, 70.4, 72.4, 80.0, 87.2, 87.2, 88.4, 88.6, 88.6, 107.6,
109.1, 114.1, 115.7, 116.4, 123.1, 124.4, 127.9, 128.7, 129.0,
131.0, 136.6, 136.6, 136.6, 143.6, 143.6, 145.8, 146.9, 154.8,
155.0, 158.0, 158.4, 159.7; 19F NMR (376.2 MHz, CD3CN):  -
77.2; HR-MS (MALDI) calcd. for C41H37N4O10F3Na [M+Na]+:
825.2354, found: 825.2364.
3-(2′-O,4′-C-methylene-β-D-ribofuranosyl)-9-(2-
aminoethoxy)-1,3-diaza-2-oxophenoxazine (6)
To a solution of 5 (45 mg, 0.084 mmol) in MeOH (10 ml) were
added Pd(OH)2/C (20 mg) and cyclohexene (8 ml), and the
mixture was refluxed for 3 h. After the reaction mixture was cooled
to room temperature, the mixture was filtrated to remove
Pd(OH)2/C. The residue was concentrated under reduced
pressure to give 6 (32 mg, 93%) as a yellow solid; [α]ꢁꢀꢄ -3.7 (c
1.0, DMSO); IR max (KBr): 1009, 1052, 1075, 1094, 1120, 1130,
1202, 1237, 1261, 1282, 1316, 1335, 1368, 1411, 1435, 1475,
1512, 1562, 1608, 1634, 1666, 2343, 2954, 3302 cm-1; 1H-NMR
(500 MHz, DMSO-d6):  2.85-2.88 (2H, m), 3.61, 3.80 (2H, ABq,
J = 6.0Hz), 3.73 (2 H, s), 3.87-3.89 (2/10 H, m), 3.91 (8/10 H, s),
3.92 (2/10 H, s), 4.03-4.06 (8/10 H, m), 4.11 (8/10 H, s), 4.12 (2/10
H, s), 5.32 (8/10 H, s), 5.35 (2/10 H, s), 6.38 (8/10 H, d, J = 4.0
Hz), 6.45 (2/10 H, d, J = 8.0 Hz), 6.56 (8/10 H, d, J = 8.0 Hz), 6.61
(2/10 H, d, J = 8.0 Hz), 6.70 (8/10 H, t, J = 8.0 Hz), 6.80 (2/10 H,
t, J = 6.0 Hz), 7.36 (8/10 H, s), 7.47 (2/10 H, s); 13C-NMR (100.5
MHz, DMSO-d6):  51.1, 55.7, 68.3, 70.9, 73.6, 78.8, 86.6, 88.8,
108.1, 109.3, 117.3, 123.1, 123.2, 126.7, 142.6, 146.9, 152.5,
153.4; HR-MS (MALDI) calcd. for C18H20N4O7Na [M+Na]+:
427.1224, found: 427.1226.
3-(3′-O-β-cyanoethyl-N,N-diisopropylphosphoramidyl-5′-O-
(4,4′-dimethoxytrityl)-2′-O,4′-C-methylene-β-D-
ribofuranosyl)-9-(2-(trifluoroacetylamino)ethoxy)-1,3-diaza-
2-oxophenoxazine (9)
Under argon atmosphere, to a solution of 8 (97 mg, 0.12 mmol) in
CH2Cl2 (2 mL) were added N,N-diisopropylethylamine (DIPEA)
(42
μL,
0.24
mmol)
and
N,N-diisopropylamino-2-
cyanoethylphosphino chloridite (32 L, 0.14 mmol) at 0 °C and
the mixture was stirred for 2 h at room temperature. After addition
of H2O at 0 °C, the mixture was extracted with AcOEt. The organic
extracts were washed with saturated aqueous NaHCO3 solution,
water, and brine, dried over Na2SO4, and then concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography (0.5% triethylamine in n-hexane/AcOEt = 3/7 to
2/8) to give 9 (93 mg, 77%) as a yellow foam; 1H-NMR (400 MHz,
CDCl3):  0.97-1.29 (12H, m), 2.38 (7/8H, t, J = 6.0 Hz), 2.55 (1/8H,
t, J = 6.0 Hz), 3.36 (8/10H, d, J = 12.0 Hz), 3.40 (2/10H, d, J =
12.0 Hz), 3.46-3.57 (5H, m), 3.76-3.82 (10H, m), 4.13-4.17 (2H,
m), 4.25 (2/10H, d, J = 8.0 Hz), 4.33 (8/10H, d, J = 8.0 Hz), 4.60
(17/20H, s), 4.64 (3/20H, s), 5.59 (3/20H, s), 5.61 (17/20H, s),
6.28 (1H, d, J = 8.0 Hz), 6.45 (1H, d, J = 8.0 Hz), 6.79 (1H, t, J =
12.0 Hz), 6.83-6.87 (4H, m), 7.22 (1H, t, J = 8.0 Hz), 7.31 (2H, t,
J = 8.0 Hz), 7.36 (4H, d, J = 8.0 Hz), 7.47 (2H, d, J = 8.0 Hz), 7.54
(1H, s), 8.29 (1H, s), 8.40 (1H, s); 19F-NMR (376.2 MHz, CDCl3):
 -76.8, -76.7; 31P-NMR (161.8 MHz, CDCl3):  149.1, 149.4; HR-
MS (MALDI) calcd. for C50H54N6O11F3NaP [M+Na]+: 1025.3433,
found: 1025.3440.
3-(5′-O-(4,4′-dimethoxytrityl)-2′-O,4′-C-methylene-β-D-
ribofuranosyl)-9-{2-(trifluoroacetylamino)ethoxy}-1,3-diaza-
2-oxophenoxazine (8)
Under argon atmosphere, to a solution of 6 (641 mg, 1.56 mmol)
in anhydrous pyridine (60 ml) was added trifluoroaceticacid
anhydride (12 ml, 3.8 mmol) at 0 °C. The mixture was stirred at
room temperature for 2 h. After the reaction mixture was
concentrated to give crude 7 (702 mg). The crude 7 was used for
next step without purification.
Synthesis of ODNs containing BAEO
Synthesis of the ODN containing BAEO was performed on an
Under argon atmosphere, to
a
solution of silver
trifluoromethanesulfonate (AgOTf) (936 mg, 3.64 mmol) in CH2Cl2
(25 mL) was added 4,4′-dimethoxy chloride (DMTrCl) (1.25 g,
3.64 mmol) in CH2Cl2 (5 mL), and the mixture was stirred at room
temperature for 1 h to give DMTrOTf solution. Then, to a solution
of 7 in anhydrous 2,6-lutidine (35 mL) was added DMTrOTf
solution (17 ml) at 0 °C, and the mixture was stirred for 2 h. After
addition of H2O at 0 °C, the mixture was extracted with AcOEt.
The organic layer was washed with saturated aqueous NaHCO3
solution and copper (II) sulfate aqueous solution (3.0 g in 20 ml
H2O) to remove 2,6-lutidine, and dried over Na2SO4, and then
concentrated under reduced pressure. The residue was purified
by silica gel column chromatography (0.5% triethylamine in n-
hexane/AcOEt = 2/8 to 0/1) to give 8 (597 mg, 47% in 2 steps) as
a yellow foam; [α]ꢀꢁꢂ -8.1 (c 1.0, CH3CN); IR max (KBr): 1017,
1057, 1081, 1104, 1155, 1190, 1228, 1261, 1287, 1329, 1363,
1383, 1418, 1446, 1480, 1517, 1566, 1612, 1676, 1726, 1969,
2048, 2177, 2300, 2359, 2428, 2548, 2601, 2837, 2949, 3091,
automated DNA synthesizer (Gene Design, nS-8) at 0.2 mole
scale.
Then,
5-[3,5-bis(trifluoromethyl)phenyl]-1H-tetrazole
(Activator 42®) was used as the activator. The coupling time of
standard phosphoramidite coupling protocol was increased from
30 s to 12 min. The BAEO phosphoramidite 9 was prepared with
0.10 M acetonitrile/tetrahydrofuran (v/v, 3:1). The oligonucleotide
synthesis was performed on the DMTr-on mode. Controlled Pore
Glass (CPG)-supported ODNs were cleaved and their protected
groups of nucleobases were removed with 50 mM K2CO3 in
MeOH solution at rt for 4 h. The DMTr group in ODNs were
detritylated and purified with a Sep-Pak® Plus C18 Cartridge and
Sep-Pak® Plus C18 Environmental Cartridge (Waters) [washed
with 10% acetonitrile aqueous solution, detritylated with 0.5%
aqueous trifluoro acetic acid solution, and eluted with 35%
aqueous MeOH solution]. ODNs were purified by reverse-phase
HPLC on a Waters XBridgeTM OST C18 2.5 m (10 × 50 mm)
using 0.1 M triethylammonium acetate (TEAA) buffer (pH: 7.0)
This article is protected by copyright. All rights reserved.

10.1002/chem.202003982
Chemistry - A European Journal
FULL PAPER
(buffer a) and acetonitrile (buffer b). The purified ODNs were
analyzed by reverse-phase HPLC on a Waters XBridgeTM OST
C18 2.5 m (4.6 × 50 mm). The structures of ODNs were
determined by MALDI-TOF-MS (Bruker Daltonics AutoflexII
TOF/TOF mass spectrometer).
Acknowledgments
This work was supported by the Core Research for Evolutional
Science and Technology (CREST) program of the Japan Science
and Technology Agency (JST), JSPS KAKENHI (grant number
15K08024 and 20K05706 (awarded to O.N.)), and the Basic
Science and Platform Technology Program for Innovative
Biological Medicine from the Japan Agency for Medical Research
and Development (AMED) under grant number JP18am0301004.
This research was also partially supported by the Platform Project
for Supporting Drug Discovery and Life Science Research from
AMED.
Thermal denaturation experiments
UV melting experiments were carried out on a Shimadzu UV-
1650PC and UV-1800 spectrometers equipped with a Tm analysis
accessory quartz cuvettes of 1 cm optical path length. The UV
melting profiles were recorded in 2 mM sodium phosphate buffer
(pH 7.2) containing 20 mM NaCl to give final concentration of
each oligonucleotide in 2 M. The samples were annealed by
heating at 100 °C followed by slow cooling to room temperature.
The melting profile was recorded at 260 nm from 5 to 90 °C at a
scan rate of 0.5 °C /min. The Tm values were calculated as the
temperature of the half dissociation of the formed duplex based
on the first derivative of the melting curve.
Conflict of Interest
The authors declare no conflict of interest.
CD spectrum measurement
CD spectra were measured using a spectrophotometer, JASCO
J-720W. These spectra were recorded at 10 °C in the quartz
cuvette of 1-cm optical path length. The samples were prepared
in the same manner as described in the UV melting experiments.
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.
Keywords: Bridged Nucleic Acid • Phenoxazine base • G-clamp
• BNAP • - Stacking interaction
[1]
[2]
[3]
N. C. Seeman, H. F. Sleiman, Nat. Rev. Materials 2017, 3, 17068.
K. Morihiro, Y. Kasahara and S. Obika, Mol. BioSyst. 2017, 13, 235–245.
a) S. Obika, Chem. Pharm. Bull. 2004, 52, 1399–140; b) F. Debart, C.
Dupouy, J. -J. Vasseur, Beilstein J. Org. Chem. 2018, 14, 436–469.
a) K. -Y.Lin, R. J. Jones, M. Matteucci, J. Am. Chem.Soc. 1995, 117,
3873–3874; b) W. M. Flanagan, R. W. Wagner, D. Grant, K. Y. Lin, M. D.
Matteucci, Nat. Biotechnol. 1999, 17, 48–52.
Molecular modeling
[4]
[5]
Molecular Dynamics (MD) calculations of duplexes containing
BAEO were conducted using MacroModel software [Schrödinger,
LLC]; OPLS3 force field (in water at 300 K for 20 ns).
a) K. -Y.Lin, M. D. Matteucci, J. Am. Chem. Soc. 1998, 120, 8531–8532;
b) W. M. Flanagan, J. J. Wolf, P. Olson, D. Grant, K.-Y. Lin, R. W.
Wagner, M. D. Matteucci, Proc. Natl. Acad. Sci. U. S. A. 1999, 96, 3513–
3518; c) S. C. Holmes, A. A. Arzumanov, M. J. Gait, Nucleic Acids Res.
2003, 31, 2759–2768; b) J. -A. Ortega, J. R. N. Blas, M. Orozco, A.
Grandas, E. Pedroso, J. Robles, Org. Lett. 2007, 9, 4503–4506; d) R.
Stanton, S. Sciabola, C. Salatto, Y. Weng, D. Moshinsky, J. Little, E.
Walters, J. Kreeger, D. DiMattia, T. Chen, T. Clark, M. Liu, J. Qian, M.
Roy, R. Dullea, Nucleic Acid Ther. 2012, 22, 344–359.
Fluorescence measurements
Fluorescence spectra were obtained with a HITACHI F-7000
fluorescence spectrophotometer. The spectra were recorded at
25 °C with a quartz cuvette of 1 cm optical path length.
Thermodynamic analysis
The UV melting profiles for thermodynamic analysis were
recorded in 2 mM sodium phosphate buffer (pH 7.2) containing
[6]
a) B. J. Rodgers, N. A. Elsharif, N. Vashisht, M. M. Mingus, M. A. Mulvahill,
G. Stengel, R. D. Kuchta, B. W. Purse, Chem. Eur. J. 2014, 20, 2010-
2015; b) C. P. Lawson, A. F. Füchtbauer, M. S. Wranne, T. Giraud, T.
Floyd, B. Dumat, N. K. Andersen, A. H. El-Sagheer, T. Brown, H. Gradén,
L. M. Wilhelmsson, M. Grøtli, Sci. Rep. 2018, 8, 13970; c) M. Bood, S.
Sarangamath, M. S. Wranne, M. Grøtli, L. M. Wilhelmsson, Beilstein J.
Org. Chem. 2018, 14, 114-129.
20 mM NaCl to achieve
a final concentration of each
oligonucleotide in 0.45, 0.74, 1.22, 2.00 M for 130 L, and 3.26
and 6.80 M for 50 L. These samples were annealed by heating
at 100 °C followed by slow cooling to room temperature. The
melting profile was recorded using the same method as that used
in thermal denaturing experiments. The van’t Hoff plots were
recorded by Tm values in each concentration, and each value of
ΔH, ΔS and ΔG was calculated according to the following
equations.
1/Tm = (R/ΔH)・ln(Ct/4) +ΔS /ΔH
ΔG =ΔH - TΔS
In these equations, R indicates gas constant, and Ct indicates
total concentration of oligonucleotides.
[7]
A. Fujii, O. Nakagawa, Y. Kishimoto, Y. Nakatsuji, N. Nozaki, S. Obika,
ChemBioChem 2020, 21, 860–864.
[8]
A. Fujii, O. Nakagawa, Y. Kishimoto, T. Okuda, Y. Nakatsuji, N. Nozaki,
Y. Kasahara, S. Obika, Chem. Eur. J. 2019, 25, 7443–7448.
Y. Kishimoto, A. Fujii, O. Nakagawa, T. Nagata, T. Yokota, Y. Hari, S.
Obika, Org. Biomol. Chem. 2017, 15, 8145–8152.
[9]
[10]
a) S. Obika, D. Nanbu, Y. Hari, K. Morio, Y. In, T. Ishida, T. Imanishi,
Tetrahedron Lett. 1997, 38, 8735–8738; b) S. Obika, D. Nanbu, Y. Hari,
J. Andoh, K. Morio, T. Doi, T. Imanishi, Tetrahedron Lett. 1998, 39,
5401–5404.
Enzymatic resistance of ODNs
[11] a) S. K. Singh, P. Nielsen, A. A. Koshkin, J. Wengel, Chem. Commun.
1998, 455–456; b) A. A. Koshkin, S. K. Singh, P. Nielsen, V. K. Rajwanshi,
R. Kumar, M. Meldgaard, C. E. Olsen, J. Wengel, Tetrahedron 1998, 54,
3607–3630.
Solutions (300 L) of 2 M ODNs-X containing 10 mM MgCl2 and
50 mM Trip-HCl buffer (pH 8.0) were reacted with 3ʹ-exonuclease
(CAVP; 0.13 g/mL) at 37 °C. 50 L of each sample was picked
up at 10, 30 and 60 min, and were heated at 90 °C for 5 min for
inactivation of 3ʹ-exonuclease. The amounts of intact ODNs were
quantified by reverse-phase HPLC.
[12]
a) P. M. McTigue, R. J. Peterson, J. D. Kahn, Biochemistry 2004, 43,
5388–5405; b) H. Kaur, A. Arora, J. Wengel, S. Maiti, Biochemistry 2006,
45, 7347–7355; c) U. Christensen, Biosci. Rep. 2007, 27, 327–333; d) H.
This article is protected by copyright. All rights reserved.

10.1002/chem.202003982
Chemistry - A European Journal
FULL PAPER
Kaur, J. Wengel, S. Maiti, Biochemistry 2008, 47, 1218–1227; e) E.
Kierzek, A. Pasternak, K. Pasternak, Z. Gdaniec, I. Yildirim, D. H. Turner,
R. Kierzek, Biochemistry 2009, 48, 4377–4387.
H. Li, J. Mårtensson, T. Brown, L. M. Wilhelmsson, B. Albinsson, Nucleic
Acids Res. 2008, 36, 157–167; g) K. Börjesson, S. Preus, A. H. El-
Sagheer, T. Brown, B. Albinsson, L. M. Wilhelmsson, J. Am. Chem. Soc.
2009, 131, 4288–4293; h) H. Gardarsson, A. S. Kale, S. T. Sigurdsson,
ChemBioChem 2011, 12, 567–575; i) B. Dumat, A. F. Larsen, L. M.
Wilhelmsson, Nucleic Acids Res. 2016, 44, e101; j) A. F. Füchtbauer, S.
Preus, K. Börjesson, S. A. McPhee, D. M. J. Lilley, L. M. Wilhelmsson,
Sci. Rep. 2017, 7, 2393.
[13] a) S. Nakano, H. Karimata, T. Ohmici, J. Kawakami, N. Sugimoto, J. Am.
Chem. Soc. 2004, 126, 14330-14331; b) D. Miyoshi, N. Sugimoto,
Biochimie 2008, 90, 1040–1051; c) S. Nakano, D. Miyoshi, N. Sugimoto,
Chem. Rev. 2014, 114, 2733-2758; d) S. Nakano, N. Sugimoto, Mol.
BioSyst. 2017, 13, 32-41; e) S. Ghosh, S. Takahashi, T. Endoh, H.
Tateishi-Karimata, S. Hazra, N. Sugimoto, Nucleic Acids Res. 2019, 47,
3284–3294.
[15] M. A. Maier, J. M. Leeds, G. Balow, R. H. Springer, R. Bharadwaj, M.
Manoharan, Biochemistry 2002, 41, 1323–1327.
[14] a) L. M. Wilhelmsson, A. Holmén, P. Lincoln, P. E. Nielsen, B. Nordén, J.
Am. Chem. Soc. 2001, 123, 2434–2435; b) L. M. Wilhelmsson, P. Sandin,
A. Holmén, B. Albinsson, P. Lincoln, B. Nordén, J. Phys. Chem. B 2003,
107, 9094–9101; c) K. C. Engman, P. Sandin, S. Osborne, T. Brown, M.
Billeter, P. Lincoln, B. Nordén, B. Albinsson, L. M. Wilhelmsson, Nucleic
Acids Res. 2004, 32, 5087–5095; d) P. Sandin, L. M. Wilhelmsson, P.
Lincoln, V. E. C. Powers, T. Brown, B. Albinsson, Nucleic Acids Res.
2005, 33, 5019–5025; e) P. Sandin, P. Lincoln, T. Brown, L. M.
Wilhelmsson, Nat. Protoc. 2007, 2, 615–623; f) P. Sandin, K. Börjesson,
[16]
a) O. Nakagawa, S. Ono, Z. Li, A. Tsujimoto, S. Sasaki, Angew. Chem.
Int. Ed. 2007, 46, 4500–4503; Angew.Chem. 2007, 119, 4584 –4587; b)
Y. Koga, Y. Fuchi, O. Nakagawa, S. Sasaki, Tetrahedron 2011, 67, 6746
–6752.
[17] C. A. M. Seidel, A. Schulz, M. H. M. Sauer, J. Phys. Chem. 1996, 100,
5541–5553.
This article is protected by copyright. All rights reserved.

10.1002/chem.202003982
Chemistry - A European Journal
FULL PAPER
Table of Contents
FULL PAPER
2′,4′-BNA/LNA with G-clamp (BAP-
Y. Kishimoto,
AEO)
incorporated
oligodeoxynucleotides (ODNs). ODNs
containing BNAP-AEO showed high
was
synthesized
and
into
O. Nakagawa*,
A. Fujii, K.Yoshioka,
T. Nagata, T. Yokota,
Y. Hari, and S. Obika*
thermal
stabilities
toward
Page No. – Page No.
complementary DNA and RNA
compared with those of ODNs
containing 2′-deoxynucleoside with G-
clamp and 2′,4′-BNA/LNA with 5-
2′,4′-BNA/LNA with 9-(2-
aminoethoxy)-1,3-diaza-
2-oxophenoxazine
efficiently forms
methylcytosine.
ODN
containing
BNAP-AEO showed high resistance to
3′-exonuclease.
duplexes and has
enhanced enzymatic
resistance
This article is protected by copyright. All rights reserved.