The reaction scheme of a biomolecule alkyne with a dye azide.
"Click Chemistry" is a term introduced by K. B. Sharpless in 2001 to describe reactions that are high in yields, wide in scope, and create only by-products that can be removed without chromatography. 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.
The reaction scheme of a biomolecule azide with a dye alkyne.
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.
Table 1. Dye Azides for Labeling Oligonucleotides and Peptides
|508||AMCA Azide||353||455||18,000||0.183 ||0.153||1 mg|
|143||Cyanine 3 azide [equivalent to Cy3® azide]||555||565||150,000||0.042||0.073||1 mg|
|153||Cyanine 5 azide [equivalent to Cy5® azide]||649||665||250,000||0.026||0.030||1 mg|
|178||Cyanine 5.5 azide [equivalent to Cy5.5® azide]||678||701||230,000||0.094||0.101||1 mg|
|163||Cyanine 7 azide [equivalent to Cy7® azide]||749||776||275,000||0.025||0.036||1 mg|
|131||5-FAM Azide||494||521||75,000||0.247||0.178||10 mg|
|133||6-FAM Azide||494||518||75,000||0.255||0.172||10 mg|
|240||6-HEX azide||533||550||74,000||0.300||0.127||5 mg|
|248||6-JOE azide||520||548||73,000||0.326||0.217||5 mg|
|494||6-ROX azide||575||602||95,000||0.307||0.179||5 mg|
Table 2. Dye Alkynes for Labeling Oligonucleotides and Peptides
|507||AMCA Alkyne||353||455||1 mg|
|144||Cyanine 3 alkyne [equivalent to Cy3® alkyne]||555||565||1 mg|
|154||Cyanine 5 alkyne [equivalent to Cy5® alkyne]||649||665||1 mg|
|179||Cyanine 5.5 alkyne [equivalent to Cy5.5® alkyne]||678||701||1 mg|
|164||Cyanine 7 alkyne [equivalent to Cy7® alkyne]||749||776||1 mg|
|132||5-FAM Alkyne||494||518||10 mg|
|134||6-FAM Alkyne||494||518||10 mg|
|241||6-HEX alkyne||533||550||5 mg|
|249||6-JOE alkyne||520||548||5 mg|
|495||6-ROX alkyne||575||602||5 mg|