Efficient Solid-Phase Chemical Synthesis of 5′-Triphosphates of DNA, RNA, and their Analogues Ivan Zlatev, †,‡ Thomas Lavergne, † Franc ¸oise Debart,* ,† Jean-Jacques Vasseur, † Muthiah Manoharan,* ,‡ and Franc ¸ois Morvan* ,† Institut des Biomole ´cules Max Mousseron (IBMM), UMR 5247 CNRS, UniVersite ´ Montpellier 1 - UniVersite ´ Montpellier 2, Place Euge `ne Bataillon, CC1704, 34095 Montpellier Cedex 5, France, and Drug DiscoVery, Alnylam Pharmaceuticals, 300 Third Street, Cambridge, Massachusetts 02142 debart@uniV-montp2.fr; morVan@uniV-montp2.fr; mmanoharan@alnylam.com Received February 23, 2010 ABSTRACT A robust, reproducible, and scalable method for the solid-phase synthesis of 5′-triphosphates of DNA, RNA, and their chemically modified analogues using 5′-H-phosphonate intermediates is described. 5′-Triphosphates of oligonucleotides with varying lengths and sequences containing different 5′-terminal nucleotides, with and without internal sugar-backbone modifications, were efficiently prepared as crude products or further purified by HPLC. DNA and RNA 5′-triphosphates (TPs) serve as important substrates for many biochemical applications. For example, DNA TPs are used in the biotechnology industry 1 as sub- strates for the preparation of synthetic genes. 2 In addition, RNA TPs are used for the ligation of RNA molecules, 3-5 the detection of viral responses via activation of the RIG-I protein, 6 the induction of antiviral immunity, 7 and in the † Universite ´ Montpellier 1 - Universite ´ Montpellier 2. ‡ Alnylam Pharmaceuticals. (1) Brownlee, G. G.; Fodor, E.; Pritlove, D. C.; Gould, K. G.; Dalluge, J. J. Nucleic Acids Res. 1995, 23, 2641–2647. (2) Xiong, A. S.; Peng, R. H.; Zhuang, J.; Gao, F.; Li, Y.; Cheng, Z. M.; Yao, Q. H. FEMS Microbiol. ReV. 2008, 32, 522–540. (3) Joyce, G. E. Angew. Chem., Int. Ed. 2007, 46, 6420–6436. (4) Ekland, E. H.; Szostak, J. W.; Bartel, D. P. Science 1995, 269, 364– 370. (5) Rohatgi, R.; Bartel, D. P.; Szostak, J. W. J. Am. Chem. Soc. 1996, 118, 3340–3344. (6) Hornung, V.; Ellegast, J.; Kim, S.; Brzozka, K.; Jung, A.; Kato, H.; Poeck, H.; Akira, S.; Conzelmann, K. K.; Schlee, M.; Endres, S.; Hartmann, G. Science 2006, 314, 994–997. ORGANIC LETTERS 2010 Vol. 12, No. 10 2190-2193 10.1021/ol1004214  2010 American Chemical Society Published on Web 04/26/2010