Preparation of Azido Containing Oligonucleotides Through Diazo Transfer Reaction

Rémy Lartia1, Laureen Bonnat1, Pierre Murat1, Eric Defrancq1

1 Département de Chimie Moléculaire, Université Joseph Fourier, Grenoble, France
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.50
DOI:  10.1002/0471142700.nc0450s50
Online Posting Date:  September, 2012
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Abstract

The use of diazo transfer reagent, imidazole‐1‐sulfonyl azide hydrochloride (ISAHC), in the presence of Cu2+ cation enables clean and efficient conversion of aminated oligodeoxyribonucleotides (ODNs) into their azido counterparts under mild conditions. ODNs bearing amino tether either at the 3′, 5′, or any internal position could be modified in this way, thus demonstrating the versatility of this reaction. The method also benefits from the commercial availability or easy access by routine automated DNA synthesis of amino‐containing oligodeoxyribonucleotide starting material. Easy access to such azido‐modified ODNs is of great interest for conjugation in particular through copper catalyzed 1,3‐dipolar cycloaddition (CuAAC reaction). Curr. Protoc. Nucleic Acid Chem. 50:4.50.1‐4.50.7. © 2012 by John Wiley & Sons, Inc.

Keywords: azide; click‐chemistry; conjugation; diazotransfer reaction; Huisgen reaction; Staudinger reaction

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis of Azido‐ODNs Starting from Fully Deprotected Aminoalkyl‐ODNs
  • Support Protocol 1: Preparation of ISAHC
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Synthesis of Azido‐ODNs Starting from Fully Deprotected Aminoalkyl‐ODNs

  Materials
  • A dried ODN bearing one aminoalkyl‐tether at the 3′ or 5′‐end, at the internucleotidic position or linked to a nucleobase and provided as ammonium or trialkylammonium salt
  • 50 mM NaHCO 3 buffer in water/MeOH (3:1; v/v) solution (pH has to be adjusted at 8.5)
  • 100 mM aqueous CuSO 4 solution
  • ISAHC reagent synthesized according to Goddard‐Borger and Stick ( ); see protocol 2
  • Methanol (MeOH), analytical grade
  • Deionized water1 M triethylammonium acetate (TEAA) aqueous solution (pH = 7.0)
  • 20‐ to 200‐µL pipettor
  • 1‐ to 20‐µL pipettor
  • Standard plastic vial closed by a screw cap
  • Vortex mixer
  • Heating block set at 60°C
  • Illustra NAP‐25 column (GE Healthcare) for size‐exclusion chromatography
  • Additional reagents and equipment for RP‐HPLC analysis and purification (see unit 10.5).
NOTE: RP‐HPLC is required only if high‐purity ODNs are needed.

Support Protocol 1: Preparation of ISAHC

  Materials
  • Acetonitrile
  • Sodium azide
  • Sulfonyl chloride
  • Imidazole
  • Ethyl acetate5% (w/v) Sodium bicarbonate solution in water
  • Anhydrous sulfate sodium
  • Ethanolic HCl solution obtained by drop‐to‐drop addition of acetyl chloride (4.3 mL) in ethanol (15 mL)
  • Crystallizer filled with ice
  • 100‐mL round‐bottom flask
  • Dropping funnel
  • Three rubber stoppers
  • Magnetic stirrer
  • Ice bath
  • Separatory funnel
  • Erlenmeyer flasks
  • Filtering funnels
  • Büchner funnel
  • Vacuum apparatus
CAUTION: The mother liquors from which ISAHC crystallizes, or any other solutions that contain ISAHC should not be concentrated. Explosions have been reported.
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Figures

Videos

Literature Cited

Literature Cited
   Amblard, F., Cho, J.H., and Schinazi, R.F. 2009. Cu(I)‐catalyzed Huisgen azide‐alkyne 1,3‐dipolar cycloaddition reaction in nucleoside, nucleotide, and oligonucleotide chemistry. Chem. Rev. 109:4207‐4220.
   Goddard‐Borger, E.D. and Stick, R.V. 2007. An efficient, inexpensive, and shelf‐stable diazotransfer reagent: Imidazole‐1‐sulfonyl azide hydrochloride. Org. Lett. 9:3797‐3800.
   Goddard‐Borger, E.D. and Stick, R.V. 2011. An efficient, inexpensive, and shelf‐stable diazotransfer reagent: Imidazole‐1‐sulfonyl azide hydrochloride. Org. Lett. 13:2514.
   Jentzsch, E. and Mokhir, A. 2009. A fluorogenic, nucleic acid directed “Click” reaction. Inorg. Chem. 48:9593‐9595.
   Lallana, E., Riguera, R., and Fernandez‐Megia, E. 2011. Reliable and efficient procedures for the conjugation of biomolecules through Huisgen azide‐alkyne cycloadditions Angew. Chem. Int. Ed. 50:8794‐8804.
   Lartia, R., Murat, P., Dumy, P., and Defrancq, E. 2011. Versatile introduction of azido moiety into oligonucleotides through diazo transfer reaction. Org. Lett. 13:5467‐5675.
   Ustinov, A.V., Stepanova, I.A., Dubnyakova, V.V., Zatsepin, T.S., Nozhevnikova, E.V., and Korshun, V.A. 2010. Modification of nucleic acids using [3+2] dipolar cycloaddition of azides and alkynes. Russ. J. Bioorg. Chem. 36:437‐481.
   van Berkel, S.S., van Eldijk, M.B., and van Hest, J.C.M. 2011. Staudinger ligation as a method for bioconjugation. Angew. Chem. Int. Ed. 50:8806‐8827.
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