Efficient synthesis and cell-based silencing activity of siRNAS that contain triazole backbone linkages

Article abstract of DOI:10.1016/j.bmcl.2011.12.104

An efficient synthesis of siRNAs modified at the backbone with a triazole functionality is reported. Through the use of 4,4×-dimethoxytrityl (DMT) phosphoramidite chemistry, triazole backbone dimmers were site-specifically incorporated throughout various siRNAs targeting both firefly luciferase and glyceraldehyde- 3-phosphate dehydrogenase (GAPDH) gene transcripts as representatives of an exogenous and endogenous gene, respectively. Following the successful silencing of the firefly luciferase reporter gene, triazole-modified siRNAs were also found to be capable of silencing GAPDH in a dose-dependent manner. Backbone modifications approaching the 3×-end on the sense strand were tolerated without compromising siRNA potency. This study highlights the compatibility of triazole-modified siRNAs within the RNAi pathway, and the modification's potential to impart favorable properties to siRNAs designed to target other endogenous genes.

Full text of DOI:10.1016/j.bmcl.2011.12.104

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1722–1726
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Efficient synthesis and cell-based silencing activity of siRNAS that
contain triazole backbone linkages
⇑
Tim C. Efthymiou, Vanthi Huynh, Jaymie Oentoro, Brandon Peel, Jean-Paul Desaulniers
Faculty of Science, University of Ontario Institute of Technology, 2000 Simcoe Street North, Oshawa, Ontario, Canada L1H 7K4
a r t i c l e i n f o
a b s t r a c t
Article history:
An efficient synthesis of siRNAs modified at the backbone with a triazole functionality is reported.
Through the use of 4,40-dimethoxytrityl (DMT) phosphoramidite chemistry, triazole backbone dimers
were site-specifically incorporated throughout various siRNAs targeting both firefly luciferase and glyc-
eraldehyde-3-phosphate dehydrogenase (GAPDH) gene transcripts as representatives of an exogenous
and endogenous gene, respectively. Following the successful silencing of the firefly luciferase reporter
gene, triazole-modified siRNAs were also found to be capable of silencing GAPDH in a dose-dependent
manner. Backbone modifications approaching the 30-end on the sense strand were tolerated without
compromising siRNA potency. This study highlights the compatibility of triazole-modified siRNAs within
the RNAi pathway, and the modification’s potential to impart favorable properties to siRNAs designed to
target other endogenous genes.
Received 2 November 2011
Revised 16 December 2011
Accepted 20 December 2011
Available online 30 December 2011
Keywords:
Short interfering RNA
Modified nucleoside
Triazole linkage
Huisgen dipolar cycloaddition
Gene silencing
Ó 2011 Elsevier Ltd. All rights reserved.
In 1998, Fire and Mello reported that short interfering RNAs
(siRNAs) could be introduced into cells in Caenorhabditis elegans
and block gene expression.1 This process involves the incorpora-
tion of siRNAs into an RNA-induced silencing complex (RISC),
which serves to select and deliver the guide strand to its target
messenger RNA (mRNA) for selective gene silencing.2 Since this
discovery, there has been considerable interest in utilizing siRNAs
as potential therapeutic agents in combating disease.3 However,
natural siRNAs are prone to degradation by nucleases and exhibit
poor cell membrane permeability due to their polyanionic charge.
There is wide interest in chemically modifying siRNAs in order to
alleviate the aforementioned problems.
A vast number of studies have thoroughly explored the proper-
ties of chemically modified oligonucleotides that contain the
unnatural architecture at the sugars4 and bases.5 However, rela-
tively very little has been done with respect to modifying the inter-
nucleotide linkage of siRNAs with nonionic moieties. With respect
to negatively charged backbone mimics, phosphorothioates (PS)
have been shown to be accommodated within the RNAi pathway;
however, ranges in potency are often observed and cytotoxicity is a
concern.6 Alternatively, functional data of siRNAs bearing borano-
phosphate linkages show RNAi compatibility and offer enhanced
potency comparable to PS with reduced cytotoxicity.7
linkage enhances thermodynamic stability and resistance to
nucleases.8 Potenza and co-workers have also illustrated that an
amide-bond at the 30-end of siRNAs acts as a substrate for the RNAi
pathway.9
The crystal structure of the RISC complex by Patel et al., com-
plexed with a duplex oligonucleotide reveals that a number of
key phosphodiester contacts are necessary for its interaction with
the protein complex; however, many of these ionic contacts are not
present on the sense strand of the oligonucleotide.10 Therefore, we
envisioned that by modifying the sense strand, we could synthe-
size novel siRNAs with uncharged backbones within the duplex
that could thus serve as appropriate substrates for the RNAi path-
way without compromising their potency.
Utilizing the well-known Cu(I)-assisted Huisgen 1,3-dipolar
cycloaddition,11 the triazole moiety can be introduced as an un-
charged oligonucleotide backbone modification with relative ease.
Although a variety of triazole-modified oligonucleotide mimics
have been reported, most constructs are DNA-based.12 Reports of
click-ligated RNAs include one from El-Sagheer and Brown,13
who were successful in synthesizing functional hairpin ribozymes
containing both cross- and intra-strand triazole linkages. Other re-
ports of triazoles within RNAs include work from Das and co-work-
ers,14a and most recently from Rozners and co-workers14b who
demonstrated site-specific incorporation of a triazole-linked nucle-
oside dimer within RNAs.
With respect to nonionic backbone modifications, two studies
have shown that these can be accommodated within the 30-over-
hangs of siRNAs. Iwase and co-workers have shown that an amide
In previous work, we have synthesized a triazole-linked nucleo-
side dimer phosphoramidite based on a PNA-like structure, and this
modification displayed compatibility when hybridized to its com-
plement.15 Despite the numerous examples of distinct nucleoside
⇑
Corresponding author. Tel.: +1 905 721 8668x3621; fax: +1 905 721 3304.
E-mail address: jean-paul.desaulniers@uoit.ca (J.-P. Desaulniers).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.12.104

T. C. Efthymiou et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1722–1726
1723
In order to generate an appropriate alkyne monomer, alkyne
linker 4 was synthesized in 71% yield by treating TBS-protected
ethanolamine with limiting equivalents of propargyl bromide. This
linker was amide-bond coupled to uracil-1-yl acetic acid18 using
EDC as the coupling reagent to produce monomer 5 in 52% yield
(Scheme 2).
With both azide (3) and alkyne (5) monomers available, 1,4-tri-
azole formation catalyzed with CuSO4 and sodium ascorbate in a
THF/H2O solution afforded compound 6 in 98% yield. TBAF depro-
tection of the silyl protective group on compound 6 afforded com-
pound 7 in 99% yield. Finally, compound 7 was treated with a
phosphitylating reagent to afford the phosphoramidite 8 in 73%
yield (Scheme 3). Both the CtU and UtU phosphoramidites were
used to synthesize a variety of triazole-modified siRNAs.
Various siRNAs were synthesized, bearing one to three triazole
backbone modifications through the use of an ABI DNA/RNA syn-
thesizer. These siRNAs were designed to target firefly luciferase
mRNA expressed within HeLa cells, in order to gauge their func-
tionality within cells-based systems (Table 1).
O
Base
N
N
N
N
O
Base
N
Figure 1. Triazole-linked nucleic acid.
backbone derivatives linked together through a triazole moiety,
none of the derivatives have been reported as substrates within
the RNAi pathway. Herein, we report the incorporation of tria-
zole-linked nucleic acids based on a PNA-type scaffold (Fig. 1) with-
in duplex siRNAs. The RNAs display effective gene silencing and
thus exhibit a high degree of compatibility as RNAi substrates.
In this paper, we expanded the scope of siRNA sequences and
modified them with a cytosine-triazole-uracil (CtU) dimer in addi-
tion to the uracil-triazole-uracil (UtU) dimer previously synthe-
sized. Synthesis of the CtU dimer phosphoramidite commenced
through the generation of 2-azidoethanamine from bromoethyl-
amine combined with sodium azide. This compound was alkylated
with 2-bromoethoxy-tert-butyldimethylsilane16 in 39% yield to af-
ford azide linker 1. Utilizing EDC as a coupling reagent, this com-
pound was amide-bond coupled to N4-benzoylcytosin-1-yl acetic
acid17 to generate monomer 2 in 62% yield. TBS-deprotection of 2
with 3HF-TEA afforded a monoalcohol, which was then protected
with a 4,40-dimethoxytrityl group (DMT) to produce compound 3
in 90% yield (Scheme 1).
To first examine the effects of our RNAs, we investigated the ef-
fect of circularly polarized light on our siRNA duplexes to deter-
mine whether our chemically modified duplexes were still able
to retain A-type helical structure. Figure S1 (Supplementary data)
illustrates that the siRNAs retain the characteristic A-form helix;
however, a loss of helicity for double-stranded RNAs that contain
the internal triazole backbone modification is observed.
In addition to the listing of luciferase-targeting siRNAs, Table 1
highlights melting temperature (Tm) data indicating that the mod-
ification can have either a destabilizing or stabilizing contribution
on the dsRNA depending on its location.
In general, as the modification approaches the 50-end of the
sense strand, its contribution is less destabilizing. For example,
RNAs 9, 10 and 12 show very little destabilization relative to
Br
TBSO
NaN3
Br
H
N
DIPEA
Br
H2N
H3N
N3
TBSO
N3
64%
39%
1
O
N
O
N
O
HN
N
HN
HN
N
N
O
O
N
O
O
O
O
1) 3HF-TEA
2) DMTCl
OH
EDC
N
N
TBSO
DMTO
N3
N3
90%
62%
2
3
Scheme 1. Synthesis of azide intermediate.
O
O
N
NH
NH
N
O
O
O
O
Br
DIPEA
71%
OH
EDC
H
N
H2N
N
OTBS
OTBS
OTBS
52%
4
5
Scheme 2. Synthesis of alkyne intermediate.

1724
T. C. Efthymiou et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1722–1726
O
O
N
O
HN
N
HN
O
N
NH
N
NH
CuSO4
Na Ascorbate
O
O
N
O
O
N
O
O
O
O
98%
N
N
N
N
N3
DMTO
OTBS
N
N
DMTO
OTBS
N
3
5
6
O
N
O
N
HN
N
O
N
HN
N
O
N
Cl(iPr2N)PO(CH2)2CN
DIPEA, DMAP
NH
NH
TBAF
99%
CN
O
O
O
O
O
O
O
O
73%
O
P
N
N
N
N
N
N
DMTO
OH
N
N
DMTO
O
N
N
N
7
8
Scheme 3. Synthesis of CtU phosphoramidite.
Figure 2. Reduction in firefly luciferase expression related to the potency of backbone-modified triazole-linked siRNAs using the Dual-luciferase reporter assay. The siRNAs
were tested at 8, 80, and 800 pM, with firefly luciferase expression normalized to Renilla luciferase.
wild-type. However, the closer the modifications lie towards the
center of the duplex, the greater the destabilizing contribution of
that modification to the duplex. For instance, siRNAs 13–16 exhibit
changes in Tm of ca. ꢀ7.5 to ꢀ13 °C. The effect of a UtU modification
on a 30-overhang increases the Tm of siRNAs 15, 17, 18, 20 and 22,
relative to the corresponding siRNA with dTdT overhangs, thus
suggesting an element of stability. This stabilizing effect has been
observed with other oligonucleotides that bear 30-overhangs.8,19
However, this trend is only observed when the modification is
not in combination with any other triazole modifications within
the same oligonucleotide strand (siRNAs 19 and 21 do not exhibit
any increase in Tm). Finally, siRNA 21 contains three UtU modifica-
tions within the sense strand. This siRNA bears a drop in Tm of
ꢀ14.1 °C. SiRNA 11 contains two CtU modifications, and exhibits
a Tm drop of ꢀ20.3 °C relative to wild-type. Overall, our Tm data
is consistent with other similar studies involving PNA–DNA chime-
ras20 and triazole-linked RNAs.14b
To examine the effects of our backbone-modified siRNAs on the
RNA interference pathway, we performed cell-based assays within
HeLa cells. The cells were co-transfected with the siRNA duplexes
identified in Table 1 at varying concentrations, along with plasmids
expressing the target firefly luciferase and a non-target Renilla
luciferase for use as an internal control. Following transfection
with the desired siRNA construct, cells were lysed after 24 h, at
which point luciferase activity was monitored (Fig. 2). The wild-
type native siRNA inhibited luciferase expression in a dose-depen-
dent manner, while the modified siRNAs triggered varying levels of
silencing activity.

T. C. Efthymiou et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1722–1726
1725
Table 1
Table 2
Sequences of anti-luciferase siRNAs and Tm Data
Sequences of anti-GAPDH siRNAs
Figure 3. Analysis of GAPDH mRNA knockdown analyzed by real-time PCR
following the treatment of HeLa cells with GAPDH siRNAs 23–26. Levels of GAPDH
expression were normalized to the internal reference gene 18S.
siRNAs is compared to their analogous RNAs lacking a 30-UtU
modification in general, a reduction in activity is observed at lower
concentrations. The effect of observing reduced activity of a large
bulky 30-modified overhang on the antisense has been previously
observed.22
SiRNAs 16, 17 and 19 contain UtU modification at various posi-
tions near the 3’-end of the sense strand. Several of these siRNAs
exhibit comparable or enhanced silencing when compared to
wild-type siRNA. With respect to thermal stability, siRNAs 16
and 19 exhibit a drop in Tm. Destabilizing this area of the duplex
along with the 30-UtU modification (siRNA 19) may offer a favor-
able response by not only promoting favorable incorporation of
the guide strand to form the active RISC-guide complex,21a but
by increasing its resistance to nucleases. To determine whether a
30-UtU modified overhang provides enhanced stability to nucleas-
es, we performed time-dependent stability assays in serum. The
analysis involving siRNAs 17, 18 and 22 illustrated that these 30-
UtU modified duplex siRNAs have reduced susceptibility to degra-
dation by nucleases relative to wild-type native siRNA (Fig. S2,
Supplementary data).
a Tms were measured in triplicate in a sodium phosphate buffer (90 mM NaCl,10 mM
Na2HPO4, 1 mM EDTA, pH 7) at 260 nm, from 10 to 95 °C.
corresponds to the
uracil-triazole-uracil modification;
corresponds to the cytosine-triazole-uracil
modification.
For siRNAs 9–12, a reduction in potency relative to wild-type is
generally observed, especially when using lower concentrations of
siRNA (Fig. 2). These RNAs are chemically modified in the area of
the 50-sense region and in general, display a decrease in Tm relative
to wild-type siRNA (Table 1). In accordance with other reports
which have identified that siRNAs exhibit thermodynamic asym-
metry,21 our observations support that destabilizing this area
may lead to decreased potency. SiRNAs 13 and 14 contain a single
CtU modification within an internal site of the sense strand, caus-
ing a large decrease in duplex thermal stability compared to
wild-type (Table 1). There is also a noticeable decrease in potency
observed with these two siRNAs relative to wild-type (Fig. 2).
RNAs 10, 15, 18, 20 and 22 contain UtU modifications at the
30-overhang of the antisense strand. When the activity of these
Finally, siRNAs 11 and 21, which contain multiple triazole mod-
ifications within their sense strands, exhibit low thermal stability
and these siRNAs manage to effectively silence the target gene.
This suggests that multiple triazoles within a duplex siRNA are

1726
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3. (a) Manoharan, M. Biochim. Biophys. Acta 1999, 1489, 117; (b) Corey, D. R. J. Clin.
Invest. 2007, 117, 3615; (c) Watts, J. K.; Deleavey, G. F.; Damha, M. J. Drug
Discovery Today 2008, 13, 842.
compatible within the RNAi pathway, albeit at reduced silencing
efficiency compared to wild-type.
To determinewhetherthis novel chemicalmodificationwould be
amenable to endogenous targets, we synthesized and investigated
the effect that our triazole backbone-modified siRNA would have
on reducing the expression of glyceraldehyde-3-phosphate dehy-
drogenase (GAPDH). We synthesized three siRNAs bearing a UtU tri-
azole modification (siRNAs 24–26). The siRNAs each contained a
single UtU modification placed either at positions 17–18, 18–19, or
at the 3’-UtU overhang of the sense strand (Table 2). The data from
Figure 3 illustrates that our triazole-linked siRNAs show a dose-
dependent downregulation of GAPDH at 10 and 20 nM comparable
to wild-type (siRNA 23). This result is significant because it high-
lights that our novel chemically modified siRNAs are capable of
silencing relevant endogenous targets.
In conclusion, we are reporting the practical synthesis of siRNAs
bearing triazoles interspersed throughout the backbone via DMT-
phosphoramidite chemistry. Monomers 3 and 5 were readily syn-
thesized through standard amide-bond coupling conditions. Via
click chemistry, the final triazole-linked CtU dimer phosphorami-
dite 8 was synthesized with an overall yield of 33% from monomers
3 and 5. All RNA duplexes are substrates for the RNAi pathway.
Therefore, a triazole in place of a phosphodiester backbone shows
compatibility within siRNA duplexes and suggests that other novel
backbone modifications may also act as proper substrates. To the
best of our knowledge, there are no other reports of nonionic
hydrophobic backbone mimics that silence gene expression when
positioned within the internal Watson–Crick double-stranded re-
gion of siRNAs.
4. (a) Dowler, T.; Bergeron, D.; Tedeschi, A.-L.; Paquet, L.; Ferrari, N.; Damha, M. J.
Nucleic Acids Res. 2006, 34, 1669; (b) Braasch, D. A.; Jensen, S.; Liu, Y.; Kaur, K.;
Arar, K.; White, M. A.; Corey, D. R. Biochemistry 2003, 42, 7967; (c) Saneyoshi,
H.; Seio, K.; Sekine, M. J. Org. Chem. 2005, 70, 10453; (d) Chiu, Y. L.; Rana, T. M.
RNA 2003, 9, 1034; (e) Elmén, J.; Thornberg, 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; (f) Ueno, Y.; Hirai, M.; Yoshikawa, K.;
Kitamura, Y.; Hirata, Y.; Kiuchi, K.; Kitade, Y. Tetrahedron 2008, 64, 11328; (g)
Manoharan, M.; Akinc, A.; Pandey, R. K.; Qin, J.; Hadwiger, P.; John, M.; Mills, K.;
Charisse, K.; Maier, M. A.; Nechev, L.; Greene, E. M.; Pallan, P. S.; Rozners, E.;
Rajeev, K. G.; Egli, M. Angew. Chem. Int. Ed. 2011, 50, 2284; (h) Zhang, N.; Tan,
C.; Cai, P.; Zhang, P.; Zhao, Y.; Jiang, Y. Bioorg. Med. Chem. 2009, 17, 2441.
5. (a) Somoza, A.; Silverman, A. P.; Miller, R. M.; Chelliserrykattil, J.; Kool, E. T.
Chemistry 2008, 14, 7978; (b) Xia, J.; Noronha, A.; Toudjarska, I.; Li, F.; Akinc, A.;
Braich, R.; Frank-Kamenetsky, M.; Rajeev, K. G.; Egli, M.; Manoharan, M. ACS
Chem. Biol. 2006, 1, 176.
6. Amarzguioui, M.; Holen, T.; Babaie, E.; Prydz, H. Nucleic Acids Res. 2003, 31, 589.
7. Hall, A. H. S.; Wan, J.; Shaughnessy, E. E.; Shaw, B. R.; Alexander, K. A. Nucleic
Acids Res. 2004, 32, 5991.
8. Iwase, R.; Toyama, T.; Nishimori, K. Nucleosides Nucleotides Nucleic Acids 2007,
26, 1451.
9. Potenza, N.; Moggio, L.; Milano, G.; Salvatore, V.; Di Blasio, B.; Russo, A.;
Messere, A. Int. J. Mol. Sci. 2008, 9, 299.
10. Wang, Y.; Juranek, S.; Li, H.; Sheng, G.; Tuschl, T.; Patel, D. J. Nature 2008, 456,
921.
11. Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew. Chem. Int. Ed.
2002, 41, 2596.
12. (a) Isobe, H.; Fujino, T.; Yamazaki, N.; Guillot-Nieckowski, M.; Nakamura, E.
Org. Lett. 2008, 10, 3729; (b) Lucas, R.; Neto, V.; Bouazza, A. H.; Zerrouki, R.;
Granet, R.; Krausz, P.; Champavier, Y. Tetrahedron Lett. 2008, 49, 1004; (c)
Fujino, T.; Yamazaki, N.; Isobe, H. Tetrahedron Lett. 2009, 50, 4101; (d) Chittepu,
P.; Sirivolu, V. R.; Seela, F. Bioorg. Med. Chem. 2008, 16, 8427; (e)
Chandrasekhar, S.; Srihari, P.; Nagesh, C.; Kiranmai, N.; Nagesh, N.; Idris, M.
M. Synthesis 2010, 21, 3710; (f) El-Sagheer, A. H.; Brown, T. J. Am. Chem. Soc.
2009, 131, 3958; (g) Chouikhi, D.; Barluenga, S.; Winssinger, N. Chem. Commun.
2010, 46, 5476; (h) Varizhuk, A.; Chizhov, A.; Florentiev, V. Bioorg. Chem. 2011,
39, 127.
13. El-Sagheer, A. H.; Brown, T. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 15329.
14. (a) Parades, E.; Das, S. R. Chembiochem 2011, 12, 125; (b) Mutisya, D.; Selvam,
C.; Kennedy, S. D.; Rozners, E. Bioorg. Med. Chem. Lett. 2011, 21, 3420.
15. Efthymiou, T. C.; Desaulniers, J.-P. J. Heterocycl. Chem. 2011, 48, 533.
16. Vader, J.; Sengers, H.; de Groot, A. Tetrahedron 1989, 45, 2131.
17. (a) Christensen, L.; Hansen, H. F.; Koch, T.; Nielsen, P. E. Nucleic Acids Res. 1998,
26, 2735; (b) Schwergold, C.; Depecker, G.; Di Giorgio, C.; Patino, N.; Jossinet, F.;
Ehresmann, B.; Terreux, R.; Cabrol-Bass, D.; Condom, R. Tetrahedron 2002, 58,
5675.
Acknowledgments
We acknowledge the National Sciences and Engineering Re-
search Council and the Canada Foundation for Innovation for
funding.
Supplementary data
18. Liu, X. J.; Chen, R. Y.; Weng, L. H.; Leng, X. B. Heteroatom Chem. 2000, 11, 422.
19. Petersheim, M.; Turner, D. H. Biochemistry 1983, 22, 256.
20. Bajor, Z.; Sági, G.; Tegyey, Z.; Kraicsovits, F. Nucleosides Nucleotides Nucleic Acids
2003, 22, 1963.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.12.104.
21. (a) Schwarz, D. S.; Hutvágner, G.; Du, T.; Xu, Z.; Aronin, N.; Zamore, P. D. Cell
2003, 115, 199; (b) Addepalli, H.; Meena; Peng, C. G.; Wang, G.; Fan, Y.;
Charisse, K.; Jayaprakash, K. N.; Rajeev, K. G.; Pandey, R. K.; Lavine, G.; Zhang,
L.; Jahn-Hofmann, K.; Hadwiger, P.; Manoharan, M.; Maier, M. A. Nucleic Acids
Res. 2010, 38, 7320.
References and notes
1. Fire, A.; Xu, S. Q.; Montgomery, M. K.; Kostas, S. A.; Driver, S. E.; Mello, C. C.
Nature 1998, 391, 806.
2. (a) Hammond, S. M.; Bernstein, E.; Beach, D.; Hannon, G. J. Nature 2000, 404,
293; (b) Nykänen, A.; Haley, B.; Zamore, P. D. Cell 2001, 107, 309.
22. Somoza, A.; Terrazas, M.; Eritja, R. Chem. Commun. 2010, 46, 4270.