Click Chemistry

"Click Chemistry" is a term introduced by K. B. Sharpless as a robust and specific method of ligating two molecules together. Two important characteristics make click chemistry attractive for assembling biomolecules. First, click reactions are bio-orthogonal, thus the click chemistry-functionalized biomolecules would not react with the natural biomolecules that lack a clickable functional group. Secondly, the reactions can proceed under mild conditions, such as at room temperature and in aqueous media. With proper modification, click reactions can run at or near biological conditions, produce stable products with minimal, non-toxic byproducts, and proceed quickly to high yield in one vial. It is now commonly used in bioconjugation to join substrates of choice with specific biomolecules. Examples of click reactions that are being used for bioconjugation are Staudinger ligation with azides, strain promoted Huisgen cycloaddition of azides, and tetrazine ligation.

 

Click chemistry reactions are stereospecific, simple to perform and can be conducted in easily removable or benign solvents. This concept was developed in parallel with the interest within the pharmaceutical, material, and other industries in capabilities of generating large libraries of compounds for screening in discovery research. Several types of reaction have been identified that fulfill these criteria. They are thermodynamically-favored reactions that lead specifically to one product, such as nucleophilic ring opening reactions of epoxides and aziridines; non-aldol type carbonyl reactions, such as formation of hydrazones and heterocycles; eletrophilic additions to carbon-carbon multiple bonds, such as oxidative formation of epoxides and Michael Additions; and cycloaddition reactions.

Click Chemistry Reactive Labels

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An examination of the azide-alkyne cycloaddition shows that it fulfills many of the prerequisites. The copper-catalyzed azide-alkyne cyloaddition is a two-step process. First, one reaction partner-either an azide or alkyne linked to a "building block" such as a peptide or an oligonucleotide, is incorporated by conventional synthesis. Subsequently, the other reaction partner-the complementary alkyne or azide linked to a fluorescent dye, biotin or other detection reagent-is "clicked" into place in the presence of catalytic copper (I). One reaction partner must be an azide derivative and the other an alkyne derivative, but functional moiety can serve as either the incorporated molecule or the detection molecule. The reaction is also regiospecific, yielding exclusively 1,4-disubstituted-1,2,3-triazole linkages. The 1,2,3-triazole linkage between a peptide or an oligonucleotide and a dye is extremely stable. It is not susceptible to hydrolysis, oxidation or reduction.

AAT Bioquest offers a variety of dye azides and alkynes for labeling peptides and oligonucleotides. These clickable reagents include both common fluorescent dyes (e.g., fluoresceins, rhodamines and cyanines) and non-fluorescent quenchers. Our Tide Fluor™ and Tide Quencher™ dyes are specifically optimized for preparing novel FRET substrates.

Assaywise Letters:

Click Chemistry Building Blocks

Optimized FastClick™ Click Reagents

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FastClick™ reagents have been developed by the scientists of AAT Bioquest for enhancing the yield and reaction speed of copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. They contain a copper-chelating ligand that significantly stabilizes the Cu(I) oxidation state and thus accelerates the click reaction. They do not require the use of an external copper-chelator (such as the common THPTA or BTTAA). The high concentration of copper chelators is known to have a detrimental effect on DNA/RNA, thus causing biocompatibility issues. The introduction of a copper-chelating moiety at the reporter molecule allows for a dramatic raise of the effective Cu(I) concentration at the reaction site and thus accelerates the reaction. Under extremely mild conditions the FastClick™ azides and alkynes react much faster in high yield compared to the corresponding conventional CuAAC reactions.

Services:

Customer Conjugation Services

References

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Metabolically active bacteria detected with click chemistry in low organic matter rainwater
Aqueous synthesis of a small-molecule lanthanide chelator amenable to copper-free click chemistry
Mechanochemical Activation of Fluorogenic CuAAC “Click” Reactions for Stress-Sensing Applications
Site-specific conjugation of 8-ethynyl-BODIPY to a protein by [2 + 3] cycloaddition