Chemical synthesis of bioactive siRNA in solution phase by using 2-(azidomethyl)benzoyl as 3′-hydroxyl group protecting group

Article abstract of DOI:10.1016/j.tetlet.2012.05.027

A protecting group AZMB was introduced to ribonucleosides 3′-hydroxyl group to facilitate solution phase synthesis of siRNA. The protection and cleavage reaction were carried out in mild conditions, that is protection by acyl chloride and cleavage by triphenylphosphine. The synthesized siRNA showed good biological activity to suppress targeted superoxide dismutase gene expression.

Full text of DOI:10.1016/j.tetlet.2012.05.027

Tetrahedron Letters 53 (2012) 3654–3657
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Chemical synthesis of bioactive siRNA in solution phase by using
2-(azidomethyl)benzoyl as 30-hydroxyl group protecting group
⇑
Jinyu Huang, Zhen Xi
Department of Chemical Biology and State Key Laboratory of Elemento-organic Chemistry, Nankai University, Tianjin 300071, China
a r t i c l e i n f o
a b s t r a c t
A protecting group AZMB was introduced to ribonucleosides 30-hydroxyl group to facilitate solution
phase synthesis of siRNA. The protection and cleavage reaction were carried out in mild conditions, that
is protection by acyl chloride and cleavage by triphenylphosphine. The synthesized siRNA showed good
biological activity to suppress targeted superoxide dismutase gene expression.
Ó 2012 Elsevier Ltd. All rights reserved.
Article history:
Received 12 March 2012
Revised 23 April 2012
Accepted 4 May 2012
Available online 11 May 2012
Keywords:
Protecting group
AZMB
Synthesis of RNA
RNA interference
RNA interference (RNAi) has showed great potential that could
be utilized for the treatment of infection,1 cancer,2 and many other
diseases.3 More than a dozen of potential drugs based on RNAi are
on different stages of clinical trials.4 Though current research
mostly focuses on off-target effects5 and drug delivery systems,6
there is still a need for progress in chemical synthesis of siRNA
both in scale and purity for therapeutic applications. The phospho-
ramidite approach7–10 for RNA synthesis, relies on stepwise addi-
tion of phosphoramidite monomers on solid support, has been
very successful in the past years. As an alternative choice, perform-
ing the synthesis procedure in solution phase through ‘block’ con-
densation strategy11,12 is suitable for large scale synthesis of a
specific sequence. And this is likely to happen if a systemic siRNA
drug is to be approved to enter the market.11 Reese and Yan13 have
reported synthesis of the first antisense deoxyoligonucleotide drug
Vitravene in solution phase, through levulinyl (Lev) protected 30-
hydroxyl group in their synthesis of oligodeoxynucleotide trimer
building blocks by H-phosphonate approach.14
is cleaved by hydrazine in pyridine-acetic acid solution, a migra-
tion of TBDMS group from 20 to 30 position may occur.12 Sekine
and co-workers29 reported 2-(azidomethyl)benzoyl (AZMB) as a
novel protecting group in deoxynucleosides. The AZMB group
could be cleaved in neutral condition by mild reducing agent, such
as triphenylphosphine. Azhayev and co-workers30 applied AZMB in
the synthesis of trimer deoxynucleotide phosphoroamidite. Here
we introduced the AZMB group into the synthesis of oligo-RNA
in solution phase for protection of the 30-hydroxyl group. With
the purpose of scale-up synthesis of biological active siRNA, we
synthesized a pair of siRNA targeting human superoxide dismutase
gene for cellular activity assay.
As shown in Scheme 1, 2-(azidomethyl)benzoyl chloride 4 was
prepared from o-methyl methyl benzoate and introduced to the 30-
hydroxyl group position of four types of ribonucleosides. o-Methyl
methyl benzoate was brominated by NBS, and then substituted by
the azido group. After hydrolization and recrystallization, pure
white product
3 was obtained in 71% yield. The 2-(azido-
In the synthesis of oligo-RNA, the additional 20-hydroxyl group
of ribonucleosides brings in much complication, suitable protect-
ing group is pivotal in the synthesis of oligo-RNA. Various kinds
of protecting groups for 20-hydroxyl group in solid phase synthesis
have been reported.15–28 The TBDMS protecting group for 20-hy-
droxyl, combined with the DMTr protecting group for 50-hydroxyl
group is the most common protection strategy, not only for their
convenience and solubility, but also for cost consideration, which
is important especially for scale-up synthesis. When the Lev group
methyl)benzoic acid was characterized by 1H NMR and element
analysis before being chlorinated to 2-(azidomethyl)benzoyl chlo-
ride 4. For neutralization of its strong acidity, 4 was firstly added
into methyl imidazole in dichloromethane solution, resulted in
pH 7–8. Then the nucleoside 5 in dichloromethane was added
dropwise. The reaction was incubated overnight at 0 °C to give 6,
and treated by acid for removing DMTr to give 50-hydroxyl group
free 7 (HO-component) as white foam. The yields for four types
of nucleoside were usually higher than 80%.
The protection strategy with AZMB protecting 30-hydroxyl
group and TBDMS protecting 20-hydroxyl group was tested for
the synthesis of siRNA in solution phase. Moreover, the phospho-
⇑
Corresponding author. Tel./fax: +86 22 23504782.
E-mail address: zhenxi@nankai.edu.cn (Z. Xi).
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2012.05.027

J. Huang, Z. Xi / Tetrahedron Letters 53 (2012) 3654–3657
3655
Scheme 1. Reagents and conditions: (a) NBS (1.1 equiv), (BzO)2 (0.02 equiv), CCl4, reflux, 1.5 h; (b) NaN3 (1.5 equiv), EtOH, overnight; (c) 2 M NaOH–CH3OH (1:1, v:v), rt,
30 min; (d) SOCl2 (2 equiv), reflux, 2 h; (e) AZMBCl (1.8 equiv), MI (2.5 equiv), CH2Cl2, 0 °C, overnight; (f) 2% TsOH (10 equiv), CH2Cl2/CH3OH (7:3, v:v), 0 °C, 10 min.
Scheme 2. The reaction cycle of synthesis, the first cycle conditions: (a) MSNT (2.8 equiv), C5H5N, rt, 2 h; (b) Ph3P (4 equiv), dioxane/H2O (9:1, v:v), rt, overnight; (c) 2% TsOH
(10 equiv), CH2Cl2/CH3OH (7:3, v:v), 0 °C, 10 min; (d) 2-chlorophenyldichlorphospate (1.6 equiv), triazole (4 equiv), C5H5N (8 equiv), CH2Cl2, 0 °C, 2 h; then wash by TEAB
(1 M).
triester approach31,32 which condenses a P-component (triethyl-
ammonium salt of nucleotide phosphate) and a HO-component
(50-hydroxyl group free nucleoside) in ambient temperature with-
out high sensitivity to moisture was our choice. As shown in
Scheme 2, the P-component 8 and HO-component 7 were coupled
by condensation reagent MSNT33 in pyridine at room temperature,
to form a fully-protected dimer 9. 50-O-DMTr of the dimer could be
removed to get a dimer HO-component 10. For cleavage of the 30-
O-AZMB, fully-protected nucleotide was dissolved in 1,4-dioxane/
water (v:v = 9:1), stirred with 4 equiv of triphenylphosphine at
room temperature overnight. The azido group was reduced to ami-
no, and subsequent intramolecular O?N acyl migration generated
isoindolin-1-one, and the 30-hydroxyl group was released. Cleavage
of the 30-O-AZMB of 9 followed by phosphorylation afforded a di-
mer P-component 12.
The newly generated P-component or HO-component could be
further coupled as the reaction cycle displayed by Scheme 2. We
synthesized different 2–4mer building blocks by adding monomers

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J. Huang, Z. Xi / Tetrahedron Letters 53 (2012) 3654–3657
Scheme 3. Construction of the 23mer oligos by building blocks: (A) sense strand; (B) antisense strand.
Table 1
Sequence of synthesized oligo-RNAs and mass identification
Name
Sequence
Calculate (M.W.)
ESI-TOF-MS
Sense
Antisense
50-GGA ACC UCA CAU CAA CGC GCA UU-30
50-UGC GCG UUG AUG UGA GGU UCC UU-30
7290
7335
[Mꢀ4H]4ꢀ1821.2296, [Mꢀ3H]3ꢀ2428.6641
[Mꢀ4H]4ꢀ1832.1865, [Mꢀ3H]3ꢀ2443.6597
with general yields 60–80% per reaction cycle and then constructed
two 23mer oligonucleotides with these building blocks according
to the strategy illustrated by Scheme 3. Synthesis of the longer
building blocks included the same condensation and cleavage reac-
tions as described above. These intermediates were identified by
mass spectra (Table S1).
Though tetramethylguanidinium salt of oxime was commonly
used for the cleavage of the chlorophenol group,34 aqueous ammo-
nia was also tried here to deprotect the synthesized oligo-RNA in a
similar way to solid-phase synthesis.35 Both procedures gave sim-
ilar results, while work-up with ammonia was cleaner.
After further cleavage of all the protecting groups, the crude
product was purified by HPLC. The two single strand oligos were
identified by ESI-TOF-MS (Table 1).
The two RNA strands were annealed to form a double stranded
siRNA, targeting on human superoxide dismutase gene. siQuant36
was used to evaluate the RNAi efficiency. After transfection of
the siRNA with Lipofectamine 2000 into HEK293A cells, luciferase
activity level out of the dual-luciferase report assay was measured
Figure 1. RNA interference activity assay was measured at 1 nM concentration, the
remaining luciferase activity: non-target negative control 85.2%, solid phase
synthesized siRNA siR-I 14.1%, solution phase synthesized siRNA siR-II 15.5%.

J. Huang, Z. Xi / Tetrahedron Letters 53 (2012) 3654–3657
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3657
for the siRNA silencing efficiency. In comparison with its solid-
phase synthesized counterpart, our solution-phase synthesized
siRNA showing the same efficiency in silencing luciferase (Fig. 1).
This implied that the solution-phase synthesis and the reagents
used in the procedures did not impact the biological activity.
Herein, we have tested the usefulness of AZMB as protecting
group for 30-hydroxyl group in oligoribonucleotide synthesis. The
mild and neutral deprotection condition used in such protection
procedure provided satisfactory results. Using phosphotriester ap-
proach, we synthesized a pair of siRNA, and the RNAi activity assay
showed the same efficiency of gene down-regulation as its solid-
phase synthesized counterpart. This approach seems suitable for
scale-up siRNA synthesis, especially for 2–4mer building blocks
that could be synthesized at multikilograms. After cleaving the
AZMB group, the building blocks could either be phosphorylated
for phosphotriester approach or H-phosphonate approach in solu-
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This work was financially supported by the Ministry of Science
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Supplementary data
Supplementary data (experimental procedures, characteriza-
tion data and 1H, 13C NMR, 31P NMR spectra and mass data) asso-
ciated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.tetlet.2012.05.027.
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