Preparation of DNA Containing 5‐Hydroxymethyl‐2′‐Deoxycytidine Modification Through Phosphoramidites with TBDMS as 5‐Hydroxymethyl Protecting Group

Qing Dai1, Chuan He1

1 Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago, Chicago, Illinois
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.47
DOI:  10.1002/0471142700.nc0447s47
Online Posting Date:  December, 2011
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Abstract

This unit describes procedures for preparation of two phosphoramidite building blocks III and IV, both containing a TBDMS as 5‐CH2OH‐protecting group. Phosphoramidites III and IV allow efficient incorporation of 5‐hmC into DNA and a “one‐step” deprotection procedure to cleanly remove all the protecting groups. A “two‐step” deprotection strategy is compatible with ultramild DNA synthesis, which enables the synthesis of 5hmC‐containing DNA with additional modifications. Methods are also presented for their incorporation into oligonucleotides by solid‐phase synthesis, subsequent deprotection, and HPLC analysis. Curr. Protoc. Nucleic Acid Chem. 47:4.47.1‐4.47.18. © 2011 by John Wiley & Sons, Inc.

Keywords: 5‐Hydroxymethylcytosine; DNA modification; oligodeoxyribonucleotide (ODN); phosphoramidite; solid‐phase synthesis; genomic DNA; epigenetic; ultramild deprotection

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Phosphoramidite III
  • Basic Protocol 2: Preparation of Phosphoramidite IV
  • Basic Protocol 3: Solid‐Phase Synthesis of DNA Using Phosphoramidites III and IV and Subsequent Deprotection
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1:

  Materials
  • 5‐Iodo‐2′‐deoxyuridine (Berry Associate)
  • N,N‐Dimethylformamide (DMF, anhydrous, 99.8%, Sigma‐Aldrich)
  • Argon
  • Di‐tert‐butylsilyl‐bis(trifluoromethanesulfonate) (97%, Sigma‐Aldrich)
  • Imidazole (>99%, Sigma‐Aldrich)
  • Ethyl acetate (EtOAc; >99%, Fisher)
  • 5% NaHCO 3 solution (see recipe)
  • Brine (see recipe)
  • Na 2SO 4
  • Silica gel (Sigma‐Aldrich)
  • Methylene chloride (CH 2Cl 2; HPLC grade, Fisher)
  • Methanol (MeOH; HPLC grade, Fisher)
  • Toluene (99.8%, anhydrous, Sigma‐Aldrich)
  • Triphenylphosphine (Ph 3P; 99%, Sigma‐Aldrich)
  • Pd 2(dba) 3 (97%, Sigma‐Aldrich)
  • Carbon monoxide (Airgas)
  • Tributyltin hydride (Bu 3SnH; 97%, Sigma‐Aldrich)
  • Methanol (MeOH; anhydrous, 99.8%, Sigma‐Aldrich)
  • Cerium(III) chloride heptahydrate (99.9%, Sigma‐Aldrich)
  • Sodium borohydride (NaBH 4; >99.9%, Sigma‐Aldrich)
  • t‐Butyldimethylsilyl chloride (TBDMS‐Cl; 97%, Sigma‐Aldrich)
  • 100‐mL round‐bottom flasks
  • Ice bath
  • Rotary evaporator connected to vacuum pump (delivering ∼100 micro max vacuum), chilled by dry‐ice
  • 2 × 40–cm glass columns
  • Self‐contained glass coupling apparatus equipped with a pressure‐equalizing addition funnel (Chemglass)
  • Silicone oil bath (Fisher)
  • Additional reagents and equipment for thin‐layer chromatography ( appendix 3D)

Basic Protocol 2: Preparation of Phosphoramidite III

  Materials
  • 3′,5′‐O‐di‐t‐butylsilyl‐t‐butyldimethylsiloxymethyl‐2′‐deoxyuridine ( S.5; see protocol 1)
  • Acetonitrile (CH 3CN)
  • Triethylamine (Et 3N; Sigma‐Aldrich)
  • Imidazole
  • 1,2,4‐Triazole (Chemgenes)
  • Phosphorus (V) oxychloride (POCl 3; 99%, Sigma‐Aldrich)
  • Dichloromethane (CH 2Cl 2; anhydrous, 99.8%, Sigma‐Aldrich)
  • 5% NaHCO 3 solution (see recipe)
  • Brine (see recipe)
  • Na 2SO 4
  • 1,4‐Dioxane (99.8%, anhydrous, Sigma‐Aldrich)
  • Ammonium hydroxide (NH 4OH; 28% to 30%, Sigma‐Aldrich)
  • Silica gel (Sigma‐Aldrich)
  • Methylene chloride (CH 2Cl 2; HPLC grade, Fisher)
  • Methanol (MeOH, HPLC grade, Fisher)
  • Pyridine (99.8%, anhydrous, Sigma‐Aldrich)
  • Benzoyl chloride (99%, Sigma‐Aldrich)
  • Argon
  • Toluene
  • Tetrahydrofuran (THF; 99.8%, anhydrous, Sigma‐Aldrich)
  • Hydrogen fluoride‐pyridine (HF; Sigma‐Aldrich)
  • DMTr‐Cl (Chemgenes)
  • 1‐Methylimidazole (>99%, Sigma‐Aldrich)
  • N,N‐Diisopropylethylamine (99.5%, Sigma‐Aldrich)
  • N,N‐diisopropylamino cyanoethyl phosphoramidic‐chloride (Chemgenes)
  • Acetone
  • 100‐mL flasks
  • Ice box
  • Rotary evaporator connected to vacuum pump (delivering ∼100 micro max vacuum), chilled by dry ice
  • 2 × 40–cm glass columns
  • 1 × 40–cm glass columns
  • Additional reagents and equipment for TLC ( appendix 3D)

Basic Protocol 3: Preparation of Phosphoramidite IV

  Materials
  • 3′,5′‐O‐di‐t‐butylsilyl‐t‐butyldimethylsiloxymethyl‐2′‐deoxyuridine ( S.5; see protocol 1)
  • Tetrahydrofuran (THF; 99.8%, anhydrous, Sigma‐Aldrich)
  • Hydrogen fluoride‐pyridine (HF; Sigma‐Aldrich)
  • Silica gel (Sigma‐Aldrich)
  • Methylene chloride (CH 2Cl 2; HPLC grade, Fisher)
  • Methanol (MeOH; HPLC grade, Fisher)
  • Pyridine (99.8%, anhydrous, Sigma‐Aldrich)
  • DMTr‐Cl (Chemgenes)
  • Argon
  • Toluene
  • 5% NaHCO 3 solution (see recipe)
  • Brine (see recipe)
  • Na 2SO 4
  • Dichloromethane (CH 2Cl 2; anhydrous, 99.8%, Sigma‐Aldrich)
  • 1‐Methylimidazole (>99%, Sigma‐Aldrich)
  • N,N‐Diisopropylethylamine (99.5%, Sigma‐Aldrich)
  • N,N‐diisopropylamino cyanoethyl phosphoramidic‐chloride (Chemgenes)
  • Acetone
  • Triethylamine (Et 3N; Sigma‐Aldrich)
  • Acetonitrile (CH 3CN; anhydrous, 99.8%, Sigma‐Aldrich)
  • 1,2,4‐Triazole (Chemgenes)
  • Phosphorus (V) oxychloride (POCl 3; 99%, Sigma‐Aldrich)
  • 100‐mL flasks
  • Rotary evaporator connected to vacuum pump (delivering ∼100 micro max vacuum), chilled by dry ice
  • 2 × 40–cm glass columns
  • 1 × 40–cm glass columns
  • Additional reagents and equipment for TLC ( appendix 3D)
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Figures

Videos

Literature Cited

Literature Cited
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