Copper‐Catalyzed Azide–Alkyne Click Chemistry for Bioconjugation

Stanislav I. Presolski1, Vu Phong Hong1, M.G. Finn1

1 The Scripps Research Institute, La Jolla, California
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch110148
Online Posting Date:  December, 2011
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Abstract

The copper‐catalyzed azide‐alkyne cycloaddition reaction is widely used for the connection of molecular entities of all sizes. A protocol is provided here for the process with biomolecules. Ascorbate is used as reducing agent to maintain the required cuprous oxidation state. Since these convenient conditions produce reactive oxygen species, five equivalents of a copper‐binding ligand are used with respect to metal. The ligand both accelerates the reaction and serves as a sacrificial reductant, protecting the biomolecules from oxidation. A procedure is also described for testing the efficiency of the reaction under desired conditions for purposes of optimization, before expensive biological reagents are used. Curr. Protoc. Chem. Biol. 3:153‐162 © 2011 by John Wiley & Sons, Inc.

Keywords: click chemistry; azides; alkynes; bioconjugation; proteins; nucleic acids; copper

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Copper‐Catalyzed Azide‐Alkyne Cycloaddition for Coupling of Cargo‐Azide to Biomolecule‐Alkyne
  • Support Protocol 1: Determining the Efficiency of Bioconjugation CuAAC with a Fluorogenic Probe
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Copper‐Catalyzed Azide‐Alkyne Cycloaddition for Coupling of Cargo‐Azide to Biomolecule‐Alkyne

  Materials
  • Biomolecule‐alkyne of interest
  • 100 mM potassium phosphate buffer, pH 7 (see Critical Parameters regarding buffers)
  • 5 mM cargo‐azide
  • 20 mM CuSO 4 in water
  • 50 mM ligand THPTA (structure 1 in Fig. ; available in small quantities from the authors, ), in water
  • 100 mM sodium ascorbate: prepare fresh just before use by adding 1 ml of water to 20 mg ascorbate
  • 100 mM aminoguanidine hydrochloride (structure 2 in Fig. ): add 1 ml of water to 11 mg aminoguanidine)
  • 2‐ml microcentrifuge tubes
  • End‐over‐end rotator

Support Protocol 1: Determining the Efficiency of Bioconjugation CuAAC with a Fluorogenic Probe

  • The materials used here are identical to those used in Basic Protocol 2, except for the “cargo‐azide,” which is compound 3 in Figure ; this molecule is available from Glen Research (http://www.glenresearch.com, cat. no. 50‐2004‐92), or it can also be prepared by following the published procedure (Sivakumar et al., ); the solid or DMSO stock solution of this compound should be stored in a refrigerator and protected from light
  • Protein X (see above) or surrogate protein, e.g., bovine serum albumin (BSA)
  • Fluorometer
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Figures

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Literature Cited

Literature Cited
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