Water‐Medium Synthesis of Nucleoside 5′‐Polyphosphates

Anaïs Depaix1, Suzanne Peyrottes1, Béatrice Roy1

1 Institut des Biomolécules Max Mousseron (IBMM), UMR 5247 CNRS, Université de Montpellier, Montpellier
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 13.16
DOI:  10.1002/cpnc.30
Online Posting Date:  June, 2017
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Abstract

This unit describes a one‐pot, two step synthesis of ribonucleoside 5′‐di‐ and 5′‐triphosphates, as well as their purification. The first step of the synthesis involves the activation of an unprotected ribonucleoside 5′‐monophosphate with 2‐chloro‐1,3‐dimethylimidazolinium hexafluorophosphate and imidazole, in a mixture of water/acetonitrile. The resulting phosphorimidazolate intermediate is then treated with inorganic phosphate or pyrophosphate to afford the corresponding nucleoside 5′‐di‐ or 5′‐triphosphates. The attractive features of this strategy include the absence of protecting groups on the starting material and convenient set up (i.e., use of water, non‐dry solvents and reagents, commercially available sodium salts). © 2017 by John Wiley & Sons, Inc.

Keywords: nucleosides; nucleotides; phosphorylation; synthetic methods; water chemistry

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis of 2‐Chloro‐1,3‐Dimethylimidazolinium Hexafluorophosphate (DMP)
  • Basic Protocol 2: One‐Pot Synthesis of Nucleoside 5′‐Diphosphates Using 2‐Chloro‐1,3‐Dimethylimidazolinium Hexafluorophosphate (DMP)
  • Basic Protocol 3: One‐Pot Synthesis of Nucleoside 5′‐Triphosphates Using 2‐Chloro‐1,3‐Dimethylimidazolinium Hexafluorophosphate (DMP)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Synthesis of 2‐Chloro‐1,3‐Dimethylimidazolinium Hexafluorophosphate (DMP)

  Materials
  • 2‐Chloro‐1,3‐dimethylimidazolinium chloride (DMC; Acros Organics, 90%)
  • Acetonitrile (CH 3CN), 99.9%, Extra dry, over molecular sieves (AcroSeal brand, Acros Organics)
  • Potassium hexafluorophosphate, 98% (KPF 6; Sigma‐Aldrich)
  • Celite 535 (Sigma‐Aldrich)
  • Diethyl ether (Et 2O; e.g., VWR)
  • Potassium hydroxide (KOH) pellets (drying agent for desiccator; e.g., Carlo Erba)
  • Acetonitrile‐d 3 (CD 3CN; Euriso‐top)
  • Argon gas
  • 50‐ and 100‐mL round‐bottom flasks
  • Magnetic stir bar
  • Magnetic stirrer
  • Rubber septum
  • 10‐mL plastic syringe (Terumo)
  • Disposable needles (0.8 × 50 mm; Terumo)
  • 250‐mL vacuum flask
  • Sintered glass filter funnel
  • Pump (Bioblock Scientific)
  • Rotary evaporator
  • Dessicator
  • Additional equipment for nuclear magnetic resonance (1H, 13C, and 31P NMR) and mass spectrometry

Basic Protocol 2: One‐Pot Synthesis of Nucleoside 5′‐Diphosphates Using 2‐Chloro‐1,3‐Dimethylimidazolinium Hexafluorophosphate (DMP)

  Materials
  • Nucleoside monophosphate (NMP) disodium salts:
    • Uridine 5′‐monophosphate disodium salt hydrate (TCI)
    • Adenosine 5′‐monophosphate disodium salt (Sigma‐Aldrich)
    • Cytidine 5′‐monophosphate disodium salt (Sigma‐Aldrich)
  • Milli‐Q water
  • Acetonitrile (CH 3CN; e.g., Sigma‐Aldrich)
  • 2‐Chloro‐1,3‐dimethylimidazolinium hexafluorophosphate (DMP)
  • Imidazole (Im; Alfa Aesar)
  • Sodium phosphate dihydrate (NaH 2PO 4·2H 2O; Merck)
  • D 2O (Euriso‐top)
  • Triethylammonium bicarbonate (TEAB), pH 7.5, (0.4 M and 1 × 10−3 M; see recipe)
  • DEAE Sephadex A‐25 ion‐exchange resin
  • 15‐mL Falcon conical centrifuge tube
  • Magnetic stir bar
  • Magnetic heating stirrer
  • Freeze dryer
  • Puriflash 430 Interchim flash chromatography apparatus
  • DEAE Sephadex A‐25 ion‐exchange column (glass column: 50 cm × 4 cm, resin: 500 mL)
  • Rotary evaporator
  • Additional equipment for nuclear magnetic resonance (1H, 13C and 31P NMR)

Basic Protocol 3: One‐Pot Synthesis of Nucleoside 5′‐Triphosphates Using 2‐Chloro‐1,3‐Dimethylimidazolinium Hexafluorophosphate (DMP)

  Materials
  • Nucleoside monophosphate disodium salts:
    • Uridine 5′‐monophosphate disodium salt hydrate (TCI)
    • Adenosine 5′‐monophosphate disodium salt (Sigma‐Aldrich)
    • Cytidine 5′‐monophosphate disodium salt (Sigma‐Aldrich)
  • Milli‐Q water
  • Acetonitrile (e.g., Sigma‐Aldrich)
  • 2‐Chloro‐1,3‐dimethylimidazolinium hexafluorophosphate (DMP)
  • Imidazole (Im; Alfa Aesar)
  • Tetrasodium pyrophosphate decahydrate (Na 4P 2O 7·10H 2O; Fluka)
  • D 2O (Euriso‐top)
  • DEAE Sephadex ion‐exchange resin
  • Triethylammonium bicarbonate (TEAB), pH 7.5, (0.6 M, 0.4 M, 1 × 10−3 M; see recipe)
  • 15‐mL Falcon conical centrifuge tube
  • Magnetic stir bar
  • Magnetic heating stirrer
  • Freeze dryer
  • Puriflash 430 Interchim flash chromatography apparatus
  • DEAE Sephadex ion‐exchange column (glass column: 50 cm × 4 cm, resin suspension: 500 mL)
  • Rotary evaporator
  • Glass column, 85 cm × 2 cm
  • Bio‐Gel P‐2 polyacrylamide gel (Bio‐Rad)
  • Isco Model UA‐6 UV‐vis detector
  • Additional equipment for nuclear magnetic resonance (1H, 13C, and 31P NMR)
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Figures

Videos

Literature Cited

Literature Cited
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