Synthesis and properties of novel 20-O-alkoxymethyl-modified nucleic acids

Article abstract of DOI:10.1016/j.bmcl.2011.09.003

Novel 2′-O-modified oligoribonucleotides with alkoxymethyl skeletons were synthesized, and their ability to hybridize complementary nucleic acids and their nuclease resistance were analyzed. The hybridization ability was improved by introducing electron-withdrawing groups and the increases in melting temperature (T_m value) was particularly high for chlorine-substituted compounds. Nuclease resistance of these 2′-O-alkoxymethylated oligomers was lower than expected, but cyano substitution resulted in a higher nuclease resistance than 2′-O-methylation.

Full text of DOI:10.1016/j.bmcl.2011.09.003

Bioorganic & Medicinal Chemistry Letters 21 (2011) 6285–6287
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and properties of novel 20-O-alkoxymethyl-modified nucleic acids
⇑
Koichiro Arai, Naoki Uchiyama, Takeshi Wada
Department of Medical Genome Sciences, The University of Tokyo, Bioscience Building 702, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562, Japan
a r t i c l e i n f o
a b s t r a c t
Novel 20-O-modified oligoribonucleotides with alkoxymethyl skeletons were synthesized, and their abil-
ity to hybridize complementary nucleic acids and their nuclease resistance were analyzed. The hybridiza-
tion ability was improved by introducing electron-withdrawing groups and the increases in melting
temperature (Tm value) was particularly high for chlorine-substituted compounds. Nuclease resistance
of these 20-O-alkoxymethylated oligomers was lower than expected, but cyano substitution resulted in
a higher nuclease resistance than 20-O-methylation.
Article history:
Received 13 May 2011
Revised 30 August 2011
Accepted 1 September 2011
Available online 7 September 2011
Keywords:
RNA analogs
20-O-Modification
20-O-Alkoxymethyl skeleton
Duplex stability
Ó 2011 Elsevier Ltd. All rights reserved.
Numerous chemically modified nucleic acids have been devel-
oped to improve the chemical and biological properties of natural
nucleic acids in drugs. In particular, 20-O-modified nucleic acids,
with modifications such as the introduction of 20-O-methyl1,
20-O-methoxyethyl2, and 20-O-aminopropyl3 groups, are known to
exhibit high duplex stability and nuclease resistance. However,
some 20-O-modifications are often difficult to introduce mainly be-
cause the alkylation of nucleobases occurs as a side reaction during
the synthesis. For this reason, the synthesis of ribonucleotide
monomers in most of studies of 20-O-modification has been limited
to those bearing pyrimidine bases, and only few studies have been
reported for the monomers bearing purine bases.4
A: 2´-O-EOM : R = CH2CH3
B: 2´-O-MCEM : R = CH2CH2Cl
C: 2´-O-DCEM : R = CH2CHCl2
D: 2´-O-TCEM : R = CH2CCl3
E: 2´-O-CEM : R = CH2CH2CN
F: 2´-O-DFEM : R = CH2CHF2
G: 2´-O-TFEM : R = CH2CF3
O
B
O
O
O
O
O
O
OR
P
Figure 1. Structure of 20-O-modifications. Abbreviations: EOM, 20-O-ethoxymethyl;
MCEM, 20-O-(2-chloroethoxy)m ethyl; DCEM, 20-O-(2,2-dichloroethoxy)methyl;
TCEM, 20-O-(2,2,2-trichlo rorethoxy)methyl; CEM, 20-O-(2-cyanoethoxy)methyl;
DFEM, 20-O-(2,2-di fuluoroethoxy)methyl; TFEM, 20-O-(2,2,2-trifluoroeth-
oxy)methyl.
The 20-O-alkoxymethyl group is a skeleton often used as a 20-OH
protecting group.5–8 Ribonucleotide monomer bearing this type of
group generally exhibits high coupling efficiency in oligomer
synthesis, and introduction of this group to the 20-position is
performed by using the same strategy for all nucleobases of with
no side reactions. These properties are practically attractive as 20-
O-modifications; however, the simplest alkoxymethyl group, the
ethoxymethyl group, was reported to destabilize a duplex when it
was introduced to the 20-position.2,9 Several 20-O-modified oligori-
bonucleotides with alkoxymethyl skeleton10,11 have been reported;
however, they either resulted in destabilization of duplexes10 or no
hybridization analysis was conducted.11
methyl skeleton. In this report, we describe the synthesis and
properties of novel 20-O-alkoxymethyl-modified oligoribonucleo-
tides bearing electron-withdrawing groups.
As an electron-withdrawing group, we introduced several
halogen atoms into the 20-O-alkoxymethyl skeleton (Fig. 1). We
also investigated the 20-O-CEM group, which was developed by
Ohgi et al. , as a 20-OH protecting group.5,6
We first synthesized various phosphoramidite monomers with
electron-withdrawing substituted 20-O-alkoxymethyl groups,
according to a previous report on the 20-OH protecting group6
(Scheme 1). The introduction of alkoxymethyl group was performed
under the acidic condition, using 20-O-methylthiomethyl uridine
and alcohols with electron-withdrawing group.
Recently, 20-O-modified RNAs with electron-withdrawing
groups at the 20-position has been reported to demonstrate high
duplex stability.4,12 These results motivated us to study the substi-
tuent effect of electron-withdrawing groups in the 20-O-alkoxy-
Next, 20-O-modified oligoribonucleotides were synthesized
using the phosphoramidite monomer 5. The synthesis was con-
ducted by an Expedite 8909 automated synthesizer (Applied Bio-
⇑
Corresponding author.
E-mail address: wada@k.u-tokyo.ac.jp (T. Wada).
systems) using a standard protocol for 0.2 lmol scale synthesis of
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.09.003

6286
K. Arai et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6285–6287
ROH
NIS
O
U
O
U
O
U
DMSO, Ac2O, AcOH
(6/5/5, v/v/v)
O
O
O
Si
O
Si
O
Si
O
TfOH
rt, 1.5 d
THF, -40 °C
O
O
S
O
OH
Si
O
O
O
OR
Si
Si
1
2
3
Cl
P
NC
DMTrO
O
U
O
N(iPr)2
DMTrO
U
O
DIPEA
NH4F
MeOH, 50°C
DMTrCl
pyridine, rt
O
O
OR
CH2Cl2, rt
NC
P
HO
O
OR
N(iPr)2
4
5
Scheme 1. General synthetic procedure for 20-O-modified phosphoramidite monomers.
RNA. After the synthesis, oligoribonucleotides were cleaved from
solid supports and purified by reversed-phase HPLC.
The hybridization properties of 20-O-modified oligoribonucleo-
tides against complementary RNAs were studied (Table 1). The
hybridization affinity of 20-O-alkokymethylated U12 for A12 was
compared with that of unmodified U12 and 20-O-methylated U12
.
As shown in Table 1, the introduction of 20-O-EOM without substi-
tutions led to decrease in the Tm value, as previously reported2,9 (A,
Table 1). In contrast, oligoribonucleotides with electron-withdraw-
ing substitution in the 20-O-alkoxymethyl groups exhibited a high-
er Tm value than that of oligoribonucleotides with the 20-O-EOM
group (B–G, Table 1). This result suggests that the introduction of
electron-withdrawing groups into the alkoxymethyl skeleton leads
to the stabilization of hybridization with complementary RNA.
Among the oligoribonucleotides with electron-withdrawing
substitution in the 20-O-alkoxymethyl groups, oligoribonucleotides
with chloro-substituted 20-O-MCEM and 20-O-DCEM groups
exhibited similar Tm values to that of oligoribonucleotides with
the 20-O-methyl group (B, C, H, Table 1, Fig. 2). This result is unex-
pected because the electron-withdrawing effect of chlorine atoms
is weaker than that of fluorine atoms or the cyano group. From
these results, the stabilizing effect caused by the 20-O-MCEM and
20-O-DCEM groups is believed to be derived from another factor,
such as hydrophobicity of chlorine atoms. On the basis of molecu-
lar modeling study, the mono- and disubstituted 20-O-ethoxy-
methyl groups were located along the minor groove of the
A-type duplex and fairly fitted to the relatively hydrophobic groove
with van der Waals contact. These structures involving hydration
would be important for stabilization of the duplex. In addition,
the electron-withdrawing substitution in the ethoxy group may af-
Figure 2. Melting curves of the 20-O-modified oligoribonucleotides with comple-
mentary RNA; sequence and conditions are same as Table 1.
fect the conformational fixation of this group by the gauche effect.
Interestingly, oligoribonucleotides with the trichloro-substituted
20-O-TCEM group did not demonstrate high affinity to the comple-
mentary RNA (D, Table 1). This observation suggests that higher-
order substitution could lead to destabilization of the duplex
because of the bulkiness of the 20-O-modification.
The hybridization properties of the 20-O-MCEM- and 20-O-DCEM-
modified U12 against the complementary DNA dA12 were also stud-
ied (Fig. 3). In these cases, no significant increase in the Tm value was
observed by the introduction of 20-O-MCEM and 20-O-DCEM groups.
These results indicate that the duplex stabilizing effect of the 20-O-
MCEM and 20-O-DCEM modifications is specific to RNA. This is
Table 1
Hybridization properties of 20-O-modified RNA against complementary RNA relative
to unmodified RNAa
b
entry
U⁄
D
Tm (°C)
D
Tm/mod.(°C)
U
0
0
A
B
C
D
E
F
G
H
20-O-EOM-U
20-O-MCEM-U
20-O-DCEM-U
20-O-TCEM-U
20-O-CEM-U
20-O-DFEM-U
20-O-TFEM-U
20-O-Me-U
ꢀ2.5
+11.0
+10.8
ꢀ0.8
+2.8
+3.5
+4.5
+12.1
ꢀ0.2
+1.0
+1.0
ꢀ0.1
+0.3
+0.3
+0.4
+1.1
a
Conditions: 10 mM sodium phosphate buffer (pH 7.0), 100 mM NaCl, 0.1 mM
EDTA, at 260 nm, 2.0 M oligoribonucleotides and complementary RNA.
Sequence: 50-U*U*U*U*U*U*U*U*U*U*U*U-30.
Figure 3. Melting curves of the 20-O-modified oligoribonucleotides with comple-
mentary DNA; sequence and conditions are same as Table 1.
l
b

K. Arai et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6285–6287
6287
In conclusion, we have synthesized and analyzed the properties
of oligoribonucleotides bearing a novel 20-O-modification with alk-
oxymethyl skeletons. Hybridization affinity of the oligoribonucleo-
tides with electron-withdrawing substituted 20-O-alkoxymethyl
groups was higher than that of unmodified 20-O-EOM oligoribonu-
cleotides. The increases in Tm value were especially high in the
20-O-MCEM and 20-O-DCEM substitutions, in which the duplex
stabilizing effect was specific to RNA. Nuclease resistance of the
20-O-alkoxymethylated oligoribonucleotides, except for the 20-O-
CEM-substitutedoligoribonucleotides, was unexpectedly lower
than that of 20-O-methylated oligoribonucleotides. Further investi-
gation of the substitution and synthesis of oligoribonucleotides
bearing other nucleobases are in progress.
Figure 4. Relative nuclease resistance of oligoribonucleotides with 20-Omodifica-
a
tions. Sequence: 50-U⁄U⁄U⁄U⁄U⁄U⁄U⁄U⁄U⁄U⁄U⁄U-30. bProcedure: 10
lM oligori-
Acknowledgments
bonucleotides were digested with snake venom phosphodiesterase (4 ꢁ 10ꢀ4 units/
ml) for 2 h in 50 mM Tris–HCl (pH 8.5), 72 mM NaCl and 14 mM MgCl2.
This work was supported by Grant-in-Aid for Scientific Re-
search on Innovative Areas (21115008).
contrast to the observed effect of the 20-O-methyl modification,
which demonstrated high affinity to the complementary strand in
both RNA and DNA (Figs. 2 and 3). From these results, it could be
stated that the 20-O-MCEM or 20-O-DCEM modification stabilizes
the duplex in a manner different from that of the 20-O-methyl
modification.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.09.003.
References and notes
Nuclease resistance of the 20-O-modified oligoribonucleo-tides
was tested by using snake venom phosphodiesterase (SVPD)
( Fig. 4). Oligoribonucleotides were treated with the nuclease for
2 h and the degradation ratio was estimated by reversed phase HPLC
analysis. Surprisingly, most of the oligoribonucleotides with 20-O-
alkoxy-methyl modifications were less resistant to the nuclease
than the oligoribonucleotides with 20-O-methyl modification,
although the former groups are bulkier than the methyl group. It is
also noteworthy that the degradation ratio increased when addi-
tional halogen atoms were substituted, except for the 20-O-TCEM
group. This result suggests that the introduction of halogen atoms,
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resistance when introduced into the 20-position. From these results,
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reduction in nuclease resistance. In contrast, 20-O-CEM-substituted
oligoribonucleotides demonstrated higher nuclease resistance than
the 20-O-methyl oligoribonucleotides. A similar improvement in
nuclease resistance was observed in the 20-O-cyanoethyl modifica-
tion4, which suggests that the introduction of the cyano group into
the 20-modification could lead to a higher nuclease resistance.
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