Attachment of Nucleosides and Other Linkers to Solid‐Phase Supports for Oligonucleotide Synthesis

Andrei P. Guzaev1, Richard T. Pon2

1 AM Chemicals LLC, Oceanside, California, 2 University of Calgary, Calgary, Alberta, Canada
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 3.2
DOI:  10.1002/0471142700.nc0302s52
Online Posting Date:  March, 2013
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Abstract

Specific step‐by‐step instructions for conversion of 5′‐O‐(4,4′‐dimethoxytrityl)‐ and base‐protected nucleosides and other mono‐O‐(4,4′‐dimethoxytrityl)‐protected diols to their hemisuccinate esters and their coupling to CPG (controlled‐pore glass) supports bearing aminopropyl or long chain aminoalkyl groups are presented. Additional guidelines are provided for selecting a coupling protocol and performing in‐process control. Curr. Protoc. Nucleic Acid Chem. 52:3.2.1‐3.2.23. © 2013 by John Wiley & Sons, Inc.

Keywords: nucleic acid chemistry; oligonucleotides; nucleosides and nucleotides; solid supports

     
 
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Table of Contents

  • Introduction
  • Strategic Planning: Selecting a Linker Arm and Coupling Protocol
  • Basic Protocol 1: Preparation of Aminopropyl CPG
  • Basic Protocol 2: Preparation of DMT‐Protected Hemiester Building Blocks
  • Basic Protocol 3: Attachment of Hemisuccinates of DMT‐Protected Building Blocks to Aminopropyl CPG
  • Alternate Protocol 1: Expedite Attachment of Hemisuccinates of DMT‐Protected Building Blocks to Aminopropyl CPG
  • Support Protocol 1: Determination of Solid Support‐Bound Amino Groups by Picric Acid Test
  • Support Protocol 2: Determination of Amino Groups by DMT Test
  • Support Protocol 3: Quantitative Determination of Surface Loading of Derivated Supports by Trityl Assay
  • Support Protocol 4: Detection of Amino Groups on Insoluble Supports by Kaiser Test
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of Aminopropyl CPG

  Materials
  • Water
  • Native CPG beads (aminopropyl‐ or LCAA CPG or aminomethyl macroporous polystyrene may be directly subjected to the loading; see recipe)
  • 3‐(Triethoxysilyl)‐1‐propanamine, reagent‐grade ( 2)
  • Methanol, reagent‐grade or ethanol
  • Ethyl acetate, optional
  • 1‐L Round‐bottom flask with overhead stirrer, reflux condenser, and thermometer
  • Heating mantle or oil bath, temperature regulator
  • Buchner funnel
  • Bunsen flask
  • Sintered glass or Whatman no. 1 filter paper
  • Vacuum oven or rotary evaporator
  • Additional reagents and equipments for picrate test (see protocol 5)

Basic Protocol 2: Preparation of DMT‐Protected Hemiester Building Blocks

  Materials
  • N‐protected 5′‐O‐DMT nucleoside or mono‐DMT‐protected diol
  • Succinic anhydride, reagent grade
  • 4‐Dimethylaminopyridine (DMAP), optional
  • N‐methylimidazole (NMI), optional
  • Anhydrous pyridine (see recipe)
  • Triethylamine, reagent grade
  • Methanol, reagent grade
  • Dichloromethane, reagent grade
  • 0.1 M Triethylammonium acetate prepared by neutralization of 0.1 M aqueous acetic acid with triethylamine to pH 7.5
  • 0.5 M citric acid
  • Na 2SO 4, anhydrous, granulated
  • Round‐bottom flasks
  • Magnetic stirrer and stirring bar
  • Silica gel TLC plates
  • Heating gun or heating plate
  • Ice bath
  • Rotary evaporator
  • Separatory funnel
  • Erlenmeyer flask
  • Oil pump
  • Additional reagents and equipment for thin‐layer chromatography (TLC; appendix 3D)

Basic Protocol 3: Attachment of Hemisuccinates of DMT‐Protected Building Blocks to Aminopropyl CPG

  Materials
  • Hemisuccinate building block 6a (see protocol 2)
  • 4‐Dimethylaminopyridine (DMAP)
  • N,N‐Diisopropylethylamine, redistilled (see recipe)
  • Acetonitrile (MeCN) anhydrous (see recipe)
  • O‐(Benzotriazol‐1‐yl)‐N,N,N′,N′‐tetramethyluronium tetrafluoroborate (TBTU; 13)
  • Aminopropyl‐ or LCAA CPG or Aminomethyl Macroporous Polystyrene
  • Anhydrous pyridine (see recipe)
  • N‐Methylimidazole
  • Acetic anhydride, reagent grade
  • Methanol, reagent grade
  • Ethyl acetate, reagent grade
  • Round‐bottom flasks
  • Shaker, wrist action or orbital
  • Pasteur pipet
  • Sintered glass filter
  • Vacuum desiccator
  • Oil or diaphragm pump
  • Bunsen flask
  • Whatman no. 1 filter paper
  • Buchner funnel
  • Screw‐capped glass vials
  • Additional reagents and equipment for trityl analysis (see protocol 7) and Kaiser test (see protocol 8)
CAUTION: Coupling agent TBTU can cause severe skin or eye irritation and allergic reactions. Use appropriate skin and eye protection, and wipe down all work areas and utensils with a wet cloth immediately after use (TBTU is water soluble).

Alternate Protocol 1: Expedite Attachment of Hemisuccinates of DMT‐Protected Building Blocks to Aminopropyl CPG

  Materials
  • Picric acid
  • Dichloromethane, reagent grade
  • Anhydrous magnesium sulfate
  • Aminoalkyl solid support
  • N,N‐diisopropylethylamine (see recipe)
  • 10% and 95% Ethanol (EtOH)
  • Tightly capped flasks
  • Disposable glass pipets with wool plugs
  • 10‐mL volumetric flasks
  • Quartz cuvettes
  • UV/VIS spectrophotometer or colorimeter

Support Protocol 1: Determination of Solid Support‐Bound Amino Groups by Picric Acid Test

  Materials
  • Aminoalkyl‐derivatized solid support
  • 0.25 M 4, 4′‐dimethoxytrityl chloride stock solution (see recipe)
  • 0.25 M tetrabutylammonium perchlorate stock solution (see recipe)
  • Methanol, reagent grade
  • Dichloromethane, reagent grade
  • Screw‐capped glass vials

Support Protocol 2: Determination of Amino Groups by DMT Test

  Materials
  • Derivatized aminopropyl CPG support (see protocol 3 and protocol 4)
  • Detritylation reagent (see recipe for TFA solution)
  • 10‐mL volumetric flask
  • Analytical balance accurate to at least 0.1 mg
  • Quartz cuvettes
  • UV/VIS spectrophotometer or colorimeter

Support Protocol 3: Quantitative Determination of Surface Loading of Derivated Supports by Trityl Assay

  • Ninhydrin, reagent‐grade
  • Phenol, reagent‐grade
  • Potassium cyanide (KCN), reagent‐grade
  • Acetonitrile (MeCN)
  • Heating block
  • Vortex mixer
  • Microcentrifuge
CAUTION: KCN is highly toxic. Ingestion of solid or solutions or inhalation of dust may result in death. Wear gloves and eye protection, and conduct any work with KCN in a fume hood.
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Figures

Videos

Literature Cited

Literature Cited
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