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: 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 Support Protocol 1)

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
  • Na2SO4, 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 Basic 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 Support Protocol 3) and Kaiser test (see Support Protocol 4)

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).

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

 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 2: Determination of Amino Groups by DMT 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 3: Quantitative Determination of Surface Loading of Derivated Supports by Trityl Assay

 Materials
  • Derivatized aminopropyl CPG support (see Basic Protocol 3 and Alternate Protocol)
  • 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 4: Detection of Amino Groups on Insoluble Supports by Kaiser Test

 Additional Materials (also see Basic Protocol 1)
  • 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

  •  FigureFigure 3.2.1 Preparation of Aminopropyl CPG, 3, by aminoalkylation of Native CPG, 1, with 3-(Triethoxysilyl)-1-propanamine, 2.
    View Image
  •  FigureFigure 3.2.2 Conversion of mono-DMT-protected diols, 4, to the respective hemisuccinates, 6a, and hemidiglycolates, 6b. Examples shown below are commonly used hemisuccinates of protected 2′-deoxynucleosides, 7, protected ribonucleosides, 8, and universal linker, 9, and hemiglycolates of protected 2′-deoxynucleosides, 10, and linker 11 for 3′-phosphorylation of oligonucleotides.
    View Image
  •  FigureFigure 3.2.3 Preparation of solid supports 12a and b using methods A and B (see Background Information). Method A described in detail in this unit, uses attachment of hemisuccinates 6a and hemiglycolates 6b to aminoalkyl-derivatized solid supports 3 with the aid of TBTU as a coupling agent. In the second step, the unreacted amino groups are capped with acetic anhydride/NMI. Protocols for practical implementation of method B can be found in various references (Damha et al., 1990; Pon et al., 1988; also see unit 3.13).
    View Image
  •  FigureFigure 3.2.4 Reaction of ninhydrin, 14, with aminoalkyl-derivatized solid support to form Ruhemann's Purple, 15, a dark-blue colored dye.
    View Image

Videos

Literature Cited

Literature Cited
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