Oligoribonucleotides with 2′‐O‐(tert‐Butyldimethylsilyl) Groups

Laurent Bellon1

1 Ribozyme Pharmaceuticals, Boulder, Colorado
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 3.6
DOI:  10.1002/0471142700.nc0306s01
Online Posting Date:  May, 2001
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Abstract

The chemical synthesis of oligoribonucleotides on solid support is routinely performed via the phosphoramidite method. However, the additional 2-OH function of the ribofuranosyl sugar requires suitable protection during oligoribonucleotide synthesis. This unit describes methods for 2-OH protection using the TBDMS group.

     
 
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Table of Contents

  • Unit Introduction
  • Basic Protocol 1: Automated Oligoribonucleotide Synthesis
  • Basic Protocol 2: Oligoribonucleotide Deprotection with Nh4OH/Ethanol and TBAF
  • Alternate Protocol 1: Oligoribonucleotide Deprotection with Aqueous Methylamine and Triethylamine Trihydrofluoride
  • Alternate Protocol 2: “One-Pot” Oligoribonucleotide Deprotection with Anhydrous Methylamine and Neat Triethylamine Trihydrofluoride
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Automated Oligoribonucleotide Synthesis

 Materials
  • Aminomethyl polystyrene (RNA primer solid support) derivatized with 5¢-O-DMTr-2¢-O-TBDMS-3¢-O-succinyl ribonucleosides (Amersham Pharmacia Biotech)
  • RNA phosphoramidites (Amersham Pharmacia Biotech; diluted on the synthesizer to 0.1 M in acetonitrile, using automated protocols)
  •     5¢-O-DMTr-N6-(phenoxyacetyl)-2¢-O-TBDMS-adenosine-3¢-O-(-cyanoethyl- N,N-diisopropyl) phosphoramidite
  •     5¢-O-DMTr-N2-(isopropylphenoxyacetyl)-2¢-O-TBDMS-guanosine-3¢-O-(- cyanoethyl-N,N-diisopropyl) phosphoramidite
  •     5¢-O-DMTr-N4-(acetyl)-2¢-O-TBDMS-cytidine-3¢-O-(-cyanoethyl-N,N- diisopropyl) phosphoramidite
  •     5¢-O-DMTr-2¢-O-TBDMS-uridine-3¢-O-(-cyanoethyl-N,N-diisopropyl) phosphoramidite.
  • 3% (v/v) TCA in methylene chloride (PE Biosystems)
  • Cap A: 10% (v/v) acetic anhydride/10% (v/v) 2,6-lutidine in THF (PE Biosystems)
  • Cap B: 16% (v/v) 1-methyl imidazole in THF (PE Biosystems)
  • Iodine solution: 16.9 mM I2/49 mM pyridine/9% (v/v) water in THF (PE Biosystems)
  • Synthesis grade acetonitrile (Burdick & Jackson)
  • Activator (prepare in acetonitrile): 0.25 M 5-ethylthio-1H-tetrazole (SET), made from solid (American International Chemical) or 0.5 M 4,5-dicyanoimidazole solution (DCI), made from solid (Proligo) or 0.45 M 1H-tetrazole (TET; Glen Research).
  • Synthesis columns for 0.2-µmol-scale syntheses (PE Biosystems)
  • ABI 394 DNA/RNA synthesizer (PE Biosystems)

Basic Protocol 2: Oligoribonucleotide Deprotection with Nh4OH/Ethanol and TBAF

 Materials
  • Oligoribonucleotide attached to solid support (see Basic Protocol 1)
  • 3:1 (v/v) 29% ammonium hydroxide (Mallinckrodt Baker)/100% ethanol (prepare immediately before use)
  • 3:1:1 (v/v/v) ethanol/acetonitrile/H2O
  • 1.0 M n-tetrabutylammonium fluoride (TBAF) in THF (Aldrich)
  • 50 mM and 2 M triethylammonium bicarbonate (TEAB), pH 7.8 (see recipe)
  • Heating blocks
  • 4-mL glass screw-top vial with Teflon lined lid (Wheaton)
  • 14-mL centrifuge tubes (Falcon)
  • Qiagen-tip 500 column (Qiagen)

Alternate Protocol 1: Oligoribonucleotide Deprotection with Aqueous Methylamine and Triethylamine Trihydrofluoride

 Additional Materials (also see Basic Protocol 2)
  • 40% (w/v) aqueous methylamine (Aldrich)
  • Triethylamine trihydrofluoride/NMP/TEA solution (see recipe)
  • 3 M aqueous sodium acetate (e.g., Fluka)
  • n-butanol
  • 70% aqueous ethanol

Alternate Protocol 2: “One-Pot” Oligoribonucleotide Deprotection with Anhydrous Methylamine and Neat Triethylamine Trihydrofluoride

 Additional Materials (also see Basic Protocol 2)
  • 1:1 (v/v) mixture of 33% ethanolic methylamine and anhydrous DMSO
  • 1.5 M ammonium bicarbonate, pH 7.5 (see recipe)
  • Acetonitrile
  • 1:1:1 (v/v/v) acetonitrile/methanol/H2O
  • RNase-free H2O/DEPC-treated
  • C18 SepPak cartridges (Waters)
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Figures

  •  FigureFigure 3.6.1 Synthesis of oligoribonucleotide with 2¢-O-TBDMS groups on solid-support via the phosphoramidite approach.
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  •  FigureFigure 3.6.2 Deprotection of chemically synthesized oligoribonucleotides with 2¢-O-TBDMS groups.
    View Image
  •  FigureFigure 3.6.3 HPLC chromatograms of a 36-mer oligoribonucleotide (5¢- GUU UUC CCU GAU GAG GCC GAA AGG CCG AAA UUC UCC -3¢) synthesized at the 0.2 µmol scale according to the basic synthesis protocol and deprotected using the “one-pot” deprotection protocol (Alternate Protocol 2). (A) 0.25 M SET, (B) 0.5 M DCI, (C) 0.45M TET. Results obtained using Dionex NucleoPac PA-100 22 × 250–mm column at 50°C. Buffer A (1 mM Tris, 20 mM NaClO4), buffer B (1 mM Tris, 300 mM NaClO4). Gradient 40% B to 70% B in 12 min, flow rate = 1.5 mL/min (see unit 10.5).
    View Image

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Literature Cited

Literature Cited
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