Synthesis of lipid-oligonucleotide conjugates for RNA interference studies

Article abstract of DOI:10.1002/cbdv.201000274

The synthesis of RNA molecules carrying lipids at their 3′-termini and 5′-termini is reported. These conjugates were fully characterized by MALDI-TOF mass spectrometry and HPLC chromatography. The ability of these conjugates to silence gene expression was

Full text of DOI:10.1002/cbdv.201000274

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Synthesis of LipidꢀOligonucleotide Conjugates for RNA Interference Studies
by Santiago Grijalvoa), Sandra M. Ocampoa), Jose´ C. Peralesb), and Ramon Eritja*a)
a) Institute for Research in Biomedicine, IQAC-CSIC, CIBER-BBN Networking Centre on
Bioengineering, Biomaterials and Nanomedicine, Edifici Helix, Baldiri Reixac 10, ES-08028 Barcelona
(phone: þ34(93)4039942; fax: þ34(93)2045904; e-mail: recgma@cid.csic.es)
b) Biophysics Unit, Department of Physiological Sciences II, Universitat de Barcelona; Feixa Llarga s/n,
ES-08907 Lꢀ Hospitalet del Llobregat, Barcelona
The synthesis of RNA molecules carrying lipids at their 3’-termini and 5’-termini is reported. These
conjugates were fully characterized by MALDI-TOF mass spectrometry and HPLC chromatography.
The ability of these conjugates to silence gene expression was evaluated in the inhibition of the tumor
necrosis factor. All the lipidꢀsiRNA derivatives were compatible with RNA interference machinery if
transfected with oligofectamine. In the absence of a transfection agent, some lipidꢀsiRNA derivatives
can exert a slight reduction of gene expression.
Introduction. – Many years ago, the use of modified nucleic acids to inhibit gene
expression by blocking translation and transcription, or by stimulating the degradation
of a particular messenger RNA (mRNA) became a promising approach in therapy.
This process uses antisense technology [1–3] and, more recently, RNA interference
(RNAi) [4]. Although both strategies inhibit the same stage of translation of a gene to
a protein, their mechanisms are different. In the case of antisense technology, synthetic
oligonucleotides are designed to selectively bind to the complementary mRNA
transcript of a targeted gene, thereby inhibiting mRNA translation to the correspond-
ing protein [5]. In contrast, the RNAi approach uses short double-stranded RNA
(dsRNA) molecules to switch genes off. Components of the RNAi machinery
specifically recognize the short interfering duplex RNAs (siRNAs) and incorporate
the guide RNA strand into a protein complex named the RNA-induced silencing
complex (RISC). The complex formed by the guide RNA strand and RISC catalyzes
the degradation of a specific mRNA, thereby repressing the synthesis of a specific
protein [6][7]. However, the use of oligonucleotides in both therapies is inefficient,
since they are rapidly degraded by exonucleases and endonucleases under physiological
conditions, and also because of their poor cellular uptake, which is primarily due to
their relatively large molecular weight and their polyanionic nature. The lack of
solutions to these two problems (especially in the case of cellular uptake) have caused a
bottleneck in the development of nucleic acids in potential therapeutics [8].
To enhance the stability of oligonucleotides to nuclease degradation and increase
cellular uptake, a major research effort is being made to design and synthesize modified
nucleic acids with similar hybridization properties but improved biological properties
[9–11]. The synthesis of modified nucleic acids such as phosphorothioate [12], methyl
phosphonate [13] and phosphoramidate oligonucleotides [14], locked nucleic acids
ꢁ 2011 Verlag Helvetica Chimica Acta AG, Zꢂrich

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[15], or peptide nucleic acids [16] has efficiently improved the stability of oligonucleo-
tides in serum. On the issue of delivery, progress has been made in the development of
new nonviral carriers such as polymers [17], lipids [18], cell-penetrating peptides [19],
or nanoparticles [20] for oligonucleotide delivery. However, despite considerable
research, efficiency and specificity results are still not satisfactory in many cases. To
date, most of the strategies for improving cellular uptake involve carriers that can be
covalently linked to ODNs using the post-synthetic conjugation [21–24] method or
through the preparation of complexes (lipoplexes, polyplexes) [25–28]. The formation
of these complexes is due to the existence of electronic interactions between
polyanionic nucleic acids and the positively charged nature of the designed carrier.
In particular, the conjugation of lipids, such as cholesterol and fatty acids, to siRNA
affords molecules with improved inhibitory properties [29–32].
As part of our ongoing interest in gene-silencing methods [33][34] and in finding
new carriers to improve inhibition and cellular uptake, we now propose a simple
strategy in which several lipid tails are covalently linked to oligonucleotides at their 3’-
end (using controlled pore glass (CPG) supports) or at 5’-end (phosphoramidite
chemistry) (Fig. 1). The target lipid molecules are based on the lipids that are used to
formulate the stable nucleic acidꢀlipid particles (SNALP) [35][36] for the systemic
administration of siRNA [37][38]. We studied the silencing properties of the
conjugates carrying the lipid modification in the passenger strand of the siRNA. Our
target for RNAi studies is the tumor necrosis factor (TNF-a) gene. The protein is
involved in several biological processes such as apoptosis, inflammation, and immunity,
and it has been implicated in the pathogenesis of many human diseases. The inhibition
Fig. 1. Proposed lipid modifications to be introduced into DNA and RNA molecules. a) Selected lipids
were introduced at the 3’-termini and b) selected lipids were introduced at the 5’-termini.

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of this cytokine is of particular relevance for the development of anti-inflammatory
drugs [24].
Results and Discussion. – 1. Synthesis of Lipid Derivatives. We considered the use of
a glycerol backbone-based structure to be a suitable scaffold for introducing our
chemical modifications, covalently linked to oligonucleotides at their 3’-end. This
strategy was successfully used to introduce the 1-O-hexyldecylglycerol unit into
antisense DNA [39]. The synthesis is outlined in Scheme 1. To prepare oligonucleotides
carrying C14 and C18 lipids at the 3’-position, CPG supports were functionalized by
reaction of the suitable trityl-lipid derivatives 4 or 9 with amino-functionalized CPG
prior to DNA/RNA synthesis. To synthesize 4, the alkylation reaction between
commercially available solketal 1 and 1-bromotetradecane with NaH (60%) in the
presence of toluene was carried out to yield the respective alkylated compound 2.
Acetonide hydrolysis (TsOH, MeOH) yielded the expected diol 3, which was
selectively protected using the 4-monomethoxy trityl (MMTr) group. This reaction
provided the desired protected alcohol 4 in high yield. We used a similar synthetic route
for the synthesis of trityl alcohol 9. Methanesulfonate 6 was used in the alkylation
reaction instead of a bromo derivative. Then, compounds 7, 8, and 9 were prepared in
good yields and were fully characterized. Finally, we coupled trityl compounds 4 and 9
to CPG supports using the succinyl linker described in the literature [40][41]. CPG
Functionalization yielded glass beads that were loaded with our proposed lipids
containing 5 (15 mmol/g) and 10 (16 mmol/g), respectively. To introduce the lipids at the
5’-position, we used the phosphoramidite approach (Scheme 2). For the synthesis of
lipid phosphoramidite 15, the selective protection of commercially available diol 11
Scheme 1. Synthesis of Lipid-Functionalized CPG Solid Supports 5 and 10 Used for the Preparation of
DNA/RNA Molecules Carrying Lipids in the 3’-Position
a) NaH (60%), 1-Bromotetradecane or 6, toluene, reflux, overnight; 2 (78%), 7 (77%). b) TsOH,
MeOH, r.t., overnight; 3 (72%), 8 (70%). c) (4-Methoxyphenyl)(diphenyl)methyl chloride (MMTr-Cl),
4-(dimethylamino)pyridine (DMAP), pyridine, 408, overnight, 4 (80%), 9 (67%). d) Controlled pore
glass (CPG) derivatization.

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Scheme 2. Synthesis of Lipophosphoramidites 15 and 19 to be Introduced into DNA/RNA Molecules in
the 5’-Position
a) Trityl chloride ([42]). b) NaH (60%), THF, reflux, overnight; 76%. c) CH2Cl2/CF3COOH (TFA)
20%, r.t., 2 h; 76%. d) NC(CH2)2OP(iPr2N)Cl, EtNiPr2 (DIEA), CH2Cl2, 08 to r.t., 1.5 h. e) 1-
Bromotetradecane ([36]); b) Pd[(PPh3)]4, N,N-dimethylbarbituric acid, THF, 908, sealed tube,
overnight; 97%; c) NC(CH2)2OP(iPr2N)Cl, DIEA, CH2Cl2, 08 to r.t., 1.5 h.
with TrCl was carried out according to the literature [42]. Then, protected alcohol 12
was subjected to an alkylation reaction with 1-bromotetradecane in the presence of
THF, which resulted in alkylated-trityl derivative 13 in high yield. Subsequently, Tr
deprotection under acidic conditions (CH2Cl2/TFA 20%) easily provided alcohol 14,

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which was transformed to the desired phosphoramidite 15. This was then directly used
for the synthesis of oligonucleotides without further purification.
Lipophosphoramidite 19 was synthesized by a double alkylation reaction of
commercially available protected triol 16 and 1-bromotetradecane, according to the
literature [36]. The removal of the allyl group of compound 17 with Pd[(PhP)3]4 in the
presence of THF yielded the desired alcohol 18, which was subsequently transformed
into its phosphoramidite derivative 19. As in the previous case, phosphoramidite 19 was
used for DNA/RNA synthesis without further purification.
2. Synthesis of OligodeoxynucleotideꢀLipid Conjugates. Solid supports and
phosphoramidites that have been previously described were used for the preparation
of oligonucleotides carrying lipids at the 3’- or 5’-ends. We selected the self-
complementary oligodeoxynucleotide sequence 5’-d(CGCGAATTCGCG)-3’ known
as the DickersonꢀDrew dodecamer, as a model compound. Oligonucleotide sequences
were assembled on a DNA synthesizer according to standard protocols. After the
assembly of the desired DNA sequence, supports were treated with concentrated NH3
at 558 for a minimum of 6 h to yield oligonucleotides 20–23, which carry lipid moieties
at the 3’- or 5’-ends. In all cases, a major peak was obtained that corresponded to the
expected molecular weight as determined by MALDI-TOF mass analysis (Exper.
Part). This indicates that the oligonucleotideꢀlipid conjugates are stable to oligonu-
cleotide synthesis conditions.
3. LipidꢀsiRNA Conjugates. Next, the synthesis of lipid conjugates of RNA was
studied. We selected a previously described siRNA duplex to inhibit the expression of
the TNF-a gene [43]. These siRNAs are being studied as potential anti-inflammatory
drugs. RNA Conjugates of passenger strands carrying lipids at the 3’- or 5’-end were
prepared (Table 1). A siRNA duplex carrying a cholesterol molecule at the 3’-end of
the passenger strand was also prepared for comparison purposes. Finally, RNA
conjugates of the guide strand carrying C14 and C18 lipids at the 3’-end of the guide
strand were prepared (Table 1). Syntheses were carried out according to standard RNA
synthesis protocols, using RNA monomers carrying the (t-Bu)Me2Si (TBDMS) group
in the 2’-position. RNA Conjugates carrying lipids in the 3’-position were purified using
DMT-on protocols, whereas RNA conjugates carrying lipids in the 5’-position were
purified according to DMT-off protocols. All modified conjugates were purified by
semipreparative HPLC chromatography. In all cases, we obtained a major peak that
was isolated and characterized by MALDI-TOF mass spectrometry (see Table 1). All
conjugates were annealed with equimolar amounts of the unmodified guide strands and
purified from EtOH precipitation (Table 1).
We examined the effect of the lipids on siRNA duplex stability. The thermal
stability of the modified and unmodified duplexes was determined in 100 mm AcOK,
2 mm (AcO)2Mg, and 30 mm HEPES-KOH at pH 7.4 (Table 2). The melting curves of
the duplexes were virtually identical (83.98, 83.68, and 84.38; for siRNA-3, siRNA-5,
and siRNA-6, resp.) compared to the siRNA-1 wild type (83.58), except for siRNA-4,
which was stabilized slightly when hybridized to its complement (86.98, with DTm ¼
3.48). This was probably due to the presence of 2’-OMe groups in the pyrimidines of the
RNA molecule [44] (Table 2). These results indicate that the presence of lipid groups in
the 3- or 5’-position of the siRNA molecule does not affect their ability to form stable
duplexes.

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Table 1. Modified and Unmodified RNA Sequences Used for the Preparation of the Corresponding siRNA
Conjugates for RNA Interference Studies
Compound Lipida)
No.
Sequence (5’-3’)b)
MSc)
siRNAd)
24
25
26
27
28
29
30
31
32
33
None, P, wt GUG CCU AUG UCU CAG CCU C-dT-dT
n.d.
siRNA-1
siRNA-2
siRNA-3
Chole), P GUG CCU AUG UCU CAG CCU C-dT-dT-Chol 7319/7310
C14-3’, P
GUG CCU AUG UCU CAG CCU C-dT-dT-C14 6909/6908
C14-3’f), P GUG CCU AUG UCU CAG CCU C-dT-dT-C14 7013 [MþNaþ ]/6993 siRNA-4
C18-3’, P
GUG CCU AUG UCU CAG CCU C-dT-dT-C18 6964/6961
siRNA-5
siRNA-6
siRNA-7
siRNA-8
siRNA-9
siRNA-10
C14N-5’, P C14N-GUG CCU AUG UCU CAG CCU C-dT-dT 6934/6937
C28N-5’, P C28-GUG CCU AUG UCU CAG CCU C-dT-dT 7112/7106
None, P, scr CAG UCG CGU UUG CGA CUG G-dT-dT
n.d.
GAG GCU GAG ACA UAG GCA C-dT-dT-C14 7123/7120
GAG GCU GAG ACA UAG GCA C-dT-dT-C18 7176/7174
C14-3’, G
C18-3’, G
a) wt: Wild type, P: passenger strand, G: guide strand, scr: scrambled. b) RNA Monomers are indicated in
uppercase letters, dT stands for thymidine. c) Mass spectra (found/expected); n.d.: not determined. d) siRNA
were prepared by hybridization with the corresponding guide strands (i.e., 24–30 with 5’-GAG GCU GAG
ACA UAG GCA C-dT-dT-3’, 31 with 5’-CCA GUC GCA AAC GCG ACU G-dT-dT-3’, and 32 and 33 with 5’-
GUG CCU AUG UCU CAG CCU C-dT-dT-3’). e) The siRNA conjugate with cholesterol at the 3’-termini was
prepared for comparison purposes. f) RNA indicated with underline contains 2’-O-methyl-RNA monomers.
Table 2. Melting Temperatures [8] of the siRNA Conjugates Prepareda)
siRNA
Lipid
Tm (DTm)b)
siRNA-1
siRNA-3
siRNA-4
siRNA-5
siRNA-6
None
C14-3’
C14-3’
C18-3’
C28-5’
83.5
83.9 (0.4)
86.9 (3.4)
83.6 (0.1)
84.3 (0.8)
a) Conditions: 100 mm AcOK, 2 mm (AcO)2Mg, and 30 mm HEPES-KOH soln. at pH 7.4. b) DTm is the
difference between the melting temperature of the lipid siRNA minus the melting temperature of the
corresponding unmodified siRNA.
Next, we studied the TNF-a inhibitory properties of the lipid-modified siRNA
duplexes [40] by two different series of experiments. In the first case, siRNA
transfection was carried out using a commercially available cationic lipid, oligofect-
amine. To this end, HeLa cells were first transfected with plasmid expressing murine
TNF-a using lipofectin. One hour later, unmodified siRNA-1 or lipid-modified siRNA
conjugates in the passenger strand (siRNA-2, siRNA-3, siRNA-4, siRNA-5, siRNA-6
and siRNA-7, resp., at 50 nm) as well as the scrambled sequence Scr were co-transfected
using oligofectamine. Cells were analyzed using an enzyme-linked immunoabsorbent
assay (ELISA) after 24 h of transfection. Inhibition results are depicted in Fig. 2.
siRNA Conjugates, unmodified and modified at the 3’-position, i.e., siRNA-2, siRNA-3,
siRNA-4, and siRNA-5, showed 90–95% inhibition of the TNF-a production
compared to the Scr duplex. The inhibition results for the modified siRNA conjugates
at the 5’-position were somewhat lower (80 and 60% for siRNA-6 and siRNA-7, resp.).

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Although we obtained better inhibition results for siRNA conjugates modified at the 3’-
position, in general, the introduction of the lipid modifications did not disrupt the
RNAi machinery. After verifying the functionality of our modifications, we evaluated
whether the presence of the lipid modifications in siRNA duplexes could improve the
cellular uptake process. After transfecting HeLa cells with the murine TNF-a plasmid,
the cells were treated with unmodified and modified siRNA conjugates (siRNA-1,
siRNA-2, siRNA-3, siRNA-4, siRNA-5, and siRNA-6 at 100 nm) in the absence of a
transfection agent (Fig. 3). The inhibition activity of TNF-a is much lower in this case,
due to the absence of oligofectamine. However, some chemically modified siRNAs
silenced the TNF-a gene expression with promising inhibitory results (19, 17, and 38%
for siRNA-5, siRNA-6, and siRNA-7, resp.) compared to the scrambled duplex
(siRNA-8). Surprisingly, the most active siRNA without a transfecting agent is the less
active siRNA with oligofectamine. These results indicate that the most interesting
siRNA derivative prepared in this work was the siRNA-7, which carries the C28 lipid at
the 5’-end.
Fig. 2. Plot of gene-specific silencing activities for unmodified (siRNA-1) and modified (siRNA-2,
siRNA-3, siRNA-4, siRNA-5, siRNA-6, siRNA-7, and siRNA-8) conjugates, along with a scrambled
sequence (Scr) (50 nm per well). Transfection of siRNA conjugates was carried out using oligofectamine
(Protocol A). Values are represented as the averageꢁES, n¼3 and are compared to a scrambled
sequence. ***: p<0.001, ANOVA Test, Bonferrini post-test.
The inhibitory properties of siRNA duplexes carrying lipids at the guide strand, i.e.,
siRNA-9 and siRNA-10, were also analyzed (Fig. 4). Lipid-modified (i.e., siRNA-9 and
siRNA-10) and unmodified siRNA (i.e., siRNA-1) against TNF-a produced an 80–
90% inhibition of the production of TNF-a compared with the scrambled control
siRNA duplex (siRNA-8). These results indicate that the introduction of the lipid at
the 3’-end of either the guide or the passenger strand of an RNA duplex does not affect
the inhibitory properties of the resulting siRNA duplex in HeLa cells.
Conclusions. – We have reported an efficient strategy for introducing novel lipid
moieties at the 3’- or 5’-position of DNA and RNA molecules through covalent bonds

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Fig. 3. Plot of gene-specific silencing activities for unmodified (siRNA-1) and modified (siRNA-2,
siRNA-3, siRNA-4, siRNA-5, siRNA-6, and siRNA-7) lipidꢀsiRNA conjugates, along with a scrambled
sequence siRNA-8 at 100 nm without transfecting agent (Protocol B)
Fig. 4. Plot of gene-specific silencing activities for unmodified (siRNA-1) and modified (siRNA-9 and
siRNA-10) lipidꢀsiRNA conjugates, along with a scrambled siRNA-8 at 50 nm with transfecting agent
(Protocol A)
that are stable to oligonucleotide synthesis conditions. The effect of these modifications
on the cellular activity does not interfere with the RNAi machinery. In the presence of a
transfection agent, we observed better RNAi inhibitory properties with siRNA
conjugates carrying lipids at the 3’-termini. However, in the absence of transfection
agent, we observed better cellular uptake, when the C28 lipid was attached at the 5’-
position of the passenger strand. These promising results will be followed up with more
detailed cellular-uptake studies using these conjugates as well as nonviral carriers to
improve gene delivery.

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This study was supported by the Spanish Ministry of Education (grant BFU2007-63287 and
CTQ2010-20541), the Generalitat de Catalunya (2009/SGR/208), and CIBER-BBN (VI National
R&D&i Plan 2008–2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions, Instituto de Salud
Carlos III with assistance from the European Regional Development Fund).
Experimental Part
General. All reactions were carried out under Ar positive pressure in anh. solvents. Commercially
available reagents and anh. solvents were used without further purification. Solvents were distilled prior
to use and dried according to standard methods. Anal. samples were homogeneous, as confirmed by TLC,
and yielded spectroscopic results that were consistent with the structures assigned. TLC: Silica gel
(Alugram Sil G/UV). NMR Spectra chemical shifts [ppm] relative to the singlet at d 7.24 ppm of CHCl3
for 1H-NMR and to the centre line of the triplet at d 77.0 ppm of CDCl3 for 13C-NMR. IR Spectra:
BOMEM MB-120. MALDI-MS: Fisons VG Tofspec spectrometer. ESI-MS: Fisons VG Platform II
spectrometer. Oligonucleotide sequences were prepared using solid-phase methodology on an Applied
Biosystems Model 3400 DNA synthesizer. Oligoribonucleotides were purified by DMT-on protocols
using reversed-phase HPLC.
HPLC Conditions. HPLC Solns.: Solvent A: 5% MeCN in 100 mm triethylammonium acetate
(TEAA), pH 6.5; and solvent B: 70% MeCN in 100 mm TEAA (pH 6.5). Column: PRP-1 (Hamilton)
250ꢂ10 mm. A flow rate of 3 ml/min linear gradient from 15 to 100% in B (DMT-on) and 0 to 50% in B
(DMT-off) was used with UV detection at 260 nm.
General Procedure for the Alkylation Reaction. Solketal 1 (100 mg, 0.757 mmol) and 60% NaH
(60.4 mg, 1.51 mmol, 2.0 equiv.) were stirred in toluene (3.0 ml) under Ar for 20 min. The corresponding
C14 alkyl bromide (420 mg, 1.51 mmol, 2.0 equiv.) or C18 alkyl methanesulfonate (520 mg, 1.51 mmol) was
dissolved in toluene (2.0 ml) and added dropwise. The mixture was refluxed under Ar overnight. It was
then diluted with more toluene (10 ml), and the mixture was cooled in an ice bath, while the reaction was
quenched via slow addition of cold H2O. The mixture was washed with H2O (5 ml) and brine (5 ml),
using EtOH to aid phase separation if necessary. The org. phase was dried (Na2SO4) and evaporated. The
crude product was purified by flash chromatography (FC; cyclohexane/AcOEt 0 to 3%).
2,2-Dimethyl-4-[(tetradecyloxy)methyl]-1,3-dioxolane (2). Yield: 78%. IR (film): 2925, 2854, 1462,
1379, 1370, 1117.4. 1H-NMR (400 MHz, CDCl3): 4.26 (q, J¼6.0, 1 H); 4.05 (dd, J¼8.2, 6.4, 1 H); 3.73 (dd,
J¼8.2, 6.4, 1 H); 3.51 (m, 2 H); 3.43 (m, 2 H); 1.60 (m, 4 H); 1.42 (s, 3 H); 1.36 (s, 3 H); 1.25 (m, 20 H);
0.88 (t, J¼6.6, 3 H). 13C-NMR (125 MHz, CDCl3): 109.6; 74.9; 72.1; 72.0; 67.1; 32.1; 30.0; 29.9; 29.8; 29.8;
29.8; 29.8; 29.7; 29.6; 29.5; 26.9; 26.2; 25.6; 22.9; 14.3. HR-ESI-MS: 351.2862 ([MþNa]þ , C20H40NaO3þ ;
calc. 351.2870), 679.5835 ([2MþNa]þ , C40H80NaO6þ ; calc. 679.5847).
2,2-Dimethyl-4-{[(10Z,13Z)-octadeca-10,13-dien-1-yloxy]methyl}-1,3-dioxolane (7). Yield: 77%. IR
1
(film): 3008, 2986, 2926, 2856, 1457, 1379, 1369, 1214, 1120, 847. H-NMR (400 MHz, CDCl3): 5.34 (m,
4 H); 4.25 (q, J¼5.8, 1 H); 4.04 (dd, J¼14.6, 8.2, 1 H); 3.71 (dd, J¼14.6, 8.2, 1 H); 3.45 (m, 4 H); 2.76 (t,
J¼5.8, 2 H); 2.00 (m, 4 H); 1.56 (m, 2 H); 1.41 (s, 3 H); 1.35 (s, 3 H); 1.29 (m, 20 H); 0.88 (t, J¼6.8, 3 H).
13C-NMR (125 MHz, CDCl3): 132.1; 128.9; 110.0; 72.7; 72.1; 70.5; 64.5; 32.1; 30.0; 29.9; 29.8; 29.8; 29.8;
29.6; 29.5; 26.3; 22.9; 14.3. HR-ESI-MS: 403.3176 ([MþNa]þ , C24H44NaO3þ ; calc. 403.3183).
General Procedure for the Deprotection Reaction. A soln. of alkylated compound 2 (1.0 equiv.) and
alkylated compound 7 (1.0 equiv.) along with TsOH·H2O (0.5 equiv.) in MeOH (5.0 ml) was stirred at
r.t. overnight. The solvent was evaporated, and the residue was purified by FC (CH2Cl2/MeOH 0 to 2%).
3-(Tetradecyloxy)propane-1,2-diol (3). Yield: 72%. IR (film): 2980, 2850, 2390, 2387. 1H-NMR
(400 MHz, CDCl3): 3.87 (m, 1 H); 3.74 (dd, J¼11.4, 3.9, 1 H); 3.67 (dd, J¼11.4, 5.0, 1 H); 3.54 (m, 2 H);
3.48 (m, 2 H); 1.60 (m, 4 H); 1.27 (m, 20 H); 0.89 (t, J¼6.8, 3 H). 13C-NMR (125 MHz, CDCl3): 72.8;
72.0; 70.5; 64.5; 32.1; 30.0; 29.9; 29.8; 29.8; 29.8; 29.6; 29.5; 26.3; 22.9; 14.3. HR-ESI-MS: 311.2555 ([Mþ
Na]þ , C17H36NaOþ3 ; calc. 311.2557).
3-[(10Z,13Z)-Octadeca-10,13-dien-1-yloxy]propane-1,2-diol (8). Yield: 70%. IR (film): 3105, 2990,
2365, 1530, 1329. 1H-NMR (400 MHz, CDCl3): 5.33 (m, 4 H); 3.83 (m, 1 H); 3.64 (m, 2 H); 3.47 (m, 4 H);
2.74 (m, 2 H); 2.01 (m, 4 H); 1.55 (m, 2 H); 1.27 (m, 18 H); 0.86 (t, J¼6.8, 3 H). 13C-NMR (125 MHz,

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CDCl3): 130.4; 130.3; 130.2; 130.2; 128.2; 128.1; 128.1; 128.0; 72.6; 72.5; 72.0; 72.0; 70.8; 70.7; 64.4; 64.3;
31.8; 31.7; 29.9; 29.8; 29.7; 29.7; 29.7; 29.6; 29.6; 29.5; 29.5; 29.4; 29.4; 29.4; 27.4; 26.3; 26.2; 25.8; 25.8;
22.8; 22.7; 14.3. HR-ESI-MS: 363.2697 ([MþNa]þ , C21H40NaO3þ ; calc. 363.2698).
General Procedure for Protection with MMTr. Diols 3 or 8 (1.0 equiv.) were co-evaporated twice with
pyridine and dissolved again in pyridine (2 ml). DMAP (0.1 equiv.) and MMTr-Cl (1.5 equiv.) were
added at r.t., and the corresponding mixtures were stirred at 408 overnight. The reaction was quenched by
addition of MeOH (5 ml). The crudes were concentrated in vacuo, and the residues were purified by FC
(CH2Cl2/MeOH/Et3N 98 :1:1).
1-[(4-Methoxyphenyl)(diphenyl)methoxy]-3-(tetradecyloxy)propan-2-ol (4). Yield: 80%. IR (film):
3200, 1630, 1521, 1232, 990. 1H-NMR (400 MHz, CDCl3): 7.37 (m, 2 H); 7.23 (m, 5 H); 7.19 (m, 5 H); 6.77
(m, 1 H); 6.74 (m, 1 H); 3.86 (m, 1 H); 3.73 (s, 3 H); 3.46 (dd, J¼9.7, 4.2, 1 H); 3.39 (dd, J¼9.7, 6.4, 1 H);
3.35 (m, 2 H); 3.11 (m, 2 H); 2.34 (br. d, J¼4.9, 1 H); 1.47 (m, 2 H); 1.19 (m, 22 H); 0.81 (t, J¼6.6, 3 H).
13C-NMR (125 MHz, CDCl3): 158.9; 158.7; 150.0; 147.3; 144.6; 135.7; 130.5; 129.4; 128.7; 128.6; 128.1;
128.0; 128.0; 127.3; 127.1; 123.9; 113.4; 113.3; 86.5; 81.9; 72.2; 71.8; 70.1; 64.7; 55.4; 55.4; 32.1; 30.0; 29.9;
29.8; 29.8; 29.8; 29.7; 29.5; 26.3; 22.9; 14.3. HR-ESI-MS: 613.7622 ([MþNa]þ , C38H54NaO5þ ; calc.
613.7623).
1-[(4-Methoxyphenyl)(diphenyl)methoxy]-3-[(10Z,13Z)-octadeca-10,13-dien-1-yloxy]propan-2-ol
(9). Yield: 67%. IR (film): 3300, 2980, 2732, 1600, 1475, 1200, 995. 1H-NMR (400 MHz, CDCl3): 7.57 (m,
1 H); 7.36 (m, 2 H); 7.20 (m, 7 H); 7.10 (d, J¼8.8, 2 H); 6.75 (d, J¼8.8, 2 H); 5.29 (m, 4 H); 3.87 (m,
1 H); 3.70 (s, 3 H); 3.45 (dd, J¼9.7, 4.2, 1 H); 3.37 (m, 1 H); 3.10 (m, 2 H); 3.01 (m, 2 H); 2.69 (t, J¼6.5,
2 H); 2.42 (br. d, J¼4.4, 1 H); 1.96 (m, 2 H); 1.46 (m, 2 H); 1.20 (m, 18 H); 0.80 (t, J¼6.7, 3 H).
13C-NMR (400 MHz, CDCl3): 158.8; 149.9; 147.4; 144.6; 139.5; 136.1; 130.5; 129.4; 128.6; 128.1; 128.0;
128.0; 127.3; 127.1; 123.9; 113.4; 86.5; 81.9; 72.3; 71.8; 70.1; 64.7; 55.4; 31.7; 29.9; 29.9; 29.8; 29.7; 29.6;
29.5; 27.5; 27.4; 26.3; 25.8; 22.8; 14.3. HR-ESI-MS: 665.8250 ([MþNa]þ , C42H58NaO5þ ; calc. 665.8249).
General Procedure for Functionalization of CPG Solid Supports, 5 and 10. MMTr-Protected alcohols
4 or 9 along with DMAP (1.5 equiv.) and succinic anhydride (1.5 equiv.) were dissolved in CH2Cl2 (1 ml).
Mixtures were stirred overnight at r.t., CH2Cl2 was added (5 ml), and the org. layers were then washed
with a 0.1m Na2HPO4 soln. The resulting org. layers were dried (Na2SO4), and the solvent was
evaporated. The residues were used in the next step without further purification. 2,2-Dithiobis(5-
nitropyridine) (0.1 mmol) dissolved in 400 ml of MeCN was mixed with a soln. of the appropriate MMTr-
protected compound (0.1 mmol) and DMAP (0.1 mmol) in MeCN (500 ml). A soln. of Ph3P (0.1 mmol)
dissolved in MeCN (200 ml) was added to the respective clear solns. at r.t. The mixtures were then
vortexed for a few s and added to a syringe containing LCAA-CPG (500 mg, 0.05 mmol amino groups).
They were allowed to react for 2 h at r.t. The reactions were quenched by adding MeOH (500 ml). The
supports were then washed with MeOH (3ꢂ10 ml), MeCN (3ꢂ10 ml), and Et2O (3ꢂ10 ml). The solid
supports were dried in vacuo and then subjected to capping of the residual amino functionalities with a
mixture of Ac2O/pyridine/THF (500 ml) and 1-methyl-1H-imidazole in THF (500 ml). After 30 min, the
resins were washed with MeOH (3ꢂ10 ml), MeCN (3ꢂ10 ml), and Et2O (3ꢂ10 ml). The solid
supports 5 and 10 were dried first in the air and then under high vacuum. Loading(5a) [mmol/g]¼15;
loading(10) [mmol/g]¼16.
N,N-Dimethyl-2-(tetradecyloxy)-3-(trityloxy)propan-1-amine (13). Protected alcohol 12 (300 mg,
0.830 mmol) and 60% NaH (133 mg, 3.32, 4.0 equiv.) were suspended in anh. THF (4.5 ml) and stirred
for 15 min at r.t. Alkyl bromide (575 mg, 2.1 mmol, 2.0 equiv.) was added dropwise. The mixture was
heated to reflux overnight. It was then cooled at 08, and H2O (1.5 ml) was added carefully. The solvent
was evaporated, and the residue was dissolved in CH2Cl2 (10 ml), and the org. layer was washed with H2O
(2ꢂ20 ml) and brine (2ꢂ20 ml). The org. layer was dried (NaSO4), and the solvent was evaporated.
Crude was purified by FC (cyclohexane/Et3N 99 :1!cyclohexane/AcOEt/Et3N 98 :1:1). Yield: 77%. IR
1
(film): 3059, 2924, 2853, 2818, 2766, 1490, 1449, 1074, 909. H-NMR (400 MHz, CDCl3): 7.39 (m, 4 H);
7.19 (m, 11 H); 3.45 (m, 1 H); 3.38 (m, 2 H); 3.08 (m, 2 H); 2.31 (s, 6 H); 1.49 (m, 2 H); 1.18 (m, 22 H);
0.81 (t, J¼6.6, 3 H). 13C-NMR (125 MHz, CDCl3): 144.4; 128.9; 127.9; 127.1; 86.7; 77.9; 70.7; 70.6; 64.9;
61.7; 60.2; 46.5; 45.7; 32.1; 30.4; 30.0; 29.9; 29.8; 29.6; 26.4; 22.9; 14.4. HR-ESI-MS: 558.4298 ([MþH]þ
,
C38H56NOþ2 ; calc. 558.4306).

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
297
3-(Dimethylamino)-2-(tetradecyloxy)propan-1-ol (14). Alcohol 13 (358 mg, 0.642 mmol) was
dissolved in a mixture of CF3COOH/CH2Cl2 2 :8. The mixture was stirred for 2 h at r.t. The org. layer
was carried to basic pH with 1m NaOH soln. and was extracted three times with CH2Cl2 (20 ml). The org.
layer was dried (NaSO4), and the solvent was evaporated. The residue was purified by FC (CH2Cl2/
MeOH/Et3N 90 :9 :1). Yield: 76%. IR (film): 3110, 2960, 2874, 1530, 1470, 1065, 864. 1H-NMR
(400 MHz, CDCl3): 3.79 (dd, J¼10.9, 4.1, 1 H); 3.68 (m, 1 H); 3.48 (m, 5 H); 2.60 (m, 2 H); 2.32 (s, 6 H);
1.54 (m, 2 H); 1.25 (m, 20 H); 0.88 (t, J¼6.6, 3 H). 13C-NMR (125 MHz, CDCl3): 75.3; 70.1; 65.6; 62.9;
46.5; 32.1; 30.3; 29.9; 29.8; 29.8; 29.8; 29.8; 29.6; 29.5; 26.3; 22.9; 14.3. HR-ESI-MS: 316.2142 ([MþH]þ
,
C19H41NOþ2 ; calc. 316.2143).
2,3-Bis(tetradecyloxy)propan-1-ol (18). A mixture of 17 (100 mg, 0.191 mmol), N,N-dimethylbarbi-
turic acid (80 mg, 0.514 mmol, 2.7 equiv.), and Pd[(PPh3)]4 catalyst (12.3 mg, 0.011 mmol, 0.05 equiv.)
was dissolved in anh. THF (2.5 ml), and the soln. was heated at 908 in a sealed-tube overnight. The
solvent was evaporated, and the residue was purified by FC (CH2Cl2/MeOH 0 to 1%) to yield a white
1
solid. Yield: 97%. H-NMR (400 MHz, CDCl3): 3.72 (m, 1 H); 3.61 (m, 2 H); 3.50 (m, 2 H); 3.30 (m,
2 H); 2.91 (m, 2 H); 2.15 (m, 2 H); 1.54 (m, 8 H); 1.26 (m, 12 H); 0.88 (t, J¼6.88, 6 H).
General Procedure for the Synthesis of Lipophosphoramidites 14 and 19. EtNiPr2 (2 equiv.) was
added dropwise at r.t. to a soln. formed by alcohol 14 or 18 (1 equiv.) in CH2Cl2 (2 ml). Subsequently,
CH2Cl2 solns. were cooled at 08, and b-cyanoethyl-N,N-diisopropylaminophosphochloride (1.0 equiv.)
was added. The resulting mixtures were removed from the ice bath and allowed to warm to r.t., with
stirring for 2 h. The mixtures were diluted with more CH2Cl2 (10 ml) and extracted with 10 ml of cold
0.5m NaHCO3. The org. layers were dried (Na2SO4) and evaporated to leave a clear colorless syrup. A
vacuum pump was used to remove the last traces of solvent and EtNiPr2. The resulting products were
used without further purification.
2-Cyanoethyl 3-(Dimethylamino)-2-(tetradecyloxy)propyl Di(propan-2-yl)phosphoramidite (15).
1
Yield: 69%. H-NMR (400 MHz, CDCl3): 4.18 (m, 2 H); 3.88 (m, 1 H); 3.56 (m, 6 H); 2.76 (m, 2 H);
2.63 (m, 2 H); 2.26 (s, 6 H); 1.56 (m, 2 H); 1.25 (m, 22 H); 1.19 (m, 12 H); 0.88 (t, J¼6.5, 3 H). 31P-NMR
(300 MHz, CDCl3): 150.1.
2,3-Bis(tetradecyloxy)propyl 2-Cyanoethyl Di(propan-2-yl)phosphoramidite (19). Yield: 80%.
1H-NMR (300 MHz, CDCl3): 4.12 (m, 2 H); 3.79 (m, 1 H); 3.47 (m, 8 H); 3.26 (m, 1 H); 2.69 (m,
2 H); 2.56 (m, 2 H); 1.48 (m, 4 H); 1.18 (m, 44 H); 1.12 (d, J¼6.7, 6 H); 1.10 (d, J¼6.7, 6 H); 0.81 (t, J¼
6.6, 6 H). 31P-NMR (300 MHz, CDCl3): 150.1.
Oligodeoxynucleotide Synthesis. Oligodeoxynucleotide sequences were prepared by solid-phase
methodology. The syntheses were carried out on an Applied Biosystems Model 3400 DNA synthesizer
using a 1-mmol scale. Oligonucleotides were purified by reversed-phase (RP) HPLC, as described above.
MS of modified dodecamers: 20 (5’-d(CGCGAATTCGCG)-C14-3’)): found 3994, expected 3996; 21 (5’-
d(CGCGAATTCGCG)-C18-3’)): found 4045, expected 4048; 22 (C28–5’-d(CGCGAATTCGCG)-3’)):
found 4196, expected 4192; 23 (C14N-5’-d(CGCGAATTCGCG)-3’)): found 4028, expected 4025.
Oligoribonucleotides. The following RNA sequences were obtained from commercial sources
(Sigma-Proligo, Dharmacon): passenger scrambled: 5’-CAG UCG CGU UUG CGA CUG G-dT-dT-3’;
guide scrambled: 5’-CCA GUC GCA AAC GCG ACU G-dT-dT-3’, guide anti-TNFa: 5’-GAG GCU
GAG ACA UAG GCA C-dT-dT-3’, and passenger anti-TNFa: 5’-GUG CCU AUG UCU CAG CCU C-
dT-dT-3’. RNA Monomers in capital letters, dT represents thymidine. The 3’-cholesterol passenger anti-
TNFa: strand 5’-GUG CCU AUG UCU CAG CCU C-dT-dT-3’-cholesterol was prepared using the
cholesterolꢀtetraethyleneglycol (TEG)-3’-CPG support from commercial sources (Glen Research).
Oligoribonucleotides were prepared on a DNA synthesizer (Applied Biosystems 3400) using 2-
cyanoethyl phosphoramidites. The following solns. were used: 0.4m 1H-tetrazol in MeCN (activation);
3% Cl3COOH in CH2Cl2 (detritylation), Ac2O/pyridine/THF 1:1:8 (capping A), 10% N-methyl-1H-
imidazole in THF (capping B), 0.01m I2 in THF/pyridine/H2O 7:2 :1 (oxidation). Guanosine was
protected with the (dimethylamino)methylidene group, cytidine was protected with the AcO group, and
adenosine with the BzO group. The 2’-OH protecting group for the RNA monomers was the (t-Bu)Me2Si
(TBDMS) group. The average coupling yield was ca. 97–98% per step. Modified RNA strands were
purified using ꢃDMT-onꢀ-based protocols: RNAs were cleaved from the support with a mixture of conc.
NH3/EtOH 3 :1 at 558 for 60 min. The solvent was evaporated to dryness, and a 1m soln. of Bu4NF in THF

298
CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
was added. RNAs were incubated for 24 h at r.t. The deprotection reaction was quenched, and modified
RNA conjugates were desalted (Sephadex, NAP-10) and purified by semi-prep. HPLC. DMTr Groups
were then deprotected (AcOH 80%, 30 min) and extracted with Et2O. Finally, chemically modified RNA
conjugates were desalted (Sephadex, NAP-5).
Melting Experiments on siRNA Duplexes. A 100 mm AcOK, 2 mm (AcO)2Mg, and 30 mm HEPES-
KOH soln. at pH 7.4 and a final concentration of ca. 0.25 OD for each strand was used for the melting
experiments. The solns. were heated to 908 and allowed to cool slowly to r.t. Melting curves were
recorded using a concentration of ca. 1 mm for each strand in 1 ml of the tested soln. in Teflon-stoppered
quartz cuvettes of 1-cm optical path length. The melting temps. were determined as the maxima of the
first derivative of absorbance vs. temp. In all cases, the complexes displayed sharp, apparently two-state
transitions. The data were analyzed by the denaturation curve-processing program.
Annealing of the siRNA Duplexes. Equimolar amounts of the corresponding passenger and guide
strands were dissolved in a buffer containing 10 mm Tris and 50 mm NaCl (100 ml), and annealed in a final
volume of 100 ml. The solns. of siRNAs were heated at 948 for 2 min and allowed to cool to r.t. Then, 3m
AcONa, pH 5.2, was added (10 ml) along with EtOH (96%) (275 ml). Samples were stirred, centrifuged at
48 (15 min, 12000 rpm), and precipitated at ꢀ208. Finally, the supernatant was removed, and the
respective pellets were carefully dried under Ar.
Cell Culture and siRNA-Conjugate Treatments. Protocol A (transfection of lipid–siRNA conjugates
using oligofectamine). HeLa Cells were cultured under standard conditions (378, 5% CO2, Dulbeccoꢀs
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 conducted at 40–60% confluence.
HeLa Cells were transfected with 250 ng of murine-expressing TNF-a plasmid using lipofectin
(Invitrogen), according to the manufacturerꢀs instructions. One h after transfection, m-TNF-a expressing
HeLa cells were then transfected with unmodified siRNA-1 and modified (i.e., siRNA-2, siRNA-3,
siRNA-4, siRNA-5, siRNA-6, siRNA-7, siRNA-9, or siRNA-10) and a scrambled sequence siRNA-8 at
50 nm per well against TNF-a using oligofectamine (Invitrogen). The TNF-a concentration was
determined from cell culture supernatant with an enzyme-linked immunoabsorbent assay kit (Bender
MedSystems) following the manufacturerꢀs instructions.
Protocol B (transfection of lipidꢀsiRNA conjugates in the absence of oligofectamine): HeLa cells
were co-transfected with murine-expressing TNF-a plasmid as in the Protocol A. One h later,
lipidꢀsiRNA conjugates siRNA-2, siRNA-3, siRNA-4, siRNA-5, siRNA-6, and siRNA-7, and a
scrambled sequence siRNA-8 at 100 nm were added. The TNF-a concentration was determined from
cell culture supernatant with an enzyme-linked immunoabsorbent assay kit (Bender MedSystems),
following the manufacturerꢀs instructions.
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Received September 23, 2010