ACKNOWLEDGEMENTS This research was supported in part by an appointment to the Postgraduate Research Participation Program at the Center for Drug Evaluation and Research administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the US Department of Energy and the US Food and Drug Administration. Funding to pay the Open Access publication charges for this article was provided by the US Food and Drug Administration. Conflict of interest statement. None declared. REFERENCES 1. Chmielewski,M.K., Marcha´n,V., Cies´lak,J., Grajkowski,A., Livengood,V., Mu¨nch,U., Wilk,A. and Beaucage,S.L. (2003) Thermolytic carbonates for potential 50-hydroxyl protection of deoxyribonucleosides. J. Org. Chem., 68, 10003–10012. 2. Grajkowski,A., Wilk,A., Chmielewski,M.K., Phillips,L.R. and Beaucage,S.L. (2001) The 2-(N-formyl-N-methyl)aminoethyl group as a potential phosphate/thiophosphate protecting group in solid-phase oligodeoxyribonucleotide synthesis. Org. Lett., 3, 1287–1290. 3. Wilk,A., Chmielewski,M.K., Grajkowski,A., Phillips,L.R. and Beaucage,S.L. (2001) The 4-oxopentyl group as a labile phosphate/ thiophosphate protecting group for synthetic oligodeoxyribonucleotides. Tetrahedron Lett., 42, 5635–5639. 4. Wilk,A., Chmielewski,M.K., Grajkowski,A., Phillips,L.R. and Beaucage,S.L. (2002) The 3-(N-tert-butylcarboxamido)-1-propyl group as an attractive phosphate/thiophosphate protecting group for solid-phase oligodeoxyribonucleotide synthesis. J. Org. Chem., 67, 6430–6438. 5. Wilk,A., Chmielewski,M.K., Grajkowski,A., Phillips,L.R. and Beaucage,S.L. (2002) The 3-(N-tert-butylcarboxamido)-1-propyl and 4-oxopentyl groups for phosphate/thiophosphate protection in oligodeoxyribonucleotide synthesis. In Beaucage,S.L., Bergstrom,D.E., Glick,G.D. and Jones,R.A. (eds), Current Protocols in Nucleic Acid Chemistry. John Wiley & Sons, Inc., New York, Vol. I, pp. 3.9.1–3.9.16. 6. Cies´lak,J. and Beaucage,S.L. (2003) Thermolytic properties of 3-(2-pyridyl)-1-propyl and 2-[N-methyl-N-(2-pyridyl)aminoethyl phosphate/thiophosphate protecting groups in solid-phase synthesis of oligodeoxyribonucleotides. J. Org. Chem., 68, 10123–10129. 7. Cies´lak,J., Grajkowski,A., Livengood,V. and Beaucage,S.L. (2004) Thermolytic 4-methylthio-1-butyl group for phosphate/thiophosphate protection in solid-phase synthesis of DNA oligonucleotides. J. Org. Chem., 69, 2509–2515. 8. Grajkowski,A., Cies´lak,J., Chmielewski,M.K., Marcha´n,V., Phillips,L.R., Wilk,A. and Beaucage,S.L. (2003) Conceptual ‘‘Heat-Driven’’ approach to the synthesis of DNA oligonucleotides on microarrays. Ann. N.Y. Acad. Sci., 1002, 1–11. 9. Wilk,A., Srinivasachar,K. and Beaucage,S.L. (1997) N-Trifluoro- acetylamino alcohols as phosphodiester protecting groups in the synthesis of oligodeoxyribonucleotides. J. Org. Chem., 62, 6712–6713. 10. Wilk,A., Grajkowski,A., Phillips,L.R. and Beaucage,S.L. (1999) The 4-[N-methyl-N-(2,2,2-trifluoroacetyl)amino]butyl group as an alternative to the 2-cyanoethyl group for phosphate protection in the synthesis of oligodeoxyribonucleotides. J. Org. Chem., 64, 7515–7522. 11. Wilk,A., Grajkowski,A., Phillips,L.R. and Beaucage,S.L. (2000) Deoxyribonucleoside cyclic N-acylphosphoramidites as a new class of monomers for the stereocontrolled synthesis of oligothymidylyl- and oligocytidylyl-phosphorothioates. J. Am. Chem. Soc., 122, 2149–2156. 12. Wilk,A., Grajkowski,A., Chmielewski,M.K., Phillips,L.R. and Beaucage,S.L. (2001) Deoxyribonucleoside phosphoramidites. In: Beaucage,S.L., Bergstrom,D.E., Glick,G.D. and Jones,R.A. (eds), Current Protocols in Nucleic Acid Chemistry. John Wiley & Sons, Inc., New York, Vol. I, pp. 2.7.1–2.712. 13. Van Houten,K.A., Heath,D.C. and Pilato,R.S. (1998) Rapid luminescent detection of phosphate esters in solution and the gas phase using (dppe)Pt{S2C2(2-pyridyl)(CH2CH2OH)}. J. Am. Chem. Soc., 120, 12359–12360. 14. Guzaev,A.P. and Manoharan,M. (2000) A novel phosphate protection for oligonucleotide synthesis: the 2-[(1-naphthyl)carbamoyloxy]ethyl (NCE) group. Tetrahedron Lett., 41, 5623–5626. Nucleic Acids Research, 2005, Vol. 33, No. 11 3559 15. Guzaev,A.P. and Manoharan,M. (2001) 2-Benzamidoethyl group—A novel type of phosphate protecting group for oligonucleotide synthesis. J. Am. Chem. Soc., 123, 783–793. 16. Zhang,S.-W. and Swager,T.M. (2003) Fluorescent detection of chemical warfare agents: functional group specific ratiometric chemosensors. J. Am. Chem. Soc., 125, 3420–3421. 17. Barber,I., Rayner,B. and Imbach,J.-L. (1995) The prooligonucleotide approach. I: Esterase-mediated reversibility of dithymidine-S-alkyl- phosphorothiolates to dithymidine phosphorothioates. Bioorg. Med. Chem. Lett., 5, 563–568. 18. Tosquellas,G., Alvarez,K., Dell’Aquilla,C., Morvan,F., Vasseur,J.-J., Imbach,J.-L. and Rayner,B. (1998) The pro-oligonucleotide approach: solid phase synthesis and preliminary evaluation of model pro-dodecathymidylates. Nucleic Acids Res., 26, 2069–2074. 19. Bologna,J.-C., Morvan,F. and Imbach,J.-L. (1999) The prooligonucleotide approach: synthesis of mixed phosphotriester and SATE phosphotriester prooligonucleotides using H-phosphonate and phosphoramidite chemistries. Eur. J. Org. Chem., 2353–2358. 20. Bologna,J.-C., Vive`s,E., Imbach,J.-L. and Morvan,F. (2002) Uptake and quantification of intracellular concentration of lipophilic pro-oligonucleotides in HeLa cells. Antisense Nucleic Acid Drug Dev., 12, 33–41. 21. Iyer,R.P., Yu,D. and Agrawal,S. (1994) Stereospecific bio-reversibility of dinucleoside S-alkyl phosphorothiolates to dinucleoside phosphorothioates. Bioorg. Med. Chem. Lett., 4, 2471–2476. 22. Iyer,R.P., Yu,D. and Agrawal,S. (1995) Prodrugs of oligonucleotides— the acyloxyalkyl esters of oligodeoxyribonucleoside phosphorothioates. Bioorg. Chem., 23, 1–21. 23. Iyer,R.P., Yu,D., Devlin,T., Ho,N.-H. and Agrawal,S. (1996) Acyloxyaryl prodrugs of oligonucleoside phosphorothioates. Bioorg. Med. Chem. Lett., 6, 1917–1922. 24. Iyer,R.P., Ho,N.-H., Yu,D. and Agrawal,S. (1997) Bioreversible oligonucleotide conjugates by site-specific derivatization. Bioorg. Med. Chem. Lett., 7, 871–876. 25. Ora,M., Ma¨ki,E., Poija¨rvi,P., Neuvonen,K., Oivanen,M. and Lo¨nnberg,H. (2001) Hydrolytic stability of nucleoside phosphotriesters derived from bis(hydroxymethyl)-1,3-dicarbonyl compounds and their congeners: toward a novel pro-drug strategy for antisense oligonucleotides. J. Chem. Soc. Perkin Trans. 2, 881–885. 26. Poija¨rvi,P., Ma¨ki,E., Tomperi,J., Ora,M., Oivanen,M. and Lo¨nnberg,H. (2002) Towards nucleotide prodrugs derived from 2,2-bis(hydroxymethyl)malonate and its congeners: hydrolytic cleavage of 2-cyano-2-(hydroxymethyl)-3-methoxy-3-oxopropyl and 3-(alkylamino)-2-cyano-2-(hydroxymethyl)-3-oxopropyl protections from the internucleosidic phosphodiester and phosphorothioate linkages. Helv. Chim. Acta, 85, 1869–1876. 27. Krieg,A.M. (2002) CpG motifs in bacterial DNA and their immune effects. Annu. Rev. Immunol., 20, 709–760. 28. Hemmi,H., Takeuchi,O., Kawai,T., Kaisho,T., Sato,S., Sanjo,H., Matsumoto,M., Hoshino,K., Wagner,H., Takeda,K. and Akira,S. (2000) A Toll-like receptor recognizes bacterial DNA. Nature, 408, 740–745. 29. Klinman,D.M., Takeshita,F., Gursel,I., Leifer,C., Ishii,K.J., Verthelyi,D. and Gursel,M. (2002) CpG DNA: recognition by and activation of monocytes. Microbes Infect., 4, 897–901. 30. Klinman,D.M. (2004) Immunotherapeutic uses of CpG oligodeoxynucleotides. Nature Rev. Immunol., 4, 249–259. 31. Klinman,D.M., Verthelyi,D., Takeshita,F. and Ishii,K.J. (1999) Immune recognition of foreign DNA: a cure for bioterrorism? Immunity, 11, 123–129. 32. Agrawal,S. and Kandimalla,E.R. (2004) Antisense and siRNA as agonists to Toll-like receptors. Nat. Biotechnol., 22, 1533–1537. 33. Iyer,R.P., Phillips,L.R., Egan,W., Regan,J.B. and Beaucage,S.L. (1990) The automated synthesis of sulfur-containing oligodeoxyribonucleotides using 3H-1,2-Benzodithiol-3-one-1,1-dioxide as a sulfur-transfer reagent. J. Org. Chem., 55, 4693–4699. 34. Regan,J.B., Phillips,L.R. and Beaucage,S.L. (1992) Large-scale Preparation of the Sulfur-Transfer Reagent 3H-1,2-benzodithiol-3-one 1,1-dioxide. Org. Prep. Proc. Int., 24, 488–492. 35. Boal,J.H., Wilk,A., Harindranath,N., Max,E.E., Kempe,T. and Beaucage,S.L. (1996) Cleavage of oligodeoxyribonucleotides from controlled-pore glass supports and their rapid deprotection by gaseous amines. Nucleic Acids Res., 24, 3115–3117.