Universal 2‐(4‐Nitrophenyl)ethyl and 2‐(4‐Nitrophenyl)ethoxycarbonyl Protecting Groups for Nucleosides and Nucleotides

Wolfgang Pfleiderer1

1 Konstanz University, Konstanz
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 2.13
DOI:  10.1002/0471142700.nc0213s30
Online Posting Date:  September, 2007
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Abstract

A universal blocking group strategy for nucleobases is described, using the 2‐(4‐nitrophenyl)ethyl (NPE) group for O4‐T‐, O4‐U‐, O6‐dG‐, and O6‐G‐protection as well as the 2‐(4‐nitrophenyl)ethoxycarbonyl (NPEOC) group for amino protection in dC, C, dA, A, dG, and G. Conversion into the corresponding 5′‐O‐dimethoxytrityl derivatives and subsequent phosphitylation to form the fully protected 3′‐O‐(2‐cyanoethyl‐N,N‐diisopropylphosphoramidites) and 3′‐O‐(2‐(4‐nitrophenyl)ethyl‐N,N‐diisopropylphosphoramidites) produces a new class of interesting building blocks for oligonucleotide synthesis. Curr. Protoc. Nucleic Acid Chem. 30:2.13.1‐2.13.25. © 2007 by John Wiley & Sons, Inc.

Keywords: NPE and NPEOC protection; Mitsunobu reaction; dimethoxytritylation; phosphitylation; fully protected building blocks

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: O4‐p‐NPE Protection of Thymidine and Uridine
  • Basic Protocol 2: N4‐p‐NPEOC Protection of 2′‐Deoxycytidine and Cytidine
  • Basic Protocol 3: N6‐p‐NPEOC Protection of Acetylated 2′‐Deoxyadenosine and Adenosine
  • Alternate Protocol 1: Mono‐N6‐p‐NPEOC Protection of Acetylated 2′‐Deoxyadenosine and Adenosine
  • Alternate Protocol 2: N6‐p‐NPEOC Protection of Trimethylsilylated 2′‐Deoxyadenosine
  • Basic Protocol 4: O6‐p‐NPE Protection of Acylated 2′‐Deoxyguanosine and Guanosine Derivatives
  • Basic Protocol 5: N2‐p‐NPEOC Protection of 2′‐Deoxyguanosine
  • Basic Protocol 6: O6‐p‐NPE and N2‐p‐NPEOC Protection of 2′‐Deoxyguanosine and Guanosine
  • Basic Protocol 7: Preparation of NPE‐ and NPEOC‐Protected 2′‐Deoxyribonucleoside 3′‐O‐Phosphoramidites
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: O4‐p‐NPE Protection of Thymidine and Uridine

  Materials
  • Sodium iodide (NaI)
  • Ethyl methyl ketone
  • 2‐(4‐Nitrophenyl)ethyl chloride
  • Acetone
  • Chloroform (CHCl 3)
  • 1% aqueous sodium hydrosulfite (Na 2S 2O 3)
  • Anhydrous sodium sulfate (Na 2SO 4)
  • Diethyl ether
  • 3′,5′‐Di‐O‐acetylthymidine (S.1; Beltz and Visser, ) or 2′,3′,5′‐tri‐O‐acetyluridine (S.2; Brown et al., )
  • Absolute toluene
  • Silver carbonate (Ag 2CO 3)
  • Silica gel 60 (220 to 440 mesh; Fluka)
  • n‐Hexane
  • 1,4‐Dioxane
  • Saturated ammonia in MeOH
  • Ethyl acetate (EtOAc)
  • Methanol (MeOH)
  • 100‐ and 250‐mL round‐bottom flasks
  • Reflux condensers
  • Oil bath
  • G4 glass filters
  • Rotary evaporator equipped with water aspirator
  • Filter paper
  • 30 × 4–cm and 11 × 3–cm chromatography columns
  • 20 × 20 × 0.2–cm TLC silica‐gel plates (silica gel PF60 254, Fluka)
  • Silica‐coated TLC‐plate with fluorescent indicator
  • UV light (254 nm)
  • Vacuum desiccator with P 2O 5

Basic Protocol 2: N4‐p‐NPEOC Protection of 2′‐Deoxycytidine and Cytidine

  Materials
  • 2‐(4‐Nitrophenyl)ethanol (Sigma‐Aldrich)
  • Methylenechloride (CH 2Cl 2, analytical grade), cold
  • 1‐(Chlorocarbonyl)benztriazole
  • Triethylamine
  • Anhydrous sodium sulfate (Na 2SO 4)
  • Benzene (analytical grade)
  • Cytidine or 2′‐deoxycytidine (Sigma‐Aldrich, Pharma Waldhof)
  • 1‐[2‐(4‐Nitrophenyl)ethoxycarbonyl]benztriazole
  • N,N‐Dimethylformamide (DMF, anhydrous)
  • 100‐ and 250‐mL round‐bottom flasks
  • Reflux condenser
  • 60°C oil bath
  • G4 glass filters
  • 50°C oven

Basic Protocol 3: N6‐p‐NPEOC Protection of Acetylated 2′‐Deoxyadenosine and Adenosine

  Materials
  • Toluene (anhydrous)
  • Phosgene
  • 2‐(4‐Nitrophenyl)ethanol (Sigma‐Aldrich)
  • Methylenechloride (CH 2Cl 2)
  • 3′,5′‐Di‐O‐acetyl‐2′‐deoxyadenosine (S.13; Hayes et al., ) or 2′,3′,5′‐tri‐O‐acetyladenosine (S.14; Bredereck, )
  • Pyridine (anhydrous)
  • Chloroform (CHCl 3)
  • Phosphate buffer, pH 7
  • Na 2SO 4
  • Toluene
  • Silica gel
  • Methanol (MeOH)
  • Concentrated aqueous ammonia
  • Ethanol (EtOH)
  • 500‐mL three‐neck round‐bottom flasks
  • 50°C oil bath
  • Condenser with drying tube
  • Gas inlet tubes
  • 20 × 4–cm chromatography column
  • 250‐ and 500‐mL round‐bottom flasks
  • Rotatory evaporator

Alternate Protocol 1: Mono‐N6‐p‐NPEOC Protection of Acetylated 2′‐Deoxyadenosine and Adenosine

  Materials
  • 2‐(4‐Nitrophenyl)ethyl chloroformate (see protocol 3)
  • Methylenechloride (CH 2Cl 2 anhydrous)
  • N‐Methylimidazole
  • N 2 atmosphere
  • 3′,5′‐Di‐O‐acetyl‐2′‐deoxyadenosine (S.13; Hayes et al., ) or 2′,3′,5′‐tri‐O‐acetyladenosine (S.14; Bredereck, )
  • 1,2‐Dichloroethane
  • Silica gel
  • Chloroform (CHCl 3)
  • Methanol (MeOH)
  • Ethyl acetate (EtOAc)
  • P 2O 5
  • Triethylamine
  • Ethanol
  • 100‐ and 500‐mL round‐bottom flasks
  • Reflux condensers
  • Drying tube
  • G4 glass filters
  • High‐vacuum pump
  • Drying pistol
  • Rotatory evaporator
  • Chromatography column (8 × 30–cm)
  • Silica‐coated TLC plate with fluorescent indicator
  • 50°C oven

Alternate Protocol 2: N6‐p‐NPEOC Protection of Trimethylsilylated 2′‐Deoxyadenosine

  Materials
  • 2′‐Deoxyadenosine or adenosine (Sigma‐Aldrich, Pharma Waldhof)
  • Dioxane (anhydrous)
  • Hexamethyldisilazane (Sigma‐Aldrich)
  • Ammonium sulfate ((NH 4) 2SO 4)
  • Toluene (anhydrous)
  • Methylenechloride (CH 2Cl 2, anhydrous)
  • 1‐Methyl‐3‐(2‐(4‐nitrophenyl)ethoxycarbonyl)imidazolium chloride
  • Triethylamine
  • Methanol (MeOH)
  • 100‐mL round‐bottom flasks
  • Reflux condenser with drying tube
  • 120°C oil bath
  • G4 glass filters
  • Rotatory evaporator
  • 50°C drying oven

Basic Protocol 4: O6‐p‐NPE Protection of Acylated 2′‐Deoxyguanosine and Guanosine Derivatives

  Materials
  • N2,3′,5′‐Triisobutyryl‐2′‐deoxyguanosine (S.23; Flockerzi et al., ), N2,2′,3′,5′‐tetraisobutyrylguanosine (S.24; Pharma Waldhof), or N2,2′,3′,5′‐tetrabenzoylguanosine (S.25; Himmelsbach et al., )
  • Triphenylphosphine
  • 2‐(4‐Nitrophenyl)ethanol (Sigma‐Aldrich)
  • 1,2‐Dioxane (anhydrous)
  • Diethyl azodicarboxylate (DEAD; Sigma‐Aldrich)
  • Chloroform (CHCl 3)
  • Methanol (MeOH)
  • n‐Hexane
  • Tetrachloromethane (CCl 4)
  • Conc. aqueous ammonia
  • Saturated methanolic ammonia
  • Ethanol (EtOH)
  • Silica‐gel PF 60 254 (Sigma‐Aldrich)
  • Toluene
  • Ethyl acetate (EtOAc)
  • Silica gel 60, 220 to 440 mesh (Sigma‐Aldrich)
  • 100‐ and 250‐mL round‐bottom flasks
  • G4 glass filters
  • 60 × 2.5–cm and 20 × 5–cm chromatography columns
  • Preparative silica‐gel plates (20 × 20 × 0.2–cm)
  • Silica‐coated TLC plate with fluorescent indicator
  • Rotatory evaporator
  • Drying pistol
  • 50°C oven

Basic Protocol 5: N2‐p‐NPEOC Protection of 2′‐Deoxyguanosine

  Materials
  • 2′‐Deoxyguanosine (S.36; Sigma‐Aldrich, Pharma Waldhof)
  • Pyridine (anhydrous)
  • Trimethylsilyl chloride (Sigma‐Aldrich)
  • 2‐(4‐Nitrophenyl)ethyl chloroformate (see protocol 3)
  • Chloroform (CHCl 3)
  • Methanol (MeOH)
  • 10% aqueous sodium bicabonate solution (NaHCO 3)
  • Diethyl ether
  • Silica‐coated TLC plate with fluorescent indicator
  • Rotatory evaporator
  • Drying pistol
  • Vacuum pump

Basic Protocol 6: O6‐p‐NPE and N2‐p‐NPEOC Protection of 2′‐Deoxyguanosine and Guanosine

  Materials
  • 3′,5′‐Di‐O‐acetyl‐2′‐deoxyguanosine (S.38; Schaller et al., ) or 2′,3′,5′‐tri‐O‐acetylguanosine (S.39; Pharma Waldhof)
  • Triphenylphosphine
  • 2‐(4‐Nitrophenyl)ethanol (Sigma‐Aldrich)
  • Dioxane (anhydrous)
  • Diethyl azodicarboxylate (Sigma‐Aldrich)
  • Pyridine (anhydrous)
  • 2‐(4‐Nitrophenyl)ethyl chloroformate (see protocol 3)
  • Chloroform (CHCl 3; anhydrous)
  • Sodium sulfate (Na 2SO 4)
  • Toluene
  • Silica gel (Merck 60)
  • Methylenechloride (CH 2Cl 2)
  • Methanol (MeOH)
  • Conc. aqueous ammonia
  • 100‐mL round‐bottom flasks
  • Rotary evaporator
  • Chromatography column (20 × 2.5–cm)
  • Silica‐coated TLC plate with fluorescent indicator
  • Drying pistol, 50°C

Basic Protocol 7: Preparation of NPE‐ and NPEOC‐Protected 2′‐Deoxyribonucleoside 3′‐O‐Phosphoramidites

  Materials
  • Protected nucleoside:
    • O4‐NPE‐thymidine (S.5)
    • N4‐NPEOC‐2′‐deoxycytidine (S.10)
    • N6‐NPEOC‐2′‐deoxyadenosine (S.19)
    • N2‐NPEOC‐2′‐deoxyguanosine (S.40)
    • N2‐NPEOC‐O6‐NPE‐2′‐deoxyguanosine (S.41)
  • Pyridine (anhydrous)
  • 4,4′‐Dimethoxytrityl chloride (Sigma‐Aldrich)
  • Methanol (MeOH)
  • Methylenechloride (CH 2Cl 2, anhydrous and acid‐free)
  • Sodium sulfate (Na 2SO 4)
  • Toluene
  • Silica‐gel (Merck 60, 63 to 200 mesh)
  • Chloroform (CHCl 3)
  • Triethylamine (Et 3N)
  • 2‐Cyanoethyl‐N,N,N′,N′‐tetraisopropylphosphordiamidite (Sigma‐Aldrich)
  • 1H‐Tetrazole (Sigma‐Aldrich)
  • Argon pressure bottle
  • Saturated NaHCO 3 solution
  • Ethyl acetate (EtOAc)
  • Trichlorophosphane (PCl 3, freshly distilled)
  • Diethyl ether (anhydrous)
  • Nitrogen pipeline
  • 2‐(4‐Nitrophenyl)ethanol (Sigma‐Aldrich)
  • N,N‐Diisopropylamine
  • Isopropanol
  • N,N‐Diisopropyltrimethylsilylamine (Sigma‐Aldrich)
  • 50‐ and 100‐mL round‐bottom flasks
  • Rotatory evaporator
  • Chromatography columns (40 × 2.5–cm, 50 × 2.5–cm, and 60 × 2.5–cm)
  • 100‐ and 250‐mL three‐neck round‐bottom flasks
  • Condenser with drying tube
  • Gas inlet tube
  • G4 glass filter
  • High‐vacuum pump
  • 100‐mL two‐neck round‐bottom flasks
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)
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Figures

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Literature Cited

Literature Cited
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