2′-O,4′-C-ethylene-bridged nucleic acids (ENA): Highly nuclease-resistant and thermodynamically stable oligonucleotides for antisense drug

Article abstract of DOI:10.1016/S0960-894X(01)00683-7

To develop antisense oligonucleotides, novel nucleosides, 2′-O,4′-C-ethylene nucleosides and their corresponding phosphoramidites, were synthesized as building blocks. The ¹H NMR analysis showed that the 2′-O,4′-C-ethylene linkage of these nucleosides restricts the sugar puckering to the N-conformation as well as the linkage of 2′-O,4′-C-methylene nucleosides which are known as bridged nucleic acids (BNA) or locked nucleic acids (LNA). The ethylene-bridged nucleic acids (ENA) showed a high binding affinity for the complementary RNA strand (ΔT_m = + 5.2 °C/modification) and were more nuclease-resistant than natural DNA and BNA/LNA. These results indicate that ENA have better properties as antisense oligonucleotides than BNA/LNA.

Full text of DOI:10.1016/S0960-894X(01)00683-7

Bioorganic & Medicinal Chemistry Letters 12 (2002) 73–76
20-O,40-C-Ethylene-Bridged Nucleic Acids (ENA):
Highly Nuclease-Resistant and Thermodynamically
Stable Oligonucleotides for Antisense Drug
Koji Morita,a Chikako Hasegawa,a Masakatsu Kaneko,a Shinya Tsutsumi,b
Junko Sone,b Tomio Ishikawa,b Takeshi Imanishic and Makoto Koizumia,*
aExploratory Chemistry Research Laboratories, Sankyo Co., Ltd., Tokyo 140-8710, Japan
bBiomedical Research Laboratories, Sankyo Co., Ltd., Tokyo 140-8710, Japan
cGraduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871, Japan
Received 13 August 2001; accepted 8 October 2001
Abstract—To develop antisense oligonucleotides, novel nucleosides, 20-O,40-C-ethylene nucleosides and their corresponding phos-
phoramidites, were synthesized as building blocks. The 1H NMR analysis showed that the 20-O,40-C-ethylene linkage of these
nucleosides restricts the sugar puckering to the N-conformation as well as the linkage of 20-O,40-C-methylene nucleosides which are
known as bridged nucleic acids (BNA) or locked nucleic acids (LNA). The ethylene-bridged nucleic acids (ENA) showed a high
binding affinity for the complementary RNA strand (ÁTm=+5.2 ꢀC/modification) and were more nuclease-resistant than natural
DNA and BNA/LNA. These results indicate that ENA have better properties as antisense oligonucleotides than BNA/LNA.
# 2001 Elsevier Science Ltd. All rights reserved.
Antisense technology has attracted more interest as a
target validation tool in the field of drug discovery
through genomics,1 and developing antisense oligonu-
cleotides as nucleic acid drugs for clinical use could lead
to a novel treatment of inveterate diseases such as can-
cer, inflammation, and viral diseases.2 Phosphoro-
thioate oligonucleotides, which are most widely used in
antisense technology, exhibit high nuclease resistance,
but have a number of limitations, such as non-sequence
specific toxicity, immune stimulation and low RNA
affinity.3 Many studies have been focused on developing
various types of modified oligonucleotides.4ꢁ9 Recently,
our and Wengel’s group independently reported the
synthesis of novel 20-O,40-C-methylene nucleosides
whose sugar puckering is fixed in the N-conformation as
in RNA, and that oligonucleotides containing these
bridged nucleosides (BNA/LNA) showed much higher
affinity toward their complementary RNA than any
other modified oligonucleotides.10ꢁ13 Moreover, Wah-
lestedt et al. showed that LNA and DNA copolymers
were useful for in vivo antisense application.14
The 20-O,40-C-methylene linkage forms a five-membered
ring with the furanose to give a bicyclic nucleoside. It
occurred to us that adding one more carbon in the
linkage could form a six-membered ring with less strain
than a five-membered ring and the resulting nucleoside
and corresponding oligonucleotide might show better
properties. Recently, Wang et al. reported the synthesis
of the 20,40-C-bridged 20-deoxynuclosides, which has six-
membered rings with 20-deoxy-type sugars and an
increase in Tm values of their corresponding oligo-
nucleotides.15 In this report, we synthesized 20-O,40-C-
ethylene nucleosides, which have six-membered rings
with ribo-type sugars, containing all possible natural
nucleobases (thymine, adenine, guanine, cytosine, ura-
cil, 5-methylcytosine) and evaluated their basic proper-
ties as antisense oligonucleotides.
*Corresponding author. Tel.:+81-3-3492-3131; fax:+81-3-5436-8570;
e-mail: koizum@shina.sankyo.co.jp
0960-894X/02/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00683-7

74
K. Morita et al. / Bioorg. Med. Chem. Lett. 12 (2002) 73–76
A novel nucleoside, 20-O,40-C-ethylene thymidine was
synthesized as shown in Scheme 1. The hydroxymethyl
group at the 4-position of compound 116 was converted
to a hydroxyethyl group via Swern oxidation, Wittig
reaction and hydroboration followed by oxidation to
give compound 4. Tosylation of 4 afforded 5, which was
converted to diacetate 6 by treatment with AcOH and
Ac2O in the presence of a catalytic amount of H2SO4.
The resulting compound 6 was coupled with a silylated
thymine by Vorbruggen’s method.17 Then, base-induced
ring closure by the treatment with 1 M NaOH/pyridine-
H2O afforded 20-O,40-C-ethylene thymidine 8. After
debenzylation of compound 8, the phosphoramidite
building block 11 was prepared by a standard proce-
amidites and natural DNA phosphoramidites, desired
oligonucleotides were synthesized on a DNA synthesizer
by the phosphoramidite method and purified by reverse-
phase HPLC. These modified oligonucleotides were
characterized by negative ion ESI mass spectroscopy.
First, we tested the affinity of an oligonucleotide con-
taining six 20-O,40-C-ethylene thymidines (eT) toward its
complementary RNA or DNA, and compared it to that
of an oligonucleotide containing six 20-O,40-C-methylene
thymidines (mT). The 20-O,40-C-ethylene-modified oli-
gonucleotide showed an excellent Tm value for RNA
(ÁTm/modification=5.2 ꢀC), that was almost identical
to that of the 20-O,40-C-methylene-modified oligonucleo-
tide (ÁTm/modification=5.5 ꢀC, Table 1). Also, Tm values
of both modified oligonucleotides for DNA were identical.
These results indicate that 20-O,40-C-ethylene nucleic
acids (ENA) have a high affinity for DNA and RNA as
do 20-O,40-C-methylene nucleic acids (BNA/LNA).
1
dure. In the H NMR analysis of the bridged nucleo-
sides (8–11),18 the coupling constant (JH1 ꢁH2 ) was 0 Hz
just as that of 20-O,40-C-methylene-bridged nucleosides,10
which indicated that the puckering of the furanose ring
was restricted to the N-conformation.19,20 Other
nucleosides containing all possible natural nucleobases
(adenine, cytosine, guanine, uracil and 5-methylcyto-
sine) were synthesized by a similar procedure (K.
Morita et al., in preparation). Using these phosphor-
0
0
Next, we evaluated the effect on stability of a 20-O,40-C-
ethylene modification against endonuclease using thy-
midine trimers (50-TpXpT-30). The middle nucleoside
Scheme 1. (i) (COCl)2, DMSO, Et3N, CH2Cl2, ꢁ78 ꢀC, 91%; (ii) Ph3P+CH3Brꢁ, NaH, DMSO, 64%; (iii) 9-BBN, THF then H2O2, NaOH, 93%;
(iv) TsCl, Et3N, CH2Cl2 97%; (v) Ac2O, H2SO4, AcOH, 86% (a:b=4:1); (vi) silylated thymine, TMSOTf, ClC2H4Cl, reflux, 67%; (vii) 1 N NaOH,
pyridine–H2O, 80%; (viii) H2, Pd(OH)2, MeOH, 77%; (ix) DMTrCl, pyridine, CH2Cl2, 81%; (x) ((iPr)2N)2P(OC2H4CN), N,N-diisopropylammo-
nium tetrazolide, 89%; (xi) DNA/RNA synthesizer.
Table 1. Tm values (ꢀC) of the modified oligonucleotides towards complementary RNA and DNA
Oligonucleotide
50-d(GCGXXXXXXGCT)-30
RNA complement
50-r(AGCAAAAAACGC)-30
DNA complement
50-d(AGCAAAAAACGC)-30
Tm (ꢀC)
ÁTm (ꢀC) /modification
Tm (ꢀC)
ÁTm (ꢀC)/modification
DNA (X=T)
BNA/LNA (X=mT)
ENA (X=eT)
43
77
75
48
61
61
5.5
5.2
2.2
2.2
Duplex concentration: 4 mM. Buffer: 100 mM NaCl, 10 mM sodium phosphate buffer (pH 7.2); mT: 20-O,40-C-methylene thymidine, eT: 20-O,40-C-
ethylene thymidine.

K. Morita et al. / Bioorg. Med. Chem. Lett. 12 (2002) 73–76
75
(X) of the trimer was substituted with 20-O,40-C-ethyl-
ene thymidine (eT) or 20-O,40-C-methylene thymidine
(mT). The trimer has two phosphodiester linkages
(position I and II, Fig. 1a) which can be cleaved by
endonuclease, nuclease P1. When the modified oligonu-
cleotides (X=eT and mT) were incubated with nuclease
P1, the products (50-TpX-30 and thymidine-50-mono-
phosphate), which would be cleaved at position I, were
never detected in the HPLC analysis even under high
concentration as 62.5 mg/mL nuclease P1 (data not
shown). On the other hand, the products (thymidine
and 50-pXpT-30), which were cleaved at position II,
were detected at 1.3 mg/mL nuclease P1. Under this
condition, both phosphodiester linkages in natural 50-
TpTpT-30 were completely hydrolyzed by nuclease P1
within 5 min (Fig. 1b). Hydrolysis rates of the phos-
phodiester linkage at position II in 50-TpXpT-30 (X=eT
and mT) were measured (Fig. 1b). The initial rate con-
stants of hydrolysis of the trimers (X=eT and mT) were
calculated as 4.6ꢂ10ꢁ3 minꢁ1 and 1.8ꢂ10ꢁ2 minꢁ1
,
respectively. From these results, we found that the 20-
O,40-C-ethylene-modified trimer was approximately 4
times more stable than the 20-O,40-C-methylene-modified
trimer.
Furthermore, we investigated the stability of the modified
oligonucleotides against 30-exonuclease (snake venom
phosphodiesterase, SVPD). Using oligothymidylates,
which contain a modified thymidine on the second
nucleotide from the 30-end (50-d(TTTTTTTTTTXT)-30,
X=T, mT or eT), the amount of the first 30-phospho-
diester cleavage by 30-exonuclease was quantified by
HPLC analysis. Although natural oligothymidylates
(X=T) and 20-O,40-C-methylene-modified oligonucleo-
tides (X=mT) were rapidly hydrolyzed by 30-exo-
nuclease, approximately 82% of 20-O,40-C-ethylene-
modified oligonucleotides still remained after 2 h (Fig.
2). We measured and calculated the observed rate con-
stants (kobs) of hydrolysis of the three oligonucleotides
(DNA:
kobs=>7.5ꢂ10ꢁ1
minꢁ1
,
BNA/LNA:
kobs=8.2ꢂ10ꢁ2 minꢁ1, ENA: kobs=1.5ꢂ10ꢁ3 minꢁ1).
These data show that one 20-O,40-C-ethylene modifica-
tion resulted in more than 500 times higher stability
than natural nucleotides, and approximately 55 times
higher stability than the 20-O,40-C-methylene nucleotide.
Figure 1. (a) Two positions of 50-TXT-30 (X=eT), which can be
recognized and cleaved by nuclease P1. (b) Stability of modified oli-
gonucleotides 50-TXT-30 (X=T, mT or eT) at position II against
nuclease P1; oligo concentration: 6.4 mg/mL, enzyme concentration:
1.3 mg/mL; buffer: 20 mM sodium acetate buffer (pH 5.2), 0.1 mM
ZnCl2, at 37 ꢀC.
In conclusion, we further optimized BNA/LNA by the
addition of one more carbon in the linkage, and syn-
thesized novel 20-O,40-C-ethylene-bridged nucleic acids
(ENA) which have high affinity for RNA and high
nuclease-resistance. Only one example of an in vivo
BNA/LNA application in the cerebrospinal fluid system
with very low levels of nuclease has already been repor-
ted.14 More highly nuclease-resistant ENA than BNA/
LNA would greatly contribute to the developments of
antisense drugs. Further antisense studies are currently
in progress.
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2. Stability
of
modified
oligonucleotides
(50-
d(TTTTTTTTTTXT)-30 (X=T, mT or eT) against SVPD; oligo con-
centration: 26 mg/mL, enzyme concentration: 0.3 mg/mL; buffer: 50
mM Tris–HCl (pH 8.0), 10 mM MgCl2, at 37 ꢀC.

76
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