Synthesis, RNAi activity and nuclease-resistant properties of apolar carbohydrates siRNA conjugates

Article abstract of DOI:10.1016/j.bmcl.2013.05.065

Oligoribonucleotide conjugates carrying apolar carbohydrates at the 5′-end and the corresponding siRNA duplexes have been prepared using phosphoramidite chemistry. All the carbohydrate-siRNA derivatives were compatible with RNA interference machinery if transfected with oligofectamine. In the absence of a transfection agent, some of them exerted certain reduction of gene expression. Double-tailed permethylated glucose conjugated to siRNA through a long spacer inhibited gene expression up to 26% compared to the scrambled duplex. Such modifications contribute positively to the stability of oligoribonucleotides against 5′-exonuclease degradation.

Full text of DOI:10.1016/j.bmcl.2013.05.065

Bioorganic & Medicinal Chemistry Letters 23 (2013) 4048–4051
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, RNAi activity and nuclease-resistant properties of apolar
carbohydrates siRNA conjugates
Empar Vengut-Climent a, Montserrat Terrazas b, Ricardo Lucas a, Matilde Arévalo-Ruiz a, Ramón Eritja b,
Juan Carlos Morales a,
⇑
a Department of Bioorganic Chemistry, Instituto de Investigaciones Químicas, CSIC-Universidad de Sevilla, Americo Vespucio 49, 41092 Sevilla, Spain
b Instituto de Química Avanzada de Cataluña, IQAC, CSIC, CIBER-BBN Networking Centre on Bioengineering, Biomaterials and Nanomedicine, Jordi Girona 18-26, E-08034
Barcelona, Spain
a r t i c l e i n f o
a b s t r a c t
Oligoribonucleotide conjugates carrying apolar carbohydrates at the 50-end and the corresponding siRNA
duplexes have been prepared using phosphoramidite chemistry. All the carbohydrate-siRNA derivatives
were compatible with RNA interference machinery if transfected with oligofectamine. In the absence of a
transfection agent, some of them exerted certain reduction of gene expression. Double-tailed permethy-
lated glucose conjugated to siRNA through a long spacer inhibited gene expression up to 26% compared to
the scrambled duplex. Such modifications contribute positively to the stability of oligoribonucleotides
against 50-exonuclease degradation.
Article history:
Received 12 April 2013
Revised 15 May 2013
Accepted 18 May 2013
Available online 28 May 2013
Keywords:
siRNA
Modified siRNA
Phosphoramidite
Carbohydrate
Lipophilic vector
Ó 2013 Elsevier Ltd. All rights reserved.
Oligonucleotides have shown great potential as therapeutic
agents by different mechanisms1 especially small interfering RNA
duplex (siRNA)2. However, the drug potential of siRNAs is still lim-
ited due to low stability and inefficient delivery.3 To increase its
stability towards nuclease degradation and increase cellular up-
take, modified oligonucleotides and conjugation of siRNA to differ-
ent delivery carriers such as antibodies, lipids, polymers, peptides
or carbohydrates have been reported.4 Among them, lipids are
being widely studied and cholesterol conjugates have been tested
in vivo showing promising results.5 Conjugation to lipophilic mol-
ecules introduces a partial hydrophobic character to the bioconju-
gates which may enhance their cellular uptake not only by
increased membrane permeability but also through receptor med-
iated endocytosis.6 Some hydrophobic modifications used on siR-
NA with some success are shown in Fig. 1.
We recently reported the synthesis of glucose oligonucleotide
conjugates where the sugar was attached to the 50-end of the
DNA strand and had different spatial presentations.7 Cell uptake
studies using flow cytometric analysis showed that conjugates
with the glucose moiety linked through long spacers (15–18 atom
distances) were better internalized than those with short linkers (4
atom distance) or than DNA control strands without sugar
modification.
Then we synthesized carbohydrate–siRNA duplexes targeting
tumor necrosis factor carrying one, two or four glucose and galact-
ose residues at the 50-end of the passenger strand. The modified
siRNA duplexes have similar inhibitory properties than unmodified
RNA duplexes in HeLa cells when transfected with oligofectamine.8
Without transfection agent, glucose–siRNA conjugates showed no
inhibition whereas galactose–siRNA duplexes on HuH-7 cells
which possess abundant asialoglycoprotein receptors displayed
up to 25% anti-TNF inhibitory properties.
Here, the idea was to examine apolar carbohydrates linked to
siRNA duplexes as new hydrophobic platforms that may improve
the oligoribonucleotide cell uptake without disrupting the RNAi
machinery during gene inhibition. The design consisted of per-
methylated glucose attached to the 50-end of the passenger strand
of the siRNA via two different spacers, a short ethylene glycol and a
long dodecanodiol (Fig. 2). A dendron scaffold was also used since
it has been demonstrated that lipid–oligonucleotide conjugates
presenting double-tailed lipid modifications show good results in
cellular uptake.9 A spacer–oligonucleotide conjugate was also pre-
pared as a negative control.
Synthesis. We synthesized carbohydrate–RNA conjugates using
standard solid-phase oligonucleotide automatic synthesis using
the corresponding apolar carbohydrate phosphoramidites
(Scheme 1). The preparation of permethylated glucose phospho-
ramidite derivative 6 containing the short spacer was carried out
following the same methodology described previously by our
⇑
Corresponding author. Tel.: +34 954489568; fax: +34 954460565.
E-mail address: jcmorales@iiq.csic.es (J.C. Morales).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.05.065

E. Vengut-Climent et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4048–4051
4049
OR
O
H
H
O
OR
H
H
O
OR
R= spacer-siRNA
OR
Figure 1. Lipophilic vectors used in the literature to improve siRNA cell uptake. Soutschek et al. (2004) used a cholesterol, Godeau et al. (2008) used a fatty alcohol, MacKellar
et al. (1992) used a double aliphatic alcohol and Nishina et al. (2008) used an alpha-tocopherol.
group.10 This strategy was extended to the apolar sugar with the
long dodecanodiol spacer 11. Briefly, glycosylation of the O-benzyl
Dual-luciferase assay. After RNA synthesis, we evaluated the ef-
fect of the above described 50-sense-carbohydrate-modified siRNAs
on the ability of the corresponding conjugate derivatives to act as
inhibitors of gene expression. Furthermore, we tested the ability of
such conjugates to impart cell uptake. We carried out separate
gene knockdown experiments in HeLa cells. In a first series of
experiments, the cells were cotransfected with two luciferase plas-
mids (Renilla and firefly; target and internal control, respectively),
with siRNA duplexes containing each of the five modified passen-
ger strands (1–4), and with the corresponding unmodified siRNA
(wt). Cotransfection of plasmids and siRNAs was carried out by
using commercial cationic liposomes (Lipofectamine 2000).
Twenty-two hours after transfection, the luciferase activities of
the samples were measured by using a luminometer. The results,
showing Renilla luciferase activity normalized to firefly luciferase,
are shown in Fig. 3. Interestingly, the presence of permethylated
glucoseC2, and permethylated glucoseC12 at the 50-end of the pas-
senger strand (1 and 2, respectively) did not disrupt RNAi activity.
The corresponding siRNA conjugates showed activities comparable
to the unmodified siRNA (wt) [(90 1)% knockdown of Renilla
expression for unmodified wt vs (90 1)% and (89 1)% for 1 and
2, respectively]. Conjugation of permethylated glucose C12-dou-
bler and C12 spacer to the 50-end of the sense strand siRNA (conju-
gates 3 and 4, respectively) caused a slight decrease in activity, but
gene silencing activity of these siRNAs was still quite remarkable:
[(71 1)% and (71 3)% for 3 and 4, respectively, vs (90 1)% for
wt]. A sequence scrambled siRNA (as negative control) was used
to ensure specificity. Cells cotransfected with the scrambled siRNA
protected dodecanodiol spacer with
a-bromoacetoglucose 7 re-
sulted in compound 8 in 62% yield. Deprotection of the acetyl
groups followed by methylation under standard conditions pro-
duced compound 9 in good overall yield (78%, two steps). Then,
hydrogenation and standard phosphoramidite preparation were
carried out yielding permethylated glucose phosphoramidite 11
(67% and 74%, respectively). We also prepared a permethylated cel-
lobiose phosphoramidite 12 and a perbutylated glucose phospho-
ramidite 13, both with the long spacer following the same
synthetic strategy. A dodecanol phosphoramidite control 14 was
also synthesized from 1-dodecanol as described previously.11
RNA synthesis. The siRNA sequences designed to target Renilla
Luciferase gene were 50-AUCUGAAGAAGGAGAAAAATT-30 as the
passenger sequence and 50-UUUUUCUCCUUCUUCAGAUTT-30 as
the guide sequence. Oligoribonucleotide conjugates (1–4) were
prepared using a DNA synthesizer. Apolar sugar phosphoramidites
were added at the last step on the RNA synthesis. Oligonucleotides
presented a major peak in HPLC chromatograms with some impu-
rities but they were easily separated by HPLC obtaining the desired
conjugates. However, isolation of siRNA conjugates containing per-
methylated cellobiose or perbutylated glucose, both with the long
spacer, was not successful due to problems either during coupling
with the corresponding phosphoramidites or during the cleavage
and deprotection steps. Oligoribonucleotide sequences carrying
apolar carbohydrates at the 50-end of the passenger strand were
hybridized to the unmodified guide strand.
OMe
4
1
O
O
O
O
O
MeO
MeO
O RNA sequence
P
O
P
O
RNA sequence
O
O
OMe
OMe
2
O
O
O
MeO
MeO
O
P
O
O
RNA sequence
OMe
OMe
O
O
O
O
MeO
O
P
MeO
O
O
NH
OMe
3
O
O
NH
P
O
OMe
O
O
RNA sequence
O
O
MeO
O
O
P
MeO
OMe
O
O
Figure 2. Structure of the carbohydrate oligoribonucleotide conjugates prepared in this study. RNA sequence is the passenger sequence: 50-AUCUGAAGAAGGAGAAAAATT-30.
The RNA sequence of the guide is: 50-UUUUUCUCCUUCUUCAGAUTT-30.

4050
E. Vengut-Climent et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4048–4051
OAc
O
OAc
a
O
AcO
AcO
O
AcO
AcO
OBn
OAc
8
AcO
7
Br
b, c
OMe
O
OMe
O
O
O
MeO
MeO
O
MeO
MeO
d
OR
OBn
OMe
OMe
10
11
R=H
R=
e
9
O
CN
P
N(iPr)2
OMe
O
OMe
N(iPr)2
MeO
O
MeO
O
O
MeO
P
MeO
MeO
CN
O
P
CN
O
O
O
MeO
O
OMe
OMe
OMe
N(iPr)2
6
12
O
N(iPr)2
O
P
O
CN
O
O
N(iPr)2
O
O
O
P
CN
O
O
13
14
Scheme 1. Synthesis of permethylated glucose phosphoramidite derivatives 6 and 11. Compound 6 was synthesized as described in Ref. 9. Reagents and conditions: (a) 1,12-
dodecanediol, monobenzyl ether, Ag2CO3, CH2Cl2; (b) Na2CO3, MeOH; (c) MeI, NaH, DMF; (d) H2, Pd(OH)2, THF/MeOH; (e) 2-cyanoethyl-N,N0-diisopropylamino-
chlorophosphoramidite, DIPEA, CH2Cl2.
Nuclease-resistant properties. Since the above experiments
showed that most of the 50-terminal modifications could mediate
RNAi activity to levels comparable to wild type siRNA, and that
some siRNA conjugates were able to penetrate the cell membrane,
it was of interest to study the 50-exonuclease resistance of these 50-
modified oligonucleotides. To carry out such studies, unmodified
(wt) or 50-modified passenger strands 1–4 were incubated with Bo-
vine Spleen Phosphodiesterase (BSP). At various time points, ali-
quots of the reactions were withdrawn and the samples were
analyzed by polyacrylamide gel electrophoresis (Fig. 5). Unmodi-
fied passenger strand showed low stability, with only ꢀ5% of the
original oligonucleotide strand remaining intact after 24 h of incu-
bation with BSP. Permethylated glucose C12-doubler-passenger 3
displayed a slight increase in stability, with 40% and 5% of the ori-
ginal oligonucleotide remaining intact through 24 and 48 h,
respectively. Very interestingly, conjugation of C12 to the 50-end
Figure 3. Reduction in Renilla luciferase activity of modified siRNAs 1–4 in Hela
cells using oligofectamine as transfecting agent for siRNA. The cells were treated
with 0.8 nM siRNA. Mean values SD from three independent experiments are
presented.
showed levels of Renilla luciferase activity similar to those of cells
transfected with plasmids alone. Thus, the results indicate that the
siRNA 1–4 do specifically induce inhibition of expression of the tar-
get gene.
Finally, in a second series of experiments we examined the abil-
ity of conjugates 1–4 to improve cell uptake in comparison to
naked siRNA (Fig. 4). Three hours after transfection of the two
luciferase plasmids, the cell medium was discarded and the cells
were incubated with fresh medium and the siRNA derivatives in
the absence of Lipofectamine 2000 (750 nM siRNA conjugates).
Twenty-two hours after transfection the luciferase activities were
measured as described above. Although gene knockdown was sig-
nificantly less efficient than that observed for siRNA-lipofectamine
complexes, results showed that siRNA conjugates 3 and 4 were
Figure 4. Reduction in Renilla luciferase activity of modified siRNAs 1–4 in Hela
cells in the absence of oligofectamine as transfecting agent for siRNA. The cells were
treated with 750 nM siRNA. Mean values SD from three independent experiments
are presented.
able to penetrate HeLa cells in the absence of a transfection agent
[(15 3)% and (26 12)% knockdown of Renilla expression for 3
and 4, respectively, vs (2 2)% for wt)].

E. Vengut-Climent et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4048–4051
4051
compared to the scrambled duplex. We also showed that conjuga-
tion of C12 spacer, permethylated glucose C2, permethylated glu-
cose C12 and the corresponding C12 doubler to the 50-end of the
passenger strand substantially enhanced resistance to 50-exonucle-
ase-catalyzed hydrolysis. Altogether, our data demonstrates that
apolar carbohydrates are a new tool for the development of nucleic
acids-based drugs with potential value in the preparation of for-
mulations for intravenous administration as described for lipid-
modified oligonucleotides.5,6
Acknowledgments
This study was supported by the Spanish Ministry of Economy
and Competitiveness (Grants CTQ2009-13705, CTQ2010-20541),
Generalitat de Catalunya (2009/SGR/208). R.L. thanks CSIC for a
JAE contract and E.V.C. thanks Ministry of Education, Culture and
Sports for a FPU Grant. M.T. acknowledges a Juan de la Cierva con-
tract (MICINN, Spain) for financial support.
Supplementary data
Supplementary data associated with this article can be found, in
the
online
version,
at
http://dx.doi.org/10.1016/
j.bmcl.2013.05.065.
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