Click Chemistry Tool

Click chemistry is a class of chemical reactions between pairs of reagents (known as click chemistry tools) that react only with each other under mild conditions and are effectively inert to naturally occurring functional groups such as amine groups. Due to its mild conditions and high selectivity, click chemistry has been widely used in bioconjugation, biomarking, and materials science in the pharmaceutical and biotechnology industries. This article focuses on several commonly used types of click chemistry tools, and illustrates the characteristics of each tool more clearly with examples.

Click on the chemical reaction category

Click chemistry can be divided into three broad categories: 1) Cu(I)-catalyzed azide-alkyne click chemistry (CuAAC); 2) strain-facilitated azide-alkyne click chemistry (SPAAC), which is biocompatible; 3) The connection between Tetrazine and an Olefin (trans-cyclooctene), this high-speed reaction does not contain copper and is ideal for cell labeling in vivo.

Azide

Azide click chemistry is a very popular chemical method that has been widely used in the pharmaceutical and biotechnology fields due to its mild conditions, high speed, and good biocompatibility. The first generation of azide click chemistry was achieved by the reaction of azide (N3) with alkynes (e.g., alkynyl propyl) in the presence of a copper catalyst to form a stable triazole bond. Second-generation azide click chemistry eliminates the need for copper through the reaction of azide molecules with structurally restricted alkynes such as DBCO (bisbenzocyclooctyne) or BCN (bicyclo[6.1.0]nonyne) bearing molecules.

Biotin-azide: N-(3-Azidopropyl)biotinamide is a form of biotin with terminal azide groups. Biotin-azide is used to prepare various biotinylated conjugates by Click Chemistry. Biotin-azide is a click chemistry that contains an azide group that undergoes a copper-catalyzed azide-alkyne cycloaddition reaction (CuAAC) with a molecule containing an alkyne group. It can also undergo a ring tension-driven alkyne-azide cycloaddition reaction (SPAAC) with molecules containing DBCO or BCN groups.

Fig.1 Chemical structural formula of Biotin-azide.

AHA: AHAs are amino acid analogs that are specifically modified with a small azide group and can be added to cultured cells and incorporated into the protein during active protein synthesis to detect azide-modified proteins using click chemistry protein assays containing TAMRA, Dapoxyl, or biotin alkynes.

The appeal of the azide functional group lies in its high selectivity and stability under most conditions. These properties are especially useful when other conjugated functional groups must be used due to limited stability, or when careful control of variables such as pH is required to ensure a high-yield reaction.

Alkynes

Alkyne reagents can react with azide-containing compounds or biomolecules via copper-catalyzed azide-alkyne click chemistry to produce stable triazole bonds. The unique properties of these alkyne reagents make them valuable in biolabeling and bioconjugation. For example, the introduction of an alkyne group into a biomolecule allows it to react with azide to form a stable triazole derivative, which is widely used in proteomics, drug development, and molecular imaging.

Fig.2 Structures of several representative alkyne reagents.

BCN

BCN reagents are a class of click chemistry reagents containing highly active BCN (bicyclo[6.1.0]nonyne) groups. The BCN series products include endo-BCN-OH, endo-BCN-PNB, endo-BCN-PEG2-NHS ester, etc. BCN reagents can react with azide-labeled molecules or biomolecules by copper-free click chemistry. It can be run in aqueous buffers or organic solvents, depending on the properties of the substrate molecule. Reagents with PEG arms increase the hydrophilicity of the compound. BCN reagents have been widely used in bioconjugation, labeling, and chemical biology.

Fig.3 Chemical structure diagram of BCN reagents.

DBCO

DBCO reagents are a class of click chemical labeling reagents that contain a very reactive DBCO (dibenzocyclooctyne) group. DBCO reagents can react with azide-labeled molecules or biomolecules by copper-free click chemistry. The reaction can be performed in an aqueous buffer or an organic solvent, depending on the nature of the substrate molecule. Reagents with PEG arms increase the hydrophilicity of the compound. DBCO reagents have been widely used in bioconjugation, labeling, and chemical biology.

Fig.4 Structures of several representative DBCO reagents.

TCO

TCO click chemistry is a third-generation click chemistry that is widely used for labeling and bioconjugation due to its speed and biocompatibility. It is a reaction by TCO (trans-cyclooctene) reagent and tetrazine moiety in the reverse electron demand Dils-Alder (IEDDA) reaction, followed by the reverse DA reaction to eliminate nitrogen. This reaction condition is mild, biocompatible, and suitable for labeling and conjugation within living cells.

Fig.5 Structures of several representative TCO reagents.

Tetrazine

Tetraazine reagents are a class of click chemical labeling reagents containing reactive tetrazine groups. The tetrazine reagent is highly reactive with TCO (trans-cyclooctene) in the reverse electron demand Diels-Alder reaction and the inverse Diels-Alder reaction to eliminate nitrogen. This is a very rapid response for bioconjugation at low concentrations in labeling live cells, molecular imaging, and other bioconjugation applications. Tetrazine reagents also have important applications in materials science and drug development, and their rapid and efficient reaction properties make their application in complex biological systems more convenient.

Fig.6 Structures of several representative Tetrazine reagents.

Click on the reaction ligand

The efficiency of the azide-alkyne click chemical reaction (CuAAC) catalyzed by copper (Cu(I)) is largely dependent on the presence of copper ions Cu(I). However, copper(I) ions are susceptible to oxidation and disproportionation, and also have cytotoxic effects on living cells. Ligands with tertiary amines and triazines stabilize Cu(I) and accelerate the reaction. Due to their biorthogonality, low cytotoxicity, and reaction rate, these ligands further expand the use of CuAAC in chemical biology.

TBTA: Tris(benzyltriazolylmethyl)amine (TBTA) is the earliest copper ion ligand for click chemical catalysis. TBTA is insoluble in water and often needs to be dissolved in DMSO or DMF first, and complexed with copper ions in DMSO/t-butanol 3:1 (v/v) solution. TBTA is suitable for click chemical labeling reactions in organic solvents or in aqueous organic solvent mixtures.

BTTAA: It is a new generation of water-soluble click chemical reaction catalyst, which is similar in structure to BTTPS, but has poor water solubility than BTTPS. Compared to previous-generation catalysts such as THPTA or TBTA, BTTAA catalyzes more efficiently and reacts faster.

Fig.7 Structures of Click Chemical copper ligands.

Summary

Click chemistry tools are widely used in pharmaceutical, biotechnology, and materials science due to their high selectivity, rapid response, and mild conditions. Through the linkage between CuAAC, SPAAC, and Tetrazine-Olefins, these chemistries provide a diverse range of solutions for different experimental needs and biological systems, facilitate the development of novel materials and drugs, and provide strong support for scientific research and technological innovation.