Liquid-Phase RNA Synthesis
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5.2, 2.8 Hz), 4.10 (1H, dd, J=4.8, 2.0 Hz), 3.98–3.91 (6H, m), 3.91–3.85
(2H, m), 3.85–3.55 (8H, m), 2.67 (4H, s), 2.46–2.35 (2H, m), 1.91 (3H,
s), 1.81–1.68 (6H, m), 1.50–1.38 (6H, m), 1.35–1.20 (84H, m), 0.86 ppm
(9H, t, J=7.0 Hz); 13C NMR (100 MHz, CDCl3): d=172.8, 170.8, 169.9,
163.8, 153.3, 150.5, 139.8, 136.5, 129.7, 111.3, 105.8, 86.0, 85.0, 75.0, 73.6,
69.4, 62.5, 37.1, 31.9, 30.3, 29.7, 29.6, 29.4, 29.3, 29.2, 27.9, 26.1, 22.7, 14.1,
12.6 ppm; IR (KBr): n˜ =2918, 2850, 1699, 1637, 1469, 1430, 1123 cmÀ1
;
HRMS (ESI+): m/z calcd for C79H138N4O11Na: 1342.0260 [M+]; found
1342.0872.
Coupling and oxidation reaction of 5’-DMTr-dT-dT-3’-ASS (7): A solu-
tion of benzylthio-1H-tetrazole/acetonitrile solution (0.25 molLÀ1, 7 mL,
1.75 mmol) was added to a solution of ASS 2a (1.32 g, 1.0 mmol) and
amidite monomer (dT-phosphoramidite, 1.12 g, 1.5 mmol) in dry di-
chloromethane (70 mL) at room temperature. After stirring the resulting
reaction mixture for 20 min, 2-butanone peroxide/dichloromethane solu-
tion (30 mL) was added. After stirring for 10 min, the resulting solution
was diluted with methanol and concentrated in vacuo. The residue was
Figure 4. MALDI-TOF mass spectrum of fully protected 10-mer RNA
oligomer containing ASS.
ed in the generation of defective sequences and reduced
yields. It should be noted that in the liquid-phase synthesis
by using ASS developed here, such side-reactions did not
occur. It was possible to avoid this side reaction by repeated
exposure to multiple reaction and separation steps, which
successfully gave the fully protected RNA oligomers con-
taining ASS. On the other hand, during removal of the pro-
tecting groups from the fully protected RNA oligomers,
some decomposition, such as phosphodiester backbone
cleavage, took place, and this diminished the total yield.
This resulted from the inherent instability of the RNA
oligomer. To overcome this issue in both the solid- and
liquid-phase approaches, the post-processing protocol for
the large-scale RNA synthesis needs to be improved.
diluted with methanol and filtrated to give the product
7 (1.98 g,
1.0 mmol, 99%) as a white solid. M.p. 1328C; 1H NMR (400 MHz,
CDCl3): d=9.36 (1H, m), 7.54 and 7.52 (1H, s), 7.40–7.20 (10H, m), 6.84
(4H, d, J=8.4 Hz), 6.59 (2H, s), 6.47–6.35 (1H, m), 6.31–6.20 (1H, m),
5.41–5.24 (1H, m), 5.23–5.13 (1H, m), 4.48–4.09 (6H, m), 4.00–3.88 (6H,
m), 3.79 (6H, s), 3.75–3.30 (10H, m), 2.80–2.74 (1H, m), 2.74–2.56 (5H,
m), 2.52–2.38 (2H, m), 2.38–2.10 (2H, m), 1.90 (3H, s), 1.84–1.68 (6H,
m), 1.53–1.42 (6H, m), 1.41 (3H, s), 1.38–1.08 (84H, m), 0.88 ppm (9H, t,
J=7.0 Hz); 13C NMR (100 MHz, CDCl3): d=172.7, 170.7, 169.8, 158.8,
153.3, 150.5, 144.0, 139.8, 135.5, 135.4, 135.0, 130.1, 129.8, 128.6, 128.1,
127.3, 116.4, 116.2, 113.4, 111.8, 111.7, 105.8, 87.3, 85.3, 85.1, 84.5, 84.3,
82.3, 79.9, 79.7, 77.3, 73.8, 73.6, 69.3, 67.8, 63.2, 62.5, 62.4, 55.3, 45.2, 42.0,
39.0, 36.7, 31.9, 29.7, 29.4, 26.1, 22.7, 19.6, 14.1, 12.6, 12.5, 11.7 ppm;
31P NMR (162 MHz, CDCl3): d=À2.854; IR (KBr): n˜ =2919, 2850, 1696,
1650, 1467, 1253, 1117, 1031 cmÀ1
;
HRMS (ESI+): m/z calcd for
C113H172N7O20PNa: 2001.2292 [M+]; found 2002.3353.
Cleavage and base deprotection: Sterile nuclease-free water was used in
all steps. The fully protected sequence (5.85 g, 0.46 mmol) was added to
ammonia/water/ethanol solution (28%, 30 mL, 3:1 v/v). The solution was
then warmed to 808C and stirred for 90 min. The reaction solution was
cooled to RT and concentrated in vacuo. NMP, TEA, and TEA-3HF
(65 mL, 6:3:4 v/v/v) were added to the residue. After stirring for 90 min
at 608C, the reaction mixture was cooled to RT. To the solution was
added triethylamine/acetic acid buffer (0.1m, 65 mL, pH 7.4). The full-
length RNA oligomer, which contains a 5’-DMTr group, were retained on
the Sep-Pak Cartridge (C18–2 g) and washed with water (10 mL). After
cleaving the DMTr by TFA (2%, 30 mL) and the reverse-phase resin was
washed with water. The full-length RNA oligomer was eluted with
MeCN solution (20%). The eluate was dried in centrifugal dryer in
vacuo to give the target RNA oligomer (1.72 g, 0.26 mmol).
Conclusion
A liquid-phase RNA synthetic system on a gram scale by
using an alkyl-chain-soluble support was reported. The dis-
persion and aggregation of the support can be effectively
controlled through a relevant solvent change, allowing for
easy separation and high recovery yields. Specifically, the
dispersion property was markedly improved by the structur-
al blocking of the hydrogen-bond interactions of the linker
moiety and by selection of dispersion media, which enabled
enhanced reactivity in the liquid-phase approach. This ap-
proach provides a practical and reliable RNA synthetic
methodology in the development of scaled-up, cost-effective
chemical RNA synthesis. We believe that the applicability
of the soluble support and liquid-phase system to the assem-
bly of RNA oligomers holds great promise for the future de-
velopment of RNA therapeutics.
Acknowledgements
This work was partially supported by a Grant-in-Aid for Scientific Re-
search from the Ministry of Education, Culture, Sports, Science and
Technology, Japan.
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Altman, Nat. Rev. Drug Discov. 2012, 11, 125–140; g) B. A. Sullen-
Experimental Section
General procedure for the liquid-phase reaction by using ASS: Detrityla-
tion reaction of compound 8: ASS 2 (1 g, 0.6 mmol) and DCA/dichloro-
methane solution (5%, 30 mL) was added to dichloromethane (90 mL).
After stirring for 1 min, the reaction mixture was diluted with methanol
and concentrated in vacuo. The residue was diluted, filtrated, and rinsed
with methanol to give ASS 2a as a white solid (0.95 g, 0.6 mmol, 99%).
M.p. 168–1698C; 1H NMR (400 MHz, CDCl3): d=8.32 (1H, m), 7.47
(1H, s), 6.57 (2H, s), 6.20 (1H, dd, J=8.0, 6.0 Hz), 5.36 (1H, ddd, J=5.6,
[2] a) S. L. Beaucage, Curr. Opin. Drug Discov. Dev. 2008, 11, 203–216;
Chem. Eur. J. 2013, 19, 8615 – 8620
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