Versatile site-specific conjugation of small molecules to siRNA using click chemistry

Article abstract of DOI:10.1021/jo101761g

We have previously demonstrated that conjugation of small molecule ligands to small interfering RNAs (siRNAs) and anti-microRNAs results in functional siRNAs and antagomirs in vivo. Here we report on the development of an efficient chemical strategy to make oligoribonucleotide-ligand conjugates using the copper-catalyzed azide-alkyne cycloaddition (CuAAC) or click reaction. Three click reaction approaches were evaluated for their feasibility and suitability for high-throughput synthesis: the CuAAC reaction at the monomer level prior to oligonucleotide synthesis, the solution-phase postsynthetic "click conjugation", and the "click conjugation" on an immobilized and completely protected alkyne-oligonucleotide scaffold. Nucleosides bearing 5′-alkyne moieties were used for conjugation to the 5′-end of the oligonucleotide. Previously described 2′-and 3′-O-propargylated nucleosides were prepared to introduce the alkyne moiety to the 3′ and 5′ termini and to the internal positions of the scaffold. Azido-functionalized ligands bearing lipophilic long chain alkyls, cholesterol, oligoamine, and carbohydrate were utilized to study the effect of physicochemical characteristics of the incoming azide on click conjugation to the alkyne-oligonucleotide scaffold in solution and on immobilized solid support. We found that microwave-assisted click conjugation of azido-functionalized ligands to a fully protected solid-support bound alkyne-oligonucleotide prior to deprotection was the most efficient "click conjugation" strategy for site-specific, high-throughput oligonucleotide conjugate synthesis tested. The siRNA conjugates synthesized using this approach effectively silenced expression of a luciferase gene in a stably transformed HeLa cell line.(Figure Presented)

Full text of DOI:10.1021/jo101761g

pubs.acs.org/joc
Versatile Site-Specific Conjugation of Small Molecules to siRNA
Using Click Chemistry
Takeshi Yamada,†,‡,§ Chang Geng Peng,†,§ Shigeo Matsuda,†
Haripriya Addepalli,† K. Narayanannair Jayaprakash,† Md. Rowshon Alam,†
Kathy Mills,† Martin A. Maier,† Klaus Charisse,† Mitsuo Sekine,‡
Muthiah Manoharan,*,† and Kallanthottathil G. Rajeev*,†
†Drug Discovery, Alnylam Pharmaceuticals, Cambridge, Massachusetts 02142, United States, and
‡Department of Life Science, Tokyo Institute of Technology, J2-12, 4259 Nagatsuta, Midoriku, Yokohama
226-8501, Japan. §Takeshi Yamada and Chang Geng Peng contributed equally to this work.
mmanoharan@alnylam.com rajeevk@alnylam.com
Received September 26, 2010
We have previously demonstrated that conjugation of small molecule ligands to small interfering
RNAs (siRNAs) and anti-microRNAs results in functional siRNAs and antagomirs in vivo. Here we
report on the development of an efficient chemical strategy to make oligoribonucleotide-ligand
conjugates using the copper-catalyzed azide-alkyne cycloaddition (CuAAC) or click reaction. Three
click reaction approaches were evaluated for their feasibility and suitability for high-throughput
synthesis: the CuAAC reaction at the monomer level prior to oligonucleotide synthesis, the solution-
phase postsynthetic “click conjugation”, and the “click conjugation” on an immobilized and com-
pletely protected alkyne-oligonucleotide scaffold. Nucleosides bearing 50-alkyne moieties were used
for conjugation to the 50-end of the oligonucleotide. Previously described 20- and 30-O-propargylated
nucleosides were prepared to introduce the alkyne moiety to the 30 and 50 termini and to the internal
positions of the scaffold. Azido-functionalized ligands bearing lipophilic long chain alkyls, cholesterol,
oligoamine, and carbohydrate were utilized to study the effect of physicochemical characteristics of
the incoming azide on click conjugation to the alkyne-oligonucleotide scaffold in solution and on
immobilized solid support. We found that microwave-assisted click conjugation of azido-functiona-
lized ligands to a fully protected solid-support bound alkyne-oligonucleotide prior to deprotection
was the most efficient “click conjugation” strategy for site-specific, high-throughput oligonucleotide
conjugate synthesis tested. The siRNA conjugates synthesized using this approach effectively silenced
expression of a luciferase gene in a stably transformed HeLa cell line.
Introduction
Due to improvements independently made to the Huisgen
1,3-dipolar azide-alkyne cycloaddition reaction2 by the
laboratories of Meldal3 and Sharpless,1,4 the click reaction
The so-called click chemistry strategy allows efficient cou-
pling of modular building blocks bearing azide and alkyne.1
1198 J. Org. Chem. 2011, 76, 1198–1211
Published on Web 02/07/2011
DOI: 10.1021/jo101761g
r
2011 American Chemical Society

Yamada et al.
JOCFeatured Article
can be performed in aqueous and nonaqueous solvent
and on solid-support.5 The inertness of azide and alkyne
toward other functional groups such as amines, carboxy-
lates, alcohols, thiols, and esters makes this strategy
advantageous for selective postsynthetic chemical liga-
tion of ligands to proteins, peptides, nucleic acids, carbo-
hydrates, and polymers.
In order to make small interfering RNAs (siRNAs) ther-
apeutically active, the molecules must be delivered into the
cytoplasm of the target cells, where the RNA-induced silen-
cing complex (RISC) is located. Various approaches, such
as chemical modifications, conjugation of small molecules,
liposomes, nanoparticles, polymers, polyamines, and cell-
penetrating peptides have been investigated to enhance
cellular uptake.27-34 Conjugation of small molecules such
as cholesterol, fatty acids, vitamin E, polycationic compounds,
and receptor specific ligand like folic acid and carbohydrates
can also improve pharmacokinetic and pharmacodynamic
properties of oligonucleotide-based therapeutic agents.35 Oli-
gonucleotide conjugates with lipophilic molecules have been
shown to exhibit improved protein binding, nuclease resis-
tance, and broader biodistribution and uptake in liver and
jejunum in vivo relative to unconjugated controls.29,30,35-38
Cholesterol-conjugated siRNAs,29,30 antisense microRNAs
(antagomirs),39 and antisense oligonucleotides40 enter cells
in the absence of cationic lipids in vivo. Recently, a report
demonstrated that siRNAs conjugated to polyspermine
entered cells in the absence of transfection reagents
in vitro.31 Conjugation of receptor-specific N-acetylgalac-
tosamine (GalNAc),41-43 folic acid,44 or R-tocopherol45 to
Click chemistry has proven very useful to the oligonucle-
otide chemistry field. The copper-catalyzed azide-alkyne
cycloaddition (CuAAC) reaction has been successfully used
for ligation and cyclization of DNA using appropriately
azido- and alkyne-functionalized DNA strands.6-8 The CuAAC
reaction has also been extensively used for synthesis of mod-
ified nucleosides,9-13 carbohydrate,14-16 fluorophore,17-21
and lipophilic22 oligonucleotide conjugates; for synthesis of
chimeras of peptide-DNA/PNA23 and oligonucleotides;12
and to explore G-quadruplex solution structures.24 A recent
report from Morvan’s laboratory demonstrated a very effi-
cient click chemistry approach for the synthesis of a dendri-
meric oligonucleotide-glycoconjugate bearing 16 galactose
moieties on its periphery.25 In addition, Best has reviewed the
usefulness of azide-alkyne cycloaddition in bioorthogonal
reactions for labeling of biomolecules, including proteins,
viruses, sugars, nucleic acids, and lipids, and its impact on
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FIGURE 1. Alkyne building blocks for postsynthetic CuAAC reaction on RNA.
antisense oligonucleotides, peptide nucleic acid, or siRNAs
elicited receptor-mediated cellular uptake of the conjugated
oligonucleotides in vitro or in vivo. However, synthesis
of oligonucleotide conjugates is challenging due to lengthy
synthetic procedures and frequent incompatibility with
solid-phase oligonucleotide synthesis and deprotection
conditions.
enables incorporation of one or more alkyne moiety to the
desired position of an oligoribonucleotide for small molecule
conjugation under CuAAC reaction conditions. The phos-
phoramidites 1-3 were designed to introduce alkyne func-
tionality to the 50-terminus of the oligonucleotide. The
phosphoramidites 4 and 6 were chosen to incorporate the
alkynes containing nucleosides at the 50-terminal or internal
positions of the oligonucleotide, whereas the solid supports 5
and 7 were selected for insertion of the alkyne containing
nucleoside moiety at the 30-terminus.
Considering the enormous potential of oligonucleotide-
ligand conjugates for delivering nucleic acid therapeutics to
organs or tissues of interest, we evaluated click chemistry for
synthesis of oligoribonucleotide/ligand conjugates. Three
strategies were evaluated. In the first one, the oligonucleotide
conjugate was obtained by solid-phase synthesis using mod-
ified building blocks which were obtained by click reaction at
the monomer stage. In the second approach, solution-phase
postsynthetic click conjugation of azido functionalized li-
gands to an alkyne-oligonucleotide scaffold bearing one or
more alkyne moieties was evaluated. In the third strategy,
azido-functionalized ligands were conjugated to a fully
protected solid-support bound alkyne-oligonucleotide.
We evaluated the effect of the physicochemical character-
istics of the incoming azide on click conjugation with azido-
functionalized ligands derived from lipophilic long chain
alkyls, cholesterol, oligoamine and carbohydrate. The siR-
NA conjugates synthesized using the CuAAC approach were
evaluated for RNAi activity in vitro using a HeLa cell line
stably transformed with the firefly and renilla luciferase gene.
The alkyne group was introduced via a carbamate linkage
at the 50 position of uridine and 20-O-methyluridine as shown
in Scheme 1. Treatment of 20,30-O-isopropylideneuridine (8)
with phenyl chloroformate in anhydrous pyridine and sub-
sequent treatment of the intermediate formed with propar-
gylamine afforded compound 9 in >90% yield. The isopro-
pylidene protection was removed by refluxing compound 9
in 80% acetic acid for 18 h to afford compound 10 in
quantitative yield. Silylation of the hydroxyl group of com-
pound 10 with tert-butyldimethylsilyl chloride (TBDMS-Cl)
in anhydrous THF in the presence of pyridine and AgNO3
yielded a mixture of 20- and 30-O-TBDMS derivatives 11 and
11a (structure not shown in the Scheme), respectively. The
isomeric ratio was approximately 7:2 (20-O-TBDMS/30-O-
1
TBDMS) as determined by H NMR analysis of the crude
compounds. Chromatography on neutral alumina using 2%
MeOH in CH2Cl2 as eluent produced good separation of the
two isomers 11 and 11a.
Results and Discussion
Phosphitylation of 20-O-silylated nucleoside 11 afforded
the phosphoramidite 1 in 71% isolated yield. The synthesis
of phosphoramidite 2 was achieved with 45% overall yield
from 30-O-acetyl-20-O-methyluridine (12)46 (Scheme 1). The
precursor 14 for the synthesis of phosphoramidite 3 was
obtained from compound 12 by Curtius rearrangement.
Addition of diphenylphosphoryl azide to a mixture of com-
pound 12 and 5-hexynoic acid in the presence of triethyl-
amine in anhydrous DMF at ambient temperature and
subsequent stirring at 100 °C for 18 h afforded com-
pound 14 in 71% yield. Deacetylation of compound 14
using NaOMe in methanol at ambient temperature followed
by phosphitylation yielded the desired phosphoramidite 3 in
31% overall yield.
Preparation of Nucleoside-Alkyne Building Blocks for
Postsynthetic Oligonucleotide Conjugation. The nucleosi-
de-alkyne building blocks shown in Figure 1 were selected
for evaluating site-specific conjugation of small molecule
ligands to siRNA under CuAAC reaction conditions. Alky-
nylation at the 20-, 30-, or 50-hydroxyl of ribonucleosides
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JOCFeatured Article
SCHEME 1. Syntheses of 50 Alkyne-Functionalized Uridine Phosphoramiditesa
aKey: (i) (a) phenyl chloroformate, pyridine, rt, 3 h, (b) propargylamine, rt, 18 h, yield 9 (91%) and 13 (>95%); (ii) 80% AcOH in MeOH, reflux, 18 h,
quant; (iii) AgNO3, pyridine, TBDMS-Cl, THF, rt, 18 h, 20%; (iv) 2-cyanoethyl N,N-diisopropylchlorophosphoramidite, N,N-diisopropylethylamine
(DIEA), CH2Cl2, rt, 4 h, yields 1 (71%), 2 (70%) and 3 (70%); (v) diphenylphosphoryl azide, 5-hexynoic acid, Et3N, DMF, rt to 100 °C, 18 h, 71%;
(vi) NaOMe, MeOH, rt, 2 h, yields 15 (67%) and 16 (65%).
The 20- and 30-alkyne-derived nucleosides were synthe-
sized following modifications of the known procedures. 50-
O-[Bis(4-methoxyphenyl)phenylmethyl] (50-O-DMTr) deri-
vatives 18 and 19 of 20- and 30-O-propargyl-5-methyluridine
were directly synthesized from 50-O-DMTr-5-methyluridine
(17) by microwave-assisted alkylation (Scheme 2). The tin-
mediated alkylation of uridine with propargyl chloride in the
presence of TBAI47 was optimized by replacing uridine with
50-O-DMTr-uridine in a mixture of benzene and CH3CN.
The microwave-assisted reaction was performed in a sealed
bottle, and the reaction was complete within 4 h with a total
isolated yield of about 70% (40% for 20- and 32% for 30-O-
propargyl derivatives). The 50-O-DMTr protection of the
nucleoside remained intact and provided a good handle for
purification after the tin-mediated alkylation. An attempt to
alkylate 17 with propargyl chloride under non-microwave
conditions resulted in partial deprotection of the 50-O-DMTr
group due to the extended alkylation time, and this made the
isolation of the product difficult. The microwave-assisted pro-
pargylation of 50-O-DMTr-uridine mediated by dibutyltin-
(IV) oxide was successful and is a promising approach for
small-scale synthesis of 20- and 30-O-propargylated pyrimi-
dines. The method was successfully extended to other ribo-
long chain alkyl amine controlled pore glass support (lcaa-
CPG) under amide coupling conditions afforded the solid
support 7 with 80 μM/g loading.
Preparation of Small Molecules Carrying an Azido Group.
The small molecules carrying azido groups shown in Figure 2
were chosen to evaluate the CuAAC reaction for high-
throughput synthesis of oligonucleotide/ligand conjugates.
The azides 20-23 are of fatty acid/alcohol origin to evaluate
the CuAAC reaction between hydrophobic compounds and
highly hydrophilic oligonucleotides in aqueous media. A
more hydrophobic azide, 24, derived from cholest-5-en-3-ol-
(3β)-(6-hydroxyhexyl)carbamate48 was also evaluated. Amino
azide 25 and its corresponding trifluoroacetamido-protected
derivative 25a were synthesized from spermine (Scheme S2,
Supporting Information) to evaluate the reaction of oligo-
cationic small molecules with alkyne-functionalized oligo-
nucleotides.49 Lastly, GalNAc derivatives 26 and 27 were
chosen to evaluate whether hydrophilic small molecules would
serve as effective reactants. GalNAc serves to deliver pay-
loads to hepatocytes.42,50,51
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nucleosides with protection of exocyclic amino groups (rABz
,
rCBz, and rGiBu) to give the corresponding 20- and 30-O-
propargylated nucleosides in good yields (Table S1, Support-
ing Information). The phosphoramidite 6 was prepared as
described above. Succinylation of 19 followed by coupling to
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SCHEME 2. Syntheses of 20- and 30-O-Propargyl-5-methyl-
synthesis and deprotection conditions to obtain modified
oligonucleotides 46 and 56 with overall yield 60 and 64%,
respectively, based on HPLC analysis.
uridine Phosphoramidites and CPG Supportsa
Conjugation of Ligands to Oligonucleotide in Solution
Using the Click Reaction. To explore the validity of the
CuAAC reaction for small-molecule conjugation to RNA
we used alkyne-modified uridine derivatives. Since the sense
strand of the model luciferase siRNA used lacked a uridine at
the 50-end, we decided to append the modified uridine mono-
mers to the 50- or 30-end of the sense strand as an additional
nucleotide. Solid-support bound alkyne-oligonucleotide scaf-
folds 32a-36a with a modified alkyne at the 50-end, and the
alkyne-oligonucleotide scaffolds 37a and 38a with alkynes
at the 30-end of the sense strand, shown in Table 1, were
synthesized using the corresponding monomers according
to the standard oligoribonucleotide synthesis conditions.
Substitution of uridine at position 12 of the sense strand
with 20- and 30-O-propargyluridine afforded the scaffolds
39a and 40a on the controlled pore glass support (CPG).
Successive coupling of phosphoramidite 4 on CPG 5 fol-
lowed by synthesis of the desired sequence on the support
under standard oligoribonucleotide synthesis conditions
afforded the solid-support bound oligonucleotide scaffold
41a with three 20-O-propargyl-5-methyluridine moieties
at the 30-end of the sense strand (Table 1). An aliquot of
each support-bound alkyne-oligonucleotide scaffold was
deprotected under standard RNA deprotection condi-
tions and analyzed by analytical HPLC and LC-MS to
establish the integrity of each modified oligonucleotide
synthesized (Table S2, Supporting Information). We used,
in this study, the alkyne-oligonucleotides 32a-41a shown
in Table 1 for evaluating the feasibility and usefulness
of CuAAC reaction for small-molecule conjugation to
siRNA.
aKey: (i) Bu2SnO (1.1 equiv), propargyl chloride (2.0 equiv), TBAI
(0.5 equiv), benzene/CH3CN, microwave at 100 °C, yields 18 40% and
19 32%; (ii) 2-cyanoethyl N,N-diisopropylchlorophosphoramidite, DIEA,
CH2Cl2, rt, 4-6 h, yield 651%; (iii) (a) succinic anhydride, DMAP, CH2Cl2,
˚
rt, 18 h, (b) HBTU, DIEA, lcaa-CPG (500 A, loading: 140 μM/g), DMF,
4 h.
Lipophilic azides52-54 20-23 (Figure 2) were synthesized
from commercially available alcohols or bromides. Treat-
ment of cholest-5-en-3-ol-(3β)-(6-hydroxyhexyl)carbamate48
(S17, Scheme S1, Supporting Information) with sodium
azide in DMF afforded the azide 24 in 84% yield. The
carbohydrate derivatives 26 and 27 were synthesized accord-
ing to reported procedures.55
Synthesis of Click-Conjugated Monomers for Solid-Phase
Oligonucleotide Synthesis. To demonstrate the click reaction
at the monomer stage, we synthesized 50-modified uridine 28
and 29 bearing linoleyl and GalNAc moieties, respectively.
The conditions reported by Jatsch et al.56 were used (Scheme 3)
for the synthesis. The reaction proceeded successfully with
>85% isolated product yield. 20 to 30 TBDMS migration in
MeOH/CH2Cl2 cosolvent was not observed during the reac-
tion, presumably due to the slight acidity provided by [(CH3-
CN)4Cu]PF6 in CH2Cl2/MeOH. Phosphitylation of com-
pounds 28 and 29 afforded the corresponding phosphoramidites
30 and 31 in 50 and 83% yields, respectively. The phosphor-
amidites 30 and 31 were subjected to standard oligonucleotide
We first evaluated the CuAAC reaction for ligand con-
jugation to alkyne-oligonucleotide scaffolds in solution. In
order to perform solution-phase CuAAC reactions on alkyne-
functionalized oligonucleotides, the oligonucleotide scaffold
32 (Table S2, Supporting Information) was obtained from
solid-support bound precursor oligonucleotide 32a (Table 1)
by following standard oligonucleotide deprotection and puri-
fication protocols. The integrity of the propargylated oligo-
nucleotide 32 was confirmed by LC-MS analysis. Reaction
of purified alkyne-oligonucleotide 32 with excess unpro-
tected azide 26a (GalNAcI-azide, Figure 2) in the presence of
catalytic amounts of CuSO4 5H2O and 3 molar excess of
sodium ascorbate (azide/CuSO4 5H2O/sodium ascorbate=
3
3
1:0.4:3 molar equiv) in MeOH/H2O (1:1, v/v) did not yield
the desired conjugate 56 (data not shown). Significant de-
gradation of the oligonucleotide 32 was observed both at
room temperature overnight and at elevated temperature
(60 °C) after 60 min. Presumably chelation of copper ions to
the phosphate backbone and 20-hydroxyl groups of un-
protected oligonucleotide caused degradation. This was not
unexpected as copper ion-mediated degradation of DNA
oligonucleotides has been reported in the literature.57,58
(52) Constantinou-Kokotou, V.; Kokotos, G.; Roussakis, C. Anticancer
Res. 1998, 18, 3439–3442.
(53) King, J. F.; Loosmore, S. M.; Aslam, M.; Lock, J. D.; McGarrity,
M. J. J. Am. Chem. Soc. 1982, 104, 7108–7122.
(54) Binder, W. H.; Kluger, C. Macromolecules 2004, 37, 9321–9330.
(55) Gambert, U.; Lio, R. G.; Farkas, E.; Thiem, J.; Bencomo, V. V.;
At higher molar equivalents of CuSO4 (10 molar equiv)
the reaction remained sluggish, and that made mass
ꢀ
Liptak, A. Bioorg. Med. Chem. 1997, 5, 1285–1291.
(57) Kanan, M. W.; Rozenman, M. M.; Sakurai, K.; Snyder, T. M.; Liu,
D. R. Nature 2004, 431, 545–549.
(58) Seela, F.; Pujari, S. S. Bioconjugate Chem. 2010, 21, 1629–1641.
(56) Jatsch, A.; Kopyshev, A.; Mena-Osteritz, E.; Baeuerle, P. Org. Lett.
2008, 10, 961.
1202 J. Org. Chem. Vol. 76, No. 5, 2011

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JOCFeatured Article
FIGURE 2. Azide monomers used for click reactions.
SCHEME 3. Syntheses of 50-Linoleyl and N-Acetylgalactosaminyl Uridine Click Monomersa
aKey: (i) 23 or 26, [(CH3CN)4Cu]PF6 (20 mol %), Cu powder (20 mol %), CH2Cl2/MeOH (4:1, v/v), rt, 4-18 h, yields 28 (98%) and 29 (88%); (ii)
2-cyanoethyl N,N-diisopropylchlorophosphoramidite, DIEA, CH2Cl2, rt, 4 h, yields 30 (50%) and 31 (83%).
spectral analysis of the product very difficult. After
desalting, the chelated copper ion interfered with HPLC
and LC-MS analysis. Treatment with EDTA prior to
desalting did not remove copper ions completely from the
product (data not shown). Use of powdered metallic
copper25 under heating (60 °C, 30 min) resulted in quan-
titative reaction; however, the product was contaminated
with copper ions. The observed m/z peak values corre-
sponded to copper adducts (Figure S1, Supporting
Information). Replacing hydrophilic azide 26a with
hydrophobic 1-docosyl azide (20) in solution-phase con-
jugation to purified alkyne-oligonucleotide 32 in the
presence of CuSO4 and sodium ascorbate was also not
effective. Moreover, solvents compatible with the unpro-
tected highly hydrophilic oligonucleotide and hydrophobic
alkyl azide that could facilitate an efficient azide-alkyne
cycloaddition reaction were difficult to identify. Recently,
copper wire has been successfully used as an efficient re-
placement for copper salt for the CuAAC reaction of
20-alkyne/azido-modified adenosine with azides or alkynes
respectively and also for the preparation of chimera of
complementary oligonucleotides functionalized with an al-
kyne and an azide.12 The authors reported green coloration
of the reaction mixture during prolonged stirring at 35 °C
suggesting contamination/release of copper ion to the reac-
tion mixture.
Microwave-assisted CuAAC click conjugation has been
successfully used for multiple labeling of solid-support
J. Org. Chem. Vol. 76, No. 5, 2011 1203

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TABLE 1. Click Reaction of Alkyne-oligonucleotide Scaffolds with Small Molecule Azides 20-27
aThe products 42-56 were obtained via CuAAC reaction of the corresponding azide and the alkyne-oligoribonucleotide precursor on solid support
unless otherwise noted. All reactions were performed under microwave conditions except for compound 55. Typically, a heterogeneous mixture of the
solid-support bound alkyne-oligoribonucleotide, the azide, CuSO4 5H2O, sodium ascorbate, and TBTA (1:3:0.4:3:3 molar equiv, respectively) in H2O/
3
MeOH/THF (1.2 mL, 2:2:1 v/v) was microwave irradiated at 60 °C in an Explorer-48 reactor for 45 min to obtain the corresponding conjugate. The 50-
oligoamine-conjugated oligonucleotide 55 was prepared under thermal conditions (1.5 equiv of CuSO4 5H2O, 5 equiv of sodium ascorbate, 1:1 t-BuOH/
H2O, 50 °C, overnight). bThe oligonucleotides 46 and 56 were also directly synthesized from the phosphoramidite 30 and 31 with 60 and 64% overall
3
yield, respectively, under solid-phase synthesis conditions. cReaction conversion was calculated from the ratio of product peak intensity to precursor
peak intensity analyzed by reversed-phase high-performance liquid chromatography (RP-HPLC). Reaction conversion was not determined due to
d
difficulty in calculating the percentage conversion after solution-phase conjugation of the azide 26 with purified oligonucleotide 32 obtained after
deprotection of 32a.
bound T12 DNA oligomer with carbohydrates.59 A variant
of this approach in solution phase in the presence of tris-
(benzyltriazolylmethyl)amine (TBTA),21,60 a copper(I)-sta-
bilizing agent, afforded the desired oligoribonucleotide con-
jugate 42. The microwave-assisted CuAAC reaction of
the alkyl azide 20 and purified alkyne-oligonucleotide 32
equiv to alkyne), sodium ascorbate, and TBTA yielded the
desired product 42. After irradiation for 45 min at 60 °C no
starting material was detected by HPLC analysis (Figure S2,
Supporting Information). The yields of the solution phase con-
jugations with and without microwave assistance were difficult
to obtain reliably using HPLC methods due to incomplete
reaction and degradation of the oligonucleotides. In addi-
tion, incompatible hydrophilic oligonucleotides and hydro-
phobic alkyl azide in a cosolvent in the presence of copper
salt made the HPLC analysis more difficult. Furthermore,
additional purification steps were required to remove TBTA
and to minimize copper ion contamination from the product.
in the presence of a large excess of CuSO4 5H2O (24 molar
3
(59) Bouillon, C.; Meyer, A.; Vidal, S.; Jochum, A.; Chevolot, Y.;
Cloarec, J.-P.; Praly, J.-P.; Vasseur, J.-J.; Morvan, F. J. Org. Chem. 2006,
71, 4700–4702.
(60) Chan, T. R.; Hilgraf, R.; Sharpless, K. B.; Fokin, V. V. Org. Lett.
2004, 6, 2853–2855.
1204 J. Org. Chem. Vol. 76, No. 5, 2011

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SCHEME 4. CuAAC Reaction of Azides 20-26 with Alkyne-Oligoribonucleotide Scaffold 32a
(i) Alkyne/azide/CuSO4 5H2O/TBTA/sodium ascorbate (1:3:0.4:3:3 mol ratio), H2O/MeOH/THF (2:2:1, v/v), 1.2 mL total volume, microwave at 60
3
°C, 45 min, yields 42 (98%), 43 (95%), 44 (89%), 45 (96%), 46 (93%) and 55 (99%); (ii) standard oligonucleotide deprotection. Yield was calculated on
the basis of RP-HPLC peak ratio of product to precursor.
TABLE 2. Optimization of CuAAC Reaction Conditions Using Azide 20 and Solid-Support Bound Fully Protected Alkyne-Oligonucleotide 32a
reaction no.
molar ratioa
solvent (2:2:1, v/v)b
temp (°C)
time (min)
conversionc (%)
1
2
3
4
5
6
7
8
9
1:16:0.4:3:0
1:16:0.4:3:3
1:3:0.4:3:3
1:3:0.1:3:3
1:3:0.4:3:3
1:3:0.4:3:3
1:3:0.4:3:3
1:3:0.4:3:3
1:3:0.4:3:3
H2O/MeOH/THF
H2O/MeOH/THF
H2O/MeOH/THF
H2O/MeOH/THF
H2O/MeOH/DMSO
H2O/MeOH/DMF
H2O/MeOH/THF
H2O/MeOH/THF
H2O/MeOH/THF
60
60
60
60
60
60
60
60
rt
45
45
45
45
45
45
45
45
48 h
no reaction
71
99
no reaction
88
79
57
60
quantitative
aAlkyne 32a/azide 20/CuSO4 5H2O/sodium ascorbate/TBTA. bThe total reaction volume is 1.2 mL except for reactions 7 and 8. For reaction 7 the
3
volume was 0.24 mL and for 8 it was 3.6 mL. cReaction conversion was monitored by HPLC.
Conjugation of Ligands to Oligonucleotide on Solid Support
Using Click Reaction. The conditions used by Morvan and
co-workers for the synthesis of various carbohydrate-DNA
conjugates by click reaction on solid-support25,59,61 were
optimized for fully protected support-bound alkyne-
oligoribonucleotide precursor 32a (Scheme 4, Table 1). The
effects of molar equivalence of azide to alkyne, Cu catalyst,
chelator (TBTA), temperature, and solvent for microwave-
assisted CuAAC reaction were investigated; the findings are
summarized in Table 2 and Figure S3, Supporting Informa-
tion. The microwave-assisted CuAAC reaction of solid-sup-
port bound 32a with the azide 20 in the presence of TBTA
afforded 71% yield of the desired conjugate 42 (Table 2,
entry 2 and Figure S4, Supporting Information), whereas in
the absence of TBTA no product was recovered (Table 2,
entry 1). Addition of TBTA was required for the postsynthetic
click reaction to occur on solid-support bound alkyne-
oligonucleotide.
product yields presumably due to aggregation of highly
hydrophobic azide 20 (Table 2, entries 2 and 3). The reaction
volume was also critical: a solvent volume of 1.2 mL was optimal
for the Explorer-48 microwave reactor with lowest volume
reaction vessel tested. Attempts to use less or more solvent
(Table 2, reactions 7 and 8, total volume of solvent 0.24 and
3.6 mL, respectively) resulted in poor yield. The optimal reaction
conditions (Table 2, entry 3) were microwave irradiation at
60 °C for 45 min with 1:3:0.4:3:3 molar ratios of alkyne-
oligonucleotide, azide, CuSO4 5H2O, TBTA, and sodium
3
ascorbate in 1.2 mL of H2O, MeOH, and THF (2:2:1, v/v).
The CuAAC reaction of 32a with the azide 20 also pro-
ceeded to completion in the absence of microwave irradia-
tion in about 48 h at ambient temperature (Table 2, entry 9).
HPLC and LC-MS analysis of the product obtained from
microwave-assisted and unassisted reactions showed forma-
tion of the expected product. The observed rate enhancement
of microwave-assisted conjugation reaction corroborated
well with the cycloaddition reaction of small molecules.62,63
When thereactionwas performedonafullyprotectedalkyne-
oligonucleotide on solid support, contamination of the pro-
duct with copper ions was minimized to below the detection
limit of our LC-MS analytical capability. Presumably poor
availability of lone-pair electrons and lack of negative charges
on the phosphate backbone for the fully protected oligonu-
cleotide 32a minimized its coordination with the metal ion.
We found that 0.4 molar equiv of CuSO4 5H2O relative to
3
the alkyne were required to achieve almost quantitative
conversion to conjugate (Table 2, entry 3). Use of 3 molar
equiv of the azide was sufficient for quantitative reaction.
THF was the most effective cosolvent tested for dissolving
lipophilic molecules in combination with water and metha-
nol to produce higher yields rather than the use of DMF and/
or DMSO in place of THF (Table 2, entries 3, 5, and 6).
Excessive amounts of lipophilic azides resulted in lower
(62) Appukkuttan, P.; Mehta, V. P.; Van der Eycken, E. V. Chem. Soc.
Rev. 2010, 39, 1467–1477.
(63) Kappe, C. O.; Dallinger, D. Mol. Diversity 2009, 13, 71–193.
(61) Pourceau, G.; Meyer, A.; Vasseur, J.-J.; Morvan, F. J. Org. Chem.
2008, 73, 6014–6017.
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SCHEME 5. Conjugation of GalNAc to the Fully Protected Alkyne-Oligonucleotide 41aa
a(i) Alkyne/azide/CuSO4 5H2O/TBTA/sodium ascorbate 1:9:1.2:9:9 (molar ratio), H2O/MeOH/THF (2:2:1, v/v, vol. 1.2 mL), microwave at 60 °C,
45 min; (ii) standard oligonucleotide deprotection
3
TABLE 3. Gene Silencing Activity of siRNAs in Dual Luciferase Assay
solid-support under the optimized conditions. All expected
products were obtained in excellent yields (Table 1, 43-46,
Figure S5, Supporting Information). A typical HPLC profile
of the deprotected precursor alkyne-oligonucleotide 32 and
the product 46 after treatment with the linoleyl azide (23)
indicated quantitative CuAAC reaction on the solid-support
under the optimized conditions. The same 50-conjugated
oligonucleotide 46 was also synthesized from the phosphor-
amidite 30 using standard solid-phase oligonucleotide synth-
esis and deprotection conditions (Table 1). LC-MS data and
HPLC profiles of the products obtained by both routes were
identical.
To evaluate the effect of the position of conjugation within
the oligonucleotide, the linoleyl azide (23) was reacted with
alkyne-oligonucleotide scaffolds carrying the alkyne moiety
at 30 or 50 terminal positions or an internal position at either
through the 20 or 30 position of the ribose moiety (Table 1,
32a-40a). The CuAAC reaction of oligonucleotide scaffolds
modified with 20-O-propargy-5-methylluridine (Table 1, 33a,
37a, and 39a) with azide 23 gave quantitative conversion to
the expected products irrespective of the location of the
modification on the oligonucleotide. Reaction with the 30-
O-propargyluridine scaffolds 34a, 38a, and 40a gave slightly
lower yields (∼90%) of the product (Table 1, 48, 52, and 54).
Reaction with the oligonucleotide scaffold 36a modified
with an alkyne at the 50-terminus gave only 84% yield of
the desired conjugate 50. The HPLC profiles of the purif-
ied products 47-54 are shown in Figure 5S, Supporting
Information.
siRNA (S/ASa)
IC50b (nM)
Tmc (°C)
59/58
42/58
43/58
44/58
45/58
46/58
47/58
48/58
49/58
50/58
51/58
52/58
53/58
54/58
0.32
0.35
0.2
73.4
74.4
72.6
nd
nd
74.4
nd
nd
nd
nd
73.4
nd
72.5
69.6
0.074
0.77
0.062
0.35
0.32
0.26
0.38
1.12
1.41
0.22
0.23
aS and AS indicate sense and antisense strands, respectively; the
unmodified duplex control was 59/58. See Table 1 for details of modified
sense strands 42-54. bIC50 values were calculated from the dose-
response curves shown in Figure 3; all IC50 were determined in the
c
presence of Lipofectamine 2000. 2 μM duplex concentration in 0.9%
physiological saline solution; nd: not determined.
Our evaluation indicated that the microwave-assisted click
reaction of solid-support bound alkyne-oligonucleotide
scaffold with azide bearing ligands is more efficient and cost-
effective for small-scale oligonucleotide-conjugate synthe-
sis than other strategies tested. The CuAAC reaction of
solid-support bound alkyne-oligonucleotide also proceeded
at ambient temperature in the absence of microwave irradia-
tion; this procedure may prove useful with more reactive
azide-bearing ligands than the ones used here.
The impact of physical properties of small molecule azides
on CuAAC reaction on an immobilized oligonucleotide-
alkyne scaffold was evaluated. Hydrophilic and cationic
small molecule azides were reacted with solid-support bound
To further evaluate the scope of the click reaction on
immobilized and protected alkyne-oligonucleotide scaf-
folds, different alkyl azides (21-24) were reacted with 32a on
1206 J. Org. Chem. Vol. 76, No. 5, 2011

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FIGURE 3. Plot of RNAi activity of various lipophile-conjugated luciferase siRNA via click chemistry at different positions on the sense
strand in the Renilla luciferase mRNA expressed HeLa cell lines. The parent duplex is unmodified control siRNA duplex 59/58, where 58
(UCGAAGUACUCAGCGUAAGdTdT) and 59 (CUUACGCUGAGUACUUCGAdTdT) represent antisense and sense strands, respec-
tively (IC50 data in Table 3). The sense strand of each siRNA conjugate was annealed with the unmodified antisense strand 58 to obtain the
corresponding siRNA.
oligonucleotide-alkyne 32a under optimized CuAAC reac-
tion conditions. Reaction with amino azide 25 did not yield
the expected product, presumably due to chelation of copper
ions to the unprotected amines. The click reaction of base-
labile TFA-protected amino azide 25a with 32a yielded the
desired product 55. The solid-support bound precursor 32a
was reacted with compound 25a in the presence of Cu-
siRNA conjugates are summarized in Table 3; IC50 values
were calculated from dose-response curves (Figure 3).
The siRNAs 44/58 and 46/58 shown in Table 3 with the
antisense strand 58 and complementary sense strands com-
prising of an added extra uridine derivative at the 50-terminus
conjugated to unsaturated lipid such as oleyl (C18, ω=1) or
linoleyl (C18, ω = 2) (44, 46, Table 1) showed 4- to 5-fold
improvement in IC50 over the control siRNA 59/58. The
most active siRNA 46/58 had a Tm 1 °C higher than that of
the control siRNA 59/58. In contrast, the siRNA 43/58
containing saturated stearyl conjugate 43 had an IC50 com-
parable to the unconjugated control. The siRNA formed
from the 50-cholesteryl conjugate (45) had a 2-fold lower IC50
than the control siRNA 59/58. Interestingly, replacement of
the “5pU” sugar linker with ‘5pU2m’ reduced activity of the
linoleyl conjugate (compare activities of 49/58 and 46/58,
Table 3).
The IC50’s calculated for “Lin-5pU” 46/58 and “Lin-
5pU2m” 49/58 were 0.062 and 0.26 nM, respectively. siR-
NAs constituted from strands with the linoleyl moiety con-
jugated through the 20- (47) or 30- (48) position of the
ribosugar (“T2p” and “T3p” linkers, Tables 1 and 3) at the
50-end of the sense strand showed comparable IC50s to the
control siRNA 59/58. The 30 (“T3p”) and 20 (“T2p”) mod-
ified siRNAs had comparable activity in all cases: 47/58 vs
48/58, 51/58 vs 52/58, and 53/58 vs 54/58). Introduction of
linoleyl to the 30-end of the sense strand through either the
“T2p” (51) or “T3p” (52) linker considerably reduced siRNA
activity.
SO4 5H2O (1.5 equiv) and sodium ascorbate (5 equiv) at
3
50 °C overnight to afford oligonucleotide conjugate 55
quantitatively. The efficiency of the reaction and the identity
of the product formed were confirmed by HPLC and LC-MS
analysis, respectively (Figure S6, Supporting Information).
Previous studies have demonstrated that conjugation of
multivalent GalNAc to drug results in binding of the con-
jugate with nM affinity to asialoglycoprotein receptor (ASGPR)
and internalization into liver hepatocytes.64 We, therefore,
explored the click reaction between GalNAc derivatives 27
carrying an azido group and an RNA oligonucleotide 41a
bearing three alkyl functionalities at the 30-end as shown in
Scheme 5. Analysis of the crude product after deprotection
showed that multiple incorporation of GalNAc onto solid-
support bound oligonucleotide was efficient. HPLC analysis
of the deprotected crude product confirmed formation of
86% of the desired tri-GalNAc product 57 with insignificant
amounts of mono- and di-GalNAc conjugate byproduct
(Figure S7, Supporting Information).
Dual-Luciferase Assay for RNA Interference Evaluation. A
dual-luciferase assay was used to evaluate gene silencing by
siRNAs formed from the modified oligonucleotides synthe-
sized. A pGL4 vector containing the Renilla and firefly lucif-
erase genes was used for the assay. The siRNA sequences
were designed to target the firefly luciferase gene. siRNAs
were transfected into cells using Lipofectamine2000. The
gene silencing abilities of unmodified siRNA (59/58) and
Placement of the bulky residues “Lin-T2p” or “Lin-T3p”
in the central region of the sense strand (53, 54) led to a small
decrease in thermal stability (Tm) of the corresponding
duplexes 53/58 and 54/58 compared to the control 59/58
(Table 3). In a recent study, we showed that local destabiliza-
tion of the central region of the sense strand can enhance the
potency of corresponding siRNA.65 Although in the current
(64) Stockert, R. J. Physiol. Rev. 1995, 75, 591–609.
J. Org. Chem. Vol. 76, No. 5, 2011 1207

Yamada et al.
JOCFeatured Article
example, the linoleyl conjugates were also placed in the
central region (position 12) of the sense strand, we only
observed a minor improvement in potency. Presumably, the
decrease in thermal stability was too small to have a sig-
nificant effect on the potency of modified siRNAs. It also is
interesting to note that insertion of an extra modified
nucleotide to the 50-end of the sense strand did not hamper
the activity of the siRNA, whereas addition of a bulky
nucleotide to the 30-end of the sense strand was not well
tolerated.
and copper-free70 azide-alkyne click reactions for synthesis
of RNA conjugates.
Experimental Section
Materials. All reagents and solvents obtained from commer-
cial suppliers were used without further purification. All reac-
tions were carried out under argon in oven-dried glassware. Thin-
layer chromatography was carried out on glass-backed silica-gel
60 F254 plates. Column chromatography was performed using
˚
silica gel (60 A, 230 ꢀ 400 mesh). Chemical shifts are given in
parts per million (ppm); J values are given in hertz (Hz). All
spectra were internally referenced to the appropriate residual
undeuterated solvent.
Conclusions
20,30-O-Isopropylidene-50-O-propargylcarbamoyluridine (9).
20,30-O-Isopropyrideneuridine (10.00 g, 35.18 mmol) in pyridine
(350 mL) was treated with phenyl chloroformate (4.8 mL, 38.70
mmol) at room temperature for 2 h. Propargylamine (7.3 mL,
175.89 mmol) was added, and the reaction mixture was stirred
at room temperature for 18 h. The reaction was monitored by
TLC (Rf = 0.60; developing solvent CH2Cl2/MeOH, 12:1, v/v).
Solvents and volatiles were removed in vacuo. The residue
was suspended in CH2Cl2 (250 mL) and washed with satd aq
NaHCO3 solution followed by standard workup. The residue
was purified by flash chromatography on silica gel (98:2
CH2Cl2/MeOH, v/v) to obtain 9 (11.73 g, 91%) as a white foam:
1H NMR (400 MHz, DMSO-d6) δ 11.41 (br, 1H), 7.73 (m, 1H),
7.65 (d, J = 8.0 Hz, 1H), 5.80 (d, J = 2.4 Hz, 1H), 5.62 (d, J =
8.0 Hz, 1H), 5.03 (dd, J = 6.4, 2.4 Hz, 1H), 4.74 (dd, J = 6.4,
3.1 Hz, 1H), 4.27-4.14 (m, 2H), 4.08 (m, 1H), 3.77 (m, 2H), 3.11
(m, 1H), 1.48 (s, 3H), 1.28 (s, 3H); 13C NMR (100 MHz, DMSO-
d6) δ 163.2, 155.5, 150.3, 142.5, 113.2, 101.8, 92.2, 84.1, 83.4,
81.2, 80.8, 73.1, 64.2, 29.8, 27.0, 25.1; MS calcd for C16H20N3O7
(MHþ) 366.13, found 366.12.
30-O-Acetyl-20-O-methyl-50-O-propargylcarbamoyluridine (13).
Compound 13 (5.03 g, 99%) was obtained as a white foam from
compound 12 (4.00 g, 13.3 mmol), phenyl chloroformate (2.15 mL,
14.64 mmol), and propargylamine (3.21 mmol, 66.5 mmol) as
described for the synthesis of 9. The residue after workup was
purified by flash chromatography on silica gel (98:2 CH2Cl2/
MeOH, v/v) to obtain compound 13: 1H NMR (400 MHz,
DMSO-d6) δ 11.49 (s, 1H), 7.91 (t, J = 5.7 Hz, 1H), 7.62 (d, J =
8.1 Hz, 1H), 5.87 (d, J = 6.6 Hz, 1H), 5.72 (d, J = 8.1 Hz, 1H),
5.22-5.16 (m, 1H), 4.26-4.12 (m, 4H), 3.85-3.79 (m, 2H),
3.28 (s, 3H), 3.18 (t, J = 2.3 Hz, 1H), 2.10 (s, 3H); 13C NMR
(100 MHz, DMSO-d6) δ 169.7, 162.9, 155.5, 150.6, 140.3, 102.7,
85.9, 81.2, 79.8, 79.4, 73.4, 70.5, 63.7, 58.2, 29.8, 20.6; MS calcd
for C16H20N3O8 (MHþ) 382.12, found 382.00.
50-O-Propargylcarbamoyluridine (10). Compound 9 (11.73 g,
32.10 mmol) in MeOH (20 mL) was refluxed with 80% AcOH
(200 mL) for 18 h. The solution was concentrated in vacuo. The
residue was coevaporated with methanol three times and then
redissolved in 50 mL of anhydrous MeOH. The MeOH solution
was sonicated for 30 min, and a white precipitate was obtained.
The precipitate was filtered on a suction funnel and washed with
CH2Cl2 (1 L). The white residue obtained was dried at 40 °C in
a vacuum oven for 18 h to obtain compound 10 (10.34 g, 99%)
1H NMR (400 MHz, DMSO-d6) δ 11.35 (br, 1H), 7.79 (m, 1H),
7.60 (d, J = 7.8 Hz, 1H), 5.78 (d, J = 5.5 Hz, 1H), 5.65 (d, J =
7.8 Hz, 1H), 5.42 (d, J = 5.5 Hz, 1H), 5.27 (d, J = 4.9 Hz, 1H),
4.33-3.72 (m, 7H), 3.13 (s, 1H); 13C NMR (100 MHz, DMSO-
d6) δ 172.1, 163.0, 155.7, 150.7, 140.7, 102.1, 87.8, 81.7, 81.2,
73.1, 72.5, 69.9, 64.0, 29.8; HRMS calcd for C13H16N3NaO7
(MNaþ) 348.0808, found 348.0812.
In this work, we developed and compared various appro-
aches to conjugate small molecules with a range of phy-
sicochemical properties to oligoribonucleotides using click
chemistry. We have demonstrated that the microwave-as-
sisted CuAAC conjugation of azido-functionalized small-
molecule ligands to solid-support bound oligoribonucleotide
allowed for efficient synthesis of oligoribonucleotide-
heteromolecule conjugates with the potential for high-through-
put applications. The procedure described here allows rapid
and economical synthesis of a wide variety of conjugates.
Using the alkyne-oligonucleotide scaffolds described here,
any azido-functionalized ligand can be introduced to a desired
site within the oligonucleotide sequence and at selected posi-
tions on the sugar moiety (20, 30, or 50 position) or in the
selected sites of nucleobase. Although we demonstrated the
conjugation using uridine derivatives, the approach will be
applicable to other nucleosides, employing the reported
derivatization protocols.66-69 We have optimized the reac-
tion conditions for conjugation of azides derived from
hydrophobic, hydrophilic, and cationic compounds to alky-
ne-oligonucleotide scaffolds. Further optimization of this
conjugation strategy for siRNA therapeutic screening with
several ligands is in progress. The very recent demonstration
of strain-promoted copper free click conjugation of ligands
to RNA that is completely devoid of heavy metal ions is
another promising approach for RNA oligonucleotide con-
jugation chemistry.70,71 However, because of simplicity of
the chemistry involved and the versatility of the CuAAC
reaction on solid-support bound alkyne-oligonucleotide, it is
a promising high-throughput strategy for oligonucleotide
conjugate synthesis for biological screening. Our laboratory
is presently engaged in optimizing both copper-assisted72
(65) Addepalli, H.; Meena; Peng, C. G.; Wang, G.; Fan, Y.; Charisse, K.;
Jayaprakash, K. N.; Rajeev, K. G.; Pandey, R. K.; Lavine, G.; Zhang, L.;
Jahn-Hofmann, K.; Hadwiger, P.; Manoharan, M.; Maier, M. A. Nucleic
Acids Res. 2010, 38, 7320–7331.
(66) Egli, M.; Minasov, G.; Tereshko, V.; Pallan, P. S.; Teplova, M.;
Inamati, G. B.; Lesnik, E. A.; Owens, S. R.; Ross, B. S.; Prakash, T. P.;
Manoharan, M. Biochemistry 2005, 44, 9045–9057.
(67) Manoharan, M.; Guinosso, C. J.; Cook, P. D. Tetrahedron Lett.
1991, 32, 7171–7174.
(68) Manoharan, M.; Tivel, K. L.; Andrade, L. K.; Cook, P. D. Tetra-
hedron Lett. 1995, 36, 3647–3650.
(69) Manoharan, M. Biochim. Biophys. Acta 1999, 1489, 117–130.
(70) Jayaprakash, K. N.; Peng, C.-G.; Butler, D.; Varghese, J. P.; Maier,
M. A.; Rajeev, K. G.; Manoharan, M. Org. Lett. 2010, 12, 5410–5413.
(71) van Delft, P.; Meeuwenoord, N. J.; Hoogendoorn, S.; Dinkelaar, J.;
Overkleeft, H. S.; van der Marel, G. A.; Filippov, D. V. Org. Lett. 2010, 12,
5486–5489.
(72) Eltepu, L.; Peng, C. G.; Jayaprakash, K. N.; Yamada, T.; Jayaraman,
M.; Kallanthottathil, G. R.; Manoharan, M. Abstracts of Papers. 240th ACS
National Meeting; Aug 22-26, 2010, Boston, MA; American Chemical Society:
Washington, DC, 2010; CARB-53.
50-O-Propargylcarbamoyl-20-O-(tert-butyldimethylsilyl)uridine
(11). Compound 10 (10.34 g, 28.30 mmol) in anhydrous THF
(30 mL) was treated with silver nitrate (5.77 g, 34.00 mmol),
pyridine (17.0 mL, 209.4 mmol), and TBDMS-Cl (4.27 g, 28.30 mmol)
1208 J. Org. Chem. Vol. 76, No. 5, 2011

Yamada et al.
JOCFeatured Article
at room temperature for 18 h. The reaction mixture was diluted
with CH2Cl2 (300 mL) and washed with water. The organic
layer was dried over anhydrous Na2SO4 and then concentrated
in vacuo. The residue was purified by chromatography on
neutral alumina (98:2 CH2Cl2/MeOH, v/v) to obtain 11 (2.45
g, 20%, Rf = 0.15 on aluminum gel TLC) as a pure isomer: 1H
NMR (400 MHz, DMSO-d6) δ 11.41 (br, 1H), 7.93-7.71 (m,
1H), 7.65 (d, J = 8.1 Hz, 1H), 5.83 (d, J = 5.1 Hz, 1H), 5.72 (dd,
J = 8.1, 2.1 Hz, 1H), 5.23 (d, J = 6.0 Hz, 1H), 4.32-4.22 (m,
2H), 4.17 (m, 1H), 4.09-3.98 (m, 1H), 3.95-3.87 (m, 1H),
3.87-3.82 (m, 2H), 3.22-3.13 (m, 1H), 0.88 (d, J = 1.5 Hz,
12H), 0.09 (s, 3H), 0.07 (s, 3H); 13C NMR (100 MHz, DMSO-d6)
δ 163.4, 163.4, 156.1, 151.0, 140.7, 102.6, 88.4, 82.5, 81.7,
75.1, 73.7, 70.0, 64.2, 30.3, 26.2, 26.1, 18.5, 18.3, -4.1, -4.3, -4.6,
-4.7; MS calcd for C19H29N3NaO7Si (MNaþ) 462.17, found
462.10.
50-O-Propargylcarbamoyl-20-O-(tert-butyldimethylsilyl)uridine-
30-O-(N,N-diisopropyl)phosphoramidite (1). Compound 11a
(452 mg, 1.02 mmol) in anhydrous CH2Cl2 (12 mL) was treated
with DIEA (612 μL, 3.41 mmol) and 2-cyanoethyl N,N-diisopro-
pylchlorophosphoramidite (380 μL, 1.71 mmol) at room tempera-
ture for 6 h. The reaction mixture was diluted with CH2Cl2
(100 mL) and washed with brine (25 mL). The organic layer was
dried over sodium sulfate and concentrated in vacuo. The residue
was purified by flash chromatography on silica gel (50:50:0.5
hexane/EtOAc/TEA, v/v) to obtain the phosphoramidite 1 (469
mg, 71%, Rf = 0.6) as a whitefoam: 1HNMR(400MHz, DMSO-
d6) δ 11.51-11.33 (m, 1H), 7.96-7.71 (m, 1H), 7.71-7.53 (m, 1H),
5.91-5.79 (m, 1H), 5.76-5.62 (m, 1H), 4.39-4.08 (m, 5H), 3.87-
3.53 (m, 6H), 3.20-3.05 (m, 1H), 2.85-2.69 (m, 2H), 1.28-1.04
(m, 12H), 0.92-0.72 (m, 9H), 0.16-0.10 (m, 6H); 31P NMR (162
MHz, DMSO-d6) δ 149.99, 148.44; HRMS calcd for C28H47N5-
O8PSi (MHþ) 640.2931, found 640.2927.
19 (270 mg, 0.45 mmol) and 2-cyanoethyl N,N-diisopropyl-
chlorophosphoramidite (200 μL, 0.88 mmol) in the presence of
DIEA (325 μL, 1.75 mmol) in anhydrous CH2Cl2 (3 mL) for 6 h
as described above for the synthesis of 1. The residue after
workup was purified by flash chromatography on silica gel (4:6
hexane/EtOAc, v/v) to obtain the phosphoramidite 6: 1H NMR
(400MHz,DMSO-d6) δ11.42-11.41 (m, 1H), 7.60-7.17 (m, 10H),
6.97-6.79 (m, 4H), 5.92-5.88 (m, 1H), 4.67-4.58 (m, 1H),
4.46-4.24 (m, 3H), 4.26-3.94 (m, 2H), 3.76 (m, 7H), 3.56 (m, 3H),
3.40-3.14 (m, 2H) 2.77-2.69 (m, 2H), 1.50-0.84 (m, 15H); 31
P
NMR (162 MHz, DMSO-d6) δ 156.06, 154.99; MS calcd for
C43H51N4NaO9P (MNaþ) 821.32, found 821.30.
20-O-TBDMS-50-O-[1-linoleyl-(1,2,3-triazo-3-yl)methyl]-
carbamoyluridine-30-O-(N,N-diisopropyl)phosphoramidite (30).
The phosphoramidite 30 (463 mg, 50%) was obtained as a white
foam from compound 28 (730 mg, 1.00 mmol) and 2-cyanoethyl
N,N-diisopropylchlorophosphoramidite (335 μL, 1.50 mmol) in
the presence of DIEA (525 μL, 3.00 mmol) in anhydrous CH2Cl2
(10 mL) for 4 h as described above for the synthesis of 1.
The residue after workup was purified by flash chromatography
on silica gel (60:30:0.5 hexane/EtOAc/TEA, v/v) to obtain the
phosphoramidite 30: 1H NMR (400 MHz, DMSO-d6) δ
11.43-11.42 (1H, br), 7.91-7.89 (2H, m), 7.66-7.59 (1H, m),
5.86-5.84 (1H, m), 5.70-5.66 (1H, m), 5.38-5.27 (3H, m),
4.32-4.13 (8H, m), 3.83-3.56 (4H, m), 2.80-2.72 (4H, m),
2.04-1.99 (3H, m), 1.79-1.76 (2H, m), 1.33-1.11 (30H, m),
0.87-0.82 (12H, m), 0.05-0.02 (6H, m); 31P NMR (162 MHz,
DMSO-d6) δ 149.98, 148.37; MS calcd for C46H79N8NaO8PSi
(MNaþ) 953.54, found 953.40.
Compound 31. The phosphoramidite 31 (403 mg, 83%) was
obtained as a white foam from compound 29 (400 mg, 0.44
mmol) and 2-cyanoethyl N,N-diisopropylchlorophosphorami-
dite (150 μL, 0.67 mmol) in the presence of DIEA (230 μL, 1.33
mmol) in anhydrous CH2Cl2 (4 mL) for 4 h as described above
for the synthesis of 1. The residue after workup was purified
by flash chromatography on silica gel (EtOAc then 2:98:0.5
MeOH/CH2Cl2/TEA, v/v) to obtain the phosphoramidite 31:
1H NMR (400 MHz, DMSO-d6) δ 11.37 (br, 1H), 7.94-7.88
(m, 1H), 7.89-7.76 (m, 2H), 7.65-7.58 (m, 1H), 5.81-5.72 (m, 1H),
5.68-5.58 (m, 1H), 5.25-5.18 (m, 2H), 4.99-4.92 (m, 1H),
4.57-4.43 (m, 3H), 4.30-4.11 (m, 5H), 4.05-3.98 (m, 4H),
3.97-3.84 (m, 2H), 3.84-3.67 (m, 3H), 3.64-3.44 (m, 3H),
2.16-2.05 (m, 3H), 2.05-1.93 (m, 3H), 1.93-1.82 (m, 3H),
20-O-Methyl-50-O-propargylcarbamoyluridine-30-O-(N,N-diiso-
propyl)phosphoramidite (2). The phosphoramidite 2 (1.18 g, 2.18
mmol, 87%, Rf = 0.1) was obtained as a white foam from
compound 15 (1.00 g, 2.51 mmol) and 2-cyanoethyl N,N-
diisopropylchlorophosphoramidite (1.12 mL, 5.01 mmol) in the
presence of DIEA (1.75 mL, 10.02 mmol) in anhydrous CH2Cl2
(25 mL) for 6 h as described above for synthesis of 1. The residue
after workup was purified by flash chromatography on silica
gel (3:7 hexane/EtOAc, v/v) to obtain the phosphoramidite 2:
1H NMR (400 MHz, DMSO-d6) δ 11.43 (br, 1H), 7.95-7.74
(m, 1H), 7.72-7.52 (m, 1H), 5.98-5.76 (m, 1H), 5.76-5.58
(m, 1H), 4.40-3.90 (m, 5H), 3.86-3.47 (m, 6H), 3.33 (br, 3H),
1.82-1.74 (m, 3H), 0.91-0.71 (m, 10H), 0.09-0.05 (m, 6H); 31
P
3.18-2.84 (m, 1H), 2.83-2.70 (m, 2H), 1.28-1.00 (m, 12H); 31
P
NMR (162 MHz, DMSO-d6) δ 148.98, 148.37; MS calcd for
C46H74N9NaO18PSi (MNaþ) 1123.17, found 1123.30.
NMR (162 MHz, DMSO-d6) δ 155.02, 154.38; HRMS calcd for
30-O-Acetyl-20-O-methyl-50-O-(4-pentynyl)carbamoyluridine (14).
5-Hexynoic acid (3.11 mL, 28.1 mmol) in DMF (150 mL) was
treated with diphenylphosphoryl azide (10.7 mL, 56.2 mmol)
and triethylamine (8.12 mL, 56.2 mmol) at ambient temperature
to 100 °C for 6 h. Then 30-O-acetyl-20-O-methyluridine (12, 2.0 g,
7.04 mmol) was added to the reaction mixture, and stirring was
continued at 100 °C for 18 h. Progress of the reaction was
monitored by TLC. The reaction mixture was cooled to ambient
temperature, diluted with EtOAc, and washed with brine. The
organic layer was dried over sodium sulfate and then concen-
trated in vacuo. The residue was purified by flash chromatogra-
phy on silica gel (98.5:1.5 CH2Cl2/MeOH, v/v) to obtain 14 (2.07
g, 71%, Rf = 0.2) as a white foam: 1H NMR (400 MHz, DMSO-
d6) δ 11.49 (s, 1H), 7.64 (d, J = 8.1 Hz, 1H), 7.44 (t, J = 5.6 Hz,
1H), 5.87 (d, J = 6.4 Hz, 1H), 5.72 (d, J = 8.1 Hz, 1H),
5.26-5.09 (m, 1H), 4.28-4.04 (m, 4H), 3.28 (s, 3H), 3.11-2.96
(m, 2H), 2.79 (t, J = 2.5 Hz, 1H), 2.21-2.11 (m, 2H), 2.13
(s, 3H), 1.61-1.46 (m, 2H); 13C NMR (100 MHz, DMSO-d6)
δ 169.6, 162.95, 155.7, 150.6, 140.3, 102.7, 86.15, 83.9, 79.8, 79.5,
71.5, 70.5, 63.3, 58.2, 28.3, 20.6, 15.2; MS calcd for C18H23N3O8
(MHþ) 410.15, found 410.00.
C23H35N5O8P (MHþ) 540.2223, found 540.2220.
20-O-Methyl-50-O-(pentynyl)carbamoyluridine-30-O-(N,N-diiso-
propyl)phosphoramidite (3). The phosphoramidite 3 (800 mg,
70%, Rf=0.2) was obtained as a white foam from compound
16 (750 mg, 2.03 mmol) and 2-cyanoethyl N,N-diisopropyl-
chlorophosphoramidite (878 μL, 2.76 mmol) in the presence of
DIEA (1.38 mL, 7.52 mmol) in anhydrous CH2Cl2 (20 mL) for
18 h as described above for synthesis of 1. The residue after
workup was purified by flash chromatography on silica gel
(30:70:0.5 hexane/EtOAc/TEA, v/v) to obtain the phosphora-
midite 3: 1H NMR (400 MHz, CD3CN) δ 9.04 (br, 1H),
7.58-7.45 (m, 1H), 5.97-5.85 (m, 1H), 5.85-5.73 (m, 1H),
5.73-5.57 (m, 1H), 4.46-4.12 (m, 4H), 3.95-3.58 (m, 5H),
3.57-3.36 (m, 3H), 3.32-3.09 (m, 2H), 2.82-2.65 (m, 2H),
2.26-2.17 (m, 2H), 2.16-2.13 (m, 1H), 1.83-1.61 (m, 2H),
1.31-1.11 (m, 12H); 31P NMR (162 MHz, CD3CN) δ 155.71,
155.47; HRMS calcd for C25H39N5O8P (MHþ) 568.2536, found
568.2550.
50-O-(4,40-Dimethoxytrityl)-30-O-propargyl-5-methyluridine-
30-O-(N,N-isopropyl)phosphoramidite (6). The phosphoramidite
6 (183 mg, 51%) was obtained as a white foam from compound
J. Org. Chem. Vol. 76, No. 5, 2011 1209

Yamada et al.
JOCFeatured Article
20-O-Methyl-50-O-propargylcarbamoyluridine (15). Compound
13 (4.35 g, 14.49 mmol) was treated with 0.5 M NaOMe in
methanol (200 mL) for 4 h. The solution was concentrated in
vacuo. The residue was purified by flash chromatography on silica
gel (95:5 CH2Cl2/MeOH, v/v) to obtain 15 (2.75 g, 67%, Rf = 0.3)
as a white foam: 1H NMR (400 MHz, DMSO-d6) δ 11.41 (br, 1H),
7.97 (dd, J = 5.7 Hz, 1H), 7.60 (d, J = 8.1 Hz, 1H), 5.87 (d, J =
5.5 Hz, 1H), 5.68 (d, J = 8.1 Hz, 1H), 5.37 (d, J = 5.8 Hz, 1H),
4.28-4.02 (m, 3H), 4.04-3.95 (m, 1H), 3.91-3.84 (m, 1H),
20.8, 16.7, 12.7, -6.0, -6.4; MS calcd for C37H63N6O7Si (MHþ)
730.45, found 731.20.
Compound 29. Compound 11 (500 mg, 1.14 mmol) in CH2Cl2/
MeOH (4:1, v/v) was treated with 26 (460 mg, 1.14 mmol),
tetrakis(acetonitrile)copper(I) hexafluorophosphate (42 mg, 0.11
mmol), and copper (7 mg, 0.11 mmol) at room temperature for
18 h. The solution was concentrated in vacuo. The residue was
directly loaded on flash silica gel column without aqueous
workup and purified (eluent: 9:1 CH2Cl2/MeOH, v/v) to obtain
29 (800 mg, 88%) as a white foam: 1H NMR (400 MHz, DMSO-
d6) δ 11.37 (s, 1H), 7.90 (s, 1H), 7.84 (dd, J = 14.7, 7.6 Hz, 2H),
7.61 (d, J = 8.1 Hz, 1H), 5.78 (d, J = 5.0 Hz, 1H), 5.64 (d, J =
8.0 Hz, 1H), 5.25-5.18 (m, 2H), 4.96 (dd, J = 11.2, 3.3 Hz, 1H),
4.57-4.43 (m, 3H), 4.30-4.11 (m, 5H), 4.05-3.98 (m, 4H),
3.97-3.84 (m, 2H), 3.84-3.67 (m, 3H), 3.64-3.44 (m, 2H), 2.09
(s, 3H), 1.99 (s, 3H), 1.89 (s, 3H), 1.76 (s, 3H), 0.83 (s, 9H), 0.03
(d, J = 11.8 Hz, 6H); 13C NMR (100 MHz, DMSO-d6) δ 170.0,
169.9, 169.6, 169.3, 162.9, 155.9, 150.5, 144.8, 140.2, 123.1,
102.1, 100.9, 87.9, 82.0, 74.7, 70.4, 69.9, 69.5, 69.2, 68.8, 68.2,
66.7, 63.5, 61.4, 49.3, 25.6, 22.8, 20.49, 20.42, 20.41, 17.8, -4.8,
-5.2; HRMS calcd for C37H58N7O17Si (MHþ) 900.3658, found
900.3660.
Loading of 50-O-(4,40-Dimethoxytrityl)-30-O-propargyl-5-
methyluridine on CPG Support (7). Compound 19 (286 mg,
0.48 mmol) in anhydrous CH2Cl2 (50 mL) was treated with
succinic anhydride (231 mg, 0.68 mmol) and DMAP (111 mg,
0.91 mmol) at ambient temperature for 18 h. The solution was
concentrated under reduced pressure. The residue was chroma-
tographed on silica (eluent: TEA/MeOH/CH2Cl2, 5:5:90) to ob-
tain the pure succinate (383 mg, 0.49 mmol, 99%, Rf = 0.6). The
succinate was dissolved in anhydrous DMF (50 mL), and DIEA
(320 μL, 1.84 mmol) and HBTU (191 mg, 0.50 mmol) were
added. The solution was agitated briefly for 2 min, lcaa-CPG
3.84-3.75 (m, 2H), 3.46 (s, 3H), 3.21 (dd, J = 2.2 Hz, 1H); 13
C
NMR (100 MHz, DMSO-d6) δ 163.0, 155.6, 150.5, 140.4, 102.3,
86.0, 82.1, 81.4, 81.3, 73.3, 68.6, 63.9, 57.5, 29.8; MS calcd for
C14H18N3O7 (MHþ) 340.11, found 340.10.
20-O-Methyl-50-O-(4-pentynyl)carbamoyluridine (16). Com-
pound 14 (296 mg, 0.72 mmol) was treated with 7 N NH3 in
methanol (5 mL) at ambient temperature for 4 h. The solution
was concentrated in vacuo and then coevaporated with CH2Cl2
to remove traces of methanol. The residue was dissolved in a
minimal amount of EtOAc/hexane/CH2Cl2 and sonicated for 5
min to give a white precipitate. The precipitate was filtered,
washed with hexane, and dried over a suction funnel. Drying in a
vacuum oven at 40 °C for 18 h gave compound 16 (173 mg,
65%): 1H NMR (400 MHz, DMSO-d6) δ 11.41 (s, 1H), 7.61 (d,
J = 8.1 Hz, 1H), 7.36 (t, J = 5.5 Hz, 1H), 5.86 (d, J = 5.4 Hz,
1H), 5.67 (d, J = 8.1 Hz, 1H), 5.35 (d, J = 5.9 Hz, 1H),
4.26-3.81 (m, 5H), 3.35 (s, 3H), 3.13-2.98 (m, 2H), 2.78 (t, J =
2.4 Hz, 1H), 2.22-2.08 (m, 2H), 1.69-1.49 (m, 2H); 13C NMR
(100 MHz, DMSO-d6) δ 162.9, 155.8, 150.5, 140.4, 102.2, 86.1,
83.9, 82.2, 81.5, 71.4, 68.6, 63.5, 57.5, 28.3, 15.2; MS calcd for
C16H22N3O7 (MHþ) 368.14, found 368.3; HRMS calcd for
C16H21N3NaO7 (MNaþ) 390.1277, found 390.1283.
General Procedure for the Propargylation of the 20/30-Hydro-
xyl Group of Ribonucleoside Derivatives. 50-O-DMTr-nucleoside
(1.00 molar equiv), dibutyltin(II) oxide (1.10 molar equiv),
tetrabutylammonium iodide (0.50-2.00 molar equiv), and pro-
pargyl chloride (2.00 molar equiv) were added to the solution of
benzene/CH3CN (1:1, 0.2 M). The suspension was microwave
irradiated at 100 °C for 1-6 h. Aqueous workup followed by
flash silica gel chromatography gave the desired propargylated
nucleosides.
50-O-(4,40-Dimethoxytrityl)-30-O-propargyl-5-methyluridine (19):
1H NMR (400 MHz, DMSO-d6) δ 11.37 (br, 1H), 7.59-7.18 (m,
10H), 6.91 (m, 4H), 5.80-5.69 (m, 1H), 5.63 (d, J = 5.99 Hz,
1H), 4.46-4.20 (m, 4H), 4.18-3.99 (m, 1H), 3.72 (s, 6H),
3.56-3.46 (m, 1H), 3.27-3.15 (m, 2H), 1.37 (d, J = 0.8 Hz,
3H); 13C NMR (100 MHz, DMSO-d6) δ 163.6, 158.2, 158.2,
150.6, 144.6, 135.5, 135.3, 135.0, 129.7, 128.0, 127.6, 126.8,
113.3, 109.5, 87.9, 86.0, 80.6, 80.1, 77.6, 76.0, 72.3, 62.9, 58.0,
55.1, 31.0, 22.1, 14.0, 11.6; HRMS calcd for C34H34N2NaO8
(MNaþ) 621.2213, found 621.2214.
˚
(pore size 500 A NH2, loading of 140 μmol/g, 3.57 g) was added
to the reaction mixture, and the slurry was agitated on a wrist-
action shaker for 4 h at ambient temperature. The CPG was
filtered, washed with CH2Cl2/MeOH (9:1, v/v, 400 mL), and
dried over suction funnel for 15 min. The remaining amino
residues on the CPG were capped by pyridine/Ac2O/TEA
(75:25:5, v/v, 100 mL) treatment for 15 min. The CPG was
filtered, washed with CH2Cl2/MeOH (9:1, v/v, 600 mL), and
then dried over a suction funnel. Finally, the CPG was dried
under vacuum at ambient temperature for 18 h to give 7 (4.08 g
CPG, loading: 80 μmol/g).
General Procedure for Solid-Phase Synthesis of Modified
siRNA Targeting the Firefly Luciferase Gene. Oligonucleotide
sequences bearing alkyne moieties at desired sites (30 or 50
termini or internal position, Table 1) and other functional
groups on the 50-terminal position (entry 46 and 56 in Table 1)
were synthesized on an ABI 394 DNA synthesizer using stan-
dard phosphoramidite chemistry with commercially available
50-O-(4,40-dimethoxytrityl)-30-O-(2-cyanoethyl-N,N-diisopropyl)-
phosphoramidite monomers of uridine (U), 4-N-acetylcytidine
(CAc), 6-N-benzoyladenosine (ABz), and 2-N-isobutyrylguano-
sine (GiBu), 20-O-tert-butyldimethylsilyl-protected phosphora-
midites, and 50-O-(4,40-dimethoxytrityl)-20-deoxythymidine-
30-O-(2-cyanoethyl-N,N-diisopropyl) phosphoramidite (dT).
Extended nucleotide coupling times (30 min) were applied
at steps incorporating modified phosphoramidites (alkyne
monomers 1, 2, 3, 4, 6, and triazoyl monomers 30 and 31). After
synthesis, a small portion of the oligonucleotide bound to
CPG was treated with 100 μL of methylamine solution (40 wt %
in water) in a 1 mL microtube at 65 °C for 10 min. The mixture
was cooled on dry ice for 5 min and the solid suspension was
spun down. Of the supernatant, 80 μL was decanted into
20-O-(tert-Butyldimethylsilyl)-50-O-[1-linoleyl(1,2,3-triazo-3-yl)-
methyl]carbamoyluridine (28). Compound 11 (100 mg, 0.17 mmol)
in CH2Cl2/MeOH (4:1, v/v) (2 mL) was treated with 23 (59 mg,
0.17 mmol), tetrakis(acetonitrile)copper(I) hexafluorophosphate
(13 mg, 0.03 mmol), and copper (2 mg, 0.03 mmol) at ambient
temperature for 3 h. The solution was concentrated in vacuo. The
residue was purified by flash chromatography on silica gel (1:3
hexane/EtOAc, v/v) to obtain 28 (122 mg, 98%) as a white foam: 1H
NMR (400 MHz, DMSO-d6) δ 11.35 (s, 1H), 7.91 (s, 1H), 7.82 (br,
1H), 7.60 (d, J = 7.8 Hz, 1H), 5.78 (d, J = 5.0 Hz, 1H), 5.63 (d, J =
7.8 Hz, 1H), 5.46-5.24 (m, 4H), 5.17 (br, 1H), 4.35-4.16 (m, 6H),
4.16-4.08 (m, 1H), 4.06-3.96 (m, 1H), 3.93-3.82 (m, 1H), 2.73
(dd, J = 6.2 Hz, 2H), 2.16-1.90 (m, 2H), 1.90-1.66 (m, 2H),
1.60-1.03 (m, 16H), 1.04-0.54 (m, 12H), 0.33-0.30 (m, 9H); 13
C
NMR (100 MHz, DMSO-d6) δ 161.7, 154.7, 139.1, 128.6, 128.5,
126.6, 101.0, 86.7, 80.9, 73.6, 68.4, 62.3, 48.1, 39.0, 38.8, 38.6, 38.0,
37.8, 34.8, 29.7, 28.6, 27.8, 27.5, 27.4, 27.2, 25.5, 25.4, 24.7, 24.4, 24.0,
another microtube and heated with 120 μL of TEA 3HF at
65 °C for 12 min. The purity of crude oligonucleotides was
analyzed by RP-HPLC or anion-exchange high-performance
3
1210 J. Org. Chem. Vol. 76, No. 5, 2011

Yamada et al.
JOCFeatured Article
liquid chromatography (IEX-HPLC) and the mass was con-
firmed by LC-MS analysis prior to postsynthetic CuAAC
reaction.
solutions in PBS buffer were measured and the duplex prepara-
tion was repeated if the optical density was not within a 10%
range of a reference sample (parent duplex).
General Procedure for Click Reaction on Solid Support. To a
0.6 μmol solid-supported alkyne-RNA phosphotriester was
added a mixture of an azide (3 equiv by alkyne, 1.8 μmol, 36
Cell Culture. HeLa SS6 cells, stably transfected with the lucif-
erase plasmids, were grown at 37 °C in 5% CO2 in Dulbecco0s
modified Eagle0s medium supplemented with 10% fetal bovine
serum (FBS) and 0.5 mg/mL Zeocin and 0.5 μg/mL puromycin
(selective for cells transfected with plasmids). The cells were
maintained in exponential growth phase.
μL of a 50 mM solution in THF), freshly prepared CuSO4 5H2O
3
(0.4 equiv, 0.24 μmol, 5 μL of a 50 mM solution in H2O), freshly
prepared sodium ascorbate (3 equiv, 1.8 μmol, 37 μL of a 50 mM
solution in H2O), and TBTA (3 equiv, 1.8 μmol, 37 μL of a
50 mM solution in THF). Water/MeOH/THF (2:2:1 v/v) was
added to obtain a total volume of 1200 μL. The resulting
preparation was heated in a sealed glass tube in an Explorer-
48 microwave synthesizer at a 100 W and a 30 s premixing time.
The temperature was monitored with an internal infrared probe
and held at 60 °C over 45 min. The solution was removed, and
the CPG supports were washed with THF and MeOH then
dried. The product on the CPG supports were cleaved and
deprotected by treating the solid-support with 100 μL of methy-
lamine solution (40 wt % in water) at 65 °C for 10 min. The
mixture was cooled on dry ice for 5 min and the solid suspension
was spun down. Of the supernatant, 80 μL was decanted into
Luciferase Assay. The cells were plated in 96-well plates (0.1 mL
medium per well) and were at about 90% confluence at transfec-
tion. The cells were grown for 24 h, and the culture medium was
changed to Opti-MEM, 0.5 mL per well. Transfection of siRNAs
was carried out with Lipofectamine 2000 as described by the
manufacturer for adherent cell lines at siRNA concentrations
ranging from 0.002 to 8.0 nM. The final volume was 150 μL per
well. The cells were harvested 24 h post transfection and lysed using
passive lysis buffer (PLB), 100 μL per well. The luciferase activities
of the samples were measured using a Victor FL Luminometer
with 20 μL of sample and 75 μL each of Luciferase assay reagent II
and Stop & Glo reagent. The inhibitory effects of the siRNAs were
expressed as normalized ratios between the activities of the reporter
(firefly) luciferase gene and the control (Renilla) luciferase gene
relative to untreated controls (no siRNA, but with Lipofectamine
2000). Values represent the mean of triplicates. The potency of the
siRNAs was determined by calculating the IC50 values from
dose-response curves using XL-Fit software.
another microtube and heated with 120 μL of TEA 3HF at
3
65 °C for 12 min. The product was analyzed by LC-MS and by
˚
RP-HPLC (C4 column, 150 ꢀ 3.9 mm i.d., 5 μm, 300 A) using a
linear gradient of 0 to 70% B over 24 min at 30 °C at a flow rate
of 1 mL/min (buffer A: 50 mM TEAA, pH 7.0; buffer B:
CH3CN). Completion of the CuAAC reaction was estimated
by comparing the purity of starting material alkyne-RNAs and
their corresponding triazole products by RP-HPLC. After base
and sugar deprotection, oligonucleotides were purified by RP-
Acknowledgment. T.Y. acknowledges the Genome Network
Project from the Ministry of Education, Culture, Sports, Science
and Technology, Japan, for a fellowship.
˚
HPLC with C4 column (300 ꢀ 7.8 mm i.d., 15 μm, 300 A) using a
linear gradient of 0 to 90% B in 40 min at room temperature at a
flow rate of 3 mL/min (buffer A: 50 mM TEAA, pH 7.0; buffer
B: CH3CN).
Supporting Information Available: Yields of 20/30-O-pro-
pargylated 50-O-DMTr nucleosides under optimized micro-
wave-assisted reaction conditions; synthetic scheme of alkyl
azides 20-24, scheme and synthetic procedures for azides 25
and 25a; NMR spectra of nucleoside derivatives and azides 24,
25, and 25a; LC-MS characterization of alkyne-oligonucleo-
tide scaffolds 32a-41a, and HPLC profiles of click reaction of
alkyne-oligonucleotides with azides and purified oligonucleo-
tide conjugates. This material is available free of charge via the
Internet at http://pubs.acs.org.
Duplex Annealing. For duplex annealing, 1 mM stock solu-
tions of oligonucleotides were prepared in deionized water. For
the cell culture experiments, 0.05 mM stock solutions of siRNA
duplexes were prepared by mixing equimolar amounts of com-
plementary sense and antisense strands in PBS buffer. The
solutions were heated at 95 °C for 5 min and then allowed to
slowly cool to room temperature. To ensure low variability in
duplex concentration, the optical densities of the final duplex
J. Org. Chem. Vol. 76, No. 5, 2011 1211