Chemistry of bisSATE Mononucleotide Prodrugs

Suzanne Peyrottes1, Christian Périgaud1

1 Université Montpellier, Montpellier
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 15.3
DOI:  10.1002/0471142700.nc1503s29
Online Posting Date:  June, 2007
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Abstract

On the basis of AZT as a nucleosidic model, the protocols herein describe the synthesis of various bis(S‐acyl‐2‐thioethyl) phosphotriester derivatives. These compounds, bearing transient phosphate‐protecting groups, were designed to liberate the corresponding 5′‐mononucleotide inside the cell through an esterase‐mediated activation process. Two synthetic approaches are presented using either phosphoramidite intermediates or esterification of a nucleoside 5′‐monophosphate.

Keywords: phosphotriester; biolabile protecting groups; pronucleotides; antiviral; esterases

     
 
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Table of Contents

  • Basic Protocol 1: Two‐Step Preparation of Sate Moiety
  • Alternate Protocol 1: One‐Pot Preparation of Sate Moiety
  • Basic Protocol 2: Preparation of bis(SATE) Phosphotriester Derivatives of AZT Using Phosphoramidite Intermediates
  • Alternate Protocol 2: Preparation of bis(SATE) Phosphotriester Derivative of AZT from the 5′‐Monophosphate
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Two‐Step Preparation of Sate Moiety

  Materials
  • Pyridine distilled from KOH
  • Acetone/dry ice bath
  • Dihydrogen sulfide gas (H 2S)
  • Pivaloyl chloride (S.1b)
  • 5 N sulfuric acid
  • Diethyl ether
  • Anhydrous Na 2SO 4
  • Thioacid: e.g., thioacetic acid (S.2a), thiopivaloic acid (S.2b), or thiobenzoic acid (S.2c)
  • Toluene
  • 1,8‐Diazabicyclo[5.4.0]undec‐7‐ene (DBU)
  • Iodoethanol
  • Dichloromethane
  • Ethyl acetate
  • 3‐L and 100‐mL separatory funnels
  • Glass funnel with a cotton bud
  • Rotary evaporator
  • High‐vacuum distillation apparatus
  • Additional reagents and equipment for column chromatography ( appendix 3E) and thin‐layer chromatography (TLC; appendix 3D)

Alternate Protocol 1: One‐Pot Preparation of Sate Moiety

  Materials
  • 2‐Mercaptoethanol
  • Anhydrous diethyl ether
  • Sodium hydride (NaH), 80% suspension in oil (Riedel‐de Haën; http://www.riedeldehaen.de/gb/index.html)
  • Pivaloyl chloride or benzoyl chloride
  • Diethyl ether
  • Anhydrous Na 2SO 4
  • 500‐mL separatory funnel
  • Glass funnel with a cotton bud
  • Rotary evaporator
  • High‐vacuum distillation apparatus
  • Additional reagents and equipment for column chromatography ( appendix 3E)

Basic Protocol 2: Preparation of bis(SATE) Phosphotriester Derivatives of AZT Using Phosphoramidite Intermediates

  Materials
  • N,N‐Diisopropylphosphoramidous dichloride (Aldrich)
  • Anhydrous tetrahydrofuran (THF, Fluka)
  • Argon source
  • Acetone/dry ice bath
  • Thioester precursor (S.3a‐c; see protocol 1 or protocol 2)
  • Triethylamine (TEA) distilled from CaH 2
  • Cyclohexane
  • Ethyl acetate (EtOAc)
  • TLC spray reagents:
    • 5% (v/v) sulfuric acid (H 2SO 4) in ethanol (for nucleosidic derivatives)
    • Molybden‐blue solution (for phosphorus‐containing derivatives; Sigma or see recipe)
  • Silica gel (Macherey‐Nagel Kieselgel 60): 0.063‐ to 0.20‐mm (for phosphoramidite intermediates) and 0.040‐ to 0.063‐mm (for phosphotriester products)
  • 3′‐Azido‐2′,3′‐dideoxythymidine (AZT)
  • Phosphorus pentoxide (P 2O 5)
  • Acetonitrile distilled from CaH 2
  • 0.45 M 1H‐tetrazole in acetonitrile (Fluka)
  • 3‐Chloroperbenzoic acid
  • Dichloromethane
  • 10% (w/v) aq. sodium sulfite (NaHSO 3)
  • Saturated aq. sodium bicarbonate (NaHCO 3)
  • Anhydrous sodium sulfate (Na 2SO 4)
  • Toluene
  • Methanol
  • TLC plate: silica‐coated aluminum plate with fluorescent indicator (Merck silica gel 60 F 254)
  • Cotton bud
  • Glass funnel
  • 3 × 10– and 3 × 20–cm chromatography columns
  • Vacuum desiccator
  • Rotary evaporator equipped with a vacuum pump
  • 100‐mL separatory funnel
  • Additional reagents and equipment for column chromatography ( appendix 3E) and thin‐layer chromatography (TLC; appendix 3D)

Alternate Protocol 2: Preparation of bis(SATE) Phosphotriester Derivative of AZT from the 5′‐Monophosphate

  Materials
  • 3′‐Azido‐2′,3′‐dideoxythymidine (AZT; S.6)
  • Pyridine
  • Trimethyl phosphate distilled from BaO
  • Phosphorus oxychloride, distilled
  • 1 M triethylammonium bicarbonate (TEAB) buffer, pH 7 (see recipe)
  • Toluene
  • Diethyl ether
  • DEAE‐Sephadex A25 gel
  • Dowex 50WX2 ion‐exchange resin (H+ form)
  • TLC spray reagents:
    • 5% (v/v) sulfuric acid (H 2SO 4) in ethanol (for nucleosidic derivatives)
    • Molybden‐blue solution (for phosphorus containing derivatives; Sigma or see recipe)
  • S‐Pivaloyl‐2‐thioethanol (S.3b; see protocol 1 or protocol 2)
  • Triisopropylbenzenesulfonyl chloride (TPS‐Cl)
  • Aq. sodium bicarbonate (NaHCO 3)
  • Dichloromethane
  • Anhydrous Na 2SO 4
  • Methanol
  • 100‐mL and 500‐mL separatory funnels
  • Rotary evaporator
  • 3 × 20–cm low‐pressure chromatography column
  • Lyophilizer
  • 1 × 20– and 5 × 30–cm chromatography columns
  • Vacuum oil pump
  • Additional reagents and equipment for thin‐layer chromatography (TLC; appendix 3D)
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Figures

Videos

Literature Cited

Literature Cited
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