O6‐(Benzotriazol‐1‐yl)inosine Derivatives for C6 Modification of Purine Nucleosides

Suyeal Bae1, Surendra Chaturvedi2, Mahesh K. Lakshman1

1 The City College and The City University of New York, New York, 2 PrimeSyn Lab, Hillsborough, New Jersey
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 1.22
DOI:  10.1002/0471142700.nc0122s36
Online Posting Date:  March, 2009
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Abstract

A new class of reactive nucleosides, O6‐(benzotriazol‐1‐yl) derivatives of inosine and 2′‐deoxyinosine, have been developed via reaction of silyl‐protected or unprotected inosine and 2′‐deoxyinosine with 1H‐benzotriazol‐1‐yloxy‐tris(dimethylamino)phosphonium hexafluorophosphate (BOP). Alternatively, the silyl‐protected O6‐(benzotriazol‐1‐yl) derivatives can be synthesized via reaction of protected inosine and 2′‐deoxyinosine with triphenylphosphine/iodine/1‐hydroxybenzotriazole. These new O6‐(benzotriazol‐1‐yl) inosine derivatives are excellent reagents for the synthesis of other nucleoside analogues via SNAr reaction with a range of nucleophiles. Curr. Protoc. Nucleic Acid Chem. 36:1.22.1‐1.22.23. © 2009 by John Wiley & Sons, Inc.

Keywords: inosine; 2′‐deoxyinosine; BOP; reactive nucleosides; convertible nucleosides; benzotriazolyl

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis of O6‐(Benzotriazol‐1‐yl)Inosine Derivatives and Subsequent C6 Modification
  • Basic Protocol 2: Synthesis of 5′‐O‐DMTr 3′‐O‐Phosphoramidite of O6‐(Benzotriazol‐1‐yl)‐2′‐Deoxyinosine
  • Alternate Protocol 1: Synthesis of Silyl‐Protected O6‐(Benzotriazol‐1‐yl)Inosine Derivatives by Reaction with Triphenylphosphine/Iodine/1‐Hydroxybenzotriazole
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Synthesis of O6‐(Benzotriazol‐1‐yl)Inosine Derivatives and Subsequent C6 Modification

  Materials
  • 2′‐Deoxyinosine (S.1a), ≥98% pure (Transgenomic or Berry & Associates)
  • Inosine (S.1b), 99% (Acros)
  • Anhydrous pyridine, distilled from KOH (stored over KOH)
  • N,N‐Dimethylformamide (DMF), anhydrous (Aldrich)
  • Imidazole, 99% pure (Sigma)
  • tert‐Butyldimethylsilyl chloride (TBDMS‐Cl), 98% pure (Acros)
  • Hexane, ∼95% n‐hexane, HPLC grade (Fisher Scientific)
  • Dichloromethane (CH 2Cl 2), anhydrous: distilled over CaH 2 for reactions and over CaCl 2 for chromatography
  • Sodium sulfate (Na 2SO 4), anhydrous, 99% pure (Spectrum)
  • 1H‐Benzotriazol‐1‐yloxy‐tris(dimethylamino)phosphonium hexafluorophosphate (BOP), ≥98% pure (Chem‐Impex)
  • Tetrahydrofuran (THF): distilled over lithium aluminum hydride (LiAlH 4) and then over sodium
  • Diisopropylethylamine (DIPEA), distilled from CaH 2
  • 200‐ to 300‐mesh silica gel (Natland)
  • Ethyl acetate (EtOAc), HPLC grade (Fisher Scientific)
  • Toluene, distilled over sodium
  • Methanol (MeOH), reagent grade, ACS (Spectrum)
  • Nitrogen gas
  • 1,2‐Dimethoxyethane (DME), anhydrous, 99.5% pure (Aldrich)
  • Bases for reaction with S.3 and S.6:
    • Cesium carbonate (Cs 2CO 3), 99% pure (Aldrich)
    • Potassium phosphate (K 3PO 4), 97% pure (Lancaster)
    • Sodium bicarbonate (NaHCO 3), 100% pure (Fisher Scientific)
  • Nucleophiles for reaction with S.3 and S.6:
    • Methanol (for S.4a)
    • Ethanol (for S.4b)
    • 2‐Propanol (for S.4c and S.5a)
    • Allyl alcohol (for S.4d)
    • Phenol (for S.4e)
    • p‐Nitrophenol (for S.4f and S.5b)
    • m‐Cyanophenol (for S.4g)
    • 1‐Naphthol (for S.4h)
    • 2‐Hydroxyfluorene (for S.4i)
    • 8‐Hydroxyquinoline (for S.4j and S.5c)
    • Morpholine (for S.4k, S.5d, S.7a, and S.8a)
    • Benzylamine (for S.4l and S.5e)
    • Imidazole (for S.4m)
    • Benzyl mercaptan (for S.4n and S.5f)
    • p‐Toluidine (for S.4o, S.5g, S.7b, and S.8b)
    • Hydroquinone (for S.4p)
    • (S)‐N‐Boc‐tyrosine methyl ester (for S.4q; Chem‐Impex)
  • Sodium hydroxide (NaOH), 97% pure (Spectrum)
  • Citric acid, 99.8% pure (J. T. Baker)
  • Saturated aqueous sodium bicarbonate (prepared from NaHCO 3 powder)
  • Diethyl ether (Et 2O)
  • 50‐ and 100‐mL round‐bottom flasks
  • Rotary evaporator equipped with a water aspirator
  • Magnetic stirrer and stir bars
  • Büchner funnel
  • Water aspirator
  • Glass funnel, plugged with cotton
  • Oil pump for vacuum drying
  • TLC plates: 250‐µm silica gel–coated glass plate with fluorescent indicator (for TLC analysis; Analtech) and 2‐mm silica gel–coated 20 × 20–cm glass plate (for purification)
  • 4‐mL clear glass vials with Teflon/rubber‐lined, closed‐top, screw caps (Wheaton)
  • Fraction collector
  • 60°C, 85°C, and 105°C temperature‐controlled sand bath
  • 125‐mL separatory funnel
  • Dual‐wavelength UV lamp (254 and 365 nm; for TLC analysis)
  • Additional reagents and equipment for performing thin‐layer chromatography (TLC; appendix 3D) and silica gel column chromatography ( appendix 3E)
NOTE: Except where indicated above, all reagents were obtained from commercial sources and used without further purification.

Basic Protocol 2: Synthesis of 5′‐O‐DMTr 3′‐O‐Phosphoramidite of O6‐(Benzotriazol‐1‐yl)‐2′‐Deoxyinosine

  Materials
  • 2′‐Deoxyinosine (S.1a), ≥98% pure (Transgenomic or Berry & Associates)
  • Anhydrous pyridine, distilled from KOH (stored over KOH)
  • 4,4′‐Dimethoxytrityl chloride (DMTr‐Cl), 98% pure (Chem‐Impex)
  • 200‐ to 300‐mesh silica gel (Natland)
  • Triethylamine (Et 3N), distilled from CaH 2
  • Methanol (MeOH), HPLC grade (Spectrum)
  • CH 2Cl 2: distilled over CaH 2 for reactions and over CaCl 2 for chromatography
  • 1H‐Benzotriazol‐1‐yloxy‐tris(dimethylamino)phosphonium hexafluorophosphate (BOP), ≥98% pure (Chem‐Impex)
  • Tetrahydrofuran (THF), distilled over lithium aluminum hydride (LiAlH 4) and then over sodium
  • Diisopropylethylamine (DIPEA), distilled from CaH 2
  • Ethyl acetate (EtOAC), HPLC grade (Fisher Scientific)
  • Sodium sulfate (Na 2SO 4), anhydrous, 99% (Spectrum)
  • Hexane, ∼95% n‐hexane, HPLC grade (Fisher Scientific)
  • 2‐Cyanoethyl N,N‐diisopropylchlorophosphoramidite, Cl 14%‐15.7% (Aldrich)
  • Saturated aqueous sodium bicarbonate (prepared from NaHCO 3 powder)
  • 50‐ and 100‐mL round‐bottom flasks
  • Rotary evaporator
  • Magnetic stirrer and stir bars
  • Balloon (filled with nitrogen gas)
  • Fraction collector
  • TLC plates: 250‐µm silica‐coated glass plate with fluorescent indicator
  • 4‐mL clear glass vials with Teflon/rubber‐lined, closed‐top, screw caps (Wheaton)
  • Inflatable glove bag (e.g., Atmosbag with tape‐seal closure; Aldrich), filled with nitrogen gas
  • 125‐mL separatory funnel
  • Additional reagents and equipment for performing thin‐layer chromatography (TLC; appendix 3D) and silica gel column chromatography ( appendix 3E)

Alternate Protocol 1: Synthesis of Silyl‐Protected O6‐(Benzotriazol‐1‐yl)Inosine Derivatives by Reaction with Triphenylphosphine/Iodine/1‐Hydroxybenzotriazole

  • Triphenylphosphine (PPh 3), 99% (Sigma‐Aldrich)
  • Iodine (I 2), >99% (J.T. Baker)
  • 1‐Hydroxybenzotriazole anhydrous (HOBt), ≥99% (Chem‐Impex)
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Figures

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Literature Cited

Literature Cited
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