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點(diǎn)擊化學(xué)Click Chemistry
Introduction
Classic Click Chemistry uses copper, Cu(I), to catalyze the 1,3-dipolar cycloaddition of an alkyne with an azide to form a 1,2,3-triazole.1,2 Sources of Cu(I) include copper(I) iodide, copper(I) bromide, copper turnings, or copper(II) sulfate (CuSO4) and a reducing agent.1 However, the thermodynamic instability of Cu(I), which readily
oxidizes to inactive Cu (II) usually requires the copper catalyst to be prepared with an appropriate chelating ligand.
An improvement in Click Chemistry uses the in situ preparation of Cu(I) from the reduction of CuSO4 with sodium ascorbate and a Cu(I) stabilizing ligand,tris-(benzyltriazolylmethyl)amine (TBTA).3 This leads to a more reliable click reaction by avoiding the oxidation of catalytic Cu(I) by dissolved oxygen. In a typical reaction, copper sulfate is pre-complexed with TBTA. This complexed catalyst is mixed with an alkyne and an azide, followed by the addition of sodium ascorbate to initiate the click reaction.
TBTA covers some of the practical applications for Click Chemistry except for aqueous conjugation reactions. The water-soluble tris(3-hydroxypropyltriazolylmethyl)amine (THPTA) click ligand further simplifies Click Chemistry by allowing the entire reaction to be run in water, affording biological compatibility for Click reactions.4 The THPTA ligand binds Cu(I), blocking the bioavailability of Cu(I) and ameliorating the potential toxic effects while maintaining the catalytic effectiveness in click conjugations. The THPTA ligand was effectively used to label live cells with high efficiency while maintaining cell viability.
In our hands, we have found THPTA to be a highly efficient ligand for click chemistry, in partially in completely aqueous reactions. Labeling is complete in as little as 15-30 minutes at room temperature. The ligand CuSO4 complex exhibits no loss of activity when frozen for at least a month.
Example Protocols:
This section contains some general protocols for click reactions. These protocols may be used as a starting point for optimization of your particular click chemistry procedures.
Labeling of Oligonucleotides and DNA
1. Prepare the following stock solutions:
2. Incubate CuSO4 with THPTA ligand in a 1:2 ratio several minutes before the reaction. This solution is stable for several weeks when frozen.
3. To the oligo/DNA solution, add an excess of azide (4-50 eq).
4. Add 25 equivalents of THPTA/CuSO4.
5. Add 40 equivalents of sodium ascorbate.
6. Let the reaction stand at room temperature for 30-60 minutes.
7. Ethanol-precipitate the oligo or purify.
Labeling of Cell Lysate
1. Prepare the following click solutions:
2. For each azide or alkyne- modified protein lysate sample, add the following to a 1.5 mL microfuge tube,
then vortex briefly to mix.
3. Add 10 µL of 100 mM THPTA solution, vortex briefly to mix.
4. Add 10 µL of 20 mM CuSO4 solution, vortex briefly to mix.
5. Add 10 µL of 300 mM sodium ascorbate solution to initiate click reaction, vortex briefly to mix.
6. Protect reaction from light and allow click reaction to incubate for 30 minutes at room temperature.
7. Proteins in lysate are now click labeled and ready for downstream processing and/or analysis.
References
1. Rostovtsev, V. V., et al (2002). A Stepwise Huisgen Cycloaddition Process: Copper(I)-Catalyzed Regioselective “Ligation” of Azides and Terminal Alkynes. Angew. Chem. Int. Ed., 41(14), 2596–2599.
2. Tornøe, C.W., et al (2002). Peptidotriazoles on Solid Phase: [1,2,3]-Triazoles by Regiospecific Copper(I)- Catalyzed 1,3- Dipolar Cycloadditions of Terminal Alkynes to Azides J. Org. Chem., 67(9), 3057–3064.
3. Chan, T.R., et al (2004). Polytriazoles as Copper(I)-Stabilizing Ligands in Catalysis. Org. Lett., 6(17), 2853- 2855.
4. (a) Presolski, S. I., et al (2011). Copper-Catalyzed Azide–Alkyne Click Chemistry for Bioconjugation. Current Protocols in Chemical Biology. 3(4), 153–162. (b) Hong, V., et al (2009). Analysis and Optimization of CopperCatalyzed Azide– Alkyne Cycloaddition for Bioconjugation. Angew. Chem. Int. Ed., 48(52), 9879–9883.
5. Hong, V., et al (2010). Labeling Live Cells by Copper-Catalyzed Alkyne−Azide Click Chemistry. Bioconjugate Chem., 21(10), 1912-1916.