Fluorous‐Assisted Synthesis of (E)‐5‐[3‐Aminoallyl]‐Uridine‐5′‐O‐Triphosphate

Anilkumar R. Kore1, Bo Yang1, Balasubramanian Srinivasan1

1 Life Sciences Solutions Group, Thermo Fisher Scientific, Austin, Texas
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 1.33
DOI:  10.1002/0471142700.nc0133s60
Online Posting Date:  March, 2015
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Abstract

An efficient, reliable method for the chemical synthesis of (E)‐5‐[3‐aminoallyl]‐uridine‐5′‐O‐triphosphate (AA‐UTP), starting from 5‐iodouridine, is described. This new strategy features the involvement of one‐pot triphosphate formation and fluorous solid‐phase extraction (F‐SPE). The one‐pot synthesis involves the mono phosphorylation of fluorous‐tagged uridine, followed by the reaction with pyrophosphate to afford the fluorous‐tagged AA‐UTP. The F‐SPE is achieved by installing a fluorous‐tag onto the uridine prior to triphosphate formation, purification via F‐SPE, and cleavage of the fluorous‐tag. It is worth mentioning that this protocol produces AA‐UTP in high yield and purity using one simple F‐SPE; no conventional column chromatography is involved. © 2015 by John Wiley & Sons, Inc.

Keywords: nucleoside triphosphates; one‐pot synthesis; fluorous separation; phosphorylation; fluorous solid‐phase extraction (F‐SPE)

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1:

  Materials
  • 5‐Iodouridine (ChemGenes)
  • Trimethyl phosphate, >97% pure (Sigma‐Aldrich)
  • Allylamine, ≥98% (Sigma‐Aldrich)
  • 0.1 M aqueous sodium acetate solution (see recipe)
  • Sodium bicarbonate (NaHCO 3), reagent grade, ≥99.5% (Sigma‐Aldrich)
  • 4.0 N acetic acid solution (see recipe)
  • Tributylamine (Sigma‐Aldrich)
  • Palladium (II) acetate, reagent grade, ≥98% (Pd(OAc) 2; Sigma‐Aldrich)
  • Acetonitrile, anhydrous (CH 3CN; Fisher Scientific)
  • Acetone, anhydrous (Fisher Scientific)
  • Methanol, anhydrous (Fisher Scientific)
  • Tetrahydrofuran (THF; Fisher Scientific)
  • Tributylammonium pyrophosphate (prepared within 1 month of use, see recipe)
  • Dichloromethane (DCM), technical grade (Sigma‐Aldrich)
  • Phosphorous oxychloride (POCl 3; Acros Organics)
  • Potassium tetrachloropalladate (II), ≥99.99% metals basis (K 2PdCl 4; Sigma‐Aldrich)
  • Triethylsilane, ≥99% (Sigma‐Aldrich)
  • Potassium fluoride (KF), ACS reagent, ≥99% (Sigma‐Aldrich)
  • Ammonium hydroxide (28% NH 3 in water; Sigma‐Aldrich)
  • N‐[4‐(1H,1H,2H,2H‐perfluorodecyl)benzyloxycarbonyloxy]succinimide (Flourous Technologies)
  • Sodium perchlorate (Sigma‐Aldrich)
  • HPLC buffer A: 5 mM ammonium phosphate monobasic, pH 2.8 (see recipe)
  • HPLC buffer B: 750 mM ammonium phosphate monobasic, pH 3.7 (see recipe)
  • FluoroFlash SPE (F‐SPE) cartridge, 20 g, 60 cc tube (Fluorous Technologies)
  • HPLC system (Waters) including:
    • Detector module
    • Hypersil SAX column (5 μm, 250 × 4.6 mm, Thermo Scientific)
  • Rotary evaporator
  • 0.2‐μm, 500‐mL Nalgene filtration unit (Thermo Scientific)
  • Vacuum manifold (nitrogen or argon gas)
  • 100‐ and 250‐mL; 1‐ and 2‐L one‐neck round‐bottom flasks, oven dried (Chemglass)
  • 500‐mL separatory funnel
  • Disposable needles
  • 1‐, 5‐, and 10‐mL sealed glass syringe
  • Magnetic stirrer (VWR)
  • Teflon‐coated magnetic stirring bars (oval shaped, Sigma‐Aldrich)
  • Rubber septa for 24/40‐glass joints (Chemglass)
  • 50‐mL centrifuge tubes
  • 250‐mL centrifuge bottle
  • Argon gas cylinder
  • RC‐3B centrifuge
  • Vacuum oven
  • Additional reagents and equipment for nuclear magnetic resonance (1H NMR and 31P NMR), and mass spectrometry
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Figures

Videos

Literature Cited

Literature Cited
  Brown, P.O. and Botstein, D. 1999. Exploring the new world of the genome with DNA microarrays. Nat. Genet. 21:33‐37.
  Curran, D.P. 2001. Fluorous reverse phase silica gel. A new tool for preparative separations in synthetic organic and organofluorine chemistry. Synlett 9:1488‐1496.
  Gladysz, J.A. and Curran, D.P. 2002. Fluorous chemistry: From biphasic catalysis to a parallel chemical universe and beyond. Tetrahedron 58:3823‐3825.
  Kore, A.R. and Muthian, S. 2012. Highly stereoselective palladium‐catalyzed Heck coupling of 5‐iodouridine‐5′‐triphosphates with allylamine: A new efficient method for the synthesis of (E)‐5‐aminoallyl‐uridine‐5′‐triphosphates. Tetrahedron Lett. 53:2530‐2532.
  Kore, A.R. and Srinivasan, B. 2013. Recent advances in the syntheses of nucleoside triphosphates. Curr. Org. Synth. 10:903‐934.
  Kore, A.R., Yang, B., and Srinivasan, B. 2013. Fluorous‐assisted synthesis of (E)‐5‐[3‐aminoallyl]‐uridine‐5′‐triphosphate. Tetrahedron Lett. 54:6264‐6266.
  Kore, A.R., Muthian, S., Annamalai, S., and Srinivasan, B. 2012. Gram‐scale chemical synthesis of 2′‐deoxynucleoside‐5′‐O‐triphosphates. Curr. Protoc. Nucleic Acid Chem. 49:13.10.1‐13.10.12.
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