Regioselective 2′‐Silylation of Purine Ribonucleosides for Phosphoramidite RNA Synthesis

Barbara L. Gaffney1, Roger A. Jones1

1 Rutgers, The State University of New Jersey, Piscataway, New Jersey
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 2.8
DOI:  10.1002/0471142700.nc0208s06
Online Posting Date:  November, 2001
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Abstract

This unit describes high‐yield procedures for protection of purine ribonucleosides based on a reaction that allows concomitant highly regioselective 2′‐silylation and 3′‐phosphitylation to give, in one step, monomers that are ready for H‐phosphonate synthesis. For preparation of phosphoramidites, the H‐phosphonate monoester is cleaved without silyl migration to give intermediates ready for phosphitylation by standard methods.

     
 
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Table of Contents

  • Basic Protocol 1: Synthesis of 5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐O‐tert‐ Butyldimethylsilyl‐6‐N‐Acyladenosine
  • Basic Protocol 2: Synthesis of 5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐O‐tert‐Butyldimethysilyl‐2‐N‐Acylguanosine
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Synthesis of 5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐O‐tert‐ Butyldimethylsilyl‐6‐N‐Acyladenosine

  Materials
  • Adenosine
  • Pyridine (reagent grade or better)
  • Dimethylformamide dimethyl acetal
  • Nitrogen source
  • Acetonitrile (anhydrous, dried over 3Å molecular sieves)
  • 0.1 M triethylammonium acetate (TEAA)
  • 4,4′‐Dimethoxytrityl chloride (DMTr‐Cl)
  • 5% (v/v) methanol in dichloromethane
  • Dichloromethane
  • Concentrated aqueous ammonium hydroxide
  • N‐Methylmorpholine
  • Trimethylchlorosilane
  • Adenosine phenoxyacetylating reagent (see recipe)
  • Sodium bicarbonate
  • Ethyl acetate
  • Petroleum ether
  • Ammonium phenyl‐H‐phosphonate (see recipe)
  • 1,8‐Diazabicyclo[5.4.0]undec‐7‐ene (DBU)
  • tert‐Butyldimethylsilyl chloride (TBDMS⋅Cl)
  • 0.5 M potassium phosphate buffer, pH 7.0 ( appendix 2A)
  • Glycerol
  • 1‐Adamantanecarbonyl chloride
  • Diisopropylammonium tetrazolide
  • Argon source
  • 2‐Cyanoethyl tetraisopropylphosphorodiamidite
  • Methylene chloride (anhydrous)
  • Triethylamine (anhydrous)
  • 250‐mL and 25‐mL round‐bottom flask
  • Rotary evaporator
  • Silica gel 60F TLC plates (Merck)
  • Waters XTerra 2.5‐µm C18 chromatography column
  • Vacuum pump
  • Septum
  • Vent needle
  • Desiccator with P 2O 5
  • Additional reagents and equipment for TLC ( appendix 3D), column chromatography ( appendix 3E), and HPLC (unit 10.5)

Basic Protocol 2: Synthesis of 5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐O‐tert‐Butyldimethysilyl‐2‐N‐Acylguanosine

  Materials
  • Guanosine
  • Pyridine
  • Nitrogen source
  • Trimethylchlorosilane
  • Guanosine phenoxyacetylating reagent (see recipe)
  • 2‐Propanol
  • Methanol
  • Dichloromethane
  • Acetonitrile (anhydrous, dried over 3Å molecular sieves)
  • 0.1 M triethylammonium acetate (TEAA)
  • Dimethylformamide dimethyl acetal
  • 4,4′‐Dimethoxytrityl chloride (DMTr‐Cl)
  • Methanol
  • Sodium bicarbonate
  • Dichloromethane
  • Ammonium phenyl‐H‐phosphonate (see recipe)
  • 1,8‐Diazabicyclo[5.4.0]undec‐7‐ene (DBU)
  • tert‐Butyldimethylsilyl chloride (TBDMS‐Cl)
  • 0.5 M potassium phosphate buffer, pH 7.0 ( appendix 2A)
  • Glycerol
  • 1‐Adamantanecarbonyl chloride
  • 5:95 to 15:85 (v/v) acetone/dichloromethane (optional; for phosphoramidite synthesis)
  • 250‐mL round‐bottom flasks
  • Magnetic stir bar
  • Rotary evaporator
  • Silica gel 60F TLC plates (Merck)
  • Waters XTerra 2.5‐µm C18 chromatography column
  • Vacuum pump
  • Septum
  • Vent needle
  • Desiccator with P 2O 5
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)
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Figures

Videos

Literature Cited

Literature Cited
   Chaix, C., Duplaa, A.M., Molko, D., and Téoule, R. 1989. Solid phase synthesis of the 5′‐half of the initiator t‐RNA from B. subtilis. Nucl. Acids Res. 17:7381‐7393.
   Hakimelahi, G.H., Proba, Z.A., and Ogilvie, K.K. 1982. New catalysts and procedures for the dimethoxytritylation and selective silylation of ribonucleosides. Can. J. Chem. 60:1106‐1113.
   Ogilvie, K.K., Beaucage, S.L., Schifman, A.L., Theriault, N.Y., and Sadana, K.L. 1978. The synthesis of oligoribonucleotides. II. The use of silyl protecting groups in nucleoside and nucleotide chemistry. VII. Can. J. Chem. 56:2768‐2780.
   Ogilvie, K.K., Schifman, A.L., and Penney, C.L. 1979. The synthesis of oligoribonucleotides. III. The use of silyl protecting groups in nucleoside and nucleotide chemistry. VIII. Can. J. Chem. 57:2230‐2238.
   Singh, K.K., and Nahar, P. 1995. An improved method for the synthesis of N‐phenoxyacetylribonucleosides. Synth. Commun. 25:1997‐2003.
   Sinha, N.D., Davis, P., Schultze, L.M., and Upadhya, K. 1995. A simple method for N‐acylation of adenosine and cytidine nucleosides using carboxylic acids activated in situ with carbonyldiimidazole. Tetrahedron Lett. 36:9277‐9280.
   Song, Q., Wang, W., Fischer, A., Zhang, X., Gaffney, B.L., and Jones, R.A. 1999. High yield protection of purine ribonucleosides for phosphoramidite RNA synthesis. Tetrahedron Lett. 40:4153‐4156.
   Wu, T., Ogilvie, K.K., and Pon, R.T. 1988. N‐Phenoxyacetylated guanosine and adenosine phosphoramidites in the solid phase synthesis of oligoribonucleotides: Synthesis of a ribozyme sequence. Tetrahedron Lett. 29:4249‐4252.
   Zemlicka, J. 1963. Reactions of dimethylformamide acetals with some heterocyclic systems. Collect. Czech. Chem. Commun. 28:1060‐1062.
   Zhang, X., Abad, J.‐L., Huang, Q., Zeng, F., Gaffney, B.L., and Jones, R.A. 1997. High yield protection of purine ribonucleosides for H‐phosphonate RNA synthesis. Tetrahedron Lett. 38:7135‐7138.
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