DNA Oligonucleotides Containing Stereodefined Phosphorothioate Linkages in Selected Positions

Barbara Nawrot1, Beata Rebowska1

1 Department of Bioorganic Chemistry, Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Lodz, Poland
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.34
DOI:  10.1002/0471142700.nc0434s36
Online Posting Date:  March, 2009
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This unit describes a method for the synthesis of DNA chimeric PO/PS‐oligonucleotides with a stereodefined phosphorothioate bond in the selected position. Diastereomerically pure 5′‐O‐DMTr‐N‐protected‐deoxyribonucleoside‐3′‐O‐(2‐thio‐spiro‐4,4‐pentamethylene‐1,3,2‐oxathiaphospholane)s obtained according to the previously described protocol (UNIT 4.17) are transformed via a stereospecific 1,3,2‐oxathiaphospholane‐ring opening condensation into the corresponding dinucleoside phosphorothioates. Such dimers cannot be introduced into an oligonucleotide chain via the phosphoramidite approach since the unprotected P‐S bond is easily oxidized during routine I2/Py/water oxidation of the phosphite function. In the methodology described here, the reversible alkylation of the PS function is applied. Subsequently, the 3′‐phosphoramidites of such PS‐protected dimers prepared in situ are used for routine synthesis of chimeric PO/PS‐oligonucleotides according to the phosphoramidite method. The presence of the alkylated PS‐function requires modified conditions for oligonucleotide deprotection and cleavage from the solid support. Detailed procedures for the synthesis of PS‐dimers and their incorporation into an oligonucleotide chain, as well as deprotection/purification steps are presented. Curr. Protoc. Nucleic Acid Chem. 36:4.34.1‐4.34.15. © 2009 by John Wiley & Sons, Inc.

Keywords: phosphorothioate oligonucleotide; PS‐oligonucleotide; stereodefined phosphorothioate; oxathiaphospholane; phosphoramidite

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Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis of Phosphoramidites of Dinucleoside Phosphorothioates from Diastereomerically Pure Nucleoside Oxathiaphospholanes
  • Basic Protocol 2: Synthesis, Deprotection, Isolation, and Characterization of Oligonucleotides Containing Stereodefined Phosphorothioate Bonds
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Synthesis of Phosphoramidites of Dinucleoside Phosphorothioates from Diastereomerically Pure Nucleoside Oxathiaphospholanes

  • Suitably protected deoxyribonucleosides (ChemGenes):
    • N6‐Benzoyl‐5′‐O‐(4,4′‐dimethoxytrityl)‐2′‐deoxyadenosine (5′‐O‐DMTr‐dABz)
    • N4‐Benzoyl‐5′‐O‐(4,4′‐dimethoxytrityl)‐2′‐deoxycytidine (5′‐O‐DMTr‐dCBz)
    • N2‐Isobutyryl‐5′‐O‐(4,4′‐dimethoxytrityl)‐2′‐deoxyguanosine (5′‐O‐DMTr‐dGi‐Bu)
  • Pyridine (Py; C 6H 5N), anhydrous, 99.8% (Aldrich)
  • Argon (or nitrogen) gas, dry
  • Isopropoxyacetyl anhydride (SinoChemexper or prepared according to Uznański et al., )
  • Chloroform (CHCl 3), HPLC grade
  • Silica gel (230 to 400 mesh)
  • Methanol (CH 3OH), HPLC grade
  • Dichloromethane
  • p‐Toluenesulfonic acid monohydrate, 98% (Aldrich)
  • Tetrahydrofurane (THF), HPLC grade, 99.9% (Aldrich)
  • Diastereoisomerically pure 5′‐O‐DMTr‐deoxynucleoside 3′‐O‐(2‐thio‐spiro‐4,4‐pentamethylene‐1,3,2‐oxathiaphospholane (OTP nucleoside S.1; unit 4.17)
  • Acetonitrile (CH 3CN), anhydrous (Fluka)
  • 1,8‐Diazabicyclo[5.4.0]undec‐7‐ene (DBU; Aldrich)
  • 1,4‐Dioxane anhydrous, 99.8% (Fluka)
  • Ammonium hydroxide, 25% (Baker)
  • 2‐Nitrobenzyl bromide, 98% (Aldrich)
  • Tiethylamine (TEA), anhydrous, HPLC grade (Aldrich)
  • Desiccant silica gel (Aldrich)
  • 2‐Cyanoethyl bis‐(N,N‐diisopropyl)phosphoramidite (prepared according to Caruthers, )
  • 5‐Ethylthio‐1H‐tetrazole, 99% (ChemGenes)
  • 50‐mL and 10‐mL round‐bottom flask
  • Rotary evaporator
  • Membrane vacuum pump
  • High‐vacuum oil pump (0.01 mmHg)
  • Magnetic stir bars and plate
  • Glass columns for chromatography:
    • 20 × 2–cm for 10 g silica gel (230 to 400 mesh)
    • 10 × 1.5–cm for 3 g silica gel (230 to 400 mesh)
  • TLC silica gel plates with UV indicator (Merck)
  • 254‐nm UV lamp
  • 3‐mL and 4‐mL vials
  • Black paper or aluminum foil
  • Rubber septum
  • Luer‐lock needle
  • Desiccator
  • Dry, gas‐tight syringe (previously dried over P 2O 5 in a desiccator for 12 hr)
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)

Basic Protocol 2: Synthesis, Deprotection, Isolation, and Characterization of Oligonucleotides Containing Stereodefined Phosphorothioate Bonds

  • 3′‐phosphoroamidite of PS‐protected dimer S.6 (see protocol 1, step 38)
  • Commercial 2‐deoxyribonucleoside 3′‐phosphoramidites: 5′‐O‐(4,4′‐Dimethoxytrityl)‐3′‐O‐[(2‐cyanoethoxy)‐(N,N‐diisopropylamino)]phosphinyl thymidine and N‐protected 2′‐deoxy‐cytidine, adenosine and guanosine (Glen Research)
  • Piperidine (Serva)
  • Acetonitrile (CH 3CN), anhydrous (Fluka)
  • Triethylamine (TEA), anhydrous, HPLC grade (Aldrich)
  • 1,4‐Dioxane anhydrous, 99.8% (Fluka)
  • Thiophenol, 99% (Merck)
  • Ethanol
  • 28% concentrated ammonium hydroxide (Baker; appendix 3C)
  • 20% Ethanolic ammonia
  • 40% aqueous methylamine (Aldrich)
  • Buffer A: 1 M triethylammonium bicarbonate (TEAB), pH 7.5
  • Buffer B: 40% acetonitrile/60% buffer A
  • 50% Aqueous acetic acid
  • 50% aqueous ethanol
  • 20% acrylamide/7 M urea gel
  • High‐vacuum oil pump (0.01 mmHg)
  • 4‐mL screw‐cap vial
  • Heat block
  • Benchtop centrifuge
  • 0.2‐µm filter
  • Speedvac evaporator
  • Analytical RP‐HPLC column (e.g., Econosphere C18, 5 µm, 250 × 4.6–mm, Alltech)
  • Vortex
  • Additional reagents and equipment for oligonucleotide synthesis ( appendix 3C), determining DNA concentration by UV spectroscopy (Brown and Brown, ), denaturing polyacrylamide gel electrophoresis ( appendix 3B), and MALDI‐TOF MS (unit 10.1)
NOTE: Solvents possessing ether bonds, such as dioxane or tetrahydrofuran, may contain peroxides. These are very reactive species, able to oxidize phosphorothioate groups into nonchiral phosphate products. Therefore, it is necessary to use highly pure peroxide‐free solvents. The solvents can be purified from peroxides by 24‐hr storage over KOH pellets, followed by careful distillation over LiAlH 4.
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Literature Cited

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