Chemical Phosphorylation of Deoxyribonucleosides and Thermolytic DNA Oligonucleotides

Cristina Ausín1, Andrzej Grajkowski1, Jacek Cieślak1, Serge L. Beaucage1

1 Center for Drug Evaluation and Research, Food and Drug Administration, Bethesda, Maryland
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 13.6
DOI:  10.1002/0471142700.nc1306s26
Online Posting Date:  October, 2006
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Abstract

The phosphorylating reagent bis[S‐(4,4′‐dimethoxytrityl)‐2‐mercaptoethyl]‐N,N‐diisopropylphosphoramidite is prepared in three steps from commercial methyl thioglycolate and diisopropylphosphoramidous dichloride. The phosphorylating reagent has been used successfully in the solid‐phase synthesis of deoxyribonucleoside 5′‐/3′‐phosphate or ‐thiophosphate monoesters and oligonucleotide 5′‐phosphate/‐thiophosphate monoesters. Bis[S‐(4,4′‐dimethoxytrityl)‐2‐mercaptoethyl]‐N,N‐diisopropylphosphoramidite has also been employed in the construction of a thermolytic dinucleotide prodrug model to evaluate the ability of the reagent to produce thermosentive oligonucleotide prodrugs under mild temperature conditions (∼25°C) for potential therapeutic applications.

Keywords: bis[S‐(4,4′‐dimethoxytrityl)‐2‐mercaptoethyl]‐N,N‐diisopropylphosphoramidite; phosphorylation; thermolytic oligonucleotide prodrugs; solid‐phase oligonucleotide synthesis

     
 
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Table of Contents

  • Basic Protocol 1: Preparation of Bis[S‐(4,4′‐Dimethoxytrityl)‐2‐Mercaptoethyl‐N,N‐Diisopropylphosphoramidite
  • Basic Protocol 2: Solid‐Phase Synthesis of 2′‐Deoxyribonucleoside 5′‐/3′‐Phosphate or ‐Thiophosphate Monoesters
  • Alternate Protocol 1: Solid‐Phase Synthesis of Oligonucleotide 5′‐Phosphate/Thiophosphate Monoesters
  • Alternate Protocol 2: Synthesis of a Dinucleotide with a Thermolytic 4‐Thiophosphato‐1‐Butyl Thiophosphate‐Protecting Group
  • Support Protocol 1: Preparation of Deoxyribonucleoside Phosphoramidite S.19
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of Bis[S‐(4,4′‐Dimethoxytrityl)‐2‐Mercaptoethyl‐N,N‐Diisopropylphosphoramidite

  Materials
  • 4,4′‐Dimethoxytrityl chloride (Aldrich)
  • Anhydrous pyridine (Aldrich)
  • Dry argon gas cylinder (Matheson)
  • Methyl thioglycolate (Aldrich)
  • Anhydrous diethyl ether (Fisher)
  • Anhydrous sodium sulfate (Baker)
  • Toluene (Fisher)
  • Anhydrous tetrahydrofuran (Aldrich)
  • Lithium aluminum hydride (LiAlH 4; Aldrich)
  • Ethyl acetate (EM Science)
  • Celite (Aldrich)
  • Hexane (Fisher)
  • Dichloromethane (CH 2Cl 2, EM Science)
  • Triethylamine (Et 3N, Aldrich)
  • Silica gel (60 Å, 230 to 400 mesh; EMD)
  • Diisopropylethylamine (DIPEA; Aldrich)
  • Anhydrous acetonitrile (MeCN; Glen Research)
  • Diisopropylphosphoramidous dichloride (Aldrich)
  • Aqueous saturated sodium hydrogen carbonate (NaHCO 3, Aldrich)
  • Benzene (Aldrich)
  • 25‐, 50‐, 100‐, 250‐, and 500‐mL round‐bottom flasks (Kontes)
  • Rubber septa for 14/20‐ and 24/40‐glass joints (Aldrich)
  • 1‐, 3‐, and 10‐mL plastic syringes (B‐D)
  • 100‐ and 250‐mL separatory funnels (Kontes)
  • 125‐, 250‐, and 500‐mL Erlenmeyer flasks (Kimax)
  • 100‐mm funnels (Nalgene)
  • Filter paper, no. 1 (Whatman)
  • Rotary evaporator (Büchi) connected to a vacuum pump (KNF)
  • 150‐mL sintered‐glass funnel (coarse porosity; Kontes)
  • 500‐mL vacuum Erlenmeyer flask
  • Water aspirator
  • 2.5 × 20–cm disposable Flex chromatography columns (Kontes)
  • 2.5 × 7.5–cm TLC plates precoated with a 250‐µm layer of silica gel 60 F 254 (EMD)
  • Vacuum desiccator
  • High vacuum oil pump (Savant)
  • Additional reagents and equipment for column chromatography ( appendix 3E) and TLC ( appendix 3D)

Basic Protocol 2: Solid‐Phase Synthesis of 2′‐Deoxyribonucleoside 5′‐/3′‐Phosphate or ‐Thiophosphate Monoesters

  Materials
  • Trichloroacetic acid (TCA; Aldrich)
  • Dichloromethane (CH 2Cl 2, EM Science)
  • Synthesis columns filled with succinyl long‐chain alkylamine controlled‐pore glass (Succ‐LCAA‐CPG, 500 Å) support functionalized with a leader 5′‐O‐DMTr‐ or 3′‐O‐DMTr‐2′‐deoxyribonucleoside (T, CBz, ABz and Gi‐Bu, 0.2 µmol; Glen Research)
  • Acetonitrile (MeCN; Acros)
  • Dry argon gas cylinder (Matheson)
  • 0.45 M 1H‐tetrazole in MeCN (Glen Research)
  • 0.1 M S.4 (see protocol 1) in dry MeCN
  • Oxidant:
    • ∼32% wt. ethyl(methyl)dioxirane (2‐butanone peroxide) in dimethyl phthalate (Aldrich; for 5′‐/3′‐phosphate monoesters)
    • 0.05 M 3H‐1,2‐benzodithiol‐3‐one‐1,1‐dioxide in MeCN (for 5′‐/3′‐thiophosphate monoesters)
  • DL‐Dithiothreitol (DTT; Aldrich)
  • Triethylamine (Et 3N; Aldrich)
  • Concentrated ammonium hydroxide (NH 4OH; Fisher Scientific)
  • 2 M triethylammonium acetate (TEAA) buffer, pH 7.0 (Applied Biosystems)
  • 1‐, 3‐, and 10‐mL syringes with Luer tips
  • 20‐G hypodermic needles with Luer‐tip adapters
  • 250‐mL vacuum Erlenmeyer flasks with rubber septa
  • Water aspirator
  • Tygon tubing (Fisher)
  • 4‐mL screw‐capped glass vials (Wheaton), one with rubber septum
  • 1‐mL gas‐tight glass syringe (Hamilton)
  • 55°C heating block
  • 5‐µm Supelcosil LC‐18S reversed‐phase HPLC column (25 cm × 4.6 mm; Supelco)

Alternate Protocol 1: Solid‐Phase Synthesis of Oligonucleotide 5′‐Phosphate/Thiophosphate Monoesters

  • Reagents recommended for manual and automated solid‐phase oligonucleotide synthesis including:
    • Synthesis columns filled with succinyl long‐chain alkylamine controlled‐pore glass (Succ‐LCAA‐CPG) support functionalized with 0.2 µmol 5′‐O‐DMTr‐N4‐benzoyl‐2′‐deoxycytidine (Glen Research)
    • Standard 5′‐O‐DMTr‐2′‐deoxyribonucleoside‐3′‐O‐(2‐cyanoethyl‐N,N‐diisopropyl) phosphoramidites (T, CBz, ABz and Gi‐Bu; Glen Research)
    • Activator solution: 1H‐tetrazole in acetonitrile (Glen Research)
    • Oxidation solution: 0.02 M iodine in THF/pyridine/water (Glen Research)
    • Cap A solution: acetic anhydride in THF/pyridine (Glen Research)
    • Cap B solution: 1‐methylimidazole in THF (Glen Research)
    • Deblocking solution: trichloroacetic acid in dichloromethane (Glen Research)
  • 1 M Tris·Cl buffer, pH 9.0 ( appendix 2A)
  • 1.0 M magnesium chloride (MgCl 2; Sigma)
  • Bacterial alkaline phosphatase (E. coli; Sigma)
  • DNA/RNA synthesizer (Applied Biosystems 392 DNA/RNA synthesizer)
  • 1.5‐mL microcentrifuge tubes
  • 37°C water bath
  • 90°C heating block
  • Additional reagents and equipment for automated DNA synthesis ( appendix 3C), HPLC (unit 10.5), and MALDI‐TOF‐MS (unit 10.1)

Alternate Protocol 2: Synthesis of a Dinucleotide with a Thermolytic 4‐Thiophosphato‐1‐Butyl Thiophosphate‐Protecting Group

  • Deoxyribonucleoside phosphoramidite S.19 (see protocol 5)
  • Acetic acid (Aldrich)
  • Anhydrous pyridine (Aldrich)
  • Hydrazine monohydrate (NH 2NH 2·H 2O; Aldrich)
  • Ammonia gas cylinder (Aldrich)
  • Pressure vessel
  • 90°C heating block
  • Additional reagents and equipment for HPLC (unit 10.5) and MALDI‐TOF‐MS (unit 10.1)

Support Protocol 1: Preparation of Deoxyribonucleoside Phosphoramidite S.19

  • Bis(N,N‐diisopropylamino)chlorophosphine (Aldrich)
  • 4‐Hydroxy‐1‐butyl levulinate (Tsukamoto and Kondo, )
  • 5% (w/v) silver nitrate in 1:1 (v/v) EtOH/H 2O
  • 5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐deoxythymidine (Chem‐Impex International)
  • Sublimed 1H‐tetrazole (Glen Research)
  • Dry‐ice/acetone bath
  • 250‐mL vacuum Erlenmeyer flask
  • 7 × 15–cm glass chromatography column (Ace Glass)
  • Cotton swab
  • Hot plate
  • 1‐ and 10‐mL glass syringe
  • 16‐G needle
  • Lyophilizer
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Figures

Videos

Literature Cited

Literature Cited
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