Nucleobase Protection with Allyloxycarbonyl

Mamoru Hyodo1, Yoshihiro Hayakawa1

1 Nagoya University, Nagoya
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 2.12
DOI:  10.1002/0471142700.nc0212s23
Online Posting Date:  January, 2006
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Abstract

This unit describes protocols for preparation of N‐allyloxycarbonyl‐protected 5′‐O‐(4,4′‐dimethoxytrityl)‐2′‐deoxyribonucleosides and 2′‐O‐(tert‐butyldimethylsilyl)‐5′‐O‐(4,4′‐dimethoxytrityl)ribonucleosides. These provide useful building blocks not only for synthesis of natural oligonucleotides but also for artificial analogs with chemically sensitive (particularly, base‐labile) modified nucleoside bases or internucleotide linkages. These protected nucleosides are stable to conditions used for conversion to the corresponding nucleoside phosphoramidites and subsequent oligonucleotide synthesis, and the N‐allyloxycarbonyl protecting group can be easily and cleanly removed by an organopalladium‐catalyzed reaction under mild, nearly neutral conditions.

Keywords: nucleoside; oligonucleotide; protecting group; allyloxycarbonayl; organopalladium

     
 
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Table of Contents

  • Basic Protocol 1: Synthesis of N6‐Allyloxycarbonyl‐5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐Deoxyadenosine
  • Basic Protocol 2: Synthesis of N4‐Allyloxycarbonyl‐5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐Deoxycytidine
  • Basic Protocol 3: Synthesis of N2‐Allyloxycarbonyl‐5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐Deoxyguanosine
  • Basic Protocol 4: Synthesis of N2‐Allyloxycarbonyl‐O6‐ALLYL‐5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐Deoxyguanosine
  • Basic Protocol 5: Synthesis of N6‐Allyloxycarbonyl‐2′‐O‐(tert‐Butyldimethylsilyl)‐5′‐O‐(4,4′‐Dimethoxytrityl)Adenosine
  • Basic Protocol 6: Synthesis of N4‐Allyloxycarbonyl‐2′‐O‐(tert‐Butyldimethylsilyl)‐5′‐O‐(4,4′‐Dimethoxytrityl)Cytidine
  • Basic Protocol 7: Synthesis of N2‐Allyloxycarbonyl‐O6‐ALLYL‐2′‐O‐(tert‐Butyldimethylsilyl)‐5′‐O‐(4,4′‐Dimethoxytrityl)Guanosine
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Synthesis of N6‐Allyloxycarbonyl‐5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐Deoxyadenosine

  Materials
  • 2′‐Deoxyadenosine ( S.1), 99% (Mitsui Chemicals)
  • Triethylamine, distilled from CaH 2
  • 4‐(Dimethylamino)pyridine (DMAP), 99% (Aldrich, TCI)
  • Pyridine, distilled from CaH 2
  • 4,4′‐Dimethoxytrityl chloride (DMTr‐Cl), 97% (Aldrich, TCI)
  • Ethyl acetate, 99% (Aldrich, Kishida)
  • Sodium sulfate, 99% (Aldrich, Nacarai Tesque)
  • N,N′‐Dimethylformamide (DMF), distilled from 4A molecular sieves
  • Imidazole, 99% (Aldrich, Nacarai Tesque)
  • tert‐Butyldimethylsilyl chloride (TBDMS‐Cl), 99% (Aldrich, Shinetsu)
  • Hexane, 99% (Aldrich, Kishida)
  • Brine: saturated solution of sodium chloride (99%; Aldrich, Wako)
  • Silica gel 60 (spherical, neutrality, particle size 75 µm; Merck, Nacarai Tesque)
  • Methanol, 99% (Aldrich, Kishida)
  • Dichloromethane, 99% (Aldrich, Nacarai Tesque)
  • 1‐(Allyloxycarbonyl)tetrazole (AOC‐Tet; see recipe)
  • Tetrahydrofuran (THF), distilled from sodium benzophenone ketyl
  • Aqueous saturated sodium hydrogencarbonate (NaHCO 3; 99%; Aldrich, Nacarai Tesque)
  • 1.0 M tetrabutylammonium fluoride in THF (Aldrich)
  • Chloroform, 99% (Aldrich, Wako)
  • Glass column
  • Diaphragm pump
  • Rotary evaporator equipped with a diaphragm pump and cooling unit
  • Vacuum oil pump
  • TLC plate: silica‐coated glass plate with fluorescent indicator (Merck silica gel 60 F 254)
  • 254‐nm UV lamp
  • Additional reagents and equipment for column chromatography ( appendix 3E) and TLC ( appendix 3D)

Basic Protocol 2: Synthesis of N4‐Allyloxycarbonyl‐5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐Deoxycytidine

  Materials
  • 2′‐Deoxycytidine hydrochloride ( S.4), 99% (Aldrich, Mitsui Chemicals)
  • Pyridine, distilled from CaH 2
  • Trimethylsilyl chloride (TMS‐Cl), 99% (Aldrich, Shinetsu)
  • Allyl 1‐benzotriazolyl carbonate (AOCOBT; see recipe)
  • Saturated aqueous sodium hydrogencarbonate (NaHCO 3), 99% (Aldrich, Nacarai Tesque)
  • Dichloromethane, 99% (Aldrich, Nacarai Tesque)
  • Sodium sulfate, 99% (Aldrich, Nacarai Tesque)
  • 4,4′‐Dimethoxytrityl chloride (DMTr‐Cl), 97% (Aldrich, TCI)
  • Hexane, 99% (Aldrich, Kishida)
  • Ethyl acetate, 99% (Aldrich, Kishida)
  • Brine: saturated aqueous sodium chloride (99%; Aldrich, Wako)
  • Silica gel 60 (spherical, neutrality, particle size 75 µm; Merck, Nacarai Tesque)
  • Chloroform, 99% (Aldrich, Wako)
  • Methanol, 99% (Aldrich, Kishida)
  • Glass column
  • Diaphragm pump
  • Rotary evaporator equipped with a diaphragm pump and cooling unit
  • Vacuum oil pump
  • TLC plate: silica‐coated glass plate with fluorescent indicator (Merck silica gel 60 F 254)
  • 254‐nm UV lamp
  • Additional reagents and equipment for column chromatography ( appendix 3E) and TLC ( appendix 3D)

Basic Protocol 3: Synthesis of N2‐Allyloxycarbonyl‐5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐Deoxyguanosine

  Materials
  • 2′‐Deoxyguanosine ( S.7), 99% (Aldrich, Mitsui Chemicals)
  • Imidazole, 99% (Aldrich, Nacarai Tesque)
  • N,N′‐Dimethylformamide (DMF), distilled from 4A molecular sieves
  • tert‐Butyldimethylsilyl chloride (TBDMS‐Cl), 99% (Aldrich, Shinetsu)
  • Ethanol, 99.5% (Aldrich, Nacarai Tesque)
  • tert‐Butylmagnesium chloride, 99% (Aldrich)
  • Tetrahydrofuran (THF), distilled from sodium benzophenone ketyl
  • Hexamethylphosphoric triamide (HMPA), 99% (Aldrich)
  • Allyloxycarbonyl chloride (AOC‐Cl), 99% (Aldrich, TCI)
  • Methanol, 99% (Aldrich, Kishida)
  • Diethyl ether, distilled from sodium benzophenone ketyl
  • 0.15 M ethylenediamine tetraacetic acid (EDTA; 99%; Aldrich, Nacarai Tesque)
  • Saturated aqueous sodium hydrogencarbonate (NaHCO 3; 99%; Aldrich, Nacarai Tesque)
  • Brine: saturated aqueous sodium chloride (99%; (Aldrich, Wako)
  • Sodium sulfate, 99% (Aldrich, Nacarai Tesque)
  • Silica gel 60 (spherical, neutrality, particle size 75 µm; Merck, Nacarai Tesque)
  • Ethyl acetate, 99% (Aldrich, Kishida)
  • Hexane, 99% (Aldrich, Kishida)
  • 1.0 M tetrabutylammonium fluoride (TBAF) in THF (Aldrich)
  • Pyridine, distilled from CaH 2
  • Dichloromethane, 99% (Aldrich, Nacarai Tesque)
  • 4,4′‐Dimethoxytrityl chloride (DMTr‐Cl), 97% (Aldrich, TCI)
  • Chloroform, 99% (Aldrich, Wako)
  • Rotary evaporator equipped with a diaphragm pump and cooling unit
  • Glass column
  • Diaphragm pump
  • Vacuum oil pump
  • TLC plate: silica‐coated glass plate with fluorescent indicator (Merck silica gel 60 F 254)
  • 254‐nm UV lamp
  • Additional reagents and equipment for column chromatography ( appendix 3E) and TLC ( appendix 3D)

Basic Protocol 4: Synthesis of N2‐Allyloxycarbonyl‐O6‐ALLYL‐5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐Deoxyguanosine

  Materials
  • N2‐Allyloxycarbonyl‐3′,5′‐di‐O‐(tert‐butyldimethylsilyl)‐2′‐deoxyguanosine ( S.9; see protocol 3)
  • Triethylamine, distilled from CaH 2
  • 4‐(Dimethylamino)pyridine (DMAP), 99% (Aldrich, TCI)
  • Dichloromethane, 99% (Aldrich, Nacarai Tesque)
  • 2‐Mesitylenesulfonyl chloride, 99% (Aldrich, TCI)
  • Saturated aqueous sodium hydrogencarbonate (NaHCO 3; 99%, Aldrich, Nacarai Tesque)
  • Sodium sulfate, 99% (Aldrich, Nacarai Tesque)
  • Trimethylamine, anhydrous gas (Aldrich)
  • Allyl alcohol, distilled from Mg
  • 1,8‐Diazabicyclo[5,4,0]undec‐7‐ene (DBU), 99% (Aldrich, Nacarai Tesque)
  • Brine: saturated aqueous sodium chloride (99%; Aldrich, Wako)
  • Silica gel 60 (spherical, neutrality, particle size 75 µm; Merck, Nacarai Tesque)
  • Ethyl acetate, 99% (Aldrich, Kishida)
  • Hexane, 99% (Aldrich, Kishida)
  • 1.0 M tetrabutylammonium fluoride (TBAF) in THF (Aldrich, Aldrich)
  • Tetrahydrofuran (THF), distilled from sodium benzophenone ketyl
  • Pyridine, distilled from CaH 2
  • 4,4′‐Dimethoxytrityl chloride (DMTr‐Cl), 97% (Aldrich, TCI)
  • Methanol, 99% (Aldrich, Kishida)
  • Chloroform, 99% (Aldrich, Wako)
  • Glass column
  • Diaphragm pump
  • Rotary evaporator equipped with a diaphragm pump and cooling unit
  • Vacuum oil pump
  • TLC plate: silica‐coated glass plate with fluorescent indicator (Merck silica gel 60 F 254)
  • 254‐nm UV lamp
  • Additional reagents and equipment for column chromatography ( appendix 3E) and TLC ( appendix 3D)

Basic Protocol 5: Synthesis of N6‐Allyloxycarbonyl‐2′‐O‐(tert‐Butyldimethylsilyl)‐5′‐O‐(4,4′‐Dimethoxytrityl)Adenosine

  Materials
  • Hexamethyldisilazane (HMDS), 99% (Aldrich, Shinetsu)
  • Adenosine ( S.13), 99% (Sigma)
  • Ammonium sulfate, 99% (Aldrich, Wako)
  • 1,4‐Dioxane, distilled from sodium benzophenone ketyl
  • Toluene, distilled from sodium benzophenone ketyl
  • Dichloromethane, 99% (Aldrich, Nacarai Tesque)
  • N‐Methylimidazole (Aldrich, Nacarai Tesque)
  • Allyloxycarbonyl chloride (AOC‐Cl), 99% (Aldrich, TCI)
  • 1.0 M phosphate buffer, pH 7
  • Sodium sulfate, 99% (Aldrich, Nacarai Tesque)
  • Methanol, 99% (Aldrich, Kishida)
  • Triethylamine, distilled from CaH 2
  • Diethyl ether, 99% (Aldrich, Nacarai Tesque)
  • Diphosphorus pentoxide (P 2O 5), 98% (Aldrich, Nacarai Tesque)
  • Pyridine, distilled from CaH 2
  • N,N′‐Dimethylformamide (DMF), distilled from 4A molecular sieves
  • 4,4′‐Dimethoxytrityl chloride (DMTr‐Cl), 97% (Aldrich, TCI)
  • Silica gel 60 (spherical, neutrality, particle size 75 µm; Merck, Nacarai Tesque)
  • Ethyl acetate, 99% (Aldrich, Kishida)
  • Hexane, 99% (Aldrich, Kishida)
  • Chloroform, 99% (Aldrich, Wako)
  • Imidazole, 99% (Aldrich, Nacarai Tesque)
  • tert‐Butyldimethylsilyl chloride (TBDMS‐Cl), 99% (Aldrich, Shinetsu)
  • Glass column
  • Diaphragm pump
  • Reflux condenser
  • Rotary evaporator equipped with a diaphragm pump and cooling unit
  • Vacuum oil pump
  • TLC plate: silica‐coated glass plate with fluorescent indicator (Merck silica gel 60 F 254)
  • 254‐nm UV lamp
  • Additional reagents and equipment for column chromatography ( appendix 3E) and TLC ( appendix 3D)

Basic Protocol 6: Synthesis of N4‐Allyloxycarbonyl‐2′‐O‐(tert‐Butyldimethylsilyl)‐5′‐O‐(4,4′‐Dimethoxytrityl)Cytidine

  Materials
  • Hexamethyldisilazane (HMDS), 99% (Aldrich, Shinetsu)
  • Cytidine ( S.17), 99% (Sigma)
  • Ammonium sulfate, 99% (Aldrich, Wako)
  • 1,4‐Dioxane, distilled from sodium benzophenone ketyl
  • Toluene, distilled from sodium benzophenone ketyl
  • Dichloromethane, 99% (Aldrich, Nacarai Tesque)
  • N‐Methylimidazole, (Aldrich, Nacarai Tesque)
  • Allyloxycarbonyl chloride (AOC‐Cl), 99% (Aldrich, TCI)
  • Methanol, 99% (Aldrich, Kishida)
  • Triethylamine, distilled from CaH 2
  • Ethyl acetate, 99% (Aldrich, Kishida)
  • Diphosphorus pentoxide (P 2O 5), 98% (Aldrich, Nacarai Tesque)
  • Pyridine, distilled from CaH 2
  • N,N′‐Dimethylformamide (DMF), distilled from 4A molecular sieves
  • 4,4′‐Dimethoxytrityl chloride (DMTr‐Cl), 97% (Aldrich, TCI)
  • Silica gel 60 (spherical, neutrality, particle size 75 µm; Merck, Nacarai Tesque)
  • Imidazole, 99% (Aldrich, Nacarai Tesque)
  • tert‐Butyldimethylsilyl chloride (TBDMS‐Cl), 99% (Aldrich, Shinetsu)
  • Sodium sulfate, 99% (Aldrich, Nacarai Tesque)
  • Hexane, 99% (Aldrich, Kishida)
  • Rotary evaporator equipped with a diaphragm pump and cooling unit.
  • Vacuum oil pump
  • TLC plate: silica‐coated glass plate with fluorescent indicator (Merck silica gel 60 F 254)
  • 254‐nm UV lamp
  • Glass column
  • Diaphragm pump
  • Additional reagents and equipment for column chromatography ( appendix 3E) and TLC ( appendix 3D)

Basic Protocol 7: Synthesis of N2‐Allyloxycarbonyl‐O6‐ALLYL‐2′‐O‐(tert‐Butyldimethylsilyl)‐5′‐O‐(4,4′‐Dimethoxytrityl)Guanosine

  Materials
  • Guanosine ( S.21), 99% (Aldrich)
  • Pyridine, distilled from CaH 2
  • N,N′‐Dimethylformamide (DMF), distilled from 4A molecular sieves
  • Acetic anhydride, 99% (Aldrich, Kishida)
  • 2‐Propanol, 99% (Aldrich, Kishida)
  • Triphenylphosphine, recrystallized from hexane
  • Allyl alcohol, distilled from Mg
  • 1,4‐Dioxane, distilled from sodium benzophenone
  • Diethyl azodicarboxylate (DEAD), 40% solution in toluene (Aldrich, TCI)
  • Dichloromethane, 99% (Aldrich, Nacarai Tesque)
  • Silica gel 60 (spherical, neutrality, particle size 75 µm; Merck, Nacarai Tesque)
  • Ethyl acetate, 99% (Aldrich, Kishida)
  • Hexane, 99% (Aldrich, Kishida)
  • Tetrahydrofuran (THF), distilled from sodium benzophenone ketyl
  • Allyloxycarbonyl chloride (AOC‐Cl), 99% (Aldrich, TCI)
  • tert‐Butylmagnesium chloride, 99% (Aldrich)
  • Methanol, 99% (Aldrich, Kishida)
  • Saturated aqueous ammonium chloride (99%; Aldrich, Kishida)
  • Aqueous saturated sodium hydrogencarbonate (NaHCO 3; 99%; Aldrich, Nacarai Tesque)
  • Brine: aqueous saturated sodium chloride
  • Ethanol, 99.5% (Aldrich, Nacarai Tesque)
  • Sodium hydroxide
  • 80% (v/v) aqueous acetic acid
  • 4,4′‐Dimethoxytrityl chloride (DMTr‐Cl), 97% (Aldrich, TCI)
  • Sodium sulfate, 99% (Aldrich, Nacarai Tesque)
  • Imidazole, 99% (Aldrich, Nacarai Tesque)
  • tert‐Butyldimethylsilyl chloride (TBDMS‐Cl), 99% (Aldrich, Shinetsu)
  • Triethylamine, distilled from CaH 2
  • Vacuum oil pump
  • Rotary evaporator equipped with a diaphragm pump and cooling unit
  • TLC plate: silica‐coated glass plate with fluorescent indicator (Merck silica gel 60 F 254)
  • 254‐nm UV lamp
  • Glass column
  • Diaphragm pump
  • Additional reagents and equipment for column chromatography ( appendix 3E) and TLC ( appendix 3D)
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Figures

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Literature Cited

Literature Cited
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