Synthetic Strategies and Parameters Involved in the Synthesis of Oligodeoxyribonucleotides According to the Phosphoramidite Method

Serge L. Beaucage1, Marvin H. Caruthers2

1 Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland, 2 University of Colorado, Boulder, Colorado
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 3.3
DOI:  10.1002/0471142700.nc0303s00
Online Posting Date:  May, 2001
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Abstract

The phosphoramidite approach has had a major impact on the synthesis of oligonucleotides. This unit describes parameters that affect the performance of this method for preparing oligodeoxyribonucleotides, as well as a number of compatible strategies. Milestones that led to the discovery of the approach are chronologically reported. Alternate strategies are also described to underscore the versatility by which these synthons can be obtained. Mechanisms of deoxyribonucleoside phosphoramidite activation, factors affecting condensation, and deprotection strategies are discussed.

     
 
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Table of Contents

  • Accounts of Chemical Research in DNA Oligonucleotide Synthesis
  • Alternate Strategies to the Preparation of Deoxyribonucleoside Phosphoramidites
  • Activation of Deoxyribonucleoside Phosphoramidites
  • Factors Affecting the Condensation Rates of Deoxyribonucleoside Phosphoramidites
  • Significance of the “Capping” Reaction in the Chemical Synthesis of Oligodeoxyribonucleotides
  • The Oxidation Reaction in the Synthesis of Oligodeoxyribonucleotides According to the Phosphoramidite Method
  • Strategies in the Deprotection of Synthetic Oligodeoxyribonucleotides
  • Alternate Strategies to the Synthesis of Oligodeoxyribonucleotides According to the Phosphoramidite Method
  • Concluding Remarks
  • Literature Cited
  • Figures
     
 
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Materials

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Figures

  •   FigureFigure 3.3.1 The phosphite triester method to oligodeoxyribonucleotide synthesis.
  •   FigureFigure 3.3.2 Application of the phosphite triester approach to solid‐phase DNA synthesis.
  •   FigureFigure 3.3.3 Preparation of deoxyribonucleoside phosphoramidite monomers.
  •   FigureFigure 3.3.4 Activation of deoxyribonucleoside phosphoramidites toward oligonucleotide synthesis.
  •   FigureFigure 3.3.5 Deoxyribonucleoside phosphoramidite monomers with improved stability properties and reagents for the deprotection of methyl phosphate triesters.
  •   FigureFigure 3.3.6 Chemoselective preparation of deoxyribonucleoside phosphoramidites in situ using bis‐(pyrrolidino)methoxyphosphine and 4,5‐dichloroimidazole.
  •   FigureFigure 3.3.7 Chemoselective preparation of deoxyribonucleoside phosphoramidites using bis‐( N, N‐diisopropyl)alkoxyphosphine and limiting amounts of 1 H‐tetrazole or its N, N‐diisopropylammonium salt.
  •   FigureFigure 3.3.8 Preparation of deoxyribonucleoside phosphoramidites using hexaethylphosphorus triamide and limiting amounts of N, N‐diethylammonium tetrazolide.
  •   FigureFigure 3.3.9 Activation of deoxyribonucleoside phosphoramidites with N, N‐dimethylaniline hydrochloride.
  •   FigureFigure 3.3.10 Mechanism of the activation of deoxyribonucleoside phosphoramidites by 1 H‐ tetrazole during solid‐phase oligonucleotide synthesis.
  •   FigureFigure 3.3.11 Model compounds used in the study of phosphoramidite activation by 1 H‐tetrazole.
  •   FigureFigure 3.3.12 Nucleoside bicyclic phosphoramidites for the preparation of P‐diastereomerically enriched oligonucleoside phosphorothioates.
  •   FigureFigure 3.3.13 Deoxyribonucleoside phosphoramidites functionalized with nucleobase bulky groups.
  •   FigureFigure 3.3.14 Nucleoside phosphoramidites functionalized with 2′‐ or 3′‐sterically demanding groups.
  •   FigureFigure 3.3.15 Efficient ribonucleoside phosphoramidites for solid‐phase RNA synthesis.
  •   FigureFigure 3.3.16 Postulated O6‐guanine adducts generated during the chain extension step of the synthesis cycle according to the phosphoramidite method.
  •   FigureFigure 3.3.17 An oxaziridine derivative as a useful oxidant in the synthesis of oligonucleotides containing iodine sensitive residues, and a benzylic deoxyribonucleoside phosphoramidite suitable for the preparation of oligonucleotide analogues.
  •   FigureFigure 3.3.18 Access to oligodeoxyribonucleotide analogues from deoxynucleoside (2‐cyano‐ 1,1‐dimethylethyl) phosphoramidites.
  •   FigureFigure 3.3.19 Preparation of oligodeoxyribonucleoside phosphorothioates according to the solid‐phase phosphoramidite method.
  •   FigureFigure 3.3.20 Solid‐phase oligonucleotide synthesis using dinucleotide phosphoramidite derivatives.
  •   FigureFigure 3.3.21 Solid‐phase synthesis of oligonucleotide analogues from dimeric phosphoramidites carrying modified internucleotidic linkages.
  •   FigureFigure 3.3.22 Trinucleotide phosphoramidite blocks for the controlled, codon‐by‐codon, construction of combinatorial gene libraries.

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Literature Cited

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