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

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Abstract
Table of Contents
Materials
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Literature Cited

Abstract

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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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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Materials

Basic Protocol 1: Preparation of the 2‐Selenothymidine Phosphoramidite

Materials

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, CH₂ Cl₂ ; Fluka, purity >99.5%)
Methanol (MeOH)
MgSO₄ (anhydrous)
Silica gel (porosity, 60 Å; particle size, 40 to 63 µm; 230 × 400 mesh)
N , N ‐Dimethylformamide (DMF; Aldrich, anhydrous, 99% pure)
Iodomethane (CH₃ I; Aldrich, 99% pure)
1,8‐Diazabicyclo[5.4.0]undec‐7‐ene (DBU; Aldrich, 98% pure)
Chloroform (CHCl₃ )
Selenium (Se; Fluka, 95% pure)
Sodium borohydride (NaBH₄ ; Aldrich, 98% pure)
Ethanol (absolute)
NaCl, aqueous, saturated
Iodopropionitrile (ICH₂ CH₂ CN; 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

Materials

Potassium iodide (Aldrich, 95%)
Acetone
Argon
3‐Bromopropionitrile (Aldrich, 99%)
Ethyl acetate (EtOAc)
MgSO₄ , 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

Materials

2‐Selenothymidine phosphoramidite ( S.6 ; protocol 1 )
Acetonitrile (CH₃ CN), 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 K₂ CO₃ in methanol
2 M triethylammonium acetate (TEAA) buffer, pH 7.0
Acetonitrile (CH₃ CN), HPLC grade
30% (v/v) trichloroacetic acid (TCA), aqueous
Argon
3‐Hydroxypicolinic acid (3‐HPA)
Diammonium citrate
NaCl
NaH₂ PO₄
Na₂ HPO₄
EDTA
MgCl₂

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; http://www.instrument.com)
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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Figures

Figure 1.23.1 Synthetic scheme of the 2‐selenothymidine phosphoramidite (S.6 ) and the Se‐DNAs (S.7 ).

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Figure 1.23.2 MALDI‐TOF mass spectrum of 2Se‐T 9‐mer (5′‐ATGGSeTGCTC‐3′); C₈₈ H₁₀₄ N₃₂ O₅₃ P₈ Se, calculated: 2794.8, observed: 2794.2.

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Figure 1.23.3 UV melting curves. (A ) Duplex of native DNAs 5′‐CTTCTTGTCCG‐3′ and 5′‐CGGACAAGAAG‐3′ ( T _m = 42.6°C). (B ) Duplex of Se‐DNA 5′‐CTTCTT^Se GTCCG‐3′ and native DNA 5′‐CGGACAACAAC‐3′ ( T _m = 42.2°C).

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Literature Cited

Literature Cited
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