Preparation of 2′‐O‐[(Triisopropylsilyl)oxy]methyl‐protected Ribonucleosides

Stefan Pitsch1, Patrick A. Weiss2

1 Institut de Chimie Organique, EPFL, Lausanne, Switzerland, 2 Xeragon AG, Zürich, Switzerland
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 2.9
DOI:  10.1002/0471142700.nc0209s07
Online Posting Date:  February, 2002
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

The [(triisopropylsilyl)oxy]methyl (TOM) group is a useful protecting group for the 2′‐OH of ribonucleosides to be used for oligoribonucleotide synthesis by the phosphoramidite method. It is completely stable to all reaction conditions applied during assembly and the first deprotection step. It does not interfere with the coupling reaction and leads to very good coupling yields under DNA‐coupling conditions. The final cleavage occurs quantitatively without concomitant destruction of the RNA product. This unit describes the synthesis and characterization of 2′‐O‐TOM‐5′‐O‐dimethoxytrityl‐N‐acetyl ribonucleosides in full detail. The TOM‐group is introduced via a dibutyltin dichloride‐mediated reaction into N‐acetylated, 5′‐O‐dimethoxytritylated ribonucleosides. Support protocols describe the synthesis of N‐acetylated, 5′‐O‐dimethoxytritylated adenosine and guanosine, as well as synthesis of the starting reagent [(triisopropylsilyl)oxy]methyl chloride (TOM‐Cl). Preparation of the phosphoramidites and their use in solid‐phase oligonucleotide synthesis are described elsewhere in the series.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: Introduction of the [(Triisopropylsilyl)Oxy]Methyl Group into N‐Acetylated, 5′‐O‐Dimethoxytritylated Ribonucleosides
  • Support Protocol 1: Preparation of N6‐Acetyl‐5′‐O‐(4,4′‐Dimethoxytrityl)Adenosine
  • Support Protocol 2: Preparation of N2‐Acetyl‐5′‐O‐(4,4′‐Dimethoxytrityl)Guanosine
  • Support Protocol 3: Preparation of [(Triisopropylsilyl)Oxy]Methyl Chloride
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Introduction of the [(Triisopropylsilyl)Oxy]Methyl Group into N‐Acetylated, 5′‐O‐Dimethoxytritylated Ribonucleosides

  Materials
  • Nitrogen source
  • 5′‐O‐Dimethoxytritylated, N‐acetylated ribonucleosides:
  • N6‐Ac‐5′‐O‐DMTr‐adenosine (S.1a; see protocol 2)
  • N2‐Ac‐5′‐O‐DMTr‐guanosine (S.1b; see protocol 3)
  • N4‐Ac‐5′‐O‐DMTr‐cytidine (S.1c; ChemGenes)
  •  5′‐O‐DMTr‐uridine (S.1d; ChemGenes)
  • 1,2‐Dichloroethane (reagent grade)
  • N‐Ethyl‐N,N‐diisopropylamine
  • n‐Dibutyltin dichloride
  • [(Triisopropylsilyl)oxy]methyl chloride (TOM⋅Cl; S.6; see protocol 4)
  • Ethyl acetate (for chromatography, technical grade)
  • Saturated aqueous sodium bicarbonate (NaHCO 3)
  • 9:1 (v/v) ethyl acetate/hexane (for A, C, and U)
  • 19:1 (v/v) dichloromethane/methanol (for G)
  • Anisaldehyde reagent (see recipe)
  • Dichloromethane (CH 2Cl 2) with and without 5% triethylamine (with TEA for G only)
  • Magnesium sulfate
  • Celite
  • Silica gel (230 to 400 mesh)
  • 6:4, 5:5, 4:6, and so on (v/v) hexane/ethyl acetate containing 2% triethylamine (for A, C, and U)
  • Sand
  • Methanol (for G)
  • 95:5:0.1 (v/v/v) dichloromethane/methanol/triethylamine
  • 500‐mL two‐neck flask equipped with a reflux condenser and stir bar
  • Balloon
  • Rubber septum
  • 80°C water bath
  • TLC plates (Merck silica gel 60, 4 × 10–cm)
  • 254‐nm UV lamp
  • 1‐L separatory funnel
  • 1‐L Erlenmeyer flask
  • 6‐cm glass filter
  • Rotary evaporator equipped with a vacuum pump or water aspirator
  • 5‐cm‐diameter chromatography column
  • Additional reagents and equipment for thin‐layer chromatography (TLC; appendix 3D) and column chromatography ( appendix 3E)

Support Protocol 1: Preparation of N6‐Acetyl‐5′‐O‐(4,4′‐Dimethoxytrityl)Adenosine

  Materials
  • Adenosine
  • Pyridine (reagent grade)
  • Nitrogen source
  • Trimethylsilyl chloride (TMS⋅Cl)
  • Acetonitrile (reagent grade), dry
  • Acetyl chloride
  • Dichloromethane, precooled
  • Magnesium sulfate
  • Toluene
  • Acetic acid
  • Dimethoxytrityl chloride (DMTr⋅Cl)
  • Triethylamine
  • Silica gel (230 to 400 mesh)
  • Sand
  • Saturated sodium bicarbonate solution in water
  • 250‐ and 500‐mL two‐neck flasks
  • Pressure‐equilizing dropping funnel
  • Balloon
  • Rubber septum
  • Stir bar
  • 1‐L separatory funnels
  • Rotary evaporator with vacuum pump
  • 1‐L Erlenmeyer flasks
  • 3‐cm glass filter
  • 3‐cm‐diameter chromatography column
  • Additional reagents and equipment for thin‐layer chromatography (TLC, appendix 3D) and column chromatography ( appendix 3E)

Support Protocol 2: Preparation of N2‐Acetyl‐5′‐O‐(4,4′‐Dimethoxytrityl)Guanosine

  • Guanosine
  • Dimethylformamide
  • Acetic anhydride
  • 1 M hydrochloric acid
  • Methanol
  • Tetrahydrofuran
  • 10 M NaOH
  • 250‐mL two‐neck flasks
  • Reflux condenser
  • 135°C bath
  • Distillation apparatus (15 × 2.9–cm Vigreux column equipped with a condenser and water aspirator)
  • 500‐mL flask
  • 4A molecular sieves

Support Protocol 3: Preparation of [(Triisopropylsilyl)Oxy]Methyl Chloride

  Materials
  • Paraformaldehyde
  • Ethanethiol
  • Nitrogen or argon gas
  • 10 M aqueous NaOH
  • Dichloromethane (CH 2Cl 2; reagent grade)
  • Imidazole
  • Triisopropylsilyl chloride (TIPS⋅Cl)
  • Hexane (technical grade)
  • 10% (w/v) NaH 2PO 4
  • Anhydrous magnesium sulfate (MgSO 4)
  • Sulfuryl chloride (SO 2Cl 2)
  • 250‐ and 1000‐mL one‐neck round‐bottom flasks
  • Balloon
  • Oil bath, 40°C
  • 1‐L separatory funnel
  • 3‐cm glass filter
  • Rotary evaporator with a vacuum pump
  • 10‐cm 14.5‐mm Vigreux distillation column
  • Vacuum distillation equipment (head, thermometer, and so on) with high‐vacuum pump
  • 250‐mL two‐neck flask
  • Rubber septum
  • Syringes
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Beaucage, S.L. and Caruthers, M.H. 1996. The chemical synthesis of DNA/RNA. In Bioorganic Chemistry: Nucleic Acids (S.M. Hecht, ed.) pp. 36‐74. Oxford University Press, Oxford.
   Gundersen, L.L., Benneche, T., and Undheim, K.A. 1989. Chloromethoxysilanes as protecting reagents for sterically hindered alcohols. Acta Chem. Scand. 43:706‐709.
   Ogilvie, K.K., Sadana, K.L., Thompson, E.A., Quilliam, M.A., and Westmore, J.B. 1974. The use of silyl protecting groups in protecting the hydroxyl functions of ribonucleosides. Tetrahedron Lett. 15:2861‐2863.
   Pitsch, S. 1997. An efficient synthesis of enantiomeric oligoribonucleotides from D‐glucose. Helv. Chim. Acta 80:2286‐2314.
   Pitsch, S., Weiss, P.A., and Jenny, L. Nov.1999. Ribonucleoside‐derivative and method for preparing the same. US Patent 5,986,084.
   Schwartz, M.E., Breaker, R.R., Asteriadis, G.T., deBear, J.S., and Gough, G.R. 1992. Rapid synthesis of oligoribonucleotides using 2′‐O‐(ortho‐nitrobenzyloxymethyl)‐protected monomers. Bioorg. Med. Chem. Lett. 2:1019‐1024.
   Stutz, A., Höbartner, C., and Pitsch, S. 2000. Synthesis of 3′‐O‐aminoacylated RNA‐fragments with novel, fluoride‐labile base‐protecting groups. Helv. Chim. Acta 83:2477‐2503.
   Wu, X. and Pitsch, S. 1998. Synthesis and pairing properties of oligoribonucleotide analogues containing a metal‐binding site attached toβ‐D‐allofuranosyl cytosine. Nucl. Acids Res. 26:4315‐4323.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library