AAT Bioquest

Click Chemistry

FastClick™ Cy5 Alkyne
The reaction (Green Bar) of FastClick™ Cy5 Alkyne with coumarin azide occurs under extremely mild conditions (e.g., [Azide] = 0.02 mM, [Alkyne] = 0.02 mM, [CuSO4] = 0.02 mM, [Sodium Ascorbate] = 5 mM, in 100 mM HEPES) under which the common Cy5 alkyne does not effectively react with the coumarin azide substrate.
"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

click chemistry bioconjugation
The five major steps of click chemistry bioconjugation. This particular example is of conjugated molecules that have been integrated into the cells, typically via incubation or other techniques. The cycloaddition reaction is between the azide and alkyne on either the labeled probe or the target biomolecule. The example above shows the eventual visualization of the targeted cell surface protein via a fluorescence-labeled click probe. Figure made in BioRender.
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 potentially suitable for labeling of DNA oligos by Click Chemistry.

Superior Alternative to
Excitation Max (nm)
Emission Max (nm)
Unit Size
Cat No.
Tide Fluor™ 1 azide [TF1 azide]EDANS3414485 mg2236
Tide Fluor™ 2 azide [TF2 azide]Fluoresceins (FAM and FITC)5035251 mg2252
Tide Fluor™ 3 azide [TF3 azide]Cy3®5545781 mg2254
Tide Fluor™ 4 azide [TF4 azide]ROX/Texas Red®5786021 mg2300
Tide Fluor™ 5WS azide [TF5WS azide]Cy5®6496641 mg2275
Tide Fluor™ 6WS azide [TF6WS azide]Cy5.56827011 mg2302
Tide Fluor™ 7WS azide [TF7WS azide]Cy7®7567801 mg2304
Tide Fluor™ 8WS azide [TF8WS azide] *Near Infrared Emission*IRDye® 8007858011 mg2306

Assaywise Letters:

Table 2. Click Chemistry Reagents

Product Name
Unit Size
1264310XHis alkyne1 mg
1264210XHis azide1 mg
203322',3'-cGAMP azide100 µg
1325-FAM Alkyne10 mg
1315-FAM Azide10 mg
4875-TAMRA alkyne5 mg
4865-TAMRA azide5 mg
1346-FAM Alkyne10 mg
1336-FAM Azide10 mg
2416-HEX alkyne5 mg
2406-HEX azide5 mg
2496-JOE alkyne5 mg
2486-JOE azide5 mg
2166-NED alkyne1 mg
2176-NED azide1 mg
2366-OG488 Azide1 mg
4956-ROX alkyne5 mg
4946-ROX azide5 mg
4916-TAMRA alkyne5 mg
4906-TAMRA azide5 mg
2456-TET alkyne5 mg
2446-TET azide5 mg
2206-VIC Alkyne1 mg
2196-VIC Azide1 mg
126296XHis alkyne1 mg
126286XHis azide1 mg
507AMCA Alkyne1 mg
508AMCA Azide1 mg
2414AzoDye-1 Alkyne1 mg
2412AzoDye-1 Azide1 mg
2415AzoDye-1 PEG10 Alkyne1 mg
2413AzoDye-1-PEG10-Azide1 mg
2434AzoDye-2 Alkyne1 mg
2432AzoDye-2 Azide1 mg
2431AzoDye-2 C2 Azide1 mg
2435AzoDye-2 PEG10 Alkyne1 mg
2433AzoDye-2-PEG10 Azide1 mg
2484AzoDye-3 Alkyne1 mg
2482AzoDye-3 Azide1 mg
2485AzoDye-3 PEG10 Alkyne1 mg
2483AzoDye-3 PEG10 Azide1 mg
3021Biotin Alkyne *CAS 773888-45-2*5 mg
3023Biotin Azide5 mg
3020Biotin C2 Azide5 mg
3019Biotin-PEG3-azide *CAS 875770-34-6*5 mg
3018Biotin PEG 4 Alkyne5 mg
20614Cal-520® alkyne100 µg
20613Cal-520® azide100 µg
20643Calbryte™-520XL azide1 mg
6910CDPI3 Alkyne [Minor Groove Binder Alkyne]1 mg
6909CDPI3 Azide [Minor Groove Binder Azide]1 mg
944Cy3B alkyne1 mg
943Cy3B azide1 mg
982Cyanine 3.5 alkyne [equivalent to Cy3.5® alkyne]1 mg
980Cyanine 3.5 azide [equivalent to Cy3.5® azide]1 mg
144Cyanine 3 alkyne [equivalent to Cy3® alkyne]1 mg
143Cyanine 3 azide [equivalent to Cy3® azide]1 mg
179Cyanine 5.5 alkyne [equivalent to Cy5.5® alkyne]1 mg
178Cyanine 5.5 azide [equivalent to Cy5.5® azide]1 mg
154Cyanine 5 alkyne [equivalent to Cy5® alkyne]1 mg
153Cyanine 5 azide [equivalent to Cy5® azide]1 mg
164Cyanine 7 alkyne [equivalent to Cy7® alkyne]1 mg
163Cyanine 7 azide [equivalent to Cy7® azide]1 mg
3505Digoxigenin alkyne1 mg
3504Digoxigenin azide1 mg
17673Hoechst 33342 alkyne1 mg
17674Hoechst 33342 azide1 mg
986ICG alkyne1 mg
985ICG azide1 mg
1094iFluor™ 405 azide1 mg
999iFluor® 488 alkyne1 mg
1000iFluor® 488 azide1 mg
1092iFluor® 555 alkyne1 mg
1093iFluor® 555 azide1 mg
1090iFluor® 647 alkyne1 mg
1091iFluor® 647 azide1 mg
1365iFluor™ 790 Alkyne1 mg
1363iFluor™ 790 Azide1 mg
833Methylene Blue Alkyne1 mg
832Methylene Blue Azide1 mg
1690mFluor™ Violet 450 Azide1 mg
39058Psoralen MOP Alkyne1 mg
39057Psoralen MOP Azide1 mg
39063Psoralen TMP Alkyne1 mg
39062Psoralen TMP Azide1 mg
3404ReadiLeave™ Reversible Biotin Alkyne1 mg
3403ReadiLeave™ Reversible Biotin Azide1 mg
21126Rhod-4™ alkyne100 µg
21125Rhod-4™ azide100 µg
39005SDA alkyne1 mg
39004SDA azide1 mg
485Texas Red® alkyne *Single Isomer*5 mg
484Texas Red® azide *Single Isomer*5 mg
2237Tide Fluor™ 1 alkyne [TF1 alkyne]5 mg
2236Tide Fluor™ 1 azide [TF1 azide]5 mg
2253Tide Fluor™ 2 alkyne [TF2 alkyne]1 mg
2252Tide Fluor™ 2 azide [TF2 azide]1 mg
2255Tide Fluor™ 3 alkyne [TF3 alkyne]1 mg
2254Tide Fluor™ 3 azide [TF3 azide]1 mg
2301Tide Fluor™ 4 alkyne [TF4 alkyne]1 mg
2300Tide Fluor™ 4 azide [TF4 azide]1 mg
2276Tide Fluor™ 5WS alkyne [TF5WS alkyne]1 mg
2275Tide Fluor™ 5WS azide [TF5WS azide]1 mg
2303Tide Fluor™ 6WS alkyne [TF6WS alkyne]1 mg
2302Tide Fluor™ 6WS azide [TF6WS azide]1 mg
2305Tide Fluor™ 7WS alkyne [TF7WS alkyne]1 mg
2304Tide Fluor™ 7WS azide [TF7WS azide]1 mg
2307Tide Fluor™ 8WS alkyne [TF8WS alkyne] *Near Infrared Emission*1 mg
2306Tide Fluor™ 8WS azide [TF8WS azide] *Near Infrared Emission*1 mg
2189Tide Quencher™ 1 alkyne [TQ1 alkyne]5 mg
2188Tide Quencher™ 1 azide [TQ1 azide]5 mg
2212Tide Quencher™ 2 alkyne [TQ2 alkyne]5 mg
2211Tide Quencher™ 2 azide [TQ2 azide]5 mg
2213Tide Quencher™ 2WS alkyne [TQ2WS alkyne]1 mg
2232Tide Quencher™ 3 alkyne [TQ3 alkyne]5 mg
2231Tide Quencher™ 3 azide [TQ3 azide]5 mg
2069Tide Quencher™ 4WS alkyne [TQ4WS alkyne]1 mg
2068Tide Quencher™ 4WS azide [TQ4WS azide]1 mg
2089Tide Quencher™ 5.1WS alkyne [TQ5.1WS alkyne]1 mg
2088Tide Quencher™ 5.1WS azide [TQ5.1WS azide]1 mg
2083Tide Quencher™ 5WS alkyne [TQ5WS alkyne]1 mg
2082Tide Quencher™ 5WS azide [TQ5WS azide]1 mg
2098Tide Quencher™ 6WS alkyne [TQ6WS alkyne]1 mg
2097Tide Quencher™ 6WS azide [TQ6WS azide]1 mg
2121Tide Quencher™ 7.1WS alkyne [TQ7.1WS alkyne]1 mg
2120Tide Quencher™ 7.1WS azide [TQ7.1WS azide]1 mg
2129Tide Quencher™ 7.2WS alkyne [TQ7.2WS alkyne]1 mg
2128Tide Quencher™ 7.2WS azide [TQ7.2WS azide]1 mg
2113Tide Quencher™ 7WS alkyne [TQ7WS alkyne]1 mg
2112Tide Quencher™ 7WS azide [TQ7WS azide]1 mg
2137Tide Quencher™ 8WS alkyne [TQ8WS alkyne]1 mg
2136Tide Quencher™ 8WS azide [TQ8WS azide]1 mg
1700XFD488 alkyne *Same Structure to Alexa Fluor™ 488 alkyne*1 mg
1701XFD488 azide *Same Structure to Alexa Fluor™ 488 azide*1 mg
1897XFD647 Alkyne1 mg
1896XFD647 Azide1 mg


Optimized FastClick™ Click Reagents

FastClick™ Biotin Alkyne
FastClick™ Biotin Alkyne contains both the CAG moiety of FastClick (for assisting click efficiency) and biotin (as the detection tag) for developing biotin-based probes. It readily reacts with an azido-containing biomolecule under extremely mild conditions.
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.

Table 3. FastClick™ Series

Product Name
Unit Size
72700FastClick™ Cy3 Azide1 mg
72702FastClick™ Cy5 Azide1 mg
72704FastClick™ Cy7 Azide1 mg
72710FastClick™ 5-FAM Azide1 mg
72711FastClick™ 6-FAM Azide1 mg
72712FastClick™ 5-TAMRA Azide1 mg
72713FastClick™ 6-TAMRA Azide1 mg
72714FastClick™ 6-ROX Azide1 mg
72730FastClick™ XFD350 Azide1 mg
72733FastClick™ XFD405 Azide1 mg
72735FastClick™ XFD488 Azide1 mg
72737FastClick™ XFD555 Azide1 mg
72740FastClick™ XFD647 Azide1 mg
72745FastClick™ XFD750 Azide1 mg
72800FastClick™ Digoxigenin (DIG) Azide1 mg
72801FastClick™ Biotin Azide1 mg
72850FastClick™ Cy3 Alkyne1 mg
72852FastClick™ Cy5 Alkyne1 mg
72854FastClick™ Cy7 Alkyne1 mg
72860FastClick™ 5-FAM Alkyne1 mg
72861FastClick™ 6-FAM Alkyne1 mg
72862FastClick™ 5-TAMRA Alkyne1 mg
72863FastClick™ 6-TAMRA Alkyne1 mg
72864FastClick™ 6-ROX Alkyne1 mg
72870FastClick™ XFD350 Alkyne1 mg
72873FastClick™ XFD405 Alkyne1 mg
72875FastClick™ XFD488 Alkyne1 mg
72877FastClick™ XFD555 Alkyne1 mg
72880FastClick™ XFD647 Alkyne1 mg
72885FastClick™ XFD750 Alkyne1 mg
72900FastClick™ Digoxigenin (DIG) Alkyne1 mg
72901FastClick™ Biotin Alkyne1 mg




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