2′-O-{[2,2-dimethyl-2-(2-nitrophenyl) acetyl] oxy} methyl protecting group for RNA synthesis

Article abstract of DOI:10.1016/j.tetlet.2013.06.001

A new 2′-OH protecting group, {[2,2-dimethyl-2-(2-nitrophenyl)acetyl] oxy} methyl (DAM), has been successfully applied to RNA oligonucleotides synthesis with high coupling efficiency. The 2′-O-DAM method is fully compatible with reagents and conditions employed in automated solid phase synthesis of RNA. Moreover, it can be easily removed via reduction in near-neutral solution.

Full text of DOI:10.1016/j.tetlet.2013.06.001

Tetrahedron Letters 54 (2013) 4281–4284
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
20-O-{[2,2-dimethyl-2-(2-nitrophenyl) acetyl] oxy} methyl protecting
group for RNA synthesis
Ke Chen a,d, Wei Wang b, Dezhong Qu a, Haoting Zhao b, Wei Xiong a, Caijie Luo b, Menghui Yin a,
Biliang Zhang a,c,
⇑
a Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China
b Guangzhou RiboBio Co., Ltd, Guangzhou 510663, China
c The State Key Laboratory of Respiratory, Guangzhou Institute of Respiratory Diseases, Guangzhou 510120, China
d Graduate University of Chinese Academy of Sciences, Beijing 100049, China
a r t i c l e i n f o
a b s t r a c t
A new 20-OH protecting group, {[2,2-dimethyl-2-(2-nitrophenyl)acetyl]oxy} methyl (DAM), has been
successfully applied to RNA oligonucleotides synthesis with high coupling efficiency. The 20-O-DAM
method is fully compatible with reagents and conditions employed in automated solid phase synthesis
of RNA. Moreover, it can be easily removed via reduction in near-neutral solution.
Ó 2013 Elsevier Ltd. All rights reserved.
Article history:
Received 8 March 2013
Revised 24 May 2013
Accepted 3 June 2013
Available online 10 June 2013
Keywords:
Chemical modification
Solid phase synthesis of RNA
20-OH protecting group
RNA oligonucleotides
SiRNA
Following the rapid development of RNA interference (RNAi)
and miRNA in biological and therapeutic fields, the demand for
synthetic RNA to be used as gene-specific therapeutics has dramat-
ically increased.1,2And the methods for chemical synthesis of RNA
have been studied accordingly. The efficiency of RNA synthesis
depends greatly on the protecting groups, especially 20-hydroxyl
protecting groups. Therefore, the design of 20-hydroxyl protecting
groups has become the focus of research in solid-phase RNA
synthesis.3–10
Fluoride-labile tert-butyldimethylsilyl (TBDMS) ether is the
most widely used 20-hydroxyl protecting group in RNA synthesis.
However, there are obvious shortcomings11–13 of 20-O-TBDMS
chemistry: (1) 20-O-TBDMS protecting group has potential to un-
dergo 20–30 isomerization during phosphitylation step of phospho-
ramidite synthesis. (2) High concentration of fluoride anions for
deprotection is unfavourable for purification. (3) Due to long
coupling time and insufficiently high coupling yields, the TBDMS
method gives rise to modest-quality RNA. Many new types of 20-
hydroxyl protecting groups14–16 have been developed and applied
in solid-phase RNA synthesis to resolve these problems. But some
of them still have the limitations, such as low compatibility with
standard 50-O-DMTr protection17 and harsh deprotection condi-
tions, which would cause degradation of RNA.18 To overcome these
limitations, we develop a new 20-hydroxyl protecting strategy,
which is compatible with standard 50-O-DMTr, stable during
ammonia treatment and can be easily removed in near-neutral
solution.
In this research, a 20-O-{[2,2-dimethyl-2-(2-nitrophenyl) acety-
l]oxy}methyl (DAM) protection strategy was introduced to chemi-
cal synthesis of RNA for the first time. Due to the steric bulkiness of
2,2-dimethyl-2-(2-nitrophenyl) acetyl,19 the DAM group is abso-
lutely stable under both basic and acid conditions.20 The inherent
flexibility of formaldehyde acetal linkers could minimize steric
hindrance as well as avoid the migration between 20-O and 30-O.
Noteworthy, DAM group could be easily removed by reducing
agents via a two-step intramolecular cyclization reaction (Scheme
1) in aqueous media at near-neutral condition. This near-neutral
condition could protect RNA from degradation that is caused by
harsh deprotection conditions. All the above indicates the potential
usefulness of the DAM strategy in solid-phase RNA synthesis.
We developed a convenient method to synthesize 20-O-DAM
protected phosphoramidites as shown in Scheme 2. Starting from
1a–d, di-tert-butyldichlorosilane (for 1a, 1c, and 1d)21 or Mark-
iewicz reagent (for 1b)22 selectively blocked the 30-OH and 50-OH
groups. Next, thioacetalization of 20-hydroxyl of nucleotides was
opted via the Pummerer reaction (DMSO, acetic anhydride and
acetic acid). We then prepared desired 20-O-DAM compounds via
⇑
Corresponding author. Tel.: +86 20 32015335; fax: +86 20 32290137.
E-mail address: Zhang_biliang@gibh.org (B. Zhang).
0040-4039/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2013.06.001

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K. Chen et al. / Tetrahedron Letters 54 (2013) 4281–4284
acid and sodium ascorbate, were applied at 60 °C for 6 h. And the
reduction was incomplete when using FeSO4 as reducing agent.
Although the DAM of 4d could be completely removed by treat-
ment of SnCl2 in DMF, we found it is difficult to remove the Tin pre-
cipitation. The best result, in terms of both yield and selectivity,
was obtained by employing a combination of reduced Fe powder
and NH4Cl (Fe–NH4Cl).
To evaluate the feasibility of DAM strategy in RNA synthesis, we
synthesized a 20mer uridine homo-oligomer (U20) and a 21mer
RNA oligoribonucleotide (Table 2) using the DAM method. This
method exhibited high-average coupling efficiencies of 98.1% and
97.7%, respectively. The 20-O-DAM fully protected oligonulceotides
(20-O-DAM-Oligo) were obtained after treatment with ammonia
ethanol. 20-O-DAM-Oligo was finally deprotected by Fe–NH4Cl
treatment and yielded a good amount of crude oligonucleotides.
The mass of oligos was confirmed by LC–MS: 6061.6[M+] and
6682.0[M+Na+], which is consistent with the calculated mass
(6061.4 [M+] and 6681.1 [M+Na+]). We then compared the purity
of the RNA oligmers with commercial 20-O-TBDMS chemistry
RNA by Ion exchange HPLC (Fig. 1). As expected, HPLC profile
showed that oligmer obtained from DAM strategy had higher pur-
ity comparing to the RNA derived from 20-O-TBDMS monomers.
The final products were further characterized by the polyacryl-
amide gel electrophoresis (PAGE) (see Figs. S1 and S2). The results
indicated that the Fe–NH4Cl mediated reduction of the 20-O-DAM
protected oligoribonucleotide as a new strategy could produce
high purity RNA oligonucleotides efficiently.
Scheme 1. Reductive deprotection of 20-O-DAM chemistry
activation of 20-thioacetal with
a slight molar excess (1.1–
1.2 equiv) of reagent Br2.23 Transacetalization was performed via
the addition of potassium 2,2-dimethyl-2-(2-nitrophenyl) acetate
in the presence of a catalytic amount of 18-crown-6. The resulting
products 3a–d were successively desilylated by Et3N-3HF. We
accomplished regioselective 50-O-dimethoxy tritylation of 4a–d
with a slight molar excess (1.2 equiv) of dimethoxytrityl chloride
in pyridine. After performing flash column purification, we ob-
tained 50-O-DMTr-protected 20-acetal (5a–d). Finally, 5a–d were
phosphitylated in anhydrous CH2Cl2 when 2-cyanoethyl N,N-diiso-
propylchloro-phosphoramidite and dry DIEA were employed. After
routine workup, we purified crude compounds (6a–d) with chro-
matography on neutral Al2O3 column and obtained solid powder
products with reasonable yields (see Supplementary data).
To evaluate the deprotection conditions, we treated 20-O-DAM
protected monomer (4d), the substrate, with various reducing
agents (see Table 1). It was shown to be no reduction when reduc-
tive mineral salts, such as Na2S, NaHS, Na2S2O3, NaHSO3, ascorbic
In conclusion, the DAM chemistry has been successfully applied
in the synthesis of RNA oligonucleotides with high yield and purity.
DAM protecting group is compatible with reagents and conditions
Scheme 2. Synthesis of 20-O-DAM protected phosphoramidites

K. Chen et al. / Tetrahedron Letters 54 (2013) 4281–4284
4283
Table 1
Deprotection conditions for removing 20-O-{[2,2-dimethyl-2-(2-nitrophenyl)acetyl]oxy}methyl group of 20-O-{[2,2-dimethyl-2-(2-nitrophenyl)acetyl]oxy} uridine (4d)
Entry
Deprotecting reagentsa
Reaction time (h)
Temperature (°C)
TLC/NMR profiles
1
2
3
4
5
6
7
8
9
10
11
12
13
14
10 equiv ascorbic acid
10 equiv sodium ascorbate
20
20
20
20
20
6
6
6
6
2
50
50
50
50
50
60
60
60
60
50
rt
rt
50 °C
55 °C
No reaction
No reaction
No reaction
40–50%
10 equiv ascorbic acid + 1 equiv FeSO4
10 equiv ascorbic acid+1 equiv FeSO4 + Fe
10 equiv FeSO4
6 equiv Na2S + 6 equiv NH4Cl
6 equiv NaHS + 6 equiv NH4Cl
6 equiv Na2S2O3 + 6 equiv NH4Cl
6 equiv NaHSO3 + 6 equiv NH4Cl
Fe + 6 equiv NH4Cl
70–80%
No reaction
No reaction
No reaction
No reaction
ꢀ100% completion
<10%
Fe + 6 equiv NH4Cl
20
20
8
10 equiv SnCl2 (DMF as solvent)
10 equiv SnCl2 (DMF as solvent)
Fe + 6 equiv NH4Cl in a sodium citrate buffer (pH 6.6)
70–80%
ꢀ100% completion
2
ꢀ100% completion
a
Except for SnCl2, all the other deprotection reactions occurred in 5 ml mixed solutions of CH3OH/H2O (1:1 v/v).
Table 2
Average coupling yield and ESI–MS profile of U20 and 21mer RNA oligoribonucleotide
a
b
c
Entry
RNA strands
Average coupling efficiency (%)
Calculated mass
Observed mass
Isolated yield (%)
1
2
U20
98.1
97.7
6061.4
6681.1[M+Na+]
6061.6
59.0
54.6
50-GGACAUAUGAGA CCUUCAAUU-30
6682.0[M+Na+]
a
b
c
The coupling efficiencies of each synthetic cycle were monitored by tracking the DMTr cation chromophoric reporter group.
ESI–MS was performed on an Oligo HTCS LC–MS system and the ProMass utility was used for deconvolution.
Isolated yield determined by the mount of desalting crude oligos/synthetic scale.
Figure 1. Ion-exchange HPLC of U20, 21mer RNA (50-GGA CAU AUG AGA CCU UCA AUU-30) made by TBDMS and DAM chemistry. (A) HPLC of commercial U20 with TBDMS
chemistry. (B) HPLC of 20-O-DAM U20. (C) HPLC of commercial 21mer RNA with TBDMS chemistry. (D) HPLC of 20-O-DAM 21mer RNA on the DNA Pac PA-200 anion-exchange
column (4.6 mm 250 mm; Dionex); buffer A:10% CH3CN, 10 mM Tris–HCl; buffer B: 1 M NaCl,10 mM Tris–HCl 10% CH3CN; gradient performed on A,B is from 2% to 20% buffer
B in 30 min; flow rate, 1 mL/min. Gradient performed on (C and D) is from 0% to 30% buffer B in 30 min; flow rate, 1 mL/min.
employed in automated solid phase synthesis of RNA as well as 50-
terminal DMTr protecting group. DAM protecting group could be
easily detached from the heterocyclic ring via reduction using re-
duced iron powder and NH4Cl in aqueous media at near-neutral
pH. This condition helps avoiding internucleotide cleavage and
migration of the internucleotide linkages.24 Altogether, our method

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K. Chen et al. / Tetrahedron Letters 54 (2013) 4281–4284
showed that using DAM as a 20-hydroxyl protecting group could
proceed readily and efficiently, which shed light on the potential
usefulness of the DAM protecting group in solid-phase RNA
synthesis.
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