Synthesis of a 4‐Selenothymidine Phosphoramidite and Incorporation into Oligonucleotides

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Abstract
Table of Contents
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Abstract

The detailed synthetic protocol for a 4?selenothymidine phosphoramidite and its use to prepare modified oligonucleotides is described here. The Se?phosphoramidite synthesis was achieved by developing a useful protection and deprotection system for the selenium functionality. The coupling reaction of the Se?phosphoramidite during solid?phase oligonucleotide synthesis is quantitative, and the oligonucleotides containing the Se?modification are stable. Based on crystal structure analysis, the selenium?modified oligonucleotides retain base?pairing like their native counterparts, and the derivatized DNA structure is virtually identical to the native structure. This achievement will present a novel opportunity for structural studies of nucleic acids and their protein complexes, because selenium can resolve the phase problem in macromolecular X?ray crystallography. In addition, this atom?specific replacement of oxygen with selenium will provide a useful tool for investigating biochemical and biophysical properties of nucleic acids and their protein complexes. Curr. Protoc. Nucleic Acid Chem. 32:1.19.1?1.19.13. © 2008 by John Wiley & Sons, Inc.

Keywords: nucleic acid; selenium; derivatization; structure determination; X?ray crystallography

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Introduction
Basic Protocol 1: Preparation of the 4‐Selenothymidine Phosphoramidite
Support Protocol 1: Synthesis of DI(2‐Cyanoethyl) Diselenide
Basic Protocol 2: Synthesis, Purification, and Characterization of Oligonucleotides Containing 4‐Selenothymidine
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol 1: Preparation of the 4‐Selenothymidine Phosphoramidite

Materials

5′‐O ‐Dimethoxytrityl‐thymidine (5′‐O ‐DMTr‐thymidine, S.1 , ChemGenes, 99.5% pure)
Acetonitrile (CH₃ CN, Fluka, anhydrous, purity >99%)
Argon
1‐(Trimethylsilyl)imidazole (TMS‐Im, Aldrich, purity >99%)
1,2,4‐Triazole (Aldrich, 98% pure)
Phosphorus oxychloride (POCl₃ , Fluka, purity >98%)
Triethylamine (Et₃ N, Aldrich, anhydrous, 99% pure)
Methanol (MeOH)
Methylene chloride (CH₂ Cl₂ , Fluka, purity >99.5%)
Ethyl acetate (EtOAc)
NaCl, aqueous, saturated
MgSO₄ (anhydrous)
Hexane
Tetrahydrofuran (THF, Fluka, purity >99%)
1.0 M triethylamine trihydrofluoride (Et₃ N·3HF, Aldrich) in THF
Sodium borohydride (NaBH₄ , Aldrich, 98% pure)
Ethanol (absolute)
Di(2‐cyanoethyl) diselenide ((NCCH₂ CH₂ Se)₂ , d = 1.8 g/mL, see protocol 2 )
Silica gel (porosity, 60 Å; particle size, 40 to 63 µm; 230 × 400 mesh)
N,N ‐Diisopropylethylamine (DIPEA, Aldrich, 99% pure)
2‐Cyanoethyl N,N ‐diisopropylchlorophosphoramidite (ChemGenes Corporation)
NaHCO₃ , saturated, aqueous
Petroleum ether

25‐, 50‐, and 100‐mL round‐bottom flasks
Separatory funnels
Vacuum oil pump
Syringe packed with cotton for filtration
Rotary evaporator
22 × 457–mm chromatography columns
Al₂ O₃ column (neutral, 20 × 4 cm)

Additional reagents and equipment for thin‐layer chromatography (TLC; appendix 3D ) and column chromatography ( appendix 3E )

NOTE: All the reactions in this protocol are carried out at room temperature unless otherwise specified.NOTE: Drying steps use a vacuum oil pump for high vacuum and a rotary evaporator for reduced pressure.

Support Protocol 1: Synthesis of DI(2‐Cyanoethyl) Diselenide

Materials

Selenium metal
Sodium borohydride (NaBH₄ , Aldrich, 98% pure)
Dioxane
Ethanol (EtOH, absolute)
Argon
3‐Bromopropionitrile (Aldrich, 99%)
10% (v/v) acetic acid
Ethyl acetate (EtOAc)
NaCl, aqueous, saturated
MgSO₄ , anhydrous
Silica gel (porosity, 60 Å; particle size, 40 to 63 µm; 230 × 400 mesh)
Methylene chloride (CH₂ Cl₂ , Fluka, purity >99.5%)
Hexane

250‐mL flask
Separatory funnel
Rotary evaporator
Chromatography column

Additional reagents and equipment for column chromatography ( appendix 3E )

Basic Protocol 2: Synthesis, Purification, and Characterization of Oligonucleotides Containing 4‐Selenothymidine

Materials

4‐Selenothymidine phosphoramidite (see protocol 1 )
Acetonitrile (CH₃ CN), anhydrous
Regular 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 mM K₂ CO₃ in methanol
20 mM 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
Nucleic Acid Mini‐Screen Kit (Hampton Research, http://www.hamptonresearch.com)

ABI392 DNA/RNA synthesizer (also see appendix 3C )
Screw‐cap tubes or vials
13‐mm syringe filter with 0.2‐µm nylon membrane (Life Sciences)
HPLC system (optional) with detector at 260 and/or 369 nm
RP‐HPLC column: 21.2 × 250–mm Zorbax RX‐C8 (Agilent Technology) or 21 × 250–mm XB‐C18 (Welch Materials; http://www.instrument.com)
Lyophilizer
UV spectrophotometer

Additional reagents and equipment for automated oligonucleotide synthesis ( appendix 3C ), and MALDI‐TOF mass spectrometry (unit 10.1 ), and determination of UV melting curves (unit 7.3 )

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Figures

Figure 1.19.1 Synthesis of the 4‐selenothymidine phosphoramidite (S.6 ) and 4‐Se‐T DNAs (S.7 ).

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Figure 1.19.2 UV thermostability study of the Se‐DNA 9‐mer 5′‐ATGT^Se TTCTC‐3′) heated at 60°C for 3 hr in buffer (5 mM NaH₂ PO₄ /Na₂ HPO₄ , pH 7.5). Reprinted from Salon et al. () with permission from the American Chemical Society.

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Figure 1.19.3 HPLC analysis of crude 5′‐ O ‐DMTr‐GCG^Se TATACGC‐3′ after cleavage from the solid support and deprotection of the nucleobases and backbone (retention time: 21.0 min). Reprinted from Salon et al. () with permission from the American Chemical Society.

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Figure 1.19.4 MALDI‐MS analysis of purified 5′‐GCG^Se TATACGC‐3′. Molecular formula: C₉₇ H₁₂₃ N₃₈ O₅₇ P₉ Se. [M+H]⁺ : 3091.6 (calculated: 3092.0). Due to MS analysis resolution, this peak represents an average‐mass peak and the Se isotopic peaks are not resolved. Reprinted from Salon et al. () with permission from the American Chemical Society.

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Figure 1.19.5 UV melting curves. (A ) Duplex of native DNAs 5′‐ATGGTGCTC‐3′ and 5′‐GAGCACCAT‐3′ ( T _m = 39.2°C). (B ) Duplex of Se‐DNA 5′‐ATGG^Se TGCTC‐3′ and native DNA 5′‐GAGCACCAT‐3′ ( T _m = 38.6°C). Reprinted from Salon et al. () with permission from the American Chemical Society.

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Videos

Literature Cited

Literature Cited
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Synthesis of a 4‐Selenothymidine Phosphoramidite and Incorporation into Oligonucleotides

Abstract

Table of Contents

Materials

Basic Protocol 1: Preparation of the 4‐Selenothymidine Phosphoramidite

Support Protocol 1: Synthesis of DI(2‐Cyanoethyl) Diselenide

Basic Protocol 2: Synthesis, Purification, and Characterization of Oligonucleotides Containing 4‐Selenothymidine

Figures

Videos

Literature Cited