Nucleoside Phosphoramidites Containing Cleavable Linkers

Richard T. Pon1

1 University of Calgary, Calgary, Alberta
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 3.12
DOI:  10.1002/0471142700.nc0312s23
Online Posting Date:  January, 2006
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Abstract

Phosphoramidite reagents (linker phosphoramidites) containing a cleavable 3′‐ester linkage between the nucleoside and the phosphoramidite group can be used to attach the first nucleoside to a solid‐phase support. Inexpensive underivatized supports such as LCAA‐CPG can then be used as universal supports for oligonucleotide synthesis. No modifications to synthesis coupling conditions and no 3′‐dephosphorylation are required. Only oligonucleotides with terminal 3′‐OH ends are produced. Phosphoramidites containing both a succinate and a sulfonyldiethanol linkage are particularly useful and create oligonucleotides with both a 3′‐OH and 5′‐phosphate. In addition, by using these reagents, one oligonucleotide sequence can be added onto the 5′‐end of another (tandem synthesis) to produce a string of multiple oligonucleotides linked end‐to‐end. Deprotection releases the oligonucleotides from each other to yield a mixture of oligonucleotides. This approach is particularly useful for making pairs of PCR primers or both strands of a double‐stranded sequence in a single operation.

Keywords: linker phosphoramidites; solid‐phase supports; tandem oligonucleotide synthesis; 96‐well plate synthesis; high‐throughput synthesis; support derivatization

     
 
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Table of Contents

  • Basic Protocol 1: Synthesis of Linker Phosphoramidites Containing a Cleavable Succinyl‐Sulfonyldiethanol Linker
  • Alternate Protocol 1: Replacement of Sulfonyldiethanol with Ethylene Glycol
  • Basic Protocol 2: Oligonucleotide Synthesis in Multi‐Well Plates on Underivatized LCAA‐CPG Supports
  • Support Protocol 1: Dry Loading of CPG Support into 96‐Well Plates
  • Basic Protocol 3: Tandem Synthesis of Multiple Oligonucleotides in a Single Synthesis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Synthesis of Linker Phosphoramidites Containing a Cleavable Succinyl‐Sulfonyldiethanol Linker

  Materials
  • Protected 2′‐deoxyribonucleosides:
    • 5′‐O‐(4,4′‐Dimethoxytrityl)‐N6‐benzoyl‐2′‐deoxyadenosine ( S.8a)
    • 5′‐O‐(4,4′‐Dimethoxytrityl)‐N4‐acetyl‐2′‐deoxycytidine or 5′‐O‐(4,4′‐dimethoxytrityl)‐N4‐benzoyl‐2′‐deoxycytidine ( S.8b)
    • 5′‐O‐(4,4′‐Dimethoxytrityl)‐N2‐isobutyryl‐2′‐deoxyguanosine ( S.8c)
    • 5′‐O‐(4,4′‐Dimethoxytrityl)thymidine ( S.8d)
  • Succinic anhydride ( S.9)
  • Dichloromethane
  • Triethylamine
  • Methanol
  • Chloroform (CHCl 3)
  • 0.5 M triethylammonium phosphate (TEAP) solution (see recipe)
  • 60% to 65% (w/v) 2,2′‐sulfonyldiethanol ( S.11) solution in water
  • Acetonitrile, anhydrous
  • Pyridine, anhydrous
  • 4‐Dimethylaminopyridine (DMAP)
  • O‐Benzotriazol‐1‐yl‐N,N,N′,N′‐tetramethyluronium hexafluorophosphate (HBTU)
  • Diisopropylethylamine (DIPEA)
  • Silica gel
  • Diisopropylamine, anhydrous
  • 2‐Cyanoethyl‐N,N,N′,N′‐tetraisopropylphosphorodiamidite ( S.13)
  • 0.45 M 1H‐tetrazole ( S.14) in anhydrous acetonitrile
  • 5% (w/v) sodium bicarbonate in water
  • Saturated aqueous sodium chloride
  • Hexanes, distilled (remove dissolved oxygen by distilling under vacuum on a rotary evaporator just prior to use)
  • 100‐, 250‐, and 500‐mL round‐bottom flasks
  • Magnetic stirrer
  • Fluorescent silica gel TLC plates, Merck 60 or similar
  • 500‐mL separatory funnels
  • Rotary evaporator and warm water bath (60°C)
  • Chromatography column, ∼3 in. (∼7.5 cm) diameter, with capacity for at least 9 in. (∼23 cm) silica gel
  • Rubber septum
  • Drying tube: an empty 10‐ or 20‐mL syringe filled with Drierite (with indicator), with a small glass wool plug at each end
  • Syringes
  • Additional reagents and equipment for TLC ( appendix 3D) and flash chromatography ( appendix 3E)

Alternate Protocol 1: Replacement of Sulfonyldiethanol with Ethylene Glycol

  • Nucleoside‐3′‐O‐succinate hemiester ( S.10; see protocol 1)
  • p‐Toluenesulfonyl chloride (p‐TsCl)
  • 1‐Methylimidazole, anhydrous (N‐methylimidazole; NMI)
  • Ethylene glycol, anhydrous
  • 250‐mL separatory funnel

Basic Protocol 2: Oligonucleotide Synthesis in Multi‐Well Plates on Underivatized LCAA‐CPG Supports

  Materials
  • Linker phosphoramidite reagents: S.4a‐d (see protocol 1) or S.16a‐d (see protocol 2)
  • Anhydrous acetonitrile
  • Reagents for oligonucleotide synthesis:
    • conventional 3′‐O‐phosphoramidites
    • activator, deprotection, oxidizing, and capping solutions ( appendix 3C)
  • Long‐chain alkylamine controlled‐pore glass (LCAA‐CPG) support, ∼100 µmol/g amino group loading, 500 Å pore size ( S.5)
  • 1:1 (v/v) ammonium hydroxide/40% (w/v) aqueous methylamine
  • Automated DNA synthesizer for multi‐well plates, with at least 8 monomer ports (e.g., MerMade 192 synthesizer, BioAutomation)
  • 96‐well Oro‐Flex OF1100 polypropylene filter plate (0.7‐mL wells; Orochem Technologies) with 96‐well collection plate (1‐mL wells)
  • Centrifuge with 96‐well plate adapters
  • Clamping apparatus for multi‐well plates (as recommended by synthesizer manufacturer)
  • 65°C oven
  • Centrifugal evaporator (e.g., Speedvac, Savant) that can accommodate 96‐well plates
  • Additional reagents and equipment for oligonucleotide synthesis ( appendix 3C) and for “dry” loading of the support (see protocol 4)

Support Protocol 1: Dry Loading of CPG Support into 96‐Well Plates

  Materials
  • Linker phosphoramidites ( S.4a‐d; see protocol 1)
  • Anhydrous acetonitrile
  • Solid support:
    • For column format: prepacked synthesis columns containing prederivatized solid‐phase support
    • For plate format: long‐chain alkylamine controlled‐pore glass (LCAA‐CPG; S.5), ∼100 µmol/g amino group loading, 500 Å pore size
  • NAP‐10 (prepacked Sephadex) column
  • Additional reagents and equipment for oligonucleotide synthesis ( appendix 3C and protocol 3)
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Figures

Videos

Literature Cited

Literature Cited
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