Synthesis of Oligodeoxyribo‐ and Oligoribonucleotides According to the H‐Phosphonate Method

Roger Strömberg1, Jacek Stawinski2

1 Karolinska Institute, Stockholm, 2 Stockholm University, Stockholm
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 3.4
DOI:  10.1002/0471142700.nc0304s19
Online Posting Date:  December, 2004
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Abstract

Oligonucleotides can be synthesized by condensing a protected nucleoside H‐phosphonate monoester with a second nucleoside in the presence of a coupling agent to produce a dinucleoside H‐phosphonate diester. This can then be converted to a dinucleoside phosphate or to a backbone‐modified analog such as a phosphorothioate or phosphoramidite. This unit discusses four alternative methods for synthesizing nucleoside H‐phosphonate monoesters. The methods are efficient and experimentally simple, and use readily available reagents. The unit describes the activation of the monoesters, as well as competing acylation and other potential side reactions.

     
 
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Table of Contents

  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1:

  Materials
  • 1,2‐Dichloroethane (DCE; BDH) over 4‐Å molecular sieves
  • Trifluoroacetic acid (TFA; Fluka)
  • Dichloroacetic acid (DCA; Lancaster, 99%), distilled
  • Acetonitrile (MeCN; Lab‐Scan) over 3‐Å molecular sieves
  • Pyridine (Py; Lab‐Scan, Anhydroscan) over 4‐Å molecular sieves
  • Protected nucleoside 3′‐H‐phosphonate building blocks (triethylammonium salts; see and unit 2.6)
  • Pivaloyl chloride (Pv‐Cl; Acros Organics, 99%), freshly distilled
  • Polystyrene support (PE Applied Biosystems) loaded at 20 to 30 µmol/g with a 5′‐O‐(4,4′‐dimethoxytrityl) (DMTr)– or 5′‐O‐(4‐monomethoxytrityl) (MMTr)–protected nucleoside succinate (or equivalent nucleoside‐loaded solid support)
  • I 2
  • Diethyl ether
  • Concentrated (28% to 32%) aqueous ammonium hydroxide (NH 4OH) or 3:1 (v/v) concentrated NH 4OH/ethanol
  • Triethylamine trihydrofluoride (for RNA synthesis with 2′‐O‐TBDMS protection)
  • n‐Butanol
  • 20 mM sodium acetate buffer, pH 6.5, containing 30% and 10% MeCN
  • LiClO 4
  • 0.1 M triethylammonium acetate (TEAA) buffer, pH 6.5
  • Automated oligonucleotide synthesizer (Gene Assembler, Pharmacia)
  • 5‐mL syringes with Luer lock
  • 1.5‐mL cryovials with screw caps
  • Glass sintered funnel of coarse porosity
  • Speedvac evaporator and a vacuum pump
  • C18 cartridge (Waters Sep‐Pac)
  • Syringe filters (Millex‐GV13 filter, 0.22‐µm, 13 mm) and disposable syringes
  • 4 × 250–mm Dionex NucleoPac PA‐100 column
  • Lyophilizer
  • 4.6 × 150–mm Supelcosil LC‐18 (3 µm) column
  • Additional reagents and equipment for automated oligonucleotide synthesis ( appendix 3C) and purification by ion‐exchange and reversed‐phase HPLC (unit 10.5)
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Figures

Videos

Literature Cited

Literature Cited
   Agrawal, S. and Tang, J.‐Y. 1990. Efficient synthesis of oligoribonucleotide and its phosphorothioate analog using H‐phosphonate approach. Tetrahedron Lett. 31:7541‐7544.
   Almer, H., Stawinski, J., and Strömberg, R. 1996. Solid support synthesis of all Rp‐oligoribonucleoside phosphorothioates. Nucl. Acids Res. 24:3811‐3820.
   Andrus, A., Efcavitch, J.W., McBride, L.J., and Giusti, B. 1988. Novel activating and capping reagent for improved hydrogen‐phosphonate DNA synthesis. Tetrahedron Lett. 29:861‐864.
   Beaucage, S.L. and Caruthers, M.H. 1981. Deoxynucleoside phosphoramidites—a new class of key intermediates for deoxypolynucleotide synthesis. Tetrahedron Lett. 22:1859‐1862.
   Beaucage, S.L. and Iyer, R.P. 1993. The synthesis of modified oligonucleotides by the phosphor‐amidite approach and their applications. Tetrahedron 49:6123‐6194.
   Chaix, C., Molko, D., and Teoule, R. 1989. The use of labile base protecting groups in oligoribonucleotide synthesis. Tetrahedron Lett. 30:71‐74.
   Corby, N.S., Kenner, G.W., and Todd, A.R. 1952. Nucleotides. Part XVI. Ribonucleoside‐5′ phosphites. A new method for the preparation of mixed secondary phosphites. J. Chem. Soc. 3669‐3675.
   Dan'kov, Y.V., Batchikova, N.V., Scaptsova, N.V., Besidsky, E.S., and Azhayev, A.V. 1988. H‐Phosphonate solid‐phase synthesis of oligodeoxyribonucleotides in syringe. Bioorg. Khim. 14:615‐620.
   Efimov, V.A. and Dubey, I.Y. 1990. Modification of the H‐phosphonate oligonucleotide synthesis on polymer support. Bioorg. Khim. 16:211‐218.
   Efimov, V.A., Dubey, I.Y., and Chakhmakhcheva, O.G. 1990. NMR study and improvement of H‐phosphonate oligonucleotide synthesis. Nucleosides Nucleotides 9:473‐477.
   Efimov, V.A., Kalinkina, A.L., and Chakhmakhcheva, O.G. 1993. Dipentafluorophenyl carbonate–a reagent for the synthesis of oligonucleotides and their conjugates. Nucl. Acids Res. 21:5337‐5344.
   Földesi, A., Balgobin, N., and Chattopadhyaya, J. 1989. Synthesis of a “branched” trinucleotide using the H‐phosphonate chemistry. Tetrahedron Lett. 30:881‐884.
   Froehler, B.C. 1986. Deoxynucleoside H‐phosphonate diester intermediates in the synthesis of internucleotide phosphate analogs. Tetrahedron Lett. 27:5575‐5578.
   Froehler, B.C. and Matteucci, M.D. 1986. Nucleoside H‐phosphonates: Valuable intermediates in the synthesis of deoxyoligonucleotides. Tetrahedron Lett. 27:469‐472.
   Froehler, B.C. and Matteucci, M.D. 1987. The use of nucleoside H‐phosphonates in the synthesis of deoxyoligonucleotides. Nucleosides Nucleotides 6:287‐291.
   Froehler, B.C., Ng, P.G., and Matteucci, M.D. 1986. Synthesis of DNA via deoxynucleoside H‐phosphonate intermediates. Nucl. Acids Res. 14:5399‐5407.
   Gaffney, B.L. and Jones, R.A. 1988. Large‐scale oligonucleotide synthesis by the H‐phosphonate method. Tetrahedron Lett. 29:2619‐2622.
   Garegg, P.J., Regberg, T., Stawinski, J., and Strömberg, R. 1985. Formation of internucleotidic bond via phosphonate intermediates. Chem. Scr. 25:280‐282.
   Garegg, P.J., Lindh, I., Regberg, T., Stawinski, J., Strömberg, R., and Henrichson, C. 1986a. Nucleoside H‐phosphonates. III. Chemical synthesis of oligodeoxyribonucleotides by the hydrogenphosphonate approach. Tetrahedron Lett. 27:4051‐4054.
   Garegg, P.J., Lindh, I., Regberg, T., Stawinski, J., Strömberg, R., and Henrichson, C. 1986b. Nucleoside H‐phosphonates. IV. Automated solid phase synthesis of oligoribonucleotides by the hydrogenphosphonate approach. Tetrahedron Lett. 27:4055‐4058.
   Garegg, P.J., Regberg, T., Stawinski, J., and Strömberg, R. 1986c. Nucleoside hydrogenphosphonates in oligonucleotide synthesis. Chem. Scr. 26:59‐62.
   Garegg, P.J., Henrichson, C., Lindh, I., Regberg, T., Stawinski, J., and Strömberg, R. 1987a. Automated solid phase synthesis of DNA and RNA fragments by the hydrogenphosphonate approach. In Biophosphates and Their Analogs—Synthesis, Structure, Metabolism and Activity (K.S. Bruzik and W.J. Stec, eds.) pp. 93‐98. Elsevier/North‐Holland, Amsterdam.
   Garegg, P.J., Regberg, T., Stawinski, J., and Strömberg, R. 1987b. Nucleoside H‐phosphonates. VII. Studies on the oxidation of nucleoside hydrogenphosphonate esters. J. Chem. Soc. Perkin Trans. I:1269‐1273.
   Garegg, P.J., Regberg, T., Stawinski, J., and Strömberg, R. 1987c. Nucleosides H‐phosphonates. V. The mechanism of hydrogenphosphonate diester formation using acyl chlorides as coupling agents in oligonucleotide synthesis by the hydrogenphosphonate approach. Nucleosides Nucleotides 6:655‐ 662.
   Garegg, P.J., Stawinski, J., et al. 1987d. Nucleoside H‐phosphonates. VI. Reaction of nucleoside hydrogenphosphonates with arenesulfonyl chlorides. J. Chem. Soc. Perkin Trans. II:1209‐1214.
   Garegg, P.J., Stawinski, J., and Strömberg, R. 1987e. Nucleoside H‐phosphonates. VIII. activation of hydrogenphosphonate monoesters by chlorophosphates and aryl sulfonyl derivatives. J. Org. Chem. 52:284‐287.
   Gryaznov, S.M. and Potapov, V.K. 1990. New approach in synthesis of natural and modified oligodeoxyribonucleotides by H‐phosphonate method. Bioorg. Khim. 16:1419‐1422.
   Hall, R.H., Todd, A., and Webb, R.F. 1957. Nucleotides. Part XLI. Mixed anhydrides as intermediates in the synthesis of dinucleoside phosphates. J. Chem. Soc. 3291‐3296.
   Hammond, P.R. 1962. A simple preparation of alkyl ammonium phosphonates and some comments on the reaction. J. Chem. Soc. 2521‐2522.
   Hata, T. and Sekine, M. 1974. Silyl phosphites. I. The reaction of silyl phosphites with diphenyl disulphides. Synthesis of S‐phenyl nucleoside phosphorothioates. J. Am. Chem. Soc. 96:7363‐7364.
   Jäger, A., Charubala, R., and Pfleiderer, W. 1987. Synthesis and characterization of deoxy and ribo H‐phosphonate dimers. Nucl. Acids Symp. Ser. 18:197‐200.
   Kung, P.‐P. and Jones, R.A. 1992. H‐ phosphonate DNA synthesis without amino protection. Tetrahedron Lett. 33:5869‐5872.
   Kuyl‐Yeheskiely, E., Spierenberg, M., van den Elst, H., Tromp, M., van der Marel, G.A., and van Boom, J.H. 1986. Reaction of pivaloyl chloride with internucleosidic H‐phosphonate diesters. Recl. Trav. Chim. Pays‐Bas 105:505‐506.
   Matteucci, M.D. and Caruthers, M.H. 1981. Synthesis of deoxyoligonucleotides on a polymer support. J. Am. Chem. Soc. 103:3185‐3191.
   Nylén, P. 1937. Die Kinetik der Verseifung von Dialkylphosphiten. II. Allgemeine Säure‐ und Basekatalyse bei der Hydrolyse von Diäthylphosphit. Svensk Kem. Tidskr. 49:79‐ 96.
   Nylén, P. 1938. Die Säure‐ und Basekatalyse bei der Reaktion zwischen Dialkylphosphiten und Jod. Z. Anorg. Allg. Chem. 235:161‐182.
   Ramzaeva, N., Mittelbach, C., and Seela, F. 1997. 7‐deazaguanine DNA: Oligonucleotides with hydrophobic or cationic side chains. Helv. Chim. Acta 80:1809‐1822.
   Regberg, T., Stawinski, J., and Strömberg, R. 1988. Nucleoside H‐phosphonates. IX. Possible side‐reactions during hydrogenphosphonate diester formation. Nucleosides Nucleotides 7:23‐35.
   Rozners, E., Kumpins, V., Rekis, A., and Bizdena, E. 1988. Solid phase synthesis of oligoribonucleotides by the H‐phosphonate method using 2′‐O‐benzoyl protective group. Bioorg. Khim. 14:1580‐1582.
   Rozners, E., Rekis, A., Kumpins, V., and Bizdena, E. 1990. Synthesis of oligoribonucleotides by the H‐phosphonate method using base‐labile 2′‐O‐protecting groups. II. Some aspects of use of 2′‐O‐benzoyl and anisoyl protecting groups. Bioorg. Khim. 16:1531‐1536.
   Rozners, E., Renhofa, R., Petrova, M., Popelis, J., Kumpins, V., and Bizdena, E. 1992. Synthesis of oligoribonucleotides by the H‐phosphonate approach using base labile 2′‐O‐protecting groups. 5. Recent progress in development of the method. Nucleosides Nucleotides 11:1579‐1593.
   Rozners, E., Westman, E., and Strömberg, R. 1994. Evaluation of 2′‐hydroxyl protection in RNA‐synthesis using the H‐phosphonate approach. Nucl. Acids Res. 22:94‐99.
   Rozners, E., Westman, E., Stawinski, J., Garegg, P.J., Bizdena, E., and Strömberg, R. 1998. Oligoribonucleotide synthesis with H‐phosphonates. An improved procedure with base labile N‐acyl protection and two alternative 2′‐O‐protecting groups. Manuscript in preparation.
   Sakatsume, O., Ohtsuka, M., Takaku, H., and Reese, C.B. 1989. Solid phase synthesis of oligoribonucleotides using the 1‐[2‐chloro‐4‐methyl)phenyl]‐4‐methoxypiperidin‐4‐yl (Ctmp) group for the protection of the 2′‐hydroxy functions and the H‐phosphonate approach. Nucl. Acids Res. 17:3689‐3696.
   Sakatsume, O., Yamane, H., Takaku, H., and Yamamoto, N. 1990. Use of new phosphonylating and coupling agents in the synthesis of oligodeoxyribonucleotides via the H‐phosphonate approach. Nucl. Acids Res. 18:3327‐3331.
   Seela, F. and Wei, C.F. 1997. 7‐Deazaisoguanine quartets: Self‐assembled oligonucleotides lacking the Hoogsteen motif. Chem. Commun. 1869‐1870.
   Seliger, H. and Rösch, R. 1990. Simultaneous synthesis of multiple oligonucleotides using nucleoside H‐phosphonate intermediates. DNA Cell Biol. 9:691‐696.
   Stawinski, J. and Strömberg, R. 1993. H‐phosphonates in oligonucleotide synthesis. Trends Org. Chem. 4:31‐67.
   Stawinski, J., Strömberg, R., Thelin, M., and Westman, E. 1988. Studies on the t‐butyldimethylsilyl group as 2′‐O‐protection in oligoribonucleotide synthesis via the H‐phosphonate approach. Nucl. Acids Res. 16:9285‐9298.
   Stawinski, J., Strömberg, R., and Westman, E. 1991a. Ribonucleoside H‐phosphonates. Pyridine vs quinoline–influence on condensation rate. Nucleosides Nucleotides 10:519‐ 520.
   Stawinski, J., Strömberg, R., and Westman, E. 1991b. Studies on reaction conditions for ribonucleotide synthesis via the H‐phosphonate approach. Nucl. Acids Symp. Ser. 24:228.
   Stein, A., Iversen, P.L., Subasinghe, C., Cohen, J.S., Stec, W.J., and Zon, G. 1990. Preparation of 35S‐labelled polyphosphorothioate oligode‐oxyribonucleotides by use of hydrogenphosphonate chemistry. Anal. Biochem. 188:11‐16.
   Strömberg, R. 1987. Nucleoside H‐phosphonates. Chemical studies directed towards oligonucleotide synthesis. Chem. Commun. Stockholm Univ. 1:1‐54.
   Strömberg, R. and Stawinski, J. 1987. Evaluation of some new condensing reagents for hydrogenphosphonate diester formation. Nucl. Acids Symp. Ser. 18:185‐188.
   Tanaka, T., Tamatsukuri, S., and Ikehara, M. 1987. Solid phase synthesis of oligoribonucleotides using the o‐nitrobenzyl group for 2′‐hydroxyl protection and H‐phosphonate chemistry. Nucl. Acids Res. 15:7235‐7248.
   Vasser, M., Ng, P.G., Jhurani, P., and Bischofberger, N. 1990. Error rates in oligodeoxynucleotides synthesized by the H‐phosphonate method. Nucl. Acids Res. 18:3089.
   Wada, T., Sato, Y., Honda, F., Kawahara, S., and Sekine, M. 1997. Chemical synthesis of oligodeoxyribonucleotides using N‐unprotected H‐phosphonate monomers and carbonium and phosphonium condensing reagents: O‐Selective phosphonylation and condensation. J. Am. Chem. Soc. 119:12710‐12721.
   Westheimer, F.H., Huang, S., and Covitz, F. 1988. Rates and mechanisms of hydrolysis of esters of phosphorous acid. J. Am. Chem. Soc. 110:181‐185.
   Westman, E. and Strömberg, R. 1994. Removal of t‐butyldimethylsilyl protection in RNA‐synthesis. Triethylamine trihydrofluoride (TEA, 3HF) is a more reliable alternative to tetrabutylammonium fluoride (TBAF). Nucl. Acids Res. 22:2430‐2431.
   Westman, E., Stawinski, J., and Strömberg, R. 1993. RNA‐synthesis using H‐phosphonates. Synchronizing 2′‐OH and N‐protection. Collect. Czech. Chem. Commun. 58:236‐237.
   Westman, E., Sigurdsson, S., Stawinski, J., and Strömberg, R. 1994. Improving the H‐phosphonate approach to oligoribonucleotide synthesis. Nucl. Acids Symp. Ser. 31:25.
   Wu, T. and Ogilvie, K.K. 1988. N‐Phenoxyacetylated guanosine and adenosine phosphoramidites in the solid phase synthesis of oligoribonucleotides: synthesis of a ribozyme sequence. Tetrahedron Lett. 29:4249‐4251.
   Wu, T. and Ogilvie, K.K. 1990. A study on the alkylsilyl groups in oligoribonucleotide synthesis. J. Org. Chem. 55:4717‐4724.
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