Selective Phosphate Protection: A Novel Synthesis of Double-Labeled Oligonucleotides Andrei P. Guzaev* and Muthiah Manoharan Department of Medicinal Chemistry, Isis Pharmaceuticals, 2292 Faraday AVenue, Carlsbad, California 92008 aguzaeV@isisph.com Received May 29, 2001 ABSTRACT A novel, selective labeling of oligonucleotides with two different reporter groups is described. The oligonucleotide is synthesized using a stable 2-(4-methoxybenzamido)ethyl protection for a selected internucleosidic thiophosphate (PS) and a labile 2-( N-isopropyl-4-methoxybenzamido)- ethyl for the 3′-terminal PS and internucleosidic phosphates. The latter group and the base protection are removed, and the 3′-terminal PS is labeled. The former protection is then cleaved by a prolonged ammonolysis, and the second reporter is introduced at the internucleosidic PS. Recently, oligonucleotides selectively labeled with two different reporter groups or other modifiers have attained widespread interest. While one of the modifications is a conjugated peptide or other pendant of biological importance, the other often is a reporter group for nonradioactive detection. 1 Alternatively, an oligonucleotide precursor can be labeled with two different reporter groups to allow a two- color detection of a product, the double-labeled oligonucle- otide. If the reporter groups are appropriate donors and acceptors, the labeled oligonucleotides display hybridization- dependent fluorescence. 2 These molecular beacons are widely used for real-time monitoring of hybridization in in vitro 3 and in vivo 4 test systems. The synthesis of double-labeled oligonucleotides is most conveniently performed with the aid of dye-labeled solid supports and phosphoramidites. 5,6 Alternatively, oligonucleo- tides that bear an amino and an activated thiol group at opposite ends are synthesized using a variety of modified phosphoramidites 7,8 and solid supports, 9,10 and the chemo- selective labeling is performed postsynthetically. 11,12 (1) (a) Astriab-Fisher, A.; Sergueev, D. S.; Fisher, M.; Ramsay Shaw, B.; Juliano, R. L. Biochem. Pharmacol. 2000, 60, 83-90. (b) Aubert, Y.; Bourgerie, S.; Meunier, L.; Mayer, R.; Roche, A. C.; Monsigny, M.; Thuong, N. T.; Asseline, U. Nucleic Acids Res. 2000, 28, 818-825. (2) Tan, W.; Fang, X.; Li, J.; Liu, X. Chem.-Eur. J. 2000, 6, 1107- 1111. Tyagi, S.; Bratu, D. P.; Kramer, F. R. Nat. Biotechnol. 1998, 16, 49-58. Tyagi, S.; Kramer, F. R. Nat. Biotechnol. 1996, 14, 303-308. (3) Sokol, D. L.; Zhang, X.; Lu, P.; Gewirtz, A. M. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 11538-11543. (4) Uchiyama, H.; Hirano, K.; Kashiwasake-Jibu, M.; Mullah, B.; Andrus, A.; Taira, K. Nucleic Acids Res. Symp. Ser. 1995, 34, 111-112. Uchiyama, H.; Hirano, K.; Kashiwasake-Jibu, M.; Taira, K. J. Biol. Chem. 1996, 271, 380-384. (5) Guzaev, A.; Salo, H.; Azhayev, A.; Lo ¨nnberg, H. Bioconjugate Chem. 1996, 7, 240-248. (6) (a) Adamczyk, M.; Chan, C. M.; Fino, J. R.; Mattingly, P. G. J. Org. Chem. 2000, 65, 596-601. (b) Mullah, B.; Livak, K. Nucleosides Nucle- otides 1999, 18, 1311-1312. (c) Mullah, B.; Andrus, A. Tetrahedron Lett. 1997, 38, 5751-5754. (7) For review, see: (a) Manoharan, M. In Antisense Research and Applications; Crooke, S. T., Lebleu, B. Eds.; CRC Press: Boca Raton, FL, 1993; pp 303-349. (b) Beaucage, S. L.; Iyer, R. P. Tetrahedron 1993, 49, 1925-1963. (8) (a) Manoharan, M.; Tivel, K. L.; Andrade, L. K.; Cook, P. D. Tetrahedron Lett. 1995, 36, 3647-3650. (b) Guzaev, A.; Hovinen, J.; Azhayev, A.; Lo ¨nnberg, H. Nucleosides Nucleotides 1995, 14, 833-837. (c) Hovinen, J.; Guzaev, A.; Azhayev, A.; Lo ¨nnberg, H. J. Chem. Soc., Perkin Trans. 1 1994, 2745-2749. (d) Manoharan, M.; Tivel, K. L.; Ross, B.; Cook, P. D. Gene 1994, 149, 147-156. (e) Manoharan, M.; Johnson, L. K.; Tivel, K. L.; Springer, R. H.; Cook, P. D. Bioorg. Med. Chem. Lett. 1993, 3, 2765-2770. (f) Manoharan, M.; Guinosso, C. J.; Cook, P. D. Tetrahedron Lett. 1991, 32, 7171-7174. (9) For review, see: Pon, R. T. In Protocols for Oligonucleotides and Analogs; Agrawal, S., Ed.; Humana Press: Totowa, New Jersey, 1993; Chapter 19, pp 465-496. ORGANIC LETTERS 2001 Vol. 3, No. 20 3071-3074 10.1021/ol016187w CCC: $20.00 © 2001 American Chemical Society Published on Web 09/11/2001