pubs.acs.

org/joc

Synthesis of Oligonucleotides Carrying Amino Lipid Groups at the 30 -End

for RNA Interference Studies
Santiago Grijalvo, Sandra M. Ocampo,, Jos
e 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 LHospitalet 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
RNA interference (RNAi) plays an important role in host
therapeutics.4 However, successful therapeutic applications
defense and regulation of gene expression. Since RNAi was
have been delayed due to delivery problems since native
discovered a decade ago by Fire et al.,1 RNA research has seen
siRNAs do not freely diffuse across the cell membrane due to
intense growth. During RNA interference, double-stranded
their relatively large molecular weight and their polyanionic
RNAs are processed into small fragments of approximately
nature. To overcome these limitations, different strategies have
21 nucleotides by the enzyme Dicer2 that are incorporated into
been employed and nonviral vectors have emerged as a promis-
the RNA-induced silencing complex (RISC) directing the
ing alternative to gene delivery.5
degradation of specific complementary mRNA sequences.3
The most used carriers for DNA and RNA oligonucleo-
The introduction of short interfering RNAs (siRNA) in mam-
tides are cationic lipids and liposomes which promote cel-
malian cells3 results in a selective silencing of the protein
lular uptake of antisense and RNAi therapies,6 although
encoded by the specific mRNA targeted by siRNA. Since then,
their mechanism is still unclear and controversial. In general,

(1) Fire, A.; Xu, S.; Montgomery, M. K.; Kostas, S. A.; Driver, S. E.;
Mello, C. C. Nature 1998, 391, 806811. (5) (a) Castanotto, D.; Rossi, J. J. Nature 2009, 457, 426433. (b) Singh,
(2) Zamore, P. D.; Tuschl, T.; Sharp, P. A.; Bartel, D. P. Cell 2000, 101, S.; Hajeri, P. B. Drug Discovery Today 2009, 14, 859865. (c) Gao, K.;
2533. Huang, L. Mol. Pharmaceutics 2009, 6, 651658. (c) Kim, D. H.; Rossi, J. J.
(3) (a) Caplen, N. J.; Parrish, S.; Imani, F.; Fire, A.; Morgan, R. A. Proc. Nat. Rev. Genet. 2007, 8, 173184. (d) Brumcot, D.; Manoharan, M.;
Natl. Acad. Sci. U.S.A. 2001, 98, 97429747. (b) Elbashir, S. M.; Harboth, J.; Koteliansky, V.; Sah, D. W. Nat. Chem. Biol. 2006, 2, 711719. (e) Mintzer,
Lendeckel, W.; Yalcin, A.; Weber, K.; Tuschl, T. Nature 2001, 411, 494498. M. A.; Simanek, E. E. Chem. Rev. 2009, 109, 259302. (f) Akhtar, S.; Benter,
(4) (a) Sibley, C. R.; Seow, Y.; Wood, M. Mol. Ther. 2010, 18, 466476. I. F. J. Clin. Invest. 2007, 117, 36233632.
(b) Singh, S. K.; Hajeri, P. B. Drug Discovery Today 2009, 14, 851858. (6) Tomkins, J. M.; Barnes, K. J.; Blaker, A. J.; Watkins, W. J.; Abell, C.
(c) Rana, T. M.; Baigude, H. ChemBioChem 2009, 10, 24492454. Tetrahedron Lett. 1997, 38, 691694.

6806 J. Org. Chem. 2010, 75, 68066813 Published on Web 09/23/2010 DOI: 10.1021/jo101143j
r 2010 American Chemical Society
Grijalvo et al.
JOC Article
SCHEME 1. Structure of the Lipid Oligonucleotide Conjugates Described in This Study

the use of targeted lipoplexes as systems to mediate siRNA expression,14 we have been looking into the possibility of
delivery has become the most reported method.7,8 These synthesizing siRNA conjugates by linking lipid molecules
units, which maintain an electrostatic association, are often with different polar and/or protonatable groups (such as
unstable9 and should be prepared before use, so other amines and triazoles) at the 30 -termini of the siRNA duplex
alternative and robust linkage methods between siRNAs (Scheme 1). We aimed to mimic the effect produced by
and nonviral vectors may be developed and validated. lipoplex complexes. As far as we know, such complex lipids
Recently, it has been demonstrated that nanoparticles can have never been introduced into siRNA. Finally, the effects
be used for the efficient delivery of siRNA.10 of such modifications on the RNAi machinery were mea-
Lipid-oligonucleotide conjugates are an interesting alter- sured through the evaluation of these chemically modified
native. Some years ago, lipid-oligonucleotide conjugates lipid-siRNA conjugates in the regulation of the tumor
were found to improve biological activity of triplex-forming necrosis factor-R (TNF-R) gene expression.
and antisense oligonucleotides.11 Recently, a few examples
on the use of siRNA covalently conjugated to lipids have Results and Discussion
been described in the literature. Cholesterol,12 fatty acid
Synthesis of the Lipid Derivatives. The introduction of lipids
derivatives,12 and R-tocopherol13 have been efficiently
to the 30 end of oligonucleotides requires the use of a linker
linked to the 50 -ends (phosphoramidite chemistry) and
that connects the lipid to the growing DNA chain. Aminodiols
30 -ends (CPG supports) of the siRNA guide and passenger
such 4-aminoprolinol12a,d and trihydroxy compounds such as
strands. Further biological evaluations of these conjugates
glycerol12c have been used for this purpose. Aminodiols are used
showed that the introduction of these groups did not affect
to incorporate fatty acids by formation of an amide bond that is
the RNAi machinery obtaining high levels of inhibition in the
stable to oligonucleotide synthesis conditions.12d Moreover,
gene expression.
aminodiols such as 4-aminoprolinol12d have the advantage of
As a part of our ongoing interest in the development of
well-defined chirality. But the use of this type of linkers needs
chemically modified DNA and siRNAs to inhibit gene
the use of fatty acids functionalized at the other end of the
hydrocarbon chain for the preparation of amino-lipids. These
(7) Zhang, S.; Zhao, B.; Jiang, H.; Wang, B.; Ma, B. J. Controlled Release
2007, 123, 110. compounds are not easily available. In contrast, trihydroxy
(8) Xie, F. Y.; Woodle, M. C.; Lu, P. Y. Drug Discovery Today 2006, 11, compounds can be used if ether or urethane bonds are used to
6773.
(9) de Fougerolles, A. R. Hum. Gen. Ther. 2008, 19, 125132.
connect the lipid to the triols. In these cases, the resulting lipid
(10) (a) Lee, J. S.; Green, J. J.; Love, K. T.; Sunshine, J.; Langer, R.; derivatives usually generate diastereoisomeric mixtures, but the
Anderson, D. G. Nano Lett. 2009, 9, 24022406. (b) Davis, M. E.; Zuckerman, presence of isomers is not considered a serious problem for
J. E.; Choi, C. H. J.; Seligson, D.; Tolcher, A.; Alabi, C. A.; Yen, T.; Heidel,
J. D.; Ribas, A. Nature 2010, 464, 10671070. biological activity of the resulting siRNA.12c Moreover, disub-
(11) (a) Gryaznov, S. M.; Lloyd, D. H. Nucleic Acids. Res. 1993, 21, 5909 stituted alkanes needed for the preparation of the lipid deriva-
5915. (b) Zelphati, O.; Wagner, E.; Leserman, L. Antiviral Res. 1994, 25, 13 tives are available. For these reasons, we considered the use of a
25. (c) LeDoan, T.; Etore, F.; Tenu, J. P.; Letourneux, Y.; Agrawal, S.
Bioorg. Med. Chem. 1999, 7, 22632269. (d) Rait, A.; Pirollo, K.; Will, D. W.; glycerol backbone (Scheme 1) as suitable linker for introducing
Peyman, A.; Rait, V.; Uhlmann, E.; Chang, E. H. Bioconjugate Chem. 2000, our chemical modifications at the 30 -end of the siRNA duplexes.
11, 153160.
(12) (a) Soutschek, J.; Akinc, A.; Bramlage, B.; Charisse, K.; Constien,
This linker, when reacted with phosphoramidites, will produce a
R.; Donoghue, M.; Elbashir, S.; Geick, A.; Hadwiger, P.; Harborth, J.; John, phosphate bond that will be stable to ammonia deprotection
M.; Kesavan, V.; Lavine, G.; Pandey, R. K.; Racie, T.; Rajeev, K. G.; R ohl, conditions used in oligonucleotide synthesis. Also, it allows the
I.; Toudjarska, I.; Wang, G.; Wuschko, S.; Bumcrot, D.; Koteliansky, V.;
Limmer, S.; Manoharan, M.; Vornlocher, H. P. Nature 2004, 432, 173178. incorporation of the lipid derivative to a solid support. The
(b) Lorenz, C.; Hadwiger, P.; John, M.; Vornlocher, H.-P.; Unverzagt, C. synthesis of lipidic solid supports 7, 15, and 16 is outlined in
Bioorg. Med. Chem. Lett. 2004, 14, 49754977. (c) Ueno, Y.; Kawada, K.; Schemes 2 and 3. All compounds were obtained from the same
Naito, T.; Shibata, A.; Yoshikawa, K.; Kim, H. S.; Wataya, Y.; Kitade, Y.
Bioorg. Med. Chem. Lett. 2008, 16, 76987704. (d) Wolfrum, C.; Shi, S.; intermediate azide 3 which was synthesized from commercially
Jayaraman, M.; Wang, G.; Pandey, R. K.; Rajeev, K. G.; Nakayama, T.; available rac-(2,2-dimethyl-1,3-dioxolan-4-yl)methanol as start-
Charisse, K.; Ndungo, E. M.; Zimmermann, T.; Koteliansky, V.; Manoharan,
M.; Stoffel, M. Nat. Biotechnol. 2007, 25, 11491157.
ing material. The synthesis started with the alkylation reaction
(13) Nishina, K.; Unno, T.; Uno, Y.; Kubodera, T.; Kanouchi, T.; between solketal 1 and commercially available 1,12-dibromodo-
Mizusawa, H.; Yokota, T. Mol. Ther. 2008, 16, 734740. decane in DMF in the presence of sodium hydride (60%), which
(14) (a) Grijalvo, S.; Terrazas, M.; Avi~
, A.; Eritja, R. Bioorg. Med.
no
Chem. Lett. 2010, 20, 21442147. (b) Avi~ no
, A.; Ocampo, S. M.; Caminal, C.; primarily yielded the alkylated compound 2. However, the
Perales, J. C.; Eritja, R. Mol. Divers. 2009, 13, 287293. presence of a side compound was also observed. This side
J. Org. Chem. Vol. 75, No. 20, 2010 6807
JOC Article Grijalvo et al.

SCHEME 2. Synthesis of Solid Support 7 Functionalized with Amino C12a

a
Reagents 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

a
Reagents and conditions: (a) selected alkynes, 10% sodium ascorbate, CuSO4 3 5H2O, tBuOH/H2O (1:1), rt, overnight; (b) (i) (for 8) DCM/TFA 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- purification by flash chromatography and without detecting any
halogenation). To avoid this, we started an alkylation reaction of trace of that impurity.
1 via di-n-butylstannylene in the presence of 2.0 equiv of cesium The nucleophilic displacement of the bromide group with
fluoride15 in the presence of DMF, providing 2 in 47% yield after sodium azide in DMF yielded the desired azide 3 in 93%
6808 J. Org. Chem. Vol. 75, No. 20, 2010
Grijalvo et al.
JOC Article
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 CGCGAATTCGCG-C12NH2 3983 3982.5 55
18 CGCGAATTCGCG-C12-triazol-CH2NH2 4066 4065 58
19 CGCGAATTCGCG-C12-triazol-(CH2)4NH2 4106 4105 57
20 CGCGAATTCGCG-C12NHC(NH)-NH2 4027 4029 61
21 CGCGAATTCGCG-C12-triazol-CH2NHC(NH)-NH2 4108 4109 56
22 CGCGAATTCGCG-C12-triazol-(CH2)4NHC(NH)-NH2 4150 4151 55
23 GUGCCUAUGUCUCAGCCUCTT-C12NH2 6896 6899 n.d.
24 GUGCCUAUGUCUCAGCCUCTT- C12-triazol-CH2NH2 6977 6991 [M Na] n.d.
25 GUGCCUAUGUCUCAGCCUCTT- C12-triazol-(CH2)4NH2 7019 7054 [M 2Na] n.d.
a
Conditions: 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 out as follows: the phthalimide 11 was removed under basic
Staudinger conditions gave the expected amine which was hydrolysis to give the expected amine, which was then
conveniently protected with Boc2O in standard conditions subsequently protected with ethyl trifluoroacetate yielding
achieving the N-Boc-protected 4 in 65% yield. Finally, the the N-protected amine 12. Finally, the selective protection of
introduction of the 4,40 -dimethoxytrityl (DMTr) protecting primary alcohol 12 with 4-monomethoxytrityl (MMTr)
group was easily obtained by removing acetonide and N-Boc afforded the corresponding protected trityl alcohol 13 in
moieties simultaneously under acidic conditions (DCM/ 39% overall yield (three steps).
TFA 3%) followed by N-protection with ethyl trifluoroace- The three trityl compounds 6, 13, and 14 were coupled
tate, thereby yielding the N-protected compound 5 in 87% with CPG supports using the succinyl linker as described.20
yield. DMTr protection with DMAP in the presence of For this purpose, the DMTr and MMTr derivatives
pyridine afforded the desired trityl derivative 6 in 58% yield described above reacted with succinic anhydride followed by
after purification by flash chromatography. coupling the resulting hemisuccinates with amino-functionalized
Click chemistry, on the other hand, is considered to be a CPG which yielded glass beads containing lipids 7 (21 mol/g),
modular approach that is increasingly found in all aspects 15 (25 mol/g), and 16 (23 mol/g), respectively. CPG functio-
of drug discovery, combinatorial chemistry, and recently, nalization was determined by the measure of the absorbance of
nucleic acid chemistry,16 easily obtaining triazolyl rings.17 the DMTr/MMTr cations released from the support upon acid
Taking this into account, we considered exploring the use of treatment.
the 1,3-dipolar cycloadditions between the previously syn- Oligodeoxyribonucleotide Synthesis. First, we synthesized
thesized azide 3 and some commercially available alkynes18 a short oligodeoxynucleotide sequence to demonstrate the
in order to study the subsequent effect that these kind of rings stability of the lipid derivative to phosphoramidite synthesis
could exercise on the RNAi machinery. Then, the click conditions. The self-complementary Dickerson-Drew
reactions were carried out under standard conditions19 to dodecamer sequence (50 -CGCGAATTCGCG-30 ) was used as
give the desired triazoles compounds 8 and 9 as only regio- a model sequence. The sequence was assembled on CPG solid
isomers in 89% and 59% yield, respectively, after purification supports 7, 15, and 16 using standard protocols in order to
by flash chromatography (Scheme 3). generate the corresponding lipid-oligonucleotide conjugates by
In order to obtain the corresponding trityl derivatives, using DMT off-based protocols. After cleavage with ammonia
compounds 8 and 9 were subjected to acetonide hydrolysis in solution (32%) followed by HPLC purification, the correspond-
different acid conditions (DCM/TFA 3% for acetonide 8; ing modified aminolipid-oligonucleotide conjugates (17-19)
p-TsOH in the presence of methanol for acetonide 9, were obtained and confirmed by MALDI-TOF mass spectro-
respectively) yielding the expected diols 10 and 11 in 72% metry (MS) (Table 1).
and 99% yield, respectively. DMTr derivative 14 was directly In addition, we studied the conversion of the amino group
obtained from protected alcohol 10 in 45% yield under the to the guanidinium group in order to extend the possibility of
same conditions used for the synthesis of compound 6. cationic charge enrichment in our synthesized aminolipid-
Finally, the synthesis of last trityl derivative 13 was carried oligonucleotide conjugates 17-19. The synthesis of cer-
tain guanidinium derivatives of nucleic acids has been
(15) (a) Fos, E.; Suesa, N.; Borras, L.; Lobato, C.; Banfi, P.; Gambetta, described in the literature,21 and some of them have been
R.; Zunino, F.; Mauleon, D.; Carganico, G. J. Med. Chem. 1995, 12161228.
(b) Leung, L. W.; Vilcheze, C.; Bittman, R. Tetrahedron Lett. 1998, 2921 synthesized through postsynthetic modification. 14a,21e
2924. (c) Gonc- alves, A. G.; Noseda, M. D.; Duarte, M. E. R.; Grindley, T. B. The guanidinium-modified ONs synthesized during this
J. Org. Chem. 2007, 72, 98969904. study are summarized in Table 1. In all cases, selective and
(16) (a) Amblard, F.; Cho, J. H.; Schinazi, R. F. Chem. Rev. 2009, 109,
42074220. (b) Mazzini, S.; Garca-Martn, F.; Alvira, M.; Avin
o, A.;
Manning, B.; Albericio, F.; Eritja, R. Chem. Biodiversity 2008, 5, 209218.
(c) Alvira, M.; Eritja, R. Chem. Biodiversity 2009, 4, 27982809. (d) Kolb, (20) (a) Gupta, K. C.; et al. Nucleosides Nucleotides 1995, 14 (3-5), 829.
H. C.; Sharpless, K. B. Drug Discov. Today 2003, 8, 11281137. (b) Pon, R. T. In Protocols for Oligonucleotides and Analogs; Agrawal, S.,
(17) Godeau, G.; Staedel, C.; Barthelemy, P. J. Med. Chem. 2008, 51, Ed.; Methods in Molecular Biology; Humana Press Inc.: Totowa, NJ, 1993;
43744376. Vol. 20, pp 465-496.
(18) A click chemistry reaction between azide 3 and N-Boc-protected (21) (a) Ohmichi, T.; Kuwahara, M.; Sasaki, N.; Hasegawa, M.; Nishikata,
propargylamine was also carried out. It is worth mentioning that we made T.; Sawai, H.; Sugimoto, N. Angew. Chem., Int. Ed. 2005, 44, 6682. (b) Maier,
several attempts to deprotect the N-Boc group and the acetonide moiety M. A.; Barber-Peoch, I.; Manoharan, M. Tetrahedron Lett. 2002, 43, 7613.
simultaneously but failed, even when the hydrolysis of this group was carried (c) Barber-Peoch, I.; Manoharan, M.; Cook, P. D. Nucleosides Nucleo-
out under neutral conditions by reaction with TMSOTf in the presence of tides 1997, 16, 1407. (d) Michel, T.; Debart, F.; Vasseur, J.-J. Tetrahedron
2,6-lutidine. For experimental details, see the Supporting Information. Lett. 2003, 44, 6579. (e) Deglane, G.; Abes, S.; Michel, T.; Prevot, P.;
(19) Rostovtsev, V. V.; Green, G. L.; Fokin, V. V.; Sharpless, K. B. Vives, E.; Debart, F.; Barvik, I.; Lebleu, B.; Vasseur, J.-J. ChemBioChem
Angew. Chem., Int. Ed. 2002, 41, 25962599. 2006, 7, 684.

J. Org. Chem. Vol. 75, No. 20, 2010 6809

JOC Article Grijalvo et al.

SCHEME 4. Synthesis of the Amine and Guanidinium Oligonucleotides

a
Reagents 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
siRNA-2 GUGCCUAUGUCUCAGCCUCTT-
C12NH2
TTCACGGAUACAGAGUCGGAG
siRNA-3 GUGCCUAUGUCUCAGCCUCTT-
C12-triazole-CH2NH2
TTCACGGAUACAGAGUCGGAG
siRNA-4 GUGCCUAUGUCUCAGCCUCTT-
C12-triazole-(CH2)4NH2
TTCACGGAUACAGAGUCGGAG FIGURE 1. Efficiency of silencing of chemically modified siRNAs
scrambled (siRNA-5) CAGUCGCGUUUGCGACUGGTT with lipids in the passenger strand. Plot of gene-specific silencing
TTGUCAGCGCAAACGCUGACC activities for native (siRNA-1), chemically modified conjugates
(siRNA-2, siRNA-3 and siRNA-4), and scrambled sequence
a
siRNAs are shown with the passenger strand above (50 - 30 ) and the
guide strand below (30 - 50 ). T stands for thymidine.
(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
quantitative guanidinylation were observed following a scrambled sequence. ***p<0.001, ANOVA Test, Bonferrini post-
classical postsynthetic approach21e on oligonucleotides test.
17-19, which were reacted with O-methylisourea for 16 h
at 55 C. After desalting, the guanidinylated compounds carried out the transfection of the aforementioned siRNA
20-22 (Scheme 4) were analyzed by analytical HPLC and derivatives with oligofectamine. Forty-eight hours after transfec-
characterized by MALDI experiments. Melting tempera- tion, the amount of TNF-R produced by the cells was analyzed
tures of the synthesized lipid-modified dodecamers were by enzyme-linked immunosorbent assay (ELISA). The silencing
higher than for the unmodified dodecamer (see Table 1). A activities are depicted in Figure 1. In general, all chemically
similar effect has been also observed recently with the modified siRNAs previously obtained maintained their abilities
dodecamer-modified with arginine and lysine residues as to down-regulate TNF-R protein expression levels to around
well as lipids.14b Preliminary experiments to insert guani- 65% at 50 nM concentration compared with the scrambled
dinium functions at the end of RNA oligomers using the control siRNA5 duplex. These results indicate that the introduc-
conditions described above yielded RNA degradation. tion of all the proposed modifications at the 30 -termini of the
Further attempts to extend such modifications in RNA passenger strand of an RNA duplex is compatible with the
oligomers are in progress. RNAi machinery in HeLa cells.
Oligoribonucleotide Synthesis. All oligoribonucleotides
Conclusions
were synthesized using solid supports 7, 15, and 16 (1.0 mol
each) using a DNA/RNA synthesizer (sequences of guide and In general, the most common method of administration of
passenger strands are shown in Tables 1 and 2). Modified RNA siRNAs in cell culture is based on the use of nonviral vectors
oligonucleotides linked to the aforementioned solid supports such as cationic lipids. The interaction between siRNA and
were released according to DMT on-based protocols, and cationic lipids is due to the existence of electrostatic interac-
then crude modified oligoribonucleotides were first purified tions and the formation of lipoplexes. Notwithstanding, the
by HPLC followed by DMTr deprotection with AcOH use of a covalent strategy between the aforementioned siRNA
80%. Finally, amino lipid-RNA conjugates 23-25 were and cationic lipids is also possible, though in most of the cases
again analyzed by reversed-phase HPLC and characterized only neutral lipids have been linked to siRNA. Following this
by MALDI-TOF mass spectrometry (Table 1). siRNA duplexes approach, we have reported a convenient synthesis by which
(siRNA2, siRNA3, and siRNA4, respectively) were obtained by glycerolipid based structures with different polar groups have
annealing of equal molar amounts of passenger (sense) and guide been efficiently synthesized and introduced into the 30 -termini
(antisense) strands, which were purified by ethanol precipitation of the siRNA sense strand being the first time that oligonu-
protocol (see the Experimental Section). cleotides carrying cationic lipids are reported. The amino
Gene Silencing by Modified siRNAs. The gene silencing group of the lipid can be used for the generation of guanidi-
effect on the TNF-R mRNA of several siRNAs: one native nium groups and also for further functionalization to pro-
siRNA1, three chemically modified siRNAs (siRNA2, teins, liposomes, nanoparticles and so on. Finally, we were
siRNA3, and siRNA4), and one scrambled siRNA5 were studied. able to confirm all these proposed modifications containing
HeLa cells were first cotransfected with plasmid pCAm TNF-R amine tails did not affect the RNAi machinery through
by using commercial cationic liposomes (lipofectin). We then silencing TNF-R gene expression.
6810 J. Org. Chem. Vol. 75, No. 20, 2010
Grijalvo et al.
JOC Article
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
Materials and Methods. All reactions were carried out under PPh3 (131 mg, 0.498 mmol; 2.0 equiv) were dissolved in 3 mL of
argon positive pressure in anhydrous solvents. Commercially anhydrous THF. The reaction was stirred for 2 h at room
available reagents and anhydrous solvents were used without temperature. Subsequently, water (500 uL) was added dropwise.
further purification. Solvents were distilled prior to use and The mixture was stirred overnight at room temperature. The
dried using standard methods. Analytical samples were homo- solvent was evaporated, and the crude was dried by pressure,
geneous as confirmed by TLC and yielded spectroscopic results yielding the anticipated amine. This amine was dissolved in
were consistent with the assigned structures. Chemical shifts are 2.0 mL of anhydrous dichloromethane (DCM); TEA (69 L,
reported in parts per million (ppm) relative to the singlet at = 0.498 mmol; 2.0 equiv) was added dropwise along with Boc2O
7.24 ppm of CHCl3 for 1H NMR and to the center line of the (82 mg, 0.373 mmol; 1.5 equiv). The reaction was stirred for 5 h at
triplet at = 77.0 ppm of CDCl3 for 13C NMR. IR spectra were room temperature. The organic layer was extracted with more
measured in film. Thin-layer chromatography (TLC) was per- DCM (5 mL) and rinsed with water (3  10 mL). The organic
formed on silica gel (Alugram Sil G/UV).
layer was dried on anhydrous Na2SO4. The solvent evaporated
HPLC Conditions. Conditions for semipreparative HPLC:
and the resulting residue was purified by flash chromatography
HPLC solutions were solvent A [5% acetonitrile (ACN) in 100
(Ch/AcOEt 4% to 9%): yield 65%; IR (film) 3020, 2935, 2850,
mM triethylammonium acetate (TEAA), pH 6.5] and solvent B
1699, 1506, 1371, 1216, 1166, 1046 cm-1; 1H NMR (400 MHz,
(70% ACN in 100 mM triethylammonium acetate, pH 6.5). CDCl3) 4.49 (broad s, 1H), 4.26 (m, 1H, CH-O), 4.04 (dd, J =
Column: PRP-1 (Hamilton) 250x 10 mm. Flow rate 3 mL/min 14.6, 8.2 Hz; 1H, O-CH-CH), 3.72 (dd, J = 14.6, 8.2 Hz; 1H,
linear gradient from 15 to 100% in B (DMTon) and 0 to 50% in O-CH-CH), 3.50 (m, 2H, CH2-O), 3.42 (m, 2H, CH2-O), 3.08 (m,
B (DMToff) was used with UV detection at 260 nm. Conditions 2H, CH2-N), 1.56 (m, 2H), 1.43 (s, 9H, 3CH3-C), 1.41 (s, 3H,
for analytical HPLC: HPLC solutions were solvent A [5% ace- CH3-C), 1.35 (s, 3H, CH3-C), 1.24 (m, 18H); 13C NMR (125
tonitrile, ACN) in 100 mM triethylammonium acetate (TEAA), MHz, CDCl3) 158.8 (CO), 110.0 (OCCH3), 75.4 (CH-O), 72.5
pH 6.5] and solvent B (70% ACN in 100 mM triethylammonium (O-CH2), 72.4 (O-CH2), 67.6 (O-CH2), 30.7 (N-CH2), 30.2, 30.1,
acetate, pH 6.5). Column: XBridge OST C18 2.5 m. Flow rate 29.9, 29.1, 27.4, 27.4, 26.7, 26.1; HR ESI MS m/z calcd for
1 mL/min linear gradient from 0 to 50% in B (DMToff) were C23H45NO5 (M H) 416.3370, found m/z 416.3370.
used with UV detection at 260 nm. N-[12-(2,3-Dihydroxypropoxy)dodecyl]-2,2,2-trifluoroacetamide,
4-[(12-Bromododecyloxy)methyl]-2,2-dimethyl-1,3-dioxolane, 5. Compound 4 (60.0 mg, 0.161 mmol) was dissolved in a mixture of
2. Alcohol 1 (200 mg; 1.51 mmol) was reacted with dibutyltin DCM/TFA 10% (5 mL) at room temperature for 20 min. The
oxide (751 mg, 3.02 mmol; 2.0 equiv) in dry methanol (10 mL) at solvent was evaporated, yielding the corresponding trifluoroacetate
reflux for 3 h, followed by removing the methanol and any traces in its salt form. The resulting oil was then dissolved in anhydrous
of water using azeotropic distillation with toluene. The reaction DCM (5.0 mL), and TEA was added dropwise (45 L, 0.322
mixture was concentrated, and then 1,12-dibromododecane mmol). The reaction was cooled at 0 C, and ethyl trifluoroacetate
(991 mg, 3.02 mmol; 2.0 equiv), cesium fluoride (458 mg, 3.02 (22.0 L, 0.177 mmol) was added. The reaction was stirred for
mmol; 2.0 equiv), and dry DMF (10 mL) were added. The 30 min, extracting the organic layer with more DCM (3  10 mL),
reaction mixture was kept at room temperature overnight. The and rinsed with water (3  10 mL). The organic layer was dried with
solvent was evaporated, and the resulting residue was purified anhydrous Na2SO4. The solvent evaporated, and the resulting oil
by flash chromatography (Ch/AcOEt 1% to 4%): yield 47%; IR was purified by flash chromatography (DCM/MeOH 2%): yield
(film) 2989, 2931, 2858, 1455, 1367, 1255, 1212, 1115, 1054, 845 87%; IR (film) 3333, 2924, 2854, 1694, 1552, 1471, 1185 cm-1;
cm-1; 1H NMR (400 MHz, CDCl3) 4.24 (q, J = 6.0 Hz, 1H, 1
H NMR (400 MHz, CDCl3) 6.42 (broad s, 1H), 3.05 (m, 1H,
CH-O), 4.03 (dd, J = 14.6, 8.2 Hz; 1H, O-CH-CH), 3.71 (dd, CH-O), 3.72 (dd, J = 11.4, 3.8 Hz; 1H, O-CH-CH), 3.65 (dd, J =
J = 8.2, 14.6 Hz; 1H, O-CH-CH), 3.49 (m, 2H, CH2-O), 3.40 (m, 11.4, 5.2 Hz; 1H, O-CH-CH), 3.51 (m, 2H, CH2-O), 3.49 (m, 2H,
2H, CH2-O), 3.38 (t, J = 6.8 Hz, 2H, CH2-Br), 1.83 (q, J = 7.1 CH2-O), 3.35 (m, 2H, CH2-NHCO), 1.57 (m, 2H), 1.26 (m, 20H);
Hz, 2H, CH2-CH2), 1.55 (m, 2H), 1.40 (s, 3H, CH3-C), 1.34 (s, 13
C NMR (125 MHz, CDCl3) 157.8 (q, J = 36.9 Hz, COCF3),
3H, CH3-C), 1.25 (m, 16H); 13C NMR (125 MHz, CDCl3) 117.9 (q, J = 287.6 Hz, CF3), 73.2 (CH-O), 72.5 (O-CH2), 71.0 (O-
109.9 (O-C-CH3), 75.4 (CH-O), 72.5 (O-CH2), 72.4 (CH2-O), CH2), 64.9 (O-CH2), 40.6 (NHCO-CH2), 30.2, 30.1, 30.1, 30.1,
67.5 (CH2-O), 34.6 (CH2-Br), 33.4, 30.2, 30.1, 30.1, 30.0, 30.0, 30.0, 29.7, 29.6, 27.3, 26.7; 19F NMR (CDCl3) -76.3 (reference
29.4, 28.8, 27.4, 26.6, 26.0; HR ESI MS m/z calcd for C18H35- CFCl3); HR ESI MS m/z calcd for C17H32F3NO4Na (M Na)
BrO3 Na (M Na) 401.1661, found m/z 401.1662; calcd for 394.2175, found m/z 394.2176.
C36H70O6NaBr2 Na (2 M Na) 779.3427, found 779.3431. N-[12-[3-[Bis(4-methoxyphenyl)(phenyl)methoxy]-2-hydroxy-
4-[(12-Azidododecyloxy)methyl]-2,2-dimethyl-1,3-dioxolane, 3. propoxy]dodecyl]-2,2,2-trifluoroacetamide, 6. Compound 5 (52
Compound 2 (100 mg, 0.264 mmol) was dissolved in 5 mL of mg, 0.140 mmol) along with 4,40 -dimethoxytrityl chloride
anhydrous DMF. NaN3 (103 mg, 1.58 mmol; 6.0 equiv) was then (DMTrCl) (48 mg, 0.141 mmol; 1.2 equiv) and DMAP (8 mg,
added, and the mixture was stirred and heated to 70 C for 48 h. 0.07 mmol; 0.5 equiv) were dissolved in 1.5 mL of anhydrous
The reaction was cooled at 0 C, and water was added carefully. pyridine. The reaction was heated at 40 C and stirred overnight.
The solvent was evaporated, and the resulting oil was then Methanol (0.5 mL) was added and the solvent evaporated. The
purified by flash chromatography (Ch/AcOEt 1% to 3%): yield resulting product was purified by flash chromatography (Hex/
93%; IR (film) 2935, 2858, 2363, 2344, 2093, 1251, 1077 cm-1; 1H AcOEt 15%): yield 58%; IR (film) 3276, 3072, 2927, 2851, 1716,
NMR (400 MHz, CDCl3) 4.26 (q, J = 6.1 Hz, 1H, CH-O), 4.06 1673, 1607, 1502, 1462, 1390, 1248, 1176, 1038 cm-1; 1H NMR
(dd, J = 14.6, 8.23 Hz; 1H, O-CH-CH), 3.72 (dd, J = 8.22, 14.6 (400 MHz, CDCl3) 7.67 (m, 3H, phenyl), 7.40 (m, 2H, phenyl),
Hz; 1H, O-CH-CH), 3.51 (m, 2H, CH2-O), 3.43 (m, 2H, CH2-O), 7.28 (m, 3H, phenyl), 7.01 (broad s, 1H), 6.81 (d, J = 8.9 Hz, 4H,
3.25 (t, J = 6.97 Hz, 2H, CH2-N3), 1.59 (m, 6H), 1.42 (s, 3H, CH3- phenyl), 3.92 (m, 1H, CH-O), 3.76 (s, 6H, 2 CH3), 3.52 (dd, J =
C), 1.36 (s, 3H, CH3-C), 1.27 (m, 14H); 13C NMR (125 MHz, 9.7 Hz, 4.2 Hz; 1H, CH-CH2-O), 3.43 (m, 3H, CH-CH2-O), 3.35
CDCl3) 109.3 (O-C-CH3), 74.7 (CH-O), 71.8 (O-CH2), 71.7 (O- (m, 2H, CH2-O), 3.17 (m, 2H, CH2-NH), 2.41 (broad d, 1H),
CH2), 66.9 (O-CH2), 51.4 (CH2-N3), 29.5, 29.4, 29.4, 29.4, 29.4, 1.85 (m, 2H), 1.57 (m, 9H), 1.25 (m, 9H); 13C NMR (125 MHz,
29.1, 28.7, 26.7, 26.6, 26.0, 25.3; HR ESI MS m/z calcd for CDCl3) 162.7, 158.6, 149.9, 145.0. 136.3, 136.2, 130.2, 128.3,
C18H36N3O3 (M) 342.2755, found m/z 342.2751; calcd for 127.9, 126.9, 123.9, 113.2, 86.2, 72.3, 71.8, 70.1, 64.7, 55.4, 55.3,
C18H35N3O3K (M K) 380.2312, found,380.2310. 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

JOC Article Grijalvo et al.

29.3, 29.1, 26.8, 26.2; 19F NMR -74.3 (reference CFCl3); HR mmol; 1.0 equiv) along with p-TsOH (5 mg, 0.026 mmol; 0.5
ESI MS m/z calcd for C38H50F3NO6Na (M Na)) 696.3482, equiv) were dissolved in methanol (1.0 mL). The reaction was
found m/z 696.3490. stirred at room temperature for 5 h. The methanol evaporated,
General Procedure for the Click Reaction (8 and 9). Azide 3 and the resulting oil was purified by flash chromatography
(1.0 equiv) and selected alkynes (1.0 equiv) were suspended in a (CH2Cl2/MeOH 2%): yield 99%; IR (film) 3349, 2924, 2850,
1:1 mixture of water and tert-butyl alcohol (1.0 mL). Sodium 1684, 1517, 1251, 1170, 1123, 1046 cm-1; 1H NMR (400 MHz,
ascorbate (10%) and copper(II) sulfate pentahydrate (1%) were CDCl3) 7.82 (m, 2H), 7.69 (m, 2H), 7.28 (s, 1H), 4.27 (t, J =
added dropwise. The heterogeneous mixture was stirred vigor- 7.2 Hz; 2H), 3.85 (m, 1H), 3.69 (m, 3H), 3.63 (m, 1H), 3.49 (m,
ously overnight. The reaction mixture was diluted with DCM, 2H), 3.44 (m, 2H), 2.74 (m, 2H), 2.53 (broad d, 2H), 1.85 (m,
and the organic layer was washed with water (3  5 mL). The 2H), 1.72 (m, 4H), 1.55 (m, 2H), 1.23 (m, 18H); 13C NMR (400
organic layer was dried with anhydrous Na2SO4 and solvent MHz, CDCl3) 168.6, 134.1, 132.3, 123.4, 72.7, 72.0, 70.6, 64.4,
evaporated. The resulting oil was purified by flash chromato- 50.4, 37.8, 30.5, 29.7, 29.6, 29.6, 29.6, 29.5, 29.5, 29.1, 28.2, 26.8,
graphy. 26.6, 26.2, 25.3; HR ESI MS m/z calcd for C29H44N4O5 (M
2-[4-[1-[12-[(2,2-Dimethyl-1,3-dioxolan-4-yl)methoxy]dodecyl]- 1) 529.3384, found m/z 529.3384; calcd for C29H44N4O5 Na
1H-1,2,3-triazol-4-yl]butyl]isoindoline-1,3-dione, 8: yield 89%; IR (M Na) 551.3190, found m/z 551.3200.
(film) 2919, 2854, 2363, 2342, 1710, 1053 cm-1; 1H NMR (400 N-[4-[1-[12-[3-[Bis(4-methoxyphenyl)(phenyl)methoxy]-2-hydro-
MHz, CDCl3) 7.81 (m, 2H, phenyl), 7.69 (m, 2H, phenyl), 7.27 xypropoxy]dodecyl]-1H-1,2,3-triazol-4-yl]butyl]-2,2,2-trifluoro-
(s, 1H, CHdC), 4.27 (t, J = 7.24 Hz; 2H, CH2-O), 4.22 (m, 1H, acetamide, 13. Compound 11 (30 mg, 0.060 mmol) was dis-
CH-O), 4.04 (dd, J = 14.6 Hz, 8.2 Hz; 1H, CH-CH2-O), 3.69 (m, solved in 1.0 mL of ethanol. Then, 80 L of MeNH2 was added
3H), 3.49 (m, 2H), 3.41 (m, 2H), 2.74 (t, J = 7.0 Hz, 2H, CH2-N), dropwise. The mixture was stirred overnight at 40 C. The
1.85 (m, 2H), 1.72 (m, 4H), 1.54 (m, 2H), 1.40 (s, 3H, CH3-C), solvent was reduced to dryness, and the resulting product was
1.34 (s, 3H, CH3-C), 1.26 (m, 16H); 13C NMR (125 MHz, CDCl3) dissolved again in anhydrous DCM (1.5 mL). TEA (38 L) was
168.6 (CO), 134.1 (phenyl), 132.3 (phenyl), 123.3 (phenyl), added dropwise at room temperature. The reaction was cooled
121.0 (CdN), 120.6 (CH-N) 109.5 (O-C-CH3), 74.9 (CH-O), 72.1 at 0 C, and EFTA (8.0 uL) was added dropwise. The reaction
(CH-CH2-O), 72.0 (CH-CH2-O), 67.1 (CH-CH2-O), 50.4 (CH2- was stirred for 30 min at 0 C. The organic layer was washed
Cd), 37.8 (CH2-NCO), 30.5 (CH2-N), 29.7, 29.7, 29.7, 29.6, 29.5, with water and brine (3  10 mL), dried with anhydrous
29.2, 28.2, 26.9, 26.8, 26.7, 26.2, 25.6, 25.3; HR ESI MS m/z calcd Na2SO4, and evaporated. The resulting compound was used
for C32H48N4O5 (M 1) 569.3697, found m/z 569.3697; calcd in the next step without further purification. The compound
for C32H48N4O5 Na (M Na) 591.3516, found m/z 591.3516. obtained along with MMTrCl (27.8 mg, 0.09 mmol; 1.5 equiv)
(9H-Fluoren-9-yl)methyl [1-[12-[(2,2-dimethyl-1,3-dioxolan- and DMAP (4.0 mg, 0.03 mmol; 0.5 equiv) were dissolved in 1.0
4-yl)methoxy]dodecyl]-1H-1,2,3-triazol-4-yl]methylcarbamate, 9: mL of pyridine. The reaction was heated at 40 C and stirred
yield 58%; IR (film) 2930, 2858, 1721,1523, 1448, 1370, 1250, overnight. Methanol was added (1.0 mL) and the solvent
1120, 1049, 758, 738 cm-1; 1H NMR (400 MHz, CDCl3) 7.74 evaporated. The product was then purified by flash chroma-
(d, J = 7.5 Hz; 2H, phenyl), 7.57 (d, J = 7.5 Hz; 2H, phenyl), 7.47 tography (DCM/MeOH/TEA 98:1:1): yield 39%; IR (film)
(s, 1H, CHdC), 7.38 (t, J = 7.3 Hz; 2H, phenyl), 7.29 (t, J = 7.4 3065, 2935, 2860, 1715, 1650, 1593, 1454, 1230, cm-1; 1H
Hz; 2H, phenyl), 5.48 (broad d, 1H, NH), 4.46 (d, J = 6.0 Hz; 2H, NMR (400 MHz, CDCl3) 7.41 (m, 2H), 7.31 (m, 4H), 7.26
CH2-OCO), 4.40 (d, J = 7.0 Hz; 2H, CH-CH2-O), 4.31 (t, J = (m, 4H), 6.82 (d, J = 8.8 Hz, 4H), 4.29 (t, J = 7.2 Hz; 2H), 3.94
7.2 Hz; 1H, O-CH-C), 4.25 (t, J = 6.0 Hz; 2H, CH2-O), 4.22 (m, (m, 1H), 3.78 (s, 6H), 3.51 (m, 2H), 3.42 (m, 4H), 3.17 (m, 2H),
2H), 4.05 (dd, J = 14.6 Hz, 8.2 Hz; 1H, CH-O-C), 3.72 (dd, J = 2.99 (m, 3H), 2.74 (t, J = 7.0 Hz, 2H), 1.87 (m, 2H), 1.75 (m,
14.6 Hz, 8.2 Hz; 1H, CH-O-C), 3.50 (m, 2H), 3.42 (m, 2H), 1.87 2H), 1.67 (m, 2H), 1.53 (m, 2H), 1.28 (m, 16H); 13C NMR (400
(m, 2H), 1.55 (m, 2H), 1.41 (s, 3H, CH3-C), 1.35 (s, 3H, CH3-C), MHz, CDCl3) 158.6, 150.0, 147.4, 145.0, 136.2, 130.2, 128.3,
1.27 (m, 16H); 13C NMR (125 MHz, CDCl3) 156.3 (CO), 143.8 128.0, 126.9, 124.0, 123.8 (q, J = 298.1 Hz, CF3), 120.9, 113.2,
(phenyl), 141.2 (phenyl), 127.6 (phenyl), 127.0 (CHdC), 125.0 86.2, 72.3, 71.8, 70.1, 64.6, 55.4, 50.4, 39.8, 30.5, 29.8, 29.7, 29.6,
(CdC), 119.9 (phenyl), 109.3 (O-C-O), 74.7 (CH-O), 71.9 (CH- 29.5, 29.2, 28.1, 26.7, 26.4, 26.3, 24.9; 19F NMR -73.5
CH2-O), 71.8 (CH-CH2-O), 66.9 (CH2-O), 50.3 (O-CH2-C), 47.2 (reference CFCl3); HR ESI MS m/z calcd for C44H59F3N4O6
(C-CH2-NCO), 36.5 (C-CH2), 30.2, 29.5, 29.5, 29.4, 29.4, 29.3, (M H) 796.4434, found m/z 796.44353; calcd for
28.9, 26.7, 26.4, 26.0, 25.4; HR ESI MS m/z calcd for C36H50N4- C44H59F3N4O6Na (M 23) 819.3745, found m/z 819.3742.
O5 (M 1) 619.3853, found m/z 619.3858; calcd for C36H50N4- (9H-Fluoren-9-yl)methyl [1-[12-[2-Hydroxy-3-[(4-methoxy-
O5 Na (M Na) 641.3673, found m/z 641.3675. phenyl)diphenylmethoxy]propoxy]dodecyl]-1H-1,2,3-triazol-4-
(9H-Fluoren-9-yl)methyl [1-]12-(2,3-Dihydroxypropoxy)dodecyl]- yl]methylcarbamate, 14. Compound 10 (32 mg; 0.055 mmol)
1H-1,2,3-triazol-4-yl]methylcarbamate, 10. Compound 9 (48 mg; along with DMAP (3 mg; 0.0275 mmol; 0.5 equiv) and
0.078 mmol) was dissolved in a mixture of CH2Cl2/TFA 3% (2.5 MMTrCl (26 mg, 0.083 mmol; 1.5 equiv) were dissolved in
mL). The reaction was stirred at room temperature for 30 min. The pyridine (1.5 mL). The reaction was heated at 40 C and stirred
solvent was reduced to dryness, washing three times with more overnight. Methanol (1.0 mL) was added, and the solvent
CH2Cl2. The resulting compound was purified by flash chromatog- was evaporated. The resulting compound was purified by flash
raphy (CH2Cl2/MeOH 2%): yield 72%; IR (film) 3329, 2926, 2858, chromatography (CH2Cl2/MeOH 1%): yield 45%; IR
1715, 1522, 1441, 1256, 1127, 1043, cm-1; 1H NMR (400 MHz, (film) 3064, 2930, 2855, 1726, 1607, 1505, 1449, 1252, 1074,
CDCl3) 7.76 (d, J = 7.5 Hz; 2H), 7.58 (d, J = 7.3 Hz; 2H), 7.38 755 cm-1; 1H NMR (400 MHz, CDCl3) 7.69 (d, J = 7.5 Hz;
(t, J = 7.3 Hz; 2H), 7.28 (m, 3H), 5.83 (broad s, 1H), 5.31 (s, 2H), 1H), 7.50 (d, J = 7.5 Hz; 1H), 7.35 (m, 4H), 7.21 (m, 4H), 7.13
4.64-4.13 (m, 5H), 3.76 (m, 4H), 3.47 (m, 4H), 1.89 (m, 2H), 1.56 (m, 1H), 6.73 (d, J = 8.9 Hz; 1H), 5.37 (m, 1H), 4.38 (dd, J =
(m, 2H), 1.26 (m, 16H); 13C NMR (400 MHz, CDCl3) 156.4, 22.9 Hz, 5.8 Hz; 4H), 4.18 (m, 4H), 3.87 (m, 1H), 3.71 (s, 3H),
143.6, 141.1, 127.5, 126.8, 124.9, 119.8, 72.3, 71.6, 70.3, 70.2, 3.45 (m, 2H), 3.38 (m, 2H), 3.10 (m, 2H), 2.48 (c, J = 7.2 Hz;
66.7, 64.0, 53.2, 50.6, 47.0, 29.9, 29.5, 29.3, 29.2, 29.2, 29.2, 2H), 1.80 (m, 2H), 1.46 (m, 2H), 1.18 (m, 16H); 13C NMR (400
29.2, 29.1, 28.7, 26.3, 25.8; HR ESI MS m/z calcd for C33H47N4O5 MHz, CDCl3) 158.8, 156.6, 144.6, 144.0, 141.5, 135.7, 130.5,
(M 1) 579.3540, found m/z 579.3539; calcd for C33H46N4O5 Na 128.7, 128.6, 128.0, 127.9, 127.2, 1271, 125.2, 122.2, 120.0,
(M Na) 601.3360, found m/z 601.3358. 113.3, 86.5, 72.3, 71.8, 70.0, 67.0, 64.7, 55.4, 50.6, 47.4, 46.4,
2-[4-]1-]12-(2,3-dihydroxypropoxy)dodecyl]-1H-1,2,3-triazol- 36.7, 30.4, 29.8, 29.7, 29.7, 29.6, 29.5, 29.2, 26.7, 26.7, 26.3; HR
4-yl]butyl]isoindoline-1,3-dione, 11. Compound 8 (30 mg, 0.052 ESI MS m/z calcd for C53H62N4O6Na (M Na) 873.4558,

6812 J. Org. Chem. Vol. 75, No. 20, 2010

Grijalvo et al.
JOC Article
found m/z 873.4555; calcd for C106H124N8O12 Na (2M 23) was quenched and modified RNA conjugates were desalted
1723.9230, found m/z 1723.9234. (Sephadex, NAP-10) and purified by semipreparative HPLC.
Synthesis of Amine Oligonucleotides (17-19). Oligonucleo- Finally, DMTr groups were deprotected (AcOH 80%, 30 min)
tide synthesis was carried out in an automated DNA synthesizer and extracted with ether, and finally, chemically modified RNA
on a 1.0 mol scale using controlled pore glass (CPG) resins 7, conjugates 24-26 were desalted (Sephadex G-25, NAP-5) and
15, 16. The synthesis followed the regular protocol for the DNA characterized by MALDI-TOF mass spectrometry.
synthesizer. After a final detritylation, the oligonucleotides were siRNA Duplexes. The chemically modified sense or passanger
removed from the universal solid support by treatment with strand and antisense or guide strand RNAs were dissolved in a
concentrated ammonia at 55 C for 20 h. After desalting buffer 10 mM TRIS 50 mM NaCl (100 L). Different aliquots
(Sephadex G-25, NAP-10), pure aminolipid-oligonucleotide were taken. For each sample, buffer was added until a final
conjugates 17-19 were obtained. volume of 100 L was reached. Modified siRNAs (siRNA-2,
Synthesis of Guanidine-Oligonucleotides 20-22. Amine oligo- siRNA-3 and siRNA-4, respectively) were heated at 94 C for
nucleotides 20-22 (about 2.0 OD) were treated with a mixture 2 min, and they were allowed to cool until room temperature was
(60 L) of a O-methylisourea chloride prepared solution (50 mg, reached. Then 3 M NaOAc pH = 5.2 was added (10 L) along
0.40 mmol) in water (50 L) and an aqueous ammonia (30%, with EtOH (96%) (275 L). Samples were stirred, centrifuged at
60 L). The reaction mixtures were incubated overnight at 4 C (15 min, 12000 rpm), and precipitated at -20 C. Finally,
55 C. The water was evaporated, and the crude was desalted the supernatant was removed, and the respective pellets were
(Sephadex G-25, NAP-5), yielding the corresponding guanidine- carefully dried with argon.
oligonucleotides 20-22 in a pure form. Cell Culture and siRNA Conjugate Treatments. HeLa cells
Melting Experiments. UV melting experiments were carried were cultured under standard conditions (37 C, 5% CO2,
out on a UV spectrometer. Samples were dissolved in a medium Dulbeccos Modified Eagle Medium, 10% fetal bovine serum,
salt buffer containing Na2HPO4 (5 mM), NaH2PO4 (10 mM), 2 mM L-glutamine, supplemented with penicillin (100 U/mL)
and NaCl (100 mM). The increase in absorbance at 260 nm as a and streptomycin (100 mg/mL). All experiments were con-
function of time was recorded while the temperature was ducted at 40-60% confluence. HeLa cells were transfected with
increased linearly from 20 to 90 C at a rate of 0.5 C/min by 250 ng of the plasmid expressing murine TNF-R plasmid using
means of a Peltier temperature programmer. The melting tem- lipofectin (Invitrogen), following the manufacturers instructions.
perature was determined by the local maximum of the first One hour after transfection, m-TNF-R-expressing HeLa cells were
derivatives of the absorbance vs temperature curve. All melting transfected with 50nM of native siRNA (23), chemically modified
curves were found to be reversible. All determinations are siRNA-2, siRNA-3, siRNA-4, and scrambled siRNA-5 against
averages of triplicates. TNF-R, using oligofectamine (Invitrogen). The TNF-R concen-
Oligoribonucleotide Synthesis. The following RNA sequences tration was determined from cell culture supernatant by enzyme-
were obtained from commercial sources (Sigma-Proligo, linked immunoabsorbent assay kit (ELISA, Bender MedSystems)
Dharmacon): sense or passanger scrambled 50 -CAGUCGCGUU- following the manufacturers instructions.
UGCGACUGGTT-30 ; antisense or guide scrambled 50 -CCAGU-
CGCAAACGCGACUGTT-30 ; sense or passenger anti-TNF-R: Acknowledgment. This research was supported by the
50 -GUGCCUAUGUCUCAGCCUCTT-30 and antisense or guide Spanish Ministry of Education (Grant Nos. BFU2007-
anti-TNFR: 50 -GAGGCUGAGAC-AUAGGCACTT-30 . Trityl 63287 and CTQ2010-20541), the Generalitat de Catalunya
derivatives 6, 13, and 14 were coupled to the CPG solid supports,
(2009/SGR/208), and the Instituto de Salud Carlos III
according to the literature.20 Modified CPG solid supports 7, 15,
and 16 were then employed in the preparation of RNAs strands (CB06_01_0019).
using a DNA/RNA synthesizer. Modified siRNAs sense strands
were purified using DMT on- based protocols: RNAs were cleaved Supporting Information Available: Complete characteriza-
from the support with a mixture of concentrated ammonia and tion data, copies of NMR spectra for all new compounds,
ethanol 3:1 at 55 C for 60 min. Solvent was evaporated to dryness and MALDI-TOF mass spectrometry of chemically modified
and 1 M solution of TBAF in THF was added. RNAs were siRNA conjugates. This material is available free of charge via
incubated 24 h at room temperature. The deprotection reaction the Internet at http://pubs.acs.org

J. Org. Chem. Vol. 75, No. 20, 2010 6813