Click Chemistry


Click Chemistry1 describes pairs of functional groups that rapidly and selectively react (“click”) with each other in mild, aqueous conditions. The concept of Click Chemistry has been transformed into convenient, versatile and reliable two-step coupling procedures of two molecules A and B1-5, that are widely used in biosciences6-8, drug discovery9 and material science10.

Principle of Click Chemistry


Advantages of Click Chemistry

1. Activation of molecule A and B
Compatible CLICK-functional groups are introduced via CLICK Reagents
2. CLICK-coupling of molecule A and B
The CLICK-activated molecules A and B form a stable conjugate
 Highly selective, low background labeling: CLICK-functional groups are inert to naturally occurring functional groups (“bioorthogonal”) such as amines
Rapid and quantitative labeling
Allows non-radioactive analysis of enzymatic activities both in vitro and in vivo: Small-sized CLICK-functional groups possess excellent substrate properties

Especially biomolecule labeling requires reaction procedures that can be performed under physiological conditions (neutral pH, aqueous solution, ambient temperature) with low reactant concentrations to ensure non-toxic, low background labeling at reasonable time scales while still preserving biological function. Among the plethora of possible reactions only a few generally fit the necessary reactivity, selectivity and biocompatibility criteria (Fig. 1):

1. Cu(I)- catalyzed Azide - Alkyne Click Chemistry reaction (CuAAC)
2. Strain-promoted Azide - Alkyne Click Chemistry reaction (SPAAC)
3. Tetrazine – trans-Cyclooctene Ligation

  Figure 1: Overview of the most common Click Chemistry reactions.
Click Chemistry reactions can be categorized into two separate groups: Copper (Cu(I))-catalyzed and Copperfree. The Cu(I)- catalyzed Azide - Alkyne Click Chemistry reaction (CuAAC) (1.) relies on the presence of Cu(I) ions whereas the Copper-free strain-promoted Azide - Alkyne Click Chemistry reaction (SPAAC) (2.) and Tetrazine – trans-Cyclooctene (TCO) Ligation (3.) efficiently proceed without metal catalysis. The well-known Copper-free Azide-Phosphine reaction (Staudinger Ligation) is hampered by the instability of phosphines and slow reaction kinetics. Recent focus therefore shifted towards strain-promoted reactions with cyclooctynes and Tetrazine – TCO Ligation, respectively.

We selected the best performing CLICK reactions in terms of selectivity, reactivity, biocompatibility and stability!

1. Cu(I)-catalyzed Azide-Alkyne Click Chemistry (CuAAC) reaction

Clearly the most prominent example of click chemistry is the Cu(I)-catalyzed Azide-Alkyne Click Chemistry (CuAAC) reaction1. An Azide-functionalized molecule A reacts with a terminal Alkyne-functionalized molecule B thereby forming a stable conjugate A-B via a Triazole moiety (Fig. 2).

Figure 2: Principle of Cu(I)-catalyzed Azide-Alkyne Click Chemistry(CuAAC).

Since terminal Alkynes are fairly unreactive towards Azides, the efficiency of a CuAAC reaction strongly depends on the presence of a metal catalyst such as copper (Cu) in the +1 oxidation state (Cu(I)). Different copper sources and reduction reagents are available however, the Cu(II) salt CuSO4 as copper source in combination with ascorbate as a reduction reagent has been recommended for most biomolecule labeling applications11,12.

The use of CuAAC reactions in live cells is hampered by the toxicity of Cu(I) ions. This problem has been partially overcome by the use of Cu(I) chelating ligands such as THPTA that serve a dual purpose: 1) Acceleration of the CuAAC reaction by maintaining the Cu(I) oxidation state and 2) Protection of the biomolecule from oxidative damage.

Presolski et. al.11 and Hong et. al.12 provide a general protocol for CuAAC reactions that may be used as a starting point for the set up and optimization of individual assays.


• Small-sized azides and alkynes possess excellent substrate properties
• Optimization of assay conditions required (type & concentration of Copper source, reduction reagent and Copper ligand)
• Suitable if potential copper toxicity does not matter (not recommended for in vivo or live cell labeling)
• Slowest reaction speed compared to 2. and 3.

2. Strain-promoted Azide-Alkyne Click Chemistry (SPAAC) reaction

The requirement of a cytotoxic copper catalyst often limits the usage of CuAAC reactions (see 2.) A Copper free and thus non-toxic labeling method of Azides is the Strain-Promoted Azide - Alkyne Click Chemistry (SPAAC) reaction3. SPAAC reactions rely on the use of strained cyclooctynes that possess a remarkably decreased activation energy in contrast to terminal Alkynes and thus do not require an exogenous catalyst13.

A number of structurally varied cyclooctyne derivatives (e.g. DIFO, BCN, DIBAC, DIBO, ADIBO) have been developed that strongly differ in terms of reaction kinetics and hydrophility. Our SPAAC conjugation chemistry is based on the reaction of Azadibenzylcyclooctyne (ADIBO = DBCO = DIBAC) (Fig. 3).

Figure 3: Principle of Strain-Promoted Azide-Alkyne Click Chemistry (SPAAC). DBCO = ADIBO = DIBAC

Azadibenzocyclooctyne (ADIBO=DBCO)-based reagents combine high reactivity with sufficient hydrophility14,15 and thus allow low background labeling of Azide-functionalized molecules16 with even greater efficiency than CuAAC reactions. Azide-DBCO reactions are furthermore highly selective and therefore ideally suited for dual labeling approaches with Tetrazine - trans-Cyclooctene Ligation (see 3.)17.


• Faster detection of small-sized Azides compared to CuAAC reactions (see 2.)
• Copper free and thus non-toxic
• No catalyst or accessory reagents and thus no extensive optimization of assay conditions required
• Suitable for dual-labeling approaches in combination with Tetrazine - trans-Cyclooctene Ligation

3. Tetrazine-trans-Cyclooctene Ligation

The Tetrazine - trans-Cyclooctene Ligation constitutes a non-toxic biomolecule labeling method of unparalleled speed that is ideally suited for in vivo cell labeling and low concentration applications. A Tetrazine-functionalized molecule A reacts with a trans-Cyclooctene (TCO)-functionalized molecule B thereby forming a stable conjugate A-B via a Dihydropyrazine moiety (Fig. 4).

Figure 4: Principle of Tetrazine - trans-Cyclooctene Ligation. R1= Phenyl, R2= H or CH3

A number of structurally varied strained alkene and tetrazine derivatives have been developed that strongly differ in terms of reaction kinetics and stability. TCO has been selected (as strained alkene) since it possesses the highest reactivity towards tetrazine18,19.

The reactivity of the tetrazine derivatives towards TCO is determined by the substituents in the 3 position (Fig. 4, R1) and 6 position (Fig. 4, R2). Two Tetrazine versions with different reactivities and stability characteristics have been selected that meet specific application requirements. Tetrazine (R1=phenyl, R2=H) reagents are the ideal choice if a rapid reaction kinetic is the key aspect, whereas 6-Methyl-Tetrazine (R1=phenyl, R2=CH3) reagents are ideally suited if an improved chemical stability is required18.


• High-speed CLICK reaction that is ideally suited for in vivo cell labeling & low concentration applications
• Copper free and thus non-toxic
• No catalyst or accessory reagents and thus no extensive optimization of assay conditions required
• Suitable for dual-labeling approaches in combination with the strain-promoted Azide - DBCO reaction17

Overview of available CLICK Reagents

Fluorescent Dyes

Quencher Dyes

Non-fluorescent Dyes

Biotinylation Reagents

Bifunctional Reagents

Trifunctional Reagents

PEGylation Reagents



Amino Acids

Agarose & Magnetic Beads


Click for products »»

If you did not find the CLICK Reagent that you are looking for, please contact our techsupport.

Selected References

1. Kolb et al. (2001) Click chemistry: diverse chemical function from a few good reactions. Angew. Chem. Int. Ed. 40(11):2004.
2. Sletten et al. (2009) Bioorthogonal Chemistry: Fishing for Selectivity in a Sea of Functionality. Angew. Chem. Int. Ed. 48:6998.
3. Jewett et al. (2010) Cu-free click cycloaddition reactions in chemical biology. Chem. Soc. Rev. 39(4):1272.
4. Best et al. (2009) Click Chemistry and Bioorthogonal Reactions: Unprecedented Selectivity in the Labeling of Biological Molecules. Biochemistry. 48:6571.
5. Lallana et al. (2011) Reliable and Efficient Procedures for the Conjugation of Biomolecules through Huisgen Azide–Alkyne Cycloadditions. Angew. Chem. Int. Ed. 50:8794.
6. Grammel et al. (2013) Chemical Reporters for biological discovery. Nature Chemical Biology 9:475.
7. Xie et al. (2013) Cell-selective metabolic labeling of biomolecules with bioorthogonal functionalities. Current Opinion in Chemical Biology 17:747.
8. Su et al. (2013) Target identification of biologically active small molecules via in situ methods. Current Opinion in Chemical Biology 17:768.
9. Zeng et al. (2013) The Growing Impact of Bioorthogonal Click Chemistry on the Development of Radiopharmaceuticals. J Nucl Med 54:829.
10. Evans et al. (2007) The Rise of Azide–Alkyne 1,3-Dipolar 'Click' Cycloaddition and its Application to Polymer Science and Surface Modification. Australian Journal of Chemistry 60(6):3.
11. Presolski et al. (2011) Copper-Catalyzed Azide-Alkyne Click Chemistry for Bioconjugation. Current Protocols in Chemical Biology 3:153.
12. Stanislav et al. (2009) Analysis and Optimization of Copper-Catalyzed Azide-Alkyne Cycloaddition for Bioconjugation. Angew. Chem. Int. Ed. 48:9879.
13. Ess et al. (2008) Transition states of strain-promoted metal-free click chemistry: 1,3-dipolar cycloadditions of phenyl azide and cyclooctynes. Org. Lett. 10:1633.
14. Debets et al. (2010) Aza-dibenzocyclooctynes for fast and efficient enzyme PEGylation via copper-free (3+2) cycloaddition. Chem. Commun. 46:97.
15. Kuzmin et al. (2010) Surface Functionalization Using Catalyst-Free Azide-Alkyne Cycloaddition. Bioconjugate Chem. 21:2076.
16. Yao et al. (2012) Fluorophore Targeting to Cellular Proteins via Enzyme-Mediated Azide Ligation and Strain-Promoted Cycloaddition. J. Am. Chem. Soc. 134:3720.
17. Liang et al. (2012) Control and Design of Mutual Orthogonality in Bioorthogonal Cycloadditions. J. Am. Chem. Soc. 134:17904.
18. Selvaraj et al. (2012) trans-Cyclooctene – a stable, voracious dienophile for bioorthogonal labeling. Current Opinion in Chemical Biology 17:753.
19. Karver et al. (2011) Synthesis and Evaluation of a Series of 1,2,4,5-Tetrazines for Bioorthogonal Conjugation. Bioconjugate Chem. 22:2263.
20. Seckute et al. (2013) Expanding room for tetrazine ligations in the in vivo chemistry toolbox. Current Opinion in Chemical Biology 17:761.

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Brands: Jena Bioscience
Product groups: Chemicals