Synthesis of Triazole‐Nucleoside Phosphoramidites and Their Use in Solid‐Phase Oligonucleotide Synthesis

Brandon J. Peel1, Tim C. Efthymiou1, Jean‐Paul Desaulniers1

1 Faculty of Science, University of Ontario Institute of Technology, Oshawa, Ontario, Canada
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.57
DOI:  10.1002/0471142700.nc0457s55
Online Posting Date:  December, 2013
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Abstract

Triazole‐backbone oligonucleotides are macromolecules that have one or more triazole units that are acting as a backbone mimic. Triazoles within the backbone have been used within oligonucleotides for a variety of applications. This unit describes the preparation and synthesis of two triazole‐nucleoside phosphoramidites [uracil‐triazole‐uracil (UtU) and cytosine‐triazole‐uracil (CtU)] based on a PNA‐like scaffold, and their incorporation within oligonucleotides. Curr. Protoc. Nucleic Acid Chem. 55:4.57.1‐4.57.38. © 2013 by John Wiley & Sons, Inc.

Keywords: triazole‐linkage; cycloaddition; phosphoramidite; solid‐phase oligonucleotide synthesis

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Uracil and Cytosine Pyrimidine Bases
  • Basic Protocol 2: Preparation of Alkyne and Azide Linkers
  • Basic Protocol 3: Preparation of Alkyne and Azide Monomers
  • Basic Protocol 4: Preparation of Uracil‐Triazole‐Uracil Phosphoramidite (24)
  • Basic Protocol 5: Preparation of Cytosine‐Triazole‐Uracil Phosphoramidite
  • Basic Protocol 6: Synthesis, Purification, and Characterization of Oligonucleotides Containing CtU or UtU Units
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of Uracil and Cytosine Pyrimidine Bases

  Materials
  • Sodium hydroxide (NaOH), ≥98%
  • Uracil (1), ≥99% pure
  • Bromoacetic acid, 97% pure
  • Methanol (MeOH), ACS grade
  • Dichloromethane (CH 2Cl 2), ACS grade
  • Concentrated hydrochloric acid (HCl), ACS grade
  • Cytosine (3), ≥99% pure
  • Dimethylformamide (DMF), anhydrous, 99.8% pure
  • Nitrogen (or argon) gas
  • Sodium hydride (NaH), 60% dispersion in mineral oil
  • Hexane, ACS grade
  • Methyl bromoacetate, 97% pure
  • Pyridine, anhydrous, ≥99% pure
  • Benzoyl chloride, 99% pure
  • Analytical balance
  • Weighing paper
  • 50‐ and 100‐mL and 1‐L round‐bottom flask
  • Stir bar
  • Hot plate magnetic stirrer
  • 45°C water bath
  • Septa
  • Disposable syringes and needles
  • TLC plates (250‐μm thick; Silicycle; cat. no. TLG‐R10011B‐2020, http://www.silicycle.com/)
  • Short‐wave UV lamp
  • pH meter
  • Vacuum pump
  • Filter paper, grade P5
  • Büchner funnels
  • 150‐, 250‐, and 500‐mL Büchner flasks
  • Oven
  • Rotary evaporator
  • Additional reagents and equipment for thin‐layer chromatography (TLC, appendix 3D)

Basic Protocol 2: Preparation of Alkyne and Azide Linkers

  Materials
  • Ethanolamine (6), 99% pure
  • Dichloromethane (CH 2Cl 2), ACS grade
  • Imidazole, ≥99.5% pure
  • Tert‐butyldimethylsilyl chloride (TBS‐Cl), >95% pure
  • Methanol (MeOH), ACS grade
  • Potassium permanganate (KMnO 4) stain (see recipe)
  • Saturated aqueous solution of sodium bicarbonate (NaHCO 3)
  • Sodium sulfate (Na 2SO 4), ACS grade
  • 2‐((tert‐butyldimethylsilyl)oxy)ethanamine (7)
  • Nitrogen (or argon) gas
  • Distilled N,N‐Diisopropylethylamine (DIPEA), 99% pure
  • 80% (w/v) 3‐bromoprop‐1‐yne (Sigma‐Aldrich, cat. no. p51001) in toluene
  • Ethyl acetate (EtOAc), ACS grade
  • Hexane, ACS grade
  • Silica gel: 40 to 63 µm (230 to 400 mesh)
  • Sodium azide (NaN 3), ≥99.5%
  • Sodium hydroxide (NaOH), ≥98%
  • 2‐bromoethylamine hydrobromide (9; Sigma‐Aldrich, cat. no. 06670)
  • Diethyl ether (Et 2O), ACS grade
  • Dimethylformamide (DMF), anhydrous, 99.8% pure
  • Nitrogen (or argon) gas
  • Distilled triethylamine (TEA), ≥99% pure
  • Ethyl 2‐bromoacetate, 98% (Sigma‐Aldrich, cat. no. 133973)
  • 2‐Bromoethanol (12; Sigma‐Aldrich, cat. no. B65586)
  • (2‐bromoethoxy)(tert‐butyl)dimethylsilane, 99% pure (13; Sigma‐Aldrich, cat. no. 428426)
  • Saturated solution of NaCl (brine)
  • 50‐, 100‐, 250‐ and 500‐mL round‐bottom flasks
  • Stir bar
  • Hot plate magnetic stirrer
  • TLC plates (250‐μm thick; Silicycle; cat. no. TLG‐R10011B‐2020, http://www.silicycle.com/)
  • Short‐wave UV lamp
  • 250‐, 500‐, and 1‐L separatory funnels
  • 100‐, 250‐, and, 500‐mL Erlenmeyer flasks
  • Rotary evaporator
  • 7 × 25–cm glass chromatography column
  • Reflux condenser
  • Additional reagents and equipment for thin‐layer chromatography (TLC, appendix 3D) and column chromatography ( appendix 3E)

Basic Protocol 3: Preparation of Alkyne and Azide Monomers

  Materials
  • Uracil‐1‐yl acetic acid (2; protocol 1)
  • N‐(2‐((tert‐Butyldimethylsilyl)oxy)ethyl)prop‐2‐yn‐1‐amine (8; protocol 2)
  • Dimethylformamide (DMF), anhydrous, 99.8% pure
  • Nitrogen (or argon) gas
  • 1‐Ethyl‐2‐(3‐dimethylaminopropyl)carbodiimide hydrochloride (EDC‐Cl; Protochem, cat. no. c1100), ≥99% pure
  • Hexane, ACS grade
  • Ethyl acetate (EtOAc), ACS grade
  • Saturated solution of NaCl (brine)
  • Sodium sulfate (Na 2SO 4), ACS grade
  • Silica gel: 40 to 63 µm (230 to 400 mesh)
  • N,NDicyclohexylcarbodiimide (DCC), 99% pure
  • 1‐Hydroxybenzotrizole (HOBt), ≥ 99% pure
  • Ethyl 2‐(2‐azidoethylamino)acetate (11; protocol 2)
  • (N4‐(Benzoyl)cytosine‐1‐yl)acetic acid (5; protocol 1)
  • Dimethylsulfoxide (DMSO), minimum 99.5% GC
  • 2‐Azido‐N‐(2‐(tert‐butyldimethylsilyloxy)ethyl)ethanamine (14; protocol 2)
  • Methanol (MeOH), ACS grade
  • Dichloromethane (CH 2Cl 2), ACS grade
  • Triethylamine trihydrofluoride (3HF/TEA; Sigma, cat. no. 344648), 98% pure
  • Pyridine, dry
  • Dimethoxytrityl chloride (DMT‐Cl), 95% pure
  • Saturated aqueous sodium bicarbonate (NaHCO 3)
  • 50‐, 100‐, 250‐ and 500‐mL round‐bottom flasks
  • Stir bar
  • Hot plate magnetic stirrer
  • TLC plates (250‐μm thick; Silicycle; cat. no. TLG‐R10011B‐2020, http://www.silicycle.com/)
  • Short‐wave UV lamp
  • 250‐, 500‐, and 1‐L separatory funnels
  • 100‐, 250‐, and, 500‐mL Erlenmeyer flasks
  • Rotary evaporator
  • 7 × 25–cm glass chromatography column
  • 250‐mL Büchner flask
  • 3.5 × 25–cm glass chromatography column
  • Aluminum foil
  • Additional reagents and equipment for thin‐layer chromatography (TLC, appendix 3D) and column chromatography ( appendix 3E)

Basic Protocol 4: Preparation of Uracil‐Triazole‐Uracil Phosphoramidite (24)

  Materials
  • N‐(2‐(tert‐Butyldimethylsilyloxy)ethyl)‐uracil‐1‐yl‐N‐(prop‐2‐ynyl)acetamide (15; protocol 3)
  • Ethyl 2‐(N‐(2‐azidoethyl)‐uracil‐1‐yl‐acetamido)acetate (16; protocol 3)
  • Tetrahydrofuran (THF), anhydrous, ≥99.9% pure
  • tert‐Butyl alcohol (t‐BuOH), ACS grade, ≥99% pure
  • (+)‐Sodium L‐ascorbate, ≥98% pure
  • Copper(II) sulfate (CuSO 4) pentahydrate, ∼99% pure
  • Ammonium hydroxide (NH 4OH) solution, ACS grade, 28.0% to 30.0% NH 3 basis
  • Methanol (MeOH), ACS grade
  • Dichloromethane (CH 2Cl 2), ACS grade
  • Silica gel: 40 to 63 µm (230 to 400 mesh)
  • Nitrogen (or argon) gas
  • 2.0 M lithium borohydride (LiBH 4) in THF
  • Pyridine, dry
  • Dimethoxytrityl chloride (DMT‐Cl), 95% pure
  • Sodium sulfate (Na 2SO 4)
  • 1.0 M tetra‐n‐butylammonium fluoride (TBAF) in tetrahydrofuran
  • N‐(2‐(tert‐Butyldimethylsilyloxy)ethyl)‐uracil‐1‐yl‐N‐((1‐(2‐(uracil‐1‐yl‐N‐(2‐
  • 4‐dimethylaminopyridine (DMAP), 99% pure
  • Saturated solution of NaCl (brine)
  • Distilled N,N‐Diisopropylethylamine (DIPEA), 99% pure
  • 13.5% to 15.5% 2‐cyanoethyl N,N‐diisopropylchlorophosphoramidite, Cl
  • Distilled triethylamine (TEA), ≥99%
  • Hexane, ACS grade
  • Acetone, ACS grade
  • 50‐, 100‐, 250‐ and 500‐mL round‐bottom flasks
  • Stir bar
  • Hot plate magnetic stirrer
  • TLC plates (250‐μm thick; Silicycle; cat. no. TLG‐R10011B‐2020, http://www.silicycle.com/)
  • Short‐wave UV lamp
  • 250‐, 500‐, and 1‐L separatory funnels
  • 100‐, 250‐, and, 500‐mL Erlenmeyer flasks
  • Filter paper, grade P5
  • 3.5 × 25–cm and 2.5 × 25–cm glass chromatography columns
  • Reflux condenser
  • Disposable syringes and needles
  • Additional reagents and equipment for thin‐layer chromatography (TLC, appendix 3D) and column chromatography ( appendix 3E)

Basic Protocol 5: Preparation of Cytosine‐Triazole‐Uracil Phosphoramidite

  Materials
  • N‐(2‐(tert‐Butyldimethylsilyloxy)ethyl)‐uracil‐1‐yl‐N‐(prop‐2‐ynyl)acetamide (15; protocol 3)
  • N‐(1‐(2‐((2‐Azidoethyl)(2‐(bis(4‐methoxyphenyl)(phenyl)methoxy)ethyl)amino)‐2‐oxoethyl)‐N4‐(benzoyl)cytosin‐1‐yl) (19; Basic Protocol 3)
  • Tetrahydrofuran (THF), anhydrous, ≥99.9% pure
  • (+)‐Sodium L‐ascorbate, ≥98% pure
  • Copper(II) sulfate (CuSO 4) pentahydrate, ∼99% pure
  • Methanol (MeOH), ACS grade
  • Dichloromethane (CH 2Cl 2), ACS grade
  • Ethyl acetate (EtOAc), ACS grade
  • Sodium sulfate (Na 2SO 4)
  • Silica gel: 40 to 63 µm (230 to 400 mesh)
  • 1.0 M tetra‐n‐butylammonium fluoride (TBAF) in tetrahydrofuran
  • Distilled N,N‐Diisopropylethylamine (DIPEA), 99% pure
  • 13.5% to 15.5% 2‐cyanoethyl N,N‐diisopropylchlorophosphoramidite, Cl
  • Distilled triethylamine (TEA), ≥99% pure
  • Acetone, ACS grade
  • Hexane, ACS grade
  • 50‐, 100‐, 250‐ and 500‐mL round‐bottom flasks
  • Stir bar
  • Hot plate magnetic stirrer
  • TLC plates (250‐μm thick; Silicycle; cat. no. TLG‐R10011B‐2020, http://www.silicycle.com/)
  • Short‐wave UV lamp
  • 250‐, 500‐, and 1‐L separatory funnels
  • 100‐, 250‐, and, 500‐mL Erlenmeyer flasks
  • 2.5 × 25–cm and 3.5 × 25–cm glass chromatography column
  • Rotary evaporator
  • Additional reagents and equipment for thin‐layer chromatography (TLC, appendix 3D) and column chromatography ( appendix 3E)

Basic Protocol 6: Synthesis, Purification, and Characterization of Oligonucleotides Containing CtU or UtU Units

  Materials
  • Phosphoramidites:
    • 2‐(N‐(2‐(4‐((N‐(2‐(Bis(4‐methoxyphenyl)(phenyl)methoxy)ethyl)‐2‐(uracil‐1‐)acetamido)methyl)‐1H‐1,2,3‐triazol‐1‐yl)ethyl)‐2‐(uracil‐1‐yl)acetami‐do)ethyl (2‐cyanoethyl) diisopropylphosphoramidite (24; Basic Protocol 4)
    • 2‐(N‐((1‐(2‐(2‐(N4‐(Benzoyl)cytosin‐1‐yl)‐N‐(2‐(bis(4‐methoxyphenyl)(phenyl)methoxy)ethyl)acetamido)ethyl)‐1H‐1,2,3‐triazol‐4‐yl)methyl)‐2‐(uracil‐1‐yl)acetamido)ethyl (2‐cyanoethyl) diisopropylphosphoramidite (27; protocol 5)
    • 2′‐TBDMS guanosine (n‐ibu) phosphoramidite, ≥98% pure (ChemGenes, cat. no. ANP‐5673)
    • 2′‐TBDMS cytidine (n‐bz) phosphoramidite, 97.4% pure (ChemGenes, cat. no. ANP‐5672)
    • 2′‐TBDMS adenosine (n‐bz) phosphoramidite, 99.3% pure (ChemGenes, cat. no. ANP‐5671)
    • 2′‐TBDMS uridine phosphoramidite, 98.8% pure (ChemGenes, cat. no. ANP‐5674)
    • 2′‐Deoxyguanosine (n‐ibu) phosphoramide, 99% pure (ChemGenes, cat. no. ANP‐5553)
    • 2′‐Deoxyadenosine (n‐bz) phosphoramidite, 99.2% pure (ChemGenes, cat. no. ANP‐5551)
    • 2′Deoxycytosine (n‐bz) phosphoramidite, 99.5% pure (ChemGenes, cat. no. ANP‐5552)
    • 5′‐DMT thymidine phosphoramidite, ≥98.0% pure (ChemGenes, cat. no. ANP‐5554)
  • 2‐[2‐(4,4′‐Dimethoxytrityloxy)ethylsulfonyl]ethyl‐(2‐cyanoethyl)‐(N,N‐diisopropyl)‐phosphoramidite (chemical phosphorylating reagent (ChemGenes, cat. no. CLP‐1544), ≥98.1% pure
  • Acetonitrile (ACN), anhydrous
  • Dichloromethane (CH 2Cl 2), anhydrous, ≥99.8%
  • Nitrogen (or argon) gas
  • Acetic anhydride/pyridine/THF (Cap A)
  • 16% N‐methylimidazole (ChemGenes, cat. no. RN‐7776) in THF (Cap B)
  • 5‐ethylthio tetrazole (activator; ChemGenes, cat. no. RN‐1466), 0.25 M in ACN
  • 0.02 M iodine/pyridine/H 2O/THF (oxidation solution)
  • 3% trichloroacetic acid/dichloromethane
  • EMAM: 1:1 mixture of 40% (w/v) methylamine in H 2O and 33% (w/v) methylamine in ethanol
  • Dimethylsulfoxide (DMSO), minimum 99.5% GC
  • Triethylamine trihydrofluoride (3HF/TEA), 98% pure
  • Ethanol (EtOH), 95%
  • 3 M sodium acetate (NaOAc), pH 5.2
  • Nuclease‐free H 2O
  • 40% acrylamide (see recipe)
  • 10× and 0.5× TBE buffer (see recipe)
  • Urea, 99.5% pure
  • 25% (w/v) ammonium persulfate (APS) (see recipe)
  • Tetramethylethylenediamine (TEMED)
  • Denaturing loading solution (see recipe)
  • Ethidium bromide, >98.0%
  • Dry ice/95% ethanol bath
  • Gel eluting buffer (see recipe)
  • Matrix solution for MALDI‐TOF (see recipe)
  • Matrix solution for ESI Q‐TOF (see recipe)
  • Sodium phosphate buffer (see recipe)
  • DNA/RNA synthesizer (e.g., Applied Biosystems 394; see appendix 3C)
  • 0.2 µM or 1.0 µM CPG 500 (with desired nucleoside bound)
  • 0.2 µM or 1.0 µM Universal III solid supports
  • 50‐mL conical centrifuge tubes (e.g., BD Falcon)
  • 10‐mL syringe
  • 1.5‐mL screw‐cap vials
  • Needle to puncture lid of screw‐cap vial
  • Speedvac evaporator
  • Parafilm
  • 65°C water bath
  • Spectrophotometer (Thermo Scientific, cat. no. 840‐208200)
  • UV‐compatible cuvette
  • Gel plates
  • Spacers
  • Gasket
  • Comb
  • Clamps
  • 100‐mL beaker
  • Stir bar
  • Gel electrophoresis apparatus (see unit 10.4 & appendix 3B)
  • Spatula
  • Shaker
  • Large TLC plate
  • Short‐wavelength UV lamp
  • Camera
  • Scalpel
  • Skinny Scoopula
  • Centrifuge pestle (Fisher Scientific cat. no. 05‐559‐26)
  • Shaker
  • 0.4‐µm syringe filter
  • MWCO 3000 cellulose centrifugal filter
  • Millipore ZipTip C18‐column micropipet tips (see unit 10.1)
  • Metal sample plate (unit 10.1)
  • Zorbax Extend C18 HPLC column (unit 10.5)
  • Quartz cuvettes, 1 mm path lengths, Teflon caps
  • Spectropolarimeter (e.g., Jasco)
  • Meltwin software version 3.5 (http://www.meltwin3.com/)
  • Additional reagents and equipment for automated solid‐phase oligonucleotide synthesis ( appendix 3C) and PAGE purification of oligonucleotides (unit 10.4 & appendix 3B), MALDI‐TOF mass spectrometry (unit 10.1), electrospray ionization mass spectrometry (unit 10.2), and HPLC purification of oligonucleotides (unit 10.5)
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Literature Cited

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