Palladium‐Catalyzed Synthesis of (E)‐5‐(3‐Aminoallyl)‐Uridine‐5′‐O‐Triphosphates

Muthian Shanmugasundaram1, Annamalai Senthilvelan1, Anilkumar R. Kore1

1 Life Sciences Solutions Group, Thermo Fisher Scientific, Austin, Texas
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 13.18
DOI:  10.1002/cpnc.42
Online Posting Date:  December, 2017
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This unit describes a simple, reliable, and efficient chemical method for the synthesis of 5‐(3‐aminoallyl)‐2′‐deoxyuridine‐5′‐O‐triphosphate (AA‐dUTP) and 5‐(3‐aminoallyl)‐uridine‐5′‐O‐triphosphate (AA‐UTP), starting from the corresponding nucleoside triphosphate. The presented strategy involves regioselective iodination of nucleoside triphosphate using N‐iodosuccinimide followed by the palladium‐catalyzed Heck coupling with allylamine to provide the corresponding (E)‐5‐aminoallyl‐uridine‐5′‐O‐triphosphate in good yields. It is noteworthy that the protocol not only provides a high‐purity product but also eliminates the use of toxic mercuric reagents. © 2017 by John Wiley & Sons, Inc.

Keywords: 5‐(3‐aminoallyl)‐2′‐deoxyuridine‐5′‐O‐triphosphate (AA‐dUTP); 5‐(3‐aminoallyl)‐2′‐uridine‐5′‐O‐triphosphate (AA‐UTP); cDNA microarrays; gene expression; labeling; reverse transcription

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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1:

  • 2′‐Deoxyuridine‐5′‐O‐triphosphate (Fisher Scientific)
  • N‐Iodosuccinimide (Fisher Scientific)
  • Sodium azide (Fisher Scientific)
  • Sodium perchlorate (Fisher Scientific)
  • Acetone
  • 28% (v/v) ammonium hydroxide (Fisher Scientific)
  • DEAE Sepharose (GE Healthcare)
  • Isopropanol (Fisher Scientific)
  • 2 M NaCl (see recipe)
  • 1 M TEAB buffer (see recipe)
  • Uridine‐5′‐O‐triphosphate (Fisher Scientific)
  • 4 M acetic acid solution (see recipe)
  • Allylamine (Across)
  • K 2PdCl 4 (Sigma‐Aldrich)
  • 0.1 M sodium acetate, pH 5.5 (see recipe)
  • HPLC mobile phase A: 5 mM ammonium phosphate monobasic, pH 2.8 (see recipe)
  • HPLC mobile phase B: 750 mM ammonium phosphate monobasic, pH 3.7 (see recipe)
  • Ammonium phosphate monobasic (Fisher Scientific)
  • Triethylamine (Fisher Scientific)
  • 500‐mL one‐neck round‐bottom flasks, oven dried (Chemglass)
  • Teflon‐coated magnetic stir bar (Fisher Scientific)
  • Rubber septa for 24/40 glass joints (Chemglass)
  • Magnetic stir plate (Fisher Scientific)
  • 500‐mL centrifuge bottles
  • Sorvall RC‐3B centrifuge (Thermo Fisher), 4°C
  • 4‐L conical flasks
  • pH meter (Thermo Fisher)
  • FPLC ÄKTA purifier (GE Healthcare) including:
  • UV detector: 260 nm and 272 nm
  • Chromatography column: (10‐cm × 78.5‐cm)
  • HPLC system (Waters) including:
  • Detector module
  • 5‐μM Hypersil SAX column (4.6‐mm × 25‐cm)
  • Rotary evaporator (Buchi)
  • Vacuum/nitrogen (or argon) gas manifold
  • Oil bath (Fisher Scientific)
  • 50‐mL Falcon tubes
  • Ice/NaCl bath (−5ο to −10οC)
  • 2‐mL sealed glass syringes (Fisher Scientific)
  • Disposable needles
  • 0.2‐µM, 1000‐mL Nalgene filters
  • Carbon dioxide gas cylinder
  • Additional reagents and equipment for proton nuclear magnetic resonance (1H NMR and 31P NMR) and mass spectrometry
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Literature Cited

Literature Cited
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