Solid‐Supported Diphosphitylating and Triphosphitylating Reagents for Nucleoside Modification

Yousef Ahmadibeni1, Keykavous Parang1

1 University of Rhode Island, Kingston, Rhode Island
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 13.8
DOI:  10.1002/0471142700.nc1308s33
Online Posting Date:  June, 2008
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Abstract

This unit describes procedures for synthesis of diphosphitylating and triphosphitylating reagents. The synthesized reagents are first immobilized on appropriate polymer‐bound linkers. Rigid and sterically hindered polymer‐bound diphosphitylating and triphosphitylating reagents are then reacted selectively with the 5′‐hydroxyl group of nucleosides in the presence of excess nucleosides. Typical oxidation with tert‐butyl hydroperoxide, deprotection, and final cleavage of the products from the resins using a trifluoroacetic acid cocktail afford various nucleoside 5′‐O‐diphosphate and nucleoside 5′‐O‐triphosphate analogs. The use of the diphosphitylating and polymer‐bound diphosphitylating reagents in preparation of oligodeoxynucleotides containing diphosphodiester internucleotide bridges is also described. This solid‐phase strategy allows for the synthesis of the phosphorylated compounds without the need for nucleoside phosphate precursors, protected nucleosides, or purification of intermediates. Curr. Protoc. Nucleic Acid Chem. 33:13.8.1‐13.8.29. © 2008 by John Wiley & Sons, Inc.

Keywords: solid‐phase synthesis; diphosphorylation; triphosphorylation; nucleosides; modified oligodeoxynucleotides; diphosphodiester

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis of Polymer‐Bound Linkers Containing p‐Hydroxybenzyl Alcohol
  • Basic Protocol 2: Synthesis and Immobilization of Diphosphitylating Reagent Bis(2‐Cyanoethyl Diisopropylphosphoramidite)
  • Basic Protocol 3: Application of Polymer‐Bound Diphosphitylating Reagents for Synthesis of Nucleoside 5′‐O‐Diphosphates
  • Basic Protocol 4: Synthesis and Immobilization of Triphosphitylating Reagent 2‐Cyanoethyl Phosphoryl‐O,O‐Bis(2‐Cyanoethyl Diisopropylphosphoramidite)
  • Basic Protocol 5: Application of Polymer‐Bound Triphosphitylating Reagents for Synthesis of Nucleoside 5′‐O‐Triphosphates
  • Basic Protocol 6: Application of Diphosphitylating Reagents for Synthesis of Modified Oligodeoxynucleotides Containing Diphosphodiester Internucleotide Linkages
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Synthesis of Polymer‐Bound Linkers Containing p‐Hydroxybenzyl Alcohol

  Materials
  • Aminomethyl polystyrene resin S.1 (100 to 200 mesh, 1% DVB; Novabiochem)
  • Dry nitrogen or argon gas (Airgas Corporation)
  • N,N‐Dimethylformamide (DMF; Fisher Scientific), anhydrous
  • 5‐formylsalicylic acid
  • 1‐Hydroxybenzotriazole (HOBt; Novabiochem)
  • 1,3‐Diisopropylcarbodiimide (DIC; Acros Organics or Sigma‐Aldrich)
  • Dichloromethane (DCM; Acros Organics)
  • Tetrahydrofuran (THF; Fisher Scientific), extra dry
  • Anhydrous methanol (MeOH; EMD‐Merck)
  • Acetic anhydride (Ac 2O; Acros Organics)
  • Anhydrous pyridine
  • Sodium borohydride (NaBH 4; Sigma‐Aldrich)
  • Isopropyl alcohol (Fisher Scientific)
  • 1‐Acetyl‐1H‐1,2,3‐triazolo[4,5‐b]pyridine (Sigma‐Aldrich)
  • 1 N sodium hydroxide (NaOH)
  • 1.0 M borane‐tetrahydrofuran complex solution in THF (Sigma Aldrich)
  • Di‐tert‐butyl dicarbonate (Novabiochem)
  • 100‐ and 250‐mL round‐bottom flasks, oven dried
  • Rubber septa
  • 25‐ and 50‐mL sealed glass syringes
  • Mechanical flask shaker or vibrator
  • 10‐mL (0.8 × 4–cm) conical polypropylene poly‐prep chromatographic columns (9‐cm high; Bio‐Rad cat. no. 731‐1550)
  • Water aspirator (∼20 mmHg)
  • 100‐mL sintered glass filtration devices
  • Vacuum oil pump (up to 10−5 torr)
  • Thermo‐Nicolet 550 FT‐IR spectrophotometer
  • Small rotator with variable speed control (2 to 83 rpm; Glas‐Col cat. no. 099A CR4012)

Basic Protocol 2: Synthesis and Immobilization of Diphosphitylating Reagent Bis(2‐Cyanoethyl Diisopropylphosphoramidite)

  Materials
  • Dry nitrogen gas (Airgas Corporation)
  • Extra‐dry and oxygen‐free tetrahydrofuran (THF; Fisher Scientific)
  • Dry ice/acetone bath
  • Phosphorus trichloride (Sigma‐Aldrich)
  • 2,6‐Lutidine (Alpha Aesar)
  • 3‐Hydroxypropionitrile (Sigma‐Aldrich)
  • Diisopropylamine (Acros Organics)
  • Deuterochloroform (NMR solvent; CDCl 3; Cambridge Isotope)
  • Polymer‐bound p‐acetoxybenzyl alcohol S.4 or polymer‐bound N‐Boc p‐acetoxybenzyl alcohol S.7
  • 1H‐tetrazole
  • Argon gas
  • Dichloromethane (DCM; Acros Organics)
  • Anhydrous methanol (MeOH; EMD‐Merck)
  • 100‐, 250‐, and 500‐mL flat‐bottom flasks
  • Magnetic stir bars and plate
  • Rubber septa
  • Hamilton sealed glass syringes (500 and 250 µL)
  • 20‐G needles
  • Water aspirator (∼20 mmHg)
  • Vacuum oil pump (up to 10−5 torr)
  • 50‐mL sealed glass syringes
  • Cannula pipets
  • Schlenk‐type filtration device or small HPLC microfilter
  • 100‐mL round‐bottom flask
  • Rotary evaporator equipped with a water aspirator
  • Mechanical flask shaker or vibrator
  • 100‐mL sintered glass filtration devices

Basic Protocol 3: Application of Polymer‐Bound Diphosphitylating Reagents for Synthesis of Nucleoside 5′‐O‐Diphosphates

  Materials
  • Polymer‐bound diphosphitylating reagent S.14A or polymer‐bound diphosphitylating reagent S.14B
  • Dry nitrogen or argon gas (Airgas Corporation)
  • Extra‐dry tetrahydrofuran (THF; Fisher Scientific)
  • Dimethyl sulfoxide (DMSO; Alfa Aesar)
  • Nucleosides (R‐OH, a‐d, Fig. )
    • Adenosine (Acros Organics)
    • Uridine (Acros Organics)
    • 2′‐Deoxythymidine (Alfa Aesar)
    • 3′‐Azido‐2′,3′‐dideoxythymidine (AZT; Euroasia's Trans Continental)
  • 1H‐Tetrazole (Acros Organics)
  • Anhydrous methanol (MeOH; EMD‐Merck)
  • 5 to 6 M tert‐butyl hydroperoxide in decane (Sigma‐Aldrich)
  • 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
  • Dioxane (Sigma‐Aldrich)
  • C 18 Sep‐Pak column (300 mg; Altech cat. no. 20924)
  • Sulfuric acid
  • Ethanol (EtOH)
  • DMSO‐d 6 (NMR reagent; Cambridge Isotope)
  • 10‐mL (0.8 × 4–cm) polypropylene conical poly‐prep chromatographic columns (9‐cm high; Bio‐Rad cat. no. 731‐1550)
  • Rubber septa
  • Small rotator with variable speed control (2 to 83 rpm; Glas‐Col cat. no. 099A CR4012)
  • Water aspirator (∼20 mmHg)
  • Vacuum oil pump (up to 10−5 torr)
  • Rotary evaporator equipped with a water aspirator
  • Aluminum silica‐gel TLC plates
  • Heat gun
  • Freeze dryer (lyophilizer)

Basic Protocol 4: Synthesis and Immobilization of Triphosphitylating Reagent 2‐Cyanoethyl Phosphoryl‐O,O‐Bis(2‐Cyanoethyl Diisopropylphosphoramidite)

  Materials
  • Dry nitrogen gas (Airgas Corporation)
  • Extra‐dry and oxygen‐free tetrahydrofuran (THF; Fisher Scientific)
  • Dry ice/acetone bath
  • Phosphorus trichloride (Sigma‐Aldrich)
  • 2,6‐Lutidine (Alpha Aesar)
  • 3‐Hydroxypropionitrile (Sigma‐Aldrich)
  • Diisopropylamine (Acros Organics)
  • Deuterochloroform (NMR solvent; CDCl 3; Cambridge Isotope)
  • 100‐, 250‐, and 500‐mL oven‐dried flat‐bottom flasks
  • Magnetic stir bars
  • Rubber septa
  • Hamilton sealed glass syringes (500 and 250 µL)
  • 20‐G needles
  • Water aspirator (∼20 mmHg)
  • Vacuum oil pump (up to 10−5 torr)
  • 50‐mL sealed glass syringes
  • Cannula pipets
  • Schlenk‐type filtration device or a small HPLC microfilter
  • Rotary evaporator equipped with a water aspirator
  • Additional reagents and equipment for immobilizing on solid support (see protocol 2)

Basic Protocol 5: Application of Polymer‐Bound Triphosphitylating Reagents for Synthesis of Nucleoside 5′‐O‐Triphosphates

  Materials
  • Polymer‐bound triphosphitylating reagent S.14C or S.14D
  • Dry nitrogen or argon gas (Airgas Corporation)
  • Extra‐dry tetrahydrofuran (THF; Fisher Scientific)
  • Dimethyl sulfoxide (DMSO; Alfa Aesar)
  • Nucleosides (R‐OH, a‐d, Fig. ):
    • Adenosine (Acros Organics)
    • Uridine (Acros Organics)
    • 2′‐Deoxythymidine (Alfa Aesar)
    • 3′‐Azido‐2′,3′‐dideoxythymidine (AZT; Euroasia's Trans Continental)
  • 1H‐Tetrazole (Acros Organics)
  • Anhydrous methanol (MeOH; EMD‐Merck)
  • 5 to 6 M tert‐butyl hydroperoxide in decane (Sigma Aldrich)
  • 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
  • Dioxane (Sigma‐Aldrich)
  • C 18 Sep‐Pak columns (Altech cat. no. 20924)
  • Sulfuric acid
  • Ethanol
  • 10‐mL (0.8 × 4–cm) polypropylene conical poly‐prep chromatographic columns (9‐cm high; Bio‐Rad cat. no. 731‐1550)
  • Rubber septa
  • Small rotator with variable speed control (2 to 83 rpm; Glas‐Col cat. no. 099A CR4012)
  • Water aspirator (∼20 mmHg)
  • Vacuum oil pump (up to 10−5 torr)
  • Rotary evaporator equipped with a water aspirator
  • Aluminum silica‐gel TLC plates
  • Heat gun
  • Freeze dryer (lyophilizer)

Basic Protocol 6: Application of Diphosphitylating Reagents for Synthesis of Modified Oligodeoxynucleotides Containing Diphosphodiester Internucleotide Linkages

  Materials
  • Polymer‐bound diphosphitylating reagent S.14A
  • Dry nitrogen or argon gas (Airgas Corporation)
  • Extra‐dry tetrahydrofuran (THF; Fisher Scientific)
  • Dimethyl sulfoxide (DMSO; Alfa Aesar)
  • 2′‐deoxynucleosides (R‐OH, a‐d, Fig. ):
    • 2′‐Deoxyadenosine (dA; MP Biomedicals)
    • 2′‐Deoxyguanosine (dG; TCI America)
    • 2′‐Deoxycytidine (dC; TCI America)
    • Thymidine (dT; Alfa Aesar)
  • 1H‐Tetrazole (Acros Organics)
  • Anhydrous methanol (MeOH; EMD‐Merck)
  • Diphosphitylating reagent S.13
  • 5 to 6 M tert‐butyl hydroperoxide in decane (Sigma Aldrich)
  • 1,8‐Diazabicyclo[5.4.0]undec‐7‐ene (DBU; Acros Organics)
  • Dichloromethane (DCM; Acros Organics)
  • Trifluoroacetic acid (TFA; Acros Organics)
  • 1,2‐Ethanedithiol (EDT; Sigma‐Aldrich)
  • Amberlite AG‐50W‐X8 resin (100 to 200 mesh, H+ form; Fisher Scientific) or a generic alternative
  • Dioxane (Sigma‐Aldrich)
  • Ammonium acetate (Fisher Scientific)
  • Acetonitrile (CH 3CN, HPLC‐grade; Fisher Scientific)
  • Ammonium citrate (Fisher Scientific)
  • 80% acetic acid
  • 10‐mL (0.8 × 4–cm) polypropylene conical poly‐prep chromatographic columns (9‐cm high; Bio‐Rad cat. no. 731‐1550)
  • Rubber septa
  • Small rotator with variable speed control (2‐83 rpm) (Glas‐Col Cat. # 099A CR4012)
  • Water aspirator (∼20 mmHg)
  • Vacuum oil pump (up to 10−5 torr)
  • Rotary evaporator equipped with a water aspirator
  • Freeze dryer (lyophilizer)
  • Phenomenex Prodigy 10‐µm ODS reversed‐phase column (2.1 × 25–cm)
  • Reversed‐phase HPLC
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Literature Cited

Literature Cited
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