Simple solid-phase synthesis and biological properties of carbohydrate - Oligonucleotide conjugates modified at the 3′-terminus

Article abstract of DOI:10.1021/bc100205v

A novel synthesis method for oligonucleotides possessing a functional moiety at the 3′-terminus was established based on solid-phase synthesis. In order to install the functional group at the 3′-terminus of the oligonucleotide, a solid support possessing

Full text of DOI:10.1021/bc100205v

Bioconjugate Chem. 2010, 21, 1685–1690
1685
Simple Solid-Phase Synthesis and Biological Properties of
Carbohydrate-Oligonucleotide Conjugates Modified at the 3′-Terminus
Yutaka Ikeda,† Daisuke Kubota,‡ and Yukio Nagasaki*
Grauate School of Pure and Applied Science, Tsukuba Research Center for Interdisciplinary Materials Science (TIMS), Center
for Tsukuba Advanced Reserch Aliance (TARA), International Center for Materials Nanoarchitectonics Satellite (MANA), and
National Institute for Materials Science (NIMS), University of Tsukuba, CREST, Japan Science and Technology Agency (JST),
and Master’s School of Medical Sciences, Graduate School of Comprehensive Human Science. Received April 28, 2010;
Revised Manuscript Received June 30, 2010
A novel synthesis method for oligonucleotides possessing a functional moiety at the 3′-terminus was established
based on solid-phase synthesis. In order to install the functional group at the 3′-terminus of the oligonucleotide,
a solid support possessing the functional group was prepared. A carbohydrate was employed in this study for the
functionalization of the oligonucleotide. To prepare a glycosylated solid support, a novel glycosyl acceptor (2)
was synthesized using 4,4-dihydroxymethyl-cyclopenta-1-ene as the starting compound. The glycosylation reaction
proceeded smoothly (yield ) 95%) to yield the suitable glycosylated compound (3). After 8 was immobilized on
the solid support, it was subjected to solid-phase oligonucleotide synthesis by the standard phosphoramidite coupling
method. An oligonucleotide possessing a sugar moiety at the 3′-terminus was obtained after the products were
deprotected and cleaved from the solid support. The stability of the carbohydrate-modified oligonucleotide was
greatly increased even in the serum buffer, indicating that the sugar moiety at the 3′-position improved the resistance
against enzymatic degradation. This technique was also applied to RNA synthesis. Galactose-ended siRNA was
prepared and was confirmed to possess enough ability, at a concentration of 10 nM, to regulate the expression of
the target gene.
fashion, the gene-silencing efficiency is retained at almost the
original level regardless of whether the modification is made
in one or both of the strands. However, the function of siRNA
is substantially impaired by modification with LNA at the 5′-
terminus of the antisense strand (5). Terminal modification of
the oligonucleotide with a biologically functional molecule is
very useful, because this type of modification can be anticipated
not only to increase the stability of the oligonucleotide, but also
to incorporate the additional biological function into the
oligonucleotide drug. For example, Soutschek et al. have
reported that the modification of the 3′-terminus of the sense
strand in siRNA with cholesterol improves its pharmacokinetic
properties in ViVo, probably due to the enhancement of the
binding of the cholesterol-ended siRNA (chol-siRNA) to serum
albumin (6). Even 24 h after the injection of the conjugate into
mice, significant levels of chol-siRNA were detected in the liver,
heart, kidney, adipose tissue, and lung (6).
Cationic cell-penetrating peptides (CPPs), such as the HIV-
derived Tat peptides and the Antennapedia peptide, are known
to be taken up by eukaryotic cells, and are therefore one
important candidate as ligands to deliver a variety of molecules
into cells by conjugation (7). Because CPPs possess a positive
charge, the design of the conjugates must be carefully consid-
ered. When an anionic oligonucleotide is conjugated with a
cationic CPP, for example, electrostatic aggregation cannot be
avoided; this lowers the cell-penetrating ability and subsequently
decreases the delivery efficiency of the conjugate (8). Uncharged
antisense oligonucleotides, such as peptide nucleic acids (PNAs)
(9) and phosphorodiamidate morpholino oligomers (PMOs) (10),
must be employed instead.
INTRODUCTION
Oligonucleotide-based drugs such as antisense, aptamer, and
siRNA drugs have attracted considerable attention as promising
therapeutic agents in the treatment of human diseases (1, 2).
Especially, siRNA is considered to be a new class of drug
because it can suppress the expression of the target gene even
at low concentrations (3). Several oligonucleotide drugs are now
being evaluated in clinical trials; however, at present, only two
drugs, Macugen and Vitramune, have been approved by the U.S.
Food and Drug Administration. This reflects the present
difficulty of exploiting therapeutic oligonucleotides. Several
issues must be overcome in the development of drugs based on
oligonucleotides, including the instability of oligonucleotides
against enzymatic degradation and the requirement that the drug
delivery system should be efficient and safe.
To increase the stability of oligonucleotide drugs, various
chemical modifications of oligonucleotides have been attempted.
Such chemical modifications must be designed so that they do
not inhibit the activity of the oligonucleotide (4). For example,
in the case of siRNA, the modifications should not interfere
with the recognition of siRNA by the RNA-induced silencing
complex (RISC), which mediates the destruction of the target
RNA in RNAi, and should also not interfere with subsequent
reactions, including the degradation of the target mRNA.
In general, the activity of oligonucleotide drugs strongly
depends on their modification sites in the sequence. For example,
when the 3′-terminus of siRNA is modified with a rigid nucleic
acid, called a “locked nucleic acid (LNA)”, in an overhang
* yukio@nagalabo.jp.
† Grauate School of Pure and Applied Science,TIMS, and TARA,
University of Tsukuba, and CREST, JST.
‡ Grauate School of Pure and Applied Science, and TIMS, University
of Tsukuba.
Another candidate for conjugation with oligonucleoptides is
carbohydrates. Carbohydrates are ubiquitous in living systems
and are present either as free carbohydrates or as glycoconju-
gates, such as glycoproteins or glycolipids. They play roles in
10.1021/bc100205v  2010 American Chemical Society
Published on Web 08/12/2010

1686 Bioconjugate Chem., Vol. 21, No. 9, 2010
Ikeda et al.
bacterial and viral adhesion, tumor cell metastasis, the regulation
of hormone and enzyme activities, and so forth (11). For
example, various membrane lectins mediate the sugar-ligand-
assisted endocytosis of complexes followed by complex dis-
sociation in the acidic environment of the endosomes (12).
Lectins also play an important role in delivering conjugates to
the right cellular compartment, such as the cytosol or the
nucleus. Since most carbohydrates are electrically neutral, the
undesirable interactions mentioned above can be avoided
through covalent conjugation with different types of biomol-
ecules. Thus, these biological activities and the chemical
properties of carbohydrates are useful in the development of
functional conjugates for drug delivery systems.
To date, several groups have reported the development of
methods to modify oligonucleotides with carbohydrates (13).
Conjugation of a reactive carbohydrate with an oligonucleotide
in solution is one of the major methods of modification (14).
For mass production, however, it is obvious that conjugation
on a solid phase during the synthesis of the oligonucleotide is
much more advantageous than conjugation in solution. One of
the most evident ways to incorporate a carbohydrate residue
into an oligonucleotide on a solid phase is to synthesize a
phosphoramidite derivative. If sugar-conjugated phosphoramid-
ite derivatives are newly prepared, the carbohydrate can be
introduced not only at the 5′-terminus, but at any site in the
oligonucleotide sequence (15, 16).
Few studies have been carried out on the modification of the
3′-terminus of the oligonucleotide, due to the rather complicated
modification reaction at this terminus. Adinolfi et al. have
reported the installation of a carbohydrate at the 3′-terminus of
an oligonucleotide (16). They used a protected carbohydrate
on a solid phase and synthesized the oligonucleotide from the
C6 position of the carbohydrate. To our knowledge, only Maier’s
group has developed the glycosylated solid support for the
synthesis of sugar-conjugated oligonucleotides, in which the C1
position of the carbohydrate is linked to the 3′ terminus of the
oligonucleotide. They prepared an oligonucleotide possessing
a carbohydrate at the 3′-terminus by using ε-aminocapronic acid
as a bifunctional linker. They prepared 1-ꢀ-aminocarbohydrate,
followed by condensation with protected ε-aminocapronic acid
(17). Recently, they developed another method (18) of conjuga-
tion based on the method previously reported by Reed et al.
(19). In their method, however, the carbohydrate has to be
converted to aminocarbohydrate before the condensation reac-
tion with the bifunctional linker, resulting in a complicated
reaction scheme for the preparation of the glycosylated solid
support. Accordingly, we tried to establish an alternative method
of modifying the oligonucleotide at the 3′-terminus with the
C1 position of the carbohydrate.
In this report, we describe our novel, simple, solid-phase
synthesis method of fabricating carbohydrate-oligonucleotide
conjugates modified at the 3′-terminus of the oligonucleotide.
We created a novel glycosylated solid support for efficient 3′-
terminus modification. In our method, a protected carbohydrate
is directly conjugated by the glycosylation reaction, without any
transformation. The modified oligonucleotide is much more
stable against enzymatic degradation. We also evaluated the
effect of the modification on the activity of siRNA. The gene-
silencing activities of the modified siRNAs were well retained
after the conjugation.
After stirring at room temperature for 1 h, benzyl bromide (306
µL, 2.6 mmol) was added and stirred at room temperature for
12 h. The reaction mixture was extracted with ethyl acetate and
saturated ammonium chloride. The organic solvent was washed
with brine, dried over MgSO4, and concentrated. Purification
on a silica gel column with hexane-ethyl acetate gave
1
compound 2 (440 mg, 86%). H NMR (CDCl3, 270 MHz) δ
7.42-7.27 (5H, m), 5.63 (2H, s), 4.56 (2H, s), 3.64 (2H, d, J
) 5.8), 3.55 (2H, s), 2.34-2.17 (4H, m). ESI TOF/MS m/z:
[M+Na]+ 241.1769.
(1-(Benzyloxymethyl)cyclopent-3-enyl)methyl 2,3,4,6-tetra-
O-acetyl-ꢀ-D-galactopyranoside (3). Compound 2 (100 mg, 0.46
mmol) and 1,2,3,4,6-penta-O-acetyl-D-galactose (179 mg,0.46
mmol) were co-evaporated 3 times with dry toluene and
dissolved in 4 mL of CH2Cl2. To the solution, boron trifluoride
diethyl etherate (84.9 µL, 0.69 mmol) was added. After stirring
at room temperature for 12 h, the reaction was quenched with
aqueous NaHCO3. The reaction mixture was extracted with ethyl
acetate. The organic solvent was washed with brine, dried over
MgSO4, and concentrated. Purification on a silica gel column
with hexane-ethyl acetate gave compound 3 (240 mg, 95%).
1H NMR (CDCl3, 500 MHz) δ 7.38-7.27 (5H, m), 5.58 (2H,
s), 5.38 (1H, dd, J ) 2.4, 3.5), 5.21 (1H, dd, J ) 8.0, 10.5),
5.00 (1H, dd, J ) 10.5, 3.5), 4.53 (2H, d, J ) 2.9), 4.44 (1H,
d, J ) 8.0), 4.15 (2H, m), 3.89 (1H, dd, J ) 6.83, 2.4), 3.85
(1H, d, J ) 9.3), 3.50 (1H, J ) 9.3), 3.40 (1H, d, 8.7), 3.34
(1H, d, 8.7), 2.22 (2H, d, 11.7), 2.17 (3H, s), 2.14 (2H, d, 11.7),
2.06 (3H, s), 2.05 (3H, s), 2.00 (3H, s). ESI TOF/MS m/z:
[M+Na]+ 571.2296.
(1-(Benzyloxymethyl)-3,4-dihydroxycyclopentyl)methyl2,3,4,6-
tetra-O-acetyl-ꢀ-D-galactopyranoside (4). Compound 3 (240 mg,
0.44 mmol) was dissolved in acetone/H2O/acetonitrile (1:1:1,
v/v/v). To the solution, 4-methylmorpholine N-oxide (103 mg,
0.87 mmol) and osmium oxide-immobilized catalyst I (Wako)
(3 mg) were added. After stirring at room temperature for 12 h,
the reaction mixture was extracted with ethyl acetate and brine.
The organic solvent was dried over MgSO4 and concentrated.
Purification on
a
silica gel column with hexane-
ethyl acetate gave compound 4 in diastereomers (170 mg, 67%).
1H NMR (CDCl3, 500 MHz) δ 7.37-7.32 (5H, m), 5.38 (1H,
d, J ) 2.9), 5.21 and 5.17 (1H, dd, J ) 7.4, 10.5 and J ) 7.9,
10.5), 5.01 (1H, dd, J ) 3.6, 10.5), 4.58 (1H, d, J ) 6.05),
4.48 (1H, d, 6.05), 4.47 and 4.39 (1H, d, J ) 7.4 and J ) 7.9),
4.17 (1H, dd, J ) 4.8, 11.2), 4.12 (1H, dd, J ) 4.4, 11.2), 4.00
(1H, m), 3.94 (1H, m), 3.86 (1H, m), 3.71 and 3.24 (1H, d, J
) 9.0 and 8.8), 3.44 and 3.22 (1H, d, J ) 9.0 and 8.8), 3.36
and 3.22 (1H, d, J ) 8.4 and 9.1), 3.30 and 3.20 (1H, d, J )
8.4, 9.1), 3.00 and 2.98 (1H, d, J ) 9.1), 2.48 and 2.44 (1H, d,
J ) 7.7), 2.17 (3H, s), 2.04 (3H, s), 2.03 (3H, s), 1.99 (3H, s),
1.88 (1H, dd, J ) 6.4, 13.8), 1.81 (1H, dd, J ) 9.1, 13.8), 1.76
(1H, dd, J ) 7.7, 13.5), 1.70 (1H, dd, J ) 5.2, 13.5). ESI TOF/
MS m/z: [M+Na]+ 605.6320.
(3-Acetoxy-1-(benzyloxymethyl)-4-hydroxycyclopentyl)meth-
yl 2,3,4,6-tetra-O-acetyl-ꢀ-D-galactopyranoside (5). Compound
4 (470 mg, 0.82 mmol) was dissolved in 8.2 mL of toluene. To
the solution, acetic anhydride (114 µL, 1.2 mmol) was added.
After stirring at room temperature for 12 h, the reaction mixture
was extracted with aqueous 1 N HCl and ethyl acetate. The
organic solvent was washed with aqueous NaHCO3 and brine,
dried over MgSO4, and concentrated. Purification on a silica
gel column with hexane-ethyl acetate gave compound 5 (292
EXPERIMENTAL PROCEDURES
1
mg, 57%) in diastereomers. H NMR (CDCl3, 500 MHz) δ
Synthesis. (1-(Benzyloxymethyl)cyclopent-3-enyl)methanol
(2). Compound 1 was synthesized according to a previously
reported method (20). Compound 1 (300 mg, 2.3 mmol) was
dissolved in 6 mL of N,N-dimethylformamide (DMF). To the
solution, NaH (140 mg, 2.3 mmol) in DMF (2 mL) was added.
7.37-7.22 (5H, m), 5.38 (d, J ) 2.8), 5.19 (1H, dd, J ) 7.8,
10.5), 4.99 (1H, dd, J ) 2.8, 10.5), 4.55 (1H, d, J ) 4.6), 4.5
(1H, d, J ) 4.6), 4.42 (1H, d, J ) 7.8), 4.22 (1H, m), 4.18 (1H,
m), 3.88 (1H, m), 3.76 and 3.37 (1H, d, J ) 9.0 and 8.4), 3.46
and 3.33 (1H, d, J ) 9.0 and 8.4), 3.35 and 3.29 (1H, d, J )

Modified Carbohydrate-Oligonucleotide Conjugates
Bioconjugate Chem., Vol. 21, No. 9, 2010 1687
10.3 and 8.8), 3.13 and 3.23 (10.3 and 8.8), 2.83 (1H, d, J )
7.7), 2.16 (3H, s), 2.10 (3H, s), 2.05 (3H, s), 2.01 (3H, s), 1.98
(3H, s), 1.93 (1H, m), 1.81 (1H, m), 1.72 (1H, m), 1.65 (1H,
m). ESI TOF/MS m/z: [M+Na]+ 647.7208.
(3-Acetoxy-4-hydroxy-1-(hydroxymethyl)cyclopentyl)methyl 2,3,
4,6-tetra-O-acetyl-ꢀ-D-galactopyranoside (6). A stirred mixture
of compound 5 (600 mg, 0.96 mmol) and palladium on carbon
(Pd/C) (488 mg) in methanol (13 mL) was hydrogenated at
ambient pressure (balloon) and temperature for 24 h. The
mixture was filtered through Celite. The solvent was evaporated,
and the residue was chromatographed on silica gel with
hexane-ethyl acetate to give compound 6 in diastereomers. This
material was used in the next step without further purification.
10% serum at 37 °C. The modified oligonucleotides, which were
labeled with fluorescence at the 5′-end of the strand and
galactose at the 3′-end of the strand, and the control oligonucle-
otide, which was labeled with fluorescence at the 5′-end of the
strand, were assayed for serum stability. Each oligonucleotide
(400 pmol) was incubated with medium (100 µL) containing
10% of serum at 37 °C for the indicated time. After the
incubation, the solution was diluted with formamide (100 µL)
and heated at 100 °C for 2 min to quench the degradation. The
samples were subjected to polyacrylamide gel electrophoresis
and visualized by fluorescence scanning on a Typhoon 9400
(GE Healthcare).
Transfection of siRNA for Surviving and Quantification
of Survivin mRNA. HeLa cells were cultured in Dulbecco’s
modified Eagle’s medium (Sigma-Aldrich, St. Louis, Mo)
supplemented with 10% heat-inactivated fetal bovine serum and
grown at 37 °C in a humidified atmosphere of 5% CO2. 1 ×104
HeLa cells were cultured in 96-well culture plates in 100 µL of
DMEM supplemented with 10% FBS, and incubated for 24 h.
The indicated amounts of siRNA were transfected by Lipo-
fectamine 2000 (Invitrogen) according to manufacturer’s pro-
tocol. siRNA was mixed with 24 µL of OPTI-MEM. 0.5 µL of
Lipofectamine 2000 was diluted in 24.5 µL of OPTI-MEM.
After 5 min of incubation, they were combined and incubated
for 20 min at room temperature. This solution was added to
the cells, and the cells were incubated for an additional 48 h.
The amounts of survivin mRNA were determined by quan-
titative RT-PCR on a Thermal Cycler Dice Real Time System
(TP-800) (TaKaRa). Isolation of the total RNA was performed
using a CellAmp Direct RNA Prep Kit for One-Step RT-PCR
(TaKaRa). Synthesis of cDNA and quantitative RT-PCR were
performed using a One-Step SYBR PrimeScript RT-PCR Kit
II (TaKaRa). The gene-specific primers used in the quantitative
PCR were as follows: survivin forward primer, 5′-AGAACTG-
GCCCTTCTTGGAGG-3′; survivin reverse primer, 5′-CTTTT-
TATGTTCCTCTATGGGGTC-3′; GAPDH forward primer,
GGTGGTCTCCTCTGACTTCAACA-3′; and GAPDH reverse
primer, 5′-GTTGCTGTAGCCAAATTCGTTGT-3′. The relative
quantification of gene expression was performed as described
in the above-mentioned user’s manual, using GAPDH as an
internal standard.
(3-acetoxy-1-O-(4,4′-dimethoxytrityl)methyl-4-hydroxy)cyclo-
pentyl)methyl 2,3,4,6-tetra-O-acetyl-ꢀ-D-galactopyranoside (7).
Compound 6 (200 mg, 0.37 mmol) was dissolved in 3 mL of
pyridine. To the solution, 4,4′-dimethoxytrityl chloride (140 mg,
0.41 mmol) was added. After stirring at room temperature for
12 h, the reaction mixture was extracted with aqueous NaHCO3
and ethyl acetate. The organic solvent was washed with brine,
dried over MgSO4, and concentrated. Purification on a silica
gel column with hexane-ethyl acetate gave compound 7 (250
mg, 70% from 5) in diastereomers. 1H NMR (CDCl3, 500 MHz)
δ 7.42-7.22 (5H, m), 6.85-6.82 (4H, m), 5.39 (1H, d, J )
3.6), 5.19 (1H, dd, J ) 7.9, 10.5), 4.99 (1H, dd, J ) 3.6, 10.5),
4.46 (1H, d, J ) 7.9), 4.19 (1H, m), 4.15 (1H, m), 4.13 (1H,
m), 4.11 (1H, m), 4.00 and 3.90 (1H, d, J ) 9.0 and 9.1), 3.47
and 3.37 (1H, d, J ) 9.0 and 9.1), 3.13 and 3.05 (1H, d, J )
9.0 and 8.6), 3.00 and 2.84 (1H, d, J ) 9.0 and 8.6), 2.16 (3H,
s), 2.12 (3H, s), 2.01 (3H, s), 1.98 (3H, s), 1.93 (3H, s), 1.89
(1H, m), 1.82 (1H, m), 1.74 (1H, m), 1.65 (1H, m). ESI TOF/
MS m/z: [M+Na]+ 859.3449.
Synthesis of the Solid Support. Compound 7 (70 mg, 0.083
mmol) was dissolved in 2 mL of CH2Cl2. To the solution,
4-(dimethylamino)pyridine (26 mg, 0.21 mmol) and succinic
anhydride (33 mg, 0.33 mmol) were added. After stirring at
room temperature for 12 h, the reaction mixture was extracted
with 10% citric acid and ethyl acetate. The organic solvent was
washed with brine, dried over MgSO4, and concentrated.
Purification on a silica gel column with hexane-ethyl acetate
gave compound 8 (77 mg, 98%) in diastereomers.
An LCAA CPG resin (915 mg) was suspended in CH2Cl2 (5
mL). To the solution, compound 8 (30 mg, 32 µmol) and N,N′-
dicyclohexylcarbodiimide (20 mg, 97 µmol) were added. After
stirring at room temperature for 24 h, the resin was filtered and
washed with CH2Cl2 and acetonitrile. A capping solution
(pyridine-acetic anhydride, 9;1, v/v) was added to the resin
and stirred at room temperature for 12 h. The resin was washed
with CH2Cl2 and acetonitrile and dried in Vacuo. The amount
of compound 8 loaded onto the solid support was calculated to
be 11 µmol/g, based on the calculation of the dimethoxytrityl
cation released by the use of a solution of 60% HClO4-EtOH
(3:2, v/v).
Oligonucleotide Synthesis. Oligonucleotides were synthe-
sized on a Nihon Techno Service NTS H-8 DNA/RNA
synthesizer. Deprotection of the bases and phosphates was
performed in concentrated NH4OH/EtOH (3:1, v/v) at 55 °C
for 5 h. In the case of RNA synthesis, the 2′-TBDMS groups
were removed by triethylamine trihydrofluoride at room tem-
perature for 24 h. The deprotected DNA and RNA were purified
with a Sep-Pak C18 cartridge, followed by denaturing 20%
polyacrylamide gel electrophoresis to afford DNAs and RNAs.
These oligonucleotides were analyzed by matrix-assisted laser
desorption/ionization time-of-flight mass spectrometry (MALDI-
TOF/MS).
RESULTS
In order to synthesize an oligonucleotide possessing a
carbohydrate the at 3′-terminus, solid-phase synthesis was
employed in this study. The synthesis procedure is summarized
in Scheme 1. Compound 1 was synthesized as previously
reported (20). Before the glycosylation reaction, we protected
one of the hydroxyl groups of compound 1 with benzyl bromide,
because the electron-donating benzyl group is known to increase
the reactivity in the glycosylation reaction. Acetylated galactose
was introduced by the glycosylation reaction via boron trifluo-
ride methyl etherate as a Lewis acid. The yield of this
glycosylation reaction was 95%. After the oxidation of com-
pound 3 with osmium oxide, one of the hydroxyl groups on
the cyclopentane was protected by acetyl chloride to afford
compound 5. The deprotection of the benzyl group of compound
5 was carried out with Pd/C, followed by protection with DMTr-
Cl. Finally, compound 8 was immobilized onto the controlled
pore glass (CPG). Carbohydrate-oligonucleotide conjugates
were synthesized by a DNA/RNA synthesizer using the glyco-
sylated CPG as the matrix. After the solid-phase synthesis, the
obtained oligomer was deprotected and cleaved from the solid
support with concentrated NH4OH/EtOH (3:1, v/v) at 55 °C
for 5 h to obtain carbohydrate-ended oligoDNA. In the case of
glycosyl-ended oligoRNA, protected RNAs were incubated with
triethylamine trihydrofluoride at room temperature for 24 h. The
Serum Stability Assay. The serum stability of the oligo-
nucleotides was evaluated by treatment with medium containing

1688 Bioconjugate Chem., Vol. 21, No. 9, 2010
Ikeda et al.
Scheme 1a
a Reagents and conditions: (a) benzyl bromide, NaH, DMF, rt; (b) pentaacetyl-D-galactose, boron trifluoride methyl etherate, CH2Cl2, rt; (c)
OsO4, 4-methylmorpholine N-oxide, H2O/acetonitrile/acetone (1:1:1, v/v), rt; (d) Ac2O, pyridine, rt; (f) H2, Pd/C, rt; (g) DMTrCl, pyridine, rt; (h)
succinic anhydride, DMAP, CH2Cl2, rt; (i) LCAA-CPG, DCC, CH2Cl2, rt.
Table 1. Synthesized Galactose-Oligonucleotide Conjugates
gated. For this objective, four samples were prepared, which
are listed in Table 2. siRNA-targeting survivin, which is a
member of the apoptosis inhibitor family (21), was transfected
into HeLa cells with Lipofectamine 2000. After incubation for
48 h, the amount of survivin mRNA was analyzed by quantita-
tive RT PCR. Figure 2 shows the silencing activities of the
siRNAs conjugated with galactose. siRNA2, which was con-
jugated with galactose at the 3′-terminus of sense strand, did
not show any significant change in activity at a concentration
of 10 nM, as compared to the unmodified siRNA (siRNA1).
Although the modification at the 3′-terminus of the antisense
strand (siRNA3) reduced the silencing activity of siRNA to some
extent, siRNA3 maintained its ability to reduce the amount of
mRNA to 20% at 10 nM. It is worth noting that, even in the
case of siRNA4, which was modified at the 3′-terminus of both
strands, the amount of mRNA was reduced to 20%, i.e., almost
the same level as that of siRNA3.
name
sequence
calcd.
found
DNA1
RNA1
Fluorescein-(dT)20-galactose
GGACCACCGCAUCUCUACAdTdT-
galactose
7011.4
6992.3
7012.1
6996
TNA2
UGUAGAGAUGCGGUGGUCCdTdT-
galactose
7145.5
7151.8
deprotected DNA and RNA thus obtained were purified with a
Sep-Pak C18 cartridge, followed by denaturing 20% polyacryl-
amide gel electrophoresis to afford sugar-ended oligomers.
These oligomers were analyzed by matrix-assisted laser de-
sorption/ionization time-of-flight mass spectrometry (MALDI-
TOF/MS). Table 1 shows the sequences and the results of the
MALDI-TOF-MS analysis of the synthesized oligonucleotide
conjugates.
The serum stability of the oligonucleotide-carbohydrate
conjugates was then investigated. The oligonucleotide which
was modified with galactose at the 3′-terminus and the control
oligonucleotide without galactose at the 3′-terminus were
incubated with medium containing 10% of serum at 37 °C. Both
oligonucleotides possessed a fluorescence probe at the 5′-
terminus. After the incubation, the samples were subjected to
polyacrylamide gel electrophoresis and visualized by fluores-
cence scanning. As shown in Figure 1, the oligonucleotide
without 3′-galactose showed a degradation band, while that with
3′-galactose showed no change in molecular mass, indicating
the serum stability of the oligonucleotide conjugated with
galactose at the 3′-terminus. It was confirmed that ca. 80% of
the incubated 3′-galactose-ended oligonucleotide remained after
180 min of incubation. It was also surprising that more than
40% of the oligonucleotide remained even after 24 h of
incubation, as shown in Figure 1b.
DISCUSSION
A safe and efficient system of delivering oligonucleotides is
in great demand for the development of oligonucleotide-based
therapy. However, research in this area suffers from the
instability of oligonucleotides in ViVo and the inefficient and
nonselective cellular uptake of oligonucleotide drugs. To
overcome these problems, we focused on glycoconjugates in
our present study, because of the biologically important proper-
ties of carbohydrates. To date, various methods of delivering
plasmid DNA or oligonucleotides into cells via molecules
modified with a carbohydrate, such as glycosylated polyamine
and glycosylated streptavidin, have been described (22). In these
systems, the glycosylated molecules can form complexes with
DNA via noncovalent interactions. In contrast to noncovalent
formation, covalent oligonucleotide-carbohydrate conjugates
have several advantages, e.g., the structural homogeneity of the
product and the low toxicity of the components of the conjugate
In order to obtain further information on the modification of
the oligonucleotide with the carbohydrate at the 3′-terminus,
the activities of carbohydrate-modified siRNAs were investi-

Modified Carbohydrate-Oligonucleotide Conjugates
Bioconjugate Chem., Vol. 21, No. 9, 2010 1689
Figure 2. Survivin mRNA level in HeLa cells. HeLa cells were
transfected with siRNA1, siRNA2, siRNA3, and siRNA4 at the
indicated concentrations. After incubation for 48 h, the amount of
survivin mRNA was measured by quantitative RT-PCR. The relative
amount of survivin mRNA from the HeLa cells without any treatment
was employed as a control.
which a carbohydrate was conjugated by condensation via a
bifunctional linker, our approach is based on the general method
of carbohydrate synthesis. Accordingly, compound 2, which is
a good glycosyl acceptor, can be easily conjugated with other
glycosyl donors that have been well studied and synthesized in
the field of oligosaccharide synthesis (23).
In an effort to increase the stability of oligonucleotides against
enzymatic degradation, several chemical modifications resulting
in the formation of compounds, such as phosphorothioate
oligonucleotides and 2′-O-methyl oligonucleotide, have been
reported. These modifications stabilize the oligonucleotides;
however, in some cases, the modified oligonucleotides have been
found to be toxic. For example, the application of phospho-
rothioate oligonucleotide to the central nervous system produces
irritation and toxicity (24). In our system, oligonucleotides can
be stabilized to a remarkable degree by modifying them only
at the 3′-terminus. In the case of oligodeoxynucleotides,
degradation usually starts to occur at the 3′-terminus of the
strand. Accordingly, protection at the 3′-terminus of the strand
is very important for the stabilization of the oligonucleotides
(25). This method can be applied to oligodeoxynucleotide-based
drugs, such as antisense, decoy DNA, and aptamer drugs.
We also evaluated the activities of RNA interference of
carbohydrate-oligonucleotide conjugates. In some cases, the
position of the modification affects the activity of the oligo-
nucleotide drugs. For example, Kitade et al. have conjugated
several lipophilic molecules at the 3′-terminus of either the sense
or the antisense stand of siRNA and evaluated the activities of
siRNA (26). In the case of modification at the 3′-terminus of
the antisense strand, the activity of siRNA was slightly reduced,
especially in the case of modification with cholesterol, and the
silencing activity was substantially impaired (26). These results
indicate that the properties of the compounds conjugated with
oligonucleotides and the site of conjugation are important factors
for efficient gene silencing. The modified siRNA in this study
maintained its ability to reduce the amount of target mRNA
to 20% at 10 nM, even in the case of modification at the 3′-
terminus of both the sense and the antisense strands of siRNA.
This indicates that two ligands can be introduced at the
3′-termini of the sense and antisense strands. Although only
galactose was employed for conjugation with the end of siRNA
in this work, it is anticipated from the obtained data that two
kinds of ligands can be introduced, one at each end of siRNA.
For example, cholesterol and a carbohydrate might be introduced
at the 3′-terminus of the sense and antisense strands of siRNA,
respectively, without great impairment of the siRNA activity.
In conclusion, a novel method of modifying oligonucleotides
at the 3′-terminus was developed based on solid-phase synthesis.
Figure 1. Comparison of the serum stabilities of the oligonucleotides.
Each oligonucleotide, which was modified or not modified with
galactose, was incubated in 10% serum at 37 °C for the durations
indicated. All oligonucleotides were labeled with fluorescein at the 5′-
end. The oligonucleotides, not conjugated with galactose (a) or
conjugated with galactose (b), were subjected to polyacrylamide gel
electrophoresis and visualized by fluorescence scanning. (c) Time-course
of the degradation of the oligonucleotides. This result indicates that
3′-end modification with galactose improved the serum stability of the
oligonucleotides.
Table 2. Sequences of the Oligonucleotides and siRNAs Used in
This Study
siRNA
ON
sequence
siRNA1
ON1
ON3
ON1
ON4
ON2
ON3
ON2
ON4
5′-GGACCACCGCAUCUCUACAdTdT-3′
3′-dTdTCCUGGUGGCGUAGAGAUGU
5′-GGACCACCGCAUCUCUACAdTdT-3′
3′-Gal-dTdTCCUGGUGGCGUAGAGAUGU
5′-GGACCACCGCAUCUCUACAdTdT-Gal3′
3′-dTdTCCUGGUGGCGUAGAGAUGU
5′-GGACCACCGCAUCUCUACAdTdT-Gal3′
3′-Gal-dTdTCCUGGUGGCGUAGAGAUGU
siRNA2
siRNA3
siRNA4
compared to those of polycationic compounds such as poly-
(L-lysine), which is toxic at concentrations above 100 µM.
In this study, we developed an easy solid-phase synthesis
method of producing carbohydrate-oligonucleotide conjugate
modified at the 3′ terminus. In the synthesis scheme, the
glycosylation reaction is the key reaction. Especially, when an
expensive carbohydrate such as a rare sugar is used for the
conjugation, the high yield of glycosylation reaction is desirable.
In general, the yield of the glycosylation reaction is dependent
on the protecting groups on the glycosyl donor and the glycosyl
acceptor. Electron-withdrawing groups, such as the acetyl or
the benzoyl group, have been found to decrease the reactivity
of the donor/acceptor, and electron-donating groups, such as
the benzyl group, have been found to increase the reactivity of
the donor/acceptor. In our case, compound 2, which is a glycosyl
acceptor, contained a benzyl group, resulting in a good yield
of the glycosylation reaction. In contrast to a previous study in

1690 Bioconjugate Chem., Vol. 21, No. 9, 2010
Ikeda et al.
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this report can be applied not just to carbohydrate modification,
but to any modification of oligonucleotides. Studies thereon are
currently being undertaken.
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