Rapid and Efficient Synthesis of Nucleoside Polyphosphates and Their Conjugates Using Sulfonyl Imidazolium Salts

Samy Mohamady1, Scott D. Taylor1

1 Department of Chemistry, University of Waterloo, Waterloo, Ontario, Canada
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 13.11
DOI:  10.1002/0471142700.nc1311s51
Online Posting Date:  December, 2012
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Abstract

This unit describes a method for preparing nucleoside polyphosphates and their conjugates such as nucleoside triphosphates, symmetrical and unsymmetrical dinucleoside polyphosphates and sugar nucleotides. The protocols employ sulfonyl imidazolium salts (SnISs) as coupling reagents. These reagents are prepared in two steps in very high yield from commercially available materials and can be stored for at least 1 year at −20°C. The tetra‐n‐butylammonium salts of nucleoside mono‐, di‐, or triphosphates are reacted with the SnISs to form reactive phosphoryl imidazolium donors. These donors are reacted with the tetra‐n‐butylammonium salts of pyrophosphate, nucleotide mono‐ or diphosphates or sugar‐1‐phosphates to give the nucleoside polyphosphates and their conjugates in excellent yields. Curr. Protoc. Nucleic Acid Chem. 51:13.11.1‐13.11.24. © 2012 by John Wiley & Sons, Inc.

Keywords: nucleoside triphosphates; dinucleoside polyphosphates; sugar nucleotides; sulfonylimidazolium salts

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis of 1‐(Phenylsulfonyl)‐1H‐Imidazole and 1‐(Phenylsulfonyl)‐2‐Methyl‐1H‐Imidazole
  • Basic Protocol 2: Synthesis of 3‐(Phenylsulfonyl)‐1‐Methylimidazolium Triflate and 3‐(Phenylsulfonyl)‐1,2‐Dimethylimidazolium Triflate
  • Basic Protocol 3: Synthesis of P1,P2‐Diadenosine 5′‐Diphosphate Disodium Salt and P1,P4‐Diuridine 5′‐Tetraphosphate Tetraammonium Salt
  • Basic Protocol 4: Synthesis of Adenosine 5′‐Triphosphate Trisodium Salt
  • Basic Protocol 5: Synthesis of P1‐Adenosine‐P4‐Uridine 5′‐Tetraphosphate Tetrasodium Salt
  • Basic Protocol 6: Synthesis of Thymidine 5′‐(β‐D‐Glucopyranosyl) Diphosphate Diammonium Salt
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Synthesis of 1‐(Phenylsulfonyl)‐1H‐Imidazole and 1‐(Phenylsulfonyl)‐2‐Methyl‐1H‐Imidazole

  Materials
  • Argon
  • Imidazole (Sigma‐Aldrich)
  • Dichloromethane (dried by distillation over calcium anhydride)
  • Benzenesulfonyl chloride (Sigma‐Aldrich)
  • Water
  • Aqueous saturated NaCl solution (saturated brine)
  • Anhydrous magnesium sulfate
  • Ethyl acetate
  • Hexane
  • 2‐Methylimidazole (Sigma‐Aldrich)
  • 1‐L three‐neck round‐bottom flask (flame‐dried)
  • Stir bar
  • Rubber septum
  • Plastic caps
  • Magnetic stir plate
  • Needle fitted with an argon‐filled balloon
  • Ice bath
  • Syringes
  • Rotary evaporator attached to a water aspirator vacuum
  • 1‐L separatory funnel
  • 1‐L Erlenmeyer flask
  • −20°C freezer
  • Vacuum pump (high vacuum) connected to a glass manifold

Basic Protocol 2: Synthesis of 3‐(Phenylsulfonyl)‐1‐Methylimidazolium Triflate and 3‐(Phenylsulfonyl)‐1,2‐Dimethylimidazolium Triflate

  Materials
  • S.1 (freshly prepared; see protocol 1) or S.2 (freshly prepared; see protocol 1)
  • Anhydrous ether (dried over sodium metal)
  • Methyl triflate (Sigma‐Aldrich)
  • 250‐mL three‐neck round‐bottom flask
  • Magnetic stir plate and stir bar
  • Rubber septum
  • Plastic caps
  • Needle fitted with an argon‐filled balloon
  • Vacuum pump (high vacuum) connected to a glass manifold
  • 250‐mL round‐bottom flasks

Basic Protocol 3: Synthesis of P1,P2‐Diadenosine 5′‐Diphosphate Disodium Salt and P1,P4‐Diuridine 5′‐Tetraphosphate Tetraammonium Salt

  Materials
  • Adenosine 5′‐monophosphate (AMP) monohydrate (Sigma‐Aldrich)
  • Distilled, deionized water (ddH 2O)
  • 10% aq. tetrabutylammonium hydroxide solution (prepared from a 40% solution of tetrabutylammonium hydroxide using ddH 2O)
  • Anhydrous acetonitrile (dried by distilling over calcium hydride)
  • Dowex 50W‐H+ resin
  • Methanol
  • 1 N sodium chloride solution prepared using ddH 2O
  • 1 N HCl prepared using ddH 2O
  • Toluene
  • Argon
  • Anhydrous N,N‐dimethylformamide (DMF; dried by distilling under aspirator pressure over calcium hydride)
  • 4Å activated molecular sieves (beads)
  • Anhydrous N‐methylimidazole (NMI; prepared by distillation under aspirator pressure over potassium hydroxide and stored in a Schlenck tube over 4Å molecular sieves)
  • Magnesium chloride trihydrate
  • S.3 (see protocol 2)
  • Buffer A: 50 mM triethylammonium acetate buffer, pH 7 (prepared by adding 7 mL HPLC‐grade triethylamine to 1 L ddH 2O and adjusting the pH to 7 with HPLC‐grade acetic acid)
  • 5% Ethylenediaminetetraacetate (EDTA) disodium salt solution (prepared by dissolving 5 g EDTA disodium in 100 mL buffer A)
  • Chloroform
  • Chelex resin (Sigma‐Aldrich)
  • 10% Aq. pyridine
  • Uridine 5′‐diphosphate (UDP) disodium salt hydrate (Mohamady and Taylor, )
  • 1 N Ammonium acetate solution prepared using ddH 2O
  • 25‐, 50‐ and 250‐mL Round‐bottom flasks
  • Magnetic stir plate and stir bar
  • pH meter
  • Pasteur pipets
  • Rotary evaporator equipped with dry ice‐acetone trap and temperature‐controlled water bath and attached to a high vacuum pump
  • Lyophilizer
  • Chromatography columns (0.5 cm in diameter)
  • Needle fitted with an argon‐filled balloon
  • Rubber septa
  • 10‐ and 25‐mL flame‐dried round‐bottom flasks
  • Ice bath
  • 50‐mL separatory funnel
  • Cotton
  • Semipreparative or preparative HPLC system equipped with a C18 semi‐preparative or preparative reversed‐phase column and a UV detector set to 255 nm
  • 10‐mL vials
  • 10‐mL conical plastic centrifuge tubes

Basic Protocol 4: Synthesis of Adenosine 5′‐Triphosphate Trisodium Salt

  Materials
  • Dowex 50W‐H+ resin
  • Methanol
  • Distilled, deionized water (ddH 2O)
  • 10% aq. Pyridine
  • Pyrophosphate tetrasodium salt (Sigma‐Aldrich)
  • 10% Tetrabutylammonium hydroxide solution (prepared from 40% solution of tetrabutylammonium hydroxide)
  • Anhydrous acetonitrile (dried by distilling HPLC‐grade acetonitrile over calcium hydride)
  • Toluene
  • Argon
  • Anhydrous N,N‐dimethylformamide (DMF; dried by distilling under aspirator pressure over calcium hydride)
  • Adenosine 5′‐monophosphate (AMP) tetrabutylammonium salt (see protocol 3)
  • 4Å activated molecular sieves (beads)
  • S.3 (see protocol 2)
  • Anhydrous diisopropylethylamine (DIPEA; Sigma‐Aldrich)
  • Chloroform
  • Buffer A: 50 mM triethylammonium acetate buffer, pH 7 (prepared by adding 7 mL HPLC‐grade triethylamine to 1 L ddH 2O and adjusting the pH to 7 with HPLC‐grade acetic acid)
  • Chromatography columns (2.0‐cm diameter)
  • 25‐, 250‐, and 500‐mL round‐bottom flasks
  • Magnetic stir plate and stir bar
  • pH meter
  • High vacuum rotary evaporator equipped with dry ice‐acetone trap and temperature controlled water bath
  • Lyophilizer
  • Flame‐dried 25‐ and 10‐mL round‐bottom flasks
  • Rubber septa
  • Needle fitted with an argon‐filled balloon
  • 50‐mL separatory funnel
  • Cotton plugs
  • Pasteur pipets
  • 15‐mL conical test tubes
  • Semipreparative or preparative HPLC system equipped with a C18 semi‐preparative or preparative reversed‐phase column and a UV detector set to 255 nm
  • 10‐mL vials

Basic Protocol 5: Synthesis of P1‐Adenosine‐P4‐Uridine 5′‐Tetraphosphate Tetrasodium Salt

  Materials
  • Dowex 50W‐H+ ion‐exchange resin
  • Methanol
  • Distilled, deionized water (ddH 2O)
  • Uridine 5′‐monophosphate (UMP) disodium salt hydrate (TCI America)
  • 10% Tetrabutylammonium hydroxide solution (prepared from 40% solution of tetrabutylammonium hydroxide using ddH 2O)
  • Anhydrous acetonitrile (dried by distilling HPLC‐grade acetonitrile over calcium hydride)
  • Toluene
  • Argon
  • Anhydrous N,N‐dimethylformamide (DMF; dried by distilling over calcium hydride)
  • 4Å activated molecular sieves (beads)
  • 10% aq. Pyridine
  • Adenosine 5′‐triphosphate (ATP) disodium salt hydrate (TCI America)
  • S.4
  • Anhydrous diisopropylethylamine (DIPEA, Sigma‐Aldrich)
  • Magnesium chloride trihydrate (Sigma‐Aldrich)
  • 5% Ethylenediaminetetraacetate (EDTA) disodium salt solution (prepared by dissolving 5 g EDTA disodium in 100 mL buffer A)
  • Buffer A: 50 mM triethylammonium acetate buffer, pH 7 (prepared by adding 7 mL HPLC‐grade triethylamine to 1 L ddH 2O and adjusting the pH to 7 with HPLC‐grade acetic acid)
  • Chloroform
  • Chelex resin
  • Chromatography columns (0.5‐cm diameter)
  • 25‐, 50‐ and 250‐mL Round‐bottom flasks
  • Magnetic stir plate and stir bar
  • pH meter
  • Rotary evaporator equipped with dry ice‐acetone trap and temperature controlled water bath and attached to a high vacuum pump
  • Lyophilizer
  • Flame‐dried 25‐mL round‐bottom flasks
  • Rubber septa
  • Needle fitted with an argon‐filled balloon
  • 50‐mL separatory funnel
  • Cotton
  • Pasteur pipets
  • 15‐mL conical test tubes
  • Semipreparative or preparative HPLC system equipped with a C18 semi‐preparative or preparative reversed‐phase column and a UV detector set to 255 nm
  • 10‐mL vials

Basic Protocol 6: Synthesis of Thymidine 5′‐(β‐D‐Glucopyranosyl) Diphosphate Diammonium Salt

  Materials
  • Dowex 50W‐H+ ion‐exchange resin
  • Methanol
  • Distilled, deionized water (ddH 2O)
  • β‐D‐glucopyranosyl‐1‐phosphate (Binch et al., )
  • 10% Tetrabutylammonium hydroxide solution (prepared from 40% solution of tetrabutylammonium hydroxide)
  • Anhydrous acetonitrile (dried by distilling HPLC‐grade acetonitrile over calcium hydride)
  • Toluene
  • Argon
  • Anhydrous N,N‐dimethylformamide (DMF; dried by distilling over calcium hydride)
  • 4Å activated molecular sieves (beads)
  • Magnesium chloride trihydrate (Sigma‐Aldrich)
  • Thymidine 5′‐monophosphate (TMP) monohydrate (Sigma‐Aldrich)
  • Anhydrous diisopropylethylamine (DIPEA; Sigma‐Aldrich)
  • S.3 (see protocol 2)
  • 5% Ethylenediaminetetraacetate (EDTA) disodium salt solution (prepared by dissolving 5 g EDTA disodium in 100 mL buffer A)
  • Buffer A: 50 mM triethylammonium acetate buffer, pH 7 (prepared by adding 7 mL HPLC‐grade triethylamine to 1 L ddH 2O and adjusting the pH to 7 with HPLC‐grade acetic acid)
  • Chloroform
  • Chelex resin (Sigma‐Aldrich)
  • Alkaline phosphatase (Calf Intestinal, New England Biolabs)
  • 25‐, 50‐, and 250‐mL Round‐bottom flasks
  • Magnetic stir plate and stir bar
  • pH meter
  • Rotary evaporator equipped with dry ice‐acetone trap and temperature controlled water bath and attached to a high vacuum pump
  • Lyophilizer
  • Flame‐dried 25‐ and 10‐mL round‐bottom flasks
  • Rubber septa
  • Needle fitted with an argon‐filled balloon
  • Pasteur pipets
  • 50‐mL separatory funnels
  • Cotton
  • 15‐mL conical test tubes
  • Semipreparative or preparative HPLC system equipped with a C18 semi‐preparative or preparative reversed‐phase column and a UV detector set to 255 nm
  • 10‐mL vials
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Figures

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Literature Cited

Literature Cited
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