Use of a Novel 5′‐Regioselective Phosphitylating Reagent for One‐Pot Synthesis of Nucleoside 5′‐Triphosphates from Unprotected Nucleosides

Julianne Caton‐Williams1, Rudiona Hoxhaj1, Bilal Fiaz1, Zhen Huang1

1 Department of Chemistry and Department of Biology, Georgia State University, Atlanta, Georgia
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 1.30
DOI:  10.1002/0471142700.nc0130s52
Online Posting Date:  March, 2013
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5′‐Triphosphates are building blocks for enzymatic synthesis of DNA and RNA. This unit presents a protocol for convenient synthesis of 2′‐deoxyribo‐ and ribonucleoside 5′‐triphosphates (dNTPs and NTPs) from any natural or modified base. This one‐pot synthesis can also be employed to prepare triphosphate analogs with a sulfur or selenium atom in place of a non‐bridging oxygen atom of the α‐phosphate. These S‐ or Se‐modified dNTPs and NTPs can be used to prepare diastereomerically pure phosphorothioate or phosphoroselenoate nucleic acids. Even without extensive purification, the dNTPs or NTPs synthesized by this method are of high quality and can be used directly in DNA polymerization or RNA transcription. Synthesis and purification of the 5′‐triphosphates, as well as analysis and confirmation of natural and sulfur‐ or selenium‐modified nucleic acids, are described in this protocol unit. Curr. Protoc. Nucleic Acid Chem. 52:1.30.1‐1.30.21. © 2013 by John Wiley & Sons, Inc.

Keywords: nucleoside 5′‐triphosphate; sulfur modification; selenium modification; phosphorothioate; phosphoroselenoate; diastereomer

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Table of Contents

  • Introduction
  • Basic Protocol 1: One‐Pot Synthesis of Native Nucleoside 5′‐Triphosphates
  • Basic Protocol 2: One‐Pot Synthesis of Nucleoside 5′‐(α‐P‐Thio)Triphosphates
  • Basic Protocol 3: One‐Pot Synthesis of Nucleoside 5′‐(α‐P‐Seleno)Triphosphates
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: One‐Pot Synthesis of Native Nucleoside 5′‐Triphosphates

  • Starting nucleosides:
    • Adenosine (5a) or 2′‐deoxyadenosine monohydrate (4a) (Sigma‐Aldrich)
    • Cytidine (5c), 2′‐deoxycytidine monohydrochloride (4c), guanosine (5g), 2′‐deoxyguanosine monohydrate (4g), or uridine (5u) (ChemGenes)
    • 2′‐Thymidine (4t), 99% (Alfa Aesar)
  • Tributylammonium pyrophosphate ( 2) (Sigma‐Aldrich)
  • 2‐Chloro‐1,3,2‐benzodioxaphosphorin‐4‐one (salicyl phosphorochloridite, 1, Sigma‐Aldrich)
  • Argon gas (dried)
  • Anhydrous N,N‐dimethylformamide (DMF, Sigma‐Aldrich)
  • Tributylamine (TBA, Sigma‐Aldrich)
  • Anhydrous dimethyl sulfoxide (DMSO, Sigma‐Aldrich)
  • Methanol (MeOH)
  • Dichloromethane (methylene chloride, CH 2Cl 2)
  • Iodine solution (Glen Research)
  • Deionized water
  • Isopropanol
  • Ammonium hydroxide (NH 4OH)
  • 3 M sodium chloride (NaCl)
  • Ethanol (200 proof, KOPTEC)
  • 20 mM triethylammonium acetate (TEAA) buffer, pH 7.1
  • Anhydrous acetonitrile, 99.8% (CH 3CN, Sigma‐Aldrich)
  • Ribonucleoside or 2′‐deoxyribonucleoside 5′‐triphosphate standard (Epicentre)
  • 5‐, 10‐, and 15‐mL oven‐dried, round‐bottom flasks
  • 8 × 1.5−mm magnetic stir bars
  • Rubber septa
  • Parafilm
  • High‐vacuum pump
  • Argon‐filled balloons: connect a deflated balloon to the top end of a 1‐mL syringe (Norm Ject) and seal the connection with Parafilm
  • 1‐ and 3‐mL syringes
  • 23‐G, 1.5‐in. (∼38‐mm) IM needles (Becton Dickinson)
  • Silica‐coated thin‐layer chromatography (TLC) plates with fluorescent indicator Kieselgel 60F 254 (Dynamic Adsorbents and Sorbent Technologies)
  • UV lamp
  • 9‐in. disposable glass pipets (Pasteur pipets)
  • 15‐ or 50‐mL Falcon tubes
  • UV‐vis spectrophotometer
  • Reversed‐phase HPLC system with 21.2 × 250−mm Welchrom (or Ultisil) C18 column
  • Lyophilizer
  • Additional reagents and equipment for thin‐layer chromatography (TLC; appendix 3D)

Basic Protocol 2: One‐Pot Synthesis of Nucleoside 5′‐(α‐P‐Thio)Triphosphates

  • 3‐[(Dimethylaminomethylidene)amino]‐3H‐1,2,4,dithiazole‐3‐thione (sulfurizing reagent II, Glen Research)
  • Pyridine (Sigma‐Aldrich)
  • 20 mM triethylammonium acetate (TEAA) buffer, pH 6.5
  • Guanosine and 2′‐deoxyguanosine 5′‐(α‐P‐thio)triphosphate standards (GTPαS and dGTPαS, TriLinks)
  • 4.6 × 250−mm Welchrom (or Ultisil) C18 RP‐HPLC column
  • Additional reagents and equipment for one‐pot synthesis of native 5′‐triphosphates (see protocol 1)

Basic Protocol 3: One‐Pot Synthesis of Nucleoside 5′‐(α‐P‐Seleno)Triphosphates

  • 3H‐1,2‐Benzothiaselenol‐3‐one (BTSe, SeNA Research)
  • Dioxane (Sigma‐Aldrich)
  • Triethylamine (TEA, Sigma‐Aldrich)
  • 20 mM triethylammonium acetate (TEAA) buffer, pH 6.5
  • 4.6 × 250−mm Welchrom (or Ultisil) C18 RP‐HPLC column
  • Additional reagents and equipment for one‐pot synthesis of native 5′‐triphosphates (see protocol 1)
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