Synthesis of Alkyne‐ and Azide‐Modified Oligonucleotides and Their Cyclization by the CuAAC (Click) Reaction

Afaf H. El‐Sagheer1, Tom Brown2

1 Chemistry Branch, Department of Science and Mathematics, Faculty of Petroleum and Mining Engineering, Suez Canal University, Suez, Egypt, 2 School of Chemistry, University of Southampton, Highfield Southampton, U.K.
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.33
DOI:  10.1002/0471142700.nc0433s35
Online Posting Date:  December, 2008
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The Cu(I)‐catalyzed alkyne‐azide cycloaddition (CuAAC) reaction has been used to synthesize cyclic mini‐DNA duplexes. The reaction is carried out on 5′‐alkyne‐3′‐azide‐labeled hairpin loop oligonucleotides and proceeds in high yield under mild conditions in as little as 5 min. The resultant duplexes have very high thermal stability and their CD spectra are characteristic of normal B‐DNA. Curr. Protoc. Nucleic Acid Chem. 35:4.33.1‐4.33.21. © 2008 by John Wiley & Sons, Inc.

Keywords: Click chemistry; CuAAC reaction; oligonucleotide cyclization; mini‐duplex

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Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis of Propargylamidohexanyl Phosphoramidite
  • Basic Protocol 2: Synthesis of Hexynyl Phosphoramidite
  • Basic Protocol 3: Synthesis of Succinimidyl‐4‐Azidobutyrate
  • Basic Protocol 4: Oligonucleotide Synthesis, Purification, Analysis, and Cyclization
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Synthesis of Propargylamidohexanyl Phosphoramidite

  • 6‐O‐TBDMS‐1‐hexanoic acid (S.1; Nicolaou et al., )
  • Anhydrous dichloromethane (DCM; distilled over CaH 2)
  • Argon gas
  • Anhydrous N,N‐diisopropylethylamine (DIPEA; distilled over CaH 2)
  • Propargylamine
  • N‐Hydroxybenzotriazole hydrate (HOBt)
  • 1‐(3‐Dimethylaminopropyl)‐3‐ethylcarbodiimide hydrochloride (EDC)
  • Saturated aqueous sodium hydrogencarbonate (NaHCO 3)
  • Sodium sulfate, anhydrous
  • Silica gel 60 (0.040 to 0.063 mm, chromatography grade; Merck)
  • Ethyl acetate (EtOAc, HPLC grade)
  • Hexane (HPLC grade)
  • Sand
  • Potassium permanganate solution (see recipe)
  • Anhydrous tetrahydrofuran (THF) (distilled over sodium metal)
  • 1.0 M tetrabutylammonium fluoride in tetrahydrofuran (TBAF/THF)
  • 2‐Cyanoethyl‐N,N‐diisopropylchlorophosphoramidite (Link Technologies)
  • Saturated aqueous potassium chloride
  • Pyridine (distilled over CaH 2)
  • Dry acetonitrile, oligonucleotide synthesis grade
  • Potassium hydroxide (KOH)
  • 50‐, 100‐, and 250‐mL single‐neck round‐bottom flasks
  • Magnetic stir bars and plate
  • Rubber septa
  • Syringe needles
  • Frit funnels
  • Rotary evaporator equipped with diaphragm pump
  • High‐vacuum pump with dual nitrogen traps
  • 50 × 4–, 50 × 3–, and 40 × 2.5–cm glass chromatography columns
  • TLC aluminium sheets (silica gel 60 F254, Merck)
  • TLC viewing cabinet with 254‐nm UV lamp
  • Hair dryer
  • 100‐mL separatory funnels
  • 100°C oven
  • Glass test tubes and stoppers
  • 45‐µm filters
  • Monomer bottles
  • Vacuum desiccator

Basic Protocol 2: Synthesis of Hexynyl Phosphoramidite

  • 5‐Hexyn‐1‐ol (S.5; Avocado)
  • Anhydrous dichloromethane (DCM; distilled over CaH 2)
  • Anhydrous N,N‐diisopropylethylamine (DIPEA; distilled over CaH 2)
  • 2‐Cyanoethyl‐N,N‐diisopropylchlorophosphoramidite (Link Technologies)
  • Argon gas
  • Saturated aqueous potassium chloride, degassed
  • Anhydrous sodium sulfate
  • Silica gel 60 F254 (Merck)
  • Triethylamine
  • Ethyl acetate
  • Hexane
  • Potassium permanganate solution (see recipe)
  • 50‐ and 250‐mL flasks
  • Magnetic stir bars and plate
  • 100‐mL separatory funnels
  • Rotary evaporator
  • Vacuum
  • 50 × 3–cm chromatography columns
  • TLC aluminium sheets

Basic Protocol 3: Synthesis of Succinimidyl‐4‐Azidobutyrate

  • 4‐Azidobutyric acid (S.7; Carboni et al., )
  • Anhydrous dichloromethane (DCM; distilled over CaH 2)
  • N‐Hydroxysuccinimide (NHS)
  • Dicyclohexylcarbodiimide (DCC)
  • Saturated aqueous potassium chloride
  • Anhydrous sodium sulfate
  • Methanol (MeOH; HPLC grade)
  • 50‐mL one‐neck round‐bottom flasks
  • Magnetic stir bars and plate
  • 100‐mL separatory funnels
  • 40 × 2–cm chromatography columns
  • High vacuum

Basic Protocol 4: Oligonucleotide Synthesis, Purification, Analysis, and Cyclization

  • 1.0‐µmol aminolink C7 DNA synthesis columns
  • 5′‐O‐DMTr‐N‐protected dA, dG, and dC monomers; also dT 2‐cyanoethylphosphoramidite monomers
  • Propargylamidohexanyl or hexynyl phosphoramidite monomer (S.4 or S.6; see protocol 1 or protocol 22, respectively)
  • Acetonitrile
  • 33% aqueous ammonia solution
  • 0.5 M sodium carbonate/sodium hydrogen carbonate buffer, pH 8.75 (Na 2CO 3/NaHCO 3)
  • Succinimidyl‐4‐azidobutyrate (S.8; see protocol 3)
  • HPLC buffer A: 0.1 M ammonium acetate, pH 7.0
  • HPLC buffer B: 0.1 M ammonium acetate, pH 7.0, with 35% acetonitrile
  • Tris‐hydroxypropyl triazole ligand (Chan et al., )
  • Sodium chloride (NaCl)
  • Argon gas
  • Sodium ascorbate
  • Copper sulfate (CuSO 4⋅5H 2O)
  • 20% polyacrylamide gel in 1× TBE buffer
  • Bromophenol blue
  • Xylene cyanol FF, molecular biology grade
  • Formamide
  • ABI 394 automated DNA synthesizer (Applied Biosystems) or equivalent
  • Screw‐cap glass vials for DNA synthesizer
  • 55°C oven or heating block
  • 50‐mL round‐bottom flasks
  • Rotary evaporator
  • 0.45‐µm filters
  • NAP‐10 and NAP‐25 disposable gel‐filtration columns (Sephadex G‐25, GE Healthcare)
  • 200‐mL Dewar flask
  • Metal tongs
  • 20‐G needles
  • Freeze drier
  • HPLC system, e.g., Gilson HPLC with Brownlee Aquapore C8 reversed‐phase HPLC column (8 × 250–mm, pore size 300 Å, Perkin Elmer)
  • 1.5‐mL microcentrifuge tubes
  • 80°C water bath
  • Spectrophotometer
  • Gel electrophoresis apparatus
  • Fluorescent TLC plates
  • UV lamp
  • Gel photography equipment
  • Additional reagents and equipment for automated DNA synthesis ( appendix 3C), HPLC purification (unit 10.5), denaturing PAGE (unit 10.4), and capillary electrophoresis (unit 10.9)
NOTE: Reagents and solvents for oligonucleotide synthesis can be obtained from Applied Biosystems, Link Technologies, Glen Research, or SAFC‐Proligo. In this study, 2‐cyanoethyl‐N,N‐diisopropylphosphoramidite monomers were used with benzoyl protection for N6 of dA and N4 of dC, and isobutyryl protection for N2 of dG.NOTE: In the cyclization protocol below, 1.0 µmol of oligonucleotide is used. To accumulate this quantity after full purification, it will be necessary to carry out five 1.0‐µmol scale syntheses.
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Literature Cited

Literature Cited
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