A direct, efficient method for the preparation of siRNAs containing ribo-like north bicyclo[3.1.0]hexane pseudosugars

Article abstract of DOI:10.1021/ol200909j

An efficient method for the preparation of siRNAs modified with ribo-like North bicyclo[3.1.0]hexane pseudosugars is described. The combined use of 2-O-(2-cyanoethoxymethyl) (CEM) and 2-O-TBDMS protection was successfully employed for RNA synthesis with the added advantage that both groups were efficiently removed in a single step. The resulting North ribo-methanocarba- modified siRNAs are compatible with the intracellular RNAi machinery and can mediate specific degradation of target mRNA.

Full text of DOI:10.1021/ol200909j

ORGANIC
LETTERS
2011
Vol. 13, No. 11
2888–2891
A Direct, Efficient Method for the
Preparation of siRNAs Containing
Ribo-like North Bicyclo[3.1.0]hexane
Pseudosugars
†
‡
,‡
~ꢀ †
Montserrat Terrazas, Anna Avino, Maqbool A. Siddiqui, Victor E. Marquez,* and
Ramon Eritja*,†
Institute for Research in Biomedicine (IRB Barcelona), Networking Center on
Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), and Institute for
Advanced Chemistry of Catalonia (IQAC), Spanish Research Council (CSIC), Cluster
Building, Baldiri i Reixac 10, E-08028 Barcelona, Spain, and Laboratory of Chemical
Biology, Center for Cancer Research, National Cancer Institute at Frederick, Frederick,
Maryland 21702, United States
marquezv@mail.nih.gov; recgma@cid.csic.es
Received April 7, 2011
ABSTRACT
An efficient method for the preparation of siRNAs modified with ribo-like North bicyclo[3.1.0]hexane pseudosugars is described. The combined
use of 20-O-(2-cyanoethoxymethyl) (CEM) and 20-O-TBDMS protection was successfully employed for RNA synthesis with the added advantage
that both groups were efficiently removed in a single step. The resulting North ribo-methanocarba-modified siRNAs are compatible with the
intracellular RNAi machinery and can mediate specific degradation of target mRNA.
Nucleotide analogues that exhibit the North-type sugar
puckering1 and possess the overall A-RNA-type confor-
mation have attracted much interest in the field of RNA
interference (RNAi) therapy.2 RNAi is a sequence-specific
RNA silencing mechanism3 triggered by double-stranded
RNA or short interfering RNA molecules (siRNA), which
are formed by a sense and a guide strand.4 Within the cells,
an RNA-induced silencing complex (RISC)5 unwinds the
siRNA duplex and uses the guide strand as a template to
find the complementary target mRNA,6 an event that
induces the endonucleolytic cleavage of the mRNA7 and
prevents its translation into protein. For efficient gene
silencing to take place, the guide siRNA:mRNA duplex
must adopt an A-type helical structure.8 To fulfill these
requirements and improve target-binding affinity, several
† IRB Barcelona.
‡ National Cancer Institute at Frederick.
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r
10.1021/ol200909j
Published on Web 05/09/2011
2011 American Chemical Society

North-locked nucleotide building blocks have been de-
signed, synthesized, and incorporated into siRNAs.9
Examples are 20-O-alkylated RNAs,8b,10 20-fluoro-
RNAs,8b,10a,11 and Locked Nucleic Acids (LNA), the
latter containing a methylene bridge between the 20 oxygen
and the 40 carbon.12 In particular, LNAs have been widely
explored, anditiswell-knownthatLNA-modifiedsiRNAs
efficiently induce the RNAi process and increase the
thermodynamic and serum stability of RNA duplexes to
a great extent.13
Another candidate for introducing new features into
siRNAs without perturbing the A-type helical structure
they require for activity is the North-locked form of
nucleotide analogues based on a carbocyclic bicyclo-
[3.1.0]hexane system [methanocarba (MC) nucleosides].
Preliminary studies on the effect of North 20-deoxy-MC
nucleosides on the RNAi process have shown that these
pseudonucleosides are accepted by the RNAi machinery.14
However, the effect of a hydroxyl group at the 20 position
of any of the modified sugars, or the carbocyclic pseudo-
sugar, on the RNAi process has never been investigated.
The design and synthesis of North ribo-MC nucleosides
(Figure 1) has been reported.15 Nevertheless, these deriva-
tives have never been incorporated into RNA strands.
access to the preparation of North ribo-MC cytidine-
modified siRNAs with potential therapeutic applications.
An important challenge in the preparation of RNA
strands modified with North ribo-MC is the protection of
the 20-OH group of the pseudoribose. Preliminary studies
on the reactivity of the 20-OH group of North ribo-MC
cytidine carried out in our group have shown that the
30-OH is much more reactive than the 20-OH group (data
not shown). Thus, in contrast to what has been reported
for a great number of nucleoside derivatives,16 the direct
protection of the 20-OH group of 50-O-protected ribo-MC
cytidine derivatives with a free 30-hydroxyl group becomes
a serious problem. The attempted strategy was to employ
a 30,50-O-disiloxan diprotected intermediate with a free
20-OH group (9, Scheme 2). Our first try involved the
protection of the free 20-OH in 9 with a benzoyl group.
However, this route was abandoned due to the rapid
migration of the 20-O-benzoyl from the 20-OH to the 30-OH
after deprotection.
Scheme 1. Synthesis of Intermediate 9
Figure 1. North ribo-methanocarba cytidine monomer (CN).
Herein we describe a synthetic strategy for the prepara-
tion of conveniently 20-O-protected phosphoramidite of
North ribo-MC cytidine (CN) (Figure 1). We also describe
its incorporation into mixed 20-O-protected RNA strands
and the removal of the 20-O-protecting groups of the
RNAs in a single step. This approach permits an easy
A wide variety of 20-O-protecting groups have been
developed for RNA synthesis.16,17 For example, fluoride-
labile protecting groups such as tert-butyldimethylsilyl
(TBDMS),17a [(triisopropylsilyl)oxy]methyl (TOM),17b and
2-cyanoethoxymethyl (CEM)17c groups, or the photolabile
[(2-nitrobenzyl)oxy]methyl group17d among others.16
TBDMS and TOM are popular 20-OH protecting groups
whose phosphoramidites are commercially available.
We decided to use the cheaper 20-O-TBDMS-protected
(9) For a review, see: Watts, J. K.; Deleavey, G. F.; Damha, M. J.
Drug Discovery Today 2008, 13, 842.
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Ferrari, N.; Damha, M. J. Nucleic Acids Res. 2006, 34, 1669. (b)
Amarzguioui, M.; Holen, T.; Babaie, E.; Prydz, H. Nucleic Acids Res.
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2003, 31, 589. (c) Czauderna, F.; Fechtner, M.; Dames, S.; Aygun, H.;
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2003, 31, 2705.
(11) Deleavey, G. F.; Watts, J. K.; Alain, T.; Robert, F.; Kalota, A.;
Aishwarya, V.; Pelletier, J.; Gewirtz, A. M.; Sonenberg, N.; Damha,
M. J. Nucleic Acids Res. 2010, 38, 4547.
(12) (a) Obika, S. Chem. Pharm. Bull. 2004, 52, 1399. (b) Singh, S. K.;
Nielsen, P.; Koshkin, A. A.; Wengel, J. Chem. Commun. 1998, 455.
(13) (a) Braasch, D. A.; Jensen, S.; Liu, Y.; Kaur, K.; Arar, K.;
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White, M. A.; Corey, D. R. Biochemistry 2003, 42, 7967. (b) Elmen, J.;
(16) For a review, see: Somoza, A. Chem. Soc. Rev. 2008, 37, 2668.
(17) (a) Sproat, B. S. In Oligonucleotide Synthesis: Methods and
Applications; Herdewijn, P., Ed.; Humana Press: NJ, 2005; Vol. 288, pp
17ꢀ31. (b) Pitsch, S.; Weiss, P. A.; Jenny, L.; Stutz, A.; Wu, X. Helv.
Chim. Acta 2001, 84, 3773. (c) Shiba, Y.; Masuda, H.; Watanabe, N.;
Ego, T.; Takagaki, K.; Ishiyama, K.; Ohgi, T.; Yano, J. Nucleic Acids
Res. 2007, 35, 3287. (d) Schwartz, M. E.; Breaker, R. R.; Asteriadis,
G. T.; deBear, J. S.; Gough, G. R. Bioorg. Med. Chem. Lett. 1992, 2,
1019. (e) Harstel, S. A.; Kitchen, D. E.; Scaringe, S. A.; Marsal, W. S. In
Oligonucleotide Synthesis: Methods and Applications; Herdewijn, P., Ed.;
Humana Press: NJ, 2005; Vol. 288, pp 33ꢀ49.
Thonberg, H.; Ljungberg, K.; Frieden, M.; Westergaard, M.; Xu, Y.;
Wahren, B.; Liang, Z.; Ørum, H.; Koch, T.; Wahlestedt, C. Nucleic
Acids Res. 2005, 33, 439.
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Eritja, R. ChemBioChem 2011, 12, 1056.
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Wihlborg, A.-K.; Erlinge, D.; Malmsjo, M.; Boyer, J. L.; Harden, T. K.;
Jacobson, K. A. J. Med. Chem. 2002, 45, 208. (b) Ravi, G.; Lee, K.; Ji,
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Org. Lett., Vol. 13, No. 11, 2011
2889

Scheme 2. Preparation of 20-O-CEM-protected Ribo-MC Cytidine Building Block
phosphoramidites of natural ribonucleosides for RNA
synthesis. However, for the protection of the 20-OH group
of the 30,50-O-disiloxan protected MC nucleoside 9, the
fluoride-labile TBDMS and TOM groups had to be
avoided since the disiloxane protecting group is also
removed by a fluoride reagent like TBAF. The CEM group
is sensitive toward fluoride anion too, but only in aprotic
solvents. In this regard, the group of Wada et al. have
found a way to prevent the removal of the CEM group
during the removal of the disiloxan group of 30,50-O-dis-
iloxan-20-O-CEM-protectednucleosides by treatment with
TBAF in the presence of AcOH.18
As for RNA synthesis, removal of the CEM group from
fully 20-O-CEM-protected RNA strands has been success-
fully achieved under aprotic conditions.19 In our search for
a synthetic method that would allow ready cleavage of
the 20-O-protecting group of the MC nucleoside and the
20-O-TBDMS protecting group of natural nucleosides
from the final RNA product in only one step, we decided
to focus on the CEM group.
The synthesis of intermediate 9 began by preparing
cyclopropane 3 from cyclopentenone 120 according to a
previously reported procedure (Scheme 1),21 which in-
volves regio- and stereoselective Luche reduction followed
by hydroxyl-directed SimmonsꢀSmith cyclopropanation.
Subsequently, the MC uridine derivative 6 was prepared
from alcohol 3 following conventional published meth-
ods:22,15a (i) conversion of alcohol 3 into amine 5 via
formation of the azido intermediate 4,22 and (ii) construc-
tion of the uracil ring by reacting amine 5 with 3-methoxy-
acryloyl isocyanate.15a Acetylation of the resulting uridine
derivative gave 6, which was converted into the unpro-
tected cytidine derivative 7 by treatment with POCl3,
triazole, and ammonia.23 Protection of the free 4-amino
group as the N-benzoyl amide 8 was followed by treatment
with 1,3-dichlorotetraisopropyl-disiloxane to give 30,
50-O-disiloxan diprotected derivative 9. As described in
Scheme 2, compound 9 was treated with 2-cyanoethyl
methylthiomethyl ether to give the desired 20-O-CEM-
30,50-O-disiloxan-protected derivative 10 in 73% yield.
Following the fluoride-catalyzed removal of the disiloxan
protecting group the desired 20-O-CEM protected diol 11
was obtained in 90% yield. The 50 hydroxyl group was then
protected by a 4,40-dimethoxytrityl group (DMT), and the
resulting 50-O-DMT-20-O-CEM-protected nucleoside 12
was converted into the desired phosphoramidite (13).
With compound 13 in hand we proceeded to incorporate
this nucleotide building block (CN) into different positions
in place of the natural ribocytidine along a 21-mer RNA
guide strand that targets the Renilla luciferase mRNA by
using a DNA/RNA synthesizer. The solid-phase synthesis
was carried out on a 0.2 μmol scale with a coupling time of
10 min. For CN-modified RNAs, P(III) to P(V) oxidation
was performed with a solution of 10% tert-butyl hydro-
peroxide in acetonitrile/water (96:4). Commercially available
succinyl polystyrene functionalized with 50-O-DMT-20-
deoxythymidine was used as the solid support and tetrazole
as the activator. Phosphoramidites of 20-O-TBDMS pro-
tected natural ribonucleosides (A, U, G, and C) were
obtained from commercial sources. We prepared RNA
guide strands containing no substitutions (14, wt), one CN
substitution at position 8 (15), and two CN substitutions
spaced-out along the guide strand at positions 8 and 15
(16). We also prepared the corresponding unmodified
sense strand (Table 1).
Table 1. Sequences of RNAs and Tm Data
a
entry
sequence
Tm
1
2
3
4
30-TTAAAAAGAGGAAGAAGUCUA (sense)
50-UUUUUCUCCUUCUUCAGAUTT (guide, 14, wt) 67.6
50-UUUUUCUCNCUUCUUCAGAUTT (guide, 15)
50-UUUUUCUCNCUUCUUCNAGAUTT (guide, 16) 64.5
65.9
a Tm’s were measured in 15 mM HEPES-KOH (pH 7.4), 1 mM
MgCl2, and 50 mM KOAc.
(18) Umemoto, T.; Wada, T. Tetrahedron Lett. 2004, 45, 9529.
(19) Ohgi, T.; Masutomi, Y.; Ishiyama, K.; Kitagawa, H.; Shiba, Y.;
Yano, J. Org. Lett. 2005, 7, 3477.
(20) Choi, W. J.; Moon, H. R.; Kim, H. O.; Yoo, B. N.; Lee, J. A.;
Shin, D. H.; Jeong, L. S. J. Org. Chem. 2004, 69, 2634.
(21) Lee, J. A.; Kim, H. O.; Tosh, D. K.; Moon, H. R.; Kim, S.;
Jeong, L. S. Org. Lett. 2006, 8, 5081.
(22) Jeong, L. S.; Buenger, G.; McCormack, J. J.; Cooney, D. A.;
Hao, Z.; Marquez, V. E. J. Med. Chem. 1998, 41, 2572.
(23) (a) Divakar, K. J.; Reese, C. B. J. Chem. Soc., Perkin Trans. 1
1982, 1171. (b) Marquez, V. E.; Siddiqui, M. A.; Ezzitouni, A.; Russ, P.;
Wang, J.; Wagner, R. W.; Matteucci, M. D. J. Med. Chem. 1996, 39,
3739.
After RNA synthesis, the DMT-on polystyrene solid
support was treated with a mixture of 33% ammonia
solution and ethanol [3:1 (v/v)] for 24 h at 35 °C17c for
the cleavage from the polystyrene solid support and the
removal of the phosphate-protecting and base-protecting
(24) Capaldi, D. C.; Gaus, H.; Krotz, A. H.; Arnold, J.; Carty, R. L.;
Moore, M. N.; Scozzari, A. N.; Lowery, K.; Cole, D. L.; Ravikumar,
V. T. Org. Process Res. Dev. 2003, 7, 832.
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Org. Lett., Vol. 13, No. 11, 2011

groups. In the next step, the 20-O-TBDMS protecting group
of unmodified guide (14) and sense strands was completely
removed by treatment with 1 M TBAF/THF for 12 h.
For the removal of 20-O-TBDMS and 20-O-CEM pro-
tecting groups of RNAs 15 and 16, 10% n-propylamine
and 1% of bis(2-mercaptoethyl) ether in 1 M TBAF/THF
was used instead of 1 M TBAF/THF to prevent formation
of CEM adducts,24 a side reaction reported by Ohgi et al.19
These authors report complete removal of 20-O-CEM
protecting groups of fully 20-O-CEM protected RNA
strands after 6 h at rt. Under these conditions, we obtained
mixtures of 20-O-protected and 20-O-deprotected RNAs
(as evaluated by HPLC). We increased the reaction time
(to 12, 15, and 24 h), and after the mixtures were shaken at
room temperature for 24 h, HPLC analysis of the crude
oligo showed a single main peak corresponding to the fully
deprotected product and only small amounts of shorter
products (Figure 2A). Fully 20-O-deprotected RNAs
(14, 15, 16, and sense) were purified by reversed-phase
semipreparative HPLC in the DMT-on mode. Finally, the
DMT-on products were treated with 80% AcOH solution
to remove the DMT group and the deprotected products
were desalted on NAP columns. The desired products were
obtained in good overall yields (15, 58%, 22 OD; 16, 35%,
13 OD).
Finally, to study the effect of the ribo North MC cytidine
substitution on the RNA interference process, we carried
out separate RNAi studies in SH-SY5Y cells with siRNA
duplexes containing each of the modified guide strands
(15 and 16), aswell as with the unmodified (wt) guide RNA
(14). Experiments were carried out in triplicate. The cells
were first transfected with dual reporter plasmids that
express Renilla luciferase (the target) and nontargeted
firefly as an internal control. The effects of the different
siRNAs on luciferase expression were evaluated using
26ꢀ0.03 nM siRNA concentrations in the cell media and
measuring luciferase responses after 22 h. The results show
that the RNAi machinery is not impaired by the North MC
riboC modification (Figure S27). The best results were
obtained for siRNA containing one substitution in the
guide strand (15), which showed activity comparable to
that of wt siRNA (14). To confirm the specificity of the
observed effects, sequence-scrambled siRNAs were used
(Table S1). As expected, scrambled sequences gave no
Renilla inhibition (Figure S27). Thermal denaturation
studies suggested that the CN modification caused a slight
destabilization of the siRNA duplex (ΔT = ꢀ1.6 °C per
substitution; Table 1). However, such small destabilization
did not significantly affect RNAi activity.
In conclusion, in this work we have reported a strategy
for (i) the preparation of North ribo-MC nucleosides
conveniently protected at the 20-OH position, (ii) their
incorporation into mixed 20-O-protected RNA strands,
and (iii) the removal of the 20-O-protecting groups of the
RNA in only one step. To the best of our knowledge, this is
the first time that the synthesis and one-step deprotection
of 20-O-TBDMS/20-O-CEM oligoribonucleotides have
been described.
Acknowledgment. This research was supported by the
Spanish Ministry of Education (BFU2007-63287 and
CTQ2010-20541-C03-01) and the Generalitat de Catalu-
nya (2009/SGR/208) and the Instituto de Salud Carlos III.
This work was funded in part by the Center for Cancer
Research, National Cancer Institute, NIH. M.T. acknowl-
edges the Juan de la Cierva contract (MICINN, Spain) for
financial support.
Figure 2. HPLC analysis of RNA 15: (A) unpurified fully
deprotected RNA 15; (B) purified fully deprotected RNA 15.
RNA strands 15 and 16 were treated with nuclease P1
and then alkaline phosphatase, and the digestion products
were analyzed by HPLC. No modified ribonucleosides
(20-O-CEM protected ribo-MC cytidine or 20-O-TBDMS
protected U, A, C, and G) were observed, showing that
complete removal of the CEM and TBDMS protecting
groups and base-protecting groups was achieved.
Supporting Information Available. Procedures, charac-
terizations, copies of NMR spectra. This material is
available free of charge via the Internet at: http://pubs.
acs.org.
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