Synthesis of oligonucleotides carrying amino lipid groups at the 3′-end for RNA interference studies

Article abstract of DOI:10.1021/jo101143j

Novel lipid derivatives carrying amino and triazolyl groups were efficiently synthesized and covalently anchored at the 3′-termini of oligonucleotides. The desired amino-lipid conjugates were fully characterized by reversed-phase HPLC and MALDI-TOF mass s

Full text of DOI:10.1021/jo101143j

pubs.acs.org/joc
Synthesis of Oligonucleotides Carrying Amino Lipid Groups at the 30-End
for RNA Interference Studies
‡
,†
Santiago Grijalvo,† Sandra M. Ocampo,†,‡ Jose C. Perales, and Ramon Eritja*
ꢀ
†Institute for Research in Biomedicine (IRB Barcelona), Networking Center on Bioengineering,
Biomaterials and Biomedicine (CIBER-BBN) and Institute for Advanced Chemistry of Catalonia (IQAC),
Spanish Research Council (CSIC), Cluster Building, Baldiri Reixac 10, E-08028 Barcelona, Spain, and
‡Biophysics Unit, Department of Physiological Sciences II, Universitat de Barcelona,
Feixa Llarga s/n, E-08907 L’Hospitalet del Llobregat, Barcelona, Spain
recgma@cid.csic.es
Received June 15, 2010
Novel lipid derivatives carrying amino and triazolyl groups were efficiently synthesized and
covalently anchored at the 30-termini of oligonucleotides. The desired amino-lipid conjugates were
fully characterized by reversed-phase HPLC and MALDI-TOF mass spectrometry. The methodo-
logy was applied to the synthesis of lipid-siRNA designed to inhibit tumor necrosis factor (TNF-R)
in order to obtain siRNAs with anti-inflammatory properties. The siRNA duplex carrying amino-
lipids at the 30-end of the passenger strand has inhibitory properties similar to those of unmodified
RNA duplexes in HeLa cells, indicating that the new lipid derivatives are compatible with the RNA
interference machinery.
Introduction
researchers have been actively looking for different approaches
to enhance this powerful tool in order to be used in
therapeutics.4 However, successful therapeutic applications
have been delayed due to delivery problems since native
siRNAs do not freely diffuse across the cell membrane due to
their relatively large molecular weight and their polyanionic
nature. To overcome these limitations, different strategies have
been employed and nonviral vectors have emerged as a promis-
ing alternative to gene delivery.5
The most used carriers for DNA and RNA oligonucleo-
tides are cationic lipids and liposomes which promote cel-
lular uptake of antisense and RNAi therapies,6 although
their mechanism is still unclear and controversial. In general,
RNA interference (RNAi) plays an important role in host
defense and regulation of gene expression. Since RNAi was
discovered a decade ago by Fire et al.,1 RNA research has seen
intense growth. During RNA interference, double-stranded
RNAs are processed into small fragments of approximately
21 nucleotides by the enzyme Dicer2 that are incorporated into
the RNA-induced silencing complex (RISC) directing the
degradation of specific complementary mRNA sequences.3
The introduction of short interfering RNAs (siRNA) in mam-
malian cells3 results in a selective silencing of the protein
encoded by the specific mRNA targeted by siRNA. Since then,
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Published on Web 09/23/2010
DOI: 10.1021/jo101143j
r
2010 American Chemical Society

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JOCArticle
SCHEME 1. Structure of the Lipid Oligonucleotide Conjugates Described in This Study
the use of targeted lipoplexes as systems to mediate siRNA
delivery has become the most reported method.7,8 These
units, which maintain an electrostatic association, are often
unstable9 and should be prepared before use, so other
alternative and robust linkage methods between siRNAs
and nonviral vectors may be developed and validated.
Recently, it has been demonstrated that nanoparticles can
be used for the efficient delivery of siRNA.10
Lipid-oligonucleotide conjugates are an interesting alter-
native. Some years ago, lipid-oligonucleotide conjugates
were found to improve biological activity of triplex-forming
and antisense oligonucleotides.11 Recently, a few examples
on the use of siRNA covalently conjugated to lipids have
been described in the literature. Cholesterol,12 fatty acid
derivatives,12 and R-tocopherol13 have been efficiently
linked to the 50-ends (phosphoramidite chemistry) and
30-ends (CPG supports) of the siRNA guide and passenger
strands. Further biological evaluations of these conjugates
showed that the introduction of these groups did not affect
the RNAi machinery obtaining high levels of inhibition in the
gene expression.
expression,14 we have been looking into the possibility of
synthesizing siRNA conjugates by linking lipid molecules
with different polar and/or protonatable groups (such as
amines and triazoles) at the 30-termini of the siRNA duplex
(Scheme 1). We aimed to mimic the effect produced by
lipoplex complexes. As far as we know, such complex lipids
have never been introduced into siRNA. Finally, the effects
of such modifications on the RNAi machinery were mea-
sured through the evaluation of these chemically modified
lipid-siRNA conjugates in the regulation of the tumor
necrosis factor-R (TNF-R) gene expression.
Results and Discussion
Synthesis of the Lipid Derivatives. The introduction of lipids
to the 30 end of oligonucleotides requires the use of a linker
that connects the lipid to the growing DNA chain. Aminodiols
such 4-aminoprolinol12a,d and trihydroxy compounds such as
glycerol12c have been used for this purpose. Aminodiols are used
to incorporate fatty acids by formation of an amide bond that is
stable to oligonucleotide synthesis conditions.12d Moreover,
aminodiols such as 4-aminoprolinol12d have the advantage of
well-defined chirality. But the use of this type of linkers needs
the use of fatty acids functionalized at the other end of the
hydrocarbon chain for the preparation of amino-lipids. These
compounds are not easily available. In contrast, trihydroxy
compounds can be used if ether or urethane bonds are used to
connect the lipid to the triols. In these cases, the resulting lipid
derivatives usually generate diastereoisomeric mixtures, but the
presence of isomers is not considered a serious problem for
biological activity of the resulting siRNA.12c Moreover, disub-
stituted alkanes needed for the preparation of the lipid deriva-
tives are available. For these reasons, we considered the use of a
glycerol backbone (Scheme 1) as suitable linker for introducing
our chemical modifications at the 30-end of the siRNA duplexes.
This linker, when reacted with phosphoramidites, will produce a
phosphate bond that will be stable to ammonia deprotection
conditions used in oligonucleotide synthesis. Also, it allows the
incorporation of the lipid derivative to a solid support. The
synthesis of lipidic solid supports 7, 15, and 16 is outlined in
Schemes 2 and 3. All compounds were obtained from the same
intermediate azide 3 which was synthesized from commercially
available rac-(2,2-dimethyl-1,3-dioxolan-4-yl)methanol as start-
ing material. The synthesis started with the alkylation reaction
between solketal 1 and commercially available 1,12-dibromodo-
decane in DMF in the presence of sodium hydride (60%), which
primarily yielded the alkylated compound 2. However, the
presence of a side compound was also observed. This side
As a part of our ongoing interest in the development of
chemically modified DNA and siRNAs to inhibit gene
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J. D.; Ribas, A. Nature 2010, 464, 1067–1070.
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Jayaraman, M.; Wang, G.; Pandey, R. K.; Rajeev, K. G.; Nakayama, T.;
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a
SCHEME 2. Synthesis of Solid Support 7 Functionalized with Amino C12
aReagents and Conditions: (a) (i) di-n-butyltin oxide, MeOH, reflux, (ii) CsF, 1,12-dibromododecane, 60 °C; (b) NaN3, DMF, 70 °C; (c) Boc2O, TEA,
DCM, rt; (d) (i) DCM/TFA 10%, rt, (ii) ethyl trifluoroacetate (ETFA), triethylamine (TEA), DCM, 0 °C; (e) DMTr, DMAP, Py, 40 °C; (f) (i) DMAP,
succinic anhydride, DCM, rt, overnight, (ii) CPG derivatization.
SCHEME 3. Click Chemistry and Synthesis of the Solid Supports 15 and 16a
aReagents and conditions: (a) selected alkynes, 10% sodium ascorbate, CuSO4 5H2O, tBuOH/H2O (1:1), rt, overnight; (b) (i) (for 8) DCM/TFA 3%,
3
rt, (ii) (for 9): pTsOH, MeOH, rt; (c) (i) MeNH2, EtOH, 40 °C, overnight, (ii) ETFA, TEA, DCM, 0 °C; (d) (for 13): DMTr, DMAP, pyridine, 40 °C,
overnight; (for 14): MMTr, DMAP, pyridine, 40 °C, overnight; (e) (i) DMAP, succinic anhydride, DCM, rt, overnight, (ii) CPG derivatization.
compound may arise from the E2 elimination of HBr (dehydro-
halogenation). To avoid this, we started an alkylation reaction of
1 via di-n-butylstannylene in the presence of 2.0 equiv of cesium
fluoride15 in the presence of DMF, providing 2in 47% yield after
purification by flash chromatography and without detecting any
trace of that impurity.
The nucleophilic displacement of the bromide group with
sodium azide in DMF yielded the desired azide 3 in 93%
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TABLE 1. Mass Spectrometry and Melting Temperatutes (Tm) of Lipid-Modified Oligonucleotides Prepared in This Study
oligonucleotide
sequence 50 f 30
MW calc
MW found
Tma (°C)
17
18
19
20
21
22
23
24
25
CGCGAATTCGCG-C12NH2
CGCGAATTCGCG-C12-triazol-CH2NH2
CGCGAATTCGCG-C12-triazol-(CH2)4NH2
CGCGAATTCGCG-C12NHC(NH)-NH2
CGCGAATTCGCG-C12-triazol-CH2NHC(NH)-NH2
CGCGAATTCGCG-C12-triazol-(CH2)4NHC(NH)-NH2
GUGCCUAUGUCUCAGCCUCTT-C12NH2
GUGCCUAUGUCUCAGCCUCTT- C12-triazol-CH2NH2
GUGCCUAUGUCUCAGCCUCTT- C12-triazol-(CH2)4NH2
3983
4066
4106
4027
4108
4150
6896
6977
7019
3982.5
4065
4105
4029
4109
4151
6899
6991 [M þ Naþ]
7054 [M þ 2Naþ]
55
58
57
61
56
55
n.d.
n.d.
n.d.
aConditions: sodium phosphate 10 mM, NaCl NaCl, pH 7.0, in these conditions unmodified Dickerson-Drew dodecamer melted at 49 °C; n.d., not
determined.
yield. Having on hand azide 3, subsequent reduction under
Staudinger conditions gave the expected amine which was
conveniently protected with Boc2O in standard conditions
achieving the N-Boc-protected 4 in 65% yield. Finally, the
introduction of the 4,40-dimethoxytrityl (DMTr) protecting
group was easily obtained by removing acetonide and N-Boc
moieties simultaneously under acidic conditions (DCM/
TFA 3%) followed by N-protection with ethyl trifluoroace-
tate, thereby yielding the N-protected compound 5 in 87%
yield. DMTr protection with DMAP in the presence of
pyridine afforded the desired trityl derivative 6 in 58% yield
after purification by flash chromatography.
Click chemistry, on the other hand, is considered to be a
modular approach that is increasingly found in all aspects
of drug discovery, combinatorial chemistry, and recently,
nucleic acid chemistry,16 easily obtaining triazolyl rings.17
Taking this into account, we considered exploring the use of
the 1,3-dipolar cycloadditions between the previously syn-
thesized azide 3 and some commercially available alkynes18
in order to study the subsequent effect that these kind of rings
could exercise on the RNAi machinery. Then, the click
reactions were carried out under standard conditions19 to
give the desired triazoles compounds 8 and 9 as only regio-
isomers in 89% and 59% yield, respectively, after purification
by flash chromatography (Scheme 3).
out as follows: the phthalimide 11 was removed under basic
hydrolysis to give the expected amine, which was then
subsequently protected with ethyl trifluoroacetate yielding
the N-protected amine 12. Finally, the selective protection of
primary alcohol 12 with 4-monomethoxytrityl (MMTr)
afforded the corresponding protected trityl alcohol 13 in
39% overall yield (three steps).
The three trityl compounds 6, 13, and 14 were coupled
with CPG supports using the succinyl linker as described.20
For this purpose, the DMTr and MMTr derivatives
described above reacted with succinic anhydride followed by
coupling the resulting hemisuccinates with amino-functionalized
CPG which yielded glass beads containing lipids 7 (21 μmol/g),
15 (25 μmol/g), and 16 (23 μmol/g), respectively. CPG functio-
nalization was determined by the measure of the absorbance of
the DMTr/MMTr cations released from the support upon acid
treatment.
Oligodeoxyribonucleotide Synthesis. First, we synthesized
a short oligodeoxynucleotide sequence to demonstrate the
stability of the lipid derivative to phosphoramidite synthesis
conditions. The self-complementary Dickerson-Drew
dodecamer sequence (50-CGCGAATTCGCG-30) was used as
a model sequence. The sequence was assembled on CPG solid
supports 7, 15, and 16 using standard protocols in order to
generate the corresponding lipid-oligonucleotide conjugates by
using DMT off-based protocols. After cleavage with ammonia
solution (32%) followed by HPLC purification, the correspond-
ing modified aminolipid-oligonucleotide conjugates (17-19)
were obtained and confirmed by MALDI-TOF mass spectro-
metry (MS) (Table 1).
In addition, we studied the conversion of the amino group
to the guanidinium group in order to extend the possibility of
cationic charge enrichment in our synthesized aminolipid-
oligonucleotide conjugates 17-19. The synthesis of cer-
tain guanidinium derivatives of nucleic acids has been
described in the literature,21 and some of them have been
synthesized through postsynthetic modification.14a,21e
The guanidinium-modified ONs synthesized during this
study are summarized in Table 1. In all cases, selective and
In order to obtain the corresponding trityl derivatives,
compounds 8 and 9 were subjected to acetonide hydrolysis in
different acid conditions (DCM/TFA 3% for acetonide 8;
p-TsOH in the presence of methanol for acetonide 9,
respectively) yielding the expected diols 10 and 11 in 72%
and 99% yield, respectively. DMTr derivative 14 was directly
obtained from protected alcohol 10 in 45% yield under the
same conditions used for the synthesis of compound 6.
Finally, the synthesis of last trityl derivative 13 was carried
(15) (a) Fos, E.; Suesa, N.; Borras, L.; Lobato, C.; Banfi, P.; Gambetta,
R.; Zunino, F.; Mauleon, D.; Carganico, G. J. Med. Chem. 1995, 1216–1228.
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(18) A click chemistry reaction between azide 3 and N-Boc-protected
propargylamine was also carried out. It is worth mentioning that we made
several attempts to deprotect the N-Boc group and the acetonide moiety
simultaneously but failed, even when the hydrolysis of this group was carried
out under neutral conditions by reaction with TMSOTf in the presence of
2,6-lutidine. For experimental details, see the Supporting Information.
(19) Rostovtsev, V. V.; Green, G. L.; Fokin, V. V.; Sharpless, K. B.
Angew. Chem., Int. Ed. 2002, 41, 2596–2599.
(21) (a) Ohmichi, T.; Kuwahara, M.; Sasaki, N.; Hasegawa, M.; Nishikata,
T.; Sawai, H.; Sugimoto, N. Angew. Chem., Int. Ed. 2005, 44, 6682. (b) Maier,
M. A.; Barber-Peoc’h, I.; Manoharan, M. Tetrahedron Lett. 2002, 43, 7613.
(c) Barber-Peoc’h, I.; Manoharan, M.; Cook, P. D. Nucleosides Nucleo-
tides 1997, 16, 1407. (d) Michel, T.; Debart, F.; Vasseur, J.-J. Tetrahedron
Lett. 2003, 44, 6579. (e) Deglane, G.; Abes, S.; Michel, T.; Prevot, P.;
Vives, E.; Debart, F.; Barvik, I.; Lebleu, B.; Vasseur, J.-J. ChemBioChem
2006, 7, 684.
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SCHEME 4. Synthesis of the Amine and Guanidinium Oligonucleotides
aReagents and conditions: (a) (i) O-methylisourea chloride, NH3 (32%), 55 °C, overnight.
TABLE 2. Sequences of Unmodified, Lipid-Modified and Scrambled
siRNA Sequences Used in This Study
siRNA
sequencesa
unmodified (siRNA-1)
GUGCCUAUGUCUCAGCCUCTT
TTCACGGAUACAGAGUCGGAG
GUGCCUAUGUCUCAGCCUCTT-
C12NH2
siRNA-2
TTCACGGAUACAGAGUCGGAG
GUGCCUAUGUCUCAGCCUCTT-
C12-triazole-CH2NH2
siRNA-3
TTCACGGAUACAGAGUCGGAG
GUGCCUAUGUCUCAGCCUCTT-
C12-triazole-(CH2)4NH2
TTCACGGAUACAGAGUCGGAG
CAGUCGCGUUUGCGACUGGTT
TTGUCAGCGCAAACGCUGACC
siRNA-4
FIGURE 1. Efficiency of silencing of chemically modified siRNAs
with lipids in the passenger strand. Plot of gene-specific silencing
activities for native (siRNA-1), chemically modified conjugates
(siRNA-2, siRNA-3 and siRNA-4), and scrambled sequence
(siRNA-5) (50 nM per well). Transfection of siRNA conjugates
was carried out by using Oligofectamine (see above). Values are
represented the average ( ES, n = 3, and are compared to a
scrambled sequence. ***p<0.001, ANOVA Test, Bonferrini post-
test.
scrambled (siRNA-5)
asiRNAs are shown with the passenger strand above (50 - 30) and the
guide strand below (30 - 50). T stands for thymidine.
quantitative guanidinylation were observed following a
classical postsynthetic approach21e on oligonucleotides
17-19, which were reacted with O-methylisourea for 16 h
at 55 °C. After desalting, the guanidinylated compounds
20-22 (Scheme 4) were analyzed by analytical HPLC and
characterized by MALDI experiments. Melting tempera-
tures of the synthesized lipid-modified dodecamers were
higher than for the unmodified dodecamer (see Table 1). A
similar effect has been also observed recently with the
dodecamer-modified with arginine and lysine residues as
well as lipids.14b Preliminary experiments to insert guani-
dinium functions at the end of RNA oligomers using the
conditions described above yielded RNA degradation.
Further attempts to extend such modifications in RNA
oligomers are in progress.
carried out the transfection of the aforementioned siRNA
derivatives with oligofectamine. Forty-eight hours after transfec-
tion, the amount of TNF-R produced by the cells was analyzed
by enzyme-linked immunosorbent assay (ELISA). The silencing
activities are depicted in Figure 1. In general, all chemically
modified siRNAs previously obtained maintained their abilities
to down-regulate TNF-R protein expression levels to around
65% at 50 nM concentration compared with the scrambled
control siRNA5 duplex. These results indicate that the introduc-
tion of all the proposed modifications at the 30-termini of the
passenger strand of an RNA duplex is compatible with the
RNAi machinery in HeLa cells.
Oligoribonucleotide Synthesis. All oligoribonucleotides
were synthesized using solid supports 7, 15, and 16 (1.0 μmol
each) using a DNA/RNA synthesizer (sequences of guide and
passenger strands are shown in Tables 1 and 2). Modified RNA
oligonucleotides linked to the aforementioned solid supports
were released according to DMT on-based protocols, and
then crude modified oligoribonucleotides were first purified
by HPLC followed by DMTr deprotection with AcOH
80%. Finally, amino lipid-RNA conjugates 23-25 were
again analyzed by reversed-phase HPLC and characterized
by MALDI-TOF mass spectrometry (Table 1). siRNA duplexes
(siRNA2, siRNA3, and siRNA4, respectively) were obtained by
annealing of equal molar amounts of passenger (sense) and guide
(antisense) strands, which were purified by ethanol precipitation
protocol (see the Experimental Section).
Conclusions
In general, the most common method of administration of
siRNAs in cell culture is based on the use of nonviral vectors
such as cationic lipids. The interaction between siRNA and
cationic lipids is due to the existence of electrostatic interac-
tions and the formation of lipoplexes. Notwithstanding, the
use of a covalent strategy between the aforementioned siRNA
and cationic lipids is also possible, though in most of the cases
only neutral lipids have been linked to siRNA. Following this
approach, we have reported a convenient synthesis by which
glycerolipid based structures with different polar groups have
been efficiently synthesized and introduced into the 30-termini
of the siRNA sense strand being the first time that oligonu-
cleotides carrying cationic lipids are reported. The amino
group of the lipid can be used for the generation of guanidi-
nium groups and also for further functionalization to pro-
teins, liposomes, nanoparticles and so on. Finally, we were
able to confirm all these proposed modifications containing
amine tails did not affect the RNAi machinery through
silencing TNF-R gene expression.
Gene Silencing by Modified siRNAs. The gene silencing
effect on the TNF-R mRNA of several siRNAs: one native
siRNA1, three chemically modified siRNAs (siRNA2,
siRNA3,andsiRNA4), and one scrambled siRNA5 were studied.
HeLa cells were first cotransfected with plasmid pCAm TNF-R
by using commercial cationic liposomes (lipofectin). We then
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Experimental Section
tert-Butyl 12-[(2,2-Dimethyl-1,3-dioxolan-4-yl)methoxy]-
dodecylcarbamate, 4. Azide 3 (85 mg, 0.249 mmol) along with
PPh3 (131 mg, 0.498 mmol; 2.0 equiv) were dissolved in 3 mL of
anhydrous THF. The reaction was stirred for 2 h at room
temperature. Subsequently, water (500 uL) was added dropwise.
The mixture was stirred overnight at room temperature. The
solvent was evaporated, and the crude was dried by pressure,
yielding the anticipated amine. This amine was dissolved in
2.0 mL of anhydrous dichloromethane (DCM); TEA (69 μL,
0.498 mmol; 2.0 equiv) was added dropwise along with Boc2O
(82 mg, 0.373 mmol; 1.5 equiv). The reaction was stirred for 5 h at
room temperature. The organic layer was extracted with more
DCM (5 mL) and rinsed with water (3 ꢀ 10 mL). The organic
layer was dried on anhydrous Na2SO4. The solvent evaporated
and the resulting residue was purified by flash chromatography
(Ch/AcOEt 4% to 9%): yield 65%; IR (film) 3020, 2935, 2850,
1699, 1506, 1371, 1216, 1166, 1046 cm-1; 1H NMR (400 MHz,
CDCl3) δ 4.49 (broad s, 1H), 4.26 (m, 1H, CH-O), 4.04 (dd, J =
14.6, 8.2 Hz; 1H, O-CH-CH), 3.72 (dd, J = 14.6, 8.2 Hz; 1H,
O-CH-CH), 3.50 (m, 2H, CH2-O), 3.42 (m, 2H, CH2-O), 3.08 (m,
2H, CH2-N), 1.56 (m, 2H), 1.43 (s, 9H, 3CH3-C), 1.41 (s, 3H,
CH3-C), 1.35 (s, 3H, CH3-C), 1.24 (m, 18H); 13C NMR (125
MHz, CDCl3) δ 158.8 (CO), 110.0 (OCCH3), 75.4 (CH-O), 72.5
(O-CH2), 72.4 (O-CH2), 67.6 (O-CH2), 30.7 (N-CH2), 30.2, 30.1,
29.9, 29.1, 27.4, 27.4, 26.7, 26.1; HR ESI MS m/z calcd for
C23H45NO5 (M þ Hþ) 416.3370, found m/z 416.3370.
Materials and Methods. All reactions were carried out under
argon positive pressure in anhydrous solvents. Commercially
available reagents and anhydrous solvents were used without
further purification. Solvents were distilled prior to use and
dried using standard methods. Analytical samples were homo-
geneous as confirmed by TLC and yielded spectroscopic results
were consistent with the assigned structures. Chemical shifts are
reported in parts per million (ppm) relative to the singlet at δ =
1
7.24 ppm of CHCl3 for H NMR and to the center line of the
triplet at δ = 77.0 ppm of CDCl3 for 13C NMR. IR spectra were
measured in film. Thin-layer chromatography (TLC) was per-
formed on silica gel (Alugram Sil G/UV).
HPLC Conditions. Conditions for semipreparative HPLC:
HPLC solutions were solvent A [5% acetonitrile (ACN) in 100
mM triethylammonium acetate (TEAA), pH 6.5] and solvent B
(70% ACN in 100 mM triethylammonium acetate, pH 6.5).
Column: PRP-1 (Hamilton) 250x 10 mm. Flow rate 3 mL/min
linear gradient from 15 to 100% in B (DMTon) and 0 to 50% in
B (DMToff) was used with UV detection at 260 nm. Conditions
for analytical HPLC: HPLC solutions were solvent A [5% ace-
tonitrile, ACN) in 100 mM triethylammonium acetate (TEAA),
pH 6.5] and solvent B (70% ACN in 100 mM triethylammonium
acetate, pH 6.5). Column: XBridge OST C18 2.5 μm. Flow rate
1 mL/min linear gradient from 0 to 50% in B (DMToff) were
used with UV detection at 260 nm.
N-[12-(2,3-Dihydroxypropoxy)dodecyl]-2,2,2-trifluoroacetamide,
5. Compound 4(60.0 mg, 0.161 mmol) was dissolved in a mixture of
DCM/TFA 10% (5 mL) at room temperature for 20 min. The
solvent was evaporated, yielding the corresponding trifluoroacetate
in its salt form. The resulting oil was then dissolved in anhydrous
DCM (5.0 mL), and TEA was added dropwise (45 μL, 0.322
mmol). The reaction was cooled at 0 °C, and ethyl trifluoroacetate
(22.0 μL, 0.177 mmol) was added. The reaction was stirred for
30 min, extracting the organic layer with more DCM (3 ꢀ 10 mL),
andrinsedwithwater(3ꢀ 10 mL). The organic layer was dried with
anhydrous Na2SO4. The solvent evaporated, and the resulting oil
was purified by flash chromatography (DCM/MeOH 2%): yield
4-[(12-Bromododecyloxy)methyl]-2,2-dimethyl-1,3-dioxolane,
2. Alcohol 1 (200 mg; 1.51 mmol) was reacted with dibutyltin
oxide (751 mg, 3.02 mmol; 2.0 equiv) in dry methanol (10 mL) at
reflux for 3 h, followed by removing the methanol and any traces
of water using azeotropic distillation with toluene. The reaction
mixture was concentrated, and then 1,12-dibromododecane
(991 mg, 3.02 mmol; 2.0 equiv), cesium fluoride (458 mg, 3.02
mmol; 2.0 equiv), and dry DMF (10 mL) were added. The
reaction mixture was kept at room temperature overnight. The
solvent was evaporated, and the resulting residue was purified
by flash chromatography (Ch/AcOEt 1% to 4%): yield 47%; IR
(film) 2989, 2931, 2858, 1455, 1367, 1255, 1212, 1115, 1054, 845
cm-1; 1H NMR (400 MHz, CDCl3) δ 4.24 (q, J = 6.0 Hz, 1H,
CH-O), 4.03 (dd, J = 14.6, 8.2 Hz; 1H, O-CH-CH), 3.71 (dd,
J = 8.2, 14.6 Hz; 1H, O-CH-CH), 3.49 (m, 2H, CH2-O), 3.40 (m,
2H, CH2-O), 3.38 (t, J = 6.8 Hz, 2H, CH2-Br), 1.83 (q, J = 7.1
Hz, 2H, CH2-CH2), 1.55 (m, 2H), 1.40 (s, 3H, CH3-C), 1.34 (s,
3H, CH3-C), 1.25 (m, 16H); 13C NMR (125 MHz, CDCl3) δ
109.9 (O-C-CH3), 75.4 (CH-O), 72.5 (O-CH2), 72.4 (CH2-O),
67.5 (CH2-O), 34.6 (CH2-Br), 33.4, 30.2, 30.1, 30.1, 30.0, 30.0,
29.4, 28.8, 27.4, 26.6, 26.0; HR ESI MS m/z calcd for C18H35-
BrO3 Na (M þ Na) 401.1661, found m/z 401.1662; calcd for
C36H70O6NaBr2 Na (2 M þ Na) 779.3427, found 779.3431.
4-[(12-Azidododecyloxy)methyl]-2,2-dimethyl-1,3-dioxolane, 3.
Compound 2 (100 mg, 0.264 mmol) was dissolved in 5 mL of
anhydrous DMF. NaN3 (103 mg, 1.58 mmol; 6.0 equiv) was then
added, and the mixture was stirred and heated to 70 °C for 48 h.
The reaction was cooled at 0 °C, and water was added carefully.
The solvent was evaporated, and the resulting oil was then
purified by flash chromatography (Ch/AcOEt 1% to 3%): yield
93%; IR (film) 2935, 2858, 2363, 2344, 2093, 1251, 1077 cm-1; 1H
NMR (400 MHz, CDCl3) δ 4.26 (q, J = 6.1 Hz, 1H, CH-O), 4.06
(dd, J = 14.6, 8.23 Hz; 1H, O-CH-CH), 3.72 (dd, J = 8.22, 14.6
Hz; 1H, O-CH-CH), 3.51 (m, 2H, CH2-O), 3.43 (m, 2H, CH2-O),
3.25 (t, J =6.97Hz, 2H, CH2-N3), 1.59 (m, 6H), 1.42(s, 3H, CH3-
C), 1.36 (s, 3H, CH3-C), 1.27 (m, 14H); 13C NMR (125 MHz,
CDCl3) δ 109.3 (O-C-CH3), 74.7 (CH-O), 71.8 (O-CH2), 71.7 (O-
CH2), 66.9 (O-CH2), 51.4 (CH2-N3), 29.5, 29.4, 29.4, 29.4, 29.4,
29.1, 28.7, 26.7, 26.6, 26.0, 25.3; HR ESI MS m/z calcd for
C18H36N3O3 (Mþ) 342.2755, found m/z 342.2751; calcd for
C18H35N3O3K (M þ K) 380.2312, found,380.2310.
87%; IR (film) 3333, 2924, 2854, 1694, 1552, 1471, 1185 cm-1
;
1H NMR (400 MHz, CDCl3) δ 6.42 (broad s, 1H), 3.05 (m, 1H,
CH-O), 3.72 (dd, J = 11.4, 3.8 Hz; 1H, O-CH-CH), 3.65 (dd, J =
11.4, 5.2 Hz; 1H, O-CH-CH), 3.51 (m, 2H, CH2-O), 3.49 (m, 2H,
CH2-O), 3.35 (m, 2H, CH2-NHCO), 1.57 (m, 2H), 1.26 (m, 20H);
13C NMR (125 MHz, CDCl3) δ 157.8 (q, J = 36.9 Hz, COCF3),
117.9 (q, J = 287.6 Hz, CF3), 73.2 (CH-O), 72.5 (O-CH2), 71.0 (O-
CH2), 64.9 (O-CH2), 40.6 (NHCO-CH2), 30.2, 30.1, 30.1, 30.1,
30.0, 29.7, 29.6, 27.3, 26.7; 19F NMR (CDCl3) δ -76.3 (reference
CFCl3); HR ESI MS m/z calcd for C17H32F3NO4Na (M þ Naþ)
394.2175, found m/z 394.2176.
N-[12-[3-[Bis(4-methoxyphenyl)(phenyl)methoxy]-2-hydroxy-
propoxy]dodecyl]-2,2,2-trifluoroacetamide, 6. Compound 5 (52
mg, 0.140 mmol) along with 4,40-dimethoxytrityl chloride
(DMTrCl) (48 mg, 0.141 mmol; 1.2 equiv) and DMAP (8 mg,
0.07 mmol; 0.5 equiv) were dissolved in 1.5 mL of anhydrous
pyridine. The reaction was heated at 40 °C and stirred overnight.
Methanol (0.5 mL) was added and the solvent evaporated. The
resulting product was purified by flash chromatography (Hex/
AcOEt 15%): yield 58%; IR (film) 3276, 3072, 2927, 2851, 1716,
1673, 1607, 1502, 1462, 1390, 1248, 1176, 1038 cm-1; 1H NMR
(400 MHz, CDCl3) δ 7.67 (m, 3H, phenyl), 7.40 (m, 2H, phenyl),
7.28 (m, 3H, phenyl), 7.01 (broad s, 1H), 6.81 (d, J = 8.9 Hz, 4H,
phenyl), 3.92 (m, 1H, CH-O), 3.76 (s, 6H, 2 CH3), 3.52 (dd, J =
9.7 Hz, 4.2 Hz; 1H, CH-CH2-O), 3.43 (m, 3H, CH-CH2-O), 3.35
(m, 2H, CH2-O), 3.17 (m, 2H, CH2-NH), 2.41 (broad d, 1H),
1.85 (m, 2H), 1.57 (m, 9H), 1.25 (m, 9H); 13C NMR (125 MHz,
CDCl3) δ 162.7, 158.6, 149.9, 145.0. 136.3, 136.2, 130.2, 128.3,
127.9, 126.9, 123.9, 113.2, 86.2, 72.3, 71.8, 70.1, 64.7, 55.4, 55.3,
55.3, 50.8, 40.2, 36.6, 31.6, 29.9, 29.8, 29.7, 29.7, 29.6, 29.6, 29.6,
J. Org. Chem. Vol. 75, No. 20, 2010 6811

Grijalvo et al.
JOCArticle
29.3, 29.1, 26.8, 26.2; 19F NMR δ -74.3 (reference CFCl3); HR
ESI MS m/z calcd for C38H50F3NO6Na (M þ Naþ)) 696.3482,
found m/z 696.3490.
mmol; 1.0 equiv) along with p-TsOH (5 mg, 0.026 mmol; 0.5
equiv) were dissolved in methanol (1.0 mL). The reaction was
stirred at room temperature for 5 h. The methanol evaporated,
and the resulting oil was purified by flash chromatography
(CH2Cl2/MeOH 2%): yield 99%; IR (film) 3349, 2924, 2850,
1684, 1517, 1251, 1170, 1123, 1046 cm-1; 1H NMR (400 MHz,
CDCl3) δ 7.82 (m, 2H), 7.69 (m, 2H), 7.28 (s, 1H), 4.27 (t, J =
7.2 Hz; 2H), 3.85 (m, 1H), 3.69 (m, 3H), 3.63 (m, 1H), 3.49 (m,
2H), 3.44 (m, 2H), 2.74 (m, 2H), 2.53 (broad d, 2H), 1.85 (m,
2H), 1.72 (m, 4H), 1.55 (m, 2H), 1.23 (m, 18H); 13C NMR (400
MHz, CDCl3) δ 168.6, 134.1, 132.3, 123.4, 72.7, 72.0, 70.6, 64.4,
50.4, 37.8, 30.5, 29.7, 29.6, 29.6, 29.6, 29.5, 29.5, 29.1, 28.2, 26.8,
26.6, 26.2, 25.3; HR ESI MS m/z calcd for C29H44N4O5 (Mþ þ
1) 529.3384, found m/z 529.3384; calcd for C29H44N4O5 Na
(M þ Naþ) 551.3190, found m/z 551.3200.
General Procedure for the Click Reaction (8 and 9). Azide 3
(1.0 equiv) and selected alkynes (1.0 equiv) were suspended in a
1:1 mixture of water and tert-butyl alcohol (1.0 mL). Sodium
ascorbate (10%) and copper(II) sulfate pentahydrate (1%) were
added dropwise. The heterogeneous mixture was stirred vigor-
ously overnight. The reaction mixture was diluted with DCM,
and the organic layer was washed with water (3 ꢀ 5 mL). The
organic layer was dried with anhydrous Na2SO4 and solvent
evaporated. The resulting oil was purified by flash chromato-
graphy.
2-[4-[1-[12-[(2,2-Dimethyl-1,3-dioxolan-4-yl)methoxy]dodecyl]-
1H-1,2,3-triazol-4-yl]butyl]isoindoline-1,3-dione, 8: yield 89%; IR
(film) 2919, 2854, 2363, 2342, 1710, 1053 cm-1; H NMR (400
N-[4-[1-[12-[3-[Bis(4-methoxyphenyl)(phenyl)methoxy]-2-hydro-
xypropoxy]dodecyl]-1H-1,2,3-triazol-4-yl]butyl]-2,2,2-trifluoro-
acetamide, 13. Compound 11 (30 mg, 0.060 mmol) was dis-
solved in 1.0 mL of ethanol. Then, 80 μL of MeNH2 was added
dropwise. The mixture was stirred overnight at 40 °C. The
solvent was reduced to dryness, and the resulting product was
dissolved again in anhydrous DCM (1.5 mL). TEA (38 μL) was
added dropwise at room temperature. The reaction was cooled
at 0 °C, and EFTA (8.0 uL) was added dropwise. The reaction
was stirred for 30 min at 0 °C. The organic layer was washed
with water and brine (3 ꢀ 10 mL), dried with anhydrous
Na2SO4, and evaporated. The resulting compound was used
in the next step without further purification. The compound
obtained along with MMTrCl (27.8 mg, 0.09 mmol; 1.5 equiv)
and DMAP (4.0 mg, 0.03 mmol; 0.5 equiv) were dissolved in 1.0
mL of pyridine. The reaction was heated at 40 °C and stirred
overnight. Methanol was added (1.0 mL) and the solvent
evaporated. The product was then purified by flash chroma-
tography (DCM/MeOH/TEA 98:1:1): yield 39%; IR (film)
1
MHz, CDCl3) δ 7.81 (m, 2H, phenyl), 7.69 (m, 2H, phenyl), 7.27
(s, 1H, CHdC), 4.27 (t, J = 7.24 Hz; 2H, CH2-O), 4.22 (m, 1H,
CH-O), 4.04 (dd, J = 14.6 Hz, 8.2 Hz; 1H, CH-CH2-O), 3.69 (m,
3H), 3.49 (m, 2H), 3.41 (m, 2H), 2.74 (t, J = 7.0 Hz, 2H, CH2-N),
1.85 (m, 2H), 1.72 (m, 4H), 1.54 (m, 2H), 1.40 (s, 3H, CH3-C),
1.34 (s, 3H, CH3-C), 1.26(m, 16H); 13C NMR (125 MHz, CDCl3)
δ 168.6 (CO), 134.1 (phenyl), 132.3 (phenyl), 123.3 (phenyl),
121.0 (CdN), 120.6 (CH-N) 109.5 (O-C-CH3), 74.9 (CH-O), 72.1
(CH-CH2-O), 72.0 (CH-CH2-O), 67.1 (CH-CH2-O), 50.4 (CH2-
Cd), 37.8 (CH2-NCO), 30.5 (CH2-N), 29.7, 29.7, 29.7, 29.6, 29.5,
29.2, 28.2, 26.9, 26.8, 26.7, 26.2, 25.6, 25.3; HR ESI MS m/z calcd
for C32H48N4O5 (Mþ þ 1) 569.3697, found m/z 569.3697; calcd
for C32H48N4O5 Na (M þ Naþ) 591.3516, found m/z 591.3516.
(9H-Fluoren-9-yl)methyl [1-[12-[(2,2-dimethyl-1,3-dioxolan-
4-yl)methoxy]dodecyl]-1H-1,2,3-triazol-4-yl]methylcarbamate, 9:
yield 58%; IR (film) 2930, 2858, 1721,1523, 1448, 1370, 1250,
1120, 1049, 758, 738 cm-1; 1H NMR (400 MHz, CDCl3) δ 7.74
(d, J = 7.5Hz; 2H, phenyl), 7.57 (d, J = 7.5Hz; 2H, phenyl), 7.47
(s, 1H, CHdC), 7.38 (t, J = 7.3 Hz; 2H, phenyl), 7.29 (t, J = 7.4
Hz; 2H, phenyl), 5.48 (broadd, 1H, NH), 4.46 (d, J = 6.0Hz; 2H,
CH2-OCO), 4.40 (d, J = 7.0 Hz; 2H, CH-CH2-O), 4.31 (t, J =
7.2 Hz; 1H, O-CH-C), 4.25 (t, J = 6.0 Hz; 2H, CH2-O), 4.22 (m,
2H), 4.05 (dd, J = 14.6 Hz, 8.2 Hz; 1H, CH-O-C), 3.72 (dd, J =
14.6 Hz, 8.2 Hz; 1H, CH-O-C), 3.50 (m, 2H), 3.42 (m, 2H), 1.87
(m, 2H), 1.55 (m, 2H), 1.41 (s, 3H, CH3-C), 1.35 (s, 3H, CH3-C),
1.27 (m, 16H); 13C NMR (125 MHz, CDCl3) δ 156.3 (CO), 143.8
(phenyl), 141.2 (phenyl), 127.6 (phenyl), 127.0 (CHdC), 125.0
(CdC), 119.9 (phenyl), 109.3 (O-C-O), 74.7 (CH-O), 71.9 (CH-
CH2-O), 71.8 (CH-CH2-O), 66.9 (CH2-O), 50.3 (O-CH2-C), 47.2
(C-CH2-NCO), 36.5 (C-CH2), 30.2, 29.5, 29.5, 29.4, 29.4, 29.3,
28.9, 26.7, 26.4, 26.0, 25.4; HR ESI MS m/z calcd for C36H50N4-
O5 (Mþ þ 1) 619.3853, found m/z 619.3858; calcd for C36H50N4-
O5 Na (M þ Naþ) 641.3673, found m/z 641.3675.
3065, 2935, 2860, 1715, 1650, 1593, 1454, 1230, cm-1 1H
;
NMR (400 MHz, CDCl3) δ 7.41 (m, 2H), 7.31 (m, 4H), 7.26
(m, 4H), 6.82 (d, J = 8.8 Hz, 4H), 4.29 (t, J = 7.2 Hz; 2H), 3.94
(m, 1H), 3.78 (s, 6H), 3.51 (m, 2H), 3.42 (m, 4H), 3.17 (m, 2H),
2.99 (m, 3H), 2.74 (t, J = 7.0 Hz, 2H), 1.87 (m, 2H), 1.75 (m,
2H), 1.67 (m, 2H), 1.53 (m, 2H), 1.28 (m, 16H); 13C NMR (400
MHz, CDCl3) δ 158.6, 150.0, 147.4, 145.0, 136.2, 130.2, 128.3,
128.0, 126.9, 124.0, 123.8 (q, J = 298.1 Hz, CF3), 120.9, 113.2,
86.2, 72.3, 71.8, 70.1, 64.6, 55.4, 50.4, 39.8, 30.5, 29.8, 29.7, 29.6,
29.5, 29.2, 28.1, 26.7, 26.4, 26.3, 24.9; 19F NMR δ -73.5
(reference CFCl3); HR ESI MS m/z calcd for C44H59F3N4O6
(M
þ
Hþ) 796.4434, found m/z 796.44353; calcd for
C44H59F3N4O6Na (Mþ þ 23) 819.3745, found m/z 819.3742.
(9H-Fluoren-9-yl)methyl [1-[12-[2-Hydroxy-3-[(4-methoxy-
phenyl)diphenylmethoxy]propoxy]dodecyl]-1H-1,2,3-triazol-4-
yl]methylcarbamate, 14. Compound 10 (32 mg; 0.055 mmol)
along with DMAP (3 mg; 0.0275 mmol; 0.5 equiv) and
MMTrCl (26 mg, 0.083 mmol; 1.5 equiv) were dissolved in
pyridine (1.5 mL). The reaction was heated at 40 °C and stirred
overnight. Methanol (1.0 mL) was added, and the solvent
was evaporated. The resulting compound was purified by flash
chromatography (CH2Cl2/MeOH 1%): yield 45%; IR
(film) 3064, 2930, 2855, 1726, 1607, 1505, 1449, 1252, 1074,
755 cm-1; 1H NMR (400 MHz, CDCl3) δ 7.69 (d, J = 7.5 Hz;
1H), 7.50 (d, J = 7.5 Hz; 1H), 7.35 (m, 4H), 7.21 (m, 4H), 7.13
(m, 1H), 6.73 (d, J = 8.9 Hz; 1H), 5.37 (m, 1H), 4.38 (dd, J =
22.9 Hz, 5.8 Hz; 4H), 4.18 (m, 4H), 3.87 (m, 1H), 3.71 (s, 3H),
3.45 (m, 2H), 3.38 (m, 2H), 3.10 (m, 2H), 2.48 (c, J = 7.2 Hz;
2H), 1.80 (m, 2H), 1.46 (m, 2H), 1.18 (m, 16H); 13C NMR (400
MHz, CDCl3) δ 158.8, 156.6, 144.6, 144.0, 141.5, 135.7, 130.5,
128.7, 128.6, 128.0, 127.9, 127.2, 1271, 125.2, 122.2, 120.0,
113.3, 86.5, 72.3, 71.8, 70.0, 67.0, 64.7, 55.4, 50.6, 47.4, 46.4,
36.7, 30.4, 29.8, 29.7, 29.7, 29.6, 29.5, 29.2, 26.7, 26.7, 26.3; HR
ESI MS m/z calcd for C53H62N4O6Na (M þ Naþ) 873.4558,
(9H-Fluoren-9-yl)methyl [1-]12-(2,3-Dihydroxypropoxy)dodecyl]-
1H-1,2,3-triazol-4-yl]methylcarbamate, 10. Compound 9 (48 mg;
0.078 mmol) was dissolved in a mixture of CH2Cl2/TFA 3% (2.5
mL). The reaction was stirred at room temperature for 30 min. The
solvent was reduced to dryness, washing three times with more
CH2Cl2. The resulting compound was purified by flash chromatog-
raphy (CH2Cl2/MeOH 2%): yield 72%; IR (film) 3329, 2926, 2858,
1
1715, 1522, 1441, 1256, 1127, 1043, cm-1; H NMR (400 MHz,
CDCl3) δ 7.76 (d, J = 7.5 Hz; 2H), 7.58 (d, J = 7.3 Hz; 2H), 7.38
(t, J = 7.3 Hz; 2H), 7.28 (m, 3H), 5.83 (broad s, 1H), 5.31 (s, 2H),
4.64-4.13 (m, 5H), 3.76 (m, 4H), 3.47 (m, 4H), 1.89 (m, 2H), 1.56
(m, 2H), 1.26 (m, 16H); 13C NMR (400 MHz, CDCl3) δ 156.4,
143.6, 141.1, 127.5, 126.8, 124.9, 119.8, 72.3, 71.6, 70.3, 70.2,
66.7, 64.0, 53.2, 50.6, 47.0, 29.9, 29.5, 29.3, 29.2, 29.2, 29.2,
29.2, 29.1, 28.7, 26.3, 25.8; HR ESI MS m/z calcd for C33H47N4O5
(Mþ þ 1) 579.3540, found m/z 579.3539; calcd for C33H46N4O5 Na
(M þ Naþ) 601.3360, found m/z 601.3358.
2-[4-]1-]12-(2,3-dihydroxypropoxy)dodecyl]-1H-1,2,3-triazol-
4-yl]butyl]isoindoline-1,3-dione, 11. Compound 8 (30 mg, 0.052
6812 J. Org. Chem. Vol. 75, No. 20, 2010

Grijalvo et al.
JOCArticle
found m/z 873.4555; calcd for C106H124N8O12 Na (2Mþ þ 23)
1723.9230, found m/z 1723.9234.
was quenched and modified RNA conjugates were desalted
(Sephadex, NAP-10) and purified by semipreparative HPLC.
Finally, DMTr groups were deprotected (AcOH 80%, 30 min)
and extracted with ether, and finally, chemically modified RNA
conjugates 24-26 were desalted (Sephadex G-25, NAP-5) and
characterized by MALDI-TOF mass spectrometry.
siRNA Duplexes. The chemically modified sense or passanger
strand and antisense or guide strand RNAs were dissolved in a
buffer 10 mM TRIS 50 mM NaCl (100 μL). Different aliquots
were taken. For each sample, buffer was added until a final
volume of 100 μL was reached. Modified siRNAs (siRNA-2,
siRNA-3 and siRNA-4, respectively) were heated at 94 °C for
2 min, and they were allowed to cool until room temperature was
reached. Then 3 M NaOAc pH = 5.2 was added (10 μL) along
with EtOH (96%) (275 μL). Samples were stirred, centrifuged at
4 °C (15 min, 12000 rpm), and precipitated at -20 °C. Finally,
the supernatant was removed, and the respective pellets were
carefully dried with argon.
Cell Culture and siRNA Conjugate Treatments. HeLa cells
were cultured under standard conditions (37 °C, 5% CO2,
Dulbecco’s Modified Eagle Medium, 10% fetal bovine serum,
2 mM L-glutamine, supplemented with penicillin (100 U/mL)
and streptomycin (100 mg/mL). All experiments were con-
ducted at 40-60% confluence. HeLa cells were transfected with
250 ng of the plasmid expressing murine TNF-R plasmid using
lipofectin (Invitrogen), following the manufacturer’s instructions.
One hour after transfection, m-TNF-R-expressing HeLa cells were
transfected with 50nM of native siRNA (23), chemically modified
siRNA-2, siRNA-3, siRNA-4, and scrambled siRNA-5 against
TNF-R, using oligofectamine (Invitrogen). The TNF-R concen-
tration was determined from cell culture supernatant by enzyme-
linked immunoabsorbent assay kit (ELISA, Bender MedSystems)
following the manufacturer’s instructions.
Synthesis of Amine Oligonucleotides (17-19). Oligonucleo-
tide synthesis was carried out in an automated DNA synthesizer
on a 1.0 μmol scale using controlled pore glass (CPG) resins 7,
15, 16. The synthesis followed the regular protocol for the DNA
synthesizer. After a final detritylation, the oligonucleotides were
removed from the universal solid support by treatment with
concentrated ammonia at 55 °C for 20 h. After desalting
(Sephadex G-25, NAP-10), pure aminolipid-oligonucleotide
conjugates 17-19 were obtained.
Synthesis of Guanidine-Oligonucleotides 20-22. Amine oligo-
nucleotides 20-22 (about 2.0 OD) were treated with a mixture
(60 μL) of a O-methylisourea chloride prepared solution (50 mg,
0.40 mmol) in water (50 μL) and an aqueous ammonia (30%,
60 μL). The reaction mixtures were incubated overnight at
55 °C. The water was evaporated, and the crude was desalted
(Sephadex G-25, NAP-5), yielding the corresponding guanidine-
oligonucleotides 20-22 in a pure form.
Melting Experiments. UV melting experiments were carried
out on a UV spectrometer. Samples were dissolved in a medium
salt buffer containing Na2HPO4 (5 mM), NaH2PO4 (10 mM),
and NaCl (100 mM). The increase in absorbance at 260 nm as a
function of time was recorded while the temperature was
increased linearly from 20 to 90 °C at a rate of 0.5 °C/min by
means of a Peltier temperature programmer. The melting tem-
perature was determined by the local maximum of the first
derivatives of the absorbance vs temperature curve. All melting
curves were found to be reversible. All determinations are
averages of triplicates.
Oligoribonucleotide Synthesis. The following RNA sequences
were obtained from commercial sources (Sigma-Proligo,
Dharmacon): sense or passanger scrambled 50-CAGUCGCGUU-
UGCGACUGGTT-30; antisense or guide scrambled 50-CCAGU-
CGCAAACGCGACUGTT-30; sense or passenger anti-TNF-R:
50-GUGCCUAUGUCUCAGCCUCTT-30 and antisense or guide
anti-TNFR: 50-GAGGCUGAGAC-AUAGGCACTT-30. Trityl
derivatives 6, 13, and 14 were coupled to the CPG solid supports,
according to the literature.20 Modified CPG solid supports 7, 15,
and 16 were then employed in the preparation of RNAs strands
using a DNA/RNA synthesizer. Modified siRNAs sense strands
were purified using DMT on- based protocols: RNAs were cleaved
from the support with a mixture of concentrated ammonia and
ethanol 3:1 at 55 °C for 60 min. Solvent was evaporated to dryness
and 1 M solution of TBAF in THF was added. RNAs were
incubated 24 h at room temperature. The deprotection reaction
Acknowledgment. This research was supported by the
Spanish Ministry of Education (Grant Nos. BFU2007-
63287 and CTQ2010-20541), the Generalitat de Catalunya
(2009/SGR/208), and the Instituto de Salud Carlos III
(CB06_01_0019).
Supporting Information Available: Complete characteriza-
tion data, copies of NMR spectra for all new compounds,
and MALDI-TOF mass spectrometry of chemically modified
siRNA conjugates. This material is available free of charge via
the Internet at http://pubs.acs.org
J. Org. Chem. Vol. 75, No. 20, 2010 6813