Oligonucleotide conjugates by means of copper-free click chemistry - Expanding the repertoire of strained cyclooctyne phosphoramidites

Article abstract of DOI:10.1055/s-0030-1260104

A set of four phosphoramidite building blocks containing a strained dibenzocyclooctyne moiety is reported, including one example equipped with a cleavable disulfide linker. Application of these amidites in solid-phase oligonucleotide synthesis yields either 5′- or 3′-cyclooctyne- modified nucleic acids. The strained cyclooctyne-bearing oligonucleotides are used in rapid and clean conjugation reactions with azide-containing (bio)molecules. Georg Thieme Verlag Stuttgart - New York.

Full text of DOI:10.1055/s-0030-1260104

2724
SPECIAL TOPIC
Oligonucleotide Conjugates by Means of Copper-Free Click Chemistry –
Expanding the Repertoire of Strained Cyclooctyne Phosphoramidites
S
ynthesis of
O
i
ligonu
e
C
o
t
njugate
e
s
r van Delft, Evert van Schie, Nico J. Meeuwenoord, Herman S. Overkleeft, Gijsbert A. van der Marel,*
Dmitri V. Filippov*
Leiden Institute of Chemistry, Leiden University, PO Box 9502, 2300 RA Leiden, The Netherlands
Fax +31(71)5274307; E-mail: filippov@chem.leidenuniv.nl; E-mail: marel_g@chem.leidenuniv.nl
Received 7 April 2011
conjugation of the oligomer to a hexadecapeptide and a
Abstract: A set of four phosphoramidite building blocks contain-
ing a strained dibenzocyclooctyne moiety is reported, including one
example equipped with a cleavable disulfide linker. Application of
these amidites in solid-phase oligonucleotide synthesis yields either
hyaluronan tetrasaccharide proceeded rapidly and in near-
quantitative fashion. These results make this strategy ap-
pealing and have guided us to the development of addi-
5¢- or 3¢-cyclooctyne-modified nucleic acids. The strained cyclooc- tional phosphoramidite building blocks. We focused on
tyne-bearing oligonucleotides are used in rapid and clean conjuga-
tion reactions with azide-containing (bio)molecules.
easily accessible building blocks for 5¢- and 3¢-conjuga-
tion. The termini are often used as conjugation sites to
Key words: oligonucleotides, strain-induced cycloaddition, conju-
gates, phosphoramidites, bioconjugates
keep the primary and secondary structures of the oligonu-
cleotide intact, as is required in the field of post-transcrip-
tional gene-silencing using RNAi or antisense
oligonucleotides. Figure 1 depicts the new building
The unique properties and biological roles of nucleic acids
have attracted considerable interest toward this class of
biomolecules. Recently, nucleic acids and their deriva-
tives have found applications in the fields of nano1,2 and
materials science.3,4 In biomedical science, the discovery
of antisense oligonucleotides5 and the process of RNA in-
terference (RNAi),6 and their associated applications,7–9
has continued to stimulate the design and synthesis of
nucleic acid derivatives. Both antisense and RNAi
techniques have been employed as tools for post-
transcriptional gene-silencing in both fundamental re-
search and clinical settings. In this respect the classic an-
tisense strategy utilizes stabilized isosters of DNA such as
phosphothioate backbones, peptide nucleic acids (PNA)10
and locked nucleic acids (LNA),11 while the RNAi-based
methodology requires double-stranded small interfering
RNAs (siRNAs).12 Although the present state of the art in-
dicates that protein translation arrest can be achieved in a
specific and effective manner, delivery of these com-
pounds across the cell membrane to their targets remains
an obstacle. In this framework several research groups
have pursued studies in which oligonucleotides are conju-
gated to fluorescent labels,13 and/or molecular entities, in
order to improve cell permeability, to enable monitoring
of cellular uptake, or both.14,15
O
O
N
N
P
O
H
O
NC
1
O
S
O
N
N
S
P
O
3
3
H
O
NC
2
O
O
N
O
N
P
H
O
O
DMTO
3
HN
We,16 and others,17 have reported the use of copper-free
alkyne–azide click chemistry based on the strained diben-
zocyclooctyne developed by Boons et al.18 in oligonucle-
otide conjugation chemistry. The compatibility of the
dibenzocyclooctyne with solid-phase nucleic acid chem-
istry was demonstrated by the successful synthesis of an
RNA 16-mer using 5¢ modifier 1 (Figure 1). Subsequent
N
O
DMTO
O
O
O
O
P
N
N
H
O
4
SYNTHESIS 2011, No. 17, pp 2724–2732
Advanced online publication: 08.07.2011
DOI: 10.1055/s-0030-1260104; Art ID: C37811SS
© Georg Thieme Verlag Stuttgart · New York
x
x.
x
x
.
2
0
1
1
Figure 1 Phosphoramidite building blocks allowing incorporation
of strained cyclooctynes into DNA or RNA oligomers (DMT = 4,4¢-
dimethoxytrityl)

SPECIAL TOPIC
Synthesis of Oligonucleotide Conjugates
2725
c
a
R
OH
BocHN
H2N
6 R = OH
7 R = SAc
5
b
Cl–
+H3N
e
d
S
S
BocHN
2
2
9
8
O
f
O
S
S
OH
O
N
N
O
N
S
H
3
H
3
3
S
S
OH
3
2
12
10
11
O2N
O
g
2
O
O
13
Scheme 1 Reagents and conditions: (a) Boc2O, CH2Cl2, sat. aq NaHCO3, 93%; (b) AcSH, Ph3P, DIAD, toluene, 0 °C, 73%; (c) KOH, EtOH,
reflux, then I2, MeOH, 80%; (d) HCl, 1,4-dioxane, 74%; (e) 13, Et3N, DMF, 88%; (f) Me3P (1 M, toluene), H2O–THF (1:9), workup, then 11,
THF, 43%; (g) 2-cyanoethoxy-N,N-diisopropylaminochlorophosphine, DIPEA, CH2Cl2, 80%.
blocks, 2–4 that we have developed for the 3¢- and 5¢- The synthesis of phosphoramidite 2 was started from
modification of nucleic acid fragments, and which are the readily available 6-aminohexan-1-ol (5). The amine moi-
subject of this work.
ety was protected as the corresponding tert-butoxycarbo-
nyl and the hydroxy group was converted into a
thioacetate under Mitsunobu22 conditions to afford com-
pound 7. Basic hydrolysis using potassium hydroxide in
ethanol followed by titration with iodine yielded symmet-
rical disulfide 8. Amine deprotection using hydrochloric
acid in 1,4-dioxane and treatment of the hydrochloride
salt thus obtained with the known p-nitrophenylcarbonate
of dibenzocyclooctyne 1318 yielded compound 10. Disul-
fide 10 was cleaved using trimethylphosphine and the re-
sulting crude thiol was reacted with disulfide 11 to furnish
asymmetric disulfide 12. Some of the starting material 10
was recovered as a result of oxidation of the intermediate
thiol during workup. Phosphitylation of the free hydroxy
group yielded target phosphoramidite 2 in 80% yield.
The new disulfide-containing phosphoramidite 2 allows
introduction of the strained cyclooctyne at the 5¢-terminus
of an oligonucleotide. The presence of the disulfide in the
linker arm of 2 allows reductive cleavage of the clicked
entities in a cellular environment. Cleavable oligonucle-
otide conjugates have been found to enhance target gene
specificity and lead to a reduced immune response.19
Phosphoramidites 3 and 4 allow 3¢ modification of the oli-
gonucleotide. Phosphoramidite 3, in combination with a
solid support containing a diethoxysulfonyl linker, gives
access to DNA fragments functionalized with a cyclooc-
tyne handle at the 3¢ end. The design of compound 4 is
based on the commonly used 3¢-TT overhang in siRNAs
for added stability towards enzymatic degradation and en-
hanced gene-silencing properties.20,21
The synthesis of both 3¢ modifiers 3 and 4 started with the
reaction of commercially available phosphine 14 with the
O
a)
b)
HN
O
N
O
OH
DMTO
O
O
18
N
O
3
3
H
N
O
Cl
N
P
N
N
O
P
N
15
H
OH
3 or 4
DMTO
OH
or
14
16
17
Scheme 2 Reagents and conditions: (a) Et3N, 1,4-dioxane, workup; (b) 1H-tetrazole, MeCN, 64% (3); 61% (4).
Synthesis 2011, No. 17, 2724–2732 © Thieme Stuttgart · New York

2726
P. van Delft et al.
SPECIAL TOPIC
known cyclooctyne 15.16 The labile bisamidite intermedi- cloaddition of strained alkyne 19 and azide 20 was execut-
ate 16 was isolated carefully and further processed by re- ed in water (at 2 mM concentration), and after 90 minutes,
action with either mono DMT-protected 1,3-propanediol LC–MS analysis revealed the complete disappearance of
17 or 5¢-O-DMT-dT (18) in the presence of 1H-tetrazole both starting materials and formation of the conjugate 21
(0.5 equiv) to yield the strained cyclooctyne building consisting of two putative regioisomers.
blocks 3 and 4 in 61% and 64% yields, respectively.
Implementation of cyclooctyne phosphoramidites 3 and 4
With our cyclooctyne amidites 2, 3 and 4 in hand, we next in oligonucleotide synthesis requires the use of a con-
undertook the synthesis of a thymidine hexamer contain- trolled pore glass (CPG) solid support containing a di-
ing the 5¢-disulfide modifier (Scheme 3). Starting from a ethoxysulfonyl linker. Whilst this type of b-eliminating
highly cross-linked polystyrene (PS) resin pre-loaded linker is commonly used to introduce a 3¢-phosphate mo-
with DMT-dT, five consecutive coupling cycles were car- noester, the use of phosphoramidites 3 and 4 would result
ried out under standard DNA synthesis conditions (five in 3¢-phosphodiesters containing a strained cyclooctyne
minutes, four equivalents of phosphoramidite, iodine oxi- handle (Scheme 5). Amidites 3 and 4 were coupled under
dation, acetic anhydride capping and dimethoxytrityl similar conditions as those used for 2, followed by five
group removal) followed by the final coupling cycle using consecutive coupling cycles with 5¢-O-DMT-dT-phos-
the disulfide-containing cyclooctyne amidite 2 under op- phoramidite, and a final cleavage step using concentrated
timized conditions (10 minutes, six equivalents). Treat- aqueous ammonium hydroxide to yield the respective 3¢-
ment of the resin with concentrated aqueous ammonium modified thymidine pentamer and hexamer. Purification
hydroxide and subsequent purification by RP-HPLC by RP-HPLC or anion exchange chromatography, or by a
yielded functionalized hexamer 19.
combination of both yielded target oligonucleotides 22
and 23.
Next, the thymidine hexamer 19 containing the cleavable
disulfide linker was conjugated to azide-functionalized To demonstrate conjugation at the 3¢-end of the oligonu-
zwitterionic tetrasaccharide 2023 (Scheme 4). The cy- cleotide we selected the thymidine hexamer 23 as a model
i) solid-phase DNA synthesis
ii) 2, BMT
iii) concd NH4OH, r.t.
O
O
PS
dT
S
O
O
N
S
P
O–
TTTTTT-3'
O
3
3
H
19
Scheme 3 Solid-phase synthesis of oligonucleotide 19 bearing a strained cyclooctyne at the 5¢-end (PS = highly crosslinked polystyrene; dT =
thymidine; T = thymidine residue, phosphodiester linked; BMT = 5-benzylmercapto-1H-tetrazole)
Scheme 4 Strain-induced alkyne–azide click reaction of thymidine hexamer 19 and tetrasaccharide 20 yielding conjugate 21, and the corre-
sponding LC–MS traces
Synthesis 2011, No. 17, 2724–2732 © Thieme Stuttgart · New York

SPECIAL TOPIC
Synthesis of Oligonucleotide Conjugates
2727
O
O
O
5'-TTTTT
O
P
O
O
O
P
O–
N
O
3
–O
H
22
or
i) 3 or 4, BMT
CPG
O
ii) solid-phase
O
N
DNA synthesis
ODMT
NH
iii) concd NH4OH,
55 °C
S
O
O
O
O
P
O–
O
5'-TTTTT-O
O
O
O
O
P
N
O
O
3
H
O–
23
Scheme 5 Solid-phase synthesis of 3¢-dibenzocyclooctyne oligonucleotides 22 and 23
compound. The oligonucleotide 23 was reacted with the tional organic solvent. After 90 minutes, quantitative con-
zwitterionic oligosaccharide 20 (Scheme 6) under identi- version of the starting materials into the target conjugates
cal conditions as described above for conjugate 21. LC– was observed. Scheme 7 depicts the LC–MS traces of the
MS analysis of the crude reaction mixture showed the reaction between 3¢-modified oligonucleotide 23 and tet-
presence of the target conjugate 24 and complete disap- ramethylrhodamine azide (25) furnishing fluorescent con-
pearance of the starting materials.
jugate 26.
The advent of copper-free click chemistry has led to the The conjugation of fluorescent dye 25 was repeated in a
commercial availability of several fluorescent labels func- similar fashion using oligonucleotide 19 to furnish
tionalized with alkyl azides, for example tetramethyl- smoothly the corresponding conjugate 27 (Scheme 8).
rhodamine (TAMRA). As mentioned previously,
In summary, we have described the synthesis of three new
fluorescently labeled oligonucleotides are often applied in
phosphoramidite building blocks containing a strained
biochemical research, and therefore we conjugated a poly-
dibenzocyclooctyne moiety. These amidites, 2, 3 and 4,
can be used in automated solid-phase synthesis of DNA
ethylene glycol (PEG) spaced tetramethylrhodamine
azide (TAMRA-N3) to both 5¢- and 3¢-oligonucleotides 19
oligomers to afford oligonucleotides bearing a 3¢- or 5¢-
and 23. Due to the water-solubility of the tetramethyl-
handle for strain-induced alkyne–azide click reactions.
rhodamine dye, conditions using 2 mM aqueous solutions
The oligonucleotides obtained were used in conjugation
could be applied without the necessity of adding an addi-
OH
–
CO2
O
O
HO
O
N3
HO
O
HO
+
NH3
OH
2
20
O
O
O
O
5'-TTTTTT
P
O–
N
H
O
3
23
OH
N
–
CO2
O
O
N
O
O
OH
O
OH
O
O
N
O
OH
5'-TTTTTT
P
O–
N
H
O
+H3N
3
HO
2
24
Scheme 6 Strain-induced alkyne–azide click reaction of thymidine hexamer 23 and tetrasaccharide 20 yielding conjugate 24
Synthesis 2011, No. 17, 2724–2732 © Thieme Stuttgart · New York

2728
P. van Delft et al.
SPECIAL TOPIC
Scheme 7 Strain-induced alkyne–azide click reaction between thymidine hexamer 23 and the fluorescent dye 25 yielding conjugate 26, and
the corresponding LC–MS traces
O
O
S
O
O
N
O
S
P
O–
TTTTTT-3'
3
H
3
19
25
Me
N
Me
N+
O
Me
Me
–O2C
N
O
N
O
S
O
O
H
N
N
P
O–
TTTTTT-3'
O
S
N
3
3
H
O
3
O
27
Scheme 8 Strain-induced alkyne–azide click reaction of thymidine hexamer 19 and the fluorescent dye 25 yielding conjugate 27
distilled over CaH2 and stored over 4 Å MS. Petroleum ether (PE)
refers to the fraction boiling in the 40–60 °C range. DIPEA was dis-
tilled and stored over KOH pellets. Compounds used in reactions re-
quiring anhyd conditions were co-evaporated with 1,4-dioxane,
pyridine or toluene three times. All reactions were performed at am-
bient temperature under an Ar atmosphere unless stated otherwise.
Oligonucleotides were synthesized on an ÄKTA Oligopilot Plus
oligonucleotide synthesizer (GE Healthcare Life Sciences). Reac-
tions were monitored by TLC on Kieselgel 60 F254 (Merck). Com-
pounds were made visual using UV light (254 nm) or by applying a
soln of (NH4)6Mo7O24·4H2O (25 g/L), (NH4)4Ce(SO4)4·2H2O (10 g/
L) or 10% H2SO4 in H2O followed by charring (ca. 150 °C). LC–MS
analyses were performed on a Jasco HPLC system (UV detection si-
multaneously at 214 and 254 nm) coupled to a PE/SCIEX API 165
single quadrupole mass spectrometer (Perkin-Elmer). An analytical
Gemini C18 column (Phenomex, 50 × 4.60 mm, 3 m) was used in
reactions with both a zwitterionic oligotetrasaccharide
and a tetramethylrhodamine fluorescent label. The conju-
gation reactions were rapid and proceeded quantitatively,
using equimolar amounts of reagents at low mM concen-
trations to furnish various conjugates including examples
with reductively cleavable disulfide linkers. The easy ac-
cessibility of the amidites together with the effectiveness
of the strain-promoted alkyne–azide click reaction dem-
onstrates the potential of our approach for future applica-
tion in the design and synthesis of constructs for
application in antisense and RNAi gene-silencing studies.
Chemicals were purchased from Acros Organics, Sigma Aldrich,
Proligo and Jena Bioscience and were used as received. CH2Cl2 was
Synthesis 2011, No. 17, 2724–2732 © Thieme Stuttgart · New York

SPECIAL TOPIC
Synthesis of Oligonucleotide Conjugates
2729
combination with eluents A: H2O, B: MeCN and C: aq NH4OAc
(0.1 M). Analytical anion exchange was performed on a GE ÄK-
TAexplorer 10 using a Dionex DNA-PAC PA-200 column (4 × 250
mm) with eluents A: NaOAc (500 mM) and NaClO4 (50 mM), and
B: NaOAc (500 mM) and NaClO4 (500 mM) using a linear gradient
(0–20%). Anion exchange purification was performed on a GE ÄK-
TAexplorer 10 using a GE Q-Sepharose HR column (260 × 10 mm)
with eluents A: NaOAc (500 mM) and NaClO4 (50 mM), and B:
NaOAc (500 mM) and NaClO4 (500 mM), followed by a desalting
procedure using a Sephadex G25 column with NH4OAc (150 mM)
as the solvent. Preparative RP-HPLC was performed on a Gilson
GX-281 HPLC system. A semi-preparative Altima C18 column
(Phenomex, 250 × 10 mm, 5 m) was used in combination with elu-
1H NMR (400 MHz, CDCl3): d = 4.71 (s, 1 H), 3.10 (dd, J = 12.9,
6.4 Hz, 2 H), 2.86 (t, J = 7.3 Hz, 2 H), 2.32 (s, 3 H), 1.63–1.52 (m,
2 H), 1.52–1.41 (m, 11 H), 1.41–1.28 (m, 4 H).
13C NMR (100 MHz, CDCl3): d = 195.8, 155.8, 78.7, 40.3, 30.5,
29.7, 29.3, 28.8, 28.3, 28.2, 26.1.
HRMS (ESI): m/z [M + Na]+ calcd for C13H25NO3SNa: 298.1447;
found: 298.1448.
Di-tert-butyl (6,6¢-Dihexyldisulfide)-1,1¢-dicarbamate (8)
Thioacetate 7 (4.13 g, 15 mmol) was dissolved in an ethanolic soln
of KOH (160 mL, 10% w/v) and heated at reflux temperature for 1
h. The mixture was cooled to 0 °C and quenched by the addition of
AcOH [17.6 mL, 1.1 equiv (relative to KOH)]. The mixture was di-
luted with H2O (200 mL) and extracted with EtOAc (3 × 200 mL).
The combined organic layer was washed with brine (150 mL), dried
(MgSO4) and concentrated under reduced pressure. The resulting
oil was dissolved in MeOH (100 mL) and titrated with a soln of I2
in MeOH (0.1 M) until the soln became pale-yellow in color. H2O
(250 mL) was added and the mixture extracted with EtOAc (3 × 200
mL). The combined organic layer was washed with sat. aq NaHCO3
soln containing Na2S2O5 (100 mL, 5% w/v) and brine (2 × 100 mL),
then dried (MgSO4) and concentrated under reduced pressure. Silica
gel column chromatography (PE–EtOAc, 85:15) of the residue af-
forded the title disulfide 8 as an oil.
1
ents A: aq Et3HN·OAc (50 mM) and B: MeCN. H, 13C and 31P
1
NMR spectra were recorded on a Bruker AV-400 instrument. H
and 13C NMR chemical shifts (d) are quoted relative to tetramethyl-
silane. 31P NMR chemical shifts (d) are quoted relative to H3PO4;
reaction mixture aliquots were measured by means of an acetone-d6
capillary. CDCl3 was neutralized by filtration over neutral Al2O3
(Merck). HRMS spectra were recorded by direct injection [2 mL of
a mM soln in H2O or MeCN and HCO2H (0.1%) or NH4OAc (0.1%)]
using a Thermo Finnigan LTQ Orbitrap equipped with an electro-
spray ion source in positive mode. IR spectra were recorded on a
Shimadzu FT-IR 8300 and are reported in cm–1. Melting points
were determined using a Stuart Scientific SMP3 melting point ap-
paratus. The yields of the oligonucleotides were determined spec-
trophotometrically using optical density (OD) measurements on a
Varian Cary 50 Bio UV-VIS Spectrophotometer at 260 nm.
Yield: 2.8 g, 6 mmol (80%); Rf = 0.3 (PE–EtOAc, 85:15).
IR (neat): 2928, 1700, 1521, 1363, 1252, 1168, 1120, 872 cm–1.
1H NMR (300 MHz, CDCl3): d = 4.59 (s, 2 H), 3.07 (dd, J = 12.8,
6.4 Hz, 4 H), 2.69–2.55 (m, 4 H), 1.71–1.54 (m, 4 H), 1.52–1.43 (m,
4 H), 1.41 (s, 18 H), 1.38–1.21 (m, 8 H).
13C NMR (75 MHz, CDCl3): d = 156.2, 79.0, 77.5, 77.3, 77.1, 76.7,
40.5, 38.9, 30.0, 29.1, 28.4, 28.1, 26.4.
ESI-MS: m/z = 464.93 [M + H]+, 487.07 [M + Na]+.
tert-Butyl (6-Hydroxyhexyl)carbamate (6)
To a stirred soln of 6-amino-1-hexanol (5) (3.5 g, 30 mmol) in
CH2Cl2–sat. aq NaHCO3 (300 mL, 3:2 v/v) was added Boc2O (9.8
g, 45 mmol, 1.5 equiv). The reaction mixture was stirred for 40 h,
after which the aq layer was separated and extracted with CH2Cl2
(100 mL). The combined organic layer was washed with H2O (2 ×
75 mL) and brine (75 mL), then dried (MgSO4) and concentrated
under reduced pressure. The resulting oil was purified by silica gel
column chromatography (PE–EtOAc, 7:3→4:6) to afford the title
compound 6 as a pale-yellow oil.
(6,6¢-Dihexyldisulfide)-1,1¢-diamine Dihydrochloride (9)
To Boc-protected disulfide 8 (1.4 g, 3 mmol) was added a soln of
HCl in 1,4-dioxane (4 M, 6 mL). The resulting mixture was stirred
for 20 min during which time a white precipitate formed. The mix-
ture was cooled to 0 °C and Et2O (3 mL) was added. The mixture
was filtered and the residue washed with Et2O (3 × 3 mL) and dried
to yield the title salt as a white solid.
Yield: 6.08 g, 28.0 mmol (93%); Rf = 0.56 (PE–EtOAc, 1:1).
IR (neat): 3363, 2930, 1683, 1510, 1362, 1245, 1165, 1059, 996
cm–1.
1H NMR (400 MHz, CDCl3): d = 4.65 (s, 1 H), 3.58 (t, J = 6.4 Hz,
2 H), 3.07 (dd, J = 12.8, 6.4 Hz, 2 H), 2.25 (s, 1 H), 1.55–1.48 (m,
2 H), 1.48–1.40 (m, 2 H), 1.40 (s, 9 H), 1.36–1.27 (m, 4 H).
13C NMR (100 MHz, CDCl3): d = 156.0, 79.0, 62.4, 40.2, 32.4,
29.9, 28.3, 26.3, 25.2.
HRMS (ESI): m/z [M + Na]+ calcd for C11H23NO3Na: 240.1570;
found: 240.1569; m/z [M + H]+ calcd for C11H24NO3: 218.1751;
found: 218.1750.
Yield: 0.75 g, 2.2 mmol (74%); mp >190 °C (decomp).
IR (neat): 2919, 1608, 1504 cm–1.
1H NMR (400 MHz, D2O): d = 2.98 (t, J = 7.5 Hz, 4 H), 2.75 (t,
J = 7.2 Hz, 4 H), 1.77–1.57 (m, 8 H), 1.49–1.33 (m, 8 H).
13C NMR (100 MHz, D2O): d = 39.4, 37.9, 28.0, 27.0, 26.5, 25.1.
HRMS (ESI): m/z [M + H]+ calcd for C12H29N2S2: 265.1767; found:
265.1767.
(6,6¢-Dihexyldisulfide)-(1-Carbonic Acid 7,8-Didehydro-
1,2:5,6-dibenzocycloocten-3-yl Ester) 1,1¢-Diamide (10)
S-{6-[(tert-Butoxycarbonyl)amino]hexyl} Ethanethioate (7)
To a stirred soln of DIAD (5.95 mL, 30 mmol, 1.2 equiv) in anhyd
toluene (30 mL) at 0 °C was added a soln of PPh3 (7.8 g, 30 mmol,
1.2 equiv) in anhyd toluene (30 mL). After 15 min, a pre-cooled (0
°C) soln of 6 (5.4 g, 25 mmol) and AcSH (2.1 mL, 30 mmol, 1.2
equiv) in anhyd toluene (300 mL) was added in one portion. The
mixture was allowed to warm to ambient temperature and stirred for
17 h. The mixture was concentrated under reduced pressure and the
remaining thick oily residue was purified by silica gel column chro-
matography (PE–EtOAc, 95:5→91.5:8.5) to afford the title com-
pound (7) as an oil.
To a slurry of disulfide 9 (675 mg, 2 mmol) and Et3N (3.37 mL, 12
mmol) in DMF (20 mL) was added carbonate 13 (4 mmol, 2 equiv)
in DMF (20 mL). An additional volume of DMF (40 mL) was added
for solubility purposes and the reaction mixture was stirred for 4 d.
The mixture was concentrated to ca. 5 mL, diluted with EtOAc (160
mL) and washed with aq NaOH (0.5 M, 2 × 50 mL), brine (2 × 50
mL), then dried (MgSO4) and concentrated under reduced pressure.
The crude residue was purified by silica gel column chromatogra-
phy (PE–EtOAc, 8:2→6:4) to afford the title disulfide 10 as an oil.
Yield: 1.34 g, 1.76 mmol (88%). Rf = 0.31 (PE–EtOAc, 7:3).
IR (neat): 3326, 2926, 1699, 1516, 1237, 1021, 750 cm–1.
Yield: 5.04 g, 18.3 mmol (73%); Rf = 0.26 (PE–EtOAc, 9:1).
IR (neat): 2931, 1683, 1506, 1363, 1247, 1169, 1134 cm–1.
Synthesis 2011, No. 17, 2724–2732 © Thieme Stuttgart · New York

2730
P. van Delft et al.
SPECIAL TOPIC
1H NMR (400 MHz, DMSO-d6): d = 7.61 (t, J = 5.7 Hz, 2 H), 7.54
(d, J = 7.7 Hz, 2 H), 7.48–7.34 (m, 14 H), 5.30 (s, 2 H), 3.17 (dd,
J = 15.0, 1.9 Hz, 2 H), 3.03–2.92 (m, 4 H), 2.77 (dd, J = 14.9, 3.9
Hz, 2 H), 2.67 (t, J = 7.2 Hz, 4 H), 1.64–1.54 (m, 4 H), 1.47–1.36
(m, 6 H), 1.36–1.22 (m, 6 H).
13C NMR (100 MHz, DMSO-d6): d = 155.7, 153.1, 151.4, 130.6,
128.9, 128.8, 127.9, 127.8, 126.6, 126.3, 124.2, 123.4, 120.9, 113.1,
110.4, 75.7, 46.0, 40.7, 40.6, 40.4, 40.2, 40.0, 39.8, 39.6, 39.4, 38.2,
29.7, 29.0, 27.9, 26.3.
6¢-(1-Carbonic Acid 7,8-Didehydro-1,2:5,6-dibenzocycloocten-
3-yl Ester 1¢-Hexamide)-disulfanyl-6-hexyl-1-(2-cyanoethoxy-
N,N-diisopropylamino)phosphoramidite (2)
To a soln of alcohol 12 (200 mg, 0.4 mmol) in anhyd CH2Cl2 (4 mL)
was added DIPEA (132 mL, 0.8 mmol, 2 equiv) and 2-cyanoethoxy-
N,N-diisopropylaminochlorophosphine (87.3 mL, 0.4 mmol, 1
equiv). The reaction mixture was stirred for 3 h, diluted with EtOAc
(15 mL, containing 1% Et3N), washed with brine (2 × 7.5 mL), dried
(Na2SO4) and concentrated under reduced pressure. Silica gel col-
umn chromatography (PE–EtOAc–Et3N, 94:5:1→91.5:7.5:1) af-
forded the title compound 2 as an oil.
HRMS (ESI): m/z [M + H]+ calcd for C46H49N2O4S2: 757.3128;
found: 757.3132.
Yield: 225 mg, 0.31 mmol (80%); Rf = 0.35 (PE–EtOAc–Et3N,
9:1:0.1).
6-(Pyridin-2-yldisulfanyl)hexan-1-ol (11)
To a stirred soln of 2,2¢-dithiodipyridine (2 g, 9 mmol, 3 equiv) in
deoxygenated MeOH (50 mL) was added dropwise 6-mercaptohex-
anol (0.4 mL, 3 mmol). The mixture was stirred for 1 h followed by
evaporation under reduced pressure. Silica gel column chromatog-
raphy of the residue (CH2Cl2–EtOAc, 95:5→8:2) afforded the title
compound 11 as an oil.
IR (neat): 2930, 1718, 1508, 1450, 1364, 1240, 1026, 975, 757
cm–1.
1H NMR (400 MHz, CD3CN): d = 7.65 (d, J = 7.8 Hz, 1 H), 7.52–
7.36 (m, 7 H), 6.02 (t, J = 5.6 Hz, 1 H), 5.43 (s, 1 H), 3.89–3.76 (m,
2 H), 3.73–3.60 (m, 4 H), 3.23 (dd, J = 15.0, 2.0 Hz, 1 H), 3.15 (dd,
J = 13.0, 6.6 Hz, 2 H), 2.89 (dd, J = 15.0, 3.9 Hz, 1 H), 2.75 (dd,
J = 15.3, 8.1 Hz, 4 H), 2.69 (t, J = 6.0 Hz, 2 H), 1.82–1.67 (m, 4 H),
1.67–1.59 (m, 2 H), 1.59–1.50 (m, 2 H), 1.50–1.34 (m, 8 H), 1.25–
1.20 (m, 12 H).
13C NMR (100 MHz, CD3CN): d = 156.4, 153.5, 152.3, 131.0,
129.2, 129.2, 128.2, 128.1, 127.1, 126.8, 124.8, 124.3, 121.8, 119.5,
113.5, 110.7, 76.8, 64.3, 64.1, 59.2, 59.1, 47.0, 43.7, 43.6, 41.4,
39.3, 39.3, 31.8, 31.7, 30.4, 29.7, 28.7, 26.9, 26.2, 24.9, 24.9, 24.9,
24.8, 21.0, 21.0.
Yield: 680 mg, 2.8 mmol (93%); Rf = 0.35 (PE–EtOAc, 6:4).
IR (neat): 3378, 2926, 2855, 1575, 1558, 1445, 1416, 1118, 1054,
758 cm–1.
1H NMR (400 MHz, CDCl3): d = 8.46 (dd, J = 4.8, 0.8 Hz, 1 H),
7.73 (d, J = 8.1 Hz, 1 H), 7.65 (td, J = 7.8, 1.8 Hz, 1 H), 7.08 (ddd,
J = 7.3, 4.9, 0.9 Hz, 1 H), 3.63 (t, J = 6.6 Hz, 2 H), 2.86–2.73 (m, 2
H), 1.87 (s, 1 H), 1.76–1.65 (m, 2 H), 1.60–1.49 (m, 2 H), 1.48–1.30
(m, 4 H).
13C NMR (100 MHz, CDCl3): d = 160.5, 149.5, 137.0, 120.5, 119.6,
62.7, 38.8, 32.5, 28.8, 28.2, 25.3.
31P NMR (160 MHz, CD3CN): d = 146.8.
HRMS (ESI): m/z [M + H]+ calcd for C38H55N3O4PS2: 712.3366;
found: 712.3370.
HRMS (ESI): m/z [M + H]+ calcd for C11H18NOS2: 244.0824;
found: 244.0823.
Carbonic Acid 7,8-didehydro-1,2:5,6-dibenzocycloocten-3-yl
Ester, 6¢-[3-O-(4,4¢-dimethoxytrityl)-propanoxy-N,N¢-diisopro-
pylamino]phosphinooxy 1¢-Amide (3)
6¢-(1-Carbonic Acid 7,8-Didehydro-1,2:5,6-dibenzocycloocten-
3-yl Ester 1¢-Hexamide)-disulfanyl-6-hexanol (12)
Et3N (180 mL, 1.3 mmol, 2.2 equiv) was added to a stirred soln of
1516 (208 mg, 0.57 mmol) in anhyd 1,4-dioxane (5 mL), followed
by the addition of bis(diisopropylamino)chlorophosphine (14) (300
mg, 1.2 mmol, 1.9 equiv). After stirring for 20 min, an aliquot of the
reaction mixture was analyzed by 31P NMR which showed complete
conversion into the desired intermediate bis-amidite 16 (d = 123
ppm). The mixture was filtered under Ar to remove triethylammo-
nium salts and concentrated under reduced pressure. A soln of 1H-
tetrazole (30 mg, 0.4 mmol, 0.7 equiv) and 3¢-O-DMT-propanol
(17) (550 mg, 1.5 mmol, 2.6 equiv) in anhyd 1,4-dioxane (5 mL)
was added to a soln of crude 16 in anhyd MeCN (5 mL). After stir-
ring for 30 min, an aliquot of the reaction mixture was analyzed by
31P NMR which showed complete conversion into amidite 3
(d = 146.5, 146.3 ppm). The reaction mixture was quenched with
sat. aq NaHCO3 soln (10 mL) and the aq layer separated and extract-
ed with EtOAc (3 × 10 mL). The combined organic layers were
dried (Na2SO4) and concentrated under reduced pressure. The crude
residue was purified by silica gel column chromatography (hexane–
CH2Cl2–Et3N, 8:2:0.3→6:4:0.3) to afford the title amidite 3 as an
oil.
To a soln of 10 (0.38 g, 0.5 mmol) in THF–H2O (7 mL, 9:1 v/v) was
added a soln of PMe3 in toluene (1 M, 2 mL, 2 mmol, 4 equiv) and
the mixture stirred for 1 h. TLC analysis (PE–EtOAc, 8:2) showed
full conversion of the starting material into a less polar product
(Rf = 0.49). The mixture was diluted with EtOAc (30 mL), washed
with brine (2 × 10 mL), dried (MgSO4) and concentrated under re-
duced pressure. The crude thiol was coevaporated with deoxygen-
ated THF (2 × 5 mL) followed by addition of a soln of alcohol 11 in
deoxygenated THF (0.5 M, 2.1 mL, 1.05 mmol, 1.05 equiv). The
mixture was stirred for 18 h and then concentrated under reduced
pressure. Silica gel column chromatography (PE–EtOAc, 7:3→6:4)
afforded the title compound 12 as an oil.
Yield: 220 mg, 0.43 mmol (43%); Rf = 0.44 (PE–EtOAc, 6:4).
IR (neat): 3324, 2926, 2855, 2363, 1701, 1560, 1522, 1450, 1418,
1254, 1130, 1040 cm–1.
1H NMR (400 MHz, DMSO-d6): d = 7.60 (t, J = 5.6 Hz, 1 H), 7.53
(d, J = 7.7 Hz, 1 H), 7.50–7.34 (m, 7 H), 5.29 (s, 1 H), 4.35 (t,
J = 5.1 Hz, 1 H), 3.40–3.35 (m, 2 H), 3.17 (dd, J = 15.0, 1.9 Hz, 1
H), 2.98 (dd, J = 12.8, 6.1 Hz, 2 H), 2.76 (dd, J = 14.9, 3.8 Hz, 1 H),
2.67 (t, J = 7.2 Hz, 4 H), 1.64–1.54 (m, 4 H), 1.46–1.21 (m, 12 H).
13C NMR (100 MHz, DMSO-d6): d = 155.2, 152.6, 150.9, 130.1,
128.5, 128.4, 127.4, 127.4, 126.2, 125.9, 123.8, 122.9, 120.4, 112.6,
110.0, 75.2, 60.6, 45.5, 40.2, 40.2, 37.7, 32.4, 29.2, 28.6, 28.5, 27.7,
27.5, 25.8, 25.1.
Yield: 320 mg, 0.37 mmol (64%); Rf = 0.3 (hexane–CH2Cl2–Et3N,
6:4:0.3).
IR (neat): 2930, 1725, 1606, 1506, 1462, 1362, 1300, 1246, 1172,
1032, 970, 826, 753 cm–1.
1H NMR (400 MHz, CD3CN): d = 7.56 (d, J = 7.5 Hz, 1 H), 7.45–
7.23 (m, 15 H), 7.19 (dd, J = 8.3, 6.2 Hz, 1 H), 6.83 (d, J = 8.9 Hz,
4 H), 5.92 (t, J = 5.7 Hz, 1 H), 5.35 (s, 1 H), 3.73 (s, 6 H), 3.71–3.59
(m, 2 H), 3.59–3.39 (m, 4 H), 3.19–3.12 (m, 1 H), 3.07 (dq, J = 19.4,
6.5 Hz, 4 H), 2.81 (dd, J = 15.0, 3.7 Hz, 1 H), 1.88–1.75 (m, 2 H),
HRMS (ESI): m/z [M + H]+ calcd for C29H38NO3S2: 512.2288;
found: 512.2286.
Synthesis 2011, No. 17, 2724–2732 © Thieme Stuttgart · New York

SPECIAL TOPIC
Synthesis of Oligonucleotide Conjugates
2731
1.64–1.37 (m, 4 H), 1.36–1.20 (m, 4 H), 1.15 (d, J = 6.0 Hz, 2 H),
1.10 (d, J = 5.8 Hz, 6 H), 1.06 (d, J = 6.8 Hz, 6 H).
tion and cleavage from the resin were accomplished using concd aq
NH4OH soln for 1 h at r.t. HPLC purification followed by anion ex-
change chromatography and consecutive desalting yielded the tar-
get oligo-2¢-deoxynucleotide (see general methods and materials)
(OD = 0.64 mmol).
13C NMR (100 MHz, CD3CN): d = 159.4, 153.5, 152.2, 146.5,
137.3, 131.0, 130.8, 129.2, 129.2, 128.8, 128.6, 128.1, 128.1, 127.5,
127.1, 126.8, 124.8, 124.3, 121.8, 113.8, 113.5, 110.7, 86.5, 76.7,
67.6, 63.8, 63.7, 61.1, 61.0, 60.9, 55.7, 46.9, 45.2, 45.1, 43.5, 43.4,
41.5, 32.6, 32.5, 32.0, 30.5, 27.1, 26.4, 25.0, 25.0, 24.9, 24.9, 23.9,
23.9, 22.7, 22.7, 14.5.
ESI-MS: m/z = 1169.4 [M + 2H]2+.
LC–MS: 9.41 min [MeCN–aq NH4OAc (10 mM), 0→50 v/v].
31P NMR (162 MHz, CD3CN): d = 145.2, 145.1.
HRMS (ESI): m/z [M + H]+ calcd for C53H64N2O7P: 871.4446;
Oligonucleic Acids 22 and 23
The syntheses of oligonucleic acids 22 and 23 were performed on 8
mmol scale using a CPG solid support containing a diethoxysulfonyl
linker. Coupling was carried out using commercially available
amidites (4 equiv) and phosphoramidite 3 or 4 (6 equiv) at a concen-
tration of 0.1 M. 5-Benzylthio-1H-tetrazole (0.3 M) was used as the
activating agent in 5 or 10 min coupling cycles (phosphoramidites
3 and 4). Oxidation and capping were performed by means of stan-
dard procedures using I2, pyridine, H2O and Ac2O, respectively.
Deprotection and cleavage from the solid support were accom-
plished using concd aq NH4OH soln overnight at 55 °C. Concentra-
tion and subsequent HPLC purification (see general methods and
materials) yielded the target oligo-2¢-deoxynucleotides 22 (OD =
0.3 mmol) and 23 (OD = 0.7 mmol).
found: 871.4451.
Carbonic Acid 7,8-Didehydro-1,2:5,6-dibenzocycloocten-3-yl
Ester, 6¢-[5¢-O-(4,4¢-Dimethoxytrityl)-3¢-thymidine-N,N¢-diiso-
propylamino]phosphinooxy 1¢-Amide (4)
Et3N (504 mL, 3.6 mmol, 2 equiv) was added to a stirred soln of 1516
(653 mg, 1.8 mmol) in anhyd 1,4-dioxane (5 mL) at r.t., followed
by addition of bis(diisopropylamino)chlorophosphine (14) (610
mg, 2.4 mmol, 1.3 equiv). After stirring for 20 min, an aliquot of the
reaction mixture was analyzed by 31P NMR which showed complete
conversion into the desired intermediate bis-amidite 16 (d = 123
ppm). The mixture was filtered under Ar to remove triethylammo-
nium salts and concentrated under reduced pressure. A soln of 1H-
tetrazole (70 mg, 1 mmol, 0.7 equiv) and 5¢-O-DMT-thymidine (18)
(1.2 g, 2.2 mmol, 1.2 equiv) in anhyd 1,4-dioxane (5 mL) was added
to a soln of crude 16 in anhyd MeCN (5 mL) at r.t. After stirring for
30 min, an aliquot of the reaction mixture was analyzed by 31P NMR
which showed complete conversion into amidite 4 (d = 148, 144). A
sat. aq soln of NaHCO3 (15 mL) was added to the mixture and the
aq layer separated and extracted with EtOAc (3 × 15 mL). The com-
bined organic layer was dried (Na2SO4) and concentrated under re-
duced pressure. The crude residue was purified by silica gel column
chromatography (hexane–CH2Cl2–Et3N, 5:5:0.3→2:8:0.3) to afford
the title compound 4 as an off-white foam.
Oligonucleic Acid 22
ESI-MS: m/z = 2024.2 [M + H]+, 1012.0 [M + 2H]2+.
LC–MS: 6.95 min [MeCN–aq NH4OAc (10 mM), 0→50 v/v].
Oligonucleic Acid 23
ESI-MS: m/z = 1095.0 [M + 2H]2+.
LC–MS: 6.97 min [MeCN–aq NH4OAc (10 mM), 0→50 v/v].
Oligonucleic Acid–Tetrasaccharide Conjugates 21 and 24
To an aq soln (50 mL) of strained alkyne oligonucleotide 19 or 23
(200 nmol) was added an aq soln (30 mL) of tetrasaccharide 20 (200
nmol). The reaction mixture was shaken for 90 min after which time
LC–MS analysis revealed conversion of all the starting material into
the corresponding conjugate.
Yield: 1.12 g, 1.09 mmol (61%); Rf = 0.3 (hexane–CH2Cl2–Et3N,
3:7:0.3).
IR (neat): 2931, 1703, 1505, 1463, 1248, 1176, 1031, 970, 826, 755
cm–1.
Conjugate 21
1H NMR (400 MHz, CD3CN): d = 7.55 (d, J = 7.5 Hz, 1 H), 7.50–
7.15 (m, 18 H), 6.84 (d, J = 8.8 Hz, 4 H), 6.23 (dt, J = 6.8, 3.0 Hz,
1 H), 5.95 (dd, J = 10.9, 5.9 Hz, 1 H), 5.35 (s, 1 H), 4.58 (ddd,
J = 10.5, 8.5, 4.4 Hz, 1 H), 4.07 (dd, J = 15.6, 3.1 Hz, 1 H), 3.77–
3.70 (m, 6 H), 3.63–3.40 (m, 4 H), 3.37–3.20 (m, 2 H), 3.13 (dd,
J = 15.0, 1.7 Hz, 1 H), 3.05 (dt, J = 13.8, 6.8 Hz, 2 H), 2.79 (dd,
J = 15.0, 3.8 Hz, 1 H), 2.43–2.23 (m, 2 H), 1.67–1.16 (m, 13 H),
1.16–1.06 (m, 8 H), 1.02 (d, J = 6.7 Hz, 3 H).
13C NMR (100 MHz, CD3CN): d = 164.7, 159.7, 156.5, 153.6,
152.3, 151.4, 145.9, 145.8, 136.7, 136.6, 136.6, 136.5, 136.5, 131.0,
129.2, 129.2, 129.0, 128.9, 128.9, 128.2, 128.1, 127.9, 127.1, 126.8,
124.9, 124.3, 121.8, 114.1, 113.5, 111.4, 111.3, 110.8, 87.4, 86.2,
86.2, 85.9, 85.4, 76.8, 74.1, 73.9, 73.7, 73.6, 64.4, 64.2, 64.2, 64.1,
55.9, 47.0, 43.8, 43.7, 41.5, 40.3, 40.1, 31.9, 30.5, 27.1, 26.4, 26.4,
25.0, 24.9, 24.8, 12.3.
ESI-MS: m/z = 1557.4 [M + 2H]2+.
LC–MS: 7.42 min, 7.60 min [MeCN–aq NH4OAc (10 mM), 0→50
v/v].
Conjugate 24
ESI-MS: m/z = 1483.2 [M + 2H]2+.
LC–MS: 5.47 min, 5.62 min [MeCN–aq NH4OAc (10 mM), 0→50
v/v].
Oligonucleic Acid–Tetramethylrhodamine Conjugates 26 and
27
To an aq soln of strained alkyne oligonucleotide 19 or 23 (200
nmol) was added an aq soln (20 mL) of tetramethylrhodamine azide
(200 nmol). The reaction mixture was shaken for 90 min after which
time LC–MS analysis revealed conversion of all the starting mate-
rial into the corresponding conjugate.
31P NMR (162 MHz, CD3CN): d = 146.86, 146.85, 146.31, 146.28.
HRMS (ESI): m/z [M + H]+ calcd for C60H70N4O10P: 1037.4824;
Conjugate 26
found: 1037.4828.
ESI-MS: m/z = 1410.8 [M + 2H]2+.
Oligonucleotide 19
LC–MS: 6.84 min, 7.13 min [MeCN–aq NH4OAc (10 mM), 0→50
The synthesis of oligonucleotide 19 was performed on 8 mmol scale
using a polystyrene solid support. Coupling was carried out using
commercially available amidites (4 equiv) and phosphoramidite 2
(6 equiv) at a concentration of 0.1 M. 5-Benzylthio-1H-tetrazole
(0.3 M) was used as the activating agent in 5 min coupling cycles.
Oxidation and capping were performed by means of standard pro-
cedures using I2, pyridine, H2O and Ac2O, respectively. Deprotec-
v/v].
Conjugate 27
ESI-MS: m/z = 1485.0 [M + 2H]2+.
LC–MS: 8.48 min, 8.65 min [MeCN–aq NH4OAc (10 mM), 0→50
v/v].
Synthesis 2011, No. 17, 2724–2732 © Thieme Stuttgart · New York

2732
P. van Delft et al.
SPECIAL TOPIC
(12) Whitehead, K. A.; Langer, R.; Anderson, D. G. Nat. Rev.
Drug Discovery 2009, 8, 129.
(13) Astakhova, I. V.; Korshun, V. A.; Jahn, K.; Kjems, J.;
Wengel, J. Bioconjugate Chem. 2008, 19, 1995.
(14) Lönnberg, H. Bioconjugate Chem. 2009, 20, 1065; and
references cited therein.
(15) Marlin, F.; Simon, P.; Saison-Behmoaras, T.;
Giovannangeli, C. ChemBioChem 2010, 11, 1493.
(16) 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.
Acknowledgment
This work was funded by the Dutch Organization for Scientific Re-
search (NWO).
References
(1) Chiu, T.-C.; Huang, C.-C. Sensors 2009, 9, 10356.
(2) Endo, M.; Sugiyama, H. ChemBioChem 2009, 10, 2420.
(3) Kwak, M.; Herrmann, A. Angew. Chem. Int. Ed. 2010, 49,
8574.
(17) Jayaprakash, K. N.; Peng, C. G.; Butler, D.; Varghese, J. P.;
Maier, M. A.; Rajeev, K. G.; Manoharan, M. Org. Lett.
2010, 12, 5410.
(18) Ning, X.; Guo, J.; Wolfert, M. A.; Boons, G.-J. Angew.
Chem. Int. Ed. 2008, 47, 2253.
(19) Jung, S.; Lee, S. H.; Mok, H. H.; Chung, J.; Park, T. G.
J. Controlled Release 2010, 144, 306.
(20) Ghosh, P.; Dullea, R.; Fischer, J. E.; Turi, T. G.; Sarver, R.
W.; Zhang, C.; Basu, K.; Das, S. K.; Poland, B. W. BMC
Genomics 2009, 10 (Suppl. 1), S17.
(4) Alemdaroglu, F. E.; Herrmann, A. Org. Biomol. Chem.
2007, 5, 1311.
(5) Stephenson, M. L.; Zamecnik, P. C. Proc. Natl. Acad. Sci.
U.S.A. 1978, 75, 285.
(6) Fire, A.; Xu, S.; Montgomery, M. K.; Kostas, S. A.; Driver,
S. E.; Mello, C. C. Nature 1998, 391, 806.
(7) Opalinska, J. B.; Gewirtz, A. M. Nat. Rev. Drug Discovery
2002, 1, 503.
(8) Dorsett, Y.; Tuschl, T. Nat. Rev. Drug Discovery 2004, 3,
318.
(21) Elbashir, S. M.; Harborth, J.; Lendeckel, W.; Yalcin, A.;
Weber, K.; Tuschl, T. Nature 2001, 411, 494.
(22) Li, Y.; Zhang, Y.; Huang, Z.; Cao, X.; Gao, K. Can. J. Chem.
2004, 82, 622.
(23) Dinkelaar, J.; Codée, J. D. C.; van den Bos, L. J.; Overkleeft,
H. S.; van der Marel, G. A. J. Org. Chem. 2007, 72, 5737.
(9) Alvarez-Salas, L. M. Curr. Top. Med. Chem. 2008, 8, 1379.
(10) Koppelhus, U.; Nielsen, P. E. Adv. Drug Delivery Rev. 2003,
55, 267.
(11) Braasch, D. A.; Corey, D. R. Biochemistry 2002, 41, 4503.
Synthesis 2011, No. 17, 2724–2732 © Thieme Stuttgart · New York