Synthesis of a 2‐Selenothymidine Phosphoramidite and Its Incorporation into Oligodeoxyribonucleotides

Wen Zhang1, Zhen Huang1

1 Georgia State University, Atlanta, Georgia
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 1.23
DOI:  10.1002/0471142700.nc0123s42
Online Posting Date:  September, 2010
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The detailed synthetic protocol for a 2‐selenothymidine phosphoramidite and its use in preparing Se‐derivatized oligonucleotides are described here. The Se‐modified phosphoramidite synthesis was achieved by activating a 2‐thiothymidine derivative, followed by introduction of selenium functionality. The coupling reaction yield of the 2‐selenothymidine phosphoramidite during solid‐phase synthesis is high (>95%), and the oligonucleotides containing the 2‐selenothymidine derivatization are stable. Curr. Protoc. Nucleic Acid Chem. 42:1.23.1‐1.23.13. © 2010 by John Wiley & Sons, Inc.

Keywords: nucleic acid; selenium; derivatization; base pairing; X‐ray crystallography

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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of the 2‐Selenothymidine Phosphoramidite
  • Support Protocol 1: Synthesis of Iodopropionitrile
  • Basic Protocol 2: Synthesis, Purification, and Characterization of Oligonucleotides Containing 2‐Selenothymidine
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Preparation of the 2‐Selenothymidine Phosphoramidite

  • 4,4′‐Dimethoxytrityl chloride
  • 2‐Thiothymidine ( S.1; ChemGenes, 99.5% pure)
  • 4‐Dimethylaminopyridine (DMAP, Aldrich, purity >99%)
  • Pyridine (Aldrich, anhydrous, purity >99%)
  • Argon
  • Ethyl acetate (EtOAc)
  • Methylene chloride (dichloromethane, CH 2Cl 2; Fluka, purity >99.5%)
  • Methanol (MeOH)
  • MgSO 4 (anhydrous)
  • Silica gel (porosity, 60 Å; particle size, 40 to 63 µm; 230 × 400 mesh)
  • N, N‐Dimethylformamide (DMF; Aldrich, anhydrous, 99% pure)
  • Iodomethane (CH 3I; Aldrich, 99% pure)
  • 1,8‐Diazabicyclo[5.4.0]undec‐7‐ene (DBU; Aldrich, 98% pure)
  • Chloroform (CHCl 3)
  • Selenium (Se; Fluka, 95% pure)
  • Sodium borohydride (NaBH 4; Aldrich, 98% pure)
  • Ethanol (absolute)
  • NaCl, aqueous, saturated
  • Iodopropionitrile (ICH 2CH 2CN; protocol 2)
  • N,N‐Diisopropylethylamine (DIPEA; Aldrich, 99% pure)
  • 2‐Cyanoethyl N,N‐diisopropylchlorophosphoramidite (ChemGenes Corporation)
  • Pentane
  • 25‐, 50‐, and 100‐mL round‐bottom flasks
  • Vacuum oil pump
  • 1‐ and 5‐mL syringes
  • Rubber septum
  • Rotary evaporator
  • Separatory funnels
  • 22 × 457–mm silica gel chromatography columns
  • Stir bar
  • 100‐mL beakers
  • Additional reagents and equipment for performing thin‐layer chromatography ( appendix 3D) and column chromatography ( appendix 3E)

Support Protocol 1: Synthesis of Iodopropionitrile

  • Potassium iodide (Aldrich, 95%)
  • Acetone
  • Argon
  • 3‐Bromopropionitrile (Aldrich, 99%)
  • Ethyl acetate (EtOAc)
  • MgSO 4, anhydrous
  • 100‐mL round‐bottom flasks
  • Stir bar
  • Condenser
  • Heating plate
  • Separatory funnel
  • Rotary evaporator

Basic Protocol 2: Synthesis, Purification, and Characterization of Oligonucleotides Containing 2‐Selenothymidine

  • 2‐Selenothymidine phosphoramidite ( S.6; protocol 1)
  • Acetonitrile (CH 3CN), anhydrous
  • Ultra‐mild phosphoramidites: Pac‐dA‐CE, iPr‐Pac‐dG‐CE, Ac‐dC‐CE, dT‐CE (Glen Research; abbreviations: Ac, acetyl; CE, cyanoethyl; iPr, isopropyl; Pac, phenoxyacetyl)
  • 50 M K 2CO 3 in methanol
  • 2 M triethylammonium acetate (TEAA) buffer, pH 7.0
  • Acetonitrile (CH 3CN), HPLC grade
  • 30% (v/v) trichloroacetic acid (TCA), aqueous
  • Argon
  • 3‐Hydroxypicolinic acid (3‐HPA)
  • Diammonium citrate
  • NaCl
  • NaH 2PO 4
  • Na 2HPO 4
  • EDTA
  • MgCl 2
  • ABI3400 DNA/RNA synthesizer
  • Screw‐cap tubes or vials
  • 13‐mm syringe filter with 0.2‐µm nylon membrane (Life Sciences)
  • RP‐HPLC column: 21.2 × 250–mm Zorbax RX‐C8 (Agilent Technology) or 21 × 250–mm XB‐C18 (Welch Materials;
  • HPLC system with detector at 260 nm
  • Lyophilizer
  • Microcentrifuge tubes
  • Microcentrifuge
  • UV spectrophotometer
  • Additional reagents and equipment for automated oligonucleotide synthesis ( appendix 3C), MALDI‐TOF mass spectrometry (unit 10.1), and determination of UV melting curves (unit 7.3)
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Literature Cited

Literature Cited
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