Solid‐Supported Reagents for Synthesis of Nucleoside Monothiophosphates, Dithiodiphosphates, and Trithiotriphosphates

Yousef Ahmadibeni1, Keykavous Parang1

1 University of Rhode Island, Kingston, Rhode Island
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 13.9
DOI:  10.1002/0471142700.nc1309s36
Online Posting Date:  March, 2009
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Abstract

This unit describes procedures for the selective synthesis of nucleoside monothiophosphates, dithiodiphosphates, and trithiotriphosphates from solid‐supported phosphitylating reagents. Rigid and sterically hindered polymer‐bound 1,3,2‐oxathiaphospholane is reacted selectively with the 5′‐hydroxyl group of nucleosides in the presence of 1H‐tetrazole. Sulfurization in the presence of Beaucage's reagent (3H‐1,2‐benzodithiole‐3‐one 1,1‐dioxide) followed by ring‐opening with 3‐hydroxypropionitrile and basic cleavage in the presence of 1,8‐diazabicyclo[5.4.0]undec‐7‐ene (DBU) through the elimination of polymer‐bound ethylene episulfide afford nucleoside monothiophosphates. Furthermore, reaction of polymer‐bound diphosphitylating and triphosphitylating reagents, prepared from polymer‐bound benzyl alcohol, with unprotected nucleosides, sulfurization with Beaucage's reagent, and acidic cleavage using trifluoroacetic acid cocktail produce nucleoside dithiodiphosphates and trithiotriphosphates in moderate yields. Curr. Protoc. Nucleic Acid Chem. 36:13.9.1‐13.9.16. © 2009 by John Wiley & Sons, Inc.

Keywords: solid‐phase reagents; nucleosides; monothiophosphorylation; dithiodiphosphorylation; trithiotriphosphorylation; oxathiaphospholane

     
 
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Table of Contents

  • Introduction
  • General Considerations for All Reactions
  • Basic Protocol 1: Preparation of Solid‐Phase Monophosphitylating Reagents
  • Basic Protocol 2: Regioselective Solid‐Phase Monothiophosphorylation of Unprotected Nucleosides
  • Basic Protocol 3: Regioselective Solid‐Phase 5′‐O‐DI‐ and Trithiophosphorylation of Unprotected Nucleosides
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of Solid‐Phase Monophosphitylating Reagents

  Materials
  • NovaSyn Tentagel bromo resin (130‐µm beads, 0.35 mmol/g, Novabiochem) or bromoacetamidomethyl NovaGel resin (S.4, 100 to 200 mesh, 0.64 mmol/g, 1% divinylbenzene [DVB], Novabiochem)
  • Dry nitrogen or argon gas (Airgas Corporation)
  • Anhydrous N‐methyl pyrrolidone (NMP; 1‐methylpyrrolidone; Sigma‐Aldrich)
  • 2,3‐Dimercapto‐1‐propanol (Acros Organics)
  • Potassium carbonate (K 2CO 3, Fisher Scientific)
  • Dichloromethane (DCM; Acros Organics)
  • Anhydrous methanol (MeOH; EMD‐Merck)
  • N,N‐Diisopropylphosphoramidous dichloride (Sigma‐Aldrich)
  • Anhydrous pyridine (EMD‐Merck)
  • 100‐ and 250‐mL round‐bottom flasks, oven dried
  • Rubber septa
  • 50‐mL sealed glass syringes
  • Mechanical flask shaker or vibrator
  • 100‐mL sintered glass filtration devices
  • Water aspirator (∼20 mmHg)
  • Vacuum oil pump (up to 10−5 torr)
  • 10‐mL polypropylene poly‐prep chromatographic columns (9‐cm high, conical 0.8 × 4–cm, Bio‐Rad cat. no. 731‐1550)
  • Small Glass‐Col rotator with variable speed control (2 to 83 rpm, cat. no. 099A CR4012)

Basic Protocol 2: Regioselective Solid‐Phase Monothiophosphorylation of Unprotected Nucleosides

  Materials
  • Polymer‐bound phosphitylating reagent S.3 or S.6 (see protocol 1)
  • Dry nitrogen gas (Airgas Corporation)
  • Dry tetrahydrofuran (THF, Fisher Scientific)
  • Dimethyl sulfoxide (DMSO, Alfa Aesar)
  • Adenosine (Acros Organics)
  • Thymidine (dT, Alfa Aesar)
  • Uridine (Acros Organics)
  • 1H‐Tetrazole (Acros Organics)
  • Anhydrous methanol (MeOH, EMD‐Merck)
  • Anhydrous acetonitrile (CH 3CN, Fisher Scientific)
  • Beaucage's reagent (Transgenomic Bioconsumables)
  • Dichloromethane (DCM, Acros Organics)
  • 1,8‐Diazabicyclo[5.4.0]undec‐7‐ene (DBU, Acros Organics)
  • 3‐Hydroxypropionitrile (Sigma‐Aldrich)
  • Amberlite AG‐50W‐X8 resin (100 to 200 mesh, H+ form, Fisher Scientific) or a generic alternative
  • Dioxane (EMD Merck)
  • C 18 Sep‐Pak column (300 mg, Altech cat. no. 20924)
  • 5:95 (v/v) concentrated sulfuric acid/ethanol
  • 10‐mL polypropylene poly‐prep chromatography columns (9‐cm high, conical 0.8 × 4–cm, Bio‐Rad cat. no. 731‐1550)
  • Rubber septa
  • Small Glass‐Col rotator with variable speed control (2 to 83 rpm, cat. no. 099A CR4012)
  • Water aspirator (∼20 mmHg)
  • Temperature‐controlled shaking incubator (controllable between 4° and 60°C, e.g., New Brunswick Scientific, C24 classic series)
  • Vacuum oil pump (up to 10−5 torr)
  • Rotary evaporator
  • Aluminum silica‐gel TLC plate
  • Heat gun

Basic Protocol 3: Regioselective Solid‐Phase 5′‐O‐DI‐ and Trithiophosphorylation of Unprotected Nucleosides

  Materials
  • Anhydrous acetonitrile (CH 3CN, Fisher Scientific)
  • Beaucage's reagent (Transgenomic Bioconsumables)
  • Extra dry tetrahydrofuran (THF, Fisher Scientific)
  • Anhydrous methanol (MeOH, EMD‐Merck)
  • 1,8‐Diazabicyclo[5.4.0]undec‐7‐ene (DBU, Acros Organics)
  • Dichloromethane (DCM, Acros Organics)
  • Trifluoroacetic acid (TFA, Acros Organics)
  • Amberlite AG‐50W‐X8 resin (100 to 200 mesh, H+ form, Fisher Scientific) or a generic alternative Adenosine (Acros Organics)
  • C 18 Sep‐Pak column (300 mg, Altech, cat. no. 20924)
  • 5:95 (v/v) concentrated sulfuric acid/ethanol
  • 10‐mL polypropylene poly‐prep chromatography columns (9‐cm high, conical 0.8 × 4–cm, Bio‐Rad cat. no. 731‐1550)
  • Small Glass‐Col rotator with variable speed control (2 to 83 rpm, cat. no.099A CR4012)
  • Temperature‐controlled shaking incubator (controllable between 4° and 60°C, e.g., New Brunswick Scientific, C24 classic series)
  • Water aspirator (∼20 mmHg)
  • Vacuum oil pump (up to 10−5 torr)
  • Rotary evaporator equipped with a water aspirator
  • Aluminum silica‐gel TLC plate
  • Heat gun
  • Freeze dryer (lyophilizer)
  • Additional reagents and equipment for preparation of aminomethyl polystyrene–bound nucleoside di‐ and triphosphates (unit 13.8)
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Figures

Videos

Literature Cited

Literature Cited
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   Ahmadibeni, Y. and Parang, K. 2005b. Selective diphosphorylation, dithiodiphosphorylation, triphosphorylation and trithiotriphosphorylation of unprotected nucleosides. Org. Lett. 7:5589‐5592.
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