Synthesis and biological properties of chemically modified siRNAs bearing 1-deoxy-d-ribofuranose in their 3′-overhang region

Article abstract of DOI:10.1016/j.bmcl.2012.01.132

To elucidate the role of the sugar moiety in the two natural nucleotides of the 3′-overhang region of small interfering RNA (siRNA), we synthesized siRNAs that incorporated two abasic nucleosides, 1-deoxy-d-ribofuranose (R ^H). We improved the method for preparing an O-protected abasic nucleoside, 1-deoxy-2,3,5-tri-O-benzoyl-β-d-ribofuranose, via the reductive cleavage of the anomeric position of 1-O-acetyl-2,3,5-tri-O-benzoyl-β-d- ribofuranose. To incorporate R^H into oligonucleotides by the standard phosphoramidite solid phase method, R^H was converted into its phosphoramidite derivative and the solid support linked to a controlled pore glass resin. Chemically modified RNAs possessing R^H at the 3′-overhang region were easily prepared in good yields. siRNAs containing R^H showed moderate nuclease-resistance and a desirable knockdown effect.

Full text of DOI:10.1016/j.bmcl.2012.01.132

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2518–2521
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological properties of chemically modified siRNAs bearing
1-deoxy-D-ribofuranose in their 30-overhang region
Kazumi Taniho a, Remi Nakashima b, Mahmoud Kandeel b, Yoshiaki Kitamura a, Yukio Kitade a,b,
⇑
a Department of Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
b United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
a r t i c l e i n f o
a b s t r a c t
To elucidate the role of the sugar moiety in the two natural nucleotides of the 30-overhang region of
small interfering RNA (siRNA), we synthesized siRNAs that incorporated two abasic nucleosides,
Article history:
Received 10 January 2012
Revised 26 January 2012
Accepted 31 January 2012
Available online 11 February 2012
1-deoxy-
1-deoxy-2,3,5-tri-O-benzoyl-b-
O-acetyl-2,3,5-tri-O-benzoyl-b-
D
-ribofuranose (RH). We improved the method for preparing an O-protected abasic nucleoside,
D
-ribofuranose, via the reductive cleavage of the anomeric position of 1-
D
-ribofuranose. To incorporate RH into oligonucleotides by the standard
phosphoramidite solid phase method, RH was converted into its phosphoramidite derivative and the solid
support linked to a controlled pore glass resin. Chemically modified RNAs possessing RH at the 30-over-
hang region were easily prepared in good yields. siRNAs containing RH showed moderate nuclease-resis-
tance and a desirable knockdown effect.
Keywords:
RNA
siRNA
RNAi
Abasic nucleoside
Deletion of nucleobase
Ó 2012 Elsevier Ltd. All rights reserved.
Chemically modified nucleosides have been utilized as biologi-
cally important compounds, for example, in virus therapy, cancer
therapy, and molecular biology.1,2 Furthermore, functional oligo-
nucleotides (ONs) containing artificial nucleic acids have been em-
ployed in diagnostic and therapeutic applications.3,4 Because
nucleic acid medicines such as antisense ONs and small interfering
and microRNAs (siRNAs and miRNAs, respectively) can be used for
sequence-dependent gene silencing,4 the development of nucleic
acid medicines has attracted considerable attention from medici-
nal chemists.
bioactive ONs. Several modified ONs containing RH have been de-
signed and synthesized.8 Several reports have described the stere-
oselective synthesis of RH;9 however, most of the existing protocols
require multiple steps. Zhou and co-workers succeeded in the di-
rect conversion of
D
-ribose into RH, but the reaction generated a
moderate yield.10
To elucidate the role of the sugar moiety at the two natural
nucleotides of the 30-overhang region, we synthesized siRNA incor-
porating an abasic nucleoside (RH) dimer at this position of each
RNA sequence (Fig. 2). We evaluated their binding activity to the
PAZ domain and knockdown effect. Furthermore, we improved
the synthetic method for preparing RH.
We recently reported the synthesis and biological properties
of modified siRNAs5 and miRNAs6 bearing hydrophobic 1,3-
bis(hydroxymethyl)benzene (B) and/or 1,3-bis(hydroxymethyl)-
pyridine (P) in their 30-overhang regions (Fig. 1). The 30-overhang
region of an antisense strand or guide strand in the case of miRNAs
is recognized by the Piwi/Argonaute/Zwille (PAZ) domain of Ago2,
and the 2 nucleotides at the 30-end are accommodated into the
binding pocket of the PAZ domain, which is composed of hydro-
phobic amino acids.7 The silencing effect of BB-modified siRNAs
was almost equal to that of normal siRNAs containing natural
nucleotides in the 30-overhang region.5 Furthermore, the analo-
gously BP-modified miRNA (miR-143/BP) showed a significant tu-
mor-suppressive effect on xenografted tumors of DLD-1 human
cancer cells.6
Scheme 1 shows a convenient method for 1 by using commer-
cially available 1-O-acetyl-2,3,5-tri-O-benzoyl-b-D-ribofuranose
(2) as a starting material. Treatment of 2 with trimethylsilyl tri-
fluoromethanesulfonate (TMSOTf) and triethylsilane (Et3SiH) in
MeCN at room temperature11 gave the desired deoxygenated prod-
uct 3 in quantitative yield. Deprotection of the benzoyl groups of 3
followed by benzylation gave 4. Removal of all benzyl groups by
catalytic hydrogenation afforded 1 (94% overall yield from 2).
To incorporate 1 into an ON by the standard phosphoramidite
solid phase method, 4 was converted into phosphoramidite deriv-
ative 8 and solid support 9 linked to controlled pore glass (CPG) re-
sin. Treatment of 1 with 4,40-dimethoxytrityl (DMTr) chloride in a
mixture of DMF and pyridine gave the corresponding 5-DMTr
1-Deoxy-D
-ribofuranose (RH; 1), an abasic nucleoside, is a useful
analog for studying the structures and mechanisms of action of
derivative 5 in 75% yield. Subsequent silylation gave the
corresponding 2-protected isomer 6 and 3-protected isomer 7 in
yields of 32% and 30%, respectively. The 2-protected isomer 6
was phosphitylated by standard procedure to produce the
⇑
Corresponding author. Tel./fax: +81 58 293 2640.
E-mail address: ykkitade@gifu-u.ac.jp (Y. Kitade).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.01.132

K. Taniho et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2518–2521
2519
Figure 1. Structures of the modified siRNAs.
Figure 2. Concept for this study.
corresponding phosphoramidite 8, quantitatively. On the other
hand, 6 was succinated to yield the corresponding succinate, which
was linked to CPG to generate solid support 9 (42 lmol/g).
(Tms) of the siRNAs. The Tm value of siRNA 11 (Tm = 77.9 °C), which
had RH in the 30-overhang region, was almost equal to that of the
unmodified siRNA 10 (Tm = 79.7 °C), which had thymidine in the
overhang region. Thus, it was found that the deletion of a nucleo-
base did not have a crucial effect on the thermal stability of the
Oligoribonucleotide synthesis: By using 8 and 9, ONs containing
RH were synthesized with a DNA/RNA synthesizer (Table 1). The
fully protected ONs linked to a solid support were treated with
concentrated NH4OH/EtOH (3:1, v/v) at room temperature for
12 h and with tetra-n-butylammonium fluoride (TBAF; 1.0 M solu-
tion in THF) at room temperature for 12 h. The ONs were purified
by denaturing 20% polyacrylamide gel electrophoresis (PAGE) to
isolate deprotected ONs bearing the abasic nucleoside RH. These
ONs were analyzed by matrix-assisted laser desorption/ionization
time-of-flight mass spectrometry (MALDI-TOF/MS), and the ob-
served molecular weights were in good agreement with their
structures.12
duplex.
14
Dual-luciferase assay:
We examined the silencing activity of
siRNAs 10 and 11 by a dual-reporter assay using a psiCHECK-2 vec-
tor in HeLa cells. The vector contains the Renilla and firefly lucifer-
ase genes, and the siRNA sequences were designed to target the
Renilla luciferase gene. HeLa cells were co-transfected with the
vector and the indicated amount of siRNAs. The levels of Renilla
luciferase were normalized to those of firefly luciferase. The silenc-
ing activity of the siRNA with the abasic nucleosides was found to
be greater than that of siRNA 10, which possessed the natural
nucleosides in the 30-overhang region (Fig. 3).
Thermal stability: 13 siRNA thermal stability was studied by ther-
mal denaturation in a buffer of 10 mM sodium phosphate (pH 7.0)
containing 100 mM NaCl. Table 2 shows the melting temperatures
Nuclease resistance: 15 Improving the nuclease resistance of syn-
thetic siRNAs is important for their therapeutic application. It was

2520
K. Taniho et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2518–2521
Scheme 1. Reagents and conditions: (a) Et3SiH, TMSOTf, MeCN, rt, quant; (b) (i) NaH, MeOH, rt; (ii) BnBr, NaH, DMF, rt, quant; (c) H2, Pd(OH)2/C, MeOH, rt, 94%; (d) DMTrCl,
DMF, pyridine, rt, 89%; (e) TBDMSCl, imidazole, DMF, rt, 32% for 6 and 31% for 7; (f) (i-Pr2N)P(Cl)O(CH2)2CN, i-Pr2NEt, THF, rt, 91%; (g) (i) succinic anhydride, DMAP, pyridine,
rt; (ii) CPG, EDCI, DMF, rt, 42 lmol/g.
Table 1
Sequences of ONs and siRNAs used in this study
No.of siRNA
No. of ON
Sequence
siRNA 10
ON 12
ON 13
50-GGCCUUUCACUACUCCUACtt-30
30-ttCCGGAAAGUGAUGAGGAUG-50
siRNA 11
ON 14
ON 15
50-GGCCUUUCACUACUCCUACRHRH-30
30-RHRHCCGGAAAGUGAUGAGGAUG-50
ꢀ
ꢀ
ON 16
ON 17
F-50-GUAGGAGUAGUGAAAGGCCtt-30
F-50-GUAGGAGUAGUGAAAGGCCRHRH-30
aCapital letters indicate ribonucleosides and small italicized letters show 20-
deoxyribonucleosides. bF denotes fluorescein.
Table 2
Figure 3. Dual-luciferase assay.
Melting temperatures (Tms) of siRNAs
No.of siRNA
Tm (°C)
siRNA 10
siRNA 11
79.7
77.9
expected that abasic nucleoside-substituted RNA would be more
resistant to nucleases than unmodified RNAs. The susceptibility
of RNAs to snake venom phosphodiesterase (SVPD), a 30-exonucle-
ase, was examined. Unmodified ON 16 and modified ON 17, which
were labeled at their 50-ends with fluorescein, were incubated with
SVPD. The reactions were analyzed using PAGE under denaturing
conditions. Unmodified ON 16 was hydrolyzed after 5 min of incu-
bation, while modified ON 17 was resistant to the enzyme (Fig. 4).
The half-life (t1/2) of unmodified ON 16 was <5 min, and that of
modified ON 17 was 10 min. ON 17 carrying an RH dimer at its
30-end was significantly more resistant to SVPD than unmodified
ON 16.
Figure 4. Nuclease resistance of ON 16 and ON 17 against SVPD.
In conclusion, we demonstrated a practical method for the prep-
aration of RH and the synthesis of siRNAs containing RH in their
30-overhang region. The silencing activity of the siRNAs was exam-
ined by using the dual-luciferase assay. It was found that the siRNA
with the abasic nucleosides in the 30-overhang region was more
effective than the siRNA possessing the natural nucleosides in this
region in an in vitro dual-luciferase experiment. Furthermore, the
modified RNA possessing RH was more resistant to nucleolytic
hydrolysis by SVPD than the unmodified RNA. Thus, the RH modifi-
cation may hold promise as a method to improve the silencing
activity and nuclease-resistance of RNA medicines.

K. Taniho et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2518–2521
2521
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Acknowledgment
This work was supported by Grants-in-Aid for Scientific Re-
search from the Japan Society for the Promotion of Science (JSPS).
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Supplementary data
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12. MALDI-TOF/MS analyses of ONs. The following spectra were obtained by MALDI-
TOF/MS (negative mode). ON 14: calculated mass, 6282.8; observed mass,
6282.9. ON 15: calculated mass, 6591.9; observed mass, 6595.3. ON 17:
calculated mass, 7130.1; observed mass, 7128.1.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2012.01.132.
13. Thermal denaturation study. Each solution containing each siRNA (3.0 lM) in a
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