Click Chemistry Reaction

Click chemistry is a type of reaction that conforms to the principles of green chemistry and is characterized by mild reaction conditions, high yields, easy separation of products, and the absence of harmful by-products. This reaction not only provides a powerful tool for the synthesis of complex molecules, but can also be carried out under biocompatible conditions, opening up new possibilities for the development of chemical biology and materials science. Several common types of click reactions will be described here, including cycloaddition, nucleophilic ring-opening, carbonylation of non-alcoholic aldehydes, carbonylation of carbon-carbon multiple bonds, azide-phosphine coupling reactions, and other common types of click chemistry.

Cycloaddition reaction

Copper-catalyzed azide-alkyne cycloaddition (CuAAC)

CuAAC is one of the most common click reactions. Sharpless and Meldal independently discovered in 2002 that cycloaddition reactions between azides and alkynes catalyzed by copper(I) in the presence of reducing agents and/or stable ligands form a stable triazole moiety similar to amide bonds [1,4-disubstituted (trans)-1,2,3-triazole], known as the CuAAC reaction.

Fig.1 The CuAAC reaction process.

Through copper-catalyzed click chemistry (CuAAC), biomolecules labeled with azide groups (-N₃), such as DNA, can be linked to alkyne-based compounds, such as fluorescent dyes containing alkyne groups. This method is widely used in fluorescent labeling and imaging studies of biomolecules, enabling scientists to track and study specific biomolecules in living cells or tissues. Due to its high efficiency and rapid kinetic properties, CuAAC reactions have been widely used in the field of biobinding and organic synthesis, including the labeling and imaging of biomolecules such as proteins, glycans, lipids, and nucleic acids, as well as the construction of bioactive molecular analogue libraries.

Inverse electron demand Diels-Alder (IEDDA)

IEDDA Click Chemistry (Inverse Electron-Demand Diels-Alder) is an efficient and rapid cycloaddition reaction that removes a molecule of nitrogen to form an intermediate through tetrazine and trans-cyclooctene derivatives, and is further isomerized to obtain a stable dihydropyridazine product, which can be performed in organic solvents, water, cell culture media or cell lysates, and is widely used in bioorthogonal chemistry and materials science. Unlike the traditional Diels-Alder reaction, the IEDDA reaction achieves cycloaddition against the electron demand by using electron-rich diene (e.g., tetrazine) and electron-defective alkynes or alkenes. This reaction does not require metal catalysts and is mild, highly selective, and biocompatible. IEDDA click chemistry has shown great potential in biomolecular labeling, drug delivery, and smart materials development, making it a powerful tool in the field of chemical biology. Its efficient response capabilities in vivo and in vitro make it promising in precision medicine and new material design.

Fig.2 The IEDDA reaction process.

Strain-promoted azide-alkyne cycloaddition (SPAAC)CuAAC

In 2004, Bertozzi's team developed a strain-promoted azide-alkyne cyclic addition reaction (SPAAC), a copper-free click chemistry reaction. It does not require the use of metal catalysts, reducing agents, and stabilizing ligands and other reagents. Instead, the reaction utilizes the cyclic strain to form a stable triazole for enthalpy released by cyclooctyne (e.g., OCT, BCN, DBCO, DIBO, and DIFO). Although the reaction kinetics of SPAAC are slower than those of CuAAC, there is no doubt about its biocompatibility in living cells. At present, this type of reaction has been widely used in the formation of hybrid and block polymers, metabolic engineering, oligonucleotide labeling and other fields. A common example of SPAAC is the use of cyclooctyne, such as BCN, to react with azide-labeled proteins. This method enables protein labeling without compromising cell viability, making it ideal for live-cell imaging and studying the localization and dynamics of proteins within cells.

Fig.3 The SPAAC reaction process.

Thiol-Ene Click Chemistry

Mercapto-alene click reactions typically include a free radical-mediated thiol-alkene reaction and a nucleophile-catalyzed thiol-Michael addition reaction. The free-radical-mediated thiol-alkene reaction involves free radical initiation, thiol radical formation, alkenyl and thiol radical reactions, and chain growth processes. The nucleophile-catalyzed thiol-Michael addition reaction is performed by sulfhydryl deprotonation and the addition of nucleophiles to alkene groups.

The thiol-ene click reaction has many advantages, the sulfhydryl reagent has a wide range of sources, and this kind of reaction method is simple, does not need to remove water and oxygen, has a high yield and few by-products, so it has a wide range of applications in the fields of organic synthesis and material preparation. Sulfhydryl-labeled polymers react with double-bonded compounds to form cross-linked polymer networks. This method is used in materials science to prepare functional materials with specific properties, such as self-healing materials and biomedical materials.

Fig.4 Common types of thiol-ene click chemistry reactions. (ZHENG Shu-juan, et al., 2021)

Azido-phosphine coupling reaction

In 1919, organic chemists Staudinger and Meyer discovered that azide benzene (Ph-N₃) reacts with triphenylphosphorus (PPh₃) in tetrahydrofuran (THF) or ether to form Aza-ylide, which is hydrolyzed to form primary amine, which is called Staudinger's reaction.

The azide-phosphine coupling reaction (Staudinger linkage) reacts azide with phosphine to produce amines and phosphorus oxides. The reaction conditions are mild and efficient, and the resulting phospiimide has the properties of Yelide, which can react with different types of substrates to form imines, primary amines, and amides, which are widely used in biomarkers and drug conjugates. Staudinger ligation is used to link azide-modified fluorescent dyes with phosphine-modified antibodies to prepare fluorescent antibodies for cell imaging.

Fig.5 The Staudinger reaction process.

Carbonylation reaction of non-alcoholic aldehydes

Such reactions include the formation of hydrazone, oxime ether and aromatic heterocycles, aldehydes or ketones form imines with primary amines, the imines formed by the reaction of ketones with hydroxylamines are called oximes, and the imines produced by the reaction of ketones with hydrazine are called hydrazones. For oxime and hydrazone, the nitrogen atom is linked to the electronegative group, which can participate in the delocalization of the imine double bond, which is more stable than other imines. By introducing carbonyl functional groups, efficient molecular ligation is achieved under mild conditions. In drug development, pyrazoles are synthesized by carbonylation of non-alcoholic aldehydes, which exhibit antitumor and anti-inflammatory activities.

Carbon-carbon multiple bond addition reaction

These include dihydroxylation and epoxidation, in which complex organic molecular structures are constructed by introducing two or more groups on carbon-carbon double or triple bonds. In the synthesis of orlistat, the dihydroxylation reaction of double bonds is used to form a key part of its active molecule.

Nucleophilic ring-opening reaction

Nucleophilic ring-opening involves the ringing of molecules such as azidine, epoxide, and cyclic sulfate under the action of nucleophiles to form new compounds. The ring-opening reaction of ethylene oxide with amines is used to synthesize polyethylene glycol (PEG)-modified drugs, which can improve the water solubility and stability of drugs.

Other types of click chemistry

SuFEx Click Chemistry

In 2014, Sharpless and Dong's team worked together to develop a next-generation click chemistry, hexavalent sulfur-fluoride exchange (SuFEx). Unlike the first-generation copper-catalyzed azide-alkyne cycloaddition (CuAAC), SuFEx can be performed under metal-free conditions, utilizing an exchange reaction between high-valent sulfur compounds and nucleophiles to form stable bonds, which is critical for biochemical applications. This reaction has a flexible link form that allows for the construction of a variety of high-valent sulfur compound libraries. Moreover, the flexible and diverse forms of linking allow different types of SuFEx linkers to be conjugated with nucleophiles to achieve the construction of high-valent sulfur compound libraries.

Fig.6 Sulfur(VI)-Fluoride exchange reaction. (Zeng Daming, et al., 2023)

SuFEx chemistry is widely used in many fields such as [18F]-tracer labeling, antibody modification, biomolecular chemistry, drug development, polymeric materials, and so on. A typical example of a SuFEx reaction is the use of a hexavalent sulfur compound, such as sulfuryl fluoride, to react with a hydroxyl group containing a compound to form a sulfuryl compound. This method is widely used in drug development and the synthesis of polymeric materials, such as for the preparation of antibody-drug conjugates and functional polymers.

Reversible Click Chemistry

Reversible click chemistry is a type of click reaction that can be carried out reversibly under specific conditions. By controlling reaction conditions such as temperature, pH, or using a specific catalyst, the reaction can be switched between generating products and dissociating reactants. This type of reaction has important applications in the study of dynamic chemistry and self-assembled materials.

A common example of reversible click chemistry is the disulfide exchange reaction. Disulfide bonds can be dissociated to form mercaptans under the action of reducing agents and re-formed disulfide bonds under the action of oxidants. This reversibility makes it an important application in the study of self-healing materials and reconfigurable materials.

Summary

Click chemistry plays an important role in chemical biology and materials science due to its high efficiency, selectivity, and biocompatibility. The variety of different click chemistry reactions provides scientists with powerful tools to synthesize and modify complex molecules, advancing drug development, biomedical materials, and functional materials research. By continuously exploring and optimizing these reaction types, the application prospects of click chemistry will be broader, bringing more possibilities for scientific research and technological innovation.