Preparation of Intrastrand {G}O6‐Alkylene‐O6{G} Cross‐Linked Oligonucleotides

Derek K. O'Flaherty1, Christopher J. Wilds1

1 Department of Chemistry and Biochemistry, Concordia University, Montréal, Québec
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 5.17
DOI:  10.1002/cpnc.14
Online Posting Date:  September, 2016
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Abstract

This unit describes the preparation O6‐2′‐deoxyguanosine‐butylene‐O6‐2′‐deoxyguanosine dimer phosphoramidites and precursors for incorporation of site‐specific intrastrand cross‐links (IaCL) into DNA oligonucleotides. Protected 2′‐deoxyguanosine dimers are produced using the Mitsunobu reaction. IaCL DNA containing the intradimer phosphodiester are first chemically phosphorylated, followed by a ring‐closing reaction using the condensing reagent 1‐(2‐mesitylenesulfonyl)‐3‐nitro‐1H‐1,2,4‐triazole. Phosphoramidites are incorporated into oligonucleotides by solid‐phase synthesis and standard deprotection and cleavage protocols are employed. This approach allows for the preparation of IaCL DNA substrates in amounts and purity amenable for biophysical characterization, and biochemical studies as substrates to investigate DNA repair and bypass pathways. © 2016 by John Wiley & Sons, Inc.

Keywords: DNA intrastrand cross‐link; chemically modified oligonucleotide; oligonucleotide synthesis; solid‐phase synthesis; DNA damage; DNA repair and bypass

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis of O6‐2′‐Deoxyguanosine‐AlkylenE‐O6‐2′‐Deoxyguanosine‐3′‐O‐Phosphoramidite Lacking the Intradimer Phosphodiester Linkage
  • Basic Protocol 2: Synthesis of O6‐2′‐Deoxyguanosine‐Alkylene‐O6‐2′‐Deoxyguanosine‐3′‐O‐Phosphoramidite Lacking the Intradimer Phosphodiester Linkage
  • Basic Protocol 3: Solid‐Phase Synthesis and Deprotection of Oligonucleotides Containing O6‐2′‐Deoxyguanosine‐Alkylene‐O6‐2′‐Deoxyguanosine Cross‐Link Modifications
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Synthesis of O6‐2′‐Deoxyguanosine‐AlkylenE‐O6‐2′‐Deoxyguanosine‐3′‐O‐Phosphoramidite Lacking the Intradimer Phosphodiester Linkage

  Materials
  • 5′‐O‐(4,4′‐Dimethoxytrityl)‐N2‐phenoxyacetyl‐2′‐deoxyguanosine (1, ChemGenes)
  • Tetrahydrofuran (THF; EMD Millipore)
  • Ice water bath
  • Phenoxyacetyl chloride (Pac‐Cl; Sigma‐Aldrich)
  • Dichloromethane (DCM; EMD Millipore, Reagent Grade)
  • 3% (w/v) Aqueous sodium bicarbonate (NaHCO 3)
  • Sodium sulfate, anhydrous (Na 2SO 4; Sigma‐Aldrich)
  • Silica gel (60 Å, 230 to 400 mesh)
  • Methanol (MeOH; EMD Millipore, Reagent Grade)
  • 1,4‐Dioxane (Sigma‐Aldrich)
  • 4‐(tert‐Butyldiphenylsilyloxy)butan‐1‐ol (UNIT 5.9; Wilds et al., )
  • Triphenylphosphine (Ph 3P; Sigma‐Aldrich)
  • Argon gas
  • Diisopropyl azodicarboxylate (Sigma‐Aldrich)
  • Hexanes (Hex; EMD Millipore, Reagent Grade)
  • Tetrabutylammonium fluoride (1 M in THF; Sigma‐Aldrich)
  • Pyridine (Sigma‐Aldrich)
  • Imidazole (Im; Sigma‐Aldrich)
  • tert‐Butyldimethylsilyl chloride (TBS‐Cl; Sigma‐Aldrich)
  • Brine (saturated NaCl aqueous solution)
  • p‐Toluenesulfonic acid monohydrate (p‐TsOH•H 2O; Sigma‐Aldrich)
  • EtOAc (EtOAc; EMD Millipore, Reagent Grade)
  • Triethylamine (NEt 3; Sigma‐Aldrich)
  • Diisopropylethylamine (DIPEA; Sigma‐Aldrich)
  • N,N‐diisopropylamino cyanoethyl phosphonamidic chloride [Cl‐P(OCE)NiPr 2; ChemGenes]
  • 10‐, 25‐, 50‐, and 100‐mL Round‐bottom flasks with a rubber septum
  • Magnetic stir plate and stir bar
  • 3‐ and 1‐mL syringes
  • 21‐G needles
  • 250‐mL separatory funnel
  • Rotary evaporator and chemical resistant dry vacuum pump
  • TLC plate, EMD silica gel 60 F 254
  • UV lamp, 254 nm
  • Vacuum manifold
  • Vacuum pump
  • Automatic dispensing pipettor

Basic Protocol 2: Synthesis of O6‐2′‐Deoxyguanosine‐Alkylene‐O6‐2′‐Deoxyguanosine‐3′‐O‐Phosphoramidite Lacking the Intradimer Phosphodiester Linkage

  Materials
  • N2‐(phenoxyacetyl)‐2′‐deoxyguanosine (10) (ChemGenes)
  • Pyridine (Sigma‐Aldrich)
  • Dichloromethane (DCM; Reagent Grade)
  • 4‐(Dimethylamino)pyridine (DMAP; Sigma‐Aldrich)
  • Imidazole
  • tert‐Butyldimethylsilyl chloride (TBS‐Cl; Sigma‐Aldrich)
  • 3% (w/v) Aqueous sodium bicarbonate (NaHCO 3)
  • Saturated NaCl aqueous solution
  • Sodium sulfate (anhydrous) (Na 2SO 4; Sigma‐Aldrich)
  • Ethyl acetate (EtOAc; Reagent Grade)
  • Hexanes (Hex; Reagent Grade)
  • Silica gel (60 Å, 230 to 400 mesh)
  • 1,4‐Dioxane (Sigma‐Aldrich)
  • Triphenylphosphine (Ph 3P; Sigma‐Aldrich)
  • Diisopropyl azodicarboxylate (Sigma‐Aldrich)
  • Argon gas
  • Tetrakis(triphenylphosphine)palladium(0) [Pd(Ph 3P) 4; Sigma‐Aldrich]
  • Formic acid (HCOOH; Sigma‐Aldrich)
  • 1‐Butylamine (n‐BuNH 2; Sigma‐Aldrich)
  • Tetrahydrofuran (THF; EMD Millipore)
  • Tetrabutylammonium fluoride (1 M in THF; Sigma‐Aldrich)
  • 1,2,4‐Triazole (Sigma‐Aldrich)
  • 2‐Chlorophenyl phosphorodichloridate (Sigma‐Aldrich)
  • Distilled water
  • Saturated NH 4Cl aqueous solution
  • 1‐(2‐Mesitylenesulfonyl)‐3‐nitro‐1H‐1,2,4‐triazole (MSNT; Sigma‐Aldrich)
  • Methanol (MeOH; Reagent Grade)
  • Diisopropylethylamine (DIPEA; Sigma‐Aldrich)
  • N,N‐diisopropylamino cyanoethyl phosphonamidic chloride [Cl‐P(OCE)NiPr 2; ChemGenes]
  • 3′‐O‐(Allyloxycarbonyl)‐5′‐O‐(4,4′‐dimethoxytrityl)‐O6‐(4‐hydroxybutyl)‐N2‐(phenoxyacetyl)‐2′‐deoxyguanosine (Compound 12) (McManus et al., ; UNIT 5.9; Wilds et al., )
  • 10‐, 25‐, 50‐, and 100‐mL round‐bottomed flasks with a rubber septum
  • Magnetic stir plate and stir bar
  • Rotary evaporator and chemical resistant dry vacuum pump
  • 250‐mL separatory funnels
  • TLC plate, EMD silica gel 60 F 254
  • UV lamp, 254 nm
  • Vacuum manifold
  • Vacuum pump
  • Automatic dispensing pipettor
  • 1‐ and 3‐mL syringes
  • Cannula
  • Sintered glass funnels
  • Whatman qualitative filter paper, Grade 2

Basic Protocol 3: Solid‐Phase Synthesis and Deprotection of Oligonucleotides Containing O6‐2′‐Deoxyguanosine‐Alkylene‐O6‐2′‐Deoxyguanosine Cross‐Link Modifications

  Materials
  • 5‐O‐(4,4‐Dimethoxytrityl)‐2′‐deoxynucleoside‐3‐O‐succinate long‐chain alkylamine controlled‐pore glass (LCAA‐CPG, 500 Å; Glen Research)
  • 2′‐Deoxyribonucleoside‐3‐phosphoramidites (Glen Research)
  • Anhydrous acetonitrile (MeCN; Fisher Scientific)
  • O6‐2′‐Deoxyguanosine‐alkylene‐O6‐2′‐deoxyguanosine phosphoramidites lacking the intradimer phosphodiester (9; protocol 1) or containing the intradimer phosphodiester (16; protocol 2)
  • 4 Å Activated molecular sieves (Sigma‐Aldrich)
  • Argon
  • Aqueous ammonium hydroxide (28% to 30%)
  • 50% (v/v) Aqueous acetonitrile
  • Screw‐capped columns
  • Brown bottles
  • DNA Synthesizer (Applied Biosystems)
  • Vacuum pump
  • 2‐mL Screw‐capped microcentrifuge tubes (Fisher Scientific)
  • Vortex mixer
  • 55°C Heated sand box
  • Automatic dispensing pipettor
  • Savant SpeedVac concentrator (Thermo Fisher Scientific)
  • 3‐mL disposable syringes (BD Biosciences)
NOTE: Prepare GG4 and GpG4 using the automated DNA synthesizer.NOTE: Carry out solid‐phase synthesis of oligonucleotides containing the O6‐2′‐deoxyguanosine‐alkylene‐O6‐2′‐deoxyguanosine modifications using phosphoramidite 9 (or16).
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Figures

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Literature Cited

Literature Cited
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