Solution‐Phase Synthesis of Di‐ and Trinucleotides Using Polymer‐Supported Reagents

François Morvan1, Jean‐Jacques Vasseur1

1 Université Montpellier II, Montpellier
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 3.14
DOI:  10.1002/0471142700.nc0314s26
Online Posting Date:  October, 2006
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Abstract

A solution‐phase phosphoramidite synthesis of short oligonucleotides using solid‐supported reagents is described. Polyvinyl pyridinium tosylate is employed as the activator in the coupling step between nucleoside‐3′‐O‐phosphoramidites and 5′‐OH nucleosides. The resulting dinucleotide phosphite triesters are oxidized or sulfurized with polymer‐bound periodate or tetrathionate to phosphotriesters or thiono phosphotriesters, respectively. Removal of the 3′‐O‐levulinyl protection is accomplished with a polymer‐supported hydrazine. In this approach, the excess reagents are removed by filtration, avoiding complicated purifications. All the polymer‐bound reagents used in this method result from salt bridge interactions with the polymers. Consequently, the resins are regenerable.

Keywords: Solution‐phase; polymer‐bound reagents; phosphoramidite; supported‐pyridinium tosylate; supported‐periodate; supported‐tetrathionate; hydrazinium‐supported

     
 
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Table of Contents

  • Basic Protocol 1: Synthesis of 5′‐OH‐3′‐O‐Levulinyl Dinucleoside Cyanoethylphosphotriesters
  • Alternate Protocol 1: Synthesis of 5′‐OH‐3′‐O‐Levulinyl Dinucleoside Cyanoethylthionophosphotriesters
  • Alternate Protocol 2: Synthesis of 3′‐OH‐5′‐O‐Dimethoxytrityl Dinucleoside Cyanoethylthionophosphotriesters
  • Alternate Protocol 3: Alternate Coupling Protocol When Using 3′‐O‐Levulinyl‐N4‐Benzoyl 2′‐Deoxycytidine
  • Support Protocol 1: Preparation of Polymer‐Supported Reagents
  • Support Protocol 2: Regeneration of Coupling Resin
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Synthesis of 5′‐OH‐3′‐O‐Levulinyl Dinucleoside Cyanoethylphosphotriesters

  Materials
  • 3′‐O‐Levulinyl nucleosides ( S.4; unit 2.11; Samchully Pharmaceuticals; Reese et al., ):
    • 3′‐O‐Levulinyl‐N6‐benzoyl‐2′‐deoxyadenosine
    • 3′‐O‐Levulinyl‐N4‐benzoyl‐2′‐deoxycytidine
    • 3′‐O‐Levulinyl‐N2‐isobutyryl‐2′‐deoxyguanosine
    • 3′‐O‐Levulinyl‐thymidine
  • Standard commercial 3′‐phosphoramidites ( S.3; e.g., Pierce, Cruachem):
    • 5′‐O‐Dimethoxytrityl‐3′‐O‐[(2‐cyanoethyl)‐N,N‐diisopropylphosphoramidite]‐N6‐benzoyl‐2′‐deoxyadenosine
    • 5′‐O‐Dimethoxytrityl‐3′‐O‐[(2‐cyanoethyl)‐N,N‐diisopropylphosphoramidite]‐N4‐benzoyl‐2′‐deoxycytidine
    • 5′‐O‐Dimethoxytrityl‐3′‐O‐[(2‐cyanoethyl)‐N,N‐diisopropylphosphoramidite]‐N2‐isobutyryl‐2′‐deoxyguanosine
    • 5′‐O‐Dimethoxytrityl‐3′‐O‐[(2‐cyanoethyl)‐N,N‐diisopropylphosphoramidite]‐ thymidine
  • Acetonitrile (<10 ppm H 2O)
  • Dichloromethane (<0.005% H 2O)
  • Poly(4‐vinylpyridinium‐p‐toluenesulfonate) resin ( S.2; Aldrich; dry 10 hr at 80°C under 30 mbar vacuum over P 2O 5; store in desiccator at room temperature up to several months under 10 mbar vacuum over P 2O 5)
  • Argon source
  • 50 mM tetraethylammonium acetate (TEAA), pH 7.5
  • Amberlyst A‐26 ion‐exchange resin, periodate form ( S.7; see protocol 5)
  • Methanol
  • p‐Toluenesulfonic acid
  • Saturated aqueous NaHCO 3 solution
  • 0.2 M sodium bisulfite/0.5 M sodium chloride
  • Na 2SO 4, anhydrous
  • Diethyl ether
  • P 2O 5
  • Shaker (Edmund Buhler SM‐30 or equivalent)
  • HPLC column: Nucleosil C18, 5 µm, 100 Å, 150 × 4.6 mm
  • Fritted‐glass funnel, 10 cm diameter, 15 cm height
  • Rotary evaporator
  • 2‐L separatory funnel
  • Vacuum desiccator
  • Additional reagents and equipment for HPLC (unit 10.5), thin‐layer chromatography (TLC; appendix 3D), and MALDI‐TOF mass spectrometry (unit 10.1)

Alternate Protocol 1: Synthesis of 5′‐OH‐3′‐O‐Levulinyl Dinucleoside Cyanoethylthionophosphotriesters

  • Amberlyst A‐26 ion‐exchange resin, tetrathionate form ( S.10; see protocol 5)
  • 2.5% (w/v) NaHCO 3
  • 2‐L separatory funnel

Alternate Protocol 2: Synthesis of 3′‐OH‐5′‐O‐Dimethoxytrityl Dinucleoside Cyanoethylthionophosphotriesters

  • Amberlyst A‐26 ion‐exchange resin, tetrathionate form ( S.10; see protocol 5)
  • Pyridine
  • Silica gel
  • Acetic acid
  • Amberlyst A‐15 ion‐exchange resin, hydrazinium form ( S.16; see protocol 5)
  • Fritted‐glass column, 5 cm diameter, 60 cm height

Alternate Protocol 3: Alternate Coupling Protocol When Using 3′‐O‐Levulinyl‐N4‐Benzoyl 2′‐Deoxycytidine

  • Dimethylformamide (DMF), anhydrous
  • 4A molecular sieves

Support Protocol 1: Preparation of Polymer‐Supported Reagents

  Materials
  • Amberlyst A‐26 ion‐exchange resin, hydroxide (OH) form (Aldrich)
  • Methanol
  • Dichloromethane
  • Sodium periodate
  • P 2O 5
  • Potassium tetrathionate (Aldrich)
  • Amberlyst A‐15 ion‐exchange resin, H+ form (Aldrich)
  • 2 M HCl
  • Hydrazine monohydrate
  • Absolute ethanol
  • Fritted‐glass filter funnel
  • 4‐L Erlenmeyer flask
  • Shaker
  • Vacuum desiccator

Support Protocol 2: Regeneration of Coupling Resin

  Materials
  • Used coupling resin S.2 [poly(4‐vinylpyridinium‐p‐toluenesulfonate) resin]
  • Acetonitrile
  • Dichloromethane
  • Methanol
  • p‐Toluenesulfonic acid
  • P 2O 5
  • 2‐L Erlenmeyer flask
  • Fritted‐glass funnel
  • Platform shaker
  • Vacuum desiccator
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Figures

Videos

Literature Cited

Literature Cited
   Beier, M. and Pfleiderer, W. 1999. Pyridinium salts: An effective class of catalysts for oligonucleotide synthesis. Helv. Chim. Acta 82:879‐887.
   Drewry, D.H., Coe, D.M., and Poon, S. 1999. Solid‐supported reagents in organic synthesis. Med. Res. Rev. 19:97‐148.
   Dueymes, C., Schönberger, A., Adamo, I., Navarro, A.E., Meyer, A., Lange, M., Imbach, J.L., Link, F., Morvan, F., and Vasseur, J.J. 2005. High‐yield solution‐phase synthesis of di‐ and trinucleotide blocks assisted by polymer‐supported reagents. Org. Lett. 7:3485‐3488.
   Efimov, V.A., Kalinkina, A.L., Chakhmakhcheva, O.G., Hill, T.S., and Jayaraman, K. 1995. New efficient sulfurizing reagents for the preparation of oligodeoxyribonucleotides phosphorothioate analogs. Nucl. Acids Res. 23:4029‐4033.
   Eleuteri, A., Capaldi, D.C., Krotz, A.H., Cole, D.L., and Ravikumar, V.T. 2000. Pyridinium trifluoroacetate/N‐methylimidazole as an efficient activator for oligonucleotide synthesis via the phosphoramidite method. Org. Proc. Res. Dev. 4:182‐189.
   Harrison, C.R. and Hodge, P. 1982. Polymer‐supported periodate and iodate as oxidizing agents. J. Chem. Soc. Perkin Trans. 1 509‐511.
   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.
   Iyer, R.P., Jiang, Z., Yu, D., Tan, W., and Agrawal, S. 1995. Improved procedure for the detritylation of DMT‐oligonucleotides: Use of Dowex. Synth. Comm. 25:3611‐3623.
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   Patil, S.V., Mane, R.B., and Salunkhe, M.M. 1994. A facile method for detritylation of 5′‐O‐dimethoxytrityl‐3′‐O‐tert‐butyldimethylsilyl‐2′‐deoxynucleosides. Synth. Comm. 24:2423‐2428.
   Reese, C.B., Song, Q.L., Rao, M.V., and Beckett, I. 1998. Preparation of an octadeoxyribonucleoside heptaphosphorothioate by the phosphotriester approach in solution. Nucleosides Nucleotides Nucl. Acids 17:451‐470.
   Rubinstein, M. and Patchornik, A. 1972. Polymers as chemical reagents: The use of poly(3,5 diethylstyrene)‐sulfonyl chloride for the synthesis of internucleotide bonds. Tetrahedron Lett. 13:2881‐2884.
   Rubinstein, M. and Patchornik, A. 1975. Poly(3,5‐diethylstyrene) sulfonyl chloride: A new reagent for internucleotide bond synthesis. Tetrahedron 31:1517‐1519.
   Sanghvi, Y.S., Guo, Z., Pfundheller, H.M., and Converso, A. 2000. Improved process for the preparation of nucleosidic phosphoramidites using a safer and cheaper activator. Org. Proc. Res. Dev. 4:175‐181.
   Sherrington, D.C. 2001. Polymer‐supported reagents, catalysts, and sorbents: Evolution and exploitation–A personalized view. J. Polym. Sci.: Polym. Chem. 39:2364‐2377.
   Tang, J.Y., Han, Y.X., Tang, J.X., and Zhang, Z.D. 2000. Large‐scale synthesis of oligonucleotide phosphorothioates using 3‐amino‐1,2,4‐dithiazole‐5‐thione as an efficient sulfur‐transfer reagent. Org. Proc. Res. Dev. 4:194‐198.
   van Boom, J.H. and Burgers, P.M.J. 1976. Use of levulinic acid in the protection of oligonucleotides via the modified phosphotriester method: Synthesis of decaribonucleotide U‐A‐U‐A‐U‐A‐U‐A‐A‐A. Tetrahedron Lett. 52:4875‐7878.
   Zhang, Z., Han, Y., Tang, J.X., and Tang, J.Y. 2002. A novel polymer‐supported sulfur‐transfer reagent for the synthesis of phosphorothioates. Tetrahedron Lett. 43:4347‐4349.
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