Scheme 1. General Synthesis of ON 5′-TPs on Solid Support
enzymatic synthesis of m7G-5′-capped RNAs.1,8,9 It was
recently demonstrated that the immune response triggered
by RNA TPs binding to RIG-I synergizes with gene silencing
mediated by small interfering RNAs (siRNAs).10 This data
emphasizes the therapeutic potential of immunostimulatory
nucleic acids,11 including siRNA TPs. Recent insights into
the nature of the RIG-I substrate were made possible using
synthetic RNA TPs rather than 5′-TP products generated by
in vitro RNA transcription.12-14 There are several advantages
to synthetic RNA TPs over those from in vitro transcriptions,
such as purity, reproducible yield (independent of RNA
sequence), the potential for large-scale synthesis, and the
ability to introduce chemical modifications into the RNA.15
syntheses of NTPs,19,22 we opted for the use of the 5′-H-
phosphonate ON (Hp-ON) as a stable and easily accessible
intermediate for triphosphate synthesis.
The general synthetic route is depicted in Scheme 1. The
solid-supported 5′-OH ON 1 was prepared by standard
automated solid-phase ON synthesis using controlled pore
glass support (CPG) and phosphoramidites.23 Treatment of
1 with a solution of diphenyl phosphite in pyridine, followed
by hydrolysis, afforded the solid-supported Hp-ON 2. The
(7) Allam, R.; Pawar, R. D.; Kulkarni, O. P.; Hornung, V.; Hartmann,
G.; Segerer, S.; Akira, S.; Endres, S.; Anders, H. J. Eur. J. Immunol. 2008,
38, 3487–3498
.
(8) Olsen, D. B.; Benseler, F.; Cole, J. L.; Stahlhut, M. W.; Dempski,
R. E.; Darke, P. L.; Kuo, L. C. J. Biol. Chem. 1996, 271, 7435–7439
(9) Peyrane, F.; Selisko, B.; Decroly, E.; Vasseur, J. J.; Benarroch, D.;
Canard, B.; Alvarez, K. Nucleic Acids Res. 2007, 35, e26
.
Despite the existence of an arsenal of methods for the
synthesis of nucleoside triphosphates (NTPs),16-19 there is
no efficient and universal method for the enzyme-free
chemical synthesis of DNA and RNA TPs. The few
approaches describing DNA and RNA TP synthesis on solid
support1,8,16,20 are dependent on oligonucleotide (ON) length
and/or sequence and require difficult separation procedures
resulting from low conversions and poor yields. These
approaches are also limited to the small-scale production of
ON TPs and have not been demonstrated with chemically
modified ONs. All these reports are based on the use of the
highly reactive classical phosphitylation reagent discovered
in 1989 by Ludwig and Eckstein.21 A versatile and scalable
method for synthesis of ON TPs is needed.
.
(10) Poeck, H.; Besch, R.; Maihoefer, C.; Renn, M.; Tormo, D.;
Morskaya, S. S.; Kirschnek, S.; Gaffal, E.; Landsberg, J.; Hellmuth, J.;
Schmidt, A.; Anz, D.; Bscheider, M.; Schwerd, T.; Berking, C.; Bourquin,
C.; Kalinke, U.; Kremmer, E.; Kato, H.; Akira, S.; Meyers, R.; Hacker, G.;
Neuenhahn, M.; Busch, D.; Ruland, J.; Rothenfusser, S.; Prinz, M.; Hornung,
V.; Endres, S.; Tuting, T.; Hartmann, G. Nat. Med. 2008, 14, 1256–1263.
(11) Barchet, W.; Wimmenauer, V.; Schlee, M.; Hartmann, G. Curr.
Opin. Immunol. 2008, 20, 389–395.
(12) Fujita, T. Immunity 2009, 31, 4–5
.
(13) Schlee, M.; Roth, A.; Hornung, V.; Hagmann, C. A.; Wimmenauer,
V.; Barchet, W.; Coch, C.; Janke, M.; Mihailovic, A.; Wardle, G.; Juranek,
S.; Kato, H.; Kawai, T.; Poeck, H.; Fitzgerald, K. A.; Takeuchi, O.; Akira,
S.; Tuschl, T.; Latz, E.; Ludwig, J.; Hartmann, G. Immunity 2009, 31, 25–
34.
(14) Schmidt, A.; Schwerd, T.; Hamm, W.; Hellmuth, J. C.; Cui, S.;
Wenzel, M.; Hoffmann, F. S.; Michallet, M. C.; Besch, R.; Hopfner, K. P.;
Endres, S.; Rothenfusser, S. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 12067–
12072
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(15) Watts, J. K.; Deleavey, G. F.; Damha, M. J. Drug DiscoV.Today
Here, we report a highly efficient and simple procedure
for the solid-phase synthesis of DNA and RNA TPs of
various lengths, sequences, and chemical modifications that
provides reasonably good yields and satisfactory purity of
the crude products so that difficult chromatography purifica-
tions can be avoided. This method uses stable, inexpensive,
commercially available reagents and is based on robust and
facile chemical reactions that are compatible with the
synthesis of natural or chemically modified DNA or RNA.
Based on several reported approaches for the solution-phase
2008, 13, 842–855.
(16) Burgess, K.; Cook, D. Chem. ReV. 2000, 100, 2047–2059.
(17) Warnecke, S.; Meier, C. J. Org. Chem. 2009, 74, 3024–3030.
(18) Crauste, C.; Pe´rigaud, C.; Peyrottes, S. J. Org. Chem. 2009, 74,
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(19) Sun, Q.; Edathil, J. P.; Wu, R.; Smidansky, E. D.; Cameron, C. E.;
Peterson, B. R. Org. Lett. 2008, 10, 1703–1706.
(20) Lebedev, A. V.; Koukhareva, I. I.; Beck, T.; Vaghefi, M. M.
Nucleos. Nucleot. Nucl. Acids 2001, 20, 1403–1409
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(21) Ludwig, J.; Eckstein, F. J. Org. Chem. 1989, 54, 631–635.
(22) Sekine, M.; Aoyagi, M.; Ushioda, M.; Ohkubo, A.; Seio, K. J. Org.
Chem. 2005, 70, 8400–8408
.
(23) Beaucage, S. L.; Iyer, R. P. Tetrahedron 1992, 48, 2223–2311.
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