Synthesis of oligonucleotides with glucosamine at the 3′-position and evaluation of their biological activity

Article abstract of DOI:10.1016/j.bmcl.2013.05.036

Short interfering RNA (siRNA) has been proven to be an utilizable tool for post-transcriptional gene silencing research. In this study, we designed and synthesized two glucosamine analogues and tried to modify the siRNA using these two glucosamine analogues at the 3′-overhang region of siRNAs to improve the nuclease resistance and to overcome some other weak points. The siRNAs modified with glucosamine analogues had almost no effect of the thermal stability and showed strong resistance to nuclease degradation. Some of them kept the same gene silencing activity level as unmodified siRNA.

Full text of DOI:10.1016/j.bmcl.2013.05.036

Bioorganic & Medicinal Chemistry Letters 23 (2013) 4157–4161
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of oligonucleotides with glucosamine at the 30-position
and evaluation of their biological activity q
Xiong Luo a, Takahiro Sugiura b, Remi Nakashima a, Yoshiaki Kitamura b, Yukio Kitade a,b,
⇑
a United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan
b Department of Biomolecular Science, Faculty of Engineering, 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
Article history:
Received 27 March 2013
Revised 1 May 2013
Accepted 10 May 2013
Available online 20 May 2013
Short interfering RNA (siRNA) has been proven to be an utilizable tool for post-transcriptional gene
silencing research. In this study, we designed and synthesized two glucosamine analogues and tried to
modify the siRNA using these two glucosamine analogues at the 30-overhang region of siRNAs to improve
the nuclease resistance and to overcome some other weak points. The siRNAs modified with glucosamine
analogues had almost no effect of the thermal stability and showed strong resistance to nuclease degra-
dation. Some of them kept the same gene silencing activity level as unmodified siRNA.
Keywords:
RNA
Ó 2013 The Authors. Published by Elsevier Ltd. All rights reserved.
siRNA
RNAi
Glucosamine analogues
Nuclease resistance
RNA interference (RNAi) has been a vital area of post-transcrip-
tional gene silencing research because of its high efficiency and se-
quence specificity in plants1 and mammalian cells.2 Moreover, a
chemically synthesized short interfering RNA (siRNA) duplex was
reported to induce an RNAi effect.3 RNAi technology, as a break-
through of gene therapy, benefits many incurable diseases;4–8
however, there are still a number of obstacles for in vivo experi-
ments and further clinical testing, such as low nuclease resistance,
poor membrane permeability, and off-target effects. In the wake of
the elucidation of the RNAi mechanism, chemical modification9–12
of siRNA is considered a potential approach to overcome these siR-
NA-related problems.
affinity and a positive charge that could be introduced into the 30-
overhang region of siRNA, which was expected to improve cell
membrane penetration, nuclease resistance, and other properties.
The amino sugar glucosamine, which is a structural element of
the polysaccharides chitosan and chitin, is considered to be a good
candidate. It is known that glucosamine shows high membrane
affinity.17 As a positively charged molecule, it can also neutralize
the negatively charged phosphates of siRNA. Furthermore, since
chitosan exists widely in organisms, such as the exoskeleton of
crustaceans and other arthropods and the cell wall of fungi,18 glu-
cosamine shows low toxicity to the human body.
Synthesis of modified glucosamine monomer units. To introduce
glucosamine into the 30-end of both sense and antisense siRNA
strands, we prepared glucosamine monomer unit 12, with a 10-C2
linker and 60-controlled pore glass (CPG) resin, as the solid support
for the DNA/RNA synthesizer from the starting material glucosa-
mine hydrochloride 1 (Scheme 1). Since directly introducing a thio-
phenyl group into the 10-position of nonprotected glucosamine
Until now, many chemical modifications of siRNAs at the 30-
overhang region or 50-end have been designed and synthe-
sized,13–16 most of which showed better thermal stability, nuclease
resistance, and other biological properties than the natural mole-
cules. In our laboratory, aromatic compounds or abasic nucleosides
were introduced at the 30-overhang region in previous studies
(Fig. 1).14–16 However, none of these modifications were aimed at
improving their affinity for the cell membrane or charge neutral-
ization. Therefore, we searched for a residue with high membrane
would result in an a/b mixture, it was necessary to protect the ami-
no and hydroxyl groups using phthalic anhydride and acetic anhy-
dride, respectively, to generate fully protected 2 in yield of 68%.
Treatment of thiophenol in the presence of a Lewis acid (BF3ꢀOEt2)
produced only b type 3 in 93% yield. Because of the failure to re-
move the phthalyl group during the post-treatment step of oligo-
nucleotide (ON) synthesis, we changed the protecting group to a
trifluoroacetyl group, which could be removed easily by aqueous
ammonia. Deprotection of the acetyl and phthalyl groups of 3 fol-
lowed by silylation of 60-OH, trifluoroacetylation of 20-NH2, and
q
This is an open-access article distributed under the terms of the Creative
Commons Attribution-NonCommercial-No Derivative Works License, which per-
mits non-commercial use, distribution, and reproduction in any medium, provided
the original author and source are credited.
⇑
Corresponding author. Tel./fax: +81 58 293 2640.
E-mail address: ykkitade@gifu-u.ac.jp (Y. Kitade).
0960-894X/$ - see front matter Ó 2013 The Authors. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.05.036

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X. Luo et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4157–4161
skipping the thiophenylation step. On account of the side reaction
of the later Mitsunobu reaction, we deprotected the acetyl groups
of 13 by treatment with MeONa in MeOH to give 14 in 88% yield.
N3-Benzoylthymine was introduced into the 60-position of 14 by
the Mitsunobu reaction to afford 15 in 84% yield. After removing
the phthalyl group of the 20-position and the benzoyl group of thy-
mine, the synthesis strategy was the same as Scheme 1 to generate
16–19 in 87, 90%, quant., and 79% yields, respectively. Subse-
quently, the CPG resin-linked solid support 20 (37
synthesized by the succination of 19.
lmol/g) was
Synthesis of glucosamine analogue-modified siRNAs. We chose the
Renilla luciferase gene as the target sequence. The basic siRNA se-
quences were as follows: Renilla sense, 50-CUUCUUCGUCGAGACC-
AUG-30; Renilla antisense 50-CAUGGUCUCGACGAAGAAG-30. By
using 12 and 20, ONs containing X or Y were synthesized using
an automatic DNA/RNA synthesizer (Fig. 2). Each of these siRNA se-
quences were specified by their 30-overhang region (0–2 molecules
of thymidine and glucosamine analogue X or Y), and fluorescent
amidites were also introduced at the 50-end of ONs 42–44 for fur-
ther assay tests.
The synthesized ONs were post-treated with the conventional
process11 and analyzed using matrix-assisted laser desorption/ion-
ization time-of-flight mass spectrometry. The observed molecular
weights of these ONs coincided with their calculated molecular
weight.19
Thermal stability. The melting temperatures (Tm) of these siRNAs
were recorded using a UV–visible spectrophotometer in a buffer of
10 mM Na2HPO4/NaH2PO4 (pH 7.0) containing 100 mM NaCl.
Absorbance at 260 nm was measured as a function of temperature
from 20 to 95 °C with a 0.5 °C/min increase. The Tm values of these
siRNAs are shown in Table 1. Neither of these 30-end modified siR-
NAs showed a significant decrease of Tm compared with the
unmodified molecule TT (Tm = 76.1 °C). Thus, we found that intro-
duction of glucosamine analogues at the 30-end of the ONs had
Figure 1. Structures of the modified siRNAs.
benzoylation of 30,40-OH afforded 4ꢁ7 in yields of 60, 86, 79, and
83%, respectively. In the presence of trifluoromethanesulfonic acid,
treatment of 2-benzyloxyethanol, N-iodosuccinimide with
molecular sieves 4 Å gave 8 in 78% yield. To remove the end of
the 2C linker, Pd(OH)2/C-catalyzed hydrogenating debenzylation
and 4,40-dimethoxytritylation were carried out to produce 9 and
10 in respective yields of 89% and 98%. Next, compound 10 was
desilylated by treatment with tetrabutylammonium fluoride to af-
ford 11 in yield of 68%. Subsequently, compound 11 was succinated
to yield the corresponding succinate linked to the CPG resin to gen-
erate solid support 12 (19 lmol/g).
Furthermore, in order to examine the difference of glucosa-
mine-modified siRNAs with/without a base moiety, we also syn-
thesized another type of glucosamine monomer unit that
included thymine at the 60-position by an improved synthesis
route (Scheme 2). In the presence of BF3ꢀOMe2, the treatment of
2-benzyloxyethanol to 2 could directly introduce a C2 linker into
the 10-position of glucosamine to give 13 in 67% yield, thereby
Scheme 1. Synthesis route of 60-abasic glucosamine monomer unit. Reagents and conditions: (a) (i) MeONa, MeOH, rt; (ii) phthalic anhydride, MeOH, rt; (iii) Ac2O, pyridine,
rt, 68%; (b) PhSH, BF3ꢀOEt2, CH2Cl2, rt, 93%; (c) ethylenediamine, MeCN, reflux, 60%; (d) TIPSCl, pyridine, rt, 86%; (e) (CF3CO)2O, pyridine, rt, 79%; (f) BzCl, pyridine, rt, 83%; (g)
2-benzyloxyethanol, NIS, TfOH, CH2Cl2, MS4 Å, rt, 78%; (h) H2, Pd(OH)2/C, THF, rt, 89%; (i) DMTrCl, pyridine, rt, 98%; (j) TBAF, THF, rt, 68%; (k) (i) succinic anhydride, DMAP,
pyridine, rt; (ii) CPG, EDC, DMF, rt, 19 lmol/g.

X. Luo et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4157–4161
4159
Scheme 2. Synthesis route of 60-basic glucosamine monomer unit. Reagents and conditions: (a) 2-benzyloxyethanol, BF3ꢀOMe2, MeCN, reflux, 67%; (b) MeONa, MeOH, rt, 88%;
(c) N3-benzoylthymine, PPh3, DEAD, THF, rt, 84%; (d) ethylenediamine, EtOH, reflux, 87%; (e) (CF3CO)2O, pyridine, rt, 90%; (f) H2, Pd(OH)2/C, THF, rt, quant; (g) DMTrCl,
pyridine, rt, 79%; (h) (i) succinic anhydride, DMAP, pyridine, rt; (ii) CPG, EDC, DMF, rt, 37 lmol/g.
Figure 2. Structures of the siRNAs in this study.
almost no effect on the thermal stability of the siRNA duplexes.
more than 95% after 3 min, in contrast, the modified ONs 43 and
44 were degraded by less than 40% after 3 min and approximately
30% still remained after 5 min. Therefore, the introduction of glu-
cosamine analogues at the 30-end of siRNAs significantly improved
their resistance against SVPD nuclease.
Gene silencing of Renilla luciferase. Furthermore, we examined
the gene silencing activity of each of these modified siRNAs using
a dual-luciferase assay in HeLa cells targeted to the Renilla lucifer-
ase gene.20 HeLa cells were co-transfected with the psiCHECK-2
vector containing Renilla luciferase and firefly luciferase and the
indicated amount of siRNA. The results were normalized by firefly
luciferase activity and the gene silencing efficacy of each siRNA
Moreover, the Tm values of the X- and Y-end siRNAs (22/23, 24/
25, and 26/27) were slightly different, which could indicate that
the nonthymine-containing glucosamine (X) siRNAs were more
thermally stable than the thymine-containing glucosamine (Y)
siRNAs.
Nuclease resistance. We tested the nuclease resistance of the 30-
end modified ONs 43 and 44 and the unmodified ON 42 against a
30-exonuclease, snake venom phosphodiesterase (SVPD). After
incubation in SVPD solution, degradation of the ONs was evaluated
using polyacrylamide gel electrophoresis analysis under denatur-
ing conditions (Fig. 3). The unmodified ON 42 was degraded by

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position) that could be introduced to the 30-overhang region of siR-
Table 1
Sequences of synthesized ONs used in this study
NAs. Compared with unmodified siRNA, glucosamine modification
of siRNAs did not affect their thermal stability. It was also found
that the X-modified siRNAs were more stable than the Y-modified
molecules. These modified siRNAs showed an obvious improve-
ment of resistance to nuclease degradation. All of these siRNAs
suppressed Renilla luciferase in a dose-dependent manner and
the Y-modified siRNAs showed better activity than the X-modified
siRNAs at 1 nM.
No. of siRNA
No. of ON
Sequence
Tm (°C)
siRNA 21
ON 28
ON 29
ON 30
ON 31
ON 32
ON 33
ON 34
ON 35
ON 36
ON 37
ON 38
ON 39
ON 40
ON 41
ON 42
ON 43
ON 44
50-CUUCUUCGUCGAGACCAUGtt-30
50-CAUGGUCUCGACGAAGAAGtt-30
50-CUUCUUCGUCGAGACCAUGX-30
50-CAUGGUCUCGACGAAGAAGX-30
50-CUUCUUCGUCGAGACCAUGY-30
50-CAUGGUCUCGACGAAGAAGY-30
50-CUUCUUCGUCGAGACCAUGtX-30
50-CAUGGUCUCGACGAAGAAGtX-30
50-CUUCUUCGUCGAGACCAUGtY-30
50-CAUGGUCUCGACGAAGAAGtY-30
50-CUUCUUCGUCGAGACCAUGttX-30
50-CAUGGUCUCGACGAAGAAGttX-30
50-CUUCUUCGUCGAGACCAUGttY-30
50-CAUGGUCUCGACGAAGAAGttY-30
F-50-CAUGGUCUCGACGAAGAAGtt-30
F-50-CAUGGUCUCGACGAAGAAGtX-30
F-50-CAUGGUCUCGACGAAGAAGtY-30
76.1
––
75.4
––
75.2
––
75.5
––
75.1
––
74.5
––
74.0
––
––
siRNA 22
siRNA 23
siRNA 24
siRNA 25
siRNA 26
siRNA 27
Besides ribose and aromatic series modifications at the 30-over-
hang region of siRNA, modification with a glucosamine analogue
represents a new direction for siRNA chemical modification re-
search. In this study, glucosamine monomer unit-modified siRNAs
showed some improvement of nuclease resistance. It is expected
that further study of glucosamine dimers or other base-containing
analogues will overcome more of the problems associated with
RNAi technology.
––
––
––
––
––
aUpper case letters indicate ribonucleosides and lower-case letters show 20-
Acknowledgment
deoxyribonucleosides.
bF denotes fluorescein.
This work was supported by Grants-in-Aid for Scientific Re-
search from the Japan Society for the Promotion of Science
(JSPS).
120
100
Supplementary data
ON 42
ON 43
ON 44
80
60
Supplementary data associated with this article can be found, in
the
online
version,
at
http://dx.doi.org/10.1016/j.bmcl.
2013.05.036.
40
20
0
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Incubation time (min)
Figure 3. Nuclease resistance of ONs 42ꢁ44 against SVPD.
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23
24
25
26
27
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Figure 4. Dual-luciferase assay.
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19. MALDI–TOF/MS analyses of ONs. ON 28: calculated mass, 6579.0; observed
mass, 6579.9. ON 29: calculated mass, 6727.5; observed mass, 6729.0. ON 30:
calculated mass, 6256.7; observed mass, 6257.9. ON 31: calculated mass,
6405.2; observed mass, 6407.0. ON 32: calculated mass, 6365.7; observed
mass, 6365.9. ON 33: calculated mass, 6514.9; observed mass, 6515.0. ON 34:
calculated mass, 6561.7; observed mass, 6562.0. ON 35: calculated mass,
6712.0; observed mass, 6711.0. ON 36: calculated mass, 6669.5; observed
mass, 6670.0. ON 37: calculated mass, 6819.0; observed mass, 6819.0. ON 38:
calculated mass, 6865.7; observed mass, 6866.0. ON 39: calculated mass,
7010.1; observed mass, 7015.1. ON 40: calculated mass, 6973.9; observed
mass, 6974.0. ON 41: calculated mass, 7122.4; observed mass, 7123.1. ON 42:
calculated mass, 7265.4; observed mass, 7266.1. ON 43: calculated mass,
was assessed as a percentage of the control sample (Fig. 4). Renilla
luciferase was suppressed by each of these siRNAs in a dose-
dependent manner. Moreover, the Y-modified (thymine-contain-
ing) siRNAs showed better activity (siRNA 23 > siRNA 22, siRNA
25 > siRNA 24, siRNA 27 > siRNA 26) than the X-modified (non-thy-
mine-containing) siRNAs at 1 nM. We supposed that the thymine
glucosamine could be a better choice for the 30-overhang region
modification of siRNAs.
In conclusion, we demonstrated a simple method for preparing
glucosamine monomer units (with/without a base moiety at the 60-

X. Luo et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4157–4161
4161
7248.3; observed mass, 7248.1. ON 44: calculated mass, 7355.7; observed
mass, 7356.1.
TransFast in Opti-MEM I reduced-serum medium (Invitrogen), and incubated
at 37 °C. Transfection without siRNA was used as a control. After 1 h, D-MEM
20. Dual-luciferase assay. HeLa cells were grown at 37 °C in
a
humidified
(100 lL) containing 10% bovine serum and antibiotics was added to each well,
atmosphere of 5% CO2 in air in D-MEM (Wako) supplemented with 10%
and the whole was further incubated at 37 °C. After 24 h, cell extracts were
frozen at ꢁ80 °C. Activities of firefly and Renilla luciferases in cell lysates were
determined with a dual-luciferase assay system (Promega). The results were
confirmed by at least three independent transfection experiments with four
cultures each and are expressed as the average from three experiments as
mean SD.
bovine serum (Sigma). 24 h before transfection, HeLa cells (4 ꢂ 104/mL) were
transferred to 96-well plates (100 lL per well). They were transfected, using
TransFast (Promega), according to instructions for transfection of adherent cell
lines. Cells in each well were transfected with a solution (35 L) of 20 ng of psi-
CHECK-2 vector (Promega), the indicated amounts of siRNAs, and 0.3 g of
l
l