Biopolym. Cell. 1998; 14(2):163-172.
Synthesis and use of disulfide-based H-phosphonate reagent for 3'- and/or 5'-oligonucleotide labelling via mercaptoalkyl linker
1Dubey I. Y., 1Fedoryak D. M.
  1. Institute of Bioorganic Chemistry and Petrochemistry, NAS of Ukraine
    1, Murmans'ka Str., Kyiv, Ukraine, 02094


Synthesis of new disulfide-based H-phosphonate reagent for solid-phase oligonucleotide functionalization with mercaptopropyl group at one or two termini is described. Using this single reagent, 3'- and 3', 5'-disulfide-linked oligonucleotides were synthesized', disulfide bonds cleaved with dithiothreitol and generated thiol groups labelled with iodoacetamidofluorescein. Tis oligonucleotides containing one or two fluorescein residues at 3'- and 5'-ends were prepared in high yields.


[1] Uhlmann E, Peyman A. Antisense oligonucleotides: a new therapeutic principle. Chem Rev. 1990;90(4):543–84.
[2] Goodchild J. Conjugates of oligonucleotides and modified oligonucleotides: a review of their synthesis and properties. Bioconjug Chem. 1990;1(3):165-87.
[3] Englisch U, Gauss DH. Chemically modified oligonucleotides as probes and inhibitors. Angew Chem Int Ed Engl. 1991;30(6):613–29.
[4] Thuong NT, H?l?ne C. Sequence-specific recognition and modification of double-helical DNA by oligonucleotides. Angew Chem Int Ed Engl. 1993;32(5):666–90.
[5] Gold L, Polisky B, Uhlenbeck O, Yarus M. Diversity of oligonucleotide functions. Annu Rev Biochem. 1995;64:763-97.
[6] Beaucage SL, Iyer RP. The Functionalization of Oligonucleotides Via Phosphoramidite Derivatives. Tetrahedron. 1993;49(10):1925–63.
[7] Korshun VA, Berlin YuA. Introduction of nonradioactive reporter groups into synthetic oligonucleotides and their detection. Bioorg Khim. 1994, 20 (6):565-616
[8] Oligonucleotides and Analogues: a Practical Approach. Ed. by F. Eckstein. Oxford: IRL press, 1991. 313 p.
[9] Protocols for Oligonucleotide Conjugates. Ed. S. Agrawal. New York: Humana Press, 1994. Vol. 26. 377 p.
[10] Haughland R. P. Handbook of Fluorescent Probes and Research Chemicals. Molecular Probes Inc. Eugene, 1996. 679 p.
[11] Zuckermann R, Corey D, Schultz P. Efficient methods for attachment of thiol specific probes to the 3'-ends of synthetic oligodeoxyribonucleotides. Nucleic Acids Res. 1987;15(13):5305-21.
[12] Bischoff R, Coull JM, Regnier FE. Introduction of 5'-terminal functional groups into synthetic oligonucleotides for selective immobilization. Anal Biochem. 1987;164(2):336-44.
[13] Chu BCF, Orgel LE. Postsynthesis functionalization of oligonucleotides. Protocols for Oligonucleotide Conjugates. Ed. S. Agrawal. New York: Humana Press, 1994. Vol. 26: 145-65.
[14] Pei D, Corey DR, Schultz PG. Site-specific cleavage of duplex DNA by a semisynthetic nuclease via triple-helix formation. Proc Natl Acad Sci U S A. 1990;87(24):9858-62.
[15] Eritja R, Pons A, Escarcellar M, Giralt E, Albericio F. Synthesis of defined peptide-oligonucleotide hybrids containing a nuclear transport signal sequence. Tetrahedron. 1991;47(24):4113–20.
[16] Ghosh SS, Kao PM, McCue AW, Chappelle HL. Use of maleimide-thiol coupling chemistry for efficient syntheses of oligonucleotide-enzyme conjugate hybridization probes. Bioconjug Chem. 1990;1(1):71-6.
[17] Ede NJ, Tregear GW, Haralambidis J. Routine preparation of thiol oligonucleotides: application to the synthesis of oligonucleotide-peptide hybrids. Bioconjug Chem. 1994;5(4):373-8.
[18] Arar K, Aubertin AM, Roche AC, Monsigny M, Mayer R. Synthesis and antiviral activity of peptide-oligonucleotide conjugates prepared by using N alpha-(bromoacetyl)peptides. Bioconjug Chem. 1995;6(5):573-7.
[19] Gupta KC, Sharma P, Sathyanarayana S, Kumar P. A universal solid support for the synthesis of 3?-thiol group containing oligonucleotides. Tetrahedron Lett. 1990;31(17):2471–4.
[20] Oberhauser B, Wagner E. Effective incorporation of 2'-O-methyl-oligoribonucleotides into liposomes and enhanced cell association through modification with thiocholesterol. Nucleic Acids Res. 1992;20(3):533-8.
[21] Kumar P, Bose NK, Gupta KC. A versatile solid phase method for the synthesis of oligonucleotide-3?-phosphates. Tetrahedron Lett. 1991;32(7):967–70.
[22] Bonfils E, Thuong NT. Solid phase synthesis of 5',3'-bifunctional oligodeoxyribonucleotides bearing a masked thiol group at the 3'-end. Tetrahedron Lett. 1991; 32(26): 3053-3056.
[23] Azhayeva E, Azhayev A, Guzaev A, Hovinen J, L?nnberg H. Looped oligonucleotides form stable hybrid complexes with a single-stranded DNA. Nucleic Acids Res. 1995;23(7):1170-6.
[24] Gao H, Yang M, Patel R, Cook AF. Circulation of oligonucleotides by disulfide bridge formation. Nucleic Acids Res. 1995;23(11):2025-9.
[25] Kelley SO, Barton JK, Jackson NM, Hill MG. Electrochemistry of methylene blue bound to a DNA-modified electrode. Bioconjug Chem. 1997;8(1):31-7.
[26] K?ster H, Schramm G. Zum Mechanismus der Nucleosidsynthese mit Polyphosphors?ure-phenylester. Chem Ber . Wiley-Blackwell; 1969;102(11):3868–76.
[27] Fieser L, Fieser M. Reagents for Organic Synthesis. New York: J. Wiley, 1967; 357 p.
[28] Froehler BC, Ng PG, Matteucci MD. Synthesis of DNA via deoxynucleoside H-phosphonate intermediates. Nucleic Acids Res. 1986;14(13):5399-407.
[29] Andrus A, Efcavitch JW, McBride LJ, Giusti B. Novel activating and capping reagents for improved hydrogen-phosphonate DNA synthesis. Tetrahedron Lett. 1988;29(8):861–4.
[30] Handbook of Biochemistry and Molecular Biology. Ed. by G. Fasman. Boca Raton: CRC, 1975. Vol. 1: 175.
[31] Harding JS, Owen LN. Dithiols. Part XIV. The alkaline hydrolysis of acetylated non-vicinal hydroxy-thiols. J Chem Soc. 1954;1536-45.
[32] Danehy JP, Hunter WE. Alkaline decomposition of organic disulfides. II. Alternative pathways as determined by structure. J Org Chem. 1967;32(7):2047–53.
[33] Sproat BS, Beijer B, Rider P, Neuner P. The synthesis of protected 5'-mercapto-2',5'-dideoxyribonucleoside-3'-O-phosphoramidites; uses of 5'-mercapto-oligodeoxyribonucleotides. Nucleic Acids Res. 1987;15(12):4837-48.
[34] Meyer KL, Hanna MM. Synthesis and characterization of a new 5-thiol-protected deoxyuridine phosphoramidite for site-specific modification of DNA. Bioconjug Chem. 1996;7(4):401-12.
[35] Fidanza JA, McLaughlin LW. Use of a thiol tether for the site-specific attachment of reporter groups to DNA. J Org Chem. 1992;57(8):2340–6.
[36] Connolly BA, Rider P. Chemical synthesis of oligonucleotides containing a free sulphydryl group and subsequent attachment of thiol specific probes. Nucleic Acids Res. 1985;13(12):4485-502.
[37] Sinha ND, Cook RM. The preparation and application of functionalised synthetic oligonucleotides: III. Use of H-phosphonate derivatives of protected amino-hexanol and mercapto-propanol or -hexanol. Nucleic Acids Res. 1988;16(6):2659-69.
[38] Sinha ND, Striepeke S. Oligonucleotides with reporter groups attached to the 5'-terminus. Oligonucleotides and Analogues: a Practical Approach. Ed. by F. Eckstein. Oxford: IRL press, 1991: 185-210.
[39] Horn T, Urdea MS. A chemical 5?-phosphorylation of oligodeoxyribonucleotides that can be monitored by trityl cation release. Tetrahedron Lett. 1986;27(39):4705–8.
[40] Dubey IY, Lyapina TV, Fedoryak DM. Study of the H-phosphonate synthesis of oligonucleotides and their derivatives. Advanced school on genes transfer and regulation of their expression in eukaryotes. Toulouse: Univ. P. Sabatier press, 1993.
[41] Theisen P, McCollum C, Andrus A. Fluorescent dye phosphoramidite labelling of oligonucleotides. Nucleic Acids Symp Ser. 1992;(27):99-100.
[42] New direct-labelling CE-phosphoramidites for use in automated DNA synthesis. CLONTECHniques. July 1992: 28.
[43] Stewart AJ, Pichon C, Midoux P et al. Fluorescent labelling of unmodified phosphorothioate oligodeoxynucleotides: synthesis and characterization. New J. Chem. 1997; 21(1):87-98.