Gram‐Scale Chemical Synthesis of Base‐Modified Ribonucleoside‐5′‐O‐Triphosphates

Muthian Shanmugasundaram1, Annamalai Senthilvelan1, Anilkumar R. Kore1

1 Life Sciences Solutions Group, Thermo Fisher Scientific, Austin, Texas
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 13.15
DOI:  10.1002/cpnc.20
Online Posting Date:  December, 2016
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This unit delineates a simple, reliable, straight‐forward, general, and efficient chemical method for the synthesis of modified nucleoside‐5′‐O‐triphosphates such as 5‐methylcytidine‐5′‐O‐triphosphate (5‐Me‐CTP), pseudouridine‐5′‐O‐triphosphate (pseudo‐UTP), and N1‐methylpseudouridine‐5′‐O‐triphosphate (N1‐methylpseudo‐UTP), starting from the corresponding nucleoside. The reaction utilizes an improved protection‐free “one‐pot, three‐step” Ludwig synthetic strategy that involves the monophosphorylation of the nucleoside with phosphorous oxychloride followed by reaction with tributylammonium pyrophosphate and subsequent hydrolysis of the resulting cyclic intermediate to furnish the corresponding ribonucleoside triphosphate (NTP) in moderate yields. It is noteworthy that the reaction affords high purity (>99.5%) NTPs after DEAE Sepharose column purification. © 2016 by John Wiley & Sons, Inc.

Keywords: ribonucleotides; chemical synthesis; one‐pot synthesis; messenger RNA; gene therapy; vaccination therapy

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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1:

  • Tributylammonium pyrophosphate (prepared as in Ludwig and Eckstein, )
  • Acetonitrile, anhydrous (e.g., Fisher Scientific)
  • Tributylamine (e.g., Fisher Scientific)
  • Nucleosides:
    • 5‐Methylcytidine (e.g., ChemGenes, cat. no. RP‐1832)
    • Pseudouridine (e.g., ChemGenes, custom synthesis)
    • N1‐methylpseudouridine (e.g., ChemGenes, custom synthesis)
  • Trimethylphosphate, >97% pure (e.g., Sigma‐Aldrich)
  • Phosphorous oxychloride (e.g., Acros)
  • Dichloromethane (DCM; e.g., Fisher Scientific)
  • Ammonium hydroxide, 28% NH 3 in water (e.g., Fisher Scientific)
  • DEAE Sepharose (e.g., GE Healthcare)
  • Isopropanol (e.g., Fisher Scientific)
  • 2.0 M NaCl (see recipe)
  • 1.0 M triethylammonium bicarbonate (TEAB) buffer (see recipe)
  • HPLC mobile phase A: 5 mM ammonium phosphate monobasic, pH 2.8 (see recipe)
  • HPLC mobile phase B: 750 mM ammonium phosphate monobasic, pH 3.7 (see recipe)
  • Sodium perchlorate (e.g., Fisher Scientific)
  • Acetone (e.g., Fisher Scientific)
  • 500‐mL and 1‐L centrifuge bottles
  • 1‐L one‐neck round‐bottom flasks, oven dried (e.g., Chemglass)
  • Rubber septa for 24/40 glass joints (e.g., Chemglass)
  • Teflon‐coated magnetic stir bar and magnetic stir plate
  • Vacuum/nitrogen (or argon) gas manifold
  • Ice bath and ice/NaCl bath (−5°C to −10οC)
  • 1‐mL, 2‐mL, and 5‐mL sealed glass syringes with disposable needles (e.g., Fisher Scientific)
  • Addition funnel
  • 2‐L separatory funnel
  • 4‐L conical flask
  • Chromatography column: 10 cm × 78.5 cm
  • FPLC ÄKTA purifier (e.g., GE Healthcare) including:
    • Gradient pump
    • UV detector: 260 nm and 272 nm
  • HPLC system (e.g., Waters) including:
    • Detector module
    • Hypersil SAX column (4.6 mm × 25 cm)
  • Rotary evaporator
  • Centrifuge (e.g., Sorvall RC‐3B)
  • Additional reagents and equipment for proton nuclear magnetic resonance (1H NMR and 31P NMR; unit 7.2), and mass spectrometry (unit 10.2)
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Literature Cited

Literature Cited
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